European Journal of Pharmacology (v.714, #1-3)

Cannabinoid system plays a pivotal role in the seizure threshold modulation which is mainly mediated through activation of the cannabinoid CB1 receptor. There is also several evidence of interaction between cannabinoid system and α2-adrenoceptors in different paradigms. Using model of clonic seizure induced by intravenous pentylenetetrazole (PTZ) in male mice, we investigated whether α2-adrenoceptors is involved in the effects of cannabinoids on the seizure threshold. Injection of the selective cannabinoid CB1 agonist ACEA (2 mg/kg) significantly (P<0.01) increased the seizure threshold which was prevented by pretreatment with the selective CB1 antagonist AM251 (1 mg/kg, i.p.). The highest doses of clonidine, a α2 receptor agonist, (1 and 5 mg/kg) showed anticonvulsant effects while yohimbine, a α2 receptor antagonist, (0.01, 0.1, 1, and 10 mg/kg) did not induce any significant effect on PTZ seizure threshold. Pretreatment with clonidine (0.1 and 0.5 mg/kg) significantly reversed the anticonvulsant effect of ACEA (2 mg/kg). Yohimbine (0.1, 1, and 10 mg/kg) pretreatment of mice enhanced the clonic seizure threshold of ACEA (1 mg/kg), significantly. Combination of non-effective doses of AM251 (0.1 mg/kg) and clonidine (0.01 mg/kg) showed additive effect in blocking the anticonvulsant effect of ACEA (2 mg/kg). In conclusion, our findings demonstrated that α2-adrenoceptors could be involved in the anticonvulsant properties of the specific cannabinoid CB1 agonist ACEA, suggesting that CB1 cannabinoid and α2 receptors have functional interactions in modulation of clonic seizure threshold.
Keywords: Cannabinoid; α2-Adrenoceptors; Pentylenetetrazole; Seizure; Mice;

Rhododendrin ameliorates skin inflammation through inhibition of NF-κB, MAPK, and PI3K/Akt signaling by Yoon-Jae Jeon; Byung-Hak Kim; Sunghwan Kim; Ikhoon Oh; Sooryun Lee; Jongheon Shin; Tae-Yoon Kim (7-14).
A wide range of active compounds isolated from nature is used in clinical applications and as a source of lead compounds for drug development. Rhododendron brachycarpum has been used as an oriental herbal medicine for skin inflammatory diseases. In this study, we isolated rhododendrin from Rhododendron brachycarpum leaves and investigated its molecular mechanisms for anti-inflammatory effect. Rhododendrin showed intracellular reactive oxygen species scavenging activity and suppressed nuclear translocation of nuclear factor-κB (NF-κB) by inhibiting phosphorylation of NF-κB, inhibitor of NF-κB(IκBα), and IκBα kinase(IKKα/β). Furthermore, rhododendrin inhibited mitogen-activated protein kinases (MAPKs), including ERK1/2, p38, and decreased c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K)/Akt signaling. As a result, rhododendrin reduced expression of pro-inflammatory mediators, such as cyclooxygenase-2 (COX-2), intracellular adhesion molecule-1 (ICAM-1), interleukin-1α (IL-1α), IL-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), chemokine (C–X–C) motif ligand 1 (CXCL1), and chemokine (C–C motif) ligand 17 (CCL17) in TNF-α/IFN-γ-stimulated keratinocytes. Notably, we demonstrated that topically applied rhododendrin alleviated skin inflammation in trinitrochlorobenzene (TNCB)-treated mouse ear skins. Collectively, these results indicate that rhododendrin is a biologically active compound that exhibits anti-inflammatory activity and is a promising candidate molecule to treat inflammatory skin diseases, such as psoriasis.
Keywords: Rhododendrin; Keratinocyte; Inflammatory skin disease; Anti-inflammatory activity;

Icariin, the main active flavonoid glucoside isolated from Herba epimedii (HEF), is an anabolic agent in bone that has been reported to prevent bone loss in ovariectomized rats and postmenopausal women. However, the molecular mechanism for this anabolic action of Icariin remain largely unknown. Here, we found that Icariin could promote MC3T3-E1 osteoblastic cell proliferation and reduce cell apoptosis, associated with increased mRNA levels of positive regulators of cell cycle gene Cyclin E and proliferating cell nuclear antigen (PCNA), decreaed mRNA level of negative regulator gene, Cyclin-dependent kinase 4 inhibitor B (Cdkn2B), and reduced caspase-3 activity. Icariin also enhanced MC3T3-E1 cell differentiation and mineralization demonstrated by increased the expression of differentiation markers, alkaline phosphatase (ALP) and collagen type I (Col I), and bone nodule formation via Alizarin red S staining. To characterize the underlying mechanisms, we examined the effect of Icariin on mitogen-activated protein kinase (MAPK) signaling. Icariin treatment rapidly induced extracellular signal-regulated kinase (ERK) and c-Jun N terminal kinase (JNK) activation but showed no effect on activation of p38 kinase. Furthermore, Icariin-mediated effects on osteoblasts were dramatically attenuated by treatment with specific inhibitors of MAPKs, U0126 (ERK inhibitor) and SP600125 (JNK inhibitor). Interestingly, treatment of osteoblasts with estrogen receptor antagonist ICI182780 attenuated Icariin-mediated effect of proliferation and mineralization, associated with suppression of ERK and JNK phosphorylation. These observations provide a potential mechanism of anabolic actions of Icariin involving ERK and JNK pathway by estrogen receptor.
Keywords: Icariin; Osteoblasts; Mitogen-activated protein kinases; Estrogen receptor; Osteoporosis;

SMND-309 is a novel derivative of salvianolic acid B, and has shown protective effects against rat cortical neuron damage in vitro and in vivo. However the molecular mechanisms through which SMND-309 affords this protection are unclear. The present study aimed to investigate the mechanisms associated with the protective activities of SMND-309 in a cerebral ischemia and reperfusion injury rat model. In this study, we used AG490, a specific inhibitor of the signaling pathway involving the Janus Kinase 2 (JAK2)/Signal Transducers and Activators of Transcription 3 (STAT3) signaling molecules and suramin, a potent inhibitor of vascular endothelial growth factor (VEGF), to investigate the mechanisms of SMND-309. The cerebral ischemia and reperfusion injury model was induced by performing middle cerebral artery occlusion (MCAO) in the rats. SMND-309 mitigated the effects of ischemia and reperfusion injury on brain by decreasing the infract volume, improving neurological function, increasing the survival of neurons and promoting angiogenesis by increasing the levels of erythropoietin (EPO), erythropoietin receptor (EPOR), phosphorylated JAK2 (P-JAK2), phosphorylated STAT3 (P-STAT3), VEGF and VEGF receptor 2 (Flk-1) in the brain. Our results suggest that SMND-309 provides significant neuroprotective effects against cerebral ischemia and reperfusion injury. The mechanisms of this protection may be attributed to the increased VEGF expression occurring from the JAK2/STAT3 pathway, activated by the increased EPO/EPOR expression in the brain.
Keywords: (2E)-2-{6-[(E)-2-carboxylvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid; Cerebral ischemia; Erythropoietin; Salviolic acid derivative; Salvia miltiorrhiza; Vascular endothelial growth factor;

Mephedrone: Public health risk, mechanisms of action, and behavioral effects by Nicholas F. Dybdal-Hargreaves; Nicholas D. Holder; Paige E. Ottoson; Melanie D. Sweeney; Tyisha Williams (32-40).
The recent shortage of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) has led to an increased demand for alternative amphetamine-like drugs such as the synthetic cathinone, 4-methylmethcathinone (mephedrone). Despite the re-classification of mephedrone as a Class B restricted substance by the United Kingdom and restrictive legislation by the United States, international policy regarding mephedrone control is still developing and interest in synthetic amphetamine-like drugs could drive the development of future mephedrone analogues. Currently, there is little literature investigating the mechanism of action and long-term effects of mephedrone. As such, we reviewed the current understanding of amphetamines, cathinones, and cocaine emphasizing the potentially translational aspects to mephedrone, as well as contrasting with the work that has been done specifically on mephedrone in order to present the current state of understanding of mephedrone in terms of its risks, mechanisms, and behavioral effects. Emerging research suggests that while there are structural and behavioral similarities of mephedrone with amphetamine-like compounds, it appears that serotonergic signaling may mediate more of mephedrone's effects unlike the more dopaminergic dependent effects observed in traditional amphetamine-like compounds. As new designer drugs are produced, current and continuing research on mephedrone and other synthetic cathinones should help inform policymakers' decisions regarding the regulation of novel ‘legal highs.'
Keywords: Mephedrone; Bath salts; Mechanism; Synthetic cathinone; Amphetamines;

Zinc (Zn) has been shown to stimulate bone formation and inhibit osteoclastic bone resorption and osteoclastogenesis. However, the effects of Zn on bone metabolism in diabetic animals remain to be clarified in vivo. Here, the effects of Zn supplementation on bone metabolism, including osteoclastogenesis and osteoblastogenesis, were investigated using streptozotocine (STZ)-induced diabetic rats. Zn-supplemented water (7.5 mg/L) was given for 1 week to diabetic rats injected with STZ (30 mg/kg body weight) 1 week earlier. The Zn supplement prevented a decrease in the activity and mRNA of alkaline phosphatase (ALP), osteocalcin mRNA, and hydroxyproline and calcium levels, and an increase in the activity and mRNA of tartrate-resistant acid phosphatase (TRAP) and cathepsin K in the proximal tibia of diabetic rats. Histological analysis revealed that the Zn supplement inhibited the diabetes-induced increase and decrease in the number of osteoclasts and osteoblasts, respectively, in the metaphysis of the proximal tibia. The increase in mRNA levels of receptor for activation of NF-κB (RANK), c-fos, c-jun, TRAP, and cathepsin K and decrease in the expression of Runx2, Dlx5, osterix, ALP, osteocalcin, and collagen were prevented by the supplement. The decrease in β-catenin, phosphorylated GSK3β, phosphorylated Akt, insulin-like growth factor 1 (IGF-1), and IGF-1 receptor (IGF-1R) protein levels in diabetic rats was also inhibited, although Zn did not affect the diabetes-increased gene and protein expression of Sost and Dkk1. These results suggested that Zn prevented the diabetes-induced increase in osteoclastogenesis and decrease in osteoblastogenesis by inhibiting RANK expression and stimulating IGF-1/IGF-1R/Akt/GSK3β/β-catenin signaling, respectively.
Keywords: Zinc; Diabetes mellitus; Osteoclastogenesis; Osteoblastogenesis; RANK; IGF-1/β-catenin signaling;

Stabilization of Snail through AKT/GSK-3β signaling pathway is required for TNF-α-induced epithelial–mesenchymal transition in prostate cancer PC3 cells by Hao Wang; Rui Fang; Xian-Feng Wang; Fan Zhang; Dan-Yang Chen; Binhua Zhou; Hong-Sheng Wang; Shao-Hui Cai; Jun Du (48-55).
Metastasis induced by chronic inflammation has been considered as a major challenge during cancer therapy. Epithelial–mesenchymal transition (EMT) is associated with cancer invasion and metastasis promoted by pro-inflammatory cytokine TNFα. However, the mechanisms underlying TNFα-induced EMT in prostate cancer cells is not entirely clear. Here we showed that EMT induced by longstanding stimulation with TNFα in prostate cancer PC3 cells is mediated by up-regulation of the transcriptional repressor Snail. TNFα-mediated EMT was characterized by acquiring mesenchymal fusiform morphology, increasing the expression of Vimentin and decreasing the expression of E-cadherin. Exposure to TNFα increased the expression of transcription factor Snail via post-transcriptional regulation process and induced Snail nuclear localization in PC3 cells. Moreover, overexpressed Snail in PC3 cells induced EMT. Conversely, suppressing Snail expression abrogated TNFα-induced EMT, suggesting that Snail plays a crucial role in TNFα-induced EMT in prostate cancer cells. Finally, we showed that TNFα time-dependently activated NF-κB, AKT, ERK, p38 MAPK signaling pathways, and elevated Snail stability by activating AKT pathway that subsequently inhibited GSK-3β activity. Taken together, these results reveal that stabilization of Snail via AKT/GSK-3β signaling pathway is required for TNFα-induced EMT in prostate cancer cells. This study offers a better understanding of TNFα-induced metastasis and provides an effective therapeutic strategy for prostate cancer treatment.
Keywords: Snail; Epithelial–mesenchymal transition; GSK-3β; Prostate cancer;

Estrogen receptor (ER)-negative breast cancers are aggressive and unresponsive to antiestrogens, and current therapeutic modalities for ER-negative breast cancer patients are usually associated with strong toxicity and side effects. Less toxic and more effective targeted therapies are urgently needed to treat this type of breast cancer. Here, we report that Broussoflavonol B, a chemical purified from the bark of the Paper Mulberry tree (Broussonetia papyrifera) exhibited potent growth inhibitory activity in ER-negative breast cancer MDA-MB-231 cells at sub-micromolar concentrations. Broussoflavonol B induced cell cycle arrest at both the G0/G1 and G2/M phases accompanied by a downregulation of c-Myc protein, a upregulation of the cell cycle inhibitory proteins p16INK4a, p19INK4D and p21WAF1/CIP1 and a down-regulation of the expression levels of the G2/M regulatory proteins such as cyclin B1, cdc2 and cdc25C. Broussoflavonol B also induced apoptotic cell death characterized by accumulation of the annexin V- and propidium iodide-positive cells, and cleavage of caspases 8, 9 and 3. In addition, Broussoflavonol B treatment also decreased the steady state levels of the epidermal growth factor receptor (EGFR) and ER-α36, a variant of estrogen receptor-α, and restricted growth of the stem-like cells in ER-negative breast cancer MDA-MB-231 cells. Our results thus indicate that Broussoflavonol B is a potent growth inhibitor for ER-negative breast cancer cells and provide a rational for preclinical and clinical evaluation of Broussoflavonol B for ER-negative breast cancer therapy.
Keywords: ER-negative breast cancer; Breast cancer stem cells; Broussoflavonol B; ER-α36; EGFR;

