European Journal of Pharmacology (v.791, #C)

Morphine-induced Straub tail reaction in mice treated with serotonergic compounds by Irina V. Belozertseva; Olga A. Dravolina; Margarita A. Tur; Marina G. Semina; Edwin E. Zvartau; Anton Yu. Bespalov (1-7).
Constitutively active 5-HT2 receptors have been suggested to contribute to motoneuronal excitability, muscle spasms and spasticity. Accordingly, 5-HT2C receptor inverse agonists have been demonstrated in pilot experiments to reduce spasticity in animal model of spasticity and patients with spinal cord injuries. Thus, 5-HT2C receptor inverse agonists may represent a novel class of anti-spasticity agents justifying a search for compounds with robust 5-HT2C receptor inverse agonist activity either among the existing medications or via a dedicated drug discovery program. Morphine-induced Straub tail response in mice is regarded as a model of transient spasticity that may be suitable for supporting such drug discovery efforts. Subcutaneous injection of morphine (10–60 mg/kg) induced a dose-dependent Straub tail reaction in male Swiss mice with maximum response obtained 15–30 min after the morphine administration. When given prior to morphine, 5-HT2B/2C receptor inverse agonists cyproheptadine (1–10 mg/kg, i.p.) and SB206553 (0.3–3 mg/kg, i.p.) diminished Straub tail reaction dose-dependently without affecting spontaneous locomotor activity. In contrast, 5-HT2B/2C receptor antagonist methysergide (1–5.6 mg/kg, i.p.) and 5-HT2C receptor antagonist SB242084 (1–5.6 mg/kg, i.p.) as well as 5-HT2A receptor inverse agonist pimavanserin (1–10 mg/kg, i.p.) had no appreciable effects on Straub tail response. Taken together, the findings indicate that constitutive activity of 5-HT2B/2C receptor may be involved in the mechanisms of morphine-induced spasticity. Thus, morphine-induced Straub tail response may be evaluated further as a candidate higher throughput test to identify 5-HT2C receptor inverse agonists with anti-spasticity effects in vivo.
Keywords: Transient spasticity; Straub tail reaction; 5-HT2 receptor; Cyproheptadine; SB206553; Mice;

Mechanistic insight of diabetic nephropathy and its pharmacotherapeutic targets: An update by Niloy Bhattacharjee; Sujata Barma; Nandita Konwar; Saikat Dewanjee; Prasenjit Manna (8-24).
Diabetic nephropathy (DN), a chronic complication of diabetes, is charecterized by glomerular hypertrophy, proteinuria, decreased glomerular filtration, and renal fibrosis resulting in the loss of renal function. Although the exact cause of DN remains unclear, several mechanisms have been postulated, such as hyperglycemia-induced renal hyper filtration and renal injury, AGEs-induced increased oxidative stress, activated PKC-induced increased production of cytokines, chemokines, and different inflammatory and apoptotic signals. Among various factors, oxidative stress has been suggested to play a major role underlying the onset and propagation of DN. It triggers several signaling pathways involved in DN, like AGEs, PKC cascade, JAK/STAT signaling, MAPK, mTOR, and SMAD. Oxidative stress-induced activation of both inflammatory and apoptotic signals are two major problems in the pathogenesis of DN. The FDA approved pharmacotherapeutic agents affecting against polyol pathway principally include anti-oxidants, like α-lipoic acid, vitamin E, and vitamin C. Kremezin and benfotiamine are the FDA approved AGEs inhibitors, another therapeutic target against DN. Ruboxistaurin, telmizartan, rapamycin, fenofibrate, aliskiren, and manidipine are some FDA approved pharmacotherapeutics effective against DN via diverse mechanisms. Beside this, some therapeutic agents are still waiting for FDA approval and few drugs without FDA approval are also prescribed in some countries for the management of DN. Despite the medications available in the market to treat DN, the involvement of multiple mechanisms makes it difficult to choose an optimum therapeutic agent. Therefore, much research is required to find out new therapeutic agent/strategies for an adequate pharmacotherapy of DN.
Keywords: Diabetic nephropathy; Hyperglycemia; Oxidative stress responsive signaling cascade; Pharmacotherapeitics;

This study shows that in spontaneously hypertensive rats (SHR) of 14-weeks-old, the sympathetically-induced, but not noradrenaline-induced tachycardic response are higher than age-matched Wistar normotensive rats. Furthermore, in SHR the sympathetically-induced tachycardic response was: (1) unaffected by moxonidine (3 μg/kg min); (2) partially inhibited by B-HT 933 (30 μg/kg min), both at the lowest doses; and (3) completely inhibited by the highest doses of B-HT 933 (100 μg/kg min), moxonidine (10 μg/kg min) or agmatine (1000 and 3000 μg/kg min) while the noradrenaline-induced tachycardic responses remained unaffected by the above compounds, except by 3000 μg/kg min agmatine. In SHR, 300 μg/kg rauwolscine failed to block the sympatho-inhibition to 100 μg/kg min B-HT 933 or 10 μg/kg min moxonidine, but 1000 μg/kg rauwolscine abolished, partially antagonized, and did not modify the sympatho-inhibition to the highest doses of B-HT 933, moxonidine, and agmatine, respectively, 3000 μg/kg AGN 192403 or 300 μg/kg BU224 given alone had no effect in the moxonidine- or agmatine-induced sympatho-inhibition, and the combination rauwolscine plus AGN 192403 but not plus BU224, abolished the sympatho-inhibition to the highest doses of moxonidine and agmatine. In conclusion, the sympathetically-induced tachycardic responses in SHR are inhibited by moxonidine and agmatine. The inhibition of moxonidine is mainly mediated by prejunctional α2-adrenoceptors and to a lesser extent by I1-imidazoline receptors, while the inhibition of agmatine is mediated by prejunctional α2-adrenoceptors and I1-imidazoline receptors at the same extent. Notwithstanding, the inhibitory function of α2-adrenoceptors seems to be altered in SHR compared with Wistar normotensive rats.
Keywords: Agmatine; α2-adrenoceptor; Hypertension; I1-imidazoline receptor; Moxonidine;

Biochemical and molecular mechanisms underlying the chemopreventive efficacy of rosmarinic acid in a rat colon cancer by Karthikkumar Venkatachalam; Sivagami Gunasekaran; Nalini Namasivayam (37-50).
To shed light on colon cancer chemoprevention, natural phytochemicals attract researchers by virtue of their beneficial biological effects. The chemopreventive potential of rosmarinic acid (RA) was tested by using the colon carcinogen, 1,2-dimethylhydrazine (DMH) by evaluating the Aberrant crypt foci (ACF), tumour incidence, lipid peroxidative byproducts, phase I & II drug metabolizing enzymes, cell proliferative and apoptotic proteins. Rats were divided into six groups and received modified pellet diet. Group 1 served as control rats, group 2 rats received RA (5 mg/kg b.w. p.o.), rats in groups 3–6 received DMH (20 mg/kg b.w., s.c.) for the first fifteen weeks. In addition to DMH, groups 4–6 received RA at the dose of 5 mg/kg b.w. during initiation, post initiation stages and also for the entire study period. DMH treated rats showed an increase in the development of ACF, tumour formation and multiplicity and decrease in lipid peroxidative byproducts. Moreover, it modulates xenobiotic enzymes and reduces the expressions of proapoptotic proteins; increases expressions of anti apoptotic proteins at the end of the study. Supplementation with RA to carcinogen treated rats protected them from the above deleterious effects caused by DMH and thus RA may be used as a potent chemopreventive agent.Display Omitted
Keywords: DMH; Apoptosis; Antioxidants; ACF; Chemoprevention;

Edaravone alleviates cisplatin-induced neurobehavioral deficits via modulation of oxidative stress and inflammatory mediators in the rat hippocampus by Ashok Jangra; Mohit Kwatra; Tavleen Singh; Rajat Pant; Pawan Kushwah; Sahabuddin Ahmed; Durgesh Dwivedi; Babita Saroha; Mangala Lahkar (51-61).
Cisplatin is a chemotherapeutic agent used in the treatment of malignant tumors. A major clinical limitation of cisplatin is its potential toxic effects, including neurotoxicity. Edaravone, a potent free radical scavenger, has been reported to have the neuroprotective effect against neurological deficits. The aim of the present study was to determine the neuroprotective effect of edaravone against cisplatin-induced behavioral and biochemical anomalies in male Wistar rats. Our results showed that cisplatin (5 mg/kg/week, i.p.) administration for seven weeks caused marked cognitive deficits and motor incoordination in rats. This was accompanied by oxido-nitrosative stress, neuroinflammation, NF-κB activation and down-regulation of Nrf2/HO-1 gene expression level in the hippocampus. Edaravone (10 mg/kg/week, i.p.) treatment for seven weeks inhibited the aforementioned neurobehavioral and neurochemical deficits. Furthermore, edaravone was found to up-regulate the gene expression level of Nrf2/HO-1 and prevented the cisplatin-induced NF-κB activation. These findings demonstrated that oxido-nitrosative stress and inflammatory signaling mediators play a key role in the development of cisplatin-induced neurobehavioral deficits which were prevented by edaravone treatment.
Keywords: Cisplatin; Edaravone; Oxido-nitrosative stress; Neurobehavioral deficits; Neuroinflammation;

Inflammatory cytokines can induce the expression of cell adhesion molecules (CAMs) in endothelial cells. The induction may play an important role in attracting circulating tumor cells (CTCs) to endothelial cells. S-nitrosocaptopril (CapNO) is known to produce vasorelaxation and interfere the hetero-adhesion of CTCs to vascular endothelium via down-regulating the expression of CAMs. To elucidate the mechanisms underlying the inhibition of CapNO on CAMs, in this study, we examined the relationship between cytokines and CAMs expression and investigated the effects of CapNO on cytokine-induced NF-кB and JAK/STAT signal pathways. The activation of CAMs by cytokines was dependent on concentrations and reaction time of cytokines, and the combination of cytokines could produce a strong synergistic effect. IL-1β induced the expression of CAMs on endothelial cells by activating NF-кB and JAK/STAT pathways. CapNO inhibited IL-1β-stimulated NF-кB pathway by down-regulating IKK-α and inducing IкB-α directly. CapNO also inhibited JAK/STAT pathway by inhibiting JAK2 and STAT3 expressions. These effects bring about down-regulating CAMs expression on endothelial cells. These results suggest that CapNO may interrupt adhesion of cancer cells to endothelium by suppressing CAMs via inhibiting the NF-кB and JAK/STAT pathways in endothelial cells.
Keywords: S-nitrosocaptopril; Cancer metastatic prevention; Cell adhesion molecules; NF-κB; JAK/STAT; Inflammation;

Imidafenacin, an antimuscarinic agent for treating overactive bladder, has an antidiuretic effect, but the detailed mechanisms of action remain unclear. The cholinergic and vasopressin systems are known to interact, for example, in the suppression of vasopressin-induced water reabsorption through muscarinic stimulation in the renal collecting duct. We, therefore, investigated whether vasopressin signaling pathway would participate in the antidiuretic effect of imidafenacin. In female Sprague-Dawley rats, urine production was measured by collecting urine from cystostomy chatheter using a Bollman restraining cage for 2 h after drug i.v. injection and water load (25 ml/kg p.o.). Both imidafenacin and a vasopressin V2 receptor agonist desmopressin acetate (desmopressin) dose-dependently suppressed urine production. The combination of imidafenacin and desmopressin at the minimum effective doses suppressed the urine production more strongly than each alone. Mozavaptan hydrochloride (mozavaptan, 3 mg/kg), a vasopressin V2 receptor antagonist, completely inhibited the antidiuretic effects of imidafenacin and desmopressin at their respective minimum effective doses. The antidiuretic effect of desmopressin emerged at the maximum antidiuretic dose level (0.1 µg/kg) even under mozavaptan-treatment, whereas that of imidafenacin (300 µg/kg) was still kept suppressed by mozavaptan. When 300 µg/kg imidafenacin was added to the combination of mozavaptan 3 mg/kg and desmopressin 0.1 µg/kg, the antidiuretic effect was further enhanced. The present study suggests that vasopressin signaling pathway participates in the antidiuretic effect of imidafenacin, and that imidafenacin exerts its antidiuretic effects by enhancing some part of the vasopressin signaling pathway in orally water-loaded rats.
Keywords: Imidafenacin; Vasopressin; Desmopressin; Antidiuretic; Water-loaded rats;

Role of dynorphin in hypoxic pulmonary hypertension by Juan Li; Xiaojie Liang; Yaguang Zhou; Shumiao Zhang; Fan Yang; Haitao Guo; Rong Fan; Na Feng; Min Jia; Yueming Wang; Mingchao Liu; Jianming Pei (78-84).
Previously study showed κ-opioid receptor stimulation with exogenous κ-opioid receptor agonist elicited a protective effect against hypoxic pulmonary hypertension (HPH). However, the effect of endogenous κ-opioid receptor agonist dynorphin A on HPH remains unclear. This study was to determine the role of dynorphin in HPH. Hypoxia for 2 weeks induced HPH. Compared with the HPH group, the HPH + nor-BNI (a selective κ-opioid receptor antagonist) group showed a significant increase in mean pulmonary arterial pressure (mPAP). Exogenous treatment with dynorphin A 1–13 significantly decreased mPAP in HPH rat. In addition, we evaluated the effect of exogenous κ-opioid receptor agonist U50,488H on mPAP. The anti-HPH effect of dynorphin A was less than that of U50,488H. Meanwhile, level of dynorphin A in serum and lung was increased during hypoxia for 2 weeks, while it decreased after hypoxia for 4 weeks. In addition, both the level of ET-1 and AngII were increased during hypoxia. Dynorphin A 1–13 and U50,488H time-dependently relaxed pulmonary artery from both normal and HPH rats. The relaxation of dynorphin A was less than that of U50,488H. Dynorphin A 1–13 inhibited the proliferation of pulmonary artery smooth muscle cells (PASMCs) during hypoxia, which was blocked by nor-BNI. κ-opioid receptor expression increased in PASMCs in both normoxia exposed to dynorphin A 1–13 and during hypoxia. Hypoxia-induced increase was enhanced by dynorphin A 1–13 and abolished by nor-BNI. In conclusion, endogenous dynorphin A released in the early stage of hypoxia plays a protective effect against HPH via stimulation of κ-opioid receptor.
Keywords: Dynorphin A; Opioid receptor; Pulmonary artery hypertension; Hypoxia;

Differential effects of R-isovaline and the GABAB agonist, baclofen, in the guinea pig ileum by Timothy Fung; Khalid A. Asseri; Yahya I. Asiri; Richard A. Wall; Stephan K.W. Schwarz; Ernest Puil; Bernard A. MacLeod (85-90).
R-isovaline is a non-proteinogenic amino acid which produces analgesia in a range of nociceptive assays. Mediation of this effect by metabotropic receptors for γ-aminobutyric acid (GABA) and glutamate, demonstrated by previous work, may depend on the type of tissue or receptor system. The objective of this study was to assess the activity of R-isovaline acting at GABAB and group II metabotropic glutamate receptors in guinea pig ileum, which is known to exhibit well-defined responses to GABAB agonists such as baclofen. The effects of bath-applied R-isovaline and RS-baclofen were examined on electrically evoked contractions of guinea pig ileum and during GABAB antagonism by CGP52432. In separate experiments, the group II metabotropic glutamate receptor agonist, LY354740 was applied to determine the functional presence of these receptors. R-isovaline (1–100 mM) decreased the amplitude of ileal muscle contractions and increased tension. RS-baclofen reduced contraction amplitude, but decreased tension. CGP52432 did not prevent the effects of R-isovaline on contraction amplitude, but antagonized effects of RS-baclofen on contraction amplitude. The group II metabotropic glutamate receptor agonist, LY354740, produced no detectable effects on evoked contractions. R-isovaline differed significantly from RS-baclofen in its actions in the guinea pig ileum, indicated in particular by the finding that CGP52432 blocked only the effects of RS-baclofen. The ileal tissue did not respond to a group II metabotropic glutamate receptor agonist, previously shown to co-mediate R-isovaline analgesia. These findings raise the possibility of a novel therapeutic target at unknown receptors for R-isovaline-like compounds in the guinea pig ileum.
Keywords: Isovaline; Baclofen; GABAB; Glutamate; Metabotropic;

Myocardial infarction (MI) and hypertension are the leading cause of death worldwide so protection of heart is focus of intense research. Rho-kinase, a downstream effector of protein involved in MI and hypertension, is inhibited by ibuprofen. This study aims to elucidate cardioprotective effect of ibuprofen in rats. MI was produced in rats with 85 mg/kg isoproterenol (ISO) administered s.c. twice at an interval of 24 h. The rats were randomized into six groups: (I) Normal; (II) ISO; (III) ISO + ascorbic acid (250 mg/kg p.o.); (IV-VI) ISO + ibuprofen (30, 60 and 90 mg/kg p.o). After the completion of the study period of 21 days, cardiac function and biomarkers were assessed. Pre-treatment with ibuprofen (30, 60 and 90 mg/kg p.o) ameliorated high BP and left ventricular dysfunction, furthermore it prevented the rise in CKMB, LDH and α-HBDH, suggesting the effect of ibuprofen in maintenance of cell membrane integrity. In addition, it also prevented alteration in the levels of electrolytes, ATPase activity and antioxidant status. Ibuprofen suppressed ISO-induced ROCK-1 mRNA expression and histological changes. Ibuprofen provided cardioprotection in a model of myocardial infarction, by restoring most of the altered physical, physiological, biochemical, haemodynamic parameters, antioxidant status, and histological changes and by inhibiting ROCK-1 mRNA expression.
Keywords: Rho kinase; Ibuprofen; Isoproterenol; ROCK-1;

Evaluation of dose-dependent effects of the proteasome inhibitor bortezomib in human platelets by Juergen Koessler; Julia Etzel; Katja Weber; Markus Boeck; Anna Kobsar (99-104).
Platelets express key proteins of the proteasome system and contain protein ubiquitination pathways. The functional role of the proteasome system in platelets, however, is still subject of studies. In addition to its role as anticancer drug, the potent and selective proteasome inhibitor bortezomib can be used for experimental proteasome research. Since it is mandatory to know exact dose-effect relationships, we intended to evaluate dose-dependent specific bortezomib effects on basal and on agonist-induced proteasome activitiy, on levels of poly-ubiquitinated proteins and on platelet aggregation.In washed platelets, unstimulated or stimulated with different agonists and pre-incubated with various bortezomib concentrations, the proteasome activity was determined by a fluorometric assay. The levels of poly-ubiquitinated proteins were assessed by an immunoassay kit. Platelet aggregation was measured by light transmission aggregometry in platelet-rich-plasma.Platelet agonists stimulate both, the proteasome activity and the accumulation of poly-ubiquitinated proteins in platelets. Bortezomib inhibits the basal and the agonist induced proteasome activity and increased the content of poly-ubiquitinated proteins in a concentration dependent manner. Bortezomib concentrations in the nM-range causing complete blockade of platelet proteasome activity do not affect agonist induced platelet aggregation, indicating that the level of platelet proteasome activity is not directly linked with the induction of platelet aggregation. Bortezomib in the µM-range may tamper platelet aggregation, possibly due to unspecific and toxic effects.
Keywords: Bortezomib; Platelet; Proteasome; Ubiquitination; Aggregation;

