European Journal of Pharmacology (v.670, #1)

The biological effect of endogenous sulfur dioxide in the cardiovascular system by Xin-Bao Wang; Hong-Fang Jin; Chao-Shu Tang; Jun-Bao Du (1-6).
Sulfur dioxide is considered a toxic gas in air pollution and detrimental to many organs, however, it can be generated endogenously in the cardiovascular system in vivo. Gaseous sulfur dioxide has an endothelium-dependent vasorelaxing effect at low concentrations, but is endothelium-independent at high concentrations and has a negative inotropic effect on cardiac function. This vasorelaxing effect is mediated by adenosine triphosphate-sensitive calcium channels and L-type calcium channels. Under pathophysiological conditions, sulfur dioxide increases anti-inflammatory response and antioxidant capacities in pulmonary hypertensive rats. Sulfur dioxide also attenuates increased blood pressure and vascular remodeling in spontaneously hypertensive and hypoxic pulmonary hypertensive rats. Recent studies suggest that endogenous sulfur dioxide is also involved in the process of myocardial ischemia–reperfusion injury and lipid metabolism. Therefore, the evidence suggests that endogenous sulfur dioxide may be a novel gasotransmitter in the cardiovascular system. The significance of sulfur dioxide on the cardiovascular system is intriguing and appealing.
Keywords: Biology; Cardiovascular; Gasotransmitter; Sulfur dioxide;

Cardiomyocyte stretching has been reported to be a major trigger for brain natriuretic peptide (BNP) release; however, an increase in circulating BNP is observed in patients with acute myocardial ischemia in the absence of increased left ventricular wall stress or cardiomyocyte stretching. In the present study, to investigate the direct and independent effects of acute myocardial ischemia on BNP expression and its mechanism, we established an in vitro glucose-free ischemia and hypoxia injured model of cultured rat cardiomyotes and proved hypoxia upregulated expressions of interleukin-6(il-6) and BNP. Further treatment with il-6 elicited dose- and time-dependent increases in BNP mRNA and protein expression as well as an upregulation in transforming growth factor-β1 (TGF-β1)/Smad2 expression, which was partially suppressed by a neutralizing antibody. In conclusion, our study showed that acute myocardial ischemia can directly upregulate BNP expression at the translational and transcriptional levels through the action of il-6, and this process is associated with the upregulation of TGF-β1/Smad2 signal path.
Keywords: Hypoxia; Brain natriuretic peptide; Interleukin-6; Transforming growth factor-β1; Smad2;

GL-V9, a newly synthetic flavonoid derivative, induces mitochondrial-mediated apoptosis and G2/M cell cycle arrest in human hepatocellular carcinoma HepG2 cells by Liwen Li; Na Lu; Qinsheng Dai; Libin Wei; Qing Zhao; Zhiyu Li; Qinghao He; Yue Dai; Qinglong Guo (13-21).
We recently established that GL-V9, a newly synthetic flavonoid derivative, is an active cytotoxic component. In this study, we demonstrated that GL-V9 inhibited cells growth via inducing apoptosis and G2/M cell cycle arrest in human hepatocellular carcinoma HepG2 cells. Following the treatment of HepG2 cells with GL-V9, we observed poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3 and caspase-9, while caspase-8 remained unchanged. The expression ratio of Bcl-2/Bax was also decreased in GL-V9-treated cells. Meanwhile, the cell cycle-related proteins, such as cyclin B1, CDK1 and cdc25 were down-regulated in GL-V9-induced G2/M cell cycle arrest. Furthermore, we showed that GL-V9-induced apoptosis in HepG2 cells was achieved through mitochondrial pathway. It also regulated changes of mitochondrial membrane potential and increased the production of intracellular reactive oxygen species. Besides, the growth inhibitory effect of GL-V9 was examined in vivo using murine implanted tumor model. These studies indicate that GL-V9 shows promise as a therapeutic agent against human heptoma.
Keywords: Apoptosis; Cell cycle arrest; Mitochondria; GL-V9; Hepatocellular carcinoma;

Karanjin from Pongamia pinnata induces GLUT4 translocation in skeletal muscle cells in a phosphatidylinositol-3-kinase-independent manner by Natasha Jaiswal; Prem P. Yadav; Rakesh Maurya; Arvind K. Srivastava; Akhilesh K. Tamrakar (22-28).
Insulin-stimulated glucose uptake in skeletal muscle is decreased in type 2 diabetes due to impaired translocation of insulin-sensitive glucose transporter 4 (GLUT4) from intracellular pool to plasma membrane. Augmenting glucose uptake into this tissue may help in management of type 2 diabetes. Here, the effects of an identified antihyperglycemic molecule, karanjin, isolated from the fruits of Pongamia pinnata were investigated on glucose uptake and GLUT4 translocation in skeletal muscle cells. Treatment of L6-GLUT4myc myotubes with karanjin caused a substantial increase in the glucose uptake and GLUT4 translocation to the cell surface, in a concentration-dependent fashion, without changing the total amount of GLUT4 protein and GLUT4 mRNA. This effect was associated with increased activity of AMP-activated protein kinase (AMPK). Cycloheximide treatment inhibited the effect of karanjin on GLUT4 translocation suggesting the requirement of de novo synthesis of protein. Karanjin-induced GLUT4 translocation was further enhanced with insulin and the effect is completely protected in the presence of wortmannin. Moreover, karanjin did not affect the phosphorylation of AKT (Ser-473) and did not alter the expression of the key molecules of insulin signaling cascade. We conclude that karanjin-induced increase in glucose uptake in L6 myotubes is the result of an increased translocation of GLUT4 to plasma membrane associated with activation of AMPK pathway, in a PI-3-K/AKT-independent manner.
Keywords: Insulin resistance; Pongamia pinnata; GLUT-4 translocation; Skeletal muscle;