Peripheral 5-HT1D and 5-HT7 serotonergic receptors modulate sympathetic neurotransmission in chronic sarpogrelate treated rats by José Ángel García-Pedraza; Mónica García; María Luisa Martín; Jesús Gómez-Escudero; Alicia Rodríguez-Barbero; Luis San Román; Asunción Morán (65-73).
5-HT2 receptor activation induces vasoconstriction, hypertension and platelet aggregation; therefore, its blocking may be useful in cardiovascular diseases, probably due to alterations in the modulation of serotonergic system. The aim of this study was to evaluate whether 5-HT2 receptor blockade changes serotonergic modulation of sympathetic neurotransmission in pithed rats. Serotonergic modulation of sympathetic neurotransmission was investigated in Wistar rats treated with sarpogrelate, a 5-HT2 receptor antagonist, during 14 days (30 mg/kg/day). After central nervous system destruction, we conducted electrical stimulation throughout the spinal cord flow to study the 5-HT-related products action on adrenergic system. 5-Hydroxytryptamine exerted inhibition of sympathetic outflow in sarpogrelate-treated pithed rats. This effect was mimicked and enhanced by 5-CT (5-HT1/7 receptor agonist). L-694,247 and AS-19, 5-HT1D and 5-HT7 receptor agonists respectively, reproduced this action. Pretreatment with LY310762+SB258719 (5-HT1D and 5-HT7 receptor antagonists, respectively) completely abolished 5-CT inhibitory action. The nature of this action was prejunctional since these agonists did not modify the pressor responses induced by exogenous noradrenaline. Western Blot analysis confirmed a higher expression of 5-HT1D receptors in sarpogrelate-treated rats. Experimental 5-HT2 receptor blockade induces changes in the 5-HT receptors involved in the serotonergic inhibition of sympathetic-induced pressor responses. Prejunctional activation of 5-HT1D and 5-HT7 receptors induces a significantly higher serotonergic inhibition on adrenergic neurotransmission in sarpogrelate-treated pithed rats. The antagonism of 5-HT2 receptors produces an enhancement of serotonergic sympathoinhibitory effect, which may explain the beneficial effects of this blockade in cardiovascular disorders where 5-hydroxytryptamine plays a crucial role.
Keywords: 5-HT; 5-CT; Sarpogrelate; 5-HT2; 5-HT1D; 5-HT7 receptors; Prejunctional sympathoinhibition; Pithed rat;

Simvastatin enhances human osteoblast proliferation involved in mitochondrial energy generation by Shu-Chun Chuang; Hsiu-Jung Liao; Ching-Ju Li; Gwo-Jaw Wang; Je-Ken Chang; Mei-Ling Ho (74-82).
Simvastatin has been shown to stimulate osteogenic cell differentiation. Our previous study showed osteoblasts on trabecular surface are increased by simvastatin treatment in animal study. However, whether simvastatin stimulates osteoblast proliferation and by what molecular mechanism have not been adequately investigated. Because the mitochondrial function is crucial for cell survival and proliferation, we hypothesize that simvastatin may promote human osteoblast (hOBs) proliferation and it may be related to mitochondrial function. Our results showed that simvastatin significantly enhanced proliferation and increased both mRNA and protein levels of cyclin D2, Bcl-2 and the ratio of Bcl-2 to Bax (Bcl-2/Bax). Furthermore, simvastatin increased mitochondrial activity and ATP content of hOBs. Most importantly, treatment with ATP synthase blocker, oligomycin, significantly decreased both simvastatin-stimulated ATP content and cell proliferation, and completely reversed the simvastatin-induced up-regulation of cyclin D2 and Bcl-2 expression in hOBs. On the other hand, rotenone, the complex I blocker, also partially blocked simvastatin-stimulated ATP content and cell proliferation, but the blocker did not suppress the effect of simvastatin on cyclin D2 and Bcl-2 expression. These results indicate that the up-regulation of cyclin D2 and Bcl-2/Bax by simvastatin depends on the intact function of ATP synthase in the mitochondria of hOBs. It suggests that simvastatin may promote hOB proliferation, at least partly, via up-regulating mitochondrial function and subsequently cyclin D2 and Bcl-2/Bax expression. The findings provide new information for the basic medical science in bone physiology and for new therapy strategy of simvastatin on bone formation in future.
Keywords: ATP; Cell cycle regulators; Mitochondria; Simvastatin; Cell survival;

Neuroprotective effect of hydroxysafflor yellow A on 6-hydroxydopamine-induced Parkinson's disease in rats by Bing Han; Jia Hu; Jingyu Shen; Yonglin Gao; Yan Lu; Tian Wang (83-88).
Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting predominantly the dopaminergic mesotelencephalic system. Enormous progress has been made in the treatment of PD. Our previous study has shown that hydroxysafflor yellow A (HSYA) could attenuate the neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. In the present work, we examined whether HSYA had the neuroprotective effect on dopaminergic neurons of substantia nigra in a rat model of PD. Adult Sprague-Dawley rats were unilaterally injected with 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. The PD rats were treated with HSYA (2 or 8 mg/kg) via caudal vein injection daily for 4 weeks. Rotational tests showed that HSYA significantly attenuated apomorphine-induced turns in 6-OHDA-induced PD rats. HSYA treatment resulted in a significant protection against the loss of tyrosine hydroxylase-positive cells. Our data showed that HSYA also increased the levels of dopamine and its metabolites, glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor in striatum of PD rats. In conclusion, these results supported a role for HSYA in preserving dopamine neuron integrity and motor function in a rodent model of PD, and implied a potential neuroprotective role for HSYA in this disease.
Keywords: Parkinson's disease; Hydroxysafflor yellow A; Glial cell line-derived neurotrophic factor; Brain-derived neurotrophic factor;

The aim of this study was to examine the therapeutic effect of oxymatrine, a monomer isolated from the medicinal plant Sophora flavescens Ait, on the hepatic lipid metabolism in non-alcoholic fatty liver (NAFLD) rats and to explore the potential mechanism. Rats were fed with high fructose diet for 8 weeks to establish the NAFLD model, then were given oxymatrine treatment (40, 80, and 160 mg/kg, respectively) for another 8 weeks. Body weight gain, liver index, serum and liver lipids, and histopathological evaluation were measured. Enzymatic activity and gene expression of the key enzymes involved in the lipogenesis and fatty acid oxidation were assayed. The results showed that oxymatrine treatment reduced body weight gain, liver weight, liver index, dyslipidemia, and liver triglyceride level in a dose dependant manner. Importantly, the histopathological examination of liver confirmed that oxymatrine could decrease the liver lipid accumulation. The treatment also decreased the fatty acid synthase (FAS) enzymatic activity and increased the carnitine palmitoyltransferase 1A (CPT1A) enzymatic activity. Besides, oxymatrine treatment decreased the mRNA expression of sterol regulatory element binding transcription factor 1(Srebf1), fatty acid synthase (Fasn), and acetyl CoA carboxylase (Acc), and increased the mRNA expression of peroxisome proliferator activated receptor alpha (Pparα), carnitine palmitoyltransferase 1A (Cpt1a), and acyl CoA oxidase (Acox1) in high fructose diet induced NAFLD rats. These results suggested that the therapeutic effect of oxymatrine on the hepatic steatosis in high fructose diet induced fatty liver rats is partly due to down-regulating Srebf1 and up-regulating Pparα mediated metabolic pathways simultaneously.
Keywords: Oxymatrine; Liver steatosis; Fatty acid synthesis; Fatty acid oxidation;

Nocistatin inhibits pregnant rat uterine contractions in vitro: Roles of calcitonin gene-related peptide and calcium-dependent potassium channel by Beáta H. Deák; Anna Klukovits; Kornélia Tekes; Eszter Ducza; George Falkay; Róbert Gáspár (96-104).
The endogenous neuropeptide nociceptin/orphanin FQ, translated from the prepronociceptin gene, exerts a contraction-inhibitory effect on the rat uterus. As nocistatin has been reported to cause functional antagonism of the pro-nociceptive effects of nociceptin, we set out to investigate its effects on the pregnant rat uterus and to elucidate its signalling pathway. The expression of prepronociceptin mRNA in the uterus and nocistatin levels in the uterus and the plasma were confirmed by RT-PCR and radioimmunoassay. The uterine levels of prepronociceptin mRNA and nocistatin were significantly increased by the last day of pregnancy, while the plasma nocistatin levels remained unchanged. In the isolated organ bath studies nocistatin inhibited the prostaglandin- and the KCl-evoked contractions in the uterus dose-dependently. This latter effect was decreased by preincubation with capsaicin. Incubation with calcitonin gene-related peptide after capsaicin treatment caused an elevation in the contraction-inhibitory effect of nocistatin. The effect of nocistatin was also decreased by the Ca2+-dependent K+ channel inhibitor paxilline, against spontaneous uterine contractions. Nociceptin potentiated the action of nocistatin. Naloxone decreased the effect of nocistatin administered either alone or in combination with nociceptin. In Ca2+-poor environment, this effect of naloxone was suspended. Enzyme immunoassay for the uterine intracellular cAMP levels partially confirmed the results of in vitro contractility studies. We conclude that nocistatin, generated locally in the uterus, exerts an inhibitory effect, the mechanism being mediated in part by Ca2+-dependent K+ channels, the elevation of cAMP levels and sensory neuropeptides.
Keywords: Uterine contractility; Nocistatin; Calcitonin gene-related peptide; Potassium channel; Pregnant rat;

Wedelolactone exhibits anti-fibrotic effects on human hepatic stellate cell line LX-2 by Yanzhe Xia; Jie Chen; Yuan Cao; Chenshu Xu; Ruiming Li; Yuhua Pan; Xiao Chen (105-111).
Wedelolactone is a major coumarin of Eclipta prostrata, which is used for preventing liver damage. However the effects of wedelolactone on hepatic fibrosis remained unexplored. The purpose of this study was to demonstrate the anti-fibrotic effects of wedelolactone on activated human hepatic stellate cell (HSC) line LX-2 and the possible underlying mechanisms by means of MTT assay, Hoechst staining, as well as real-time quantitative PCR and western blot. The results showed that wedelolactone reduced the cellular viability of LX-2 in a time and dose-dependent manner. After treatment of wedelolactone, the expressions of collagen I and α-smooth muscle actin, two biomarkers of LX-2 activation, were remarkably decreased. The apoptosis of LX-2 cells was induced by wedelolactone accompanied with the decreasing expression of anti-apoptotic Bcl-2 and increasing expression of pro-apoptotic Bax. In addition, phosphorylated status of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was up-regulated, but not in p38. Moreover, wedelolactone significantly repressed the level of phosphorylated inhibitor of nuclear factor κB (IκB) and p65 in nucleus in spite of tumor necrosis factor-α stimulation. In conclusion, wedelolactone could significantly inhibit the activation of LX-2 cells, the underlying mechanisms of which included inducing Bcl-2 family involved apoptosis, up-regulating phosphorylated status of ERK and JNK expressions, and inhibiting nuclear factor-κB (NF-κB) mediated activity. Wedelolactone might present as a useful tool for the prevention and treatment of hepatic fibrosis.
Keywords: Wedelolactone; Liver fibrosis; Hepatic stellate cell; NF-κB; Apoptosis;

Protective effects of neferine on amiodarone-induced pulmonary fibrosis in mice by Chang-He Niu; Yong Wang; Jing-Di Liu; Jia-Ling Wang; Jun-Hua Xiao (112-119).
The effects of neferine, a bisbenzylisoquinline alkaloid extracted from the Chinese traditional medicine seed embryo of Nelumbo nucifera Gaertn, on amiodarone-induced pulmonary fibrosis in mice were evaluated. Adult Kunming mice were induced to develop pulmonary fibrosis through intratracheal instillation of amiodarone (6.25 mg/kg) on the 1st, 3rd and 5th day. Mice were treated orally with saline, neferine (20 mg/kg), prednisolone (15 mg/kg), pirfenidone (100 mg/kg) twice a day after the third amiodarone instillation. On Day 21, all the lung tissues were collected for hydroxyproline measurement and the histological examination by hematoxylin–eosin and Masson staining. All the blood sample were collected for surfactant protein-D (SP-D) levels assay, Th1/Th2 balance valuation, CD4+CD25+ regulatory T cells (Tregs) analysis by Enzyme-linked immunosorbent assay and flow cytometry. Our data showed that neferine significantly restored the significant reductions in body weights, the increased levels of lung index and hydroxyproline, the abnormal histological findings, the serum SP-D increase, the Th1/Th2 imbalance by decreasing IL-4 and increasing IFN-γ levels and the increases in the population of CD4+CD25+ Tregs associated with amiodarone instillation in mice. Similar changes were also observed in the prednisolone or pirfenidone treated mice. In conclusion, these results indicated that neferine possessed a significant inhibitory effect on amiodarone-induced pulmonary fibrosis, probably due to its properties of anti-inflammation, SP-D inhibition and restoring increased CD4+CD25+ Tregs which may modulate Th1/Th2 imbalance by suppressing Th2 response (from Th2 polarity toward a Th1 dominant response).
Keywords: Neferine; Amiodarone; Pulmonary fibrosis; Surfactant protein-D; Th1/Th2 balance; CD4+CD25+ regulatory T cells;

The changes induced by cyclophosphamide in intestinal barrier and microflora in mice by Jin Yang; Kai-xiong Liu; Jie-ming Qu; Xiao-dan Wang (120-124).
Infection is one of the most commonly encountered complication during chemotherapy treatment, and recent studies showed that such infections are aroused primarily from the intestinal microflora through bacterial translocation. We aimed to investigate the alterations of mucosal barrier and colonization resistance in mouse treated with cyclophosphamide (CTX) to further understand the translocation mechanism. Male Balb/c mice were administered intraperitoneally with CTX at 25 mg/kg, 50 mg/kg and 100 mg/kg for 5 days. We found that pretreatment with CTX, especially at high dose, increased the potentially pathogenic bacteria counts (Escherichia coli, enterobacteraceae, Pseudomonas and enterococci) and the intestinal permeability, which was associated with the reduction of tight junctions and adherens junctions. Our results suggested that disruption of mucosal barrier and colonization resistance may be partly responsible for the bacterial translocation during chemotherapy. Thus, modulation of mechanical mucosal barrier and colonization resistance might represent a new opportunity for applications in cancer patients to reduce infectious complications.
Keywords: Cyclophosphamide; Bacterial translocation; Tight junction; Intestinal microflora;

