European Journal of Pharmacology (v.718, #1-3)

The protective and therapeutic effects of alpha-solanine on mice breast cancer by Maryam Mohsenikia; Ali Mohammad Alizadeh; Saeed Khodayari; Hamid Khodayari; Seyed Amin kouhpayeh; Aliasghar Karimi; Mina Zamani; Saleh Azizian; Mohammad Ali Mohagheghi (1-9).
Alpha-solanine, a naturally steroidal glycoalkaloid, is found in leaves and fruits of plants as a defensive agent against fungi, bacteria and insects. Herein, we investigated solanine toxicity in vitro and in vivo, and assessed its protective and the therapeutic effects on a typical animal model of breast cancer. The study conducted in three series of experiments to obtain (i) solanine effects on cell viability of mammary carcinoma cells, (ii) in vivo toxicity of solanine, and (iv) the protective and therapeutic effects of solanine on animal model of breast cancer. Alpha-solanine significantly suppressed proliferation of mouse mammary carcinoma cells both in vitro and in vivo (P<0.05). Under the dosing procedure, 5 mg/kg solanine has been chosen for assessing its protective and therapeutic effects in mice breast cancer. Tumor take rate in the solanine–treated group was zero compared with a 75% rate in its respective control group (P<0.05). The average tumor size and weight were significantly lower in solanine-treated animals than its respective control ones (P<0.05). Proapoptotic Bax protein expression increased in breast tumor by solanine compared with its respective control group (P<0.05). Antiapoptotic Bcl-2 protein expression found to be lower in solanine-treated animals (P<0.05). Proliferative and angiogenic parameters greatly decreased in solanine-treated mice (P<0.05). Data provide evidence that solanine exerts a significant chemoprotective and chemotherapeutic effects on an animal model of breast cancer through apoptosis induction, cell proliferation and angiogenesis inhibition. These findings reveal a new therapeutic potential for solanine in cancer.A potential mechanism of solanine that can facilitate the opening of the mitochondrial permeability transitional pores (mPTP) by lowering the membrane potential, leading to release of Ca2+ from mitochondria. This result in turn leads to in an increase in the concentration of Ca2+ in the cell, thus triggering Bax and Bcl-2, and the occurrence of apoptosis.Display Omitted
Keywords: Solanine; Breast cancer; Mice; Apoptosis; Angiogenesis;

A review on antiepileptic drugs-dependent fatigue: Pathophysiological mechanisms and incidence by Antonio Siniscalchi; Luca Gallelli; Emilio Russo; Giovambattista De Sarro (10-16).
Fatigue represents a common side effect of several drugs, however, the underlying mechanisms have not been well identified. A depression of the central nervous system (CNS) and/or changes in peripheral processes have been associated with the development of fatigue. Antiepileptic drugs (AEDs), generally decreasing CNS excitability, are used in the treatment of seizures as well as other neurological and psychiatric diseases. Fatigue is certainly a common AEDs' side effect, although a high degree of variability exists depending on both patients' characteristics and the drug used. Here, we delineate the pathophysiological central and peripheral mechanisms by which AEDs may cause fatigue also reviewing the available clinical data in order to assess a possible AEDs rank and highlight each AEDs related risk. It appears that drugs acting on the GABAergic system have the highest incidence (with tiagabine exception) of fatigue followed by Gabapentin and Levetiracetam whereas drugs mainly inhibiting sodium channels (Carbamazepine, Eslicarbazepine, Lamotrigine, Phenytoin and Valproate) have the lowest. However, the dose used, AEDs related side effects and patients' characteristics might influence the degree of fatigue observed.
Keywords: AED; Side effect; Fatigue; Central mechanism; Peripheral mechanism;

Effect of metabotropic glutamate 5 receptor antagonists on morphine efficacy and tolerance in rats with neuropathic pain by Quanhong Zhou; Jing Wang; Xin Zhang; Lulu Zeng; Li Wang; Wei Jiang (17-23).
The metabotropic glutamate 5 (mGlu5) receptor is involved in both pain processing and modulation of µ-opioid induced antinociception and antihyperalgesia. Systemic mGlu5 receptor antagonists 2-methyl-6-phenylethynylpyridine (MPEP) or 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine (MTEP) provide antihyperalgesic effects in various pain models, but few studies have shown their interaction with morphine in neuropathic pain models. The aim of this study is to compare the effects of systemic and intrathecal MPEP/MTEP on morphine efficacy and tolerance in rats with chronic neuropathic pain. L5-6 spinal nerve ligation (SNL) was used to establish neuropathic pain model in rats. The Von Frey test and the hot water tail flick test were employed as behavior tests. Low, medium and high doses of MPEP/MTEP were tested for their effect on both acute morphine efficacy and chronic morphine tolerance. SNL provides sustained neuropathic pain on the ipsilateral hind paw of rats. Both systemic and intrathecal MPEP/MTEP had antiallodynia effects and boosted morphine's efficacy in a dose-dependent manner in the Von Frey tests but not in the tail flick tests. In fact, high doses of MTEP and MPEP attenuated morphine's antinociceptive effect in the latter test. After intrathecal chronic co-administration with morphine, low-doses of MTEP/MPEP attenuated morphine tolerance in both tests. Systemically, only MTEP attenuated morphine tolerance, and only in the Von Frey tests, not in the tail flick tests, whereas MPEP had no effect on morphine tolerance in either tests. The therapeutic use of mGlu5 receptor antagonists may have distinct effects in different pain models.
Keywords: Neuropathic pain; Morphine; Efficacy; Tolerance; Metabotropic glutamate 5 receptor;

Guggulsterone regulates the function and expression of P-glycoprotein in rat brain microvessel endothelial cells by Xian-Zhen Chen; Hong-Bin Xu; Lu-Zhong Xu; Xia-Ping Mao; Ling Li (24-29).
Our previous studies found that guggulsterone could inhibit P-glycoprotein-mediated multidrug resistance in P-glycoprotein over-expressed human cancer cell lines. However, the effects of guggulsterone on the ;P-glycoprotein function and expression in rat brain microvessel endothelial cells (rBMECs) are poorly understood. In the present study, we investigated whether guggulsterone has a modulative effect on the function and expression of P-glycoprotein in rBMECs. rhodamine 123 acts as a good substrate for P-glycoprotein, and agents that block P-glycoprotein have been found to increase the retention of rhodamine in cells. The results showed that the accumulation of rhodamine 123 in rBMECs was potentiated in a time-dependent manner after incubation with 30, 100 μM guggulsterone (P<0.05). Efflux of intracellular rhodamine 123 was decreased in a time-dependent manner from after 30, 100 μM guggulsterone treatment. The inhibitory effect of guggulsterone on P-glycoprotein function was reversible and remained at 120 min after removal of 30, 100 μM guggulsterone from the medium. Further results showed that guggulsterone (30, 100 μM) down-regulated the expression of P-glycoprotein, and had no influence on the expression of breast cancer resistance protein in rBMECs. In addition, the present study revealed that guggulsterone promoted the activity of P-glycoprotein ATPase in a dose-dependent manner. These results indicated that guggulsterone suppressed the function and expression of P-glycoprotein in rBMECs primary cultures.
Keywords: Guggulsterone; Rat brain microvessel endothelial cell; P-glycoprotein;

Platelet-activating factor (PAF) is a potent lipid mediator that is implicated in numerous inflammatory diseases. Under inflammatory conditions, PAF is biosynthesized through the remodelling pathway and elicits many inflammatory responses through binding to its specific PAF receptor. Endogenous bioactive endothelins (ETs: ET-1, -2, and -3) are also considered potent inflammatory mediators that play a critical role in many inflammatory diseases. In this perspective, we provide a brief overview of possible common mechanisms in ETs and PAF receptor function for inflammatory responses. Accumulating evidence strongly suggests that ET-3, but not ET-1 and ET-2, can attenuate PAF-induced inflammation through direct binding of the Tyr–Lys–Asp (YKD) region in the peptide to PAF and its metabolite/precursor lyso-PAF, followed by inhibition of binding between PAF and its receptor. Additionally, YKD sequence-containing peptides may be useful as a novel type of anti-inflammatory drugs targeting this mechanism. These findings should lead to new treatment strategies for numerous inflammatory diseases by targeting the common mechanism in ET and PAF receptor function.
Keywords: Platelet-activating factor; Endothelin-3; Inflammation; PAF receptor; Anti-inflammatory drug;

Synergistic anti-proliferative effect of resveratrol and etoposide on human hepatocellular and colon cancer cell lines by Fatemehsadat Amiri; Amir-Hassan Zarnani; Hamid Zand; Fariba Koohdani; Mahmood Jeddi-Tehrani; Mohammadreza Vafa (34-40).
Resveratrol is an active component of grape, which has been shown to inhibit proliferation of a wide variety of tumor cells. The ability of resveratrol to enhance anti-proliferative effects of etoposide in wild type p53 liver carcinoma (HepG2) and colon cancer (HCT-116) cells was investigated with focusing on p53 activation. HepG2 cells and HCT-116 cells were treated with resveratrol and/or etoposide in a time- and dose-dependent manner and their proliferative response was evaluated by XTT assay. The expression of p53 protein was assessed using Western blot. Resveratrol exerted anti-proliferative activity on both cell types in a dose (25–100 μM)- and time (24–72 h)-dependent manner. Interestingly in HepG2 cells, resveratrol exhibited the same levels of cytotoxicity as etoposide (10 μM) when the cells treated with ≥25 μM for 48–72 h. In contrast to HepG2, resveratrol significantly enhanced anti-proliferative effects of etoposide in HCT-116 cells. P53 expression was up-regulated by resveratrol and etoposide and pre-incubation of both cells with resveratrol increased levels of etoposide-induced p53 expression. In line with cytotoxicity effect, combination therapy showed stronger activation of p53 in HCT-116 compared to HepG2. It seems that resveratrol exerts differential synergistic effect with etoposide on proliferation of cancer cells from different origin which is mainly accompanied by p53 activation. Our data represent a future strategy to provide much safer and more effective treatment for colon cancer.
Keywords: Resveratrol; Etoposide; HepG2; HCT-116; Cytotoxicity; p53;

Transferrin modified PEG-PLA-resveratrol conjugates: In vitro and in vivo studies for glioma by Wanhua Guo; Aimei Li; Zhijun Jia; Yi Yuan; Haifeng Dai; Hongxiu Li (41-47).
Glioblastoma is one of the most malignant brain tumors with a poor prognosis. In this study, we examined the effects of transferrin (Tf)-modified poly ethyleneglycol-poly lactic acid (PEG-PLA) nanoparticles conjugated with resveratrol (Tf-PEG-PLA-RSV) to glioma therapy in vitro and in vivo. The cell viability of Tf-PEG-PLA-RSV on C6 and U87 glioma cells was determined by the MTT assay. In vivo biodistribution and antitumor activity were investigated in Brain glioma bearing rat model of C6 glioma by i.p. administration of RSV-polymer conjugates. We found that the average diameter of each Tf-PEG-PLA-RSV is around 150 nm with 32 molecules of Tf on surface. In vitro cytotoxicity of PEG-PLA-RSV against C6 and U87 cells was higher than that of free RSV, and further the modification of Tf enhanced the cytotoxicity of the RSV-polymer conjugates as a result of the increased cellular uptake of the RSV-modified conjugates by glioma cells. In comparison with free RSV, RSV conjugates could significantly decrease tumor volume and accumulate in brain tumor, which resulted in prolonging the survival of C6 glioma-bearing rats. These results suggest that Tf-NP-RSV had a potential of therapeutic effect to glioma both in vitro and in vivo and might be a potential candidate for targeted therapy of glioma and worthy of further investigation.
Keywords: Glioma; Transferrin; Resveratrol; PEG-PLA; In vitro and in vivo;

Evaluation of the effect of kaempferol in a murine allergic rhinitis model by Hyun-A Oh; Na-Ra Han; Myong-Jo Kim; Hyung-Min Kim; Hyun-Ja Jeong (48-56).
Kaempferol (KP) is a major compound of Naju Jjok (Polygonum tinctorium Lour.). The effect of KP on allergic rhinitis (AR) has not been elucidated. Here, we report the effects and mechanisms of KP on new and predominant mediators of AR using an eosinophil cell line, Eol-1 and an ovalbumin (OVA)-induced AR mouse model. KP significantly inhibited the production of interleukin (IL)-32 and IL-8 and activation of caspase-1 in Eol-1 cells. Allergic symptoms and predominant mediators (IgE and histamine) in the KP-administered group were significantly lower than in the AR group. The levels of interferon-γ were enhanced while the levels of IL-4 were reduced in the KP group. KP significantly reduced the levels of IL-32 and thymic stromal lymphopoietin (TSLP) compared with the AR mice. KP reduced the levels of inflammation-related proteins. In the KP-administered groups, the infiltrations of eosinophils and mast cells increased by OVA were decreased. In addition, KP significantly reduced caspase-1 activity in nasal mucosa tissue of AR mice. Our findings indicate that KP has an anti-allergic effect through the regulation of the production of IL-32 and TSLP and caspase-1 activity in allergic diseases including AR.
Keywords: Kaempferol; Allergic rhinitis; Interleukin-32; Thymic stromal lymphopoietin; Caspase-1;

Anti-arthritic and anti-inflammatory activity of combined pioglitazone and prednisolone on adjuvant-induced arthritis by Sanvidhan G. Suke; Harsh Negi; P.K. Mediratta; B.D. Banerjee; K.K. Sharma (57-62).
The aim of the present study was to evaluate the effect of combined treatment of pioglitazone (PGZ) and prednisolone (PDL) on the progression of adjuvant-induced arthritis in rats. Adjuvant arthritis was induced by single intra-dermal injection of 0.1 ml Freund's complete adjuvant (0.05% w/v Mycobacterium butyricum in mineral oil) into foot pads of left hind paws of Wistar rats of either sex. There were six experimental groups: Group I was healthy animals as control, Group II was arthritic animals without drug treatment, Group III was arthritic animals treated with a standard non-steroidal anti-inflammatory drug aspirin (100 mg/kg), Group IV was arthritic animals received PGZ (10 mg/kg) alone, Group V was arthritic animals received PDL (10 mg/kg) alone, and Group VI was arthritic animals treated with a combined suspension of PGZ and PDL (20 mg/kg). Drugs were administered daily orally at day 0 and continued upto 28th day after induction of arthritis. Induction of arthritis significantly increased hind paw volume (HPV), loss of body weight (BW), enhanced tibiotarsal joint thickness (TJT), and increased plasma tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 levels. Treatment with aspirin or combined suspension of PGZ and PDL in the arthritic animals produced significant reductions in HPV and TJT, normalized BW, and significantly decreased plasma levels of TNF-α and IL-6. These observations suggest that the combined administration of PGZ and PDL was effective in modulating the inflammatory response and suppress arthritis progression in experimental animal model. These findings may help to improve the treatment of rheumatoid arthritis.The data were expressed as mean±S.D. Each group contained 8–10 animals. Statistical significance was evaluated applying Student t-test for independent variable. Comparisons were made between: a—Group I vs Group II–V and VI. b—Group II vs Group III–VI. c—Group III vs Group VI. d—Group VI vs Group IV and V. Symbols represent statistical significance: * (P<0.001), # (P<0.01) and (P<0.05).Display Omitted
Keywords: Arthritis; Inflammation; Pioglitazone; Prednisolone; Freund's complete adjuvant;