The purpose of this study is to investigate the antitumor activity of a new derivative of lupeol-3β-O-succinyl-lupeol (LD9-4) and the molecular mechanism underlying cell death in human non-small cell lung cancer A549 cells. The results revealed that LD9-4 inhibited A549 cell proliferation in a time- and dose-dependent manner, with an IC50 value of 5.78 ± 0.48 μM after cells exposed to LD9-4 for 72 h. Markers indicative of apoptosis (cell cycle arrest, phosphatidylserine externalization and Hoechst33258 staining) were uniformly negative in LD9-4 exposed cells. Interestingly, transmission electron microscope, MDC staining and LC3 level determination all confirmed that autophagy was induced in LD9-4 treated A549 cells. Furthermore, we found that LD9-4-induced autophagy in A549 cells was associated with the increase of intracellular reactive oxygen species and the decrease of phosphorylated mTOR and p70S6K levels. In the meanwhile, both mRNA and protein levels of Beclin 1 were up-regulated in a time-dependent manner. Our data suggest that autophagy is induced by LD9-4 in A549 cells, and the accumulating reactive oxygen species, up-regulation of Beclin 1 and inhibition of the mTOR signaling pathway are involved in this process.
Keywords: LD9-4; Apoptosis; Autophagy; Beclin 1; mTOR; A549;

Effect of sorafenib on the energy metabolism of hepatocellular carcinoma cells by Luigi Fiume; Marcella Manerba; Marina Vettraino; Giuseppina Di Stefano (39-43).
Recent data demonstrated that sorafenib impaired the oxidative phosphorylation of a rat myogenic cell line and suggested that this biochemical lesion can contribute to the cardiac toxicity caused by the drug. With the experiments reported here, we verified whether sorafenib inhibits oxidative phosphorylation also in cells from human hepatocellular carcinomas (HCCs), which are treated with this drug. By using the HCC cell lines PLC/PRF/5 and SNU-449 we studied the effects of the drug on ATP cellular levels, oxygen consumption and aerobic glycolysis, a metabolic pathway generally used by neoplastic cells to meet their energy demand. The effect of sorafenib on ATP cellular levels was also studied in cells grown in a glucose-free medium, which only derive their energy from oxidative phosphorylation. We found that at clinically relevant concentrations sorafenib hindered oxidative phosphorylation, whereas at the same time stimulated aerobic glycolysis in glucose-grown cells, thus attenuating the cellular ATP depletion. These results support the impairment of oxidative phosphorylation as a mechanism contributing to the antineoplastic activity of sorafenib in the treatment of HCCs.
Keywords: Sorafenib; Hepatocellular carcinoma chemotherapy; Aerobic glycolysis; Tumor cell metabolism;

Structure–function relationships of citrus limonoids on p38 MAP kinase activity in human aortic smooth muscle cells by Jinhee Kim; Guddadarangavvanahally K. Jayaprakasha; Mariappan Muthuchamy; Bhimanagouda S. Patil (44-49).
Limonoids, abundantly present in citrus fruits, have potential role in reducing risk of different type of cancer. In the present study, we hypothesized that seven structurally different limonoids would involve in inflammatory pathway via modulating p38 MAP kinase activity at various extent in vascular smooth muscle cells. Results demonstrated that the different functional groups containing limonoids had differential effects on the p38 MAP kinase activity in human aortic smooth muscle cells. Among seven limonoids, nomilin exhibited the highest (38%) inhibition of p38 MAP kinase activity, followed by limonin (19%), deacetyl nomilin (19%), and defuran nomilin (17%). While defuran limonin and methyl nomilinate showed no significant decrease in p38 MAP kinase activity, obacunone significantly increased the p38 MAP kinase activity by 38%. Furthermore, TNF-α induced p38 MAP kinase activity in the smooth muscle cells was completely inhibited by nomilin. Thus our data provide the first evidence that nomilin is the potent natural inhibitor for p38 MAP kinase activity in human aortic smooth muscle cells. These data also suggest that a seven-membered A ring with acetoxy group, present in nomilin, seems to be essential for its inhibitory activity on p38 MAP kinase.Display Omitted
Keywords: Citrus limonoids; HASMC; p38 MAPK; Structure–function relationship; TNF-α;

Impact of cisplatin administration on protein expression levels in renal cell carcinoma: A proteomic analysis by Radovan Vasko; Gerhard A. Mueller; Ann-Kristin von Jaschke; Abdul R. Asif; Hassan Dihazi (50-57).
Renal cell carcinoma (RCC) is the most common renal neoplasm in adults. Considering that chemoresistance is a typical feature of RCC, every effort should be made in order to identify mechanisms of drug resistance. We used two-dimensional gel electrophoresis and mass spectrometry to study changes in protein expression levels that occur in primary resistant LN78 RCC cells when treated with therapeutic concentrations of cisplatin. Expression differences of selected proteins were confirmed by immunoblot. Up-regulation of heat-shock proteins can block apoptosis indirectly by altered protein folding and by direct interaction with apoptosis regulatory proteins. Cyclophilin A and stratifin can modify cell cycle control and enable tumor cells to escape and further proliferate despite DNA damage caused by cisplatin. Increased activity of glycolytic enzymes reflect metabolic adaptations to increased energy requirements as well as converting to alternative energy sources because of cisplatin-induced disturbed mitochondrial oxidation. Changes in cytoskeletal proteins may change the handling of cisplatin by altering transport and increasing cellular efflux of the drug. Repression of vimentin and disturbance of antioxidative mechanisms may represent vulnerable points in tumor cellular defense against cisplatin. The involvement of these proteins in cisplatin resistance and their potential as therapeutic targets requires further evaluation.
Keywords: Renal cell carcinoma; Proteomics; Cisplatin; Chemotherapy; Chemoresistance;