Effect of 18β-glycyrrhetinic acid and hydroxypropyl γcyclodextrin complex on indomethacin-induced small intestinal injury in mice by Tsukasa Ishida; Ikuya Miki; Toshihito Tanahashi; Saori Yagi; Yasuyuki Kondo; Jun Inoue; Shoji Kawauchi; Sin Nishiumi; Masaru Yoshida; Hideko Maeda; Chisato Tode; Atsuko Takeuchi; Hirokazu Nakayama; Takeshi Azuma; Shigeto Mizuno (125-131).
Non-steroidal anti-inflammatory drugs (NSAIDs)-induced small intestinal injury is a serious clinical event with recent advances of diagnostic technologies, but a successful therapeutic method to treat such injuries is still lacking. Licorice, a traditional herbal medicine, and its derivatives have been widely used for the treatment of a variety of diseases due to their extensive biological actions. However, it is unknown whether these derivatives have an effect on NSAIDs-induced small intestinal damage. Previously, the anti-inflammatory effects of three compounds extracted from the licorice root, glycyrrhizin, 18β-glycyrrhetinic acid, and dipotassium glycyrrhizinate, were compared in vitro cell culture. The most prominent inhibitory effect on the tumor necrosis factor-α (TNF-α) production was observed with the administration of 18β-glycyrrhetinic acid as an active metabolite of glycyrrhizin. In this study, a complex compound of 18β-glycyrrhetinic acid and hydroxypropyl γcyclodextrin was examined to improve the oral bioavailability. After administration of this complex to indomethacin treated mice, a significantly high plasma concentration of 18β-glycyrrhetinic acid was detected using the tandem mass spectrometry coupled with the HPLC. Furthermore, the complex form of 18β-glycyrrhetinic acid and hydroxypropyl γcyclodextrin reduced mRNA expressions of TNF-α, interleukin (IL)-1β, and IL-6, which was histologically confirmed in the improvement of indomethacin-induced small intestinal damage. These results suggest that the complex of 18β-glycyrrhetinic acid and hydroxypropyl γcyclodextrin has the potential therapeutic value for preventing the adverse effects of indomethacin-induced small intestinal injury.
Keywords: Glycyrrhizin; 18β-Glycyrrhetinic acid; Hydroxypropyl γcyclodextrin; Indomethacin; Small intestinal injury;

Some MDMA (3,4-methylenedioxymethamphetamine) users develop dependence as a result of chronic consumption. The present study evaluated the role of 5-hydroxytryptamine 5-HT3 receptors in the acquisition, expression and reinstatement of the conditioned place preference (CPP) induced by MDMA. Adolescent male mice were conditioned with 10 mg/kg of MDMA and then treated with 1 or 3 mg/kg of the 5-hydroxytryptamine 5-HT3 antagonist MDL72222 during acquisition of conditioning (experiment 1), before expression of CPP in a post-conditioning test (experiment 2) or before a reinstatement test (experiment 3). MDL72222 was devoid of motivational effects but blocked acquisition of the MDMA-induced CPP. Moreover, following extinction, the low dose of MDL72222 blocked reinstatement of the CPP induced by priming with MDMA. Acute MDMA reduced levels of dihydroxypheylacetic acid (DOPAC) in the striatum and levels of acid 5-hydroxyindoleacetic (5-HIAA) in the cortex. Acute MDMA+MDL72222 also reduced striatal DOPAC. The repeated co-administration of MDMA plus MDL72222 (on PND 32–34–36–38) increased dopamine and decreased DOPAC in the striatum, and increased cortical serotonin and enhanced transporters of dopamine and serotonin. The acute administration (on PND ±55) of MDMA or MDL72222 increased levels of dopamine and reduced those of DOPAC in the striatum and co-administration of MDMA plus MDL72222 increased striatal serotonin. Our results confirm that 5-hydroxytryptamine 5-HT3 receptors are involved in the acquisition of conditioned rewarding effects of MDMA and demonstrate that these receptors are also involved in reinstatement after extinction.
Keywords: 5-hydroxytryptamine 5-HT3 receptor; Conditioned place preference; MDMA; Adolescence; (Mouse);

An endothelin-3-related synthetic biotinylated pentapeptide as a novel inhibitor of platelet-activating factor by Akira Sato; Tasuku Suzuki; Kazunori Oikawa; Raiki Ohta; Keiichi Ebina (142-147).
Platelet-activating factor (PAF), a potent proinflammatory mediator, is involved in many inflammatory diseases. We recently reported that synthetic biotinylated peptides having a Tyr-Lys-Asp-Gly sequence inhibit PAF-induced inflammation by directly binding to PAF. In this study, we investigated the effect of two synthetic biotinylated peptides, both of which have a sequence similar to Tyr-Lys-Asp-Gly—an endothelin-3 (ET-3)-related biotinylated pentapeptide (Tyr-Lys-Asp-Lys-Glu, BPET3) and a scavenger receptor CD36-related biotinylated tetrapeptide (Tyr-Lys-Gly-Lys, BPCD36)—on PAF-induced inflammation by using a rat model of hind paw oedema. BPET3 markedly inhibited PAF-induced oedema in a dose-dependent manner, and the dose that caused 50% inhibition was estimated to be approximately 2.64 nmol/paw. The inhibitory effect of BPCD36 on PAF-induced paw oedema was less than that of BPET3, while a synthetic biotinylated pentapeptide (Lys-Lys-Tyr-Asp-Glu) shuffling amino acid sequence of BPET3, an ET-1-related synthetic biotinylated pentapeptide (Leu-Met-Asp-Lys-Glu), or an ET-2-related synthetic biotinylated pentapeptide (Trp-Leu-Asp-Lys-Glu) did not inhibit PAF-induced paw oedema. Furthermore, intrinsic tryptophan fluorescence studies demonstrated that ET-3 specifically interacted with both PAF and its metabolite/precursor lyso-PAF. These results provide evidence that the Tyr-Lys-Asp region in both ET-3 and BPET3 is essential for marked inhibition of the peptide on PAF-induced inflammation, and strongly suggest that BPET3 may be useful as a novel anti-inflammatory drug targeting PAF.
Keywords: Synthetic peptide; Endothelin-3; Platelet-activating factor; PAF; PAF inhibitor; Anti-inflammatory drug;

The protective effect of low-dose methotrexate on ischemia–reperfusion injury of the rabbit spinal cord by Hayri Kertmen; Bora Gürer; Erdal Reşit Yılmaz; Ahmet Metin Şanlı; Mehmet Sorar; Ata Türker Arıkök; Mustafa Fevzi Sargon; Mehmet Ali Kanat; Berrin Imge Ergüder; Zeki Şekerci (148-156).
Methotrexate was developed as a cytostatic agent, but at low doses, it has shown potent anti-inflammatory activity. Previous studies have demonstrated that the anti-inflammatory effects of methotrexate are primarily mediated by the release of adenosine. In this study, we hypothesized that low-dose methotrexate has protective effects in spinal cord ischemia–reperfusion injury. Rabbits were randomized into the following four groups of eight animals each: group 1 (control), group 2 (ischemia), group 3 (methylprednisolone) and group 4 (methotrexate). In the control group only a laparotomy was performed. In all the other groups, the spinal cord ischemia model was created by the occlusion of the aorta just caudal to the renal artery. Neurological evaluation was performed with the Tarlov scoring system. Levels of myeloperoxidase, malondialdehyde and catalase were analyzed, as were the activities of xanthine oxidase and caspase-3. Histopathological and ultrastructural evaluations were also performed. After ischemia–reperfusion injury, increases were found in the serum and tissue myeloperoxidase levels, tissue malondialdehyde levels, xanthine oxidase activity and caspase-3 activity. In contrast, both serum and tissue catalase levels were decreased. After the administration of a low-dose of methotrexate, decreases were observed in the serum and tissue myeloperoxidase levels, tissue malondialdehyde levels, xanthine oxidase activity and caspase-3 activity. In contrast, both the serum and tissue catalase levels were increased. Furthermore, low-dose methotrexate treatment showed improved results concerning the histopathological scores, the ultrastructural score and the Tarlov scores. Our results revealed that low-dose methotrexate exhibits meaningful neuroprotective activity following ischemia–reperfusion injury of the spinal cord.
Keywords: Adenosine; Ischemia–reperfusion; Methotrexate; Methylprednisolone; Neuroprotection; Spinal cord;

Treatment of tuberculous meningitis or tuberculoma has become complicated because of adverse drug interactions found amongst antitubercular and anticonvulsant drugs. The aim of the study is to evaluate the effect of simultaneously administered isoniazid (300 mg/day) and phenytoin (300 mg/day) on 60 patients with tuberculous meningitis or tuberculoma having seizures. Plasma samples were analyzed for isoniazid, acetylated-isoniazid (AcINH) and phenytoin levels by high performance liquid chromatography at 3 h of drugs administration and patients were classified as rapid or slow acetylator on the basis of metabolic ratio of isoniazid (Rm) and percentage of acetylated-isoniazid (%AcINH). Out of 60 patients studied, 23 were slow acetylators and 37 were rapid acetylators. Slow acetylators revealed higher plasma isoniazid levels and lower plasma AcINH levels, metabolic ratio and %AcINH as compared to rapid acetylators. Plasma phenytoin levels were found to be significantly higher (above therapeutic range) in slow acetylators as compared to rapid acetylators. Plasma phenytoin concentration was moderately strong, negatively correlated with metabolic ratio (r=−0.439, P<0.001) and %AcINH (r=−0.729, P<0.001). Eight comatose patients (34.8%) also showed significantly higher plasma phenytoin levels. Our results suggest that assessment of acetylator status and plasma phenytoin level is critical for dose optimization of isoniazid and phenytoin and to predict the patients at risk of intoxication.
Keywords: Tuberculous meningitis; Seizures; Isoniazid; Phenytoin; Adverse drug interactions;

Effects of allicin on hyperhomocysteinemia-induced experimental vascular endothelial dysfunction by De-shan Liu; Wei Gao; Er-shun Liang; Shu-li Wang; Wei-wei Lin; Wei-dong Zhang; Qing Jia; Rui-chen Guo; Ji-dong Zhang (163-169).
This study was designed to investigate the effect and mechanism of allicin on hyperhomocysteinemia-induced experimental vascular endothelial dysfunction in rats. Fifty male Wistar rats were randomly divided into five groups: the normal control rats (NC), the high-methionine-diet rats (Met), the high-methionine-diet rats treated with folic acid, vitaminB6 and vitaminB12 (Met+F), or with low-dose allicin (Met+L), or with high-dose allicin (Met+H). After 6 weeks, we collected blood samples of all groups to determine plasma endothelin (ET), serum homocysteine (Hcy), nitric oxide (NO), superoxide dismutase (SOD), malondialdehyde (MDA), and detected the expression of basic fibroblast growth factors (bFGF), transforming growth factor beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and intercellular adhesion molecule-1 (ICAM-1) in the aorta. The Hcy and the expression of TGF-β in both the Met+L and Met+H groups were significantly lower than the Met and Met+F groups. The ET, ET/NO ratio and the MDA levels of the Met+L and Met+H groups were significantly lower than the Met group. The SOD and NO levels and the expression of bFGF, TNF-α and ICAM-1 of the Met+L and Met+H groups were significantly higher than the Met group. Our data indicate that allicin inhibits lipid peroxidation induced by hyperhomocysteinemia and regulates the excretion and equilibrium of ET and NO, and suggest that allicin might be useful in the prevention of endothelial dysfunction caused by hyperhomocysteinemia.Display Omitted
Keywords: Allicin; Homocysteine; Endothelial function; Nitric oxide; Oxidative stress;

Autophagy is a degradation pathway for cytoplasmic proteins and organelles in eukaryotes. Although the mechanisms of autophagy regulation are not completely understood, the target of rapamycin (TOR) signaling pathway plays a major role in controlling the induction of autophagy. Cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase A (PKA) has also been shown to regulate autophagy in yeast and mammalian cells. In an effort to elucidate the role of the cAMP/PKA pathway in autophagy, we used the PKA inhibitor N-[2-p-bromocinnamylamino-ethyl]-5-isoquinolinesulphonamide (H89) to treat mammalian cells. Our data demonstrated that H89 induced autophagy at 10 μM, which is a commonly used concentration for PKA inhibition, but PKA inhibition was not involved in the induction of autophagy. The effects of cAMP on autophagy seemed to be dependent on the cell type and the culture conditions. In addition, we investigated which protein kinase was involved in H89-induced autophagy because several kinases other than PKA have been shown to be inhibited by 10 μM of H89. There was no protein kinase largely responsible for autophagy induction, although the inhibition of Akt, which is a downstream effector protein kinase of phosphatidylinositol-3-kinase, appeared to be partially associated. Furthermore, H89-induced autophagy was independent of TOR. H89 is a widely used PKA inhibitor, but PKA-independent effects have been reported. Therefore, it is suggested that autophagy induction is a nonspecific effect of H89, and H89-induced autophagy is independent of the cAMP–PKA and the TOR pathways.
Keywords: Autophagy; Protein kinase A; H89; Akt;

2-Aryl- and 2-amido-benzothiazoles as multifunctional vasodilators on rat artery preparations by Fabio Fusi; Miriam Durante; Giampietro Sgaragli; Nguyen Manh Cuong; Phan Thi Phuong Dung; Nguyen Hai Nam (178-187).
The neuroprotective agent riluzole [2-amino-6-(trifluoromethoxy)benzothiazole] has been shown to antagonize neuronal high-voltage activated Ca2+ currents. In the search for novel scaffolds leading to potential antihypertensive agents, a series of 2-aryl- and 2-amido-benzothiazoles (HUP) were assessed for their vasorelaxing property on rat aorta rings and for their L-type Ba2+ currents [I Ba(L)] blocking activity on single myocytes isolated from the rat tail artery.HUP5 and HUP30, the most potent of the series, inhibited phenylephrine-induced contraction with IC50 values in the range 3–6 µM. The presence of endothelium did not modify their spasmolytic activity. Both HUP5 and HUP30 increased tissue levels of cGMP and shifted to the left the concentration–response curve to sodium nitroprusside. In rings precontracted by phenylephrine, tetraethylammonium or 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) shifted to the right the concentration–relaxation curves of HUP5 and HUP30. The antispasmodic effect of HUP5 and HUP30 was more marked on rings stimulated with 25/30 mM than with 60 mM K+. HUP5 and HUP30 antagonized both extracellular Ca2+ influx and Ca2+ mobilization from intracellular stores in response to phenylephrine: this effect was not modified by the presence of ODQ. I Ba(L) was partly inhibited by HUP5 and blocked by HUP30 in a concentration-dependent as well as ODQ-independent manner.In conclusion, HUP5 and HUP30 are vasorelaxing agents that stimulate soluble guanylyl cyclase, activate K+ channels, and block extracellular Ca2+ influx. The present benzothiazole derivatives form a novel class of multifunctional vasodilators which may give rise to effective antihypertensive agents.
Keywords: Benzothiazoles; Rat aorta; Soluble guanylyl cyclase; cGMP; L-type Ca2+ channel;