Effects of propofol on GABAergic and glutamatergic transmission in isolated hippocampal single nerve-synapse preparations by Masahito Wakita; Naoki Kotani; Kiku Nonaka; Min-Chul Shin; Norio Akaike (63-73).
We evaluated the effects of propofol on synaptic transmission using a mechanically dissociated preparation of rat hippocampal CA3 neurons to allow assays of single bouton responses evoked from retained functional native nerve endings. We studied synaptic and extrasynaptic GABAA and glutamate receptor responses in a preparation in which experimental solutions rapidly accessed synaptic terminals. Whole-cell responses were evoked by bath application of GABA and glutamate. Synaptic inhibitory and excitatory postsynaptic currents (IPSC and EPSC) were measured as spontaneous and evoked postsynaptic responses. Evoked currents were elicited by focal electrical stimulation. Propofol (1–100 μM) enhanced extrasynaptic GABAA-receptor mediated responses but the increase at clinically relevant concentrations (1 μM) were minor. In contrast, 1 μM propofol significantly increased both the amplitude and frequency of spontaneous IPSCs (sIPSCs) and increased the amplitudes of evoked IPSCs (eIPSCs) while decreasing failure rates (Rf) and paired-pulse ratios (PPR). Decay times of sIPSCs and eIPSCs were significantly prolonged. Although propofol had no effect on extrasynaptic glutamate responses, only supra-clinical propofol concentrations (≥10 µM) increased the spontaneous EPSCs (sEPSCs, amplitudes and frequencies) but suppressed evoked EPSCs (eEPSCs decreased amplitudes with increased Rf and PPR). The decay phases of sEPSCs and eEPSCs were not changed. The propofol-induced changes in sEPSCs and eEPSCs resulted from presynaptic GABAA receptor-mediated depolarization, because these actions were blocked by bicuculline. These results suggest that propofol acts at presynaptic and postsynaptic GABAA receptors within GABAergic synapses, but also increases extrasynaptic GABA responses. Our results expand the locus of propofol actions to GABAergic and glutamatergic synapses.
Keywords: Propofol; Synaptic transmission; GABA; Glutamate; ‘Synaptic-bouton’ preparation; Focal electrical stimulation;

Hydrogen sulfide may protect multiple organ systems against ischemic–reperfusion injuries. It is unknown if treatment with sodium hydrosulfide (NaHS, a hydrogen sulfide donor) will improve myocardial function and minimize oxidative stress in hypoxic-reoxygenated newborn piglets. Mixed breed piglets (1–5 day, 1.5–2.5 kg) were anesthetized and acutely instrumented for the measurement of systemic, pulmonary and regional (carotid, superior mesenteric and renal) hemodynamics and blood gas parameters. The piglets were induced with normocapnic alveolar hypoxia (10–15% oxygen, 2 h) followed by reoxygenation with 100% (1 h) then 21% oxygen (3 h). At 10 min of reoxygenation, either NaHS (10 mg/kg, 5 ml) or saline (5 ml) was administered intravenously for 30 min (5 min bolus followed by 25 min of continuous infusion) in a blinded, block-randomized fashion (n=7/group). Plasma lactate and troponin I levels and tissue markers of myocardial oxidative stress were also determined. Two hours hypoxia caused cardiogenic shock (45±3% of respective normoxic baseline), reduced regional perfusion with metabolic acidosis (pH 6.94±0.02). NaHS infusion significantly improved recovery of cardiac index (84±3% vs. 72±5% in controls), systemic oxygen delivery (84±3% vs. 72±5% in controls) and systemic oxygen consumption (102±5% vs. 84±6% in controls) at 4 h of reoxygenation. NaHS had no significant effect on systemic and pulmonary blood pressures, regional blood flows, plasma lactate and troponin I levels. The myocardial glutathionine ratio was reduced in piglets treated with NaHS (vs. controls, P<0.05). Post-resuscitation administration of NaHS improves cardiac function and systemic perfusion and attenuates myocardial oxidative stress in newborn piglets following hypoxia-reoxygenation.
Keywords: Hydrogen sulfide; Neonatal asphyxia; Reoxygenation; Swine; Oxidative stress; Hemodynamics;

Gender difference in epileptogenic effects of 2-BFI and BU224 in mice by Jia-Wei Min; Bi-Wen Peng; Xiaohua He; Yanan Zhang; Jun-Xu Li (81-86).
Imidazoline I2 receptors are involved in pain modulation and psychiatric disorders and its ligands may represent a new therapeutic strategy against pain and depression. In particular, 2-BFI and BU224 are the two most widely studied I2 receptor ligands and have antinociceptive and antidepressant-like activities in rodents. However, little is known of the toxicological effects and potential gender differences of these I2 receptor ligands. This study examined the epileptogenic activities of 2-BFI and BU224 in male and female mice and also examined their underlying receptor mechanisms. 2-BFI (10–40 mg/kg, i.p.) and BU224 (10–40 mg/kg) produced epileptic seizures in a dose-related manner, as did the epileptogenic agent, pentylenetetrazole (PTZ, 15–60 mg/kg). However, female mice were significantly more sensitive than male mice in all the measures. The commonly used I2 receptor antagonist, idazoxan (10 mg/kg), did not block the onset and magnitude of the epileptic seizures or lethality induced by 2-BFI and BU224. When studied in combination, PTZ potentiated the epileptogenic effect of 2-BFI and BU224. The lack of antagonism by idazoxan of the epileptogenic activities of 2-BFI and BU224 suggests that the epileptogenic effects of 2-BFI and BU224 are mediated by non-imidazoline I2 receptors and that I2 receptors remain a viable therapeutic target for neurological disorders such as pain.
Keywords: 2-BFI; BU224; Imidazoline I2 receptor; Idazoxan; Seizure; Mice;

Compound ICA-105574 prevents arrhythmias induced by cardiac delayed repolarization by Jing Meng; Chenxia Shi; Lin Li; Yumin Du; Yanfang Xu (87-97).
Impaired ventricular repolarization can lead to long QT syndrome (LQT), a proarrhythmic disease with high risk of developing lethal ventricular tachyarrhythmias. The compound ICA-105574 is a recently developed hERG activator and it enhances I Kr current with very high potency by removing the channel inactivation. The present study was designed to investigate antiarrhythmic properties of ICA-105574. For comparison, the effects of another compound NS1643 was in-parallel assessed, which also acts primarily to attenuate channel inactivation with moderate potency. We found that both ICA-105574 and NS1643 concentration-dependently shortened action potential duration (APD) in ventricular myocytes, and QT/QTc intervals in isolated guinea-pig hearts. ICA-105574, but not NS1643, completely prevented ventricular arrhythmias in intact guinea-pig hearts caused by I Kr and I Ks inhibitors, although both ICA-105574 and NS1643 could reverse the drug-induced prolongation of APD in ventricular myocytes. Reversing prolongation of QT/QTc intervals and antagonizing the increases in transmural dispersion of repolarization and instability of the QT interval induced by I Kr and I Ks inhibitors contributed to antiarrhythmic effect of ICA-105574. Meanwhile, ICA-105574 at higher concentrations showed a potential proarrhythmic risk in normal hearts. Our results suggest that ICA-105574 has more efficient antiarrhythmic activity than NS1643. However, its potential proarrhythmic risk implies that benefits and risks should be seriously taken into consideration for further developing this type of hERG activators.
Keywords: Antiarrhythmic activity; hERG activator; ICA-105574; NS1643; Long QT syndrome; Action potential; Proarrhythmic risk;

The serotonin-2 receptor modulator, (-)-trans-PAT, decreases voluntary ethanol consumption in rats by James Kasper; Rajiv Tikamdas; Myong Sang Kim; Kaley MacFadyen; Richard Aramini; Joseph Ladd; Sarah Bisceglia; Raymond Booth; Joanna Peris (98-104).
Serotonin (5-HT) 5-HT2C receptor agonists have shown promise as novel alcoholism pharmacotherapies, but developing selective agonists has been problematic. Female Sprague Dawley rats were given ethanol in a palatable gel vehicle during operant sessions. 5-HT2C receptor modulators (Ro60-0175, SB242,084, and (-)-trans-PAT) were administered before operant sessions. As a control for the effects of 5-HT2C receptor agonism on caloric intake, drugs were also tested using non-ethanol containing gelatin. Ro60-0175, a 5-HT2 family receptor agonist, decreased both ethanol and vehicle responding while (-)-trans-PAT, a 5-HT2C receptor agonist with 5-HT2A-2B receptor inverse agonist activity, selectively reduced only ethanol responding. The effect of 5-HT2C receptor agonists on self-administration after reinstatement of ethanol after a three week deprivation was also determined. (-)-trans-PAT eliminated increases in ethanol intake following ethanol deprivation whereas Ro60-0175 had no effect. These results emphasize the need for caloric controls and further support the idea that selective modulation of 5-HT2 family receptors is a potential pharmacotherapeutic approach in the treatment of alcoholism.
Keywords: Ethanol; Serotonin 2C receptor; Alcoholism; Operant self-administration; Alcohol deprivation;

Protective effects of sitagliptin on myocardial injury and cardiac function in an ischemia/reperfusion rat model by Guanglei Chang; Peng Zhang; Lin Ye; Kai Lu; Ying Wang; Qin Duan; Aihua Zheng; Shu Qin; Dongying Zhang (105-113).
The purpose of this study is to investigate the effects and the underlying mechanisms of sitagliptin pretreatment on myocardial injury and cardiac function in myocardial ischemia/reperfusion (I/R) rat model. The rat model of myocardial I/R was constructed by coronary occlusion. Rats were pretreated with sitagliptin (300 mg/kg/day) for 2 weeks, and then subjected to 30 min ischemia and 2 h reperfusion. The release of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB), cardiac function and cardiomyocyte apoptosis were evaluated. The levels of malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in heart and glucagon-like peptide-1 (GLP-1) level in plasma were measured. Western blot analysis was performed to detect the target proteins of sitagliptin. Our results showed that sitagliptin pretreatment decreased LDH and CK-MB release, and MDA level in I/R rats. More importantly, we revealed for the first time that sitagliptin pretreatment decreased cardiomyocyte apoptosis while increased the levels of GSH-Px and SOD in heart. Sitagliptin also increased GLP-1 level and enhanced cardiac function in I/R rats. Furthermore, sitagliptin pretreatment up-regulated Aktserine473 and Badserine136 phosphorylation, reduced the ratio of Bax/Bcl-2, and decreased expression levels of cleaved caspase-3 and caspase-3. Interestingly, the above observed effects of sitagliptin were all abolished when co-administered with GLP-1 receptor antagonist exendin-(9-39) or PI3K inhibitor LY294002. Taken together, our data indicate that sitagliptin pretreatment could reduce myocardial injury and improve cardiac function in I/R rats by reducing apoptosis and oxidative damage. The underlying mechanism might be the activation of PI3K/Akt signaling pathway by GLP-1/GLP-1 receptor.
Keywords: DPP4 inhibitor; Sitagliptin; Ischemia/reperfusion; Myocardial injury; Cardiomyocyte apoptosis; Cardiac function;

Coagulation factor Xa induces an inflammatory signalling by activation of protease-activated receptors in human atrial tissue by Alicja Bukowska; Ines Zacharias; Sönke Weinert; Kerstin Skopp; Christian Hartmann; Christof Huth; Andreas Goette (114-123).
Activated factor X (FXa) is an important player in the coagulation cascade responsible for thrombin generation, which is activated during atrial fibrillation. Increasing evidence suggests that FXa influences cell signalling in various cell types by activating protease-activated receptors (PARs). It is so far not known if molecular effects of FXa affect atrial signal transduction. To study the effects of FXa, human atrial tissue slices were cultivated with FXa up to 24 h. Additionally, rapid pacing was applied at 4 Hz to resemble atrial fibrillation. The inhibitory impact of FXa antagonist (Rivaroxaban), protease-activated receptor 1 antagonist (SCH79797), and protease-activated receptor 2 antagonist (GB83) were analysed under experimental conditions. The exposure of atrial tissue to FXa resulted in the 1.7 fold upregulation of PAR2-mRNA, activation of MAP kinases (ERK1/2) and NF-κB signalling. Furthermore FXa increased the expression of adhesion molecule ICAM-1 (1.82±0.20), chemokine IL-8 (1.94±0.20), as well as prothrombotic molecule PAI-1 (1.52±0.17). The combination of rapid pacing and FXa caused significant upregulation of PAR1 (2.82±0.22), PAR2 (2.66±0.40), ICAM-1 (2.13±0.25), IL-8 (2.22±0.24), LOX-1 (2.59±0.35), and PAI-1 (2.65±0.52) at the mRNA level. Rivaroxaban and GB83 prevented upregulation of PARs, ICAM-1, LOX-1, IL-8, and activation of MAP kinases. The elevation in the expression of PAI-1 was hindered in the presence of SCH79797, or Rivaroxaban. The present study indicates that FXa mediates inflammatory signalling in atrial tissue. Importantly, FXa and tachyarrhythmia act synergistically to increase expression of protease-activated receptors and inflammatory mediators. Rivaroxaban prevented effectively FXa-induced molecular effects in human atrial tissue particularly during rapid pacing.
Keywords: Atrial fibrillation; FXa-induced signal transduction; FXa antagonist; PAR1 antagonist; PAR2 antagonist;

Vasorelaxant effect of propentofylline in isolated equine digital veins by Nasr Kabbesh; Marc Gogny; Gérard Chatagnon; Jacques Noireaud; Jean-Claude Desfontis; Mohamed Yassine Mallem (124-130).
We evaluated the vasorelaxant effect of propentofylline (PPF), a methylxanthine derivative, and its mechanism of action in equine digital veins (EDVs). Cumulative concentration-response curves to PPF (1 nM–300 µM) were recorded in phenylephrine-precontracted EDV rings under different experimental conditions. PPF-induced relaxation was partially inhibited by endothelium removal, but was unaltered by CGS-15943 (an adenosine receptor antagonist; 3 µM). PPF-induced relaxation was partially inhibited in the presence of L-NAME (a nitric oxide (NO) synthase inhibitor; 100 µM), ODQ (an inhibitor of soluble guanylyl cyclase; 30 µM) or Rp-8-Br-PET-cGMP-S (a protein kinase G inhibitor; 3 µM). It was not modified by indomethacin (a non-selective cyclooxygenase (COX) inhibitor; 10 µM), and was slightly potentiated by H-89 (a protein kinase A inhibitor; 2 µM). In endothelium-intact EDVs, PPF-induced relaxation was associated with a 2.4- and 24.1-fold increase in the tissue cGMP and cAMP content respectively. PPF (100 μM) did not shift the concentration–response curve to phenylephrine (1 nM–300 µM) but reduced the maximal effect. To investigate whether PPF can affect cAMP- and cGMP-induced relaxations, relaxation curves to forskolin (an activator of adenylate cyclase) and to sodium nitroprusside (SNP, a NO donor) were recorded in EDV rings pretreated with PPF (100 µM). PPF only slightly potentiated the forskolin-induced relaxation without affecting the SNP-induced relaxation. We demonstrated that PPF-induced relaxation in EDVs is partially endothelium-dependent. The PPF-induced relaxation partially occurred via NO release and both cAMP and cGMP generation, through COX-independent mechanisms but could also result from the inhibition of cAMP-phosphodiesterase activity for the highest concentrations.Display Omitted
Keywords: Propentofylline; Endothelium; Vasorelaxation; EDVs; Nitric oxide;