p38β-regulated induction of the heat shock response by carbon monoxide releasing molecule CORM-2 mediates cytoprotection in lung cells in vitro by Nils Schallner; Sven Schwemmers; Christian I. Schwer; Christian Froehlich; Patrick Stoll; Matjaz Humar; Heike L. Pahl; Alexander Hoetzel; Torsten Loop; Ulrich Goebel (58-66).
The carbon monoxide releasing molecule tricarbonyldichlororuthenium (CORM-2) displays protective actions like carbon monoxide. The molecular mechanism underlying this effect remains controversial. We hypothesized that CORM-2 mediates cytoprotection via induction of heat shock proteins through activation of p38 mitogen-activated kinase. Embryonic bovine lung cells were incubated with CORM-2. Apoptosis was induced by staurosporine and analyzed by flow cytometry following annexin-V staining, caspase-3 activity assay, and by Western Blot for caspase-3 cleavage. Heat shock response was assessed by DNA-binding activity of heat shock factor 1 and by reporter gene activity. Cells were transfected with siRNA targeting p38 isoforms. Data were analyzed with ANOVA and post-hoc Holm-Sidak test. CORM-2 inhibited staurosporine-induced apoptosis (% annexin-V positive cells: staurosporine = 60 ± 4% vs. CORM-2 10 μM = 48 ± 4%, CORM-2 25 μM = 42 ± 5%, CORM-2 50 μM = 40 ± 4% and CORM-2 100 μM = 38 ± 2%, mean ± S.D., P < 0.001; caspase-3 activity: staurosporine = 92 ± 15 RFUs vs. CORM-2 50 μM = 60 ± 14 RFUs, mean ± S.D. P < 0.001). CORM-2 induced phosphorylation of p38 MAPK, but not of JNK and ERK1/2. CORM-2 induced DNA-binding of heat shock factor 1 and elicited a 4-fold induction of gene activity (P < 0.05). Incubation with the Hsp inhibitors KNK437 attenuated and 17-AAG abolished the anti-apoptotic effect of CORM-2 (P < 0.001). p38 inhibition and silencing of p38β attenuated the anti-apoptotic effect of CORM-2 (P < 0.05), most likely by abolishing CORM-2-induced HSF-1 binding activity. These findings suggest that CORM-2-mediated cytoprotection is caused by induction of the heat shock response and by p38 activation. Furthermore, the p38β isoform activation may represent an upstream mechanism of heat shock response induction.
Keywords: CORM-2; Heat shock response; p38 mitogen-activated protein kinase;

Raloxifene protects cultured human chondrocytes from IL-1β induced damage: A biochemical and morphological study by Laura Tinti; Silvia Niccolini; Antonello Lamboglia; Nicola A. Pascarelli; Roberto Cervone; Antonella Fioravanti (67-73).
It is well known that estrogens are implicated in the pathogenesis of osteoarthritis. Raloxifene is a selective estrogen receptor modulator used in the treatment of osteoporosis, though little is known about the possible effects of raloxifene on cartilage metabolism. The aim of our study was to evaluate the possible in vitro effects of raloxifene in human osteoarthritis chondrocytes cultivated in the presence or absence of Interleukin-1 beta (IL-1β) (5 ng/ml). The effects of 0.1 μM and 1 μM of raloxifene in the culture medium were assessed using an immuno-enzymatic method for proteoglycans and metalloproteinase-3 (MMP-3), and the Griess method for nitrite. Gene expression of inducible Nitric Oxide Synthase (iNOS) was detected by real-time PCR. A morphological analysis was performed by transmission electron microscopy (TEM). Cell viability was significantly (P < 0.01) reduced by the IL-1β, and restored to basal levels by raloxifene at both of the concentrations used. The presence of IL-1 β led to a significant decrease (P < 0.01) in proteoglycan levels as well as a significant increase of MMP-3 and NO (P < 0.01). When the cells were co-incubated with IL-1β and raloxifene, a significant and dose-dependent increase in proteoglycans and a reduction of MMP-3 and nitric oxide (NO) were detected. iNOS was noticeably expressed in IL-1β stimulated chondrocytes, while raloxifene decreased in a very significant manner the gene expression of iNOS at both of the concentrations used. The results of the biochemical evaluation were confirmed by TEM. Our data suggest that raloxifene may have a potential chondroprotective role in osteoarthritis.
Keywords: Raloxifene; Chondrocyte; Interleukin-1 beta; Proteoglycan; Metalloproteinase-3; Nitric oxide;