2, 3, 4′, 5-tetrahydroxystilbene-2–0-β-D glucoside (TSG) could inhibit cardiac remodeling in response to pressure overload. Peroxisome proliferator-activated receptor gamma (PPAR-γ) has been recognized as a potent, endogenous antifibrotic factor and maintaining a proper expression level in myocardium is necessary for assuring that structure and function of heart adapt to pressure overload stress. The aim of the present study was to investigate whether PPAR-γ is involved in the beneficial effect of TSG on pressure overload-induced cardiac fibrosis. TSG (120 mg/kg/day) or TSG (120 mg/kg/day) plus the PPAR-γ antagonist GW9662 (1 mg/kg/day) was administered to rats with pressure overload induced by abdominal aortic banding. 30 days later, pressure overload-induced hypertension, cardiac dysfunction and fibrosis were significantly inhibited by TSG. TSG also significantly reduced collagen I, collagen III, fibronectin and plasminogen activator inhibitor (PAI)−1 expression, as makers of myocardial fibrosis. Theses anti-fibrotic effects of TSG in pressure overloaded hearts could be abrogated by co-treatment with GW9662. Accordingly, upregulated PPAR-γ protein expression by TSG in pressure overloaded hearts was also reversed by co-treatment with GW9662. Additionally, the inhibitory effects of TSG on angiotensin II induced cardiac fibroblasts proliferation, differentiation and expression of collagen I and III, fibronectin and PAI-1 were abrogated by PPAR-γ antagonist GW9662 and PPAR-γ silencing. Furthermore, TSG directly increased PPAR-γ gene expression at gene promoter, mRNA and protein level in angiotensin II-treated cardiac fibroblats in vitro. Our results suggested that upregualtion of endogenous PPAR-γ expression by TSG may be involved in its beneficial effect on pressure overload-induced cardiac fibrosis.
Keywords: 2,3,4',5-Tetrahydroxystilbene-2-O-beta-D-glycoside; Cardiac fibrosis; Peroxisome proliferator-activated receptor gamma;

In vitro and in vivo pharmacological characterization of SSD114, a novel GABAB positive allosteric modulator by Alessandra Porcu; Carla Lobina; Daniela Giunta; Maurizio Solinas; Claudia Mugnaini; M. Paola Castelli (115-123).
Positive allosteric modulators (PAMs) of the GABAB receptor have emerged as a novel approach to the pharmacological manipulation of the GABAB receptor, enhancing the effects of receptor agonists with few side effects. Here, we identified N-cyclohexyl-4-methoxy-6-(4-(trifluoromethyl)phenyl)pyrimidin-2-amine (SSD114) as a new compound with activity as a GABAB PAM in in vitro and in vivo assays. SSD114 potentiated GABA-stimulated [35S]GTPγS binding to native GABAB receptors, whereas it had no effect when used alone. Its effect on GTPγS stimulation was suppressed when GABA-induced activation was blocked with CGP54626, a competitive antagonist of the GABAB receptor. SSD114 failed to potentiate WIN55,212,2-, morphine- and quinpirole-induced [35S]GTPγS binding to cortical and striatal membranes, respectively, indicating that it is a selective GABAB PAM. Increasing SSD114 fixed concentrations induced a leftward shift of the GABA concentration-response curve, enhancing the potency of GABA rather than its efficacy. SSD114 concentration-response curves in the presence of fixed concentrations of GABA (1, 10, and 20 μM) revealed a potentiating effect on GABA-stimulated binding of [35S]GTPγS to rat cortical membranes, with EC50 values in the low micromolar range. Bioluminescence resonance energy transfer (BRET) experiments in Chinese Hamster Ovary (CHO)-cells expressing GABAB receptors showed that SSD114 potentiates the GABA inhibition of adenylyl-cyclase mediated by GABAB receptors. Our compound is also effective in vivo potentiating baclofen-induced sedation/hypnosis in mice, with no effect when tested alone. These findings indicate that SSD114, a molecule with a different chemical structure compared to known GABAB PAMs, is a novel GABAB PAM with potential usefulness in the GABAB-receptor research field.
Keywords: GABAB receptors; Positive allosteric modulator; GTPγS binding; Baclofen-induced sedation/hypnosis;

After spinal cord injury (SCI), there is an acute phase of alternatively activated (M2) macrophage infiltration, followed by a long-lasting phase of classically activated (M1) macrophage accumulation in the wound, which is believed to derail healing and compromize organ functions. Thus, agents which are able to modulate macrophage phenotypes may provide significant benefits to SCI patients. In the present study, we demonstrate that the niacin receptor HCA2 is specifically expressed on the cell surface of M1 but not M2 macrophages. Treatment of M1 macrophages with niacin (300 μM) resulted in down-regulation of the p65 NF-κB phosphorylation, associated with a marked decrease in the levels of M1 markers, including CD86, IL-12, and IL-6, and a significant increase in the expressions of M2 markers, such as CD206, IL-10, and IL-13, suggesting that niacin causes a shift of M1 to M2. Moreover, treatment of the M1-oligodendrocyte precursor cell (OPC) co-cultures with niacin markedly promoted the expression of myelin binding protein (MBP). After SCI in C57/BL6 mice for a week, a marked accumulation of M1 macrophages, which expressed HCA2 receptor, was evident in the wound. Treatment of the SCI mice with niacin (100 mg/kg) resulted in a dramatic decrease in the number of M1 macrophages and a significant increase in the number of M2 macrophages in the wound. This was associated with a robust inflammation resolution, attenuation of demyelination and neurofilament loss, and significant improvement of locomotor function. Thus, HCA2 receptor may serve as a therapeutic target to promote post-SCI recovery.
Keywords: Niacin; HCA2 receptor; Spinal cord injury; Inflammation; Macrophages;

Anti-cancer activity and potential mechanism of a novel aspirin derivative by Ming Zong; Dan-Dan Fan; Shan Lin; Yan-Ping Song; Ze-Yu Wang; Xiao-Liang Ma; Wei-Hua Qiu; Yu-Hua Bai; Lei Li; Sen Li (137-146).
Aspirin has been used in the treatment and chemoprevention of many malignant cancers. The mechanism of its anti-cancer activity mainly involves the inhibition of cyclooxygenase-2 (COX-2). However, the application of aspirin is limited by the serious gastric mucosal damage that accompanies its usage. We have previously reported the preparation of a novel aspirin derivative that we named Ca-Asp, and showed that it causes less damage to gastric mucosa of rat and inhibits the expression of COX-2 to higher degree than Asp. However, the anti-cancer effect and mechanism of Ca-Asp was not demonstrated. In this study, the anti-cancer effect of Ca-Asp was investigated and compared with those of Asp and Hydroxyapatite (Hap) at the cell level. The results showed that treatment of SGC-7901 cells (human gastric cancer cell line) with 200–400 μg/ml Ca-Asp resulted in significant reduction in cell viability, compared to treatment with either Asp or Hap, and at a higher concentration (500 μg/ml). Subsequent investigation into the possible underlying mechanism showed that Ca-Asp induced apoptosis and caused cell cycle arrest at the G1 phase. Ca-Asp also up-regulated the levels of caspase-3 and p53, but down regulated the level of cyclin D1, NF-κB, COX-2 and PGE2. Furthermore, simultaneous treatment of SGC-7901 cells with Ca-Asp and exogenous PGE2 reduced the anti-proliferative effect of Ca-Asp on the cells. Taken together, the results suggested that Ca-Asp might act as a potential anti-cancer drug, and that its suppression of PGE2 production might constitute an important part of its anti-cancer activity.
Keywords: Ca-Asp; Aspirin-derivative; Anticancer effect; Mechanism;

Thyroid hormones have important role in metabolism and impairment of glucose metabolism and insulin secretion has been shown in hypothyroid rats but the exact mechanisms for this defect are poorly understood. The aim of this study was to investigate the effect of hypothyroidism on oxidative stress parameters, insulin secretory pathway and histomorphometric changes of pancreas.In the isolated islets of the control and methimazole -treated hypothyroid insulin secretion and content, ATP production, Glucokinase, and hexokinase specific activity and kATP and L-type channels sensitivity were assayed. In order to determine oxidative stress parameters, antioxidant enzymes and lipid peroxidation were measured in pancreatic homogenates. Histomorphometric changes and histochemistry of the islet in both groups were compared.Results showed that plasma glucose and insulin concentration and their area under the curve during IPGTT in hypothyroid group were respectively higher and lower than the controls. In the hypothyroid islets, glucose stimulated insulin secretion, ATP production, hexokinase and glucokinase activities were decreased. Hypothyroid induced a significant increased lipid peroxidation, and decreased the antioxidant enzyme activity. Compared with the control group, insulin antibody positivity, the total volume of the pancreas, islets, and the total number as well as the mean volume of the beta cells were also significantly decreased in the hypothyroid group.These findings indicate that oxidative stress produced under hypothyroidism could have a role in progression of pancreatic β-cell dysfunction, reduced beta cell mass and decreased glucokinase activity, impairing glucose tolerance and insulin secretion.
Keywords: Hypothyroidism; Oxidative stress; Beta cell mass; Insulin secretion; Histomorphometry;

Associations between autophagy, the ubiquitin-proteasome system and endoplasmic reticulum stress in hypoxia-deoxygenation or ischemia-reperfusion by Tao Fan; Zhixin Huang; Lei Chen; Wei Wang; Boyou Zhang; Yao Xu; Shize Pan; Zhangfan Mao; Hao Hu; Qing Geng (157-167).
The activation of autophagy has been demonstrated to exert protective roles during hypoxia-reoxygenation (H/R)-induced brain injuries. This study aimed to investigate whether and how preconditioning with a proteasome inhibitor (MG-132), a proteasome promoter (Adriamycin, ADM), an autophagy inhibitor (3-methyladenine, 3-MA) and an autophagy promoter (Rapamycin, Rap) affected endoplasmic reticulum stress (ERS), the ubiquitin-proteasome system (UPS), autophagy, inflammation and apoptosis. Ubiquitin protein and 26S proteasome activity levels were decreased by MG-132 pretreatment but increased by ADM pretreatment at 2 h, 4 h and 6 h following H/R treatment. MG-132 pretreatment led to the increased expression of autophagy-related genes, ER stress-associated genes and IκB but decreased the expression levels of NF-κB and caspase-3. ADM pretreatment led to the decreased expression of autophagy-related genes, ERS-associated genes and IκB but increased the expression of NF-κB and caspase-3. Pretreatment with 3-MA reduced the expression of autophagy-related genes, autophagy and UPS co-related genes, as well as apoptosis-related although the latter was increased by Rap pretreatment at 2 h, 4 h and 6 h following H/R treatment. In vivo, pretreatment of rats with ADM, MG-132, 3-MA or Rap followed by ischemia-reperfusion (I/R) treatment resulted in similar changes. Proteasome inhibition preconditioning strengthened autophagy and ER stress but decreased apoptosis and inflammation. Autophagy promotion preconditioning exhibited similar changes. The combination of a proteasome inhibitor and an autophagy promoter might represent a new possible therapy to treat H/R or I/R injury-related diseases.
Keywords: Endoplasmic reticulum stress; Autophagy; Proteasome; Hypoxia-reoxygenation injury; Ischemia-reperfusion injury; Alveolar macrophage;

Propofol inhibits hERG K+ channels and enhances the inhibition effects on its mutations in HEK293 cells by Sheng-Na Han; Ying Jing; Lin-Lin Yang; Zhao Zhang; Li-Rong ZHANG (168-178).
QT interval prolongation, a potential risk for arrhythmias, may result from gene polymorphisms relevant to cardiomyocyte repolarization. Another noted cause of QT interval prolongation is the administration of chemical compounds such as anesthetics, which may affect a specific type of cardiac K+ channel encoded by the human ether-a-go-go-related gene (hERG).hERG K+ current was recorded using whole-cell patch clamp in human embryonic kidney (HEK293) cells expressing wild type (WT) or mutated hERG channels. Expression of hERG K+ channel proteins was evaluated using western blot and confirmed by fluorescent staining and imaging. Computational modeling was adopted to identify the possible binding site(s) of propofol with hERG K+ channels. Propofol had a significant inhibitory effect on WT hERG K+ currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC50) of 60.9±6.4 μM. Mutations in drug-binding sites (Y652A or F656C) of the hERG channel were found to attenuate hERG current blockage by propofol. However, propofol did not inhibit the trafficking of hERG protein to the cell membrane. Meanwhile, for the three selective hERG K+ channel mutant heterozygotes WT/Q738X-hERG, WT/A422T-hERG, and WT/H562P-hERG, the IC50 of propofol was calculated as 14.2±2.8 μM, 3.3±1.2 μM, and 5.9±1.9 μM, respectively, which were much lower than that for the wild type.These findings indicate that propofol may potentially increase QT interval prolongation risk in patients via direct inhibition of the hERG K+ channel, especially in those with other concurrent triggering factors such as hERG gene mutations.
Keywords: Propofol; QT prolongation; Human ether-a-go-go-related gene (hERG); Protein trafficking; Gene mutation;

Trovafloxacin, a fluroquinolone antibiotic, was recently found to be an inhibitor of pannexin-1 channels through which ATP is released as “find-me” signals in apoptotic Jurkat cells. Our interest in the role of pannexin-1 channels in α1-adrenoceptor-mediated vasoconstriction led us to the novel finding reported here. Concentration-response curves to methoxamine and phenylephrine were competitively antagonised by trovafloxacin (1–30 µM) with a pKB of 5.54 and 5.32, respectively, in rat mesenteric small arteries isolated for myography. In comparison, prazosin (1–10 nM) antagonised methoxamine concentration-response curves with a pKB of 9.76. Trovafloxacin (1–30 µM) had no effect on either the thromboxane mimetic (U46619) or endothelin-1 concentration-contraction curves. Interestingly, the concentration range is similar for trovafloxacin antagonising the 3 distinct pharmacological targets: (i) fourth generation fluroquinolone antibiotic, (ii) pannexin-1 channel inhibitor in apoptotic cells, and now (iii) as an α1-adrenoceptor antagonist. When trovafloxacin was in use clinically, CNS side effects of dizziness, flushing and headache consistent with α1-adrenoceptor antagonism were common. We conclude that trovafloxacin with its quinolone moiety is a weak α1-adrenoceptor competitive antagonist in comparison with prazosin.
Keywords: α1-adrenoceptors; Pannexin-1 channels; Vascular contraction; Trovafloxacin; Prazosin;

The potential to promote neovascularization in ischemic tissues using exogenous agents is an attractive avenue for therapeutics. To identify novel pro-angiogenic small-molecule compound, we screened a series of resveratrol methylated derivatives and identified 3,3′,4,4′, 5,5′-hexamethoxy-trans-stilbene (3,3′,4,4′,5,5′-HMS) potently promotes proliferation, migration, invasion and tube formation of human umbilical vein VECs (HUVECs) in vitro. Furthermore, 3,3′,4,4′,5,5′-HMS accelerates neo-vessels sprouting of rat aortic rings ex vivo, and neovascularization of chick chorioallantoic membrane (CAM) and mouse matrigel plugs in vivo. Microarray analyses show that the level of early growth response 1 (EGR-1), an inducible pro-angiogenic gene regulatory factor, was upregulated. The upregulation of EGR-1 was confirmed by semiquantitative RT-PCR, quantitative real-time PCR and western blotting analyses. In addition, the levels of several pro-angiogenic factors including transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), nitric oxide (NO), and the activity of endothelial NO synthase (eNOS) were elevated in 3,3′,4,4′,5,5′-HMS-treated HUVECs. Inhibition of NO synthase by l-NAME blocked the pro-angiogenic effects of 3,3′,4,4′,5,5′-HMS. Our research shows that 3,3′,4,4′,5,5′-HMS dramatically promoted angiogenesis in vitro, ex vivo and in vivo, which might represent a novel potential agent for the development of therapeutic drugs to treat ischemic diseases.
Keywords: 3,3′,4,4′,5,5′-hexamethoxy-trans-stilbene; Angiogenesis; Early growth response-1; Vascular endothelial cell;

Design, synthesis and pharmacological evaluation of new anti-inflammatory compounds by Amanda F. Cidade; Patrícia A. Vasconcelos; Daiany P.B. Silva; Iziara F. Florentino; Géssica A. Vasconcelos; Boniek G. Vaz; Elson A. Costa; Luciano M. Lião; Ricardo Menegatti (195-204).
Inflammatory diseases and pain are among the main problems that significantly influence the lifestyle of millions of people and existing therapies are not always effective and can cause several adverse effects. In this context, the molecular modifications or synthesis of compounds continue being the best strategies for the identification of new compounds for the treatment of pain and inflammation. The aim of this study was to evaluate the analgesic and anti-inflammatory activities of new analogues of pyrazole compounds containing subunits N-phenyl-1-H-pirazoles and 1,3,4-oxadiazole-2(3H)-thione, LQFM-146, LQFM-147 and LQFM-148. In the acetic acid-induced abdominal writhing test, treatments with LQFM-146, LQFM-147 or LQFM-148 at doses 89, 178 and 356 µmol/kg p.o. reduced the abdominal writhing in a dose-dependent manner. In the formalin test, these compounds at dose 178 µmol/kg p.o. reduced the licking time only in inflammatory phase of this test, suggesting an antinociceptive effect dependent of the anti-inflammatory effect. The treatment with the three compounds in intermediate dose (178 µmol/kg p.o.) reduced the edema at all tested time points in the carrageenan-induced paw edema test and reduced polymorphonuclears cell migration, activity myeloperoxidase and TNF-α levels in the carrageenan-induced pleurisy test. Our date suggest that the new compounds LQFM-146, LQFM-147 and LQFM-148 possess satisfactory anti-inflammatory and antinociceptive effects that involves the reduction of pro-inflammatory cytokines and inhibition of the myeloperoxidase enzyme.
Keywords: Pyrazole compounds; LQFM-146; LQFM-147, LQFM-148; Inflammation;

Discovery of a new structural class of competitive hDHODH inhibitors with in vitro and in vivo anti-inflammatory, immunosuppressive effects by Wenbin Shen; Xiao Ren; Jingtong Zhu; Yan Xu; Jie Lin; Yeying Li; Feng Zhao; Haizhou Zheng; Ruolan Li; Xiaolan Cui; Xuexia Zhang; Xinhua Lu; Zhihui Zheng (205-212).
Human dihydroorotate dehydrogenase (hDHODH) is an inner mitochondrial membrane enzyme that involves in the fourth step of the biosynthesis of pyrimidine base. Inhibitors of hDHODH have been proven efficacy for the treatments of inflammation, rheumatoid arthritis, multiple sclerosis and cancer. In the present study, ascochlorin (ASC) and its derivatives, natural compounds from fungal metabolites, were discovered as hDHODH inhibitors by high-throughput screening. Enzyme kinetics studies showed that ASC competitively binds to hDHODH at the site of coenzyme Q substrate. In ex vivo study, ASC significantly inhibited the ConA-stimulated T lymphocytes proliferation and interleukin-2, interferon-γ production. Furthermore, ASC showed significant in vivo anti-inflammatory and immunosuppressive effects on the mice ears swelling, allogenic skin grafts and rat collagen-induced arthritis animal disease models. ASC significantly reduced ears edema level of mice, increased the survival time of allogenic skin implanted on the mice and attenuated arthritis severity of rat model. In conclusion, ASC was identified as a new structural class of hDHODH inhibitors with efficient anti-inflammatory, immunosuppressive activity, and may be a promising candidate for the development of new therapy in the treatment of autoimmune diseases.
Keywords: Ascochlorin; DHODH inhibitor; Immunosuppression; Anti-inflammatory; Rheumatoid arthritis;

Apelin-36 is protective against N-methyl-D-aspartic-acid-induced retinal ganglion cell death in the mice by Kenji Sakamoto; Yuta Murakami; Shohei Sawada; Hiroko Ushikubo; Asami Mori; Tsutomu Nakahara; Kunio Ishii (213-220).
Retinal ganglion cell death in glaucoma is caused at least in part by a large Ca2+ influx through N-methyl-D-aspartic acid (NMDA) receptors. Apelin is a peptide originally found in the tissue extracts of bovine stomach. Recent studies have been shown that apelin protects against the ischemic-reperfused injury in the brain. We examined whether apelin had protective effects on the NMDA-induced retinal ganglion cell (RGC) death using B6.Cg-TgN(Thy1-CFP)23Jrs/J transgenic mice, which express the enhanced cyan fluorescent protein in RGCs in the retina, in vivo. The mice were anesthetized by ketamine and xylazine, and NMDA (40 nmol/eye) was intravitreally injected. We evaluated the effects of apelin-13, [Glp1]-apelin-13, a potent agonist of apelin receptor, and apelin-36 on the NMDA-induced retinal ganglion cell death. NMDA-induced retinal ganglion cell loss was clearly seen 7 days after NMDA injection. Intravitreal apelin-36 (0.33 nmol/eye), but not apelin-13 (1 nmol/eye) nor [Glp1]-apelin-13 (1 nmol/eye), simultaneously injected with NMDA significantly reduced the cell loss. The protective effect of apelin-36 was not reduced by ML221 (0.1 nmol/eye; 5-[(4-Nitrobenzoyl)oxy]-2-[(2-pyrimidinylthio)methyl]-4H-pyran-4-one), an apelin receptor antagonist, GF109203X (0.03 nmol/eye), a protein kinase C inhibitor, U0126 (0.2 nmol/eye), a MAPK/ERK kinase inhibitor, LY294002 (0.1 nmol/eye), a phosphoinositide 3-kinase inhibitor, Akti 1/2 (0.05 nmol/eye), an Akt inhibitor, or 4,5,6,7-tetrabromobenzotriazole (0.2 nmol/eye), a casein kinase-2 inhibitor. In addition, human apelin-36 did not affect the kainic-acid (20 nmol/eye)-induced ganglion cell death. The present study suggests that apelin-36 protects against the NMDA-induced ganglion cell death independently of the activation of apelin receptor in the murine retina in vivo.
Keywords: Retina; N-methyl-D-aspartic acid; Neuronal cell death; Apelin; Apelin receptor;