Tetrodotoxin-dependent effects of menthol on mouse gastric motor function by Antonella Amato; Sara Baldassano; Rosa Serio; Flavia Mulè (131-137).
Menthol, the main active constituent of peppermint oil, exerts gut spasmolytic effects, although its mechanism of action remains unclear. We investigated the effects of menthol on gastric emptying and spontaneous- or evoked- mechanical activity of whole murine stomach. Gastric emptying was calculated after i.p. administration of menthol (50 mg/Kg). Responses induced by menthol on gastric intraluminal pressure and evoked-cholinergic contractions were analyzed in vitro. Menthol decreased the gastric emptying rate. In vitro, menthol (0.3–30 mM) produced a concentration-dependent relaxation of whole stomach, that was significantly reduced by tetrodotoxin or ω-conotoxin GVIA. The gastric relaxant responses were not affected by N ω -nitro-l-arginine methyl ester, inhibitor of nitric oxide-synthase, apamin or [Lys1,Pro2,5,Arg3,4,Tyr6] vasoactive intestinal peptide (VIP)7-28, a VIP receptor antagonist, but they were significantly antagonized by atropine or guanethidine, a blocker of adrenergic neurotransmission. The joint application of atropine and guanethidine did not produce any additive effects on menthol effects. Phentolamine, an α-adrenoceptor antagonist, but not propranolol, a β-adrenoceptor antagonist, significantly reduced menthol responses and the contemporary administration of both adrenergic antagonists did not produce additive effects. Menthol (1–100 μM) produced a reduction of the electrically-evoked cholinergic contractions, which was prevented by guanethidine. Menthol did not affect the contractions induced by carbachol. In conclusion, menthol in mouse, is able to reduce the rate of gastric emptying and to relax the stomach in vitro. The latter effect appears due, almost in part, to neural mechanisms, with involvement of α-adrenoceptors leading to reduction of tonic ongoing release of acetylcholine.
Keywords: Menthol; Enteric nervous system; Stomach; Gastric emptying; Acetylcholine; Noradrenergic pathway;

ISO-1, a macrophage migration inhibitory factor antagonist, prevents N-methyl-d-aspartate-induced retinal damage by Taeko Naruoka; Tsutomu Nakahara; Yo Tsuda; Yuki Kurauchi; Asami Mori; Kenji Sakamoto; Jun Nishihira; Kunio Ishii (138-144).
Macrophage migration inhibitory factor (MIF) has been shown to play an important role in a variety of inflammatory and immune-mediated diseases. The inflammatory responses contribute to retinal neuronal degeneration. However, the role of MIF in the progression of retinal degeneration has not yet been elucidated. In this study, we determined whether pharmacological inhibition of MIF protects against the retinal damage induced by N-methyl-d-aspartate (NMDA) in rats. Intravitreal injection of NMDA (200 nmol) resulted in (1) cell loss in the ganglion cell layer and reduction in the thickness of the inner plexiform layer, (2) an increase in apoptotic cells, (3) a decrease in parvalbumin-positive amacrine cells, (4) accumulation of leukocytes, and (5) microglia activation. Injection of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1, 100 nmol), a MIF antagonist, significantly attenuated these NMDA-induced responses. These findings suggest that ISO-1 exerts protective effects against retinal injuries and that MIF may be a target for neuroprotective intervention in retinal diseases associated with glutamate-induced excitotoxicity.
Keywords: Amacrine cells; Excitotoxicity; Glutamate; Macrophage migration inhibitory factor; Neuronal cells;

Ulcerative colitis (UC) is a chronic inflammatory bowel disorder (IBD) that has an elevated risk of developing into colon cancer. In trials to develop new therapeutic alternatives for UC, it is important to fulfill modifying effects on pathogenic targets and to reach the colon in a high concentration. Thus, the current work has investigated a colon-specific delivery formula of resveratrol in targeting sphingosine kinase 1 (SphK1) and apoptotic pathways to control pathogenesis and its progression to any expected neoplasm. This work was conducted on 40 Wister albino rats equally divided into 4 groups where group I served as the normal control group. The untreated oxazolone-induced colitis in group II exhibited significant increase in SphK1 activity as well as activity of both myeloperoxidase (MPO) and caspase-3 with concomitant mild DNA fragmentation in colonic tissue. Colonic SphK1 activity showed significant positive correlation with the disease activity index (DAI) and histopathological score in this group. Comparable with treatment by the native resveratrol formula, nRes (group III), treatment by the colon-specific delivery resveratrol formula, cRes (group IV) caused significant decrease in the activity of SphK1 and MPO with massive DNA fragmentation in colonic tissue and non significant change in caspase-3 activity. The lowest DAI and histopathological score have been recorded in the group treated by the colon-specific delivery resveratrol formula. In conclusion, the anti-inflammatory and apoptotic effects of resveratrol could be attributed to its inhibitory effect on sphingosine kinase 1 (SphK1) providing a useful therapeutic tool to break the link between inflammation and carcinogenesis risk in ulcerative colitis.Inhibition of sphingosine kinase 1 (SphK1) by colon-specific delivery formula of resveratrol provides anti-inflammatory, apoptotic, and anti-carcinogenic activity for the treatment of experimental ulcerative colitis in rats Display Omitted
Keywords: Ulcerative colitis; Sphingosine kinase; Sesveratrol; Colon-specific delivery formula;

Anticancer activity of anandamide in human cutaneous melanoma cells by Barbara Adinolfi; Antonella Romanini; Alessia Vanni; Enrica Martinotti; Andrea Chicca; Stefano Fogli; Paola Nieri (154-159).
Cannabinoids are implicated in the control of cell proliferation, but little is known about the role of the endocannabinoid system in human malignant melanoma. This study was aimed at characterizing the in vitro antitumor activity of anandamide (AEA) in A375 melanoma cells. The mRNA expression of genes that code for proteins involved in the metabolism and in the mechanism of AEA action was assessed by RT-PCR. Cell viability was tested using WST-1 assay and the apoptotic cell death was determined by measuring caspase 3/7 activities. A375 cells express high levels of fatty acid amide hydrolase (FAAH), cyclooxygenase (COX)-2, cannabinoid receptor 1 (CB1), transient receptor potential cation channel subfamily V member 1 (TRPV1) and G-protein-coupled receptor 55 (GPR55) genes. AEA induced a concentration-dependent cytotoxicity with an IC50 of 5.8±0.7 µM and such an effect was associated to a caspase-dependent apoptotic pathway. AEA cytotoxicity was potentiated by FAAH inhibition (2-fold increase, p<0.05) and mitigated by COX-2 or lipoxygenase (LOX) inhibition (5- and 3-fold decrease, respectively; p<0.01). Blocking CB1 receptors partially decreased AEA cytotoxicity, whereas selective antagonism on the TRPV1 barely affected the mechanism of AEA action. Finally, methyl-β-cyclodextrin, a membrane cholesterol depletory, completely reversed the cytotoxicity induced by the selective GPR55 agonist, O-1602, and AEA. Overall, these findings demonstrate that AEA induces cytotoxicity against human melanoma cells in the micromolar range of concentrations through a complex mechanism, which involves COX-2 and LOX-derived product synthesis and CB1 activation. Lipid raft modulation, probably linked to GPR55 activation, might also have a role.
Keywords: Anandamide; Endocannabinoid system; Melanoma; Cytotoxicity; Apoptosis;

Protective effects of aliskiren on ischemia–reperfusion-induced renal injury in rats by Zhen Wang; Yukai Liu; Yu Han; Weiwei Guan; Xun Kou; Jinjuan Fu; Di Yang; Hongmei Ren; Duofen He; Lin Zhou; Chunyu Zeng (160-166).
The protective effect of aliskiren on ischemia–reperfusion (I/R) injury in the heart and brain has been reported. Whether or not this protective effect extends into the alleviation of renal I/R injury is not known. Therefore, we investigated the protective effect of aliskiren in the kidney in this study. Sprague-Dawley rats were randomly divided into four groups: sham control group; sham control with aliskiren pretreatment; I/R group and I/R with aliskiren pretreatment. Aliskiren (3 mg/kg) or vehicle was administrated intravenously via vena cava. Blood samples and the left kidneys were then collected to check for renal function, angiotensin II (Ang II), apoptosis and oxidative stress levels. Compared with the sham rats, serum creatinine (SCR) and blood urea nitrogen (BUN) were significantly increased in the I/R rats, accompanied by histopathological damage to the kidney, which included tubular cell swelling, desquamation, and cast formation. There were also more apoptotic cells and leukocyte infiltration in the I/R rats than in the sham rats. Pretreatment with aliskiren ameliorated I/R induced renal injury, i.e. reduced SCR and BUN levels, ameliorated renal histopathological changes, and decreased the apoptosis of cells and leukocyte infiltration in kidney. I/R injury also decreased superoxide dismutase (SOD) and glutathione (GSH-reduced form) levels, which were blocked with the aliskiren pretreatment. Aliskiren pretreatment exerts a protective effect on ischemia/reperfusion injury in the kidney, via amelioration of oxidative stress, and reduction in leukocyte infiltration and cellular apoptosis.
Keywords: Aliskiren; Renal ischemia–reperfusion; Angiotensin II; Oxidative stress; Apoptosis;

Effects of the sazetidine-a family of compounds on the body temperature in wildtype, nicotinic receptor β2−/− and α7−/− mice by Edward D. Levin; Hannah G. Sexton; Karen Gordon; Christopher J. Gordon; Yingxian Xiao; Kenneth J. Kellar; Venkata Mahidhar Yenugonda; Yong Liu; Michael P. White; Mikell Paige; Milton L. Brown; Amir H. Rezvani (167-172).
Nicotine elicits hypothermic responses in rodents. This effect appears to be related to nicotinic receptor desensitization because sazetidine-A, an α4β2 nicotinic receptor desensitizing agent, produces marked hypothermia and potentiates nicotine-induced hypothermia in mice. To determine the specificity of sazetidine-A induced hypothermia to β2 subunit-containing nicotinic receptors, we tested its efficacy in β2 knockout (β2−/−) mice. These effects were compared with wildtype (WT) and α7 knockout (α7−/−) mice. Confirming our earlier results, sazetidine-A elicited a pronounced and long-lasting hypothermia in WT mice. In comparison, sazetidine-A induced a much attenuated and shorter hypothermic response in β2−/− mice. This indicates that the greater proportion of sazetidine-A induced hypothermia is mediated via actions on β2-containing nicotinic receptors, while a smaller component of hypothermia induced by sazetidine-A is mediated by non-β2 receptors. Similar to WT mice, α7−/− mice showed the full extent of the sazetidine-A effect, suggesting that the hypothermia produced by sazetidine-A did not depend on actions on α7 nicotinic receptor subtype. Three other novel nicotinic receptor desensitizing agents derived from sazetidine-A, triazetidine-O, VMY-2-95 and YL-1-127 also produced hypothermia in WT and α7−/− mice. Furthermore, unlike sazetidine-A, triazetidine-O and YL-1-127 did not show any hint of a hypothermic effect in β2−/− mice. VMY-2-95 like sazetidine-A did show a residual hypothermic effect in the β2−/− mice. These studies show that the hypothermic effects of sazetidine-A and the related compound VMY-2-95 are mainly mediated by nicotinic receptors containing β2 subunit, but that a small component of the effect is apparently mediated by non-β2 containing receptors.
Keywords: Nicotinic; Temperature regulation; Sazetidine-A; Desensitization; Knockout;

Protective effect of moxonidine on ischemia/reperfusion-induced acute kidney injury through α2/imidazoline I1 receptor by Hidenobu Tsutsui; Takahiro Sugiura; Kentaro Hayashi; Tokihito Yukimura; Mamoru Ohkita; Masanori Takaoka; Yasuo Matsumura (173-180).
Enhancement of renal sympathetic nerve activity during renal ischemia and norepinephrine overflow from the kidney after reperfusion play important roles in the development of ischemic acute kidney injury. Recently, we have found that moxonidine, an α2/imidazoline Ι1-receptor agonist, has preventive effects on ischemic acute kidney injury by suppressing the excitation of renal sympathetic nervous system after reperfusion. In the present study, to clarify the renoprotective mechanisms of moxonidine (360 nmol/kg, i.v.) against ischemic acute kidney injury, we investigated the effect of intravenous (i.v.) and intracerebroventricular (i.c.v.) injection of efaroxan, an α21 receptor antagonist, on the moxonidine-exhibited actions. Ischemic acute kidney injury was induced by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. The suppressive effect of moxonidine on enhanced renal sympathetic nerve activity during renal ischemia was not observed in the rat treated with either i.v. (360 nmol/kg) or i.c.v. (36 nmol/kg) of efaroxan. Furthermore, i.v. injection of efaroxan eliminated the preventive effect of moxonidine on ischemia/reperfusion-induced kidney injury and norepinephrine overflow, and i.c.v. injection of efaroxan did not completely inhibit the moxonidine's effects. These results indicate that moxonidine prevents the ischemic kidney injury by sympathoinhibitory effect probably via α21 receptors in central nervous system and by suppressing the norepinephrine overflow through α21 receptors on sympathetic nerve endings.
Keywords: Moxonidine; Renal sympathetic nerve activity; Norepinephrine; Ischemia/reperfusion; Acute kidney injury;

Phorbaketal A inhibits adipogenic differentiation through the suppression of PPARγ-mediated gene transcription by TAZ by Mi Ran Byun; Cham Han Lee; Jun-Ha Hwang; A Rum Kim; Sung Ah Moon; Mi Kyung Sung; Jung-Rae Roh; Eun Sook Hwang; Jeong-Ho Hong (181-187).
Obesity causes several metabolic diseases, including diabetes. Adipogenic differentiation is an important event for fat formation in obesity. Natural compounds that inhibit adipogenic differentiation are frequently screened to develop therapeutic drugs for treating obesity. Here we investigated the effects of phorbaketal A, a natural marine compound, on adipogenic differentiation of mesenchymal stem cells. Phorbaketal A significantly inhibited adipogenic differentiation as indicated by less fat droplets and decreased expression of adipogenic marker genes. The expression of TAZ (transcriptional coactivator with PDZ-binding motif), an inhibitor of adipogenic differentiation, significantly increased during adipogenic differentiation in the presence of phorbaketal A. Phorbaketal A increased the interaction of TAZ and PPARγ to suppress PPARγ (peroxisome proliferator-activated receptor γ) target gene expression. TAZ-depleted cells showed higher adipogenic potential than that of control cells even in the presence of phorbaketal A. During cellular signaling induced by phorbaketal A, ERK (extracellular signal-regulated kinase) played an important role in adipogenic suppression; an inhibitor of ERK blocked phorbaketal A-induced adipogenic suppression. Thus, the results show that phorbaketal A inhibits adipocyte differentiation through TAZ.
Keywords: Adipogenesis; Phorbaketal A; TAZ; PPARγ; ERK;