Treatment with exendin-4 improves the antidiabetic efficacy and reverses hepatic steatosis in glucokinase activator treated db/db mice by Nirav Dhanesha; Amit Joharapurkar; Gaurang Shah; Samadhan Kshirsagar; Vishal Patel; Kartik Patel; Rajesh Bahekar; Mukul Jain (188-192).
The glucokinase activators improve the fasting as well as postprandial glucose control and are important investigational drugs for the treatment of diabetes. However, recent studies have implicated that continuous activation of glucokinase with a small molecule activator can increase hepatic triglycerides and the long term glucose control is not achieved. In this study, we investigated the effect of combination of glucokinase activator (GKA, Piragliatin) with GLP-1 receptor agonist exendin-4 (Ex-4) in male db/db mice. Twelve weeks combination treatment in the db/db mice resulted in a significant decrease in body weight gain, food consumption, random glucose and %HbA1c. The decrease in serum glucose and %HbA1c in combination group was more profound and significantly different than that of individual treatment (GKA or Ex-4) group. GKA treatment increased hepatic triglycerides, whereas combination of Ex-4 with GKA attenuated hepatic steatosis. The combination of GKA with Ex-4 reduced the hepatic lipid accumulation, improved the insulin sensitivity, and reduced hepatic glucose production in db/db mice. Overall, our data indicate that combination of GKA and GLP-1 receptor agonist Ex-4 improves glucose homeostasis, shows antiobesity activity, without causing harmful side effects like fatty liver.
Keywords: Glucokinase; Exendin-4; Diabetes; db/db mice; Steatosis; Obesity;

Folic acid modulates eNOS activity via effects on posttranslational modifications and protein–protein interactions by Sarah Y. Taylor; Hannah M. Dixon; Shobana Yoganayagam; Natalie Price; Derek Lang (193-201).
Folic acid enhances endothelial function and improves outcome in primary prevention of cardiovascular disease. The exact intracellular signalling mechanisms involved remain elusive and were therefore the subject of this study. Particular focus was placed on folic acid-induced changes in posttranslational modifications of endothelial nitric oxide synthase (eNOS). Cultured endothelial cells were exposed to folic acid in the absence or presence of phosphatidylinositol-3' kinase/Akt (PI3K/Akt) inhibitors. The phosphorylation status of eNOS was determined via western blotting. The activities of eNOS and PI3K/Akt were evaluated. The interaction of eNOS with caveolin-1, Heat-Shock Protein 90 and calmodulin was studied using co-immunoprecipitation. Intracellular localisation of eNOS was investigated using sucrose gradient centrifugation and confocal microscopy. Folic acid promoted eNOS dephosphorylation at negative regulatory sites, and increased phosphorylation at positive regulatory sites. Modulation of phosphorylation status was concomitant with increased cGMP concentrations, and PI3K/Akt activity. Inhibition of PI3K/Akt revealed specific roles for this kinase pathway in folic acid-mediated eNOS phosphorylation. Regulatory protein and eNOS protein associations were altered in favour of a positive regulatory effect in the absence of bulk changes in intracellular eNOS localisation. Folic acid-mediated eNOS activation involves the modulation of eNOS phosphorylation status at multiple residues and positive changes in important protein–protein interactions. Such intracellular mechanisms may in part explain improvements in clinical vascular outcome following folic acid treatment.
Keywords: Vascular biology; Endothelium; Folic acid; Nitric oxide synthase; Kinase; Phosphorylation;

Stimulation of either α1-adrenoceptor or angiotensin type 1 receptor (AT1 receptor) induces proliferation of mouse induced pluripotent stem (iPS) cells. Both α1-adrenoceptor and AT1 receptor are guanine nucleotide-binding protein q polypeptide (Gq)-coupled receptors. However, it is not fully understood whether stimulation of these Gq-coupled receptors exert a similar effect in human iPS cells, i.e. proliferation of human iPS cells. In this study, we evaluated the involvement of α1-adrenoceptor and AT1 receptor in the DNA synthesis of human iPS cells. Treatment with either l-phenylephrine (a selective α1-adrenoceptor agonist) or angiotensin II (Ang II) significantly increased DNA synthesis in human iPS cells. Enhanced DNA synthesis was significantly inhibited by pretreatment with protein kinase C (PKC) inhibitors, mitogen-activated protein kinase kinase (MEK) inhibitor, or phosphatidylinositol-3 phosphate kinase (PI3K) inhibitor. Treatment with either l-phenylephrine or Ang II significantly increased Akt and p44/42 MAPK phosphorylation. Short interfering RNA (siRNA) directed against Gq significantly inhibited DNA synthesis and phosphorylation of Akt and p44/42 MAPK enhanced by l-phenylephrine or Ang II. These results suggest that stimulation of α1-adrenoceptor or AT1 receptor may enhance DNA synthesis in human iPS cells via Gq-coupled receptor-dependent signaling pathways.
Keywords: α1-adrenoceptor; Angiotensin type 1 receptor; Mitogen-activated protein kinase; Akt; Gq-coupled receptor; Induced pluripotent stem cell;

Thapsigargin (TG), an inhibitor of Ca2+ ATPase pumps in the endoplasmic reticulum (ER), inhibits replication of human vascular smooth muscle cell (hVSMC) at low nM concentrations. TG blocks replication of other cell types through promotion of ER stress (ERS). In order to determine whether ERS may mediate the cytostatic effect of TG in hVSMCs, the effect of TG on ERS in hVSMCs was studied by assessing markers of ERS: Immunoglobulin Heavy Chain Binding Protein (BiP), growth inhibitory transcription factor, GADD153, phosphorlylated eukaryotic initiation factor 2α (p-eIF2α) and phosphorlylated protein kinase R (p-PKR). hVSMCs derived from saphenous veins were rendered quiescent with serum-free medium for 96 h incubated with 10 nM TG at 37° C for 24 h, then washed free of TG and incubated with 10% foetal calf serum (FCS) for a further 24 h. At selected times, BiP, GADD153, p-eIF2α, p-PKR and cyclin D1 expression was assessed. TG promoted a marked increase in BiP and GADD153, but suppressed cyclin D1 mRNA and protein expression. Under serum-free conditions p-eIF2α and p-PKR expression was not enhanced by TG. 15–24 h After removal of TG all these factors returned to levels seen in control cells. These data demonstrate that the inhibitory effect of 10 nM TG on hVSMC replication is mediated through induction of ERS and associated factors that cessate replication and is reversible. These observations have implications in the aetiology and treatment of diseases that include atherogenesis, vein graft failure and restenosis.
Keywords: Vascular; Proliferation; Thapsigargin; Endoplasmic reticular stress;

The procyanidin trimer C1 induces macrophage activation via NF-κB and MAPK pathways, leading to Th1 polarization in murine splenocytes by Nak-Yun Sung; Mi-So Yang; Du-Sup Song; Eui-Baek Byun; Jae-Kyung Kim; Jong-Heum Park; Beom-Seok Song; Ju-Woon Lee; Sang-Hyun Park; Hyun-Jin Park; Myung-Woo Byun; Eui-Hong Byun; Jae-Hun Kim (218-228).
Numerous studies have shown various relationships between foods with a high nutritional value and a robust immune response, particularly studies that have focused on host protection and cytokine networks. This study aimed to clarify the role played by the procyanidin trimer C1 in innate and adaptive immunity. Procyanidin C1 did not exert cytotoxicity at concentrations ranging from 7.8 to 62.5 μg/ml in macrophage cells; therefore, concentration of 62.5 μg/ml was used as the maximum dose of procyanidin C1 throughout subsequent experiments. Procyanidin C1 enhanced inducible nitric oxide synthase-mediated nitric oxide production in a concentration-dependent manner. In addition, procyanidin C1 functionally induced macrophage activation by augmenting the expression of cell surface molecules (CD80, CD86, and MHC II) and proinflammatory cytokine production (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) via activation of mitogen-activated protein kinase (MAPK), e.g., p38, ERK, and JNK and nuclear factor (NF)-κB signaling pathways. Interestingly, procyanidin C1 effectively polarized T helper type 1 (Th1) by secreting Th1-mediated cytokines (interferon-γ, IL-12p70, and IL-2) and inducing splenocyte proliferation, indicating that procyanidin C1 contributes to Th1 polarization of the immune response. Accordingly, these findings confirms that the procyanidin C1 induces macrophage activation via NF-κB and MAPK pathways, leading to Th1 polarization in murine splenocytes, which suggests that procyanidin C1 regulates innate and adaptive immunity by macrophage activation and Th1 polarization.
Keywords: Bone marrow-derived macrophage; Procyanidin C1; RAW 264.7 cells; Murine splenocyte; Th1 polarization;

Stress-induced increase in intracellular calcium (Ca2+) has been demonstrated to produce various deleterious effects in the body. The rise in intracellular Ca2+ (particularly neuronal) in response to stress has been mainly attributed to opening of voltage gated L-type Ca2+ channels. The role of P/Q-, N-, R- and T-type Ca2+channels, and plasma membrane localized exchangers such as Na+/Ca2+ exchanger and Ca2+ ATPase has also been implicated in increasing intracellular Ca2+ in response to stress. Stress-induced changes in Ca2+ currents has been mainly attributed to increased release of corticosterone (activation of glucocorticoid receptors in the hippocampus) and catecholamine release as a consequence of activation of Hypothalamic–Pituitary–Adrenal (HPA) axis and sympathetic neural system, respectively. Stress-induced increase intracellular Ca2+ may trigger various deleterious signaling pathway including free radical generation, apoptosis, increased synaptic release of glutamate and synthesis/release of cytotoxic cytokines that may be responsible for damaging effects associated with stress. The present review discusses the mechanisms involved in stress-induced rise in intracellular Ca2+ levels and subsequent implications of increased Ca2+ levels in stress.
Keywords: Stress; Calcium; L-type calcium channel; Na+/Ca2+ exchanger;

Several natural products and their derivatives, either in purified or structurally identified form, exhibit immense pharmacological and biological properties, some of them showing considerable anticancer potential. Although the molecular mechanisms of action of some of these products are yet to be elucidated, extensive research in this area continues to generate new data that are clinically exploitable. Recent advancement in molecular biology, high throughput screening, biomarker identifications, target selection and genomic approaches have enabled us to understand salient interactions of natural products and their derivatives with cancer cells vis-à-vis normal cells. In this review we highlight the recent approaches and application of innovative technologies made to improve quality as well as efficiency of structurally identified natural products and their derivatives, particularly in small molecular forms capable of being used in “targeted therapies” in oncology. These products preferentially involve multiple mechanistic pathways and overcome chemo-resistance in tumor types with cumulative action. We also mention briefly a few physico-chemical features that compare natural products with drugs in recent natural product discovery approaches. We further report here a few purified natural products as examples that provide molecular interventions in cancer therapeutics to give the reader a glimpse of the current trends of approach for discovering useful anticancer drugs.
Keywords: Natural products; High throughput screening; Structure activity relationship; Targeted cancer therapy; Chemo-resistance; Drug formulation;

Effects of cordycepin on Y-maze learning task in mice by Zhao-Lin Cai; Chun-Yang Wang; Zhong-Jiao Jiang; Hai-Hang Li; Wen-Xiao Liu; Liang-Wei Gong; Peng Xiao; Chu-Hua Li (249-253).
Cordycepin (3′-deoxyadenosine) is the major bioactive component of Cordyceps militaris that has been widely used in oriental countries as a Traditional Chinese Medicine and healthy food for preventing early aging, improving physical performance and increasing lifespan. Cordyceps militaris extracts other than cordycepin have been reported to improve cognitive function. Although cordycepin is one of the most utilized Cordyceps militaris components, it remains unknown whether cordycepin could improve learning and memory. Here we investigated effects of cordycepin on learning and memory in healthy and ischemic mice using Y-maze test. We found that oral cordycepin administration at dose of 10 mg/kg significantly improved Y-maze learning performance both in healthy and ischemic mice. However, cordycepin at dose of 5 mg/kg enhanced Y-maze learning only in ischemic mice but not healthy mice. In this study, simultaneously, we found that orally administrated cordycepin significantly decreased the neuronal loss induced by ischemia in hippocampal CA1 and CA3 regions. Collectively, our results can provide valuable evidence that cordycepin may act as a nootropic product or potential clinical application in improving cognitive function of patients with ischemic stroke in the future.
Keywords: Cordycepin; Y-maze test; Healthy; Ischemia; Neuronal loss; Hippocampus;

Phytoestrogenic molecule desmethylicaritin suppressed adipogenesis via Wnt/β-catenin signaling pathway by Xin-Luan Wang; Nan Wang; Li-Zhen Zheng; Xin-Hui Xie; Dong Yao; Ming-Yan Liu; Zhi-Hong Yao; Yi Dai; Ge Zhang; Xin-Sheng Yao; Ling Qin (254-260).
Epimedium flavonoids inhibit extravascular lipid deposition during prevention of steroid-associated osteonecrosis. Desmethylicaritin is a bioactive metabolite of Epimedium flavonoids in serum. As it is well known that estrogen inhibits aidpogenesis, so we hypothesized that desmethylicaritin as a phytoestrogen might have the potential to inhibit lipid deposition. This study was designed to investigate the effect of desmethylicaritin on adipogenesis and its underlying mechanism in vitro. Adipogenesis was assessed by Oil Red O staining in 3T3-L1 preadipocytes. Bromodeoxyuridine was used to test the clonal expansion. Further, the mRNA level and protein expression of adipgenic and related factors were detected by qRT-PCR and western blot, respectively. The nuclear location of β-catenin was identified using immunofluoresence assay. Our results showed that desmethylicaritin suppressed the adipogenesis in 3T3-L1 cells in a dose-dependent manner. In addition, desmethylicaritin inhibited clonal expansion during adipogenesis. Desmethylicaritin did not affect CCAAT/enhancer binding protein δ and β mRNA expression, but decreased the mRNA expression of CCAAT/enhancer binding protein α, peroxisome proliferator-activated receptor γ, adipocyte lipid-binding protein and lipoprotein lipase. Desmethylicaritin up-regulated the mRNA expression of Wnt10b that was however down-regulated after adipogenic induction. Desmethylicaritin increased the protein expression of β-catenin both in the cytoplasm and nuclei and immunofluorescence results confirmed that desmethylicaritin increased nuclear translocation of β-catenin. Above findings implied that desmethylicaritin was able to inhibit adipogenesis and Wnt/β-catenin signaling pathway was regulated by desmethylicaritin in the process of suppression of adipogenesis. Above findings supported desmethylicaritin as a novel phytochemical agent for potential prevention of disorders involving lipid metabolism.Display Omitted
Keywords: Desmethylicaritin; Adipogenesis; Wnt/β-catenin; PPARγ;