A novel muscarinic receptor-independent mechanism of KCNQ2/3 potassium channel blockade by Oxotremorine-M by Ruud Zwart; Hannah Reed; Sophie Clarke; Emanuele Sher (221-228).
Inhibition of KCNQ (Kv7) potassium channels by activation of muscarinic acetylcholine receptors has been well established, and the ion currents through these channels have been long known as M-currents. We found that this cross-talk can be reconstituted in Xenopus oocytes by co-transfection of human recombinant muscarinic M1 receptors and KCNQ2/3 potassium channels. Application of the muscarinic acetylcholine receptor agonist Oxotremorine-methiodide (Oxo-M) between voltage pulses to activate KCNQ2/3 channels caused inhibition of the subsequent KCNQ2/3 responses. This effect of Oxo-M was blocked by the muscarinic acetylcholine receptor antagonist atropine. We also found that KCNQ2/3 currents were inhibited when Oxo-M was applied during an ongoing KCNQ2/3 response, an effect that was not blocked by atropine, suggesting that Oxo-M inhibits KCNQ2/3 channels directly. Indeed, also in oocytes that were transfected with only KCNQ2/3 channels, but not with muscarinic M1 receptors, Oxo-M inhibited the KCNQ2/3 response. These results show that besides the usual muscarinic acetylcholine receptor-mediated inhibition, Oxo-M also inhibits KCNQ2/3 channels by a direct mechanism. We subsequently tested xanomeline, which is a chemically distinct muscarinic acetylcholine receptor agonist, and oxotremorine, which is a close analogue of Oxo-M. Both compounds inhibited KCNQ2/3 currents via activation of M1 muscarinic acetylcholine receptors but, in contrast to Oxo-M, they did not directly inhibit KCNQ2/3 channels. Xanomeline and oxotremorine do not contain a positively charged trimethylammonium moiety that is present in Oxo-M, suggesting that such a charged moiety could be a crucial component mediating this newly described direct inhibition of KCNQ2/3 channels.
Keywords: KCNQ2/3 channels; M1 muscarinic acetylcholine receptors; M-current; Recombinant; Oxotremorine-M; Xenopus oocytes;

Tenuigenin exhibits protective effects against LPS-induced acute kidney injury via inhibiting TLR4/NF-κB signaling pathway by Haiyan Fu; Zhansheng Hu; Xingwei Di; Qiuhong Zhang; Rongbin Zhou; Hongyang Du (229-234).
Tenuigenin (TNG) has been reported to have various pharmacological activities, such as anti-oxidative and anti-inflammatory activities. However, the protective effects of TNG on lipopolysaccharides (LPS)-induced acute kidney injury (AKI) are still not clear. The aim of this study was to investigate the protective effects and mechanism of TGN on LPS-induced AKI in mice. The kidney histological change, levels of blood urea nitrogen (BUN), and creatinine were measured to assess the protective effects of TNG on LPS-induced AKI. The levels of TNF-α, IL-1β, and IL-6 in serum and kidney tissues were detected by ELISA. The extent of nuclear factor kappa-B (NF-κB) p65 and the expression of Toll-like receptor-4 (TLR4) were detected by western blot analysis. The results showed that TNG markedly attenuated the histological alterations, BUN and creatinine levels in kidney. TNG also suppressed LPS-induced TNF-α, IL-1β, and IL-6 production. Furthermore, the expression of TLR4 and NF-κB activation induced by LPS were markedly inhibited by TNG. In conclusion, this study demonstrated that TNG protected against LPS-induced AKI by inhibiting TLR4/NF-κB signaling pathway.
Keywords: Acute kidney injury; Tenuigenin; LPS; NF-κB;

Activation of AMPK α2 inhibits airway smooth muscle cells proliferation by Lu Liu; Yilin Pan; Yang Song; Xiaofan Su; Rui Ke; Lan Yang; Li Gao; Manxiang Li (235-243).
The aims of the present study were to examine the effect of adenosine monophosphate-activated protein kinase (AMPK) activation on airway smooth muscle cells (ASMCs) proliferation and to address its potential mechanisms. Platelet derived growth factor (PDGF) activated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, and this in turn up-regulated S-phase kinase-associated protein 2 (Skp2) and consequently reduced cyclin dependent kinase inhibitor 1B (p27) leading to ASMCs proliferation. Pre-incubation of cells with metformin, an AMPK activator, blocked PDGF-induced activation of mTOR and its downstream targets changes of Skp2 and p27 without changing Akt phosphorylation and inhibited ASMCs proliferation. Transfection of ASMCs with AMPK α2-specific small interfering RNA (siRNA) reversed the effect of metformin on mTOR phosphorylation, Skp2 and p27 protein expression and cell proliferation. Our study suggests that activation of AMPK, particularly AMPK α2, negatively regulates mTOR activity to suppress ASMCs proliferation and therefore has a potential value in the prevention and treatment of asthma by negatively modulating airway remodeling.
Keywords: AMPK; Airway smooth muscle cells; MTOR; Skp2; P27;

Ramipril restores PPARβ/δ and PPARγ expressions and reduces cardiac NADPH oxidase but fails to restore cardiac function and accompanied myosin heavy chain ratio shift in severe anthracycline-induced cardiomyopathy in rat by Hana Cernecka; Gabriel Doka; Jasna Srankova; Lenka Pivackova; Eva Malikova; Kristina Galkova; Jan Kyselovic; Peter Krenek; Jan Klimas (244-253).
We hypothesized that peroxisome proliferator-activated receptors (PPARs) might be involved in a complex protective action of ACE inhibitors (ACEi) in anthracyclines-induced cardiomyopathy. For purpose of study, we compared effects of ramipril on cardiac dysfunction, cardiac failure markers and PPAR isoforms in moderate and severe chronic daunorubicin-induced cardiomyopathy. Male Wistar rats were administered with a single intravenous injection of daunorubicin: 5 mg/kg (moderate cardiomyopathy), or 15 mg/kg (severe cardiomyopathy) or co-administered with daunorubicin and ramipril (1 mg/kg/d, orally) or vehicle for 8 weeks. Left ventricular function was measured invasively under anesthesia. Cardiac mRNA levels of heart failure markers (ANP, Myh6, Myh7, Myh7b) and PPARs (alpha, beta/delta and gama) were measured by qRT-PCR. Protein expression of NADPH subunit (gp91phox) was measured by Western blot. Moderate cardiomyopathy exhibited only minor cardiac dysfunction what was corrected by ramipril. In severe cardiomyopathy, hemodynamic dysfunction remained unaltered upon ramipril although it decreased the significantly up-regulated cardiac ANP mRNA expression. Simultaneously, while high-dose daunorubicin significantly decreased PPARbeta/delta and PPARgama mRNA, ramipril normalized these abnormalities. Similarly, ramipril reduced altered levels of oxidative stress-related gp91phox. On the other hand, ramipril was unable to correct both the significantly decreased relative abundance of Myh6 and increased Myh7 mRNA levels, respectively. In conclusion, ramipril had a protective effect on cardiac function exclusively in moderate chronic daunorubicin-induced cardiomyopathy. Although it normalized abnormal PPARs expression and exerted also additional protective effects also in severe cardiomyopathy, it was insufficient to influence impaired cardiac function probably because of a shift in myosin heavy chain isoform content.
Keywords: Anthracycline; Chronic cardiomyopathy; Angiotensin-converting enzyme inhibitor; Peroxisome proliferator-activated receptors; Myosin heavy chain isoforms;

Hepatocyte Nuclear Factor-4α (HNF-4α) is a key nuclear receptor protein required for liver development. miR-122 is a predominant microRNA expressed in liver and is involved in the regulation of cholesterol and fatty acid metabolism. HNF-4α is know to regulate expression of miR-122 in liver. We examined how HNF-4α regulated gluconeogenesis and lipid metabolism through miR-122 in vivo and in vitro. Expression of miR-122, HNF-4α, phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), sterol response elementary binding protein-1 (SREBP-1), fatty acid synthase-1 (FAS-1), carnitine palmitoyltransferase-1 (CPT-1) and acetyl Coenzyme A carboxylase alpha (ACCα) were determined in livers of Type 2 diabetic mice and in insulin resistant palmitate-treated HepG2 cells. CPT-1 and phosphorylated ACCα expression were significantly decreased in livers of Type 2 diabetic mice and in palmitate-treated HepG2 cells compared to controls. In contrast, expression of miR-122, HNF-4α, PEPCK, G6Pase, SREBP-1, FAS-1 and ACCα were significantly elevated in liver of Type 2 diabetic mice and in palmitate-treated HepG2 cells compared to controls. Expression of HNF-4α increased whereas siRNA knockdown of HNF-4α decreased miR-122 levels in HepG2 cells compared to controls. In addition, expression of HNF-4α in HepG2 cells increased PEPCK, G6Pase, SREBP-1, FAS-1, ACCα mRNA and protein expression and decreased CPT-1 and p-ACCα mRNA and protein expression compared to controls. Addition of miR-122 inhibitors attenuated the HNF-4α mediated effect on expression of these gluconeogenic and lipid metabolism proteins. The results indicate that HNF-4α regulated miR-122 contributes to development of the gluconeogenic and lipid metabolism alterations observed in Type 2 diabetic mice and in palmitate-treated HepG2 cells.Display Omitted
Keywords: Type 2 diabetes; Gluconeogenesis; Lipid metabolism; MiR122; HNF-4α;

Antiarrhythmic gene therapy – will biologics replace catheters, drugs and devices? by Patrick Lugenbiel; Patrick A. Schweizer; Hugo A. Katus; Dierk Thomas (264-273).
The clinical management of heart rhythm disorders still constitutes a major challenge. The development of alternatives to current approaches is of significant interest in order to establish more effective therapies that increase quality of life and reduce symptoms and hospitalizations. Over the past two decades the mechanistic understanding of pathophysiological pathways underlying cardiac arrhythmias has advanced profoundly, opening up novel avenues for mechanism-based therapeutic approaches. In particular, gene therapy offers greater selectivity than small molecule-based or interventional treatment. The gene of interest is packaged into viral or non-viral carriers and delivered to the target area via direct injection or using catheter-based techniques, providing the advantage of site-restricted action in contrast to systemic application of drugs. This work summarizes the current knowledge on mechanistic background, application strategies, and preclinical outcome of antiarrhythmic gene therapy for atrial fibrillation, ventricular tachycardia, and modulation of sinus node function.
Keywords: Antiarrhythmic drug; Atrial fibrillation; Cardiac arrhythmia; Gene therapy; Sinus rhythm;

Inhibition of tachykinin NK1 receptor using aprepitant induces apoptotic cell death and G1 arrest through Akt/p53 axis in pre-B acute lymphoblastic leukemia cells by Samaneh Bayati; Davood Bashash; Shahin Ahmadian; Ava Safaroghli-Azar; Kamran Alimoghaddam; Ardeshir Ghavamzadeh; Seyed H. Ghaffari (274-283).
Increasing number of genetic and cancer biology studies indicated a prominent role for tachykinin NK1 receptor (NK1R) in cancer cell growth and survival. Considering the fact that neoplastic lymphoid precursors in acute lymphoblastic leukemia (ALL) carry a three- to four-fold NK1R expression as compared to normal lymphocytes, using NK1R antagonist seems to be noteworthy in the treatment of ALL patients. In this study, we found that inhibition of NK1R with aprepitant, a selective high-affinity antagonist of the human NK1R, exerts cytotoxic and anti-proliferative effects against pre-B ALL-derived Nalm-6 cells either as single drug or in combination with doxorubicin. Our data showed that treatment of the cells with the inhibitor resulted in apoptotic cell death, at least partly, through abrogation of PI3K/Akt pathway, as revealed by the reduction of phospho/total Akt ratio. In agreement with the inhibitory effect on Akt, we also found that aprepitant increased the expression level of p21 and p27, which in turn leads to the induction of G1 cell cycle arrest. Overall, this study recommends mechanistic pathways by which inhibition of NK1R can augment apoptotic cell death through a plausible p53-dependent pathway rather than NF-κB-depended mechanism in pre-B ALL cells; however, further studies are needed to better characterize the application of NK1R inhibition in clinical cancer treatment.
Keywords: Acute lymphoblastic leukemia; Aprepitant; P53; Nalm-6 cells; Tachykinin NK1 receptor;

Neurochemical factors associated with the antidepressant-like effect of flavonoid chrysin in chronically stressed mice by Carlos Borges Filho; Cristiano Ricardo Jesse; Franciele Donato; Lucian Del Fabbro; Marcelo Gomes de Gomes; André Tiago Rossito Goes; Leandro Cattelan Souza; Renata Giacomeli; Michelle Antunes; Cristiane Luchese; Silvane Souza Roman; Silvana Peterini Boeira (284-296).
Chrysin is a flavonoid which is found in bee propolis, honey and various plants. Antidepressant-like effect of chrysin in chronically stressed mice was previously demonstrated by our group. Conversely, neurochemical factors associated with this effect require further investigations. Thus, we investigated the possible involvement of pro-inflammatory cytokines, kynurenine pathway (KP), 5-hydroxytryptamine (5-HT) metabolism and caspases activities in the effect of chrysin in mice exposed to unpredictable chronic stress (UCS). UCS applied for 28 days induced a depressive-like behavior, characterized by decrease in the time of grooming in the splash test and by increase in the immobility time in the tail suspension test. Oral treatment with chrysin (5 or 20 mg/kg, 28 days), similarly to fluoxetine (10 mg/kg, positive control), culminated in the prevention of these alterations. UCS elevated plasma levels of corticotropin-releasing hormone and adrenocorticotropic hormone, as well the tumor necrosis factor-α, interleukin-1β, interleukin-6 and kynurenine levels in the prefrontal cortex (PFC) and hippocampus (HP). UCS induced the decrease in the 5-HT levels in the HP and the increase in the indoleamine-2,3-dioxygenase, caspase 3 and 9 activities in the PFC and HP. Treatment with chrysin, similarly to fluoxetine, promoted the attenuation of these alterations occasioned by UCS. These results corroborated with the antidepressant potential of chrysin in the treatment of psychiatric diseases. Furthermore, this work indicated the association of pro-inflammatory cytokines synthesis, KP, 5-HT metabolism and caspases activities with the action exercised by chrysin in mice exposed to UCS.
Keywords: Flavonoid; Depression; Chronic stress; Antidepressant-like;

Theanine from tea and its semi-synthetic derivative TBrC suppress human cervical cancer growth and migration by inhibiting EGFR/Met-Akt/NF-κB signaling by Jiannan Liu; Yuping Sun; Huarong Zhang; Dexin Ji; Fei Wu; Huihui Tian; Kun Liu; Ying Zhang; Benhao Wu; Guoying Zhang (297-307).
Cervical cancer is the third most prevalent cancer among women worldwide. Theanine from tea and its derivatives show some anticancer activities. However, the role of theanine and its derivatives against human cervical cancer and the molecular mechanisms of action remain unclear. Thus, in this study, we aim to investigate the anticancer activities and underlying mechanisms of theanine and a theanine derivative, ethyl 6-bromocoumarin-3- carboxylyl L-theanine (TBrC), against human cervical cancer. In vitro and in vivo assays for cancer cell growth and migration have confirmed the inhibition of the cell growth and migration by TBrC and theanine in highly-metastatic human cervical cancer. TBrC displays much stronger activity than theanine on inhibition of the cell growth and migration as well as induction of apoptosis and regulation of related protein expressions in the human cervical cancer cells. TBrC and theanine greatly reduced endogenous and exogenous factors-stimulated cell migration and completely repressed HGF- and EGF+HGF-activated EGFR/Met-Akt/NF-κB signaling by reducing the phosphorylation and expressions of EGFR, Met, Akt, and NF-κB in cervical cancer cells. The enhancer of zeste homolog 2 (EZH2) knockdown decreased the cancer cell migration and NF-κB expression. The NF-κB knockdown reduced the cancer cell migration. TBrC and theanine reduced the EZH2 expression by more than 80%. In addition, TBrC and theanine significantly suppressed human cervical tumor growth in tumor-bearing nude mice without toxicity to the mice. Our results suggest that TBrC and theanine may have the potentials of the therapeutic and/or adjuvant therapeutic application in the treatment of human cervical cancer.
Keywords: TBrC; Theanine; Cervical cancer; Migration; Tumor growth; EGFR/Met-Akt/NF-κB pathways;

Salvianolic acid B improves vascular endothelial function in diabetic rats with blood glucose fluctuations via suppression of endothelial cell apoptosis by Younan Ren; Shanjun Tao; Shuguo Zheng; Mengqiu Zhao; Yuanmei Zhu; Jieren Yang; Yuanjie Wu (308-315).
Vascular endothelial cell injury is an initial event in atherosclerosis. Salvianolic acid B (Sal B), a main bioactive component in the root of Salvia miltiorrhiza, has vascular protective effect in diabetes, but the underlying mechanisms remain unclear. The present study investigated the effect of Sal B on vascular endothelial function in diabetic rats with blood glucose fluctuations and the possible mechanisms implicated. The results showed that diabetic rats developed marked endothelial dysfunction as exhibited by impaired acetylcholine induced vasodilation. Supplementation with Sal B resulted in an evident improvement of endothelial function. Phosphorylation (Ser 1177) of endothelial nitric oxide synthase (eNOS) was significantly restored in Sal B treated diabetic rats, accompanied by an evident recovery of NO metabolites. Sal B effectively reduced vascular endothelial cell apoptosis, with Bcl-2 protein up-regulated and Bax protein down-regulated markedly. Treatment with Sal B led to an evident amelioration of oxidative stress in diabetic rats as manifested by enhanced antioxidant capacity and decreased contents of malondialdehyde in aortas. Protein levels of NOX2 and NOX4, two main isoforms of NADPH oxidase known as the major source of reactive oxygen species in the vasculature, were markedly decreased in Sal B treated groups. In addition, treatment with Sal B led to an evident decrease of serum lipids. Taken together, this study indicates that Sal B is capable of improving endothelial function in diabetic rats with blood glucose fluctuations, of which the underlying mechanisms might be related to suppression of endothelial cell apoptosis and stimulation of eNOS phosphorylation (Ser 1177).
Keywords: Sal B; Diabetes; Endothelial dysfunction; Oxidative stress; Apoptosis;

Pioglitazone inhibits EGFR/MDM2 signaling-mediated PPARγ degradation by Juanjuan Shi; Wenbo Zhang; Mengli You; Ying Xu; Yongzhong Hou; Jianhua Jin (316-321).
Aberrant activation of the epidermal growth factor receptor (EGFR) signaling is involved in many cancer events. Although peroxisome proliferator-activated receptor γ (PPARγ) has been implicated in inhibition of inflammation and cancer, EGFR/MDM2 signaling induces PPARγ phosphorylation and degradation. Here we found that cancer cells in response to EGF reduced PPARγ protein levels by inducing its phosphorylation, ubiquitination and degradation, but PPARγ agonist pioglitazone reversed this event. More importantly, pioglitazone increased cancer cell sensitivity to chemotherapy drugs. Therefore, our study revealed a novel mechanism that pioglitazone inhibited EGFR/MDM2-mediated cancer cell chemoresistance, which provides a novel strategy for cancer treatment.
Keywords: Pioglitazone; PPARγ; EGFR; Ubiquitination; Degradation;