Investigation of the metabolic effects of chronic clozapine treatment on CCK-1 receptor deficient Otsuka Long Evans Tokushima Fatty (OLETF) rats by Csaba Hegedűs; Diána Kovács; László Drimba; Réka Sári; Angelika Varga; József Németh; Zoltán Szilvássy; Barna Peitl (188-196).
Clozapine increases meal size and meal duration, effects similar to the pharmacological blockade or congenital deficiency of CCK-1 receptor. We aimed to investigate the role of CCK-1 receptor in clozapine-induced weight gain and insulin sensitivity in CCK-1 receptor deficient, male Otsuka Long Evans Tokushima Fatty rats (OLETF). Long Evans Tokushima Otsuka (LETO) rats served as healthy control. Animals were orally treated with either clozapine (10 mg/kg) or its vehicle over 25 days. Daily metabolic parameters were measured by metabolic cages. The insulin sensitivity was determined by hyperinsulinaemic euglycaemic glucose clamping (HEGC). Adiposity was determined by measuring the perirenal, intraabdominal and epididymal white adipose tissue fat pads. Hypothalamic mRNA expression of CCK-1 and CCK-2 receptor was measured by real-time PCR, plasma insulin by radioimmunoassay. Clozapine failed to increase weight gain or daily food intake, but it increased adiposity, 1st meal size and duration and decreased insulin sensitivity both in OLETF or LETO rats. The glucose infusion rate during the steady state of the HEGC was unaltered, but the metabolic clearance rate of insulin was reduced by the clozapine treatment. Hypothalamic mRNA of CCK-1 and CCK-2 receptor was elevated in LETO rats, but the mRNA of CCK-2 receptor was reduced by clozapine in OLETF rats. Our results suggest that the CCK-1 receptor has no direct role in the clozapine-induced adiposity and insulin resistance. We also demonstrated that atypical antipsychotic treatment can induce insulin resistance in the absence of manifest obesity in male rats.
Keywords: Cholecystokinin; Atypical antipsychotics; Weight gain; Adiposity; Insulin resistance; Feeding pattern;

Reduction of carrageenan-induced acute pulmonary inflammation in mice by novel thiazolidinedione derivative LPSF/RA-4 by Karla P.S. Barbosa; Laise A.M. Santos; Edlene L. Ribeiro; Ingrid T. Fragoso; Sura W.S. Rocha; Ana K.S. Nunes; Maria E.R. França; Bruna S. Silva; Amanda K.S.e. Silva; Mariana A.M. Donato; Fabiana O.S. Gomes; Teresinha G. Silva; Ivan R. Pitta; Marina R. Pitta; Maria C.A. Lima; Flávia D.T. Uchôa; Suely L. Galdino; Christina A. Peixoto (197-205).
A number of studies have demonstrated the biological activities of peroxisome proliferator-activated receptors. However, few studies have addressed the effects of the agonists of these receptors on lung diseases. The aim of the present study was to evaluate the anti-inflammatory action of a novel synthetic thiazolidine derivative (5Z)-3-benzyl-5-(1H-indol-3-ylmethylene)-thiazolidine-2,4-dione (LPSF/RA-4) on acute lung inflammation (pleurisy) induced by carrageenan. Forty mice were randomly allocated to the following groups: (I) saline control group (sham); (II) carrageenan (CAR) group; (III) CAR+LPSF/RA-4 group treated with LPSF/RA-4 (60 μmol/kg); and (IV) INDO group treated with indometacin (5 mg/kg). Total cell counts and the measure of nitric oxide (NO) were performed in pleural exudates. Lung fragments were processed for light microscopy, transmission electron microscopy, immunohistochemistry and Western blotting. The influx of leucocytes and NO levels were significantly reduced following treatment with LPSF/RA-4 and INDO. Histopathological and ultrastructural analyses of the CAR group revealed evident tissue alterations, such as oedema, infiltrates of inflammatory cells and emphysema. These alterations were significantly reduced in the groups treated with LPSF/RA-4 or INDO. Immunohistochemistry revealed an increase in inflammatory markers (COX-2, iNOS, TNF-α and IL-1β) in the lung tissue of the CAR group, whereas the groups treated with LPSF/RA-4 and INDO exhibited significant reductions in such immunomarkers. Western blot analysis revealed an increased expression of COX-2 and IL-1 in the CAR group, which was reduced by treatment with LPSF/RA-4. The present findings demonstrate the potent anti-inflammatory action of the novel derivative thiazolidinedione LPSF/RA-4 in acute lung injury induced by carrageenan.
Keywords: Peroxisome proliferator-activated receptor; Pleurisy; Acute lung injury; Thiazolidinedione;

Protective effect of ischemic preconditioning on ischemia/reperfusion-induced acute kidney injury through sympathetic nervous system in rats by Hidenobu Tsutsui; Ryosuke Tanaka; Masayo Yamagata; Tokihito Yukimura; Mamoru Ohkita; Yasuo Matsumura (206-212).
We have found that a series of brief renal ischemia and reperfusion (preconditioning), before the time of ischemia significantly attenuated the ischemia/reperfusion-induced acute kidney injury through endothelial nitric oxide synthase. In this study, we examined the effects of ischemic preconditioning on renal sympathetic nervous system and kidney function in ischemia/reperfusion-induced acute kidney injury with or without nitric oxide synthase inhibitor. Ischemia/reperfusion-induced acute kidney injury was made by clamping the left renal artery and vein for 45-min followed by reperfusion, 2 weeks after the contralateral nephrectomy. Ischemic preconditioning, consisting of three cycles of 2-min ischemia followed by 5-min reperfusion, was performed before the 45-min ischemia. Ischemic preconditioning suppressed the enhanced renal sympathetic nerve activity during ischemia and the elevated renal venous plasma norepinephrine level after reperfusion, and attenuated renal dysfunction and histological damage. The renoprotective effect of ischemic preconditioning was diminished by NG-nitro-L-arginine methyl ester (0.3 mg/kg, i.v.), a nonselective nitric oxide synthase inhibitor, 5 min before the start of ischemic preconditioning. Thus, ischemic preconditioning decreased renal sympathetic nerve activity and norepinephrine release probably through activating nitric oxide production, thereby improving ischemia/reperfusion-induced acute kidney injury.
Keywords: Ischemic preconditioning; Renal sympathetic nerve activity; Norepinephrine; Ischemia/reperfusion; Acute kidney injury;

The aim of the present study was to evaluate the protective effects of diosmin on experimentally induced myocardial infarcted rats. Diosmin (5 and 10 mg/kg body weight) was administered orally as pretreatment daily for a period of 10 days. Then isoproterenol (100 mg/kg) was injected subcutaneously into rats at an interval of 24 h for 2 days (on 11th and 12th day). Isoproterenol-induced myocardial infarcted rats showed significant changes in electrocardiogram and an increase in the levels of cardiac markers, compared with normal rats. Additionally, increased plasma lipid peroxidation products and altered lipid metabolism in the plasma were observed in the isoproterenol-induced myocardial infarcted rats. Pretreatment with diosmin (5 and 10 mg/kg body weight) minimized the electrocardiographic changes, decreased the levels of serum cardiac marker enzymes reduced plasma lipid peroxidation and minimized the alterations in the lipid metabolism of isoproterenol-induced myocardial infarcted rats. Also, diosmin inhibited the enhanced activity of liver HMG CoA reductase. The in vitro study revealed the free radical scavenging activity of diosmin. The free radical scavenging and anti-hyperlipidaemic effects are the reasons for the cardioprotective effects of diosmin.
Keywords: Diosmin; Electrocardiogram; Isoproterenol; Myocardial infarction; Lipids; Lipo proteins;

The study was performed to investigate the improved effect of curcumin on gastrointestinal function in streptozotocin-induced diabetic rats. Curcumin was administrated intragastrically at a dose of 100, 200 and 400 mg/kg/day to diabetic rats. After 28 days of treatment, the activity of superoxide dismutase (SOD), catalase (CAT), the content of reduced glutathione (GSH) and malondialdehyde (MDA) in gastric mucosa was assayed. Furthermore, the gastric function was evaluated by hormone secretion and gastric emptying tests. The results indicated that: (i) the diabetic rats exhibited significant decreases in the above mentioned antioxidative enzymes activities and GSH level and exhibited a high level of MDA. After administration of curcumin, the parameters were ameliorated to a large extent; (ii) curcumin treatment dose-dependently augmented the ghrelin levels of stomach and plasma, which were earlier depleted in the diabetic control rats. Conversely, the expression of ghrelin mRNA was decreased after curcumin treatment; and (iii) the gastric emptying in curcumin treated diabetic rats was notably accelerated compared with the diabetic control rats. These findings showed an improved effect of curcumin against streptozotocin-induced gastroparesis possibly by its antioxidant property.
Keywords: Curcumin; Diabetic gastroparesis; Ghrelin; Gastric emptying; Oxidative stress;

The dual effect of PNU-120596 on α7 nicotinic acetylcholine receptor channels by Bopanna I. Kalappa; Victor V. Uteshev (226-234).
PNU-120596 (1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea), a Type-II positive allosteric modulator of α7 nicotinic acetylcholine receptors inhibits α7 desensitization and robustly prolongs openings of α7 channels. However, these effects may render α7 channels more accessible to positively charged molecules and thus, more susceptible to voltage-dependent open-channel-block-like inhibition. To test this hypothesis, choline chloride (i.e., choline), a selective endogenous α7 agonist, and bicuculline methochloride (i.e., bicuculline), a competitive α7 antagonist, were used as membrane voltage-sensitive probes in whole-cell voltage-clamp recordings from hippocampal CA1 interneurons in acute brain slices in the absence and presence of PNU-120596. PNU-120596 enhanced voltage-dependent inhibition of α7 responses by bicuculline and choline. In the presence of PNU-120596, α7 channels favored a burst-like kinetic modality in the presence, but not absence of bicuculline and bursts of α7 openings were voltage-dependent. These results suggest that PNU-120596 alters the pharmacology of α7 channels by making these channels more susceptible to voltage-dependent inhibitory interactions with positively charged drugs at concentrations that do not potently inhibit α7 channels without PNU-120596. This inhibition imitates α7 nicotinic receptor desensitization and compromises the potentiating anti-desensitization effects of PNU-120596 on α7 nicotinic receptors. This unexpected dual action of PNU-120596, and possibly other Type-II positive allosteric modulators of α7 nicotinic receptors, may lead to unanticipated α7 channel-drug interactions and misinterpretation of α7 single-channel data.
Keywords: PNU-120596; PNU120596; α7 Nicotinic receptor; Desensitization; Channel block; Choline; Bicuculline;

Galangin inhibits proliferation of HepG2 cells by activating AMPK via increasing the AMP/TAN ratio in a LKB1-independent manner by Haitao Zhang; Ning Li; Jun Wu; Lijuan Su; Xiaoyi Chen; Biyun Lin; Hui Luo (235-244).
Galangin, a flavonol derived from Alpinia officinarum Hance and used as food additives in southern China, induces apoptosis and autophagy to suppress the proliferation of HepG2 cells. In this study, we demonstrated that galangin induced autophagy by increasing the ratio of AMP/TAN in HepG2 cells. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and LKB1, but inhibited the phosphorylation of AKT and mTOR. Inhibition of AMPK activation suppressed the dephosphorylation of mTOR to block galangin-induced autophagy. AMPK activation by galangin appeared to be independent of the LKB1 signaling pathway because the down-regulation of LKB1 by its siRNA failed to affect galangin-induced autophagy. Collectively, the findings demonstrated a novel mechanism of how galangin induces autophagy via activating AMPK in a LKB1- independent manner. The induction of autophagy can thus reflect the anti-proliferation effect of galangin in HCC cells.
Keywords: Galangin; Autophagy; AMPK; LKB1; mTOR;

The α2-adrenoceptors mediating inhibition of the vasopressor sympathetic outflow in pithed rats: Pharmacological correlation with α2A, α2B and α2C subtypes by Ma. Trinidad Villamil-Hernández; Oscar Alcántara-Vázquez; Araceli Sánchez-López; David Centurión (245-252).
α2-Adrenoceptors were first described as presynaptic receptors inhibiting the release of various transmitters from neurons in the central and peripheral nervous systems. In vitro studies have confirmed that α2A, α2B and α2C subtypes inhibited noradrenaline release from postganglionic sympathetic neurons but no study has been reported their involvement in the vasopressor sympathetic outflow in vivo. Thus, this study analysed the subtype(s) involved in the inhibition produced by the α2-adrenoceptor agonist, B-HT 933, on the vasopressor sympathetic outflow. Male Wistar pithed rats were pre-treated with i.v. bolus injections of gallamine (25 mg/kg) and desipramine (50 µg/kg) and prepared to stimulate the vasopressor sympathetic outflow (T7–T9) or to receive i.v. bolus of exogenous noradrenaline. Sympathetic stimulation or exogenous noradrenaline produced, respectively, frequency-dependent and dose-dependent vasopressor responses. I.v. continuous infusion of B-HT 933 (30 μg/kg min) failed to modify the vasopressor responses to exogenous noradrenaline and inhibited those induced by preganglionic stimulation of the vasopressor sympathetic outflow at all frequencies of stimulation (0.03–3 Hz). The sympatho-inhibition elicited by B-HT 933 was: (i) unaffected by vehicles (1 ml/kg); (ii) partially antagonised by BRL44408 (300 μg/kg; α2A), imiloxan (3000 μg/kg; α2B) and/or JP-1302 (300 μg/kg; α2C) given separately; and (iii) completely blocked by rauwolscine (300 μg/kg) or the combination of BRL44408 (300 μg/kg)+imiloxan (3000 μg/kg)+JP-1302 (300 μg/kg). The above doses of antagonists did not modify per se the sympathetically-induced vasopressor responses. These results suggest that the vasopressor sympatho-inhibition to B-HT 933 is primarily mediated by activation of α2A/2B/2C-adrenoceptors in pithed rats.
Keywords: α2-Adrenoceptors; B-HT 933; BRL44408; Imiloxan; JP-1302; Sympatho-inhibition;

Capacities of metabotropic glutamate modulators in counteracting soman-induced seizures in rats by Trond Myhrer; Espen Mariussen; Siri Enger; Pål Aas (253-260).
Current treatment of nerve agent poisoning with ionotropic drugs proves inadequate, and alternative treatment strategies are searched for. Based on positive findings with metabotropic glutamate modulators in microinfusion studies, the present study was initiated to examine anticonvulsant effects of MPEP (2-Methyl-6-(phenylethynyl)pyridine hydrochloride), a metabotropic glutamate receptor 5 antagonist, and DCG-IV ((2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine), a metabotropic glutamate receptor 2/3 agonist, when administered systemically in combinations with HI-6 (1-[([4-(aminocarbonyl)pyridino]methoxy)methyl]-2-[(hydroxyimino)methyl]pyridinium) and procyclidine or HI-6 and levetiracetam relative to the combination of HI-6, procyclidine, and levetiracetam. The results showed that MPEP or DCG-IV combined with HI-6 and procyclidine resulted in substantial antidotal efficacy when administered 20 min after onset of seizures elicited by soman. MPEP or DCG-IV combined with HI-6 and levetiracetam did not terminate seizures and preserve lives. When given 20 min before challenge with soman, DCG-IV in combination with HI-6 and procyclidine provided protection, whereas MPEP combined with HI-6 and procyclidine did not. Combinations with metabotropic glutamate receptor modulators did not achieve the same high level of antidotal efficacy as the combination of HI-6, procyclidine, and levetiracetam. MPEP alone inhibited pseudocholinesterase activity in the brain markedly. A positive correlation was found between latency to seizure onset or full protection and level of pseudocholinesterase activity in brain. MPEP and DCG-IV can serve as effective anticonvulsants against nerve agent poisoning when combined with HI-6 and procyclidine. Metabotropic glutamate receptor modulators may represent an alternative or supplement to treatment with ionotropic drugs.
Keywords: DCG-IV; HI-6; Levetiracetam; MPEP; Procyclidine; Soman;