MC70 potentiates doxorubicin efficacy in colon and breast cancer in vitro treatment by Amalia Azzariti; Anna E. Quatrale; Letizia Porcelli; Nicola A. Colabufo; Mariangela Cantore; Giuseppe Cassano; Giuseppe Gasparre; Giuseppina Iannelli; Stefania Tommasi; Maria A. Panaro; Angelo Paradiso (74-84).
A major limitation of cancer treatment is the ability of cancer cells to develop resistance to chemotherapeutic drugs, by the establishment of multidrug resistance. Here, we characterize MC70 as ABC transporters inhibitor and anticancer agent, alone or with chemotherapy. MC70 was analyzed for its interaction with ABCB1, ABCG2 and ABCC1 by specific transport assays. In breast and colon cancer cell lines, cell growth and apoptosis were measured by MTT assay and DNA laddering Elisa kit, respectively. Cell cycle perturbation and cellular targets modulation were analyzed by Flow-cytometry and Western blotting, respectively. MC70 interacted with ABC transporters. In breast cancer cells, MC70 slightly inhibited cell proliferation strongly enhancing doxorubicin effectiveness. By contrast, MC70 was found to inhibit cell growth in colon cancer cells without affecting doxorubicin efficacy and in combination with topoisomerase I inhibitors it could be a promising therapeutic approach. What is more, it was also observed that MC70 induced apoptosis, canceled in favor of necrosis when given in combination with high doses of doxorubicin. MC70 inhibited cell migration probably through its interaction with sigma-1 receptor. Modulations of i) cell cycle, ii) pAkt and the phosphorylation of the three MAPKs were highlighted, while any activity was excluded at transcription level, thus accounting for the phenotypic effects observed. MC70 might be considered as a new potential anticancer agent capable to i) enhance chemotherapy effectiveness and ii) to play a contributory role in the treatment of chemotherapy resistant tumors.
Keywords: MC70; Doxorubicin; Topotecan; CPT-11; Breast cancer; Colon cancer; ABCB1; ABCG2;

Effect of thymol on Ca2+ homeostasis and viability in human glioblastoma cells by Shu-Shong Hsu; Ko-Long Lin; Chiang-Ting Chou; An-Jen Chiang; Wei-Zhe Liang; Hong-Tai Chang; Jeng-Yu Tsai; Wei-Chuan Liao; Fong-Dee Huang; Jong Khing Huang; I-Shu Chen; Shuih-Inn Liu; Chun-Chi Kuo; Chung-Ren Jan (85-91).
The effect of the natural essential oil thymol on cytosolic Ca2+ concentrations ([Ca2+]i) and viability in human glioblastoma cells was examined. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Thymol at concentrations of 400–1000 μM induced a [Ca2+]i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca2+. Thymol-induced Ca2+ signal was not altered by nifedipine, econazole, SK&F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca2+ was removed, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished thymol-induced [Ca2+]i rise. Incubation with thymol also abolished thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished thymol-induced [Ca2+]i rise. At concentrations of 200–800 μM, thymol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that thymol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. Collectively, in human glioblastoma cells, thymol induced a [Ca2+]i rise by inducing phospholipase C- and protein kinase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via non store-operated Ca2+ channels. Thymol induced cell death that may involve apoptosis.
Keywords: Ca2+; Human glioblastoma; Fura-2; Thymol;

Altered liver α1-adrenoceptor density and phospholipase C activity in the human hepatocellular carcinoma by Woubet T. Kassahun; Bianca Günl; Sven Jonas; Fritz R. Ungemach; Getu Abraham (92-95).
The human hepatocellular carcinoma (HCC) is a common cancer with high mortality rate. We examined the density and coupling to phospholipase C (PLC) of the α1-adrenoceptors. In HCC liver, the α1-adrenoceptor density – as assessed by [³H]-Prazosin binding – was significantly reduced to about 75% when compared to non-adjacent non-tumorous liver (NA-NL) (P = 0.0002). The decrease in maximal α1-adrenoceptor concentration (Bmax) was accompanied by a significant reduction in noradrenaline-stimulated PLC activity (P < 0.032 versus NA-NL) (assessed by [³H]-PIP2 hydrolysis). GTPγS-stimulated PLC activity in HCC livers did not statistically differ from NA-NL livers. NaF, which activates all G-proteins, stimulated PLC in both HCC and NA-NL livers to a similar extent. The altered noradrenaline-induced functional responsiveness of HCC livers was not reflected by changes in the binding affinity of [³H]-Prazosin for α1-adrenoceptors (NA-NL: 0.066 ± 0.010 pmol/l; tumour: 0.067 ± 0.020 pmol/l). These results demonstrate that human HCC causes profound alteration of the hepatic α1-adrenoceptor signal transduction pathway and may account for a negative cancer related metabolism of carbohydrates and wasting syndrome in tumour patients.
Keywords: Hepatocellular carcinoma; α-adrenoceptor; Phospholipase C;