Visfatin triggers the in vitro migration of osteosarcoma cells via activation of NF-κB/IL-6 signals by Guang-Ji Wang; Ning-Jiang Shen; Liang Cheng; Yehan Fang; Hui Huang; Kang-Hua Li (322-330).
Pulmonary metastasis is the major challenge for clinical treatment of osteosarcoma patients. Recent studies indicated that visfatin, a 52 kDa adipocytokine, can trigger the cell motility of various cancers, while its role in the progression of osteosarcoma remains not clear. Our present study revealed that visfatin can significantly promote the in vitro migration and invasion of osteosarcoma MG-63 and HOS cells and up regulate the expression of matrix metalloproteinase-2 (MMP-2) and fibronectin (FN). Furthermore, visfatin treatment also increased the expression of IL-6 in both MG-63 and HOS cells via a time dependent manner, while anti-IL-6 antibody can significantly attenuate visfatin induced cell invasion and up regulation of MMP-2 and FN. It suggested that up regulation of IL-6 mediated visfatin induced in vitro motility of osteosarcoma cells. Visfatin treatment can increase the phosphorylation of both p65 and ERK1/2 in MG-63 and HOS cells, while only the inhibitor of NF-κB, BAY 11–7082, can abolish visfatin induced up regulation of IL-6. BAY 11–7082 also attenuated visfatin induced upregulation of MMP-2 and FN in MG-63 cells. Western blot analysis revealed that visfatin treatment can significantly increase the phosphorylation of IκBα and IKKβ in MG-63 cells. ACHP, the inhibitor of IKK-β, blocked visfatin induced expression of IL-6 mRNA in both MG-63 and HOS cells. Collectively, our data suggested that visfatin can increase the motility of osteosarcoma cells via up regulation of NF-κB/IL-6 signals. It indicated that visfatin might be a potential therapeutic target of osteosarcoma treatment.
Keywords: Visfatin; Migration; Osteosarcoma; IL-6; NF-κB;

Aspirin prevents bone loss with little mechanical improvement in high-fat-fed ovariectomized rats by Sien Lin; Wayne Y.W. Lee; Meiling Huang; Ziwei Fu; Yanlong Liang; Haiyou Wu; Liangliang Xu; Chun Wai Suen; Jianping Huang; Tie Wu; Liao Cui; Gang Li (331-338).
Obesity and osteoporosis are often concurrently happened in the menopausal women. Obesity in menopausal women is not only related to a high risk of cardiovascular disease, but also results in a detrimental effect on bone health. This study aimed to investigate the effects of aspirin, a popular anti-thrombosis drug, on bone quantity and quality in the high-fat-fed animal model. Adult female rats were subjected to either sham operations or ovariectomized operations. The ovariectomized rats were orally administered with deionized water or standardized high fat emulsion with or without aspirin. All rats were injected with calcein before killed for the purpose of double in vivo labeling. Biochemistry, histomorphometry, micro-computed tomography analysis, mechanical test, and component analysis were performed after 12 weeks. In vitro cell culture was also performed to observe the effect of aspirin in osteogenesis. We found that high fat remarkably impaired bone formation and bone biomechanics. Aspirin treatment significantly prevented bone loss by increasing bone formation. In vitro studies also validated the enhancement of osteogenic differentiation. However, aspirin presented no significant improvement in bone mechanical properties. Component analysis shown aspirin could significantly increase the content of mineral, but had limited effect on the content of collagen. In conclusion, aspirin is beneficial for the prevention of bone loss; meanwhile, it may cause an imbalance in the components of bone which may weaken the mechanical properties. The current study provided further evidence that aspirin might not be powerful for the prevention of fracture in osteoporotic patients.
Keywords: Aspirin; Osteoporosis; Ovariectomy; High fat diet; Biomechanics; Inflammation;

YZG-331 is a synthetic adenosine analogue which exhibits the sedative and hypnotic effects by binding to the adenosine receptor. The present study was designed to investigate the effects of P-glycoprotein (P-gp) on the intestine and brain distribution of YZG-331 in vitro and in vivo as well as related binding mechanisms. The activity of P-gp ATPase was both induced by YZG-331 and verapamil, a typical P-gp inhibitor, but affinity of YZG-331 for P-gp was lower than that of verapamil. The docking analyses further elucidated the binding relationship of YZG-331 and P-gp. The directional transport of YZG-331 was disappeared in Caco-2 and MDCK-MDR1 cells when the P-gp activity was blocked. However, the penetration of digoxin, a P-gp known substrate, was not change in MDCK-MDR1 cells along with YZG-331. In the everted intestinal sac model, the influx of YZG-331 was significantly reduced in the presence of verapamil in all the segments except for the colon. In the in situ and in vivo study, the brain exposure of YZG-331 was promoted after co-administered of verapamil. Furthermore, the Kp value changed from 0.03 to 0.05 after drug combination. Taken together, these results indicated that YZG-331 is a substrate but may not an inhibitor of P-gp. The intestine and brain permeability of YZG-331 can be restricted, at least in part, by P-gp. The drug interactions should be awarded when YZG-331 and other P-gp-related drugs used together.Display Omitted
Keywords: YZG-331; Intestine; Brain; P-gp; ATPase;

Protective role of 6-Hydroxy-1-H-Indazole in an MPTP-induced mouse model of Parkinson's disease by Liang Xiao-feng; Zhu Wen-ting; Xu Yuan-yuan; Lai Chong-Fa; Zheng Lu; Rao Jin-jun; Wang Wen-ya (348-354).
This study aimed to explore the neuroprotective role of 6-hydroxy-1H-indazole on dopaminergic neurons in a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). Forty 12-week-old C57BL/6 male mice were were randomized divided into 4 groups. Mice were treated with 2 mg/kg and 4 mg/kg 6-hydroxy-1H-indazole (i.p.) 1 d before the initiation of MPTP administration (30 mg/kg), and the 6-hydroxy-1H-indazole were daily injected half an hour before MPTP treatment in the following 5 days. The MPTP group was given normal saline on day 1 (i.p.), followed by 30 mg/kg MPTP treatment in the following 5 days. Control group received an equivalent volume of normal saline. Ten days after the final injection of MPTP, the mice were killed. The results showed that MPTP decreased the dopaminergic neurons in the substantia nigra and dopamine in the striatum, downregulated the expression of tyrosine hydroxylase (TH), induced the impairment of behavior and hyperphosphorylation of tau, However, 6-hydroxy-1-H-indazole decreased the loss of dopaminergic neurons, increased dopamine concentration and TH expression, alleviated the behavioral damage and level of phosphor-tau in the MPTP-induced model of PD in C57BL/6 mice. These findings showed that 6-hydroxy-1-H-indazole-mediated neuroprotection was related to the inactivation of tau. In addition, 6-hydroxy-1-H-indazole may be a potential drug candidate for PD.Display Omitted
Keywords: Dopaminergic neurons; 6-hydroxy-1-H-indazole; MPTP; Parkinson's disease; Tau;

Intracellular zinc status influences cisplatin-induced endothelial permeability through modulation of PKCα, NF-κB and ICAM-1 expression by Vijaya Lakshmi Bodiga; Santhi Priya Inapurapu; Praveen Kumar Vemuri; Madhukar Rao Kudle; Sreedhar Bodiga (355-368).
Platinum-based chemotherapeutic regimen induces vascular dysfunction. Action of cisplatin on endothelial cells is mediated by protein kinase C (PKC-α), which further activates nuclear factor-κB (NF-κB) and induces canonical transient receptor potential channel (TRPC1) and intercellular adhesion molecule (ICAM-1) expression. Increased ICAM-1 contributes to hyperadhesion of monocytes and endothelial dysfunction. PKC-α is also involved in phosphorylation of TRPC1, resulting in store-operated calcium entry (SOCE) and further activation of NF-κB. Although the role of altered intracellular zinc status is not known in cisplatin-induced vascular dysfunction, because of the ability of zinc to modulate PKC-α, NF-κB activity, we hypothesized that zinc can ameliorate the extent of endothelial dysfunction induced by cisplatin. Human umbilical vein endothelial cells treated with cisplatin (8.0 μg/ml) showed lowered intracellular free zinc, concomitant with enhanced activation of PKC-α, NF-κΒ activation, TRPC1, SOCE and ICAM-1 levels. Zinc deficiency per se induced using membrane permeable chelator (TPEN) mimicked the cisplatin-induced PKC-α, NF-κB activation and ICAM-1 expression, but also activated Activator Protein-1 (AP-1). Zinc supplementation (2.0–10.0 μM) to the endothelial cells during cisplatin treatment or TPEN-induced zinc deficiency suppressed PKC-α, NF-κB, TRPC1, SOCE activation and lowered the ICAM-1 expression. Zinc supplementation thereby effectively decreased the cisplatin-induced endothelial permeability and adherence of the activated endothelial cells to U937 monocytes.
Keywords: Cisplatin; Zinc; PKC; NF-κB; TRPC1; SOCE; ICAM-1;

The effect of nitrazepam on depression and curiosity in behavioral tests in mice: The role of potassium channels by Vahid Nikoui; Sattar Ostadhadi; Pardis Azhand; Samira Zolfaghari; Shayan Amiri; Mehrdad Foroohandeh; Manijeh Motevalian; Ali Mohammad Sharifi; Azam Bakhtiarian (369-376).
Evidence show that gamma-aminobutyric acid (GABA) receptors are involved in depression, so the aim of this study was to investigate the effect of nitrazepam as agonist of GABAA receptors on depression and curiosity in male mice and the role of potassium channel in antidepressant-like response. For this purpose, we studied the antidepressant-like properties of fluoxetine, nitrazepam, glibenclamide, and cromakalim by both forced swimming test (FST) and tail suspension test (TST). Animals were injected by various doses of nitrazepam (0.05, 0.1, and 0.5 mg/kg). Nitrazepam at dose of 0.5 mg/kg significantly decreased the immobility time compared to control group in both FST and TST. Fluoxetine also showed such a response. Co-administration of nitrazepam (0.05 mg/kg) with glibenclamide in TST (1 mg/kg) and in FST (0.3, 1 mg/kg) also showed antidepressant-like response. Beside, cromakalim (0.1 mg/kg) could reverse the antidepressant-like effect of nitrazepam (0.5 mg/kg) in both FST and TST, while cromakalim and glibenclamide alone could not change the immobility time compared to control group (P>0.05). The hole-board test revealed that nitrazepam at doses of 0.5 and 0.1 mg/kg could increase the activity of the animal’s head-dipping and boost the curiosity and exploration behavior of mice. The results of this study revealed that nitrazepam may possess antidepressant-like properties and this effect is dependent to potassium channels in both FST and TST.
Keywords: Nitrazepam; Potassium channel; Forced swimming test; Tail suspension test; Hole-board test; Mice;

Participation of pro- and anti-nociceptive interleukins in botulinum toxin A-induced analgesia in a rat model of neuropathic pain by Magdalena Zychowska; Ewelina Rojewska; Wioletta Makuch; Siro Luvisetto; Flaminia Pavone; Sara Marinelli; Barbara Przewlocka; Joanna Mika (377-388).
Botulinum neurotoxin serotype A (BoNT/A) shows antinociceptive properties, and its clinical applications in pain therapy are continuously increasing. BoNT/A specifically cleaves SNAP-25, which results in the formation of a non-functional SNARE complex, thereby potently inhibiting the release of neurotransmitters and neuropeptides, including those involved in nociception. The aim of the present study was to determine the effects of BoNT/A (300 pg/paw) on pain-related behavior and the levels of glial markers and interleukins in the spinal cord and dorsal root ganglia (DRG) after chronic constriction injury (CCI) to the sciatic nerve in rats. Glial activity was also examined after repeated intraperitoneal injection of minocycline combined with a single BoNT/A injection. Our results show that a single intraplantar BoNT/A injection did not influence motor function but strongly diminished pain-related behaviors in naïve and CCI-exposed rats. Additionally, microglial inhibition using minocycline enhanced the analgesic effects of BoNT/A. Western blotting results suggested that CCI induces the upregulation of the pronociceptive proteins IL-18, IL-6 and IL-1β in the ipsilateral lumbar spinal cord and DRG, but no changes in the levels of the antinociceptive proteins IL-18BP, IL-1RA and IL-10 were observed. Interestingly, BoNT/A injection suppressed the CCI-induced upregulation of IL-18 and IL-1β in the spinal cord and/or DRG and increased the levels of IL-10 and IL-1RA in the DRG. In summary, our results suggest that BoNT/A significantly attenuates pain-related behavior and microglial activation and restores the neuroimmune balance in a CCI model by decreasing the levels of pronociceptive factors (IL-1β and IL-18) and increasing the levels of antinociceptive factors (IL-10 and IL-1RA) in the spinal cord and DRG.Display Omitted
Keywords: Neuropathic pain model; Botulinum neurotoxin A; Minocycline; Glia; Interleukins;

Central inhibitory effects on feeding induced by the adipo-myokine irisin by Claudio Ferrante; Giustino Orlando; Lucia Recinella; Sheila Leone; Annalisa Chiavaroli; Chiara Di Nisio; Rugia Shohreh; Fabio Manippa; Adriana Ricciuti; Michele Vacca; Luigi Brunetti (389-394).
Irisin, the soluble secreted form of fibronectin type III domain containing 5 (FNDC5)-cleaved product, is a recently identified adipo-myokine that has been indicated as a possible link between physical exercise and energetic homeostasis. The co-localization of irisin with neuropeptide Y in hypothalamic sections of paraventricular nucleus, which receives NPY/AgRP projections from the arcuate nucleus, suggests a possible role of irisin in the central regulation of energy balance. In this context, in the present work we studied the effects of intra-hypothalamic irisin (1 μl, 50–200 nmol/l) administration on feeding and orexigenic [agouti-related peptide (AgRP), neuropeptide Y (NPY) and orexin-A] and anorexigenic [cocaine and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC)] peptides in male Sprague-Dawley rats. Furthermore, we evaluated the effects of irisin on hypothalamic dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) concentrations and plasma NE levels. Compared to vehicle, irisin injected rats showed decreased food intake, possibly mediated by stimulated CART and POMC and inhibited DA, NE and orexin-A, in the hypothalamus. We also found increased plasma NE levels, supporting a role for sympathetic nervous system stimulation in mediating increased oxygen consumption by irisin.
Keywords: Irisin; DA; NE; CART; Orexin-A; POMC;

The roles of miRNAs as potential biomarkers in lung diseases by Shamila D. Alipoor; Ian M. Adcock; Johan Garssen; Esmaeil Mortaz; Mohammad Varahram; Mehdi Mirsaeidi; Aliakbar Velayati (395-404).
MicroRNAs (miRNAs) are small non-coding RNAs which can act as master regulators of gene expression, modulate almost all biological process and are essential for maintaining cellular homeostasis. Dysregulation of miRNA expression has been associated with aberrant gene expression and may lead to pathological conditions. Evidence suggests that miRNA expression profiles are altered between health and disease and as such may be considered as biomarkers of disease. Evidence is increasing that miRNAs are particularly important in lung homeostasis and development and have been demonstrated to be the involved in many pulmonary diseases such as asthma, COPD, sarcoidosis, lung cancer and other smoking related diseases. Better understanding of the function of miRNA and the mechanisms underlying their action in the lung, would help to improve current diagnosis and therapeutics strategies in pulmonary diseases. Recently, some miRNA-based drugs have been introduced as possible therapeutic agents. In this review we aim to summarize the recent findings regarding the role of miRNAs in the airways and lung and emphasise their potential therapeutic roles in pulmonary diseases.
Keywords: MicroRNA; Sarcoidosis; Lung cancer; Asthma; COPD; Lung disease;

β-N-oxalyl-L-α, β- diaminopropionic acid induces HRE expression by inhibiting HIF-prolyl hydroxylase-2 in normoxic conditions by Ravi Kumar Eslavath; Deepshikha Sharma; Nabil A.M. Bin Omar; Rajasekhar Chikati; Mahesh Kumar Teli; G.K. Rajanikant; Surya S. Singh (405-411).
Hypoxia inducible factor (HIF)-1α, a subunit of HIF transcription factor, regulates cellular response to hypoxia. In normoxic conditions, it is hydroxylated by prolyl hydroxylase (PHD)-2 and targeted for proteosomal degradation. Drugs which inhibit PHD-2 have implications in conditions arising from insufficient blood supply. β-ODAP (β-N- oxalyl-L-α, β- diaminopropionic acid), a non-protein excitatory amino acid present in Lathyrus sativus, is an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor agonist known to activate conventional protein kinase C and stabilize HIF-1α under normoxic conditions. However, the mechanism of HIF-1α stabilization by this compound is unknown. In silico approach was used to understand the mechanism of stabilization of HIF-1α which revealed β-ODAP interacts with key amino acid residues and Fe2+ at the catalytic site of PHD-2. These results were further corroborated with luciferase HRE (hypoxia response element) reporter system in HeLa cells. Different chemical modulators of PHD-2 activity and HIF-1α levels were included in the study for comparison. Results obtained indicate that β-ODAP inhibits PHD-2 and facilitates HIF dependent HRE expression and hence, might be helpful in conditions arising from hypoxia.
Keywords: β-ODAP; Prolyl hydroxylase-2; Hypoxia response elements;

HHX-5, a derivative of sesquiterpene from Chinese agarwood, suppresses innate and adaptive immunity via inhibiting STAT signaling pathways by Zhixiang Zhu; Yunfang Zhao; Huixia Huo; Xiaoli Gao; Jiao Zheng; Jun Li; Pengfei Tu (412-423).
Induction of excessive, prolonged, or dysregulated immune responses causes immunological disorders, such as inflammatory diseases, autoimmune diseases, allergic diseases, and organ-graft rejections. In the present study, we investigated the inhibitory effects of HHX-5, a derivative of sesquiterpene from Chinese agarwood, on innate and adaptive immunity for revealing its potential to treat above immunological disorders. The results showed that HHX-5 significantly inhibited the activation of macrophages and neutrophils which play important roles in innate immunity. Furthermore, HHX-5 strongly suppressed adaptive immunity via inhibiting differentiation of naive CD4+ T cells into Th1, Th2, and Th17 cells and suppressing activation, proliferation and differentiation of CD8+ T cells and B cells. The mechanism study showed that HHX-5 significantly inhibited STAT1 signaling pathway in macrophages and suppressed STAT1, STAT4, STAT5, and STAT6 signaling pathways in naive CD4+ T cells. In conclusion, we demonstrated that HHX-5 can strongly inhibit innate and adaptive immunity via suppressing STAT signaling pathways and has potential to be developed into therapeutic drug for treat immunological disorders.
Keywords: Immunological disorder; HHX-5; Sesquiterpene; Innate immunity; Adaptive immunity; STAT signaling pathways;

Transplantation of pancreatic islets is the most reliable treatment for Type 1 diabetes. However cell death mediated by hypoxia is considered as one of the main difficulties hindering success in islet transplantation. The aim of our experiment was to investigate the role of small molecules in survival of Islet like cell aggregates (ICAs) engineered from umbilical cord matrix under oxygen deprived condition (<5% O2). ICAs were analyzed for cell death via fluoroscein diacetate/propidium iodide (FDA/PI) staining, estimation of Caspase 3 and free radical release in presence and absence of small molecules. The samples were also analyzed for the presence of hypoxia inducible factor 1α (HIF1α) at both transcriptional and translational level. The addition of small molecules showed profound defensive effect on ICAs under hypoxic environment as evidenced by their viability and insulin secretion compared to untreated ICAs. The combinations of Eicosapentaenoic acid (EPA), Docosahexaenoic acid(DHA) and metformin and EPA, DHAandγ amino butyric acid (GABA) acted as anti-apoptotic agents for human ICAs when exposed to 1% O2 for 48 h. The combinations of the small molecules reduced the total reactive oxygen species and malonaldehyde (MDA) levels and enhanced the production of glutathione peroxidise (GPx) enzyme under hypoxic conditions. Finally the increase in HIF1α at both protein and gene level confirmed the defensive effect of the additives in hypoxia. These results suggest that the combination of small molecules maintained the viability and functionality of the ICAs in hypoxia by up-regulating HIF1α expression and down regulating the Caspase 3 activity.
Keywords: Islet like cell aggregates (ICA's); Eicosapentaenoic acid (EPA); Docosahexaenoic acid (DHA); γ amino butyric acid (GABA); Metformin; Hypoxia;