Local infiltration of neuropeptide Y as a potential therapeutic agent against apoptosis and fibrosis in a swine model of hypercholesterolemia and chronic myocardial ischemia by Robina Matyal; Sruthi Sakamuri; Angela Wang; Eitezaz Mahmood; Michael P. Robich; Kamal Khabbaz; Philip E. Hess; Frank W. Sellke; Feroze Mahmood (261-270).
While the angiogenic effects of Neuropeptide Y (NPY) in myocardial ischemia and hypercholesterolemia have been studied, its effects on altering oxidative stress, fibrosis and cell death are not known. We hypothesized that local infiltration of NPY in a swine model of chronic myocardial ischemia and hypercholesterolemia will induce nerve growth and cell survival, while reducing oxidative stress and fibrosis. Yorkshire mini-swine (n=15) were fed a high cholesterol diet for 5 weeks. Three weeks after surgical induction of focal myocardial ischemia, an osmotic pump was implanted, which delivered NPY (n=8, high cholesterol treated, HCT) or the vehicle (n=7, high cholesterol control, HCC) for 5 weeks. Then myocardium was harvested for analysis. Assessment of myocardial function and perfusion was made the last intervention. Immunoblotting demonstrated significantly decreased levels of MMP-9 (p=0.001) and TGF-β (p=0.05) and significantly increased levels of Ang-1 (p=0.002), MnSOD (p=0.006) and NGF (p=0.01) in HCT. Immunohistochemistry results revealed significantly decreased TUNEL staining (p=0.005) and GLUT4 translocation (p=0.004) in HCT. The functional data showed significantly improved blood flow reserve (p=0.02) and improved diastolic function –dP/dt (p=0.009) in the treated animals. Local infiltration of NPY results in positive remodeling in ischemic myocardium in the setting of hypercholesterolemia. By initiating angio and neurogenesis, NPY infiltration improves blood flow reserve and restoration of fatty acid metabolism. The associated increased cell survival and decreased fibrosis result in improved myocardial diastolic function. NPY may have a potential therapeutic role in patients with hypercholesterolemia associated coronary artery disease.
Keywords: Chronic ischemia; Oxidative stress; Fibrosis; Neuropeptide Y;

Exposure of cardiomyocytes to angiotensin II induces over-activation of monoamine oxidase type A: Implications in heart failure by Maria Elena Manni; Marina Zazzeri; Claudia Musilli; Elisabetta Bigagli; Maura Lodovici; Laura Raimondi (271-276).
Several evidences indicate that increased cardiac mitochondrial monoamine oxidase type A (MAO-A) activity associates with a failing phenotype. Till now, the mechanism underlying such relation is largely unknown. We explored the hypothesis that exposure of cardiomyocytes to AT-II caused activation of MAO-A and also of catalase and aldehyde dehydrogenase activities, enzymes involved in degrading MAO's end products.Left ventricular cardiomyocytes were isolated from normoglycemic (N) and streptozotocin-injected (50 mg/kg) rats (D) treated or not treated with losartan (20 mg/kg/day in drinking water; DLos and NLos, respectively), a type 1 receptor (AT1) antagonist, for 3 weeks. In each group of cells, MAO, catalase and aldehyde dehydrogenase activities were measured radiochemically and spectrophotometrically. The same enzymes were also measured in HL-1 immortalized cardiomyocytes not exposed and exposed to AT-II (100 nM for 18 h) in the absence and in the presence of irbesartan (1 μM), an AT1 antagonist. MAO-A catalase and aldehyde dehydrogenase activities were found significantly higher in D, than in N cells. MAO-A positively correlated with catalase activity in D cells. MAO-A and aldehyde dehydrogenase but not catalase over-activation, were prevented in DLos cells. Similarly, MAO-A activity, but not catalase and aldehyde dehydrogenase increased significantly in HL-1 cells acutely exposed to AT-II and this increase was prevented when irbesartan, an AT1 antagonist was present. Over-activation of cardiomyocyte MAO-A activity is among acute (18 h) and short-term (2-weeks of diabetes) cardiac effects of AT-II and a novel target of AT1 antagonists, first line treatments of diabetic cardiomyopathy.Display Omitted
Keywords: Monoamine oxidase; Heart failure; Diabetic cardiomyopathy; Angiotensin-II; Angiotensin type 1 receptor antagonist;

The role of different cyclooxygenase (COX) metabolites released after antigen exposure has been difficult to assess due to simultaneous release of dominant constrictors such as histamine and cysteinyl-leukotrienes (CysLT). In addition prostaglandin E2 (PGE2) also has a powerful effect on basal tone. The aim was to exclude PGE2, histamine and CysLTs from the antigen-induced contraction to define the possible involvement of remaining COX metabolites. Isometric force was measured in guinea pig trachea after exposure to cumulatively increasing concentrations of ovalbumin (OVA; 0.1 ng/ml to 0.1 mg/ml) in the absence or presence of biosynthesis inhibitors and receptor antagonists. Challenge with OVA induced a concentration-dependent contraction that reached 75% of maximal tissue response. COX-inhibition or a combination of EP1 and EP2 receptor antagonism (ONO-8130 and PF-04418948) completely abolished the tone, resulting in an augmented antigen response. COX inhibition in combination with either antihistamines or antileukotrienes (FLAP inhibitor or CysLT1–2 receptor antagonist) displayed no difference compared to COX inhibition alone. However, a combination of all three classes reduced the contraction to 30%, revealing an unknown contractile component. Exchanging COX inhibition with EP1–2 receptor antagonists together with antihistamines and antileukotrienes could not decrease the contraction more than to 50%. However, when a TP receptor antagonist (SQ-29,548) was further included, the maximal antigen contraction reached 30%, similar as previously, clearly revealing a TP-mediated contractile component. PGE2 primarily regulate the basal tone via EP1 and EP2, whereas prostanoids, such as TXA2 and PGD2, contribute as mediator of the antigen-response by activation of the TP receptor.
Keywords: Ovalbumin; Guinea pig trachea; Eicosanoids; TP receptor; EP receptor; Organ bath;

Upregulation of thrombomodulin expression by activation of farnesoid X receptor in vascular endothelial cells by Xie He; Zhizhen Xu; Bin Wang; Yingru Zheng; Wei Gong; Gang Huang; Li Zhang; Yuan Li; Fengtian He (283-289).
Thrombomodulin (TM) serves as a vasoprotective molecule on the surface of vascular endothelial cells (VECs) to maintain the endothelial microenvironment by suppressing cellular proliferation, adhesion and inflammatory responses. Farnesoid X receptor (FXR), a nuclear receptor (NR) and originally considered as a bile acid-activated transcriptional factor, not only regulates metabolism homeostasis, but also influences cholesterol transport, vascular tension, and inflammation. Recent studies have shown that TM expression is upregulated by several NRs. However, it is unknown whether there is a link between FXR and TM. Our studies demonstrated that TM expression and activity were up-regulated by FXR activation in VECs. Reporter assays showed that FXR activation significantly enhanced the transcriptional activity of human TM gene promoter. Elecrophoretic mobility-shift and chromatin immunoprecipitation assays indicated that FXR induced TM expression by binding to a novel FXR-responsive element (FXRE), an inverted repeat DNA motif, IR8 (-503 AGGTCCtcccaaagTGCCCT-484) in the promoter region of TM gene. These results suggest that FXR may serve as a novel molecular target for manipulating TM expression and activity in VECs, which may be helpful for designing the therapeutic strategies to the treatment of associated diseases by targeting FXR/TM pathway.
Keywords: Farnesoid X receptor; Thrombomodulin; Vascular endothelial cell; Inflammation; Gene regulation;

Mineralocorticoid receptor antagonists attenuate pulmonary inflammation and bleomycin-evoked fibrosis in rodent models by Gissela B Lieber; Xiomara Fernandez; Garfield G Mingo; Yanlin Jia; Michael Caniga; Malgorzata A. Gil; Shanil Keshwani; Janice D. Woodhouse; Milenko Cicmil; Lily Y Moy; Nancy Kelly; Johanna Jimenez; Yvette Crawley; John C Anthes; Joel Klappenbach; Yu-Lu Ma; Robbie L. McLeod (290-298).
Accumulating evidence indicates protective actions of mineralocorticoid antagonists (MR antagonists) on cardiovascular pathology, which includes blunting vascular inflammation and myocardial fibrosis. We examined the anti-inflammatory and anti-fibrotic potential of MR antagonists in rodent respiratory models. In an ovalbumin allergic and challenged Brown Norway rat model, the total cell count in nasal lavage was 29,348±5451, which was blocked by spironolactone (0.3–60 mg/kg, p.o.) and eplerenone (0.3–30 mg/kg, p.o.). We also found that MR antagonists attenuated pulmonary inflammation in the Brown Norway rat. A series of experiments were conducted to determine the actions of MR blockade in acute/chronic lung injury models. (1) Ex vivo lung slice rat experiments found that eplerenone (0.01 and 10 µM) and spironolactone (10 µM) diminished lung hydroxyproline concentrations by 55±5, 122±9, and 83±8%. (2) In in vivo studies, MR antagonists attenuated the increases in bronchioalveolar lavage (BAL) neutrophils and macrophages caused by lung bleomycin exposure. In separate studies, bleomycin (4.0 U/kg, i.t.) increased lung levels of hydroxyproline by approximately 155%, which was blocked by spironolactone (10–60 mg/kg, p.o.). In a rat Lipopolysaccharide (LPS) model, spironolactone inhibited acute increases in BAL cytokines with moderate effects on neutrophils. Finally, we found that chronic LPS exposure significantly increased end expiratory lung and decreased lung elastance in the mouse. These functional effects of chronic LPS were improved by MR antagonists. Our results demonstrate that MR antagonists have significant pharmacological actions in the respiratory system.
Keywords: Mineralocorticoid; Eplerenone; Pulmonary fibrosis; Pulmonary inflammation;

Menadione (Vitamin K3) decreases melanin synthesis through ERK activation in Mel-Ab cells by Eun-Hyun Kim; Myo-Kyoung Kim; Hye-Young Yun; Kwang Jin Baek; Nyoun Soo Kwon; Kyoung-Chan Park; Dong-Seok Kim (299-304).
Menadione is a synthetic vitamin K3 derivative. Here, we examined the effects of menadione on melanogenesis and its related signaling pathways. Our results showed that melanin content was significantly reduced after menadione treatment in a dose-dependent manner. However, menadione treatment did not reduce tyrosinase activity directly. Wnt signaling is known to play a major role in the control of melanin synthesis. Thus, we tested the effects of menadione treatment on GSK3β and β-catenin signaling, but found that menadione did not influence either of these signaling pathways. We also investigated changes in the phosphorylation of ERK, which is related to melanin regulation. These results indicated that menadione treatment led to the phosphorylation of ERK. Additionally, menadione treatment reduced both MITF and tyrosinase protein levels. Treatment with PD98059, a specific ERK pathway inhibitor, restored menadione-induced melanin reduction and also prevented MITF and tyrosinase downregulation by menadione. These results suggest that the hypopigmentary action of menadione is due to MITF and tyrosinase downregulation by ERK activation.
Keywords: Menadione; Melanogenesis; MITF; Tyrosinase; ERK;

Histamine inhibits high mobility group box 1-induced adhesion molecule expression on human monocytes by Hideo Takahashi; Hiroshi Sadamori; Kiyoshi Teshigawara; Atsuko Niwa; Keyue Liu; Hidenori Wake; Shuji Mori; Tadashi Yoshino; Masahiro Nishibori (305-313).
Cell–cell interaction through binding of adhesion molecules on monocytes to their ligands on T-cells plays roles in cytokine production and lymphocyte proliferation. High mobility group box 1 (HMGB1), an abundant and conserved nuclear protein, acts in the extracellular environment as a primary pro-inflammatory signal. HMGB1 induces expression of intercellular adhesion molecule (ICAM), B7.1, B7.2 and CD40 on monocytes, resulting in production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α production and lymphocyte proliferation in human peripheral blood mononuclear cells (PBMCs). Histamine inhibits pro-inflammatory cytokine production via histamine H2-receptors; however, it is not known whether histamine inhibits HMGB1 activity. This study was designed to study the inhibitory effect of histamine on HMGB1 activity. We examined the effect of histamine on HMGB1-induced expression of ICAM-1, B7.1, B7.2 and CD40 on monocytes, production of IFN-γ and TNF-α and lymphocyte proliferation in PBMCs. Histamine inhibited HMGB1 activity in a concentration-dependent manner. The effects of histamine were partially ablated by the H2-receptor antagonist, famotidine, and mimicked by the H2/H4-receptor agonists, dimaprit and 4-methylhistamine. Histamine induced cyclic adenosine monophosphate (cAMP) production in the presence and absence of HMGB1. The effects of histamine were reversed by the protein kinase A (PKA) inhibitor, H89, and mimicked by the membrane-permeable cAMP analog, dibutyryl cAMP (dbcAMP), and the adenylate cyclase activator, forskolin. These results together indicated that histamine inhibited HMGB1 activity
Keywords: High mobility group box 1; Histamine; H2 receptor; Monocyte; Adhesion molecule; Cyclic adenosine monophosphate;

Bee venom phospholipase A2-induced phasic contractions in mouse rectum: Independent roles of eicosanoid and gap junction proteins and their loss in experimental colitis by Ryouya Nomura; Madoka Yanagihara; Hiromi Sato; Kenjiro Matsumoto; Kimihito Tashima; Shunji Horie; Shuonan Chen; Hiromichi Fujino; Kouichi Ueno; Toshihiko Murayama (314-322).
Various events including digestion and inflammation are regulated by secreted phospholipase A2 (sPLA2) in gastrointestinal tissues, however, the role of sPLA2 on contractile activity has not been elucidated. We investigated the effect of bee venom PLA2 (bvPLA2), which is homologous to the central domain of group III sPLA2, on contractile activity in mouse rectum. The longitudinal preparations of rectum showed rhythmic phasic contractions (RPCs) with varied amplitude and high frequency. Treatment with bvPLA2 at 1 μg/ml increased amplitudes of RPCs without marked changes in frequency and basal tone. RPCs by bvPLA2 were affected neither by atropine nor by inhibition of nitric oxide synthase, and partly inhibited by dual inhibition of the cyclooxygenase and lipoxygenase pathways. Pretreatment of bvPLA2 with dithiothreitol, which inhibits the enzyme activity, partly reduced bvPLA2-induced RPCs, and arachidonic acid-increased RPCs were completely abolished by cyclooxygenase/lipoxygenase inhibition. Phasic contractions have been shown to be regulated by gap junction and to be decreased in gastrointestinal tissues with experimental colitis. Treatment with inhibitors of gap junction proteins, 50 μM 18β-glycyrrhetinic acid and 100 μM carbenoxolone, partly and almost completely reduced bvPLA2-induced RPCs without and with the cyclooxygenase/lipoxygenase inhibitors, respectively, but not arachidonic acid-induced RPCs. In rectum from mouse having colitis, where total levels and modified forms of connexin43 increased, bvPLA2-induced RPCs were markedly decreased. Our results suggest that both arachidonic acid metabolism and gap junction proteins independently regulated the sPLA2-induced RPCs in mouse rectum. An increased expression and/or modification of connexin43 may influence sPLA2-induced RPCs in rectum with colitis.Display Omitted
Keywords: Rhythmic phasic contractions; Bee venom PLA2; Gap junction proteins; Arachidonic acid; Ulcerative colitis;