CHM-1 induces apoptosis via p38-mediated upregulation of DR5 expression in human ovarian cancer SKOV3 cells by Jang-Chang Lee; Li-Chen Chou; Chi-Hung Huang; Jing-Gung Chung; Li-Jiau Huang; Kuo-Hsiung Lee; Mien-Chie Hung; Tzong-Der Way; Sheng-Chu Kuo (96-104).
Ovarian cancer is a leading cause of death due to neoplasm of the female genital tract. Treatment for advanced-stage disease remains limited, and an effective drug for ovarian cancer is urgently needed today. In the present study, MTT assay was used to evaluate the antiproliferative effect of the 2-(substituted phenyl)-6,7-methylenedioxyquinolin-4-one derivatives for developing new anti-ovarian cancer drugs. CHM-1 was the most active compound, and it exhibited potent antiproliferative activity against human ovarian cancer cells. CHM-1 inhibited the growth of SKOV3 cells and induced apoptosis in a concentration-dependent manner, but it was less cytotoxic to human diploid skin fibroblast Detroit 551 cells. The western blot experiments showed that CHM-1 caused the upregulation of death receptor (DR) 5 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, CHM-1-mediated cellular apoptosis was found to be closely involved with the p38-mediated upregulation of DR5 expression. In an SKOV3 subcutaneous xenograft model, both CHM-1 and its phosphate, CHM-1-P caused a significant dose- and time-dependent tumor regression. Furthermore, CHM-1 inhibited tumor growth and prolonged the lifespan in the SKOV3 ip1/luc orthotopic xenograft model. Intravenous administration of CHM-1-P significantly prolonged the survival time in the SKOV3/ICR-Foxn1nu orthotopic xenograft model. Based on their excellent antitumor activity with the interesting mechanism of action, CHM-1 and CHM-1-P were considered new anti-ovarian cancer drug candidates.
Keywords: Apoptosis; CHM-1; Death receptor 5; Ovarian cancer; p38;

The binding site within the L-type Ca2+ channel Cav1.2 for neutral dihydropyridines is well characterized. However, the contributions of the alkylamino side chains of charged dihydropyridines such as amlodipine and nicardipine to channel block are not clear. We tested the hypothesis that the distinct locations of the charged side chains on amlodipine and nicardipine would confer distinct properties of channel block by these two drugs. Using whole-cell voltage clamp, we investigated block of wild type Cav 2.1, wild type Cav1.2, and Cav1.2/Dihydropyridine insensitive, a mutant channel insensitive to neutral DHPs, by amlodipine and nicardipine. The potency of nicardipine and amlodipine for block of closed (stimulation frequency of 0.05 Hz) Cav1.2 channels was not different (IC50 values of 60 nM and 57 nM, respectively), but only nicardipine block was enhanced by increasing the stimulation frequency to 1 Hz. The frequency-dependent block of Cav1.2 by nicardipine is the result of a strong interaction of nicardipine with the inactivated state of Cav1.2. However, nicardipine block of Cav1.2/Dihydropyridine insensitive was much more potent than block by amlodipine (IC50 values of 2.0 μM and 26 μM, respectively). A mutant Cav2.1 channel containing the neutral DHP binding site (Cav2.1/Dihydropyridine sensitive) was more potently blocked by amlodipine (IC50  = 41 nM) and nicardipine (IC50  = 175 nM) than the parent Cav2.1 channel. These data suggest that the alkylamino group of nicardipine and amlodipine project into distinct regions of Cav1.2 such that the side chain of nicardipine, but not amlodipine, contributes to the potency of closed-channel block, and confers frequency-dependent block.
Keywords: Calcium channel blocker; Dihydropyridine; Cav1.2; Amlodipine; Nicardipine; Voltage clamp;

The influence of acute and chronic treatments with intraperitoneal venlafaxine, a selective serotonin/norepinephrine reuptake inhibitor, on the anticonvulsant activity of selected antiepileptic drugs was studied in the maximal electroshock test in mice. Venlafaxine (12.5 and 25 mg/kg), given either acutely or chronically, significantly increased the electroconvulsive threshold. Moreover, both acute and chronic venlafaxine, applied at the highest subprotective dose of 6.25 mg/kg, enhanced the anticonvulsant effect of valproate, without affecting the protective action of carbamazepine, phenobarbital and phenytoin. The antidepressant did not affect brain concentration of valproate, indicating that the interaction between the two drugs seems pharmacodynamic in nature. Despite the lack of effect on the antielectroshock action of the remaining antiepileptics, acute venlafaxine increased the brain concentration of phenobarbital, while chronic venlafaxine reduced the brain level of phenytoin. In terms of adverse effects, acute/chronic venlafaxine and antiepileptic drugs alone, as well as their combinations, did not produce significant motor or long-term memory deficits in mice. Summing up, it seems that venlafaxine may be considered as a safe drug for the clinical use in patients with epilepsy and depressive disorders.
Keywords: Venlafaxine; Antiepileptic drug; Electroshock maximal; Pharmacokinetic interaction;

Effects of flavone 6-substitutions on GABAA receptors efficacy by Lihuan Ren; Wing Man Chan; Feng Wang; Zhiwen Xu; Cunyou Zhao; Wai Kin Mat; Yanwen Chai; J. Tze-Fei Wong; Shui Ying Tsang; Hong Xue (121-129).
Flavones have been studied for their activities via benzodiazepine site on the type-A γ-aminobutyric acid (GABAA) receptors, for which knowledge on structure–efficacy relationships has been rather limited in comparison to that on structure–affinity relationships. The present study focused on flavone 6-substitution, implied in previous studies being relevant to efficacy. Structure analogs, each varying only at position 6, were compared, including 6-fluoroflavone, 6-chloroflavone, 6-bromoflavone, and 2′-hydroxyflavone analyzed in the present study, as well as 6,2′-dihydroxyflavone reported earlier. Radio-ligand binding assays, whole-cell patch-clamp, and mouse behavioral experiments were performed. In consistent with a previous report, the present whole-cell patch-clamp and animal behavior experiments demonstrated 6-bromoflavone to be a positive modulator at GABAA receptors acting through flumazenil-sensitive high-affinity benzodiazepine site. In contrast, the other two 6-haloflavones were both neutralizing modulators. In vitro electrophysiological and in vivo animal experiments showed that 2′-hydroxyflavone was a neutralizing modulator, different in efficacy from its structural analog, 6,2′-dihydroxyflavone, a negative modulator of GABAA receptors. The fact that flavone analogs differing only at position 6 showed drastically different pharmacological properties clearly points to 6-substitution being an important determinant of efficacy. The results suggest that a large width of the first atom on the 6-substituent favors a high binding affinity of the 6-substituted flavone, whereas a large overall volume of the 6-substituent favors positive modulator activity, which could be modified by, e.g., 2′-hydroxyl substitution. These findings have contributed to the understanding of quantitative structure–efficacy relationships for flavones acting at GABAA receptors, and hence facilitation of flavone-based drug development.
Keywords: Benzodiazepine site; Flavone; Positive modulator; Neutralizing modulator; Negative modulator; Structure–efficacy relationship;