Ester to amide substitution improves selectivity, efficacy and kinetic behavior of a benzodiazepine positive modulator of GABAA receptors containing the α5 subunit by Tamara Timić Stamenić; Michael M. Poe; Sabah Rehman; Anja Santrač; Branka Divović; Petra Scholze; Margot Ernst; James M. Cook; Miroslav M. Savić (433-443).
We have synthesized and characterized MP-III-022 ((R)−8-ethynyl-6-(2-fluorophenyl)-N,4-dimethyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide) in vitro and in vivo as a binding- and efficacy-selective positive allosteric modulator of GABAA receptors containing the α5 subunit (α5GABAARs). By approximation of the electrophysiological responses which the estimated free rat brain concentrations can induce, we demonstrated that convenient systemic administration of MP-III-022 in the dose range 1–10 mg/kg may result in a selective potentiation, over a wide range from mild to moderate to strong, of α5βγ2 GABAA receptors. For eliciting a comparable range of potentiation, the widely studied parent ligand SH-053-2′F-R-CH3 containing an ester moiety needs to be administered over a much wider dose range (10–200 mg/kg), but at the price of activating non-α5 GABAARs as well as the desired α5GABAARs at the highest dose. At the dose of 10 mg/kg, which elicits a strong positive modulation of α5GABAARs, MP-III-022 caused mild, but significant muscle relaxation, while at doses 1–10 mg/kg was devoid of ataxia, sedation or an influence on the anxiety level, characteristic for non-selective benzodiazepines. As an amide compound with improved stability and kinetic properties, MP-III-022 may represent an optimized tool to study the influence of α5GABAARs on the neuronal pathways related to CNS disorders such as schizophrenia, Alzheimer's disease, Down syndrome or autism.
Keywords: Amide compound; Receptor efficacy; Binding assay; Free brain concentration; Rat kinetics; Basic behavior;

PARP-1 inhibition alleviates diabetic cardiac complications in experimental animals by Esraa M. Zakaria; Hany M. El-Bassossy; Nabila N. El-Maraghy; Ahmed F. Ahmed; Abdelmoneim A. Ali (444-454).
Cardiovascular complications are the major causes of mortality among diabetic population. Poly(ADP-ribose) polymerase-1 enzyme (PARP-1) is activated by oxidative stress leading to cellular damage. We investigated the implication of PARP-1 in diabetic cardiac complications. Type 2 diabetes was induced in rats by high fructose-high fat diet and low streptozotocin dose. PARP inhibitor 4-aminobenzamide (4-AB) was administered daily for ten weeks after diabetes induction. At the end of study, surface ECG, blood pressure and vascular reactivity were studied. PARP-1 activity, reduced glutathione (GSH) and nitrite contents were assessed in heart muscle. Fasting glucose, fructosamine, insulin, and tumor necrosis factor alpha (TNF-α) levels were measured in serum. Finally, histological examination and collagen deposition detection in rat ventricular and aortic sections were carried out.Hearts isolated from diabetic animals showed increased PARP-1 enzyme activity compared to control animals while significantly reduced by 4-AB administration. PARP-1 inhibition by 4-AB alleviated cardiac ischemia in diabetic animals as indicated by ECG changes. PARP-1 inhibition also reduced cardiac inflammation in diabetic animals as evidenced by histopathological changes. In addition, 4-AB administration improved the elevated blood pressure and the associated exaggerated vascular contractility, endothelial destruction and vascular inflammation seen in diabetic animals. Moreover, PARP-1 inhibition decreased serum levels of TNF-α and cardiac nitrite but increased cardiac GSH contents in diabetic animals. However, PARP-1 inhibition did not significantly affect the developed hyperglycemia.Our findings prove that PARP-1 enzyme plays an important role in diabetic cardiac complications through combining inflammation, oxidative stress, and fibrosis mechanisms.
Keywords: PARP-1; Type 2 DM; Diabetic cardiovascular complications; 4-aminobenzamide; Olaparib;

5-Acetyl goniothalamin suppresses proliferation of breast cancer cells via Wnt/β-catenin signaling by Nittaya Boonmuen; Natthakan Thongon; Arthit Chairoungdua; Kanoknetr Suksen; Wilart Pompimon; Patoomratana Tuchinda; Vichai Reutrakul; Pawinee Piyachaturawat (455-464).
Styryl lactones are plant-derived compounds from genus Goniothalamus with promising anti-proliferation and anticancer properties. However, the exact mechanism and the target for their activities remained unclear. In the present study, we investigated the effect of 5-acetyl goniothalamin (5GTN) from Goniothalamus marcanii on Wnt/β-catenin signaling pathway which is a key regulator in controlling cell proliferation in breast cancer cells (MCF-7 and MDA-MB-231). 5GTN, a naturally occurring derivative of goniothalamin (GTN) mediated the toxicity to MCF-7 and MDA-MB-231 cells in a dose- and time- related manner, and was more potent than that of GTN. 5GTN strongly inhibited cell proliferation and markedly suppressed transcriptional activity induced by β-catenin in luciferase reporter gene assay. In consistent with this view, the expression of Wnt/β-catenin signaling target genes including c-Myc, cyclin D1 and Axin2 in MCF-7 and MDA-MB-231 cells were suppressed after treatment with 5GTN. It was concomitant with cell cycle arrest at G1 phase and cell apoptosis in MCF-7 cells. In addition, 5GTN enhanced glycogen synthase kinase (GSK-3β) activity and therefore reduced the expression of active form of β-catenin protein in MCF-7 and MDA-MB-231 cells. Taken together, 5GTN exhibited a promising anticancer effect against breast cancer cells through an inhibition of Wnt/β-catenin signaling. This pathway may be served as a potential chemotherapeutic target for breast cancer by 5GTN.Display Omitted
Keywords: 5-Acetyl goniothalamin; Anticancer; Antiproliferation; Breast cancer; Styryl lactone; Wnt signaling;

The present study was carried out to evaluate the effect of nebivolol vs. bisoprolol treatment on the intrauterine fetal growth, mortality and postnatal development in N ω-Nitro-l-arginine methyl ester hydrochloride (l-NAME)-induced hypertensive rats. Hypertension was induced in normotensive pregnant Wistar rats by daily administration of l-NAME (100 mg/kg/day, in the drinking water) for the period of pregnancy. After 9 days of l-NAME treatment, rats with systolic and diastolic blood pressure (SBP and DBP) more than 140/90 mmHg were considered hypertensive. Then, some of them were treated from day 11 to day 18 of pregnancy with nebivolol (8 mg/kg/day) or bisoprolol (10 mg/kg/day) via oral gavage. SBP, DBP and heart rate (HR) were re-evaluated by tail cuff method on day 19 of pregnancy and morphometrical or histological studies were performed on day 20. In addition, the mortality and postnatal development of newborn pups were assessed in all groups. The l-NAME administration during pregnancy induced an increase in SBP and DBP while HR did not change. Nebivolol or bisoprolol treatment completely prevented the elevation of SBP and DBP induced by l-NAME with a reduction in HR in pregnant and non-pregnant rats. The intra-uterine fetal growth and the postnatal development of newborn rats in nebivolol-treated hypertensive group were significantly lower vs. control and higher vs. bisoprolol-treated group with a higher mortality in the both types of treatments vs. control rats. The nebivolol and bisoprolol administration produce adverse effects on fetal growth and postnatal development, that limits their therapeutic use in females during pregnancy.
Keywords: Nebivolol; l-NAME; Hypertension; Pregnancy; Fetal growth; Mortality;

Pharmaceutical evaluation of naftopidil enantiomers: Rat functional assays in vitro and estrogen/androgen induced rat benign prostatic hyperplasia model in vivo by Jun-Jun Huang; Yi Cai; Yan-Zhen Yi; Min-Yi Huang; Liu Zhu; Fei He; Xia-Wen Liu; Bi-Yun Huang; Mu Yuan (473-481).
Naftopidil (NAF) is a α 1D/1A adrenoceptor selective drug used for the treatment of both benign prostatic hyperplasia and lower urinary tract symptoms (BPH/LUTS). However, NAF is used as a racemate in clinic. To compare the differences and similarities among two enantiomers and racemate, pharmacological activities were evaluated through rat functional assays in vitro and estrogen/androgen (E/T) induced rat BPH model in vivo. NAF and the two enantiomers showed similar blocking activity on α 1 receptor. S -NAF exhibited more α 1D/1A adrenoceptor subtype selectivity than R -NAF and the racemate. The selectivity ratios pA2 (α 1D)/pA2 (α 1B) and pA2 (α 1A)/pA2 (α 1B) were 40.7- and 16.2-fold, respectively. NAF and its enantiomers effectively prevented the development of rat prostatic hyperplasia via suppressing the increase of the prostatic wet weight, visually. The quantitative analysis of the relative acinus volume, relative stroma volume, relative epithelial volume, epithelial height and expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were carried out. S -NAF showed an advantage on the effect of inhibiting prostate wet weight and stroma volume over R -NAF and racemate NAF (P<0.05). Nevertheless, no other significant difference was observed between these two enantiomers. In conclusion, both R -NAF and S -NAF not only relax prostate muscle but also inhibit the prostate growth, thus relieve BPH.Display Omitted
Keywords: Benign prostatic hyperplasia; Naftopidil enantiomers; α 1 adrenoceptor blocker; Estrogen/androgen;

Induction of heat shock protein 27 by bicyclol attenuates d-galactosamine/lipopolysaccharide-induced liver injury by Hui-Juan Dai; Da-Wei Li; Ya-Xiang Wang; Ai-Jun Sun; Yi-Xian Lu; Xin- Ding; Ming- Zhang; Yong-Gang Song; Xuan-Dong Huang (482-490).
Heat shock proteins (Hsps) are critical for cell survival under adverse environmental conditions. Bicyclol is a novel hepatoprotectant that has been shown to protect against liver injury by inducing Hsps, including Hsp27 and Hsp70. Although the role of Hsp70 in protecting against acute hepatic failure has been clearly explored, the precise function of Hsp27 in this setting is poorly defined. This study was undertaken to evaluate the role of Hsp27 in bicyclol-mediated hepatoprotection. Both primary hepatocytes and bone marrow-derived macrophages were subjected to bicyclol treatment, followed by detection of Hsp27 expression. Adenoviruses containing the mouse Hsp27 coding sequence or shRNA interference sequence targeting Hsp27 were used to manipulate Hsp27 expression in the liver before the mice were treated with bicyclol and/or confronted with D-galactosamine/lipopolysaccharide (Galn/LPS)-induced acute liver damage. Only hepatocytes increased their Hsp27 expression after bicyclol treatment and the time course of bicyclol-induced Hsp27 expression in hepatocytes was in line with the in vivo results. Although high-dose bicyclol could protect against liver failure without Hsp27, the effect of bicyclol given at a low dose was dependent on Hsp27 induction. Adenovirus-mediated transduction of Hsp27 protected against acute liver damage and partially replicated the protective effect afforded by bicyclol. These results demonstrated that bicyclol induced Hsp27 expression in hepatocytes, which was essential to bicyclol-mediated hepatoprotection. Overexpression of Hsp27 in hepatocytes could confer remarkable protection against acute liver damage.
Keywords: Acute liver failure; Bicyclol; Hepatocyte; Hsp27; Macrophage;

Matrine ameliorates adriamycin-induced nephropathy in rats by enhancing renal function and modulating Th17/Treg balance by Yixiao Xu; Hongzhou Lin; Wenjie Zheng; Xiaohua Ye; Lingfang Yu; Jieqiu Zhuang; Qing Yang; Dexuan Wang (491-501).
Matrine (MAT) is an active alkaloid extracted from Radix Sophora flavescens. The present study was to investigate whether MAT could effectively treat Adriamycin-induced nephropathy (AIN). AIN was induced in rats using a single injection of Adriamycin (ADR). Renal interleukin-6 (IL-6), IL-10, IL-17 and transforming growth factor-β (TGF-β) levels, and the expression of forkhead box protein 3 (Foxp3) and retinoid-related orphan nuclear receptor γt (Rorγt) was measured. AIN rats developed severe albuminuria, hypoalbuminaemia, hyperlipidaemia and podocyte injury. Daily administration of MAT (100 mg/kg or 200 mg/kg) significantly prevented ADR-induced podocyte injury, decreased AIN symptoms and improved renal pathology manifestations. Of note, treatment with MAT (100 mg/kg) plus prednisone (Pre, 5 mg/kg) had equivalent efficacy to that of Pre alone (10 mg/kg). Additional findings showed that ADR triggered a disordered cytokine network and abnormal expression of Foxp3 and Rorγt in rats, as reflected by increased levels of IL-6, IL-10, TGF-β, Rorγt and decreased levels of IL-10 and Foxp3. Interestingly, MAT weakened the disordered cytokine network and normalized the expression of Foxp3 and Rorγt. In addition, a significant negative correlation was observed between the values of Foxp3/Rorγt and renal pathology scores. Finally, MAT normalized regulatory T cells (Treg)/ T-helper17 cells (Th17) ratio in peripheral blood mononuclear cells of AIN rats. These data indicate MAT prevents AIN through the modification of disordered plasma lipids and recovery of renal function, and this bioactivity is at least partly attributed to the suppression of renal inflammation and the regulation of the Treg/Th17 imbalance.
Keywords: Matrine; Adriamycin-induced nephropathy; Proteinuria; Treg cells; Th17 cells;

Short-term esmolol attenuates remodeling of the thoracic aorta in hypertensive rats by decreasing concentrations of ADMA down-regulated by oxidative stress by Begoña Quintana-Villamandos; María Carmen González; María Jesús Delgado-Martos; Luis Condezo-Hoyos; Rainer H. Böger; Nicole Lüneburg; Laia Pazó-Sayós; Perla Yareli Gutiérrez-Arzapalo; Emilio Delgado-Baeza (502-509).
Esmolol produces early regression of left ventricular hypertrophy and improves coronary artery remodeling, although the impact of short-term treatment with this beta-blocker on remodeling in large arteries has not yet been studied. We hypothesized that even a short (48 h) course of esmolol might alter remodeling of the aorta in the spontaneously hypertensive rat (SHR). Fourteen-month-old male SHRs were treated intravenously with vehicle (SHR, n=8) or esmolol (SHR-E, n=8) (300 μg/kg/min). Age-matched, vehicle-treated male Wistar-Kyoto rats (WKY, n=8) served as controls. After 48 h, we studied the structure, volume density of elastic fibers, and passive mechanical properties of the aorta. Determination of asymmetrical dimethylarginine concentrations and total protein carbonyls in the aorta were analyzed. Esmolol significantly attenuated abnormal aortic wall thickness, cross-sectional area, wall-to-lumen ratio, volume density of elastic fibers, and wall stiffness. The protective effect of esmolol could be related to a decrease in asymmetrical dimethylarginine levels after down-regulation by oxidative stress. These findings could play a key role in the selection of antihypertensive therapy in patients with hypertension and aortic remodeling.
Keywords: Aorta; Asymmetrical dimethylarginine; Esmolol; Hypertension; Spontaneously hypertensive rats; Vascular remodeling;

GADD45β (Growth Arrest and DNA Damage inducible protein) is a stress activated protein which plays an important role in regulating apoptosis, proliferation, DNA repair and potentially may have a role in cancer. In this study we examined the role of anti-oxidative stress on the expression of GADD45β in glioma stem-like cells (GSC). We show that patient derived GSCs have high survival in the absence of exogenous growth factors. Addition of D609 (Tricyclodecan-9-yl-xanthogenate), a known anti-oxidative compound, to GSCs reduced the cellular ATP content with significant effects observed when GSCs were cultured in growth factor free medium. D609 exposure also resulted in a decrease in the protein and an increase in mRNA of GADD45β with a concomitant decline in the survival of cells. However, under similar conditions the phosphorylation of p38 MAP kinase (stress activated MAP kinase), a downstream target of GADD45β, was significantly enhanced in response to D609. Therefore it appears that GADD45β might play a role in glioma stem cell survival and that p38 MAP kinase may not be directly activated by GADD45β. Together these observations suggest that anti-oxidative compounds like D609 can target GADD45β which may be one strategy to curtail the growth of glioma stem like cells.
Keywords: GADD45β; Glioma; Tumor stem cells; Survival;

Poly(ADP-ribose) polymerase is not involved in the neuroprotection exerted by azithromycin against ischemic stroke in mice by Francesco Petrelli; Mirko Muzzi; Alberto Chiarugi; Giacinto Bagetta; Diana Amantea (518-522).
Repurposing azithromycin has recently emerged as a promising strategy for the acute treatment of ischemic stroke. The mechanism of neuroprotection depends on the ability of this macrolide to promote polarization of microglia/macrophages towards beneficial M2 phenotypes. The immunomodulatory and anti-inflammatory effects of azithromycin, well documented in chronic inflammatory airway diseases, have been ascribed to the inhibition of the transcription factors nuclear factor (NF)-κB and activator protein (AP)−1. Since these inflammatory transcription factors are positively regulated by poly(ADP-ribose) polymerase (PARP)−1, an enzyme actively involved in ischemic brain injury, we have investigated whether the neuroprotective properties of azithromycin in ischemic stroke involve upstream modulation of PARP-1. Administration of a single dose of this macrolide antibiotic upon reperfusion reduced, to a similar extent in wild type and PARP-1 knockout mice, infarct brain damage produced by transient occlusion of the middle cerebral artery. Moreover, we demonstrated the lack of effects of azithromycin on PARP-dependent death of HeLa cells, as well as on activity of purified PARP-1 and PARP-2. Thus, azithromycin protects mice against ischemic stroke injury through a mechanism independent of PARP activation.
Keywords: Azithromycin; drug repurposing; ischemic stroke; neuroprotection; PARP;

Pharmacokinetic-pharmacodynamic influence of N-palmitoylethanolamine, arachidonyl-2′-chloroethylamide and WIN 55,212-2 on the anticonvulsant activity of antiepileptic drugs against audiogenic seizures in DBA/2 mice by Rita Citraro; Emilio Russo; Antonio Leo; Roberto Russo; Carmen Avagliano; Michele Navarra; Antonio Calignano; Giovambattista De Sarro (523-534).
We evaluated the effects of ACEA (selective cannabinoid (CB)1 receptor agonist), WIN 55,212-2 mesylate (WIN; non-selective CB1 and CB2 receptor agonist) and N-palmitoylethanolamine (PEA; an endogenous fatty acid of ethanolamide) in DBA/2 mice, a genetic model of reflex audiogenic epilepsy. PEA, ACEA or WIN intraperitoneal (i.p.) administration decreased the severity of tonic-clonic seizures. We also studied the effects of PEA, WIN or ACEA after co-administration with NIDA-41020 (CB1 receptor antagonist) or GW6471 (PPAR-α antagonist) and compared the effects of WIN, ACEA and PEA in order to clarify their mechanisms of action. PEA has anticonvulsant features in DBA/2 mice mainly through PPAR-α and likely indirectly on CB1 receptors, whereas ACEA and WIN act through CB1 receptors. The co-administration of ineffective doses of ACEA, PEA and WIN with some antiepileptic drugs (AEDs) was examined in order to identify potential pharmacological interactions in DBA/2 mice. We found that PEA, ACEA and WIN co-administration potentiated the efficacy of carbamazepine, diazepam, felbamate, gabapentin, phenobarbital, topiramate and valproate and PEA only also that of oxcarbazepine and lamotrigine whereas, their co-administration with levetiracetam and phenytoin did not have effects. PEA, ACEA or WIN administration did not significantly influence the total plasma and brain levels of AEDs; therefore, it can be concluded that the observed potentiation was only of pharmacodynamic nature. In conclusion, PEA, ACEA and WIN show anticonvulsant effects in DBA/2 mice and potentiate the effects several AEDs suggesting a possible therapeutic relevance of these drugs and their mechanisms of action.Display Omitted
Keywords: PEA; Cannabinoid compounds; PPAR-α receptors; Audiogenic seizure model; Antiepileptic drugs; Drug interaction;