Acamprosate, the calcium salt of bis(3-acetamidopropane-1-sulfonate), contributes to the maintenance of abstinence in alcohol-dependent patients, but its mechanism of action in the central nervous system is unclear. Here, we report the effect of acamprosate on ethanol-drinking behavior in standard laboratory Wistar rats, including voluntary ethanol consumption and the ethanol-deprivation effect. After forced ethanol consumption arranged by the provision of only one drinking bottle containing 10% ethanol, the rats were given a choice between two drinking bottles, one containing water and the other containing 10% ethanol. In rats selected for high ethanol preference, repeated oral administration of acamprosate diminished voluntary ethanol drinking. After three months of continuous access to two bottles, rats were deprived of ethanol for three days and then presented with two bottles again. After ethanol deprivation, ethanol preference was increased, and the increase was largely abolished by acamprosate. After exposure of primary neuronal cultures of rat cerebral cortex to ethanol for four days, neurotoxicity, as measured by the extracellular leakage of lactate dehydrogenase (LDH), was induced by incubation with glutamate for 1 h followed by incubation in the absence of ethanol for 24 h. The N-methyl-d-aspartate receptor blocker 5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine, the metabotropic glutamate receptor subtype 5 antagonist 6-methyl-2-(phenylethynyl)pyridine and the voltage-gated calcium-channel blocker nifedipine all inhibited glutamate-induced LDH leakage from ethanol-exposed neurons. Acamprosate inhibited the glutamate-induced LDH leakage from ethanol-exposed neurons more strongly than that from intact neurons. In conclusion, acamprosate showed effective reduction of drinking behavior in rats and protected ethanol-exposed neurons by multiple blocking of glutamate signaling.
Keywords: Acamprosate; Glutamate signals; Alcohol dependence; Ethanol; N-Methyl-d-aspartate receptor; Voltage-gated calcium channels; Metabotropic glutamate receptor subtype 5;

Levosimendan inhibits interleukin-1β-induced cell migration and MMP-9 secretion in rat cardiac fibroblasts by Muneyoshi Okada; Atsushi Suzuki; Hideyuki Yamawaki; Yukio Hara (332-339).
Cell migration and matrix metalloproteinases (MMPs) secretion in cardiac fibroblasts are important processes in the cardiac remodeling during the development of cardiac diseases and are regulated by proinflammatory cytokines. Although levosimendan, a novel inotropic agent, is expected to have some beneficial influences on preventing cardiac remodeling, its effects on proinflammatory cytokines-induced functional changes in cardiac fibroblasts have not been clarified. Therefore, we investigated the effects of levosimendan on interleukin (IL)-1β−induced MMP-9 secretion and migration in adult rat cardiac fibroblasts. Primary cardiac fibroblasts were isolated from adult male Wistar rats. MMP-9 secretion in culture medium and extracellular signal-regulated kinase (ERK) phosphorylation in cell lysate were measured by using Western blotting. Gelatin zymography was performed to measure activity of secreted MMP-9. MMP-9 mRNA expression in the cell was measured by using reverse transcription polymerase chain reaction. Boyden chamber assay was performed for detection of migration. Levosimendan (3–100 μM) concentration-dependently inhibited IL-1β (4 ng/ml)-induced MMP-9 secretion, activity and mRNA expression. Levosimendan inhibited IL-1β (4 ng/ml)-induced ERK phosphorylation. Levosimendan (10 and 100 μM) inhibited IL-1β-induced migration, and CTTHWGFTLC peptide (10 μM), an MMP inhibitor, or PD98059 (50 μM), an ERK inhibitor, also suppressed it. The present study for the first time demonstrated in adult rat cardiac fibroblasts that levosimendan inhibits IL-1β-induced migration at least partly through the inhibition of MMP-9 secretion via suppressing ERK phosphorylation.
Keywords: Cardiac fibroblasts; Interleukin-1β; Levosimendan; Migration; Matrix metalloproteinase-9;

Bryostatin 5 induces apoptosis in acute monocytic leukemia cells by activating PUMA and caspases by Yiwei Wang; Jinbao Zhang; Qixia Wang; Tao Zhang; Yang Yang; Yanghua Yi; Guangxun Gao; Hongjuan Dong; Huafeng Zhu; Yue Li; Houwen Lin; Haifeng Tang; Xiequn Chen (340-349).
Acute leukemia is a malignant clonal hematopoietic stem cell disease. In the current study, we examined the effects of bryostatin 5 on acute monocytic leukemia cells in vitro and in vivo. We also explored the mechanisms and pathways underlying the increase in apoptosis induced by bryostatin 5. Bryostatin 5 inhibited the growth of primary acute monocytic leukemia cells and U937 cells in a dose- and time-dependent manners. Bryostatin 5 also induced an increase in apoptosis and a decrease in the mitochondrial membrane potential (MMP) in U937 cells. Transmission electron microscopy (TEM) revealed that bryostatin 5-treated cells displayed typical apoptotic characteristics (chromatin condensation, karyopyknosis and formation of crescents and apoptotic bodies). In addition, bryostatin 5 increased the expression of P53 upregulated modulator of apoptosis (PUMA) and slightly increased P53 expression. Bryostatin 5 also significantly decreased Bcl-XL expression and significantly increased the expression levels of Bak, Bax, cleaved caspase 9 and cleaved caspase 3. The pro-apoptotic activity of bryostatin 5 in U937 cells was inhibited by PUMA siRNA and z-LEHD-fmk (a specific caspase 9 inhibitor). In addition, the PUMA siRNA significantly affected the expression of cleaved caspase 9, whereas z-LEHD-fmk had little effect on the expression of PUMA. The results suggest that PUMA is located upstream of caspase 9 in this apoptotic signaling pathway. These novel findings provide mechanistic insight into the induction of apoptosis by bryostatin 5 and might facilitate the development of clinical strategies to enhance the therapeutic efficacy of treatments for acute monocytic leukemia.
Keywords: Acute monocytic leukemia; Bryostatin 5; Apoptosis; PUMA; Caspase;

Dual effect of capsaicin on cell death in human osteosarcoma G292 cells by Chi-Sheng Chien; Kuo-Hsing Ma; Herng-Sheng Lee; Pei-Shan Liu; Yui-Huei Li; Yu-Shiuan Huang; Sheau-Huei Chueh (350-360).
Thirty percent of osteosarcoma patients die within 5 years. New agents that induce apoptosis of osteosarcoma cells might be therapeutically useful. Here, we characterized the apoptotic mechanism induced by capsaicin in G292 osteosarcoma cells. Our results show that capsaicin induces an increase in the cytosolic Ca2+ concentration which is independent of the extracellular Ca2+ concentration and depletes intracellular Ca2+ stores, suggesting the presence of endoplasmic reticulum transient receptor potential vanilloid receptor type 1. Capsaicin also activates the mitochondrial caspase 3-dependent death cascade. Rapamycin, an inhibitor of mammalian target of rapamycin, evokes autophagy, as do capsaicin or thapsigargin, a sarco(endo)plasmic reticulum Ca2+ ATPase inhibitor that causes Ca2+ store depletion. Capsaicin-induced cell death is completely inhibited by co-treatment with the pan-caspase inhibitor Z-VAD-fmk and increased by the autophagy inhibitor 3-methyladenine, suggesting the existence of an autophagy-dependent anti-apoptotic mechanism. Capsaicin also induces ERK phosphorylation, which acts as a downstream effector of autophagy. 3-Methyladenine or PD98059, an ERK kinase inhibitor, restores capsaicin-induced cell death in the presence of Z-VAD-fmk, suggesting that inhibition of autophagy activates a second cell death pathway that is caspase-independent. Taken together, our data show that capsaicin causes Ca2+ depletion of intracellular Ca2+ stores and simultaneously activates the mitochondrial caspase-dependent death cascade and autophagy-dependent ERK activation and that the latter counteracts a second death signaling pathway that is caspase-independent.
Keywords: Capsaicin; Transient receptor potential vanilloid receptor; Autophagy; Osteosarcoma; Apoptosis;

Didox potentiates the cytotoxic profile of doxorubicin and protects from its cardiotoxicity by Ahmed M. Al-Abd; Fahad A. Al-Abbasi; Gihan F. Asaad; Ashraf B. Abdel-Naim (361-369).
The use of adjuvant therapies in cancer treatment is rationalized by potentiating the efficacy and/or protecting from the major side effects of chemotherapeutics. Didox, besides its antioxidant properties, is an inhibitor for DNA synthesis and repair which might recommend its use as adjuvant therapy. Herein, we have studied the effect of didox in potentiating the efficacy of doxorubicin (DOX) against liver cancer cells and protecting from its dose-limiting cardiotoxic effects. Didox combination with DOX significantly decreased in the IC50 of DOX to half its original value in Huh7 and HepG2 liver cancer cell lines. The calculated combination index (CI-value) indicated additive type of drug interaction (CI-value ranged from 0.81 to 0.9). Both didox and DOX significantly blocked the cell cycle in S-phase and their combination significantly increased cell cycle blockade. Also, didox combination significantly increase the caspase-3 level compared to DOX treatment alone. On the other hand, didox (150 mg/kg daily) significantly protected the cardiomyocyte membrane integrity and decreased the intra-cardiac oxidative stress induced by DOX treatment (15 mg/kg). This protective effect was reflected in reverting the cardiomegaly and cardio-pathological features induced by DOX treatment. Also didox prolonged the median survival time of mice treated with DOX and decreased the mortality risk by 3.7 folds. In conclusion, didox significantly potentiated the cytotoxicity of DOX in liver cancer cells and protected from its cardiotoxicity.Display Omitted
Keywords: Didox; Liver cancer; Doxorubicin; Cardiotoxicity;

Comparison of the behavioral effects of bupropion and psychostimulants by Tomohisa Mori; Masahiro Shibasaki; Yuki Ogawa; Mayuna Hokazono; Tzu-Chueh Wang; Mahardian Rahmadi; Tsutomu Suzuki (370-375).
Psychostimulant abuse has been a serious social problem worldwide for a long time. Bupropion, which is used as an antidepressant and to aid smoking cessation in the US, is considered to have psychostimulant-like activity. Although activation of the dopaminergic system induces several behavioral effects and bupropion can activate the dopaminergic system, the abuse potential and other behavioral effects of bupropion have not been fully investigated. Therefore, in this study we compared the behavioral effects of bupropion to those of psychostimulants in mice. Both methamphetamine and bupropion induced sensitization to locomotor activity, and cross-sensitized each other. Methamphetamine and bupropion also induced robust rewarding effects as measured by the conditioned place preference paradigm, although the conditioned reward with bupropion extinguished faster than that with methamphetamine. Furthermore, unlike psychostimulants, bupropion did not disrupt prepulse inhibition, even in bupropion-sensitized mice. These findings constitute evidence that bupropion and methamphetamine have similar pharmacological profiles, particularly with regard to dopamine-related behaviors. However, bupropion has lower abuse potential and side effects than methamphetamine.
Keywords: Rewarding effect; Sensitization; Bupropion; Methamphetamine; Dopamine;

Olanzapine induces glucose intolerance through the activation of AMPK in the mouse hypothalamus by Megumi Ikegami; Hiroko Ikeda; Yoko Ishikawa; Masahiro Ohsawa; Takahiro Ohashi; Misa Kai; Atsuko Kamei; Junzo Kamei (376-382).
Treatment with atypical antipsychotic drugs is known to increase the risk of glucose intolerance and diabetes. However, the mechanism of this effect is unclear. Since central adenosine 5′-monophosphate-activated protein kinase (AMPK) plays an important role in regulating nutrient homeostasis, the present study was performed to examine the involvement of central AMPK in the glucose intolerance induced by olanzapine, an atypical antipsychotic drug, in mice. Acute intraperitoneal treatment with olanzapine dose-dependently increased blood glucose levels in the glucose tolerance test. Intracerebroventricular administration of olanzapine also increased blood glucose levels in the glucose tolerance test. The glucose intolerance induced by both intraperitoneal and intracerebroventricular treatment with olanzapine was significantly attenuated by intracerebroventricular pretreatment with the AMPK inhibitor compound C. Intracerebroventricular treatment with the AMPK activator AICAR increased blood glucose levels in the glucose tolerance test, and this increase was inhibited by compound C. Moreover, the hypothalamic level of phosphorylated AMPK after glucose injection was significantly increased by intracerebroventricular pretreatment with olanzapine. Olanzapine did not affect plasma glucagon and insulin levels. Our results indicate that acute treatment with olanzapine causes glucose intolerance through the activation of hypothalamic AMPK. The present study suggests that the inhibition of central AMPK activity may have a therapeutic effect on the metabolic disturbance induced by atypical antipsychotic drugs.
Keywords: Atypical antipsychotic drug; Glucose tolerance test;

tCFA15, a trimethyl cyclohexenonic long-chain fatty alcohol, affects neural stem fate and differentiation by modulating Notch1 activity by Julien Bouissac; Jeremy Garwood; Céline Girlanda-Jungès; Bang Luu; Pascal Dollé; Eliane Mohier; Marie Paschaki (383-392).
We have investigated the effects of tCFA15, a non-peptidic compound, on the differentiation of neural stem cell-derived neurospheres, and have found that tCFA15 promotes their differentiation into neurons and reduces their differentiation into astrocytes, in a dose-dependent manner. This response is reminiscent of that resulting from the loss-of-function of Notch signaling after inactivation of the Delta-like 1 (Dll1) gene. Further analysis of the expression of genes from the Notch pathway by reverse transcriptase-PCR revealed that tCFA15 treatment results in a consistent decrease in the level of Notch1 mRNA. We have confirmed this result in other cell lines and propose that it reflects a general effect of the tCFA15 molecule. We discuss the implications of this finding with respect to regulation of Notch activity in neural stem cells, and the possible use of tCFA15 as a therapeutic tool for various pathologies that result from impairment of Notch signaling.
Keywords: Brain; Neural stem cell; Neuronal differentiation; Neurosphere; Notch signaling;