Beneficial effects of desacyl-ghrelin, hexarelin and EP-80317 in models of status epilepticus by Giuseppe Biagini; Antonio Torsello; Carla Marinelli; Fabio Gualtieri; Riccardo Vezzali; Silvia Coco; Elena Bresciani; Vittorio Locatelli (130-136).
It has been reported that ghrelin exerts anticonvulsive effects in models of epilepsy. In this study we aimed to characterize the anticonvulsive activity of ghrelin and other growth hormone secretagogue receptor 1a (GHSR1a) ligands in rats exposed to status epilepticus induced by pilocarpine or kainate. Firstly, in three independent experiments, before receiving pilocarpine (380 mg/kg, i.p.), rats were pretreated with one among ghrelin (1.5 mg/kg), desacyl-ghrelin (1.5 mg/kg), hexarelin (330 μg/kg), EP-80317 (330 μg/kg), JMV-1843 (330 μg/kg), JMV-2959 (330 μg/kg) or saline. Secondly, in the fourth experiment, rats were pretreated with i.p. ghrelin, desacyl-ghrelin, hexarelin, EP-80317 or saline, followed by kainate (15 mg/kg, i.p.). We evaluated: induction of generalized seizures, latency to generalized seizures, status epilepticus, latency to status epilepticus (the time lag between the first tonic–clonic convulsion and the switch to continuous seizures) and mortality. In the pilocarpine model, 60% of rats pretreated with EP-80317 (P  < 0.05) showed no seizure. Hexarelin and EP-80317 were both able to prevent progression to status epilepticus in pilocarpine-treated rats (P  < 0.05). When status epilepticus was induced by kainate, seizures developed with few exceptions. However, latency to status epilepticus was significantly (P  < 0.01) longer in rats pretreated with desacyl-ghrelin, whereas hexarelin and EP-80317 did not display any effect. Almost all GHSR1a ligands prevented pilocarpine-induced mortality, which was observed only in rats pretreated with saline or JMV-2959. After kainate administration, all rats survived to status epilepticus. These findings demonstrate that desacyl-ghrelin, hexarelin and EP-80317 but not other GHSR1a ligands display relevant anticonvulsive properties in models of limbic seizures.
Keywords: Desacyl-ghrelin; Epilepsy; Ghrelin; Growth hormone secretagogue; Kainate; Pilocarpine;

The present study examined the effect of dexmedetomidine, an α2 adrenoceptor agonist, on endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes). We also explored the possible mechanism that triggers dexmedetomidine to act. Dexmedetomidine dose-dependently inhibited the release of glutamate evoked by the K+ channel blocker 4-aminopyridine. Presynaptic α2A adrenoceptors were involved in this release inhibition, with the α2A antagonist (but not by the α2B/C antagonist) blocking the dexmedetomidine-mediated inhibition. The effect of dexmedetomidine on the evoked glutamate release was prevented by the chelating extracellular Ca2+ ions, and by the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate did not have any effect on the action of dexmedetomidine. Dexmedetomidine decreased the degree of depolarization-induced increase in the intrasynaptosomal Ca2+ levels, but did not affect the synaptosomal membrane potential. The inhibitory effect of dexmedetomidine on evoked glutamate release was abolished by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but was insensitive to the endoplasmic reticulum ryanodine receptors or mitochondrial Na+/Ca2+ exchange. In addition, the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors prevented dexmedetomidine from inhibiting glutamate release. Further, western blotting showed that dexmedetomidine decreased the 4-aminopyridine-induced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase 1 and 2 and synapsin I, the main presynaptic target of mitogen-activated protein kinase. Thus, we concluded that dexmedetomidine acts at α2A adrenoceptors present on cerebrocortical nerve terminals inhibit the release of glutamate. We further concluded that this effect is linked to the suppression of voltage-dependent Ca2+ channels and mitogen-activated protein kinase activity.
Keywords: Dexmedetomidine; α2 adrenoceptor agonist; Glutamate release; Voltage-dependent Ca2+ channel; Mitogen-activated protein kinase; Synaptosome;