Heart failure is the consequence of sustained, abnormal neurohormonal and mechanical stress and remains a leading cause of death worldwide. The aim of this work was to identify whether blockade of receptor for advanced glycation end products (RAGE) protected against systolic overload-induced heart failure and investigate the possible underlying mechanism. It was found that RAGE mRNA and protein expression was up-regulated in cardiac tissues from mice subjected to pressure overload by transverse aortic constriction (TAC). Importantly, inhibition of RAGE by treatment with soluble RAGE (sRAGE) or FPS-ZM1 (a high-affinity RAGE-specific inhibitor) for 8 weeks attenuated cardiac remodeling (including cardiac hypertrophy and fibrosis), and dysfunction in mice exposed to TAC. Furthermore, treatment of TAC mice with sRAGE or FPS-ZM1 enhanced phosphorylation of AMPK and reduced phosphorylation of mTOR and protein expression of NFκB p65 in cardiac tissues. In addition, treatment of TAC mice with sRAGE or FPS-ZM1 abated oxidative stress, attenuated endoplasmic reticulum stress, and suppressed inflammation in cardiac tissues. These data demonstrated the benefits of blocking RAGE on the progression of systolic overload-induced heart failure in mice, which was possibly through modulating AMPK/mTOR and NFκB pathways.
Keywords: Receptor for advanced glycation end products; Transverse aortic constriction; Heart failure; AMPK/mTOR; NFκB;

Inhibition of human equilibrative nucleoside transporters by 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine by Philip C.T. Tang; Cui Yang; Rachel Wai-Sum Li; Simon Ming-Yuen Lee; Maggie Pui-man Hoi; Shun-Wan Chan; Yiu-Wa Kwan; Chung-Ming Tse; George Pak-Heng Leung (544-551).
Equilibrative nucleoside transporters (ENTs) play a crucial role in the transport of nucleoside and nucleoside analogues, which are important for nucleotide synthesis and chemotherapy. In addition, ENTs regulate extracellular adenosine levels in the vicinity of its receptors and hence influence adenosine-related functions. The clinical applications of ENT inhibitors in the treatment of cardiovascular diseases and cancer therapy have been explored in numerous studies. However, all ENT inhibitors to date are selective for ENT1 but not ENT2. In the present study, we investigated the novel compound 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) as an inhibitor of ENT1 and ENT2. Nucleoside transporter-deficient PK15NTD cells stably expressing ENT1 and ENT2 showed that FPMINT inhibited [3H]uridine and [3H]adenosine transport through both ENT1 and ENT2 in a concentration-dependent manner. The IC50 value of FPMINT for ENT2 was 5-10-fold less than for ENT1, and FPMINT could not be displaced with excess washing. Kinetic studies revealed that FPMINT reduced V max of [3H]uridine transport in ENT1 and ENT2 without affecting K M. Therefore, we conclude that FPMINT inhibits ENTs in an irreversible and non-competitive manner. Although already selective for ENT2 over ENT1, further modification of the chemical structure of FPMINT may lead to even better ENT2-selective inhibitors of potential clinical, physiological and pharmacological importance.
Keywords: Cancer; Cardiovascular disease; Equilibrative nucleoside transporters; Inhibitor;

Action of Pitolisant on the stimulant and rewarding effects of cocaine in mice by Christian Brabant; Yana Charlier; Maria Elisa Serrano Navacerrada; Livia Alleva; Ezio Tirelli (552-559).
Previous studies have demonstrated that the histamine H3 receptor inverse agonist thioperamide potentiates the stimulant and rewarding effects of cocaine. However, these potentiating effects of thioperamide do not necessarily result from H3 receptor blockade since thioperamide is an imidazole-based compound capable of enhancing plasma cocaine concentrations by blocking cytochrome P450 activity. In contrast, Pitolisant is a non-imidazole H3 receptor inverse agonist that has already been tested in clinical trials but it remains to be determined whether this compound also potentiates the behavioral effects of cocaine. The present study tested the effects of Pitolisant on locomotion, on cocaine-induced hyperactivity and on the development of conditioned place preference induced by cocaine (2 and 8 mg/kg, i.p.) in male C57BL/6J mice. Pitolisant was injected 30 min before each cocaine-pairing session. Locomotion recorded on the first cocaine-pairing session was used to test the effects of Pitolisant on the locomotor effects of cocaine. Our results show that doses of Pitolisant higher than 10 mg/kg depressed locomotion. When injected alone at doses that did not affect locomotion, Pitolisant (2.5–10 mg/kg, i.p.) had no rewarding properties in the place conditioning technique. Additionally, Pitolisant did not significantly alter cocaine-induced hyperactivity and cocaine-induced conditioned place preference. Taken together, our study indicates that Pitolisant has no addictive properties alone. Moreover, this compound does not significantly affect the stimulant and rewarding effects of cocaine. These results add further evidence to support the hypothesis that a pharmacokinetic interaction is involved in the ability of thioperamide to potentiate cocaine's psychomotor effects.
Keywords: Pitolisant; Cocaine; Histamine H3 receptor; Conditioned place preference; Reward; Locomotion;

SYP-5, a novel HIF-1 inhibitor, suppresses tumor cells invasion and angiogenesis by Li-Hui Wang; Xiao-Rui Jiang; Jing-Yu Yang; Xue-Fei Bao; Jun-Li Chen; Xing Liu; Guo-Liang Chen; Chun-Fu Wu (560-568).
Hypoxia-inducible factor-1 (HIF-1) plays an essential role in carcinogenesis. The overexpression of HIF-1 induced by hypoxia is closely associated with metastasis, poor prognosis and high mortality. In this study, a novel HIF-1 inhibitor SYP-5 was first observed by the luciferase reporter assay. Western blots results showed SYP-5 inhibited hypoxia-induced upregulation of HIF-1. Moreover, the proteins of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP)-2 that are targets of HIF-1, were down-regulated by SYP-5. Furthermore, in the tube formation assay, SYP-5 suppressed angiogenesis induced by hypoxia and VEGF in vitro. Additionally, using Transwell and RTCA assays, we found that SYP-5 also retarded the Hep3B and Bcap37 cells migration and invasion induced by hypoxia and FBS. Last, we also detected the upstream pathways related to HIF-1 and found both PI3K/AKT and MAPK/ERK were involved in the SYP-5 mediated invasive inhibition of Bcap37 cells. These results indicates that SYP-5 inhibits tumor cell migration and invasion, as well as tumor angiogenesis, which are mediated by suppressing PI3K/AKT- and MAPK/ERK-dependent HIF-1 pathway. It suggests that SYP-5 might be a potential HIF-1 inhibitor as an anticancer agent.
Keywords: HIF-1; SYP-5; Angiogenesis; Invasion; PI3K/AKT pathway; MAPK pathway;

Pharmacological characterization of a series of aryl-sulfonamide derivatives that potently and selectively inhibit monoacylglycerol acyltransferase 2 by Ryutaro Adachi; Tsuyoshi Ishii; Kazumasa Ogawa; Shinichi Matsumoto; Takuya Satou; Junichi Sakamoto; Kenjiro Sato; Tomohiro Kawamoto (569-577).
Monoacylglycerol acyltransferase (MGAT) 2 is an endoplasmic reticulum membrane enzyme that catalyzes the synthesis of diacylglycerol (DAG) from fatty acyl-CoA and monoacylglycerol as substrates. It is important for the resynthesis of triacylglycerol in the intestine. We have identified a series of aryl-sulfonamide MGAT2 inhibitors and demonstrated pharmacological inhibition of MGAT2 improved hyperlipidemia, obesity, and diabetes in animal models. However, its mechanism of action has not been elucidated in molecular and cellular levels. In the present study, we have characterized a series of aryl-sulfonamide derivatives that potently and selectively inhibit human MGAT2 and determined their pharmacological profiles. Analyses on the molecular mechanism of a representative aryl-sulfonamide MGAT2 inhibitor revealed a reversible inhibitory activity and a binding activity to MGAT2. The aryl-sulfonamide derivatives exhibited potent inhibitory activities against both human and mouse intestinal MGAT activities, which were correlated to those determined using recombinant human and mouse MGAT enzymes. We have developed a cellular assay using Liquid Chromatography-Mass Spectrometry and confirmed that the aryl-sulfonamide derivatives suppressed DAG synthesis in the cellular context. We have thus elucidated their pharmacological profiles and provided the fundamental clues for understanding the molecular and cellular actions of the aryl-sulfonamide MGAT2 inhibitors.Display Omitted
Keywords: Monoacylglycerol acyltransferase 2; Intestinal microsomes; Affinity Selection Mass Spectrometry; Cellular LC/MS assay; Compound B;

The reactive oxygen species(ROS)/mitogen-activated protein kinase (MAPK) destroyed autophagy and the reactive oxygen species/mitogen-activated protein kinase (MAPK) pathway are considered closely related to ethanol-induced hepatocellular injury. Previous work indicated that corosolic acid, the natural extracts of leaves of the banaba tree, Lagerstroemia speciosa L., could protect the liver against ethanol-induced damage, but the underlying mechanism is unclear. In the study we found that corosolic acid significantly inhibited ethanol-induced apoptosis, increased level of tumor necrosis factor-α(TNF-α) and reactive oxygen species accumulation in vitro. Corosolic acid inhibited ethanol-activated p38 and c-Jun N-terminal kinase MAPK signaling in BRL-3A and HepG2 cells as well as in experimental rats. Corosolic acid restored the ethanol-suppressed expression of autophagy-related genes, including beclin-1 and the ratio of microtubule-associated protein light chain 3II/I (LC3II/I) via AMP-activated protein kinase (AMPK) activation both in vitro and in vivo. In experimental rats, corosolic acid ameliorated the detrimental histopathological findings. Corosolic acid may protect the liver against ethanol-induced injury by modulation of MAPK signaling and autophagy activation. These findings suggested that corosolic acid might be a promising agent in treatment of alcoholic liver diseases.
Keywords: Corosolic acid; Ethanol; Apoptosis; Reactive oxygen species /MAPKs; Autophagy; AMPK;

Efficacy of CoenzymeQ10 in inhibiting monosodium urate crystal-induced inflammation in rats by B. Udhaya Lavinya; Ishita Bardhan; Sabina Evan Prince (589-594).
Gout is a type of arthritis, which could result from the deposition of monosodium urate crystals in joints. It can cause redness, burning pain, inflammation of joints especially in big toe. In this study, we have looked for anti-arthritic effect of coenzyme Q10 (CoQ10) on monosodium urate crystal-induced inflammation in rats and compared it with that of the non-steroidal anti-inflammatory drug, indomethacin. The evaluation was done by measuring the paw volume, antioxidant status, lipid peroxidation, lysosomal enzymes (β-glcuronidase, β-galactosidase, N-acetyl-β-d-glucosaminidase, acid phosphatase) activities and histopathological studies. Paw volume, the levels of lysosomal enzymes, lipid peroxidation were significantly (P<0.05) increased and the antioxidant activity status was in turn decreased in monosodium urate crystal-induced rats. CoQ10 (10 mg/kg/b.w. orally) treated monosodium urate crystal-induced rats showed near normal activities of lysosomal enzymes, reduced levels of lipid peroxidation, near normal paw volume and antioxidant status. CoQ10 was also able to minimize mononuclear cell infiltration and damage to articular cartilage. Current study indicates that CoQ10 possesses anti-inflammatory effect against gouty arthritis and can be used to treat acute form of gouty arthritis.
Keywords: Gouty arthritis; Inflammation; Coenzyme Q10; Antioxidant;

Myocardial infarction continues to be a major public health problem. Reduction in mortality rate and prevention of myocardial infarction are of utmost importance. Inflammation and thrombosis play an important role in the pathogenesis of myocardial infarction. The anti-inflammatory and anti-thrombotic effects of zingerone were evaluated in isoproterenol induced myocardial infarcted rats. Rats were pretreated with zingerone (6 mg/kg body weight) daily for 14 days and were then induced myocardial infarction with isoproterenol (100 mg/kg body weight) on 15th and 16th day. Isoproterenol induced myocardial infarcted rats showed significant (P<0.05) increase in the levels/ activities of cardiac troponin-I (cTnI), high sensitive C-reactive protein (Hs CRP), lysosomal hydrolases in the serum and concentration of heart lysosomal lipid peroxidation (LPO) products. RT-PCR study revealed over expression of myocardial tumour necrosis factor - alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) genes in the myocardial infarcted rats. Histopathology of heart and coronary artery revealed marked inflammation and coronary thrombosis. Zingerone pretreatment significantly (P<0.05) decreased serum cTnI, Hs CRP, lysosomal hydrolases and heart lysosomal LPO and down regulated myocardial TNF-α, IL-1β and IL-6 genes and prevented coronary thrombosis in isoproterenol induced myocardial infarcted rats. The observed effects of zingerone could be attributed to its anti-inflammatory and anti-thrombotic properties.Display Omitted
Keywords: Adenosine triphosphate (PubChem CID: 5957); Ammonium sulfate (PubChem CID: 6097028); Isoproterenol (PubChem CID: 5807); Magnesium sulfate (PubChem CID: 24083); Magnesium chloride (PubChem CID: 5360315); Potassium chloride (PubChem CID: 4873); P-nitrophenol (PubChem CID: 980); Sodium carbonate (PubChem CID: 10340); Thio barbituric acid (PubChem CID: 2723628); Zingerone (PubChem CID: 31211); Troponin-I; Zingerone; Isoproterenol; Myocardial infarction; Heart; C- reactive protein;

MicroRNA-127 is a tumor suppressor in human esophageal squamous cell carcinoma through the regulation of oncogene FMNL3 by Xuhui Gao; Xuelian Wang; Kaican Cai; Wujun Wang; Qun Ju; Xiyao Yang; Haofei Wang; Hua Wu (603-610).
In this study, we investigated the expression patterns and functional roles of microRNA 127 (miR-127) and its target gene Formin-Like 3 (FMNL3) in human esophageal squamous cell carcinoma (ESCC). Quantitative RT-PCR (qRT-PCR) was used to compare miR-127 expression between ESCC cell lines and normal esophageal epithelium cell line, as well as paired ESCC tumors and adjacent normal esophageal tissues in 33 patients. We found miR-127 was aberrantly downregulated in both ESCC cell lines and human ESCC tumors. In ESCC cell lines TE-1 and ECA109 cells, lentiviral-induced miR-127 upregulation markedly inhibited cancer proliferation and migration in vitro, and tumorigenicity in vivo. Through dual-luciferase assay and qRT-PCR, FMNL3 was confirmed to be the downstream target gene of miR-127 in ESCC. Finally, FMNL3 was downregulated by siRNA in TE-1 and ECA109 cells. And we discovered that SiRNA-induced FMNL3 downregulation had tumor suppressive effect in ESCC, inhibiting cancer proliferation, migration in vitro, and tumorigenicity in vivo. These results suggest that miR-127 is downregulated and acting as tumor suppressor in ESCC. Inversely, FMNL3, the target gene of miR-127, is upregulated and acting as an oncogene in ESCC.
Keywords: Esophageal squamous cell carcinoma; MiR-127; Cancer in vitro growth; Cancer in vivo growth, FMNL3;

Bergenin, isolated from Bergenia ligulata is a potent antioxidant and antilithiatic agent. Present work was designed to establish the biochemical role of bergenin on mitochondrial dysfunction in the ethylene glycol induced hyperoxaluric rat model.Bergenin was administrated at a dose of 10 mg/kg body wt i.p. from 14th day of establishing the 28 days hyperoxaluria rat model. α-Tocopherol was given as positive control at a dose of 100 mg/kg body wt i.p. Mitochondrial dysfunction was studied by evaluating the activities of respiratory chain complexes, mitochondrial membrane potential and reactive oxygen species. Histopathological analysis of the kidney tissue was done after Pizzolato staining. Also, expression of monocyte chemoattractant protein −1(MCP-1) and kidney injury marker protein (KIM-1) were studied and the levels of IL-1β were evaluated in kidney tissue homogenate.Mitochondrial dysfunction during stone crystallization was evident by decreased activities of electron transport chain complexes I, II and IV and augmented mitochondrial oxidative stress in hyperoxaluric rats. Bergenin treatment significantly (P<0.05) restored the activities of these complexes. Moreover, it curtailed the lipid peroxidation and up regulated antioxidant levels, ameliorating the state of mitochondrial dysfunction. The protective role of bergenin was also reinforced by reducing IL-1β production and expression of KIM-1 and MCP-1 in the renal tissue.The findings of the present study provide evidence that bergenin exerted protective effects in hyperoxaluria through mitochondrial protection that involves attenuation of oxidative stress. Hence, it presented itself as an effective remedy in combating urolithiasis.
Keywords: Hyperoxaluria; Renal injury; Mitochondrial dysfunction; Bergenin; Oxidative stress;

Chronic LPSF/GQ-02 treatment attenuates inflammation and atherosclerosis development in LDLr−/− mice by Amanda Karolina Soares e Silva; Fabiana Oliveira dos Santos Gomes; Bruna dos Santos Silva; Edlene Lima Ribeiro; Amanda Costa Oliveira; Shyrlene Meyre da Rocha Araújo; Ingrid Tavares de Lima; Anne Gabrielle Vasconcelos Oliveira; Martina Rudnicki; Dulcineia S.P. Abdalla; Maria do Carmo Alves de Lima; Ivan da Rocha Pitta; Christina Alves Peixoto (622-631).
Atherosclerosis is a complex disorder with a multifactorial pathogenesis. We previously indicated that the new TZD LPSF/GQ-02 inhibits hepatic steatosis and inflammation, which are reported as risk factors for atherosclerosis development. Here, we explored the effects of LPSF/GQ-02 on atherosclerosis in LDLr−/− mice comparing two treatment periods.LDLr−/− mice were fed a high-fat diet for 10 and 12 weeks and received oral treatment with LPSF/GQ-02 (30 mg/kg/day) or pioglitazone (20 mg/kg/day) for 15 and 30 days, respectively. Both treatment protocols with LPSF/GQ-02 resulted in lower collagen density in the atherosclerotic lesions. In addition, the treatment for 15 days also decreased mRNA levels of CD40, MCP-1, ABCG1 and upregulated PPARα, whereas the 30-days treatment reduced the protein levels of LOX-1, p-IκBα and p-NFκB.This study provides evidence that LPSF/GQ-02 affects the composition and growth of atherosclerotic lesions in LDLr−/− mice. Moreover, our data also support previous findings showing anti-inflammatory properties of LPSF/GQ-02 and reinforce the therapeutic potential of this TZD for treating atherosclerosis and inflammation-related disorders.
Keywords: Atherosclerosis; Inflammation; LPSF/GQ-02; Thiazolidinediones;

Tanshinone IIA (Tan) exerts potential protective effects against cardiovascular diseases. Oxidative stress and inflammation are involved in cardiac hypertrophy. Activation of silent information regulator 1 (SIRT1) signaling has been suggested to attenuate cardiac hypertrophy. This study aims to evaluate the antioxidative and anti-inflammatory effects of Tan treatment in pressure overload-induced myocardial remodeling and elucidated its potential mechanisms. Sprague-Dawley rats were treated with Tan in the absence or presence of the SIRT1 inhibitor sirtinol (Snl) and then subjected to transverse aortic constriction (TAC). Tan conferred cardioprotective effects by improving cardiac function, reducing apoptosis and myocardial remodeling, upregulating SIRT1, Bcl-2 expressions, and downregulating Bax and caspase-3 expressions. Snl attenuated these effects by inhibiting SIRT1 signaling. Tan treatment also reduced myocardium malondialdehyde (MDA) content, and cardiac inflammatory cytokines (TNF-α and IL-6) and increased myocardium superoxide dismutase (SOD) level. However, these effects were also abolished by Snl. In conclusion, these results indicate that Tan significantly attenuates TAC-induced myocardial remodeling possibly due to its strong anti-oxidative and anti-inflammatory activity. Importantly, SIRT1 signaling activation is involved in this process.
Keywords: Myocardium remodeling; Cardiac hypertrophy; Tanshinone IIA; Oxidative stress; Inflammation;