The multi-targeted therapy for liver cancer has been considered as a novel strategy to fight hepatocellular carcinoma. In this study, we first found that sprengerinin C, a naturally derived compound strongly suppressed tumor angiogenesis in human umbilical vein endothelial cells. A mechanism study revealed that sprengerinin C blocked vascular endothelial growth factor receptor 2-dependent phosphoinositide 3-kinase/Akt/mTOR/matrix metalloproteinase and p38 MAPK/matrix metalloproteinase pathways, two major pathways for tumor angiogenesis. Moreover, sprengerinin C inhibited vascular endothelial growth factor release, a vital event for early angiogenesis response, from hypoxic HepG-2/BEL7402 cells by suppressing hypoxia-inducible factor-1α transcriptional activity. Furthermore, sprengerinin C induced HepG-2/BEL7402 cell apoptosis by activating NADPH oxidase/reactive oxygen species-dependent caspase apoptosis pathway and suppressed HepG-2/BEL7402 cell growth through p53-mediated G2/M-phase arrest. Sprengerinin C also showed a significant anti-tumor effect in the nude mouse xenograft model of human hepatocellular carcinoma. These results provide new insights into development of potent candidate compounds for liver cancer through affecting multiple tumor progression steps of angiogenesis, apoptosis and proliferation.
Keywords: Sprengerinin C; Angiogenesis; Proliferation; Apoptosis; Hepatocellular carcinoma;

Quercetin antagonism of GABAAρ1 receptors is prevented by ascorbic acid through a redox-independent mechanism by Cecilia I. Calero; Andrea N. Beltrán González; Javier Gasulla; Silvia Alvarez; Pablo Evelson; Daniel J. Calvo (274-280).
Quercetin is a natural flavonoid widely distributed in plants that acts as a neuroprotective agent and modulates the activity of different synaptic receptors and ion channels, including the ionotropic GABA receptors. GABAAρ1 receptors were shown to be antagonized by quercetin, but the mechanisms underlying these antagonistic actions are still unknown. We have analyzed here if the antagonistic action produced by quercetin on GABAAρ1 receptors was related to its redox activity or due to alternative mechanism/s.Homomeric GABAAρ1 receptors were expressed in frog oocytes and GABA-evoked responses electrophysiologically recorded. Quercetin effects on GABAAρ1 receptors were examined in the absence or presence of ascorbic acid. Chemical protection of cysteines by selective sulfhydryl reagents and site directed mutagenesis experiments were also used to determine ρ1 subunit residues involved in quercetin actions.Quercetin antagonized GABAAρ1 receptor responses in a dose-dependent, fast and reversible manner. Quercetin inhibition was prevented in the presence of ascorbic acid, but not by thiol reagents that modify the extracellular Cys-loop of these receptors. H141, an aminoacidic residue located near to the ρ1 subunit GABA binding site, was involved in the allosteric modulation of GABAAρ1 receptors by several agents including ascorbic acid. Quercetin similarly antagonized GABA-evoked responses mediated by mutant H141DGABAAρ1 and wild-type receptors, but prevention exerted by ascorbic acid on quercetin effects was impaired in mutant receptors. Taken together the present results suggest that quercetin antagonistic actions on GABAAρ1 receptors are mediated through a redox-independent allosteric mechanism.
Keywords: GABA receptor; Flavonoid; Quercetin; Ascorbic acid; Allosteric modulator;

Involvement of riboflavin transporter RFVT2/Slc52a2 in hepatic homeostasis of riboflavin in mice by Yoshiaki Yao; Atsushi Yonezawa; Hiroki Yoshimatsu; Tomohiro Omura; Satohiro Masuda; Kazuo Matsubara (281-287).
Riboflavin (vitamin B2) acts as an intermediary during various biochemical oxidation–reduction reactions in the liver. Hepatic riboflavin homeostasis is suggested to be maintained through its transporter(s). Riboflavin transporters, RFVT2/Slc52a2 and RFVT3/Slc52a3, have been identified in rodents. However, the role of each RFVT in the hepatic homeostasis of riboflavin has not yet been fully clarified. In this study, we assessed the contribution of each RFVT to riboflavin uptake into the liver using in vitro and in vivo studies. The uptake of riboflavin by mouse primary hepatocytes increased in a time-dependent and a concentration-dependent manner. Riboflavin transport was independent of extracellular Na+. However, the uptake decreased slightly along with the extracellular pH increases. Real-time PCR analysis revealed that the mRNA level of Slc52a2, or coding for mouse (m)RFVT2, in the mouse liver was 10 times higher than that of Slc52a3 (coding for mRFVT3). The uptake of riboflavin at pH 7.4 by primary hepatocytes was significantly decreased by the transfection of Slc52a2-small interfering RNA (siRNA), but not Slc52a3-siRNA. Furthermore, we also confirmed the contribution of riboflavin transporters in vivo. The riboflavin concentrations in plasma, but not in the liver, were significantly decreased in mice fed on a riboflavin-deficient diet for 8 weeks. The expression of Slc52a2 mRNA was significantly upregulated by riboflavin deprivation. These results strongly suggest that mRFVT2 was involved in hepatic riboflavin homeostasis.
Keywords: Vitamin B2; Carrier-mediated transport; Hepatic homeostasis;

Piperine exerts anti-seizure effects via the TRPV1 receptor in mice by Chang-Yuan Chen; Wen Li; Kun-Peng Qu; Chang-Rui Chen (288-294).
The mechanisms involved in the anti-seizure property of piperine (1-[5-(1,3-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]-(E,E)-piperidine, C17H19NO3) are still unclear. Piperine could activate transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor, and the rapid activation of whole-cell currents is antagonized by the competitive TRPV1 antagonist capsazepine. Interestingly, recent studies have reported that TRPV1 may be a novel anti-epileptogenic target which led us to hypothesize that the anti-seizure property of piperine involves the TRPV1 receptor. To test this hypothesis, we examined the effect of piperine on seizures induced in mice and identified the receptors involved in the suppression of seizure caused by maximal electroshock (MES) and pentylenetetrazol (PTZ) models. Piperine, administered at doses of 40 and 80 mg/kg, significantly delayed the onset of myoclonic jerks and generalized clonic seizures, and decreased the seizure stage and mortality compared with the vehicle-treated animals. Piperine also significantly reduced the incidence of MES-induced tonic hindlimb extension (THE) and PTZ-induced Fos immunoreactivity in the dentate gyrus. The anti-seizure effects of piperine were blocked by a TRPV1-selective antagonist capsazepine. Taken together, these data support the further investigation of piperine as a TRPV1 agonist for anti-seizure therapy.
Keywords: Piperine; Seizure; TRPV1 receptor; Pentylenetetrazol; Capsazepine;

It is clear that activated β3-adrenoceptor can improve disorders of lipid metabolism, however there are few reports concerning the anti-atherosclerotic effects of β3-adrenoceptor in the artery of apolipoprotein E knockout (Apoe −/− ) mice. In the present study, we aimed at investigating the effects of β3-adrenoceptor on lipids, atherosclerosis plaques, scavenger receptor class B type 1 and its signal transduction in Apoe −/− mice. Ten C57BL/6J mice were used as a control, and fifty age-matched Apoe −/− mice were randomly divided into five groups: atherosclerotic model (saline), positive control (atorvastatin), low-dose β3-adrenoceptor agonist, high-dose β3-adrenoceptor agonist and β3-adrenoceptor antagonist groups. After 26 weeks on the high-fat diet, the mice received the above treatments for 12 weeks. Thoracic aortas, serum lipids, SR-B1, P-MeK1/2, P-ErK1/2 and protein kinase Cα(PKCα) activity were detected. We found that the levels of serum total cholesterol, triglyceride, very low-density lipoprotein/low-density lipoprotein cholesterol and the area of atherosclerotic plaques were significantly decreased in β3-adrenoceptor agonist-treated mice (P<0.01), while the levels of high-density lipoprotein cholesterol, thoracic aortic lumen area, activity of liver PKCα, the protein expression of SR-B1, P-MeK1/2 and P-ErK1/2 were significantly increased (P<0.01), compared with the atherosclerotic model mice. Effects of the high-dose agonist were superior to those of the low-dose (P<0.01). These findings suggest that activation of β3-adrenoceptor reduce the plaque area in the thoracic aorta and play an important anti-atherosclerotic role by regulating lipid metabolism disorders and the SR-B1 signal transduction pathway.
Keywords: β3 adrenoceptors; Lipids; SR-B1; Signal transduction;

Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke by Teng Guan; Qian Liu; Yisong Qian; Haopeng Yang; Jiming Kong; Junping Kou; Boyang Yu (303-311).
Transient cerebral ischemia initiates a complex series of inflammatory events, which has been associated with an increase in behavioral deficits and secondary brain damage. Ruscogenin is a major steroid sapogenin in the traditional Chinese herb Ophiopogon japonicus that have multiple bioactivities. Recent studies have demonstrated that Ruscogenin is involved in down-regulation of intercellular adhesion molecule-1 (ICAM-1) and nuclear factor-κB (NF-κB) activation in anti-inflammatory pathways. We hypothesized that Ruscogenin protects against brain ischemia by inhibiting NF-κB-mediated inflammatory pathway. To test this hypothesis, adult male mice (C57BL/6 strain) were pretreated with Ruscogenin and then subjected to transient middle cerebral artery occlusion (MCAO)/reperfusion. After 1 h MCAO and 24 h reperfusion, neurological deficit, infarct sizes, and brain water content were measured. Ruscogenin markedly decreased the infarct size, improved neurological deficits and reduced brain water content after MCAO. The activation of NF-κB Signaling pathway was observed after 1 h of ischemia and 1 h of reperfusion, and Ruscogenin significantly inhibited NF-κB p65 expression, phosphorylation and translocation from cytosol to nucleus at this time point in a dose-dependent manner. NF-κB DNA binding activity, and the expression of NF-κB target genes, including ICAM-1, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), were also suppressed by Ruscogenin pretreatment after 1 h MCAO and 24 h reperfusion. The results indicated that Ruscogenin protected the brain against ischemic damage caused by MCAO, and this effect may be through downregulation of NF-κB-mediated inflammatory responses.
Keywords: Ruscogenin; Stroke; Middle cerebral artery occlusion; NF-κB; Inflammation;

Effect of fatty acids on the phosphate binding of TRK-390, a novel, highly selective phosphate-binding polymer by Junko Nakaki; Shinichi Yamaguchi; Yuichi Torii; Atsushi Inoue; Satoshi Minakami; Takami Kanno; Masanori Murakami; Masahiro Tsuzuki; Hidenori Mochizuki; Kazuharu Suyama; Mitsuko Miyamoto (312-317).
Phosphate binders are used for the treatment of hyperphosphatemia in hemodialysis patients with chronic kidney disease. Sevelamer, a phosphate-binding polymer, has been reported to bind bile acids or fatty acids and thereby decrease its phosphate-binding capacity. The novel phosphate binder TRK-390 is a poly (allylamine) polymer that has been shown to have enhanced phosphate selectivity, with low bile-acid-binding. In this study we evaluated the effect of fatty acids on the phosphate-binding capacity of TRK-390. In the absence of fatty acids and bile acids, the phosphate-binding capacity of TRK-390 was similar to that of sevelamer. In the presence of fatty acids and bile acids, the phosphate-binding capacity of TRK-390 was reduced to 83%; in contrast, that of sevelamer was reduced to 35%. TRK-390 and sevelamer showed a similar effect in lowering urinary phosphate excretion in normal rats fed a normal diet. However, urinary phosphate excretion of rats treated with TRK-390 was reduced by about one half of that obtained with sevelamer, when given with a high-fat diet that had a fat content similar to the diet of hemodialysis patients. TRK-390 was superior in terms of phosphate selectivity in the presence of fatty acids and bile acids in vitro, and the phosphate-binding capacity of TRK-390 in vivo was shown to be less affected by fat in comparison with that of sevelamer. Thus, TRK-390 is expected to be useful as a novel highly selective phosphate binder.
Keywords: Hyperphosphatemia; Phosphate binder; Phosphate absorption; Poly (allylamine) polymer;

Ozone oxidative postconditioning ameliorates joint damage and decreases pro-inflammatory cytokine levels and oxidative stress in PG/PS-induced arthritis in rats by Jaqueline Dranguet Vaillant; Angela Fraga; María Teresa Díaz; A. Mallok; Renate Viebahn-Hänsler; Ziad Fahmy; Ariana Barberá; Liván Delgado; Silvia Menéndez; Olga Sonia León Fernández (318-324).
Rheumatoid Arthritis (RA) is the most prevalent chronic condition present in ~1% of the adult population. Many pro-inflammatory mediators are increased in RA, including Reactive Oxygen Species such as nitric oxide NO, pro-inflammatory cytokines as tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β) and other molecules. Ozone oxidative postconditioning has regulatory effects on some pathological targets associated with RA. Thus, the aim of this study was to investigate the efficacy of ozone therapy in PG/PS-induced arthritis in rats in point of joints inflammation and morphology. Moreover, cytokines, nitric oxide and oxidative stress levels in spleen homogenates were evaluated. Ozone treatment ameliorated joint damage, reduced TNF-α concentrations as well as TNF-α and IL-1β mRNA levels. Besides, cellular redox balance, nitric oxide and fructolysine levels were reestablished after ozone oxidative postconditioning. It was concluded that pleiotropic ozone′s effects clarify its therapeutic efficacy in RA. Decreasing inflammation and joint injury, reduction of pro-inflammatory cytokines, TNF-α and IL-1β transcripts and re-establishment of cellular redox balance after ozone treatment were demonstrated.
Keywords: Oxidative stress; Ozone oxidative postconditioning; Arthritis; Inflammation;