Maslinic acid, a natural triterpenoid compound from Olea europaea, protects cortical neurons against oxygen–glucose deprivation-induced injury by Yisong Qian; Teng Guan; Xuzhen Tang; Longfei Huang; Menghao Huang; Yunman Li; Hongbin Sun (148-153).
Maslinic acid is a triterpenoid compound present in plants of Olea europaea. This compound has been reported to have potent antioxidant, anti-cancer, anti-HIV and anti-inflammatory activities. In this study, we investigated the neuroprotective effect of maslinic acid and its mechanism of action. With presence or absence of maslinic acid, cortical neurons were subjected to 1 h of oxygen–glucose deprivation and 24 h of reoxygenation. Cell injury was determined by lactate dehydrogenase (LDH) measurement and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Neuronal apoptosis was evaluated by flow cytometry assay, caspase-3 expression/activity, caspase-9 activity and Bcl-2/Bax ratio. Nitric Oxide (NO) production and inducible nitric oxide synthase (iNOS) expression were also detected. Results showed that maslinic acid dose-dependently ameliorated neuron injury and apoptosis. Maslinic acid treatment normalized the caspase expression/activation and increased the Bcl-2/Bax ratio. In addition, maslinic acid inhibited oxygen–glucose deprivation-induced NO production and iNOS expression. These results indicated that maslinic acid has beneficial effects on hypoxic neurons by suppressing iNOS activation, which may, in turn, provide neuroprotection.
Keywords: Maslinic acid; Pentacyclic triterpene; Oxygen–glucose deprivation; Neuron; iNOS; Apoptosis;

In animal models, N-methyl-d-aspartate (NMDA) receptors antagonists inhibit physical dependence and the reinforcing effects of ethanol. The group I metabotropic glutamate (mGlu) receptors antagonists (mGlu1 and mGlu5) attenuate excitatory effect of glutamate by functional modulation of the glutamate/NMDA receptors. The objective of the present study was to evaluate the effects of a selective mGlu5 receptors antagonist—MTEP, and mGlu1 receptors antagonist—EMQMCM, on two processes relevant to alcohol addiction: the expression of ethanol-induced conditioned place preference (CPP) paradigm, and ethanol withdrawal audiogenic seizures in rats. Our experiments indicated that EMQMCM at the doses of 5 and 10 mg/kg, and MTEP at the doses of 2.5 and 5 mg/kg, significantly attenuated the expression of ethanol CPP. Furthermore, both group I mGlu receptor antagonists, i.e. EMQMCM at the dose of 10 mg/kg and MTEP at the dose of 5 mg/kg, attenuated audiogenic seizures induced by the sound stimulus 12 h after withdrawal of ethanol in dependent rats. Our study shows the importance of mGlu5 and mGlu1 receptors for the expression of ethanol-induced CPP and withdrawal seizures, although mGlu5 receptors antagonist (MTEP) was more potent than the antagonist of mGlu1 receptors (EMQMCM).
Keywords: Ethanol; Expression of CPP; Withdrawal seizure; Group I mGlu receptors antagonist; EMQMCM; MTEP;

To clarify the mechanisms by which compound 48/80 (C48/80) induces scratching behavior, the involvement of dopamine D1 receptors was investigated. The intracisternal (i.t.) administration of SCH23390 (1.0 μg), a selective dopamine D1 receptor antagonist, significantly decreased C48/80-induced scratching behavior in mice. These results suggest that dopamine D1 receptors contribute to scratching behavior or the itch sensation induced by subcutaneous injection of C48/80 in mice. Co-administration of SCH23390 and C48/80 enhanced c-fos immunoreactivities in the peduncular part of the lateral hypothalamus (PLH), whereas the immunoreactivities in the other groups were unchanged. The dopaminergic system may be playing an important role in the suppression of C48/80-induced scratching behavior by SCH23390.
Keywords: Compound 48/80; Scratching behavior; SCH23390; c-fos;

Effects of gestational isoflurane exposure on postnatal memory and learning in rats by Feijuan Kong; Linhao Xu; Daqiang He; Xiaoming Zhang; Huishun Lu (168-174).
A maternal fetal rat model was developed to study the effects of gestational isoflurane exposure on postnatal memory and learning and investigate the potential mechanisms. Pregnant rats at gestational day 14 were exposed to 1.3% isoflurane for 4 h. Spatial learning and memory of the offspring were examined using the Morris Water Maze. The expression levels of C/EBP homologous transcription factor protein (CHOP) and caspase-12 in the hippocampus of the pups were determined by immunohistochemistry and western blot analysis. Simultaneously, the ultrastructure changes of synapse in the hippocampal CA1 and dentate gyrus region were also observed by transmission electron microscopy (TEM). Prenatal exposure to isoflurane impaired postnatal spatial memory and learning in the offspring rats as shown by the longer escape latency and the fewer times of original platform crossing in the Morris Water Maze test. The number of CHOP and caspase-12 positive neurons significantly increased by 138% and 147% respectively in the hippocampus of isoflurane-exposed pups, as well as the levels of CHOP and caspase-12 protein. Furthermore, TEM studies showed changes of synaptic ultrastructure in isoflurane-exposed hippocampus characterized by the decreased synapse number, the widened synaptic cleft and the thinned postsynaptic densities. These results demonstrate that gestational exposure to a clinically relevant concentration of isoflurane could cause neuron apoptosis, changes of synaptic structure, and postnatal spatial memory and learning impairments in offspring. Our study further showed that the up-regulation of CHOP and caspase-12 may contribute to isoflurane-induced neuron apoptosis.
Keywords: Isoflurane; Memory and learning impairment; Hippocampus; C/EBP homologous protein; Caspase-12; Neuron apoptosis;