Antispasmodic effect of selected Citrus flavonoids on rat isolated jejunum specimens by Marta Mendel; Magdalena Chłopecka; Natalia Dziekan; Wojciech Karlik (640-646).
Citrus flavonoids are acknowledged for numerous pharmacological activities, including the myorelaxant effect on various smooth muscles. However, there is no data on their effect on jejunum contractility. Therefore, the aim of the study at hand was to evaluate the impact of hesperetin and diosmetin along with their glycosides on the motoric activity of intestine and to verify the possible mechanism of hesperetin-induced effect. The experiments were performed on rat isolated jejunum strips and were conducted under isometric conditions.Hesperetin and diosmetin, but not hesperidin and diosmin, dose-dependently (10–100 µM) and reversibly inhibited acetylcholine (1 µM) and KCl (80 mM) induced contractile activity. The antispasmodic effect of hesperetin was partially blocked by 4-aminopyridine (100 µM), glibenclamide (100 µM) and NG-nitro-L-arginine methyl ester (L-NAME, 100 µM). By contrast, apamin (0.1 µM), tetraethylammonium (500 µM) and methylene blue (10 µM) did not affect the magnitude of hesperetin-induced myorelaxant effect. Indomethacin (10 µM) increased the force of hesperetin-evoked reaction.In conclusion, hesperetin and diosmetin are potent myorelaxant agents. The antispasmodic effect of hesperetin is partially mediated by fast current low-voltage activated K+ channels, voltage-independent K+ channels and involves the nitric oxide pathway. Finally, hesperetin shows a synergistic effect with indomethacin towards jejunal KCl-precontracted smooth muscle.Display Omitted
Keywords: Hesperetin; Diosmetin; Jejunum; Antispasmodic effect; Potassium channels; Nitric oxide;

Inhibitory effect of recombinant human endostatin on the proliferation of hypertrophic scar fibroblasts in a rabbit ear model by Yong-Fang Gong; Xiao-Ming Zhang; Fei Liu; Zhen-Zhen Wang; Xue-Fei Deng; Yi Jiao; Xiao-Jing Li; Xue-Ying Huang (647-654).
Hypertrophic scar (HS) is a pathological scar that particularly occurs after traumatic injuries, surgical procedures and burning. Abnormal activation of hypertrophic scar fibroblasts (HSFs) intensifies fibrosis during wound healing. Our previous studies demonstrated that recombinant human endostatin (rhEndostatin) prevented synovial thickening in adjuvant arthritis (AA) rats via inhibition of proliferation and enhancement of apoptosis in synovial fibroblasts. However, the effect of this protein on HSF proliferation is not known. This study investigated the inhibitory effect of rhEndostatin on the proliferation of cultured HSFs in a rabbit ear model. MTT assay and flow cytometric detection were performed to investigate HSF proliferation and cell cycle progression, respectively. The expression levels of p53, p21, cyclinD1, cyclin-dependent kinase 4 (CDK4) and proliferating cell nuclear antigen (PCNA) in HSFs were detected using real-time PCR and Western blotting. Our data revealed that HSFs treated with rhEndostatin were significantly inhibited in a concentration-dependent manner with an IC50 value of 100 mg/L. Also, rhEndostatin (100 mg/L) primarily induced G0/G1 and partially G2/M cell cycle arrest of HSFs. There were significant decreases in the expression levels of p53, p27, CDK4, cyclinD1 and PCNA in HSFs treated with rhEndostatin. In conclusion, rhEndostatin inhibited HSF proliferation via G0/G1 and/or G2/M phase arrest of the cell cycle, which was partially due to the down-regulation of cyclinD1, CDK4 and PCNA. These findings suggest that rhEndostatin may reduce scar hypertrophy in vivo via inhibition of HSF proliferation and may be a novel agent for HS treatment.
Keywords: Recombinant human endostatin; Hypertrophic scar; Fibroblast; Proliferation;

The α2β3γ2 GABAA receptor preferring agonist NS11394 aggravates dystonia in the phenotypic dt sz model by Christine Spröte; Franziska Richter; Anne Bauer; Julia Gerstenberger; Angelika Richter (655-658).
Dystonia is a movement disorder, characterized by involuntary muscle contractions resulting in abnormal movements and/or postures. Antidystonic effects of benzodiazepines in patients with different types of dystonia could be replicated in the dt sz mutant hamster, a phenotypic model of paroxysmal dystonia. Compounds with preferred binding at specific subunits of the gamma aminobutyric acid type A (GABAA) receptor may provide a more beneficial spectrum of effects in comparison with benzodiazepines. We therefore examined the effects of the α1β3γ2 GABAA receptor preferring compound zolpidem (2.0–10.0 mg/kg i.p.) and of the α2β3γ2 GABAA receptor preferring compound NS11394 (3.0–30 mg/kg i.p.) on the severity of dystonia in the dt sz mutant in comparison with the benzodiazepine clonazepam (0.5–1.0 mg/kg i.p.). As expected, clonazepam exerted pronounced antidystonic effects. While zolpidem showed moderate beneficial effects, NS11394 significantly increased the severity of dystonia. The present results indicate for the first time that positive GABAA receptor modulators show contrary effects on dystonia dependent on their preference for alpha-subunits. The potential link between alterations in GABAA receptor subunits and GABAergic disinhibition in dystonia deserves further attention in research on the pathophysiology and therapeutic targets.
Keywords: Dyskinesia; Movement disorders; Dystonia; GABA; Subunits;

The calcilytics Calhex-231 and NPS 2143 and the calcimimetic Calindol reduce vascular reactivity via inhibition of voltage-gated Ca2+ channels by Harry Z.E. Greenberg; Kazi S. Jahan; Jian Shi; W.-S. Vanessa Ho; Anthony P. Albert (659-668).
The present study investigates the effect of commonly used negative and positive allosteric modulators of the calcium-sensing receptor (CaSR) on vascular reactivity. In wire myography studies, increasing [Ca2+]o from 1 mM to 6 mM induced concentration-dependent relaxations of methoxamine-induced pre-contracted rabbit mesenteric arteries, with 6 mM [Ca2+]o producing almost complete relaxation. [Ca2+]o-induced relaxations were attenuated in the presence of the calcilytics Calhex-231 and NPS 2143, and abolished by the removal of the endothelium. In addition to their calcilytic effects, Calhex-231 and NPS 2143 also produced concentration-dependent inhibitions of methoxamine- or KCl-induced precontracted tone, which were unaffected by removal of the endothelium and unopposed in the presence of the calcimimetic Calindol. In vessels with depleted Ca2+ stores, contractions mediated by Ca2+ influx via voltage-gated Ca2+ channels (VGCCs) were inhibited by Calhex231. In freshly isolated single rabbit mesenteric artery smooth muscle cells, Calhex-231 and NPS 2143 inhibited whole-cell VGCC currents. Application of Calindol also inhibited methoxamine- and KCl-induced pre-contracted tone, and inhibited whole-cell VGCC currents. In conclusion, in addition to their CaSR-mediated actions in the vasculature, Calhex-231, NPS 2143 and Calindol reduce vascular contractility via direct inhibition of VGCCs.
Keywords: Calcium-sensing receptor; Calcilytic; Calcimimetic; Calhex-231; NPS 2143; Calindol;

Palmitoylethanolamide reduces inflammation and itch in a mouse model of contact allergic dermatitis by Massimo Vaia; Stefania Petrosino; Daniele De Filippis; Luana Negro; Andrea Guarino; Rosa Carnuccio; Vincenzo Di Marzo; Teresa Iuvone (669-674).
In mice, 2,4-dinitrofluorobenzene (DNFB) induces contact allergic dermatitis (CAD), which, in a late phase, is characterized by mast cell (MC) infiltration and angiogenesis. Palmitoylethanolamide (PEA), an endogenous anti-inflammatory molecule, acts by down-modulating MCs following activation of the cannabinoid CB2 receptor and peroxisome proliferator-activated receptor-α (PPAR-α). We have previously reported the anti-inflammatory effect of PEA in the early stage of CAD. Here, we examined whether PEA reduces the features of the late stage of CAD including MC activation, angiogenesis and itching. After sensitization to DNFB, female C57BL/6J mice underwent to three DNFB challenges at days 5, 12 and 19 and treatments were given at each challenge and for two more days. CAD was expressed as Δ increase in ear thickness between challenged and un-challenged mice. PEA (5 mg/kg/i.p.) reduced: i) the DNFB-induced Δ increase; ii) the number of MCs per tissue area; iii) the expression of VEGF and its receptor Flk-1. These effects were reversed by co-administration of AM630 (1 mg/kg/i.p.), a CB2 antagonist, but not GW6471 (1 mg/kg/i.p.), a PPAR-α antagonist. Finally, PEA reduced the number of ear scratchings 48 h after DNFB challenge and this effect was reversed by both CB2 and PPAR-α antagonists, suggesting the involvement of both receptors. PEA, by reducing the features of late stage CAD in mice, may be beneficial in this pathological condition.
Keywords: Angiogenesis; Contact allergic dermatitis (CAD); Dinitrofluorobenzene (DNFB); Itch; Mast cells (MCs); Palmitoylethanolamide (PEA);

Gestational carbenoxolone exposure inhibits placental 11β-hydroxysteroid dehydrogenase (11β-HSD), the physiological barrier for glucocorticoids, which increases fetal exposure to glucocorticoids and induces intrauterine growth restriction (IUGR). We hypothesized that carbenoxolone exposure influences the expression of placental estrogen receptors-α and β (ERα & ERβ) and p53 leading to inhibited fetal and placental growth. Pregnant Sprague-Dawley rats were injected twice daily with either carbenoxolone (10 mg/kg; s.c.) or vehicle (control group) from gestational days (dg) 12 onwards. Maternal blood and placentas were collected on 16 dg, 19 dg and 21 dg. The expression of ERα, ERβ and p53 were studied in placental basal and labyrinth zones by RT-PCR, Western blotting and immunohistochemistry. Carbenoxolone did not affect placental and fetal body weights, but ELISA showed decreased estradiol levels on 19 dg and 21 dg, and increased maternal luteinizing hormone levels on all dg. The follicle stimulating hormone levels decreased on 16 dg and 19 dg, and increased on 21 dg. Carbenoxolone decreased ERα mRNA levels on 16 dg in both zones and its protein level on 19 dg in the labyrinth zone. However, carbenoxolone increased ERβ mRNA levels on 19 dg and 21 dg and protein levels on 16 dg and 19 dg in the labyrinth zone. The p53 mRNA levels increased on all dg, but its protein levels increased on 21 dg in both zones. In conclusion, carbenoxolone exposure changes placental p53, ERα, ERβ expression in favor of cell death but these changes do not induce IUGR in rats.
Keywords: Apoptosis; Carbenoxolone; P53; Estrogen receptor; Intrauterine growth restriction;

Pyrazole derivatives were originally suggested as selective blockers of the transient receptor potential cation 3 (TRPC3) and channel. In particular, pyr3 and 10 selectively inhibit TRPC3, whereas pyr2 (BTP2) and 6 inhibit ORAI1. However, their effects on background K+ channel activity have not been elucidated. In this study, the effects of BTP2, pyr3, pyr6, and pyr10 were studied on cloned human TWIK-related K+ channels (TREKs) and TWIK-related acid-sensitive K+ channel 2 (TASK-2) channels, which modulate Ca2+ signaling by controlling membrane potential, in HEK293T-overexpressing cells by using a whole-cell patch clamp technique. Pyr3 potently inhibited TREK-1 (ITREK1), TREK-2 (ITREK2), and TASK2 current (ITASK-2) with half-maximal inhibitory concentrations (IC50) of 0.89±0.27, 1.95±1.44, and 2.42±0.39 µM, respectively. BTP2 slightly inhibited ITASK-2 (80.3±2.5% at 100 μM). In contrast, pyr6 at 100 µM potentiated ITREK1 and ITREK2 by approximately 2.6- and 3.6-fold compared to the control and inhibited ITASK2 (38.7±9.2%). Pyr10 showed a subtype-specific inhibition of ITREK1 but not ITREK2. It also inhibited ITASK2 (70.9±3.1% at 100 μM). To the best of our knowledge, this study is the first to describe the differential modulation of TREKs and TASK2 channels by pyrazole derivatives, previously used as inhibitors of TRPC3 and ORAI1. Therefore, studies using these drugs should consider their modulation of other channels such as TREK and TASK-2.
Keywords: Pyrazole derivative; TREK-1; TREK-2; TASK-2; Calcium signaling; Potassium channels;

The role of histamine H1, H2 and H3 receptors of ventral posteromedial nucleus of thalamus in modulation of trigeminal pain by Esmaeal Tamaddonfard; Amir Erfanparast; Hamid Ghasemi; Farzin Henareh-Chareh; Mansoor Hadidi; Navideh Mirzakhani; Sahar Seyedin; Mina Taati; Reza Salighedar; Sara Salimi; Farshad Safaei (696-702).
Histamine receptors are involved in supraspinal modulation of pain. In the present study, we investigated the effects of microinjection of histamine H1, H2 and H3 receptor antagonists and agonists into the ventral posteromedial (VPM) nucleus of the thalamus on two models of trigeminal pain. Right and left sides of VPM were implanted with two guide cannulas. Corneal pain was induced by local corneal surface application of hypertonic saline and the number of eye wipes was recorded. The duration of face rubbing, as an orofacial pain measure, was recorded after subcutaneous (s.c.) injection of capsaicin into the vibrissa pad. 2-pyridylethylamine (2-PEA, a histamine H1 receptor agonist, 4 µg/site) and dimaprit (a histamine H2 receptor agonist, 1 and 4 µg/site) suppressed corneal and orofacial pains. Mepyramine (a histamine H1 receptor antagonist) and ranitidine (a histamine H2 receptor antagonist) at the similar doses of 0.5, 2 and 8 µg/site alone had no effects on trigeminal pain. Prior microinjection of mepyramine and ranitidine at a similar dose of 8 µg/site inhibited the antinociceptive effects of 2-PEA (4 µg/site) and dimaprit (4 µg/site), respectively. Immepip (a histamine H3 receptor agonist, 1 and 4 µg/site) increased, and thioperamide (a histamine H3 receptor antagonist, 2 and 8 µg/site) attenuated nociceptive responses. Prior microinjection of thioperamide (8 µg/site) prevented immepip (4 µg/site)-induced nociception. These chemicals did not change locomotor behavior. It is concluded that post-synaptic histamine H2, and to a lesser extent H1, receptors and pre-synaptic histamine H3 receptor may be involved in VPM modulation of trigeminal pain.
Keywords: Histamine receptors; Ventral posteromedial nucleus; Trigeminal pain; Rats;

Insulin resistance is associated with accelerated atherosclerosis. Although high fructose is known to induce insulin resistance, it remains unclear as to how fructose regulates insulin receptor signaling and proliferative phenotype in vascular smooth muscle cells (VSMCs), which play a major role in atherosclerosis. Using human aortic VSMCs, we investigated the effects of high fructose treatment on insulin receptor substrate-1 (IRS-1) serine phosphorylation, insulin versus platelet-derived growth factor (PDGF)-induced phosphorylation of Akt, S6 ribosomal protein, and extracellular signal-regulated kinase (ERK), and cell cycle proteins. In comparison with PDGF (a potent mitogen), neither fructose nor insulin enhanced VSMC proliferation and cyclin D1 expression. d-[14C(U)]fructose uptake studies revealed a progressive increase in fructose uptake in a time-dependent manner. Concentration-dependent studies with high fructose (5–25 mM) showed marked increases in IRS-1 serine phosphorylation, a key adapter protein in insulin receptor signaling. Accordingly, high fructose treatment led to significant diminutions in insulin-induced phosphorylation of downstream signaling components including Akt and S6. In addition, high fructose significantly diminished insulin-induced ERK phosphorylation. Nevertheless, high fructose did not affect PDGF-induced key proliferative signaling events including phosphorylation of Akt, S6, and ERK and expression of cyclin D1 protein. Together, high fructose dysregulates IRS-1 phosphorylation state and proximal insulin receptor signaling in VSMCs, but does not affect PDGF-induced proliferative signaling. These findings suggest that systemic insulin resistance rather than VSMC-specific dysregulation of insulin receptor signaling by high fructose may play a major role in enhancing atherosclerosis and neointimal hyperplasia.
Keywords: Fructose; Insulin receptor substrate; Insulin; PDGF; Vascular smooth muscle cells; Proliferation;

Galacto-N-biose is neuroprotective against glutamate-induced excitotoxicity in vitro by Yo Shinoda; Yui Nakajima; Hirotoshi Iguchi; Satoshi Tatsumi; Motomitsu Kitaoka; Masahiro Nakajima; Tsutomu Takahashi; Yasuyuki Fujiwara; Teiichi Furuichi (711-717).
Galacto-N-biose (GNB: Galβ1-3GalNAc) is an O-glycan disaccharide core moiety that is a core component of mucin in the gastrointestinal tract; however, the physiological properties of GNB are not well understood. Glutamate excitotoxicity causes neuronal death in acute neurological disorders including stroke, trauma, and neurodegenerative disease. Therefore the discovery of drugs to treat glutamate excitotoxicity is an important goal. Here, we report that GNB is neuroprotective against glutamate-induced excitotoxicity. We treated 14–15 days in vitro cultured rat cortical neurons with 0.1–1000 nM GNB together with 30 µm glutamate for various durations. Short-term (3 h) GNB treatments showed a modest neuroprotective effect against glutamate neurotoxicity, however, long-term (24 h) GNB treatment conferred significant neuroprotective effects, as shown by both MTT and immunocytochemical assays. Prolonged GNB treatment did not alter glutamate-induced calcium influx, but did induce antioxidant-related gene expression. Furthermore, GNB treatment did not induce cell death or alter synaptic connections. These data suggest that GNB is a potential candidate drug that protects against glutamate excitotoxicity without affecting cell viability and synaptic connections.
Keywords: GNB; Excitotoxicity; Glutamate; Cortical neuron;

Helium postconditioning regulates expression of caveolin-1 and -3 and induces RISK pathway activation after ischaemia/reperfusion in cardiac tissue of rats by Moritz Flick; Martin Albrecht; Gezina T.M.L. Oei; Renske Steenstra; Raphaela P. Kerindongo; Coert J. Zuurbier; Hemal H. Patel; Markus W. Hollmann; Benedikt Preckel; Nina C. Weber (718-725).
Caveolae, lipid enriched invaginations of the plasma membrane, are epicentres of cellular signal transduction. The structural proteins of caveolae, caveolins, regulate effector pathways in anaesthetic-induced cardioprotection, including the RISK pathway. Helium (He) postconditioning (HePoc) is known to mimic anaesthetic conditioning and to prevent damage from myocardial infarction. We hypothesize that HePoc regulates caveolin-1 and caveolin-3 (Cav-1 and Cav-3) expression in the rat heart and activates the RISK pathway.Male Wistar rats (n=8, each group) were subjected to 25 min of cardiac ischaemia followed by reperfusion (I/R) for 5, 15 or 30 min (I/R 5/15/30). The HePoc groups underwent I/R with 70% helium ventilation during reperfusion (IR+He 5/15/30 min). Sham animals received surgical treatment without I/R. After each protocol blood and hearts were retrieved. Tissue was obtained from the area-at-risk (AAR) and non-area-at-risk (NAAR) and processed for western blot analyses and reverse-transcription-real-time-polymerase-chain-reaction (RT-qPCR).Protein analyses revealed increased amounts of Cav-1 and Cav-3 in the membrane of I/R+He15 (AAR: Cav-1, P<0.05; Cav-3, P<0.05; both vs. I/R15). In serum, Cav-3 was found to be elevated in I/R+He15 (P<0.05 vs. I/R15). RT-qPCR showed increased expression of Cav-1 in IR+He15 in AAR tissue (P<0.05 vs. I/R15). Phosphorylation of RISK pathway proteins pERK1/2 (AAR: P<0.05 vs. I/R15) and pAKT (AAR: P<0.05; NAAR P<0.05; both vs. I/R15) was elevated in the cytosolic fraction of I/R+He15.These results suggest that 15 min of HePoc regulates Cav-1 and Cav-3 and activates RISK pathway kinases ERK1/2 and AKT. These processes might be crucially involved in HePoc mediated cardioprotection.
Keywords: Cardiac postconditioning; Helium; Caveolin; RISK pathway; Ischaemia reperfusion; Noble gases;