Beraprost sodium improves survival rates in anti-glomerular basement membrane glomerulonephritis and 5/6 nephrectomized chronic kidney disease rats by Shinichi Yamaguchi; Chifumi Inada; Mitsutaka Tamura; Nahoko Sato; Masateru Yamada; Shoichi Itaba; Seiji Okazaki; Hirotoshi Matsuura; Shigeo Fujii; Fuko Matsuda; Yasufumi Goto; Hidenori Mochizuki; Hajimu Kurumatani; Mitsuko Miyamoto (325-331).
Beraprost sodium, a stable prostacyclin analog, was showed to improve survival rates in two different rat models, anti-glomerular basement membrane (GBM) glomerulonephritis (GN) and 5/6 nephrectomized (Nx) chronic kidney disease (CKD) rats. In the anti-GBM rat, beraprost sodium (0.2 and 0.6 mg/kg/day) improved survival rate (hazard ratio for beraprost sodium 0.6 mg/kg/day group, 0.10; 95% confidence interval, 0.01 to 0.68). Subsequently, in the 5/6 Nx CKD rat, beraprost sodium (0.6 mg/kg/day) improved survival rate (hazard ratio for beraprost sodium, 0.46; 95% confidence interval, 0.23 to 0.92), serum creatinine doubling time and the slope of the reciprocal of serum creatinine. In the anti-GBM GN rats, beraprost sodium suppressed the serum accumulation of representative uremic toxins such as indoxyl sulfate. Furthermore, beraprost sodium inhibited human aortic endothelial cell (HAEC) injury induced by indoxyl sulfate, indicating that beraprost sodium might have a protective effect against cardiovascular damage due to CKD. These results show that beraprost sodium can improve the survival rates in two rat models of anti-GBM GN and 5/6 Nx CKD rats by protecting endothelial cells and thereby ameliorating decreased renal function. Therefore, clinical studies are needed in patients with chronic kidney failure to determine whether beraprost sodium will become a useful medication in CKD.
Keywords: Chronic kidney disease; Anti-glomerular basement membrane glomerulonephritis; 5/6 nephrectomized; Survival rate; Uremic toxin; Indoxyl sulfate; Endothelial cell protection; Beraprost sodium;

Mechanisms involved in abdominal nociception induced by either TRPV1 or TRPA1 stimulation of rat peritoneum by Gabriela Trevisan; Mateus F. Rossato; Carin Hoffmeister; Sara M. Oliveira; Cássia R. Silva; Filipe C. Matheus; Gláucia C. Mello; Edson Antunes; Rui D.S. Prediger; Juliano Ferreira (332-344).
Abdominal pain is a frequent symptom of peritoneal cavity irritation, but little is known about the role of the receptors for irritant substances, transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), in this painful condition. Thus, we investigated the abdominal nociception caused by peritoneal stimulation with TRPV1 (capsaicin) and TRPA1 (allyl isothiocyanate, AITC) agonists and their mechanisms in rats. The intraperitoneal (i.p.) injection of either capsaicin or AITC (0.03–10 mg/kg) induced short-term (up to 20 min) and dose-dependent abdominal nociception, and also produced c-fos expression in spinal afferents of the dorsal horn. TRPV1 antagonism prevented (94±4% inhibition) nociception induced by capsaicin but not by AITC. In contrast, the TRPA1 antagonism almost abolished AITC-induced nociception (95±2% inhibition) without altering the capsaicin response. Moreover, nociception induced by either capsaicin or AITC was reduced by the desensitisation of TRPV1-positive sensory fibres with resiniferatoxin (73±18 and 76±15% inhibitions, respectively) and by the NK1 receptor antagonist aprepitant (56±5 and 53±8% inhibitions, respectively). Likewise, the i.p. injections of capsaicin or AITC increased the content of substance P in the peritoneal fluid. Nevertheless, neither the mast cell membrane stabiliser cromoglycate, nor the H1 antagonist promethazine, nor depletion of peritoneal macrophages affected abdominal nociception induced either by capsaicin or AITC. Accordingly, neither capsaicin nor AITC increased the histamine content in the peritoneal fluid or provoked peritoneal mast cell degranulation in vitro. Collectively, our findings suggest that TRPV1 and TRPA1 stimulation in the peritoneum produces abdominal nociception that is mediated by sensory fibres activation.
Keywords: Capsaicin; Allyl isothiocyanate; Substance p; Mast cells; Resiniferatoxin; Pain;

Corrigendum to “Neuroprotective effects of salvianolic acid B on an Aβ25–35 peptide-induced mouse model of Alzheimer׳s disease” [Eur. J. Pharmacol. 704 (1–3) (2013) 70–77] by Young Woo Lee; Dong Hyun Kim; Su Jin Jeon; Se Jin Park; Jong Min Kim; Jun Man Jung; Hyung Eun Lee; Shin Gil Bae; Hee Kyong Oh; Kun Ho Son; Jong Hoon Ryu (345).

Dexketoprofen and tramadol, alone or in combination, were evaluated after oral or intra-articular administration on knee osteoarthritis nociception induced by intra-articular (i.ar.) monosodium iodoacetate (MIA, 1 mg/25 µl) in the rat right knee while the left knee received saline (25 µl). Seven days after MIA treatment, dexketoprofen, tramadol, their combination or the vehicle were administered. Nociception was evaluated as alteration in hind limb weight distribution with Incapacitance tester at different time-points after drug administration. Oral dexketoprofen (0.1–1 mg/kg) or tramadol (0.5–5 mg/kg) induced maximal antinociception at 1 and 5 mg/kg, respectively. Their combination dose-dependently increased the intensity and duration of antinociception, that was additive and lasted up to 3 days. Also the intra-articular administration of dexketoprofen or tramadol (10–100 µg/25 µl) inhibited MIA-induced nociception, and the combination of the lower doses (10 µg/25 µl) produced a long lasting more than additive antinociceptive effect indicating a synergistic interaction between the two drugs. This effect was significantly reduced by naloxone (10 μg/25 μl, i.ar.) co-administered with both compounds. The intra-articular administration of both drugs at 10 µg/25 µl in the contralateral control knee joint provoked a marked synergistic antinociceptive effect indicating significant systemic diffusion through synovial membrane. The oral or intra-articular combination of dexketoprofen and tramadol produced additive or synergistic antinociceptive effects, respectively, in the model of MIA-induced osteoarthritis in rats, that might allow to obtain therapeutic advantages with lower side effects.
Keywords: Osteoarthritis; Monosodium iodoacetate; Nociception; Incapacitation; Drug interaction; Antinociceptive effect;

There are several studies carried out to test the effect of cholestasis on memory impairment and anxiolytic-like behaviors. Some previous studies have shown that cholestasis alters the activity of opioidergic and dopaminergic systems. The aim of the present study is however to investigate the role of mu opioid, D1 and D2 dopamine ventral hippocampal (CA3) receptors upon cholestasis-induced anxiolytic-like behaviors in hole-board task. Male mice weighing 25–30 g were used. Cholestasis was induced by ligation of the main bile duct. Our data indicated that cholestasis can induce anxiolytic-like response. Furthermore, the results showed that the intra-CA3 injection of naloxone, a mu receptor antagonist at 0.25 and 0.5 µg/mouse, SCH23390, a D1 dopamine receptor antagonist or sulpiride, as a D2 dopamine receptor antagonist, 5 min before testing, reversed the cholestasis-induced anxiolytic-like behaviors seven days after bile duct ligation (BDL). Unlike the higher dose of SCH23390 (0.5 µg/mouse) which induced anxiogenic-like behaviors, other doses of the above drugs did not alter the exploratory behaviors in examined mice. Based on our findings, co-administration of the subthreshold dose of naloxone (0.125 µg/mouse), SCH23390 or sulpiride, and SCH23390 with sulpiride, neither altered exploratory behaviors in animals nor reversed the cholestasis-induced anxiolytic-like behaviors, seven days post BDL. Current results demonstrated firstly, the anxiolytic-like behaviors are evident in cholestatic mice seven days post BDL; secondly, there are plausible mechanisms governing the involvement of the CA3 opioidergic and dopaminergic systems in this phenomenon and thirdly, there seem to be no interaction between these systems.
Keywords: Cholestasis; Naloxone; SCH23390; Sulpiride; Anxiety; Hole-board task; Mice;

Electrophysiological evidence for rapid 5-HT1A autoreceptor inhibition by vilazodone, a 5-HT1A receptor partial agonist and 5-HT reuptake inhibitor by Charles R. Ashby; John H. Kehne; Gerd D. Bartoszyk; Matthew J. Renda; Maria Athanasiou; Kerri A. Pierz; Christoph A. Seyfried (359-365).
This study examined the effect of vilazodone, a combined serotonin (5-HT) reuptake inhibitor and 5-HT1A receptor partial agonist, paroxetine and fluoxetine on the sensitivity of 5-HT1A autoreceptors of serotonergic dorsal raphe nucleus neurons in rats. These effects were assessed by determining the intravenous dose of (±)-8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) required to suppress the basal firing rate of these neurons by 50% (ID50) in anesthetized rats using in vivo electrophysiology. 5-HT uptake inhibition was determined by the ability of the compounds to reverse (±)-p-chloroamphetamine (PCA)-induced rat hypothalamic 5-HT depletion ex vivo. Acute vilazodone administration (0.63 and 2.1 µmol/kg, s.c.), compared with vehicle, significantly increased (2–3-fold) the ID50 of 8-OH-DPAT at 4 h, but not 24 h after administration. Subchronic administration (3 days) significantly increased the ID50 value at 4 h (3–4-fold) and at 24 h (~2-fold). In contrast, paroxetine and fluoxetine at doses that were supramaximal for 5-HT uptake inhibition did not significantly alter the ID50 value of 8-OH-DPAT after acute or subchronic administration. Vilazodone antagonized the action of PCA 3.5 h and 5 h after a single dose (ID50 1.49 and 0.46 µmol/kg, s.c., respectively), but was inactive 18 h post-administration, corroborating the electrophysiological results at 24 h following acute administration. The results are consistent with the concept of rapid and, following repeated treatment, prolonged inhibition of 5-HT1A autoreceptors by vilazodone. This effect could occur by either direct interaction with, or desensitization of, these receptors, an effect which cannot be ascribed to vilazodone's 5-HT reuptake inhibiting properties.
Keywords: Vilazodone; 5-HT reuptake inhibitor; 5-HT1A receptor partial agonist; In vivo electrophysiology; PCA model; Antidepressant;

The use of carbetapentane for spinal anesthesia and use-dependent block of sodium currents by Yuk-Man Leung; Jann-Inn Tzeng; Chang-Shin Kuo; Yu-Wen Chen; Chin-Chen Chu; Jhi-Joung Wang (366-372).
Although carbetapentane produces skin (peripheral) infiltrative analgesia, the underlying mechanism of carbetapentane in local anesthesia is not well understood. The purpose of the study was to examine the effect of carbetapentane on voltage-gated Na+ channels and its efficacy on spinal (central) anesthesia. We evaluated the effects of carbetapentane on rat motor and pain behavior (when administered intrathecally) and on voltage-gated sodium channels in differentiated neuronal NG108-15 cells. Carbetapentane exhibited dose-dependent spinal blockade with a more sensory-selective action over motor blockade (P<0.05). Carbetapentane was more potent than lidocaine (P<0.05) in spinal anesthesia. Intrathecal 5% dextrose (vehicle) elicited no spinal anesthesia. Lidocaine, used as a positive control, demonstrated concentration- and state-dependent effects on tonic block of voltage-gated Na+ currents (IC50 of 49.6 and 194.6 µM at holding potentials of −70 and −100 mV, respectively). Carbetapentane was more potent (IC50 of 36.3 and 62.2 µM at holding potentials of −70 and −100 mV, respectively). Carbetapentane showed a much stronger frequency-dependence of block than lidocaine: with high frequency stimulation (3.33 Hz), 50 µM lidocaine produced an additional 30% blockade, while the same concentration of carbetapentane produced 70% more block. These results revealed carbetapentane had a more potent and prolonged spinal blockade with a more sensory/nociceptive-selective action over motor blockade in comparison with lidocaine. Spinal anesthesia with carbetapentane could be through inhibition of voltage-gated Na+ currents.
Keywords: Intrathecal; Carbetapentane; Lidocaine; Sodium currents; Spinal anesthesia;

Tramadol, an analgesic used alone or combined with acetaminophen, has a complex mechanism of action involving opioid and amine mechanisms. In this study, we explored the involvement of spinal and peripheral adenosine A1 receptors in antinociception by tramadol, and determined whether spinal serotonin 5-HT7 receptors were linked to spinal A1 receptor actions. Antinociception was examined using the 2% formalin test in mice. Tramadol was administered systemically (intraperitoneal) or peripherally (intraplantar). Caffeine (non-selective A1/A2A receptor antagonist) and SCH58261 (selective A2A receptor antagonist) were given systemically, while DPCPX (selective A1 receptor antagonist) was given systemically, spinally (lumbar puncture), or peripherally. Systemic tramadol 35 mg/kg produced antinociception against phase 2 formalin-evoked flinching behaviors, particularly in the earlier parts (phase 2A). Systemic caffeine (10 mg/kg) and DPCPX (1 mg/kg), but not SCH58261 (3 mg/kg), inhibited antinociception by systemic tramadol. Spinal DPCPX 3 μg also inhibited the action of systemic tramadol. Spinal SB269970 (selective 5-HT7 receptor antagonist) 3–10 μg did not alter the effect of systemic tramadol. Intraplantar tramadol produced antinociception against flinching behaviors, and this action was reversed by intraplantar DPCPX 4.5 μg administered on the ipsilateral, but not contralateral, side. Intraplantar DPCPX also reversed antinociception by systemic tramadol. These results indicate that adenosine A1 receptors contribute to antinociception by tramadol in the mouse formalin model, and that spinal and peripheral sites are involved in these actions. 5HT7 receptors in the spinal cord do not appear to be involved in the recruitment of A1 receptor mechanisms when tramadol is given systemically in this model.
Keywords: Tramadol; Adenosine; Formalin;

RETRACTED: PBOX-15 induces apoptosis and improves the efficacy of oxaliplatin in human colorectal cancer cell lines by Giuseppina Gangemi; Patrizia Gazzerro; Donatella Fiore; Maria Chiara Proto; Stefania Butini; Sandra Gemma; Alice Casagni; Chiara Laezza; Mario Vitale; Alessia Ligresti; Vincenzo Di Marzo; Daniela M. Zisterer; Seema Nathwani; D. Clive Williams; Giuseppe Campiani; Maurizio Bifulco (379-387).
This article has been retracted at the request of the authors.Upon close inspection, the co-authors realized that the first author (Giuseppina Gangemi) substantially manipulated some of the western blot figures reported in the paper. None of the other authors are responsible for this occurrence. Even though the main conclusions are not affected by such manipulations, they wish to retract this publication, and apologize for the confusion and inconvenience caused