Mechanisms underlying the antinociceptive effect of mangiferin in the formalin test by Teresa Izquierdo; Antonio Espinosa de los Monteros-Zuñiga; Claudia Cervantes-Durán; María Concepción Lozada; Beatriz Godínez-Chaparro (393-400).
The purpose of this study was to investigate the possible antinociceptive effect of mangiferin, a glucosylxanthone present in Mangifera indica L., in inflammatory pain. Furthermore, we sought to investigate the possible mechanisms action that contributes to these effects. The ipsilateral local peripheral (1–30 µg/paw), intrathecal (1–30 µg/rat) and oral (1–30 mg/kg) administration of mangiferin produced a dose-dependent reduction in formalin-induced nociception. The antinociceptive effect of this drug was similar to that induced by diclofenac after oral and local peripheral administration. Furthermore, mangiferin was also able to reduce 0.1% capsaicin- and serotonin-induced nociceptive behavior. The local peripheral antinociceptive effect of mangiferin in the formalin test was blocked by naloxone (50 μg/paw), naltrindole (1 μg/paw), 5-guanidinonaltrindole (5-GNTI, 1 μg/paw), N G-l-nitro-arginine methyl ester (L-NAME, 100 µg/paw), 1H-(1,2,4)-oxadiazolo [4,2-a]quinoxalin-1-one (ODQ, 50 µg/paw) and glibenclamide (50 μg/paw), but not by methiothepin (30 μg/paw). These results suggest that the antinociceptive effects induced by mangiferin are mediated by the peripheral opioidergic system involving the activation of δ, κ, and probably µ, receptors, but not serotonergic receptors. Data also suggests that mangiferin activates the NO–cyclic GMP-ATP-sensitive K+ channels pathway in order to produce its local peripheral antinociceptive effect in the formalin test. Mangiferin may prove to be effective in treating inflammatory pain in humans.
Keywords: Mangiferin; Antinociception; Opioid receptors; K+ channels; Formalin test;

Correction of vascular hypercontractility in spontaneously hypertensive rats using shRNAs-induced delta protein kinase C gene silencing by Tetiana Novokhatska; Sergey Tishkin; Victor Dosenko; Alexey Boldyriev; Irina Ivanova; Ievgen Strielkov; Anatoly Soloviev (401-407).
Potassium conductance in vascular smooth muscle (VSM) is known to be altered in arterial hypertension. High level of protein kinase C (PKC) activity is a common feature for hypertension of different genesis. The main goal of this study was to investigate the efficacy of the RNA interference (RNAi) technique targeting PKC delta-isoform gene as a possible pharmacological tool to restore vasodilator potential in spontaneously hypertensive rats (SHR). Experimental design of the study comprised RNAi and patch-clamp techniques, RT-PCR analysis and standard acetylcholine test. Total outward currents and acetylcholine-induced endothelium-dependent relaxant responses were blunted in SHR. BKCa alpha subunit mRNA expression in SHR was unchanged whereas KV and KATP mRNA expression appeared significantly increased. PKC inhibitor, chelerythrine (100 nM), restored potassium channels activity in SHR. PKC-delta-isoform protein expression and PKC-delta-isoform mRNA expression are 2.5–4 fold increased in VSM from SHR. PKC gene silencing with the short hairpin RNAs (shRNAs)-plasmid delivery system administered intravenously led to an increment in maximal amplitude of acetylcholine-relaxation, restored outward K+ currents and PKC-delta-isoform mRNA and protein expression. Arterial blood pressure in SHR was normalized following shRNAs administration. We conclude that BKCa channels are likely to be the most PKC-dependent member of K+ channels family responsible for vascular hypercontractility in SHR while Kv and KATP channels may constitute a reserve mechanism for the maintenance of vasodilator potential under BKCa channelopathy. It is likely that RNAi technique is a good therapeutic approach to inactivate PKC gene and to normalize vascular functions and high arterial blood pressure in SHR.Display Omitted
Keywords: Protein kinase C; Potassium channel; Vascular smooth muscle; RNA-interference; Arterial hypertension;

Increased expressions of cyclooxygenase-2 (COX-2) and its downstream metabolite, prostaglandin E2 (PGE2), are well documented events in the development of colorectal cancer. Interestingly, PGE2 itself can induce the expression of COX-2 thereby creating the potential for positive feedback. Although evidence for such a positive feedback has been previously described, the specific E-type prostanoid (EP) receptor subtype that mediates this response, as well as the relevant signaling pathways, remain unclear. We now report that the PGE2 stimulated induction of COX-2 expression in human colon cancer HCA-7 cells is mediated by activation of the prostanoid EP4 receptor subtype and is followed by coupling of the receptor to Gαi and the activation of phosphatidylinositol 3-kinase. Subsequent activation of metalloproteinases releases membrane bound heparin-binding epidermal growth factor-like growth factor resulting in the transactivation of epidermal growth factor receptors and the activation of the extracellular signal-regulated kinases and induction of COX-2 expression. This induction of COX-2 expression by PGE2 stimulation of the prostanoid EP4 receptor may underlie the upregulation of COX-2 during colorectal cancer and appears to be an early event in the process of tumorigenesis.
Keywords: Prostaglandin E2; PGE2; Human prostanoid EP4 receptor; Gαi; Phosphatidylinositol 3-kinase; Cyclooxygenase-2; Colon cancer;

Beclomethasone dipropionate and formoterol reduce oxidative/nitrosative stress generated by cigarette smoke extracts and IL-17A in human bronchial epithelial cells by Angela Marina Montalbano; Giulia Anzalone; Giusy Daniela Albano; Caterina Di Sano; Rosalia Gagliardo; Anna Bonanno; Loredana Riccobono; Gabriele Nicolini; Eleonora Ingrassia; Mark Gjomarkaj; Mirella Profita (418-427).
Interleukin-17A (IL-17A), cigarette smoke and oxidative/nitrosative stress are involved in inflammatory airway diseases, and the mechanisms behind these processes are still poorly understood. We investigated whether recombinant human IL-17A (rhIL-17A), in combination with cigarette smoke extracts (CSE), increases the levels of inducibile nitric oxide synthase (iNOS), reactive oxygen species, nitrotyrosine (NT) and the activation of signal transducer and activator of transcription 1 (STAT-1) in normal human bronchial epithelial cells (16HBE). The effect of beclomethasone dipropionate (BDP), formoterol and their combination was also evaluated. We demonstrated that rhIL-17A or CSE alone increases iNOS expression, reactive oxygen species and NT production and STAT-1 downstream signalling activation in terms of STAT-1ser727 and STAT-1tyr701 phosphorylation. The combination of both stimuli further increased iNOS, ROS, NT and STAT-1ser727 phosphorylation. The silencing of STAT-1 expression partially reduced the levels of iNOS, reactive oxygen species and NT generated by rhIL-17A and inhibited the effect of CSE alone in 16HBE cells. The treatment of the cells with the MEK1/2 inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto butadiene) abolished the expression of iNOS and STAT-1ser727 phosphorylation generated by rhIL-17A. 16HBE treated with BDP or formoterol alone partially suppressed the effect of IL-17A or CSE on ROS, NT, and STAT-1 activation. Furthermore the use of the drugs in combination showed an additive effect in 16HBE. Our findings demonstrate that IL-17A increases oxidative/nitrosative markers, likely via ERK1/2 downstream signalling and STAT-1 pathway activation in human bronchial epithelial cells. BDP and formoterol treatment reduces this effect showing an additive effect used in combination.
Keywords: Epithelial cells; Oxidative/nitrosative stress; Cigarette smoke; Interleukin-17A (IL-17A);

N-formyl-methionyl-leucyl-phenylalanine (fMLF), its methyl ester fMLF-OMe and interleukin 8 (IL8) play a pivotal role in neutrophil chemotaxis regulation in the latter and early stages, respectively, but the mechanisms through which the signal transduction pathways activate this function are not yet completely understood. Compounds 3l and 3r, a new class of arylcarbamoyl-imidazo-pyrazoles derivatives, were described as the first example of compounds able to inhibit human neutrophil chemotaxis induced by both fMLF-OMe and IL8. Here, we report their effects on superoxide production and lysozyme release. No inhibition was observed, thus they could be defined as “pure” chemotactic antagonists. Therefore, such molecules were used to highlight specific kinases involved in neutrophil chemotaxis. Our data provide support that compounds 3l and 3r strongly inhibit p38 MAPK with either fMLF-OMe or IL8 chemoattractants, while they show different signaling pathways regarding PKC isoforms suggesting that a fine tuning of the neutrophil activation occurs through differences in the activation of signaling pathways. Neither fMLF-OMe nor IL8 were able to obtain activation of the PI3K/Akt pathway. Since anomalous activation of neutrophil recruitment is one of the causes of many inflammatory diseases, the good versatility of our derivatives could represent the most important characteristic of these new molecules in the development of novel therapeutics.
Keywords: Neutrophil chemotactic inhibitors; IL8; fMLF-OMe; Chemotaxis; Signaling pathways.;

Proton pump inhibitors as also inhibitors of atrial fibrillation by Kun Lin; Xinpei Chen; Li Zhang; Yutang Wang; Zhaoliang Shan (435-440).
Proton pump inhibitors (PPIs) are widely used for the treatment of acid-related upper digestive diseases, including gastric and duodenal ulcer and gastroesophageal reflux disease (GERD). Remarkably, several small clinical trials have shown that these drugs also reduce the symptoms and frequency of atrial fibrillation (AF) episodes in patients treated for comorbid acid reflux. Although the mechanism remains unclear, the effect might pinpoint a connection between GERD and AF. To this end, it is known that both oxidants and inflammation affect initiation and maintenance of AF, and PPIs may reduce symptoms and frequency of AF episodes through their antioxidant and anti-inflammatory effects. This review focuses on the anti-AF effects of PPIs beyond their inhibition of gastric acid production.
Keywords: Proton pump inhibitors; Atrial fibrillation; Gastroesophageal reflux disease;

We undertook this study to investigate the influence of lacidipine on endoplasmic reticulum stress (ERS) and myocardial remodeling under pressure overload conditions. Thirty male Sprague Dawley rats were randomly divided into three groups: sham, transverse aortic constriction (TAC), and TAC+lacidipine groups. Invasive hemodynamics was measured. The structure of the left ventricle was observed via echocardiography. ERS parameters such as calreticulin (CRT) and caspase-12 expressions in cardiomyocytes were examined by immunohistochemistry. TUNEL staining was used to detect cardiomyocyte apoptosis. Left ventricular end-diastolic pressure (LVEDP), interventricular septal thickness (IVST), left ventricular posterior wall thickness (LVPWT), left ventricular weight index (LVWI), CRT and caspase-12 expression in cardiomyocytes were evaluated. The incidence of cardiomyocyte apoptosis significantly increased in the TAC group compared with the sham group (P<0.01). Meanwhile, left ventricular end-systolic pressure (LVESP), ejection fraction (EF), and early diastolic blood maximum flow rate of mitral valve/late diastolic blood flow velocity of mitral valve (E/A) values decreased significantly (P<0.01). LVEDP, IVST, LVPWT, and LVWI values and the incidence of cardiac apoptosis in the TAC+lacidipine group decreased significantly compared with those in the TAC group. CRT and caspase-12 expressions in cardiomyocytes were also significantly downregulated (P<0.05). On the other hand, LVESP, EF, and E/A values in the TAC+lacidipine group substantially increased (P<0.05). Our results suggest that lacidipine attenuates hypertrophied myocardial remodeling accompanied by inhibiting CRT and caspase-12 expression and cardiomyocyte apoptosis in rats subjected to TAC.
Keywords: Lacidipine; Myocardium hypertrophy; CRT; Caspase-12; Apoptosis;

The numerous mediators of pain and inflammation are products of injury-induced gene expression that lead to changes in the nervous system and immune responses. These multiple molecules and mechanisms suggest novel strategies that could be used for analgesic drug development. The present study investigated the possible anti-hyperalgesic effects of anomalin in complete Freund′s adjuvant (CFA)-induced acute and chronic inflammatory pain models. Acute pretreatment of mice with anomalin (10 and 50 mg/kg, i.p.) produced a significant anti-nociceptive effect against CFA- and carrageenan-induced mechanical hyperalgesia and allodynia. In a chronic pain model, administration of anomalin inhibited CFA-induced hyperalgesia, and it did not cause any apparent toxicity. Another set of experiments observed that anomalin inhibited CFA- and carrageenan-induced paw edema in acute and chronic models. To elucidate the molecular mechanism underlying the anti-nociceptive effect of anomalin, the various pain signaling pathways [NF-κB, cAMP response element-binding protein (CREB), and mitogen activated protein kinase (MAPKs)/AP-1] that are involved were examined. Intraperitoneal (i.p.) pretreatment of anomalin exhibited potent inhibitory effects on direct mediators of hyperalgesia (iNOS and COX-2). The release of CFA-induced plasma nitrite and paw tissue hyperalgesic cytokine (TNF-α) was reduced remarkably. In addition, the adenosine 5'-triphosphate (ATP) in plasma and substance P (SP) in paw tissue were markedly suppressed by anomalin. These results demonstrate that anomalin exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of the NF-κB, CREB, and MAPKs/AP-1 signaling pathways.
Keywords: Anomalin; Hyperalgesia; Allodynia; NF-κB; CREB; MAPK;

PHII-7 inhibits cell growth and induces apoptosis in leukemia cell line K562 as well as its MDR- counterpart K562/A02 through producing reactive oxygen species by Hongwei Peng; Xiangfei Yuan; Ruizan Shi; Xiaohua Wei; Simei Ren; Cihui Yan; Yahui Ding; Yang Lin; Dongmei Fan; Ming Yang; Yanjun Zhang; Dongsheng Xiong (459-468).
Multidrug resistance (MDR) is a major obstacle that hinders the efficacy of chemotherapy in many human malignancies. PHII-7 is a derivative of indirubin, which was designed and synthesized by our laboratory. Our preliminary work indicated its potent antitumor activities in vitro and in vivo. Furthermore, based on the model of MDR cell line, we found its powerful effects in inhibiting the expression of P-glycoprotein (P-gp) and killing multidrug-resistant (MDR) cells with the detailed mechanism remained to be explored. Reactive oxygen species are known for high reactive activity as they possess unmatched electrons. In this study, we showed that PHII-7 generated equal reactive oxygen species in parental K562 and its counterpart MDR K562/A02 cells. Pre-incubation with thiol antioxidants glutathione or N-acetyl-cysteine(NAC) almost abolished the cytotoxicity of PHII-7. Moreover, NAC abrogated DNA damage, cell cycle arrests and apoptosis induced by PHII-7. Our results collectively indicated that reactive oxygen species production induced by PHII-7 contributed to both apoptosis and cell cycle arrets in MDR K562/A02 cells, thus extending our prior related findings. Notably, JNK phosphorylation was also induced by PHII-7 and pre-incubated of K562/A02 cells with NAC or inhibitor of JNK(SP006125) eliminated P-gp downregulation. Taken together, our results may provide a detailed biochemical basis for further clinical application of PHII-7.
Keywords: Reactive oxygen species; PHII-7; DNA damage; P-glycoprotein (P-gp); Apoptosis; Cell cycle arrest;