Esculin attenuates endotoxin shock induced by lipopolysaccharide in mouse and NO production in vitro through inhibition of NF-κB activation by Weifeng Li; Yu Wang; Xiumei Wang; Zehong He; Fang Liu; Wenbing Zhi; Hailin Zhang; Xiaofeng Niu (726-734).
Esculin, a coumarin compound derived from the traditional Chinese herbs such as Cortex Fraxini, has long been used for treating inflammatory and vascular diseases. In present study, we analyzed the role of esculin against macrophages and endotoxin shock induced by lipopolysaccharide (LPS) in mice. Here, we demonstrated that esculin suppressed inflammatory reactions in macrophages and protected mice from LPS-induced endotoxin shock. We found that esculin significantly inhibited the production of nitric oxide (NO) production via the inhibition of nuclear factor-κB (NF-κB) activation in macrophages. In animal model, esculin pretreatment significantly improved the survival rate of mice. LPS-induced increase of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in serum, lung, liver and kidney were markedly inhibited by esculin. IL-10, an anti-inflammatory cytokine, was up-regulated by esculin. Moreover, the histopathological analyses showed that esculin significantly attenuated the tissues injury of lung, liver, kidney in endotoxic mice. In addition, esculin significantly diminished the protein expression of NF-κB p65 in lung, liver, kidney, which resulted in lower levels of inflammatory mediators. These results suggest that esculin may be a potential drug for treatment of various inflammatory diseases.Display Omitted
Keywords: Esculin; Endotoxin shock; Macrophages; Pro-inflammatory cytokines; Nuclear factor-kappa B;

Liraglutide attenuates lipopolysaccharide-induced acute lung injury in mice by Feng Zhou; Ying Zhang; Jing Chen; Xuemei Hu; Yancheng Xu (735-740).
Liraglutide, an effective drug for the treatment of diabetes, has been proven to demonstrate anti-inflammatory and immunomodulatory effects. Hence, this study explored the effects and mechanism of action of liraglutide on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male BALB/c mice were pre-conditioned with liraglutide or saline prior to intraperitoneal LPS or saline administration. Histopathological examination of lung, the wet/dry (W/D)weight ratio, protein content, inflammatory cell numbers and pro-inflammatory cytokine levels in broncho-alveolar lavage fluid (BAL fluid) were conducted. The effects of liraglutide on the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome signalling pathway were assessed by Western blot. Pre-treatment with liraglutide decreased the wet-to-dry weight ratio and protein concentrations in BAL fluid and neutrophil infiltration in the lung tissues. Liraglutide also significantly reduced the interleukin-1β and interleukin-18 levels in BAL fluid, as well as effectively inhibited the expression of NLRP3 inflammasome. These results indicated that liraglutide pre-treatment attenuated LPS-induced ALI by inhibiting the NLRP3 inflammasome pathway.
Keywords: Liraglutide; Lipopolysaccharide; Acute lung injury; Inflammation; NLRP3 inflammasome;

Vasodilation and hypotension of a novel 3-benzylquinazolin- 4(3H)-one derivative via the inhibition of calcium flux by Sen Li; Sai-jie Zuo; Lei Cao; Dong-zheng Liu; San-qi Zhang; Yong-xiao Cao (741-750).
A novel 3-benzylquinazolin-4(3H)-one derivative Z32, namely 6,7-dimethoxy-3-(3-chloro-4-(4-fluorobenzyloxy)benzyl)quinazolin-4(3H)-one was synthesized. The vasorelaxant and antihypertensive effects of Z32 and its underlying mechanisms were investigated. The following methods were used. The isometric tension of artery ring segments was recorded using an in vitro myography system. Changes in the calcium influx in mesenteric arteries were surveyed using a real-time confocal microscopy. The arterial pressure of spontaneously hypertensive rats was measured in vivo using a non-invasive tail cuff blood pressure system. The results showed that Z32 can relax rat mesenteric arteries pre-constricted by KCl or phenylephrine in a concentration-dependent manner. The vasorelaxant effects were not affected by the removal of the endothelium, blockade of potassium channels by tetraethylammonium chloride, or inhibition of either guanylate cyclase by ODQ, nitric oxide synthase by l-NAME, or cyclooxygenase by indomethacin. In Ca2+-free conditions, Z32 did not affect the constriction evoked by caffeine, however, significantly reduced the constrictions induced (1) by phenylephrine, (2) by CaCl2 in either phenylephrine (in the presence of verapamil) or KCl stimulated arteries, (3) by extracellular Ca2+ restoration in thapsigargin-treated mesenteric arteries, and (4) by the activator of protein kinase C phorbol-12, 13-dibutyrate, and the inhibitor of protein tyrosine phosphatase sodium orthovanadate. Further, Z32 decreased the systolic and diastolic arterial pressure of spontaneously hypertensive rats in a dose-dependent manner. In conclusion, Z32 lowers the arterial pressure and induces vasorelaxation through the inhibition of calcium flux, probably via a protein tyrosine phosphorylation-dependent way.
Keywords: 3-benzylquinazolin-4(3H)-one; Calcium flux; Mesenteric artery; Vasodilation; Hypertensive rats; Hypotension;

Synergistic effect of piperine and paclitaxel on cell fate via cyt-c, Bax/Bcl-2-caspase-3 pathway in ovarian adenocarcinomas SKOV-3 cells by Manish Kumar Pal; Shyama Pyari Jaiswar; Ajeet Kumar Srivastav; Shruti Goyal; Ashish Dwivedi; Ankit Verma; Jyoti Singh; Anumesh Kumar Pathak; Pushpa Lata Sankhwar; Ratan Singh Ray (751-762).
Ovarian cancer is fourth most common and lethal among all gynecologic malignancies. The chemotherapy usually requires in all stages of ovarian cancer but drugs have several side effects. We hypothesized that use of combination therapy of paclitaxel (PTX) and phytochemical piperine (PIP) may reduce the PTX dose as well as toxicity. The human ovarian adenocarcinomas SKOV3 cell treated with PTX-5 nM and PIP-10 µm after determination of IC50 by MTT assay. Reactive oxygen species generation, mitochondrial membrane potential (MMP), DNA damage, cell death pathway markers as release of cyt-c, Bax/Bcl2-caspase-3 and cell cycle arrest were analyzed. The dose dependent treatment of SKOV-3 cells showed IC50 and synergism at combination of 5 nM-PTX and 10 µm-PIP in cell viability assay. PTX and PIP increases the accumulation of reactive oxygen species which subsequently leading to increase in JC-1 and fragmented nuclei in mitotracker/DAPI staining. Comet assay showed 4.4-fold increase of tail formation in combined treated cells as compared to control. PTX-PIP arrests the cell cycle in sub-G1 phase. Immunocytochemistry of Bax showed increase in red fluorescence intensity whereas decrease in green fluorescence i.e Bax/Bcl-2 ratio increased. Moreover morphological EB/AO and Hoechst staining confirmed the enhanced apoptosis in combined treatment. Significant upregulation of apoptotic genes, cyt-c (3.4 fold) Bax (2.8 fold), caspase-3 (3.6 fold) whereas no change occurred in Bcl2 mRNA expression and protein expressions. The combination of PTX with PIP produces synergistic effects in SKOV-3 cells via the modulation of pro and anti-apoptotic gene and may compensate the toxicity and side effects of PTX.
Keywords: Paclitaxel; Piperine; DNA damage; Apoptosis; Caspase-3;

Cytokine production by PBMC and serum from allergic and non-allergic subjects following in vitro histamine stimulation to test fexofenadine and osthole anti-allergic properties by Natalia Karolina Kordulewska; Elżbieta Kostyra; Anna Cieślińska; Ewa Fiedorowicz; Beata Jarmołowska (763-772).
FXF is a third-generation antihistamine drug and osthole is assumed a natural antihistamine alternative. This paper compares peripheral blood mononuclear cell (PBMC) incubation with FXF and osthole, by studying FXF, osthole and histamine cytokine secretion in PBMC in vitro cultures. Mabtech kits determined the interleukins IL-1β, IL-4, IL-10, IL-13 and TNF-α. The influence of the above active substances on cytokine secretion in PBMC's and serum was assessed: cytokines were IL-1β, IL-4, IL-10, IL-13 and TNF-α; and cytokine levels secreted by untreated PBMCs in pure culture medium formed the absolute control (ctrl).We determined that osthole affects PBMC cytokine secretion to almost precisely the same extent as FXF (IL-1β, IL-4, IL-10 and TNF). In addition osthole had greater IL-13 blocking ability than FXF. Moreover, we observed significantly decreased IL-4 level in histamine/osthole theatment compared to histamine alone. Meanwhile, FXF not significantly decrease the level of IL-4 increased by histamine. This data indicates osthole's strong role in allergic inflamation.All results confirm our hypothesis that osthole is a natural histamine antagonist and therefore can be beneficially used in antihistamine treatment of conditions such as allergies.
Keywords: Allergic disease; Antihistamine drugs; Cultures in vitro; Cytokine secretion; Interleukin;

The effects of ZD0947, a novel ATP-sensitive K+ channel (KATP channel) opener, on the activity of reconstituted KATP channels were investigated using cell-attached recordings. KATP channels were studied in HEK 293 cells by co-expression of inwardly rectifying-6 family K+ channel subunits (Kir6.x: Kir6.1 and Kir6.2) with 3 different types of sulphonylurea receptors (SUR.x: SUR1, SUR2A and SUR2B). ZD0947 (100 µM) activated SUR2B/Kir6.2 channels in a concentration-dependent manner, but caused only weak activation of SUR1/Kir6.2 channels and SUR2A/Kir6.2 channels expressed in HEK 293 cells. ZD0947 reversibly suppressed diazoxide-elicited SUR1/Kir6.2 channels activity and pinacidil-elicited SUR2A/Kir6.2 channel activity. However, ZD0947 did not affect SUR2B/Kir6.2 channels fully activated by 100 µM pinacidil. ZD0947 had little inhibitory effects on the activity of Kir6.2ΔC26 channels (a truncated isoform of Kir6.2) or its mutant channels (i.e. Kir6.2ΔC26C166A) expressed in HEK 293 cells. ZD0947 also elicited activity in SUR2B/Kir6.1 channels expressed in HEK 293 cells, in a concentration-dependent manner. Therefore, ZD0947 is a relatively effective activator of smooth muscle-type KATP channels (SUR2B/Kir6.1 and SUR2B/Kir6.2) but is a partial antagonist of pancreatic-type KATP channels (i.e. SUR1/Kir6.2) and cardiac-type KATP channels (i.e. SUR2A/Kir6.2). These results suggest that a pharmacological agent can possess either agonist or antagonist actions on the activity of KATP channels, depending on the subtype of SUR.x.
Keywords: ATP-sensitive K+ channels; K+ channel opener; Smooth muscle; ZD0947;

L-Arginine supplementation improves insulin sensitivity and beta cell function in the offspring of diabetic rats through AKT and PDX-1 activation by Diego Soares Carvalho; Marilia Melo Diniz; André Abour Haidar; Maria de Fátima Cavanal; Eduardo da Silva Alves; Angelo Rafael Carpinelli; Frida Zaladek Gil; Aparecida Emiko Hirata (780-787).
Maternal hyperglycemia can result in defects in glucose metabolism and pancreatic β-cell function in offspring. The purpose of this study was to evaluate the impact of maternal diabetes mellitus on pancreatic islets, muscle and adipose tissue of the offspring, with or without oral l-Arginine supplementation. The induction of diabetes was performed using streptozotocin (60 mg/kg). Animals were studied at 3 months of age and treatment (sucrose or l-Arginine) was administered from weaning. We observed that l-Arg improved insulin sensitivity in the offspring of diabetic mothers (DA), reflected by higher insulin-induced phosphorylation of Akt in muscle and adipose tissue. Insulin resistance is associated with increased oxidative stress and the NADPH oxidase enzyme plays an important role. Our results showed that the augmented interaction of p47PHOX with gp91PHOX subunits of the enzyme in skeletal muscle tissue in the offspring of diabetic rats (DV) was abolished after l-Arg treatment in DA rats. Maternal diabetes caused alterations in the islet functionality of the offspring leading to increased insulin secretion at both low (2.8 mM) and high (16.7 mM) concentrations of glucose. l-Arg reverses this effect, suggesting that it may be an important modulator in the insulin secretory process. In addition it is possible that l-Arg exerts its effects directly onto essential molecules for the maintenance and survival of pancreatic islets, decreasing protein expression of p47PHOX while increasing Akt phosphorylation and PDX-1 expression. The mechanism by which l-Arg exerts its beneficial effects may involve nitric oxide bioavailability since treatment restored NO levels in the pancreas.
Keywords: Fetal programming; Maternal diabetes; Pancreatic islets; Insulin; l-Arginine;

Effects of thyroid hormones on aortic tissue after myocardial infarction in rats by Vanessa D. Ortiz; Alexandre L. de Castro; Cristina Campos; Rafael O. Fernandes; Jéssica H.P. Bonetto; Rafaela Siqueira; Adriana Conzatti; Tânia R.G. Fernandes; Adriane Belló-Klein; Alex S.R. Araujo (788-793).
Studies have shown a cardioprotective role of thyroid hormones (THs) in cardiac remodeling after acute myocardial infarction (MI). However, there is no data in the literature examining the influence of TH administration on the aortic tissue in an animal model of MI. This study aimed to evaluate the effects of thyroid hormones on the aorta after MI. Male Wistar rats were divided into a sham group (SHAM), infarcted group (AMI), sham+TH (SHAMT) and AMI+TH (AMIT). After MI, the animals received T3 and T4 (2 and 8 μg/100 g/day, respectively) by oral gavage for 12 days. Later, the animals underwent echocardiography and euthanasia and the aorta was collected for molecular and biochemical analysis. T3 and T4 administration increased the expression of the pro-angiogenic proteins vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1α (HIF-1α) in the aorta of AMIT rats when compared with AMI. With respect to TH receptors, AMI rats presented a decrease in TRβ levels, which was prevented by the hormonal administration. In AMIT rats, both TRα and TRβ levels were increased when compared with the AMI group. Reactive oxygen species levels and NADPH oxidase activity were decreased in both treated groups when compared with the non-treated animals. TH administration after MI may improve angiogenic signaling in the aorta as well as the responsiveness of this vessel to T3 and T4. These positive effects in the aorta may result in additional protection for the cardiovascular system in the context of cardiac ischaemic injury.
Keywords: Angiogenesis; T3; T4;

Pain-depression dyad induced by reserpine is relieved by p,p'-methoxyl-diphenyl diselenide in rats by Carla Elena Sartori Oliveira; Marcel Henrique Marcondes Marcondes Sari; Vanessa A. Zborowski; Vinicius Costa Prado; Cristina Wayne Nogueira; Gilson Zeni (794-802).
Depression and pain comorbidity represent a neuropsychiatric condition with substantial socioeconomic impact to society. The commonly used antidepressants and analgesics to treat this comorbidity have shown restricted clinical efficacy. In this way, the aim of this study was to investigate the behavioral, biochemical and neurochemical effects of a p,p'-methoxyl-diphenyl diselenide (OMePhSe)2 supplemented diet on pain-depression dyad induced by reserpine in rats. Adult Wistar rats were fed with 10 mg (MeOPhSe)2 per kg of rat chow supplemented diet for 30 days. Pain-depression dyad was induced by daily subcutaneous reserpine injection (0.5 mg/kg for three consecutive days) from 22 to 24 day of (MeOPhSe)2 supplementation. The results showed that the reserpine injected rats had behavior phenotypes typical of depression-pain dyad and the (MeOPhSe)2-supplemented diet protected against these modifications. Furthermore, the (MeOPhSe)2 dietary supplementation was effective against the increase in the prefrontal cortical MDA levels caused by reserpine. (MeOPhSe)2-supplemented diet triggered a per se augmentation of Nrf-2 levels. The [3H] serotonin uptake, [3H] glutamate uptake and release and MAO activity were not altered in the prefrontal cortices of rats from any experimental group. Therefore, the results indicate that protective effects of a (MeOPhSe)2-supplemented diet can be mediated, at least in part, by its antioxidant property.
Keywords: Selenium; Pain-depression dyad; Reserpine; Organoselenium; Antioxidant;

New 2-Aminothiazoline derivatives lower blood pressure of spontaneously hypertensive rats (SHR) via I1-imidazoline and alpha-2 adrenergic receptors activation by Renan B. Ferreira; Mariana G. de Oliveira; Edson Antunes; Wanda P. Almeida; Badr M. Ibrahim; Abdel A. Abdel-Rahman (803-810).
2-Aminothiazolines share an isosteric relationship with imidazolines and oxazolines with antihypertensive activity mainly mediated by the imidazoline I 1-receptor. In the present work, we have prepared five aminothiazolines, following a previously described synthetic pathway. Aminothiazolines derived from dicyclopropylmethylamine (ATZ1) and cyclohexylamine (3) are unprecedented in the literature. Competitive radioligand assay was carried out with all synthetic compounds, and the I 1 receptor affinity in comparison to rilmenidine in PC12 cells was determined. Surprisingly, the rilmenidine isoster (ATZ1) showed no I 1-receptor interaction. Diethyl (ATZ4) and 2-ethyl-hexylamine (ATZ5) derivatives bind to the receptor with 11.98 and 10.94 nmol/l, respectively. These compounds were selected for in vivo experiments. Both compounds reduced the blood pressure of spontaneously hypertensive rats (SHR). The hypotensive effect of these compounds was abrogated in the presence of α2 adrenergic (yohimbine) and I 1 (efaroxan) receptor antagonists suggesting that both aminothiazolines bind to the adrenergic and imidazoline receptors. Lipinski's descriptors of the synthesized aminothiazolines were calculated and are similar to the known imidazoline I 1 receptor ligands. 3D-Similarity between ATZ5 and agmatine, the natural imidazoline receptor ligand, was also observed.
Keywords: Imidazoline receptor; Aminothiazoline; Hypertension; PC12 cells; Binding assay;

Conjugated Alpha-Alumina nanoparticle with vasoactive intestinal peptide as a Nano-drug in treatment of allergic asthma in mice by Seyyed Shamsadin Athari; Zahra Pourpak; Gert Folkerts; Johan Garssen; Mostafa Moin; Ian M. Adcock; Masoud Movassaghi; Mehdi Shafiee Ardestani; Seyed Mohammad Moazzeni; Esmaeil Mortaz (811-820).
Asthma is a chronic respiratory disease characterized by airway inflammation, bronchoconstriction, airway hyperresponsiveness and recurring attacks of impaired breathing. Vasoactive intestinal peptide (VIP) has been proposed as a novel anti-asthma drug due to its effects on airway smooth muscle relaxation, bronchodilation and vasodilation along with its immunomodulatory and anti-inflammatory properties. In the current study, we investigated the therapeutic effects of VIP when conjugated with α-alumina nanoparticle (α-AN) to prevent enzymatic degradation of VIP in the respiratory tract. VIP was conjugated with α-AN. Balb/c mice were sensitized and challenges with ovalbumin (OVA) or PBS and were divided in four groups; VIP-treated, α-AN-treated, α-AN-VIP-treated and beclomethasone-treated as a positive control group. Specific and total IgE level, airway hyperresponsiveness (AHR), bronchial cytokine expression and lung histology were measured. α-AN-VIP significantly reduced the number of eosinophils (Eos), serum IgE level, Th2 cytokines and AHR. These effects of α-AN-VIP were more pronounced than that seen with beclomethasone or VIP alone (P<0.05). The current data indicate that α-AN-VIP can be considered as an effective nano-drug for the treatment of asthma.
Keywords: Alpha-Alumina nanoparticle; Vasoactive intestinal peptide; Allergic asthma;