Enhancement of PLGF production by 15-(S)-HETE via PI3K-Akt, NF-κB and COX-2 pathways in rheumatoid arthritis synovial fibroblast by Ming-Yueh Wu; Rong-Sen Yang; Tzu-Hung Lin; Chih-Hsin Tang; Yung-Cheng Chiu; Houng-Chi Liou; Wen-Mei Fu (388-396).
Metabolites from arachidonic acids play the pivotal roles in inflammatory arthritis. Arachidonic acid could be metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) to produce the bioactive eicosanoids. Although the down-stream products of COX including prostaglandin E2 are well-known inflammatory stimulators, the role of LOX products in inflammatory arthritis is still unclear. Here we found that the downstream product of 15-LOX, 15-S-hydroxyeicosatetraenoic acid (15-(S)–HETE), can enhance the expression of placenta growth factor (PLGF), which is recently considered to play an important role in rheumatoid arthritis. 15-(S)-HETE increased the expression of PLGF in human rheumatoid arthritis synovial fibroblasts in a time-dependent and concentration-dependent manner. PI3K-Akt, NF-κB signaling pathways were involved in the potentiation effects of 15-(S)–HETE. In addition, COX-2 was up-regulated by the treatment of 15-(S)-HETE and the increase of COX-2 expression participated in 15-(S)-HETE-induced PLGF expression, which was confirmed by COX-2 shRNA or pharmacological COX-2 inhibitor. Moreover, it was found that treatment of prostaglandin E2 (PGE2), which was the main down-stream metabolite of COX-2, increased the expression of PLGF. EP1, EP2, EP3 and EP4 agonists could up-regulate PLGF as well. In animal studies, we found that the adjuvant-induced expression of PLGF and COX-2 was inhibited in 15-LOX knockout mice. These results indicated that PLGF up-regulation by 15-LOX downstream product may be involved in inflammatory arthritis.
Keywords: COX-2; 15-lipoxygenase; Inflammation; PLGF; Synovial fibroblasts;

Sex differences in ischemia/reperfusion-induced acute kidney injury are dependent on the renal sympathetic nervous system by Ryosuke Tanaka; Hidenobu Tsutsui; Mamoru Ohkita; Masanori Takaoka; Tokihito Yukimura; Yasuo Matsumura (397-404).
Resistance to ischemic acute kidney injury has been shown to be higher in female rats than in male rats. We found that renal venous norepinephrine overflow after reperfusion played important roles in the development of ischemic acute kidney injury. In the present study, we investigated whether sex differences in the pathogenesis of ischemic acute kidney injury were derived from the renal sympathetic nervous system using male and female Sprague-Dawley rats. Ischemia/reperfusion-induced acute kidney injury was achieved by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function was impaired after reperfusion in both male and female rats; however, renal dysfunction and histological damage were more severe in male rats than in female rats. Renal venous plasma norepinephrine levels after reperfusion were markedly elevated in male rats, but were not in female rats. These sex differences were eliminated by ovariectomy or treatment with tamoxifen, an estrogen receptor antagonist, in female rats. Furthermore, an intravenous injection of hexamethonium (25 mg/kg), a ganglionic blocker, 5 min before ischemia suppressed the elevation in renal venous plasma norepinephrine levels after reperfusion, and attenuated renal dysfunction and histological damage in male rats, and ovariectomized and tamoxifen-treated female rats, but not in intact females. Thus, the present findings confirmed sex differences in the pathogenesis of ischemic acute kidney injury, and showed that the attenuation of ischemia/reperfusion-induced acute kidney injury observed in intact female rats may be dependent on depressing the renal sympathetic nervous system with endogenous estrogen.
Keywords: Sex difference; Ischemia/reperfusion; acute kidney injury; Norepinephrine overflow;

Functional characterization of acetylcholine receptors and calcium signaling in rat testicular capsule contraction by Edilson Dantas da Silva Júnior; Bruno Palmieri de Souza; Juliano Quintela Dantas Rodrigues; Afonso Caricati-Neto; Aron Jurkiewicz; Neide Hyppolito Jurkiewicz (405-413).
The motor activity of mammalian testicular capsule (TC) contributes to male fertility and infertility, but the acetylcholine receptors related to the contractions induced by cholinergic drugs are poorly known. Indeed to characterize the acetylcholine receptors and cellular signaling by Ca2+ involved in TC motor activity of rats, the potency of agonists (pD2) and antagonists (pA2) of acetylcholine receptors, and effects of Ca2+ cellular transport blockers on the cholinergic contractions were evaluated. pD2 values of acetylcholine (5.98) were ten-fold higher than that of carbachol (4.99). Efficacy (E max) of acetylcholine and carbachol to induce contractions corresponded to 95% and 97% of E max for KCl, but E max for nicotine was very low (8% of E max for KCl). Further, physostigmine did not affect the acetylcholine potency. Contractions induced by acetylcholine or carbachol were antagonized by muscarinic but not nicotinic antagonist. The order of pA2 values obtained for muscarinic antagonists, namely atropine>4-DAMP>AF-DX116>pirenzepine, corresponded to a typical profile of M3 receptors. Contractions induced by acetylcholine or carbachol were inhibited by blockers of Ca2+ influx through voltage-dependent calcium channels (nifedipine and Ni2+), Ca2+ reuptake by sarco-endoplasmic reticulum (cyclopiazonic acid) and mitochondria (FCCP). The protein kinase C (PKC) inhibitor chelerythrine only affected the acetylcholine-induced contraction. These results suggest that TC motor activity of rats are mediated mainly by M3 receptors followed by the increase of cytosolic Ca2+ concentration regulated by voltage-dependent calcium channels, sarco-endoplasmic reticulum and mitochondria. Furthermore, the differential effects of chelerythrine in the acetylcholine or carbachol-induced contractions are discussed.
Keywords: Cholinergic agonists; Muscarinic acetylcholine receptors; Calcium; Rat testicular capsule.;

Pregnancy is a physiological state that involves an increase in uterine blood flow, which is mediated in part by nitric oxide (NO) liberated from the endothelium and nitrergic neurons. The main focus of this review article is to provide information about how endogenous NO regulates uterine and placental blood flow and vascular tone in experimental animals and humans in vivo or in vitro in non-pregnant and pregnant states as well as pregnancy with pre-eclampsia. Uterine arteries from non-pregnant women respond to NO liberated from the endothelium and nitrergic nerves with relaxations, and the release of endothelial NO is influenced by the phase of the estrous cycle, with its enhanced release at the follicular phase when the estrogen level is high. NO bioavailability in the uteroplacental circulatory system is gradually increased during pregnancy. Pre-eclamptic pregnancies with or without intrauterine growth restriction show impaired uteroplacental blood flow accompanied by reduced NO synthesis due to down-regulation of eNOS as well as asymmetric dimethylarginine accumulation and by augmented NO degradation by oxidative stress. Further studies are expected to provide new mechanistic insights into the fascinating process of maternal uterine adaptation in humans and novel prophylactic and therapeutic measures against pre-eclampsia.
Keywords: Uterine artery; Uteroplacental blood flow; Endothelium; Nitrergic nerve; Pregnancy; Pre-eclampsia;

Agmatine attenuates neuropathic pain in sciatic nerve ligated rats: Modulation by hippocampal sigma receptors by Nandkishor Ramdas Kotagale; Saurabh Haridas Shirbhate; Pradeep Shukla; Rajesh Ramesh Ugale (424-431).
Present study investigated the influence of the sigma (σ 1 and σ 2) receptors within hippocampus on the agmatine induced antinociception in neuropathic rats. Animals were subjected to sciatic nerve ligation for induction of neuropathic pain and observed the paw withdrawal latency in response to thermal hyperalgesia, cold allodynia and the mechanical hyperalgesia. Intrahippocampal (i.h.) as well as intraperitoneal (i.p.) administration of agmatine attenuated neuropathic pain in sciatic nerve ligated rats. Intrahippocampal administration of σ 1 agonist (+)-pentazocine or σ 2 agonist PB28 sensitized whereas, σ 1 antagonist BD1063 or σ 2 antagonist SM21 potentiated antinociceptive effect of agmatine. The behavioral effects correlated with hippocampal tumor necrosis factor-α (TNF-α) levels observed by western blot analysis. These results suggest that both the σ 1 and σ 2 receptor subunits within hippocampus play an important role in antinociceptive action of agmatine against neuropathic pain.
Keywords: Agmatine; Allodynia; Hyperalgesia; Neuropathic pain; Sigma Receptor; Hippocampus; TNF-α;

Pentoxifylline (PTX) is a methylxanthine derivative currently being used in the treatment of peripheral vascular diseases. Recently, we had evaluated its action in human MDA-MB-231 breast cancer cells. PTX exhibited anti-metastatic activity by affecting key processes such as proliferation, adhesion, migration, invasion and apoptosis. In light of the preliminary findings, the present work accounts for the possible mechanistic insights of the pathways affected by PTX. Aberrant Focal Adhesion Kinase (FAK) signaling forms a key determinant in breast cancer and in view of this fact we had investigated downstream processes regulated by FAK. PTX at sub-toxic doses lowers the level of activated FAK, Extracellular Regulated Kinase or Mitogen Activated Protein Kinase (ERK/MAPK), Protein Kinase B (PKB/Akt) affecting cellular proliferation and survival. It blocks G1/S phase of cell cycle by inhibiting the expression of Cyclin D1/Cdk6. Further, it modulates the activities of RhoGTPases and alters actin organization resulting in decreased motility. PTX also delays tumor growth and inhibited blood vessel formation in vivo. In purview of these findings, PTX surely qualifies as a suitable prospect in the intervention of breast cancer.
Keywords: Pentoxifylline; MDA-MB-231 Breast cancer cells; Focal Adhesion Kinase; RhoGTPases; Proliferation; Apoptosis;

Sexual dysfunction is a common and underestimated effect of antidepressants. However, the mechanism by which these drugs cause erectile dysfunction is unclear. We investigated the reactivity of the corpus cavernosum of rabbits that were treated with either chronic imipramine, which is a tricyclic agent; agomelatine, which is a melatonergic agonist and serotonin 5HT2c antagonist; or moclobemide, which is a reversible inhibitor of monoamine-oxidase A. Twenty rabbits were randomly divided into four groups: the control group (n=5), the imipramine-treated group (n=5), which received i.p. injections of 10 mg/kg/day of imipramine, the moclobemide-treated group (n=5), which received i.p. injections of 20 mg/kg/day of moclobemide, and the agomelatine-treated group (n=5), which was orally administered 10 mg/kg/day of agomelatine. The reactivities of corpus cavernosum tissue obtained from the antidepressant-treated and the control groups were studied in organ chambers after the animals were subjected to 21 days of drug administration. The acetylcholine-induced endothelium-dependent and the electrical field stimulation (EFS)-induced neurogenic relaxation of the corpus cavernosum of the imipramine-treated group was significantly decreased compared with the control group. However, neither the acetylcholine- nor EFS-induced relaxation was changed in the moclobemide- or agomelatine-treated groups. There were no change in the relaxant response to the nitric oxide (NO) donor sodium nitroprusside and contractile response to KCl between the groups. This study suggests that chronic imipramine treatment but not agomelatine and moclobemide treatments causes significant functional changes in the penile erectile tissue of rabbits and that these changes may contribute to the development of impotence.
Keywords: Penis; Corpus cavernosum; Imipramine; Agomelatine; Moclobemide; Erectile dysfunction;

Increasing evidence has established causative links between obesity, chronic inflammation and insulin resistance; the core pathophysiological feature in type 2 diabetes mellitus. This study was designed to examine whether the combination of l-cysteine and metformin would provide additional benefits in reducing oxidative stress, inflammation and insulin resistance in streptozotocin-induced type 2 diabetes in rats. Male Wistar rats were fed a high-fat diet (HFD) for 8 weeks to induce insulin resistance after which they were rendered diabetic with low-dose streptozotocin. Diabetic rats were treated with metformin (300 mg/kg/day), l-cysteine (300 mg/kg/day) and their combination along with HFD for another 2 weeks. Control rats were fed normal rat chow throughout the experiment. At the end of treatment, fasting blood glucose, fasting serum insulin, homeostasis model assessment-insulin resistance index (HOMA-IR) and serum free fatty acids (FFAs) were measured. Serum levels of the inflammatory markers; monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP) and nitrite/nitrate were also determined. The liver was isolated and used for determination of malondialdehyde (MDA), reduced glutathione (GSH), caspase-3 and cytochrome c levels. The hypoglycemic effect of the combination therapy exceeded that of metformin and l-cysteine monotherapies with more improvement in insulin resistance. All treated groups exhibited significant reductions in serum FFAs, oxidative stress and inflammatory parameters, caspase-3 and cytochrome c levels compared to untreated diabetic rats with the highest improvement observed in the combination group. In conclusion, the present results clearly suggest that l-cysteine can be strongly considered as an adjunct to metformin in management of type 2 diabetes.
Keywords: l-Cysteine; Metformin; Streptozotocin-induced type 2 diabetes; Insulin resistance;

Outcome improvement of cellular cardiomyoplasty using triple therapy: Mesenchymal stem cell+erythropoietin+vascular endothelial growth factor by Fatemeh Tavakoli; Seyed Nasser Ostad; Vahid Khori; Ali Mohammad Alizadeh; Anita Sadeghpour; Amir Darbandi Azar; Majid Haghjoo; Asghar Zare; Mohsen Nayebpour (456-463).
To improve cellular cardiomyoplasty efficacy after myocardial infarction (MI), we postulated that combining mesenchymal stem cells (MSCs) transplantation with anti-apoptotic and angiogenic effects of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) may provide better prognosis in an infarcted heart 48 rats, underwent left anterior descending artery ligation, were divided into eight groups and treated as follows: Group 1: MSC+EPO+VEGF, Group 2: MSC+EPO, Group 3: MSC+VEGF, Group 4: MSC, Group 5: EPO+VEGF, Group 6: EPO, Group 7: VEGF and Group 8: Control. After MI induction, EPO and VEGF were injected subcutaneously at the dose of 3000 U/kg and 3 µg/kg respectively. MSCs were transplanted one week after MI. In the fourteenth and sixteenth days after infarction, EPO was injected again. Echocardiography demonstrated that all treatments improved left ventricular function significantly (before vs. after treatment) but in control group ejection fraction deteriorated over the 2-months period. Percent of ejection fraction recovery in all treatment groups were significantly greater than control (P<0.05). Compared with the control group, all treatments attenuated cell death in peri-infarct areas significantly, except groups 6 and 7. Vascular density of all treatment groups were more than control group but this superiority was statistically significant only in group1 (P<0.01). All of our treatments had beneficial effects to some extent but MSC transplantation combined with EPO and VEGF administration resulted in superior therapeutic outcome in enhancing cell survival and neovascularization.
Keywords: Heart failure; Myocardial infarction; Mesenchymal stem cell; Erythropoietin; Vascular endothelial growth factor;