The effect of alpha-lipoic acid in ovariectomy and inflammation-mediated osteoporosis on the skeletal status of rat bone by Beyzagul Polat; Zekai Halici; Elif Cadirci; Abdulmecit Albayrak; Emre Karakus; Yasin Bayir; Habip Bilen; Ali Sahin; Tugba Nurcan Yuksel (469-474).
Osteoporosis is a high mortality and morbidity ranged skeletal disease and results in high costs of medical care in the European Union. We evaluated the possible protective effect of alpha-lipoic acid (ALA) on rat bone metabolism in ovariectomy and inflammation-mediated osteoporosis models. Groups were designed as: (1) sham; (2) sham+inflammation; (3) ovariectomy (OVX); (4) ovariectomy+ALA—25 mg/kg; (5) ovariectomy+ALA—50 mg/kg; (6) ovariectomy+inflammation; (7) ovariectomy+inflammation+ALA—25 mg/kg; and (8) ovariectomy+inflammation+ALA—50 mg/kg groups. OVX groups were allowed to recover for two months. Then, inflammation was induced in inflammation groups by subcutaneous talc injection. ALA—25 mg/kg and 50 mg/kg were administered to drug groups chronically. The skeletal response was assessed by bone mineral density (BMD), osteopontin and osteocalcin measurements. Pro-inflammatory cytokine measurements (interleukin (IL)-1beta, interleukin-6, and tumor necrosis factor-alpha) were performed to observe inflammatory process. In OVX, INF and OVX+INF groups, BMD levels were lowest and osteocalcin, osteopontin, IL-1beta, IL-6, and TNF-alpha levels were highest when compared to sham group. ALA administration increased BMD levels and decreased osteocalcin, osteopontin, IL-1beta, IL-6, and TNF-alpha levels versus OVX and OVX+INF control groups. Both in senile and postmenopausal osteoporosis, the balance in coupling were destroyed on behalf of bone resorption. ALA had a protective effect on both senile and postmenopausal osteoporosis. The positive effect of this drug in these osteoporosis models might originate from its positive effects on bone turnover markers and cytokine levels. From this perspective, ALA may be a candidate for radical osteoporosis treatment both in senile and postmenopausal types clinically at the end of advanced studies.
Keywords: Alpha-lipoic acid; Bone mineral density; Ovariectomised rat; Inflammation; Magnesium silicate;

Adenosine prevents isoprenaline-induced cardiac contractile and electrophysiological dysfunction by Yangzhen Shao; Björn Redfors; Lillemor Mattson-Hultén; Margareta Scharing Täng; Elma Daryoni; Mohammed Said; Elmir Omerovic (475-483).
Excessive levels of catecholamines are believed to contribute to cardiac dysfunction in a variety of disease states, including myocardial infarction and heart failure, and are particularly implicated in stress-induced cardiomyopathy, an increasingly recognized cardiomyopathy associated with significant morbidity and mortality. We have previously shown that a high dose of isoprenaline induces reversible regional dysfunction of the left ventricle in mice. We now hypothesize that adenosine can prevent cardiac dysfunction in this mouse model of stress-induced cardiomyopathy. Hundred male C57BL/6 mice were injected with 400 mg/kg isoprenaline and then randomized to either 400 mg/kg adenosine or saline. Cardiac function was evaluated by echocardiography at baseline and 2, 24, 48, 72, 96 and 120 min post isoprenaline. Myocardial fibrosis was quantified after 10 days. Intracellular lipid accumulation was quantified after 2 and 24 h. Electrophysiological parameters and degree of lipid accumulation were evaluated in cultured HL1 cardiomyocytes. Two hours post isoprenaline treatment, echocardiographic parameters of global and posterior wall regional function were significantly better in adenosine-treated mice (P<0.05). This difference persisted at 24 h, but saline-treated mice gradually recovered over the next 96 h. Intracellular lipid accumulation was also significantly lower in adenosine mice. We found no sign of fibrosis in the adenosine mice, whereas the extent of fibrosis in isoprenaline mice was 1.3% (P<0.05). Furthermore, adenosine-treated HL1 cells showed preserved electrophysiological function and displayed less severe intracellular lipid accumulation in response to isoprenaline. In conclusion, adenosine attenuates isoprenaline-induced cardiac dysfunction in mice and cells.
Keywords: Cardiac dysfunction; Isoprenaline; Adenosine; Mouse; Stress-induced cardiomyopathy;

High dose remifentanil increases myocardial oxidative stress and compromises remifentanil infarct-sparing effects in rats by Bin Mei; Tingting Wang; Yuan Wang; Zhengyuan Xia; Michael G. Irwin; Gordon T.C. Wong (484-492).
Chronic administration of high dose opioids such as morphine is known to create intracellular oxidative stress via an opioid receptor dependent mechanism and this can interfere with cellular function. This study aimed at examining whether such changes can occur following short term exposure to high concentration of remifentanil, a potent short acting opioid. We conducted a experimental study using rat myocardium and systematically quantified tissue levels of superoxide anions, malondialdehyde (MDA) and nitrotyrosine following exposure to increasing duration (15 min, 1 or 2 h) or escalating doses of remifentanil (1 μg, 5 μg, 10 μg or 20 μg/kg/min). Concurrently the susceptibility of the heart to ischaemia reperfusion injury was assessed under the similar conditions. For any given duration of remifentanil infusion, there was increasing superoxide anions generated as the dose of remifentanil was increased. MDA concentrations were significantly increased when the animal was exposed to 10 μg/kg/min for 2 h or 20 μg/kg/min for any duration. There was a trend towards an increased nitrotyrosine concentration with increasing dose of remifentanil, becoming significant when the dose was 20 μg/kg/min. The infarct limiting ability of remifentanil was compromised when the dihydroethidium fluorescence positive cell percentage exceeded 50%, MDA concentration greater than 2 nmol/mg of protein and nitrotyrosine content exceeding 1.5 μg/mg of protein. Short term high dose opioid exposure can induce oxidative changes seen previously only with chronic opioid use and this high oxidative stress environment corrupts the heart's sensitivity to be preconditioned by opioids.
Keywords: Remifentanil; Oxidative stress; Nitrosative stress; Opioids; Cardiac protection;

Riluzole attenuates the effects of chemoconvulsants acting on glutamatergic and GABAergic neurotransmission in the planarian Dugesia tigrina by Latha Ramakrishnan; Zachary Dalhoff; Samantha L. Fettig; Michael R. Eggerichs; Briegette E. Nelson; Bibita Shrestha; Amira H. Elshikh; Pratima Karki (493-501).
Planarians, the non-parasitic flatworms, display dose-dependent, distinct (C-like and corkscrew-like) hyperkinesias upon exposure to 0.001–10 mM aqueous solutions of glutamatergic agonists (l-glutamate and N-methyl-d-aspartate (NMDA)) and 0.001–5 mM concentrations of the glutamate decarboxylase (GAD) inhibitor (semicarbazide). In the planarian seizure-like activity (PSLA) experiments the three chemoconvulsants displayed the following order of potency (EC50): l-glutamate (0.6 mM)>NMDA (1.4 mM)>semicarbazide (4.5 mM). Planarian hyperkinesias behavior counting experiments also revealed that riluzole (0.001 to 1 mM), an anti-convulsive agent, displayed no significant behavioral activity by itself, but attenuated hyperkinesias elicited by the three chemoconvulsants targeting either glutamatergic or GABAergic neurotransmission with the following order of potency (IC50): NMDA (44.7 µM)>semicarbazide (88.3 µM)>l-glutamate (160 µM). Further, (+)-MK-801, a specific NMDA antagonist, alleviated 3 mM NMDA (47%) or 3 mM l-glutamate (27%) induced planarian hyperkinesias. The results provide pharmacological evidence for the presence of glutamatergic receptor-like and semicarbazide sensitive functional GAD enzyme-like proteins in planaria in addition to demonstrating, for the first time, the anti-convulsive effects of riluzole in an invertebrate model. High performance liquid chromatography coupled with fluorescence detection (HPLC-F) analysis performed on planarian extracts post no drug treatment (control) or treatment with 3 mM semicarbazide, combination of 3 mM semicarbazide and 0.1 mM riluzole, or 0.1 mM riluzole revealed that 3 mM semicarbazide induced 35% decrease in the GABA levels and a combination of 3 mM semicarbazide and 0.1 mM riluzole induced 42% decrease in glutamate levels with respect to the control group.

Intracellular pathways of antipsychotic combined therapies: Implication for psychiatric disorders treatment by Andrea de Bartolomeis; Livia Avvisati; Felice Iasevoli; Carmine Tomasetti (502-523).
Dysfunctions in the interplay among multiple neurotransmitter systems have been implicated in the wide range of behavioral, emotional and cognitive symptoms displayed by major psychiatric disorders, such as schizophrenia, bipolar disorder or major depression. The complex clinical presentation of these pathologies often needs the use of multiple pharmacological treatments, in particular (1) when monotherapy provides insufficient improvement of the core symptoms; (2) when there are concurrent additional symptoms requiring more than one class of medication and (3) in order to improve tolerability, by using two compounds below their individual dose thresholds to limit side effects. To date, the choice of drug combinations is based on empirical paradigm guided by clinical response. Nonetheless, several preclinical studies have demonstrated that drugs commonly used to treat psychiatric disorders may impact common intracellular target molecules (e.g. Akt/GSK-3 pathway, MAP kinases pathway, postsynaptic density proteins). These findings support the hypothesis that convergence at crucial steps of transductional pathways could be responsible for synergistic effects obtained in clinical practice by the co-administration of those apparently heterogeneous pharmacological compounds. Here we review the most recent evidence on the molecular crossroads in antipsychotic combined therapies with antidepressants, mood stabilizers, and benzodiazepines, as well as with antipsychotics. We first discuss clinical clues and efficacy of such combinations. Then we focus on the pharmacodynamics and on the intracellular pathways underpinning the synergistic, or concurrent, effects of each therapeutic add-on strategy, as well as we also critically appraise how pharmacological research may provide new insights on the putative molecular mechanisms underlying major psychiatric disorders.
Keywords: Mood stabilizer; Antidepressant; Benzodiazepine; Schizophrenia; Bipolar disorder; Postsynaptic density;

Metabonomic profiling in studying anti-osteoporosis effects of strontium fructose 1,6-diphosphate on estrogen deficiency-induced osteoporosis in rats by GC/TOF-MS by Bo Ma; Xiaotian Li; Qi Zhang; Di Wu; Guangji Wang; Jiye A; Jianguo Sun; Jing Li; Yinhui Liu; Yonglu Wang; Hanjie Ying (524-532).
A novel strontium salt compound strontium fructose 1, 6-diphosphate (FDP-Sr) has been proved to have highly effective for bone loss via dual effects of stimulating bone formation and suppressing bone absorption. In the present study, metabolomic approach was used to identify and study potential biomarkers associated with the effect and safety of FDP-Sr. The metabolomic profiles of bone loss induced by estrogen deficiency in a rat model was described to attain a system-level map of the shift on the metabolic response in plasma using GC/TOF-MS, after FDP-Sr was orally administered at the dose of 110 mg/kg/day for the prevention and 220 mg/kg/day for the treatment. Meanwhile, bone turnover biomarkers and bone mineral density were investigated to identify the specific changes of potential anti-osteoporosis effects of FDP-Sr. The differences in metabolic profiles between osteoporosis rats and FDP-Sr treated rats were well observed by the partial least squares-discriminant analysis (PLS-DA) to the MS spectra. Some metabolites including homocysteine, arachidonic acid, alanine, and hydroxyproline, which significantly changed during osteoporosis progression could be effectively reversed after FDP-Sr therapy. Of course some metabolites such as uric acid, glyceric acid, octadecadienoic acid, docosahexaenoic acid, oleic acid, and hexadecanoic acid were not found to reverse significantly after FDP-Sr administration. These results delineated the FDP-Sr effects-related metabolic alterations in the bone loss rats, suggesting that metabonomic analysis could provide helpful information on the new potential biomarkers relating to the mechanism of anti-osteoporosis action and side effects of FDP-Sr against estrogen deficiency induced bone loss.Display Omitted
Keywords: Metabonomic; GC/TOF-MS; Osteoporosis; Strontium fructose 1; 6-diphosphate (FDP-Sr);

The role of insulin–thyroid hormone interaction on β-adrenoceptor-mediated cardiac responses by Ebru Arioglu-Inan; Isil Ozakca; Gizem Kayki-Mutlu; Aylin Sepici-Dincel; Vecdi Melih Altan (533-543).
β-adrenoceptor-mediated responses are known to be attenuated in diabetic rat hearts, related to decreased receptor sensitivity and density. These impaired responses were improved with insulin in diabetic rats, but not in thyroidectomized diabetic rats. We aimed to investigate the possible interaction between insulin and thyroid hormones to restore diabetes-induced alterations on β-adrenoceptor-mediated responses. Male Sprague-Dawley rats were divided into seven groups: control (C), diabetic (D), insulin-treated diabetic (DI), thyroidectomized diabetic (TxD), insulin-treated thyroidectomized diabetic (TxDI), insulin+low dose 3,3′,5-triiodo-L-thyronine (T3) treated (TxDIT2.5) or insulin+high dose T3 (TxDIT5) treated thyroidectomized diabetic rats. Diabetes was induced with 38 mg/kg streptozotocin. Cardiac function was assessed through pressure-volume analysis and papillary muscle experiments. QPCR and western blot experiments were performed to evaluate cardiac gene expressions. Hemodynamic parameters were impaired in diabetes, and were mostly corrected in DI and TxDIT5 groups. Isoprenaline- and BRL37344-induced contractile responses were also decreased in diabetes. Isoprenaline responses were improved significantly in DI and TxDIT5 groups, whereas BRL 37344-mediated responses were increased slightly. Reduced β 1-adrenoceptor and SERCA 2A mRNA levels in diabetes were corrected in DI and TxDIT5 groups. Decreased SERCA 2A and increased β 3-adrenoceptor protein levels in diabetes were improved in DI and TxDIT5 groups. No significant changes were found in phospholamban or endothelial nitricoxide synthase protein levels. These results show that the beneficial effects of insulin on β-adrenoceptor-mediated responses in diabetic rats are dependent upon adequate concentrations of thyroid hormones.
Keywords: Heart; β-adrenoceptor; Diabetes; Insulin; Thyroid hormone;

Toll-like receptor (TLR) 7 decreases and TLR9 increases the airway responses in mice with established allergic inflammation by Mikael Adner; Magnus Starkhammar; Susanna Kumlien Georén; Sven-Erik Dahlén; Lars-Olaf Cardell (544-551).
Toll-like receptor (TLR) 7 and TLR9 recognise microbial products of viral descent. Since viruses are a common trigger of asthma exacerbations these TLRs have emerged as interesting therapeutic targets. Even though their effects on allergic inflammation have been evaluated in several models their effects on established allergic airway inflammation remains to be described. Therefore, mice with an on-going ovalbumin (OVA)-induced allergic airway inflammation were given R848 or CpG (TLR7 and TLR9 agonists, respectively) intranasally during four consecutive days. At day five, the R848 treatment had reduced OVA-induced airway hyperresponsiveness (measured as the increased resistance to methacholine), counteracted the accompanying influx of eosinophils and macrophages, and decreased the OVA-enhanced release of interleukin (IL)-5 and leukotriene (LT) B4 in bronchoalveolar lavage fluid. CpG, which by itself caused airway hyperresponsiveness, did not influence the OVA-induced airway hyperresponsiveness, and release of IL-5 and LTB4, but decreased the OVA-induced influx of cells in bronchoalveolar lavage fluid, and increased the amount of pro-inflammatory mediators like IL-12, CXCL1 and CXCL9. To conclude, TLR7 dampens the allergic airway reactivity and local inflammation, whereas TLR9 that causes airway hyperresponsiveness and increased cellular response per se, do generally not interfere with the effects induced by allergic inflammation.
Keywords: Airway mechanics; Allergy; Asthma; Innate immunity; Viral infection;