Antinociceptive activity of CC44, a biotinylated improgan congener by Paul Hoerbelt; Julia W. Nalwalk; James G. Phillips; Mark P. Wentland; Zhixing Shan; Lindsay B. Hough (464-471).
Improgan, a non-opioid, antinociceptive drug, activates descending analgesic circuits following brain administration, but the improgan receptor remains unidentified. Since biotinylation of drugs can enhance drug potency or facilitate discovery of new drug targets, a biotinylated congener of improgan (CC44) and several related compounds were synthesized and tested for antinociceptive activity. In rats and mice, intracerebroventricular (i.c.v.) administration of CC44 produced dose-dependent reductions in thermal nociceptive (tail flick and hot plate) responses, with 5-fold greater potency than improgan. CC44 also robustly attenuated mechanical (tail pinch) nociception in normal rats and mechanical allodynia in a spinal nerve ligation model of neuropathic pain. Similar to the effects of improgan, CC44 antinociception was reversed by the GABAA agonist muscimol (consistent with activation of analgesic circuits), and was resistant to the opioid antagonist naltrexone (implying a non-opioid mechanism). Also like improgan, CC44 produced thermal antinociception when microinjected into the rostral ventromedial medulla (RVM). Unlike improgan, CC44 (i.c.v.) produced antinociception which was resistant to antagonism by the cannabinoid CB1 antagonist/inverse agonist rimonabant. CC44 was inactive in mice following systemic administration, indicating that CC44 does not penetrate the brain. Preliminary findings with other CC44 congeners suggest that the heteroaromatic nucleus (imidazole), but not the biotin moiety, is required for CC44's antinociceptive activity. These findings demonstrate that CC44 is a potent analgesic compound with many improgan-like characteristics. Since powerful techniques are available to characterize and identify the binding partners for biotin-containing ligands, CC44 may be useful in searching for new receptors for analgesic drugs.
Keywords: Improgan; Analgesia; Pain; Neuropathic pain; Biotin;

Enalaprilat increases PPARβ/δ expression, without influence on PPARα and PPARγ, and modulate cardiac function in sub-acute model of daunorubicin-induced cardiomyopathy by Hana Cernecka; Katarina Ochodnicka-Mackovicova; Dana Kucerova; Jana Kmecova; Viera Nemcekova; Gabriel Doka; Jan Kyselovic; Peter Krenek; Peter Ochodnicky; Jan Klimas (472-477).
Anthracycline therapy is limited by a cardiotoxicity that may eventually lead to chronic heart failure which is thought to be prevented by ACE inhibitors (ACEi). However, the protective effect of ACEi in early stages of this specific injury remains elusive. Activated nuclear transcription factors peroxisome proliferator-activated receptors (PPAR) regulate cellular metabolism, but their involvement in anthracycline cardiomyopathy has not been investigated yet. For this purpose, Wistar rats were administered with daunorubicin (i.p., 3 mg/kg, in 48 h intervals) or co-administered with daunorubicine and enalaprilat (i.p., 5 mg/kg in 12 h intervals). Control animals received vehicle. Left ventricular function was measured invasively under anesthesia. Cell-shortening was measured by videomicroscopy in isolated cardiomyocytes. Expression of PPARs mRNA in cardiac tissue was measured by Real-Time PCR. Although the hemodynamic parameters of daunorubicin-treated rats remained altered upon ACEi co-administration, ACEi normalized daunorubicin-induced QT prolongation. On cellular level, ACEi normalized altered basal and isoproterenol-stimulated cardiac cell shortening in daunorubicine-treated group. Moreover, anthracycline administration significantly up-regulated heart PPARα mRNA and its expression remained increased after ACEi co-administration. On the other hand, the expression of cardiac PPARβ/δ was not altered in anthracycline-treated animals, whereas co-administration of ACEi increased its expression. Conclusively, effect of ACEi can be already detected in sub-acute phase of anthracycline-induced cardiotoxicity. Altered expression of heart PPARs may suggest these nuclear receptors as a novel target in anthracycline cardiomyopathy.
Keywords: Anthracycline; Cardiomyopathy; ACEi (Angiotensin conversing enzyme inhibitor); PPAR (Peroxisome proliferator-activated receptors);

The role of nitric oxide in the PKA inhibitor induced spatial memory deficits in rat: Involvement of choline acetyltransferase by Sheyda Najafi; Borna Payandemehr; Kaveh Tabrizian; Marjan Shariatpanahi; Ehsan Nassireslami; Kian Azami; Mojdeh Mohammadi; Farideh Asadi; Ali Roghani; Mohammad Sharifzadeh (478-485).
Several lines of evidence show that cAMP-PKA signaling pathway plays critical role in memory functions and suggest nitric oxide as an important modulator in learning and memory. In this study, we assessed the effects of intra-hippocampal infusion of H-89, a selective PKAII inhibitor, and 1400 W, a selective inducible nitric oxide synthase (iNOS) inhibitor, on spatial memory in rats. By using the Morris water maze, spatial memory retention parameters were examined 48 h after the infusions through measuring escape latency, traveled distance, and swimming speed. The rats receiving intra-hippocampal infusions of 1400 W (100 µM/side) showed a significant reduction ( P<0.05) in escape latency and traveled distance in comparison with the control saline group. In contrast, a significant increase (⁎⁎ P<0.01) in escape latency and traveled distance was observed after infusion of 10 µM H-89. Moreover, among combination groups, co-administration of 1400 W (400 µM/side) with 10 µM/side of H-89 caused a significant reduction ( P<0.05) in escape latency and traveled distance in comparison with the H-89 group. Also, we evaluated the molecular effects of 1400 W on the expression of choline acetyltransferase (ChAT), a cholinergic marker, in the CA1 region of the hippocampus and medial septal area (MSA). Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400 W revealed a significant increase in ChAT immunoreactivity levels in both the CA1 and the MSA regions. Overall, the results suggest that 1400 W has protective effect against H89-induced spatial memory impairment. Moreover, the observed memory improvements caused by 1400 W infusions, might be due to interaction of iNOS with the cholinergic system.
Keywords: Inducible nitric oxide synthase; Spatial memory; Morris water maze; Protein kinase A;

Phosphodiesterases: Regulators of cyclic nucleotide signals and novel molecular target for movement disorders by Sorabh Sharma; Kushal Kumar; Rahul Deshmukh; Pyare Lal Sharma (486-497).
Movement disorders rank among the most common neurological disorders. During the last two decades substantial progress has been made in understanding of the pathological basis of these disorders. Although, several mechanisms have been proposed, downregulation of cyclic nucleotide mediated signaling cascade has consistently been shown to contribute to the striatal dysfunctioning as seen in movement disorders. Thus, counteracting dysregulated cyclic nucleotide signaling has been considered to be beneficial in movement disorders. Cyclic nucleotide phosphodiesterases (PDEs) are the enzymes responsible for the breakdown of cyclic nucleotides and upregulation in PDE activity has been reported in various movement disorders. Thus, PDE inhibition is considered to be a novel strategy to restore cerebral cyclic nucleotide levels and their downstream signalling cascade. Indeed, various PDE inhibitors have been tested pre-clinically and were reported to be neuroprotective in various neurodegenerative disorders associated with movement disabilities. In this review, we have discussed a putative role of PDE inhibitors in movement disorders and associated abnormalities.
Keywords: Cyclic nucleotides; Dyskinesia's; Huntington's disease; Movement disorders; Parkinson's disease; Phosphodiesterase inhibitors;

The role of endogenous glucocorticoids in glucose metabolism and immune status of MIF-deficient mice by Ivana Nikolic; Milica Vujicic; Tamara Saksida; Timea Berki; Stanislava Stosic-Grujicic; Ivana Stojanovic (498-506).
Macrophage migration inhibitory factor (MIF)-deficient mice develop glucose intolerance and hyperglycemia, but remain entirely responsive to exogenous insulin in adult age. Furthermore, as a consequence of MIF deficiency, the immune response in these mice is predominantly anti-inflammatory. Since MIF is a natural counter-regulator of glucocorticoid action, and it is known that excessive concentration of glucocorticoids contribute both to beta cell dysfunction and immunosuppression, we hypothesized that MIF absence enables elevation of glucocorticoids which in turn caused the observed condition. Our results confirm that MIF-knockout (MIF-KO) mice possess higher levels of circulating corticosterone, but lower expression of glucocorticoid receptor in pancreatic islets, liver and adipose tissue to the one observed in wild type (WT) mice. A significant up-regulation of glucocorticoid receptor expression was however noticed in MIF-deficient lymph node cells. The inhibition of glucocorticoid receptor by RU486 improved tolerance to glucose in MIF-KO mice and restored euglycemia. Although RU486 treatment did not alter the level of glucose receptor GLUT2, it enhanced insulin secretion and up-regulated insulin-triggered Akt phosphorylation within hepatic tissue. Finally, inhibition of glucocorticoid receptor changed anti-inflammatory phenotype of MIF-KO lymphocytes toward a physiological profile. Our results indicate that deregulated glucocorticoid secretion and glucocorticoid receptor expression in the absence of MIF possibly contributes to the development of glucose intolerance and immunosuppression in MIF-KO mice. However, since MIF-KO mice respond normally to insulin and their beta cell function is within physiological range, additional cause for glucose intolerance could be sought in the possible malfunction of their insulin.
Keywords: Macrophage migration inhibitory factor; Glucocorticoids; Diabetes; Insulin; Inflammation;

Ileal apical sodium-dependent bile acid transporter protein levels are down-regulated through ubiquitin-dependent protein degradation induced by bile acids by Masaaki Miyata; Hiroki Yamakawa; Kenjiro Hayashi; Hideaki Kuribayashi; Yasushi Yamazoe; Kouichi Yoshinari (507-514).
The ileal apical sodium-dependent bile acid transporter (ASBT or SLC10A2) has a crucial role in intestinal bile acid absorption. We previously reported that enterobacteria-mediated bile acid conversion was involved in the alteration of ileal ASBT expression levels. In the present study, to investigate the hypothesis that ileal ASBT protein levels are post-translationally regulated by enterobacteria-associated bile acids, alteration of ileal ASBT protein levels was analysed in mice 12 h and 24 h after anti-bacterial drug ampicillin (ABPC) treatment (100 mg/kg, single shot) that altered bile acid composition in the intestinal lumen. In ABPC-treated mice, enterobacteria-biotransformed bile acid, taurodeoxycholic acid (TDCA) and cholic acid (CA) levels were decreased, whereas taurocholic acid (TCA) and tauro-β-muricholic acid levels were increased in the intestinal lumen. Ileal ASBT protein levels in brush-border membrane vesicles (BBMVs), but not ileal Asbt mRNA levels, were significantly increased in the ABPC-treated mice, and the extent of ubiquitination of the ileal ASBT protein was reduced in the ABPC-treated mice. Treatment of ABPC-pretreated mice with CA or TDCA, but not TCA, significantly decreased ileal ASBT protein levels and increased the extent of ubiquitination of ileal ASBT protein. Treatment of mice with the lysosome inhibitor, chloroquine, or the proteasome inhibitor, MG132, increased ileal ASBT protein levels in BBMVs. CA-mediated reduction of ASBT protein levels in the ABPC-pretreated mice was attenuated by co-treatment with chloroquine or MG132. These results suggest that ileal ASBT protein is degraded by a ubiquitin-dependent pathway in response to enterobacteria-associated bile acids.Display Omitted
Keywords: ASBT; Ubiquitination; Bile acid; Enterobacteria; Antibiotics; MG132;

Neuroprotective role of PDE4 and PDE5 inhibitors in 3-nitropropionic acid induced behavioral and biochemical toxicities in rats by Tarun Thakur; Sorabh Sharma; Kushal Kumar; Rahul Deshmukh; Pyare Lal Sharma (515-521).
Phosphodiesterase inhibitors have been reported to be beneficial in cognitive and motor disorders. In the present study, we have investigated the effects of RO 20-1724 (PDE4 inhibitor) and sildenafil (PDE5 inhibitor) in 3-nitropropionic acid (3-NP) induced experimental Huntington's disease in rats. 3-Nitropropionic acid was administered for 14 days (10 mg/kg i.p.) 1 h following 3-NP administration, the rats were treated with either vehicle, RO 20-1724 (0.25 and 0.5 mg/kg i.p.) or sildenafil (2 and 4 mg/kg i.p.) for 14 days. Cognitive functions were assessed by using Morris water maze whereas, motor functions were assessed by spontaneous locomotor activity, limb withdrawal and suspended wire test at different time points. Biochemically, markers of oxidative stress and cell damage, such as reduced glutathione, malondialdehyde, nitrite and lactate dehydrogenase levels were assessed terminally in the brain homogenate. Chronic administration of 3-NP produced significant decrease in body weight, showed marked abnormalities in cognitive and motor functions. Further, significant oxidative–nitrosative stress and cell damage was also observed. Chronic administration of RO 20-1724 and sildenafil in 3-NP treated rats significantly and dose dependently attenuated 3-NP induced behavioral and biochemical abnormalities in rats. Both these drugs were equally effective in attenuating 3-NP induced neurotoxicity. These results suggesting that the inhibition of PDE4 and PDE5 would be therapeutic in neurodegenerative disorders associated with cognitive and motor dysfunction.
Keywords: PDE inhibitor; Motor disorder; Oxidative stress; 3-Nitropropionic acid; Huntington's disease;

Adenosine participates to asthma physiopathology by signaling through more than just one receptor subtype. Defining the role of each receptor is complicated by evidence that often results obtained on rodents do not coincide with human studies, but what emerges is that an important condition to establish hyperresponsiveness to adenosine in any species of sensitized animals is the exposure to allergen; this feature appears to be very similar to the human situation, since allergic humans regularly undergo exposure to allergen. Furthermore, A2B in humans, but A3 receptor in rodents, would mediate, indirectly, the bronchoconstriction in response to adenosine and would play the main role in adenosine-induced airway inflammation and airway hyperreactivity. On the other hand, A1 receptor over-expressed on asthmatic airways would mediate a direct adenosine bronchoconstrictor effect. Antagonists and agonists to adenosine receptors have been considered as antiasthmatic drugs but often their development has been limited by unwanted effects. Preventing adenosine accumulation in airways should be considered as a novel promising antiasthmatic strategy.
Keywords: Adenosine; Airway; Asthma; CD39; CD73; Hyperreactivity;