Glycyrrhetinic acid prevents cutaneous scratching behavior in mice elicited by substance P or PAR-2 agonist by Yuko Akasaka; Tsuyoshi Yoshida; Michiko Tsukahara; Akira Hatta; Hideo Inoue (175-179).
Although glycyrrhetinic acid (GA) has been used for the prevention of itch in chronic dermatitis, the mechanism underlying the antipruritic effects of GA is still unclear. Recently, several mediators other than histamine, such as substance P and tryptase, were found to participate in chronic itch. Here, we investigated the effect of GA on pruritus induced by various pruritic agents including histamine in mice. We also determined the level of leukotriene (LT)B4 in mouse skin injected with substance P in an effort to uncover part of the antipruritic mechanism of GA. Scratching events were counted for 10 min after intradermal injection of histamine, substance P (100 nmol per site each), protease-activated receptor-2 (PAR-2) agonistic peptide (50 nmol per site), or LTB4 (0.03 nmol per site) with or without GA (4 nmol per site) into male ICR mice. Levels of LTB4 in the skin after injection of substance P were determined by ELISA. GA did not suppress scratching behavior induced by histamine and LTB4, but markedly and dose-dependently suppressed that induced by substance P and PAR-2 agonistic peptide. LTB4 levels in skin elevated by substance P were lowered by GA. These data support the efficacy of GA in counteracting itch in chronic dermatitis because GA reduced scratching behavior induced by substance P and PAR-2 agonistic peptide. GA may exert antipruritic effects via inhibition of LTB4 production in skin.
Keywords: Glycyrrhetinic acid; Substance P; Protease-activated receptor (PAR)-2; Histamine; Leukotriene (LT)B4; Scratching behavior;

Effects of opioidergic systems upon anxiolytic-like behaviors induced in cholestatic rats by Delaram Eslimi; Sharhbano Oryan; Mohammad Nasehi; Mohammad Reza Zarrindast (180-185).
Cholestasis is associated with endotoxinemia and elevated serum levels of bile acids, cholesterol, bile salts and opioids. Opioid systems have been reported to modulate anxiety. In the present study, the possible involvement of opioidergic system on anxiolytic-like behaviors induced by cholestasis was investigated. Cholestasis was induced in male Wistar rats by ligation of the main bile duct using two ligatures and transecting the duct at the midpoint between them. A standard elevated plus-maze was used to determine anxiety levels in animals. The data indicated that there is an increase, 13 days (but not 10 days) after bile duct ligation, in the percentage of open arm time (%OAT) and open arm entries (%OAE) but not locomotor activity, grooming, rearing and defecation, showing anxiolytic-like effects of cholestasis. Intraperitoneal (i.p.) injection of a subthreshold dose of morphine (4 mg/kg), 15 min before testing 10 days after bile duct ligation, showed an increase in %OAT and %OAE, suggesting an anxiolytic-like effect for the drug. Furthermore, injection of subthreshold doses of naloxone (0.4 and 0.6 mg/kg, i.p.), 15 min before testing 13 days after bile duct ligation, decreased %OAT and %OAE. This indicates that naloxone blocks anxiolytic-like behaviors induced by cholestasis. Also, injection of a subthreshold dose of naloxone (0.4 mg/kg, i.p.), 15 min before the injection of a subthreshold dose of morphine (4 mg/kg, i.p.), 10 days after bile duct ligation, decreased %OAT and %OAE but not other behaviors. In conclusion, the results show the involvement of opioidergic system in anxiolytic-like behaviors induced by cholestasis.
Keywords: Cholestasis; Morphine; Naloxone; Anxiolytic-like response; Elevated plus-maze; Rat;

Pharmacological characterization and expression of VIP and PACAP receptors in isolated cranial arteries of the rat by Michael Baun; Anders Hay-Schmidt; Lars Edvinsson; Jes Olesen; Inger Jansen-Olesen (186-194).
Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) are potent vasodilators in animals and humans. PACAP infusion but not VIP infusion precipitates migraine attacks in migraine patients. The vascular effects of VIP and the two varieties of PACAP (PACAP-27 and PACAP-38) were investigated versus selective antagonists in segments of rat middle cerebral arteries (MCA), basilar arteries (BA) and middle meningeal arteries (MMA) using myographs. The luminal and abluminal effects of VIP were studied using perfusion myograph. mRNA expression of the relevant receptors (VPAC1, VPAC2 and PAC1) was examined by in situ hybridization. There was no significant difference in relaxant potency of the peptides in the MCA. In BA the relaxant potency was VIP > PACAP-27 = PACAP-38. Relaxant responses were either absent or very weak in MMA. VIP was found to be somewhat more potent in BA than in the MCA. Maxadilan, a selective PAC1-receptor agonist, showed no relaxant effect in either vessel. The VPAC2-antagonist PG 99-465 alone proved ineffective in the MCA, while it had a weak effect on BA. The VPAC1-antagonist PG 97-269 inhibited relaxation induced by both VIP and the PACAPs in cerebral vessels. In combination, the two antagonists demonstrated better effect than either alone. VIP applied luminally via perfusion myograph caused no dilatation, indicating lack of endothelial involvement. In situ hybridization demonstrated the presence of mRNA for all three receptors in the smooth muscle cells of the vessels. In conclusion, migraine-like headache induced by PACAP-38 infusion is unlikely to be caused by direct vasodilator action on intracranial vessels.
Keywords: PACAP; VIP; Myograph; Perfusion myograph; In situ hybridization; Migraine; Neuropeptide; VPAC1; VPAC2; PAC1;

C-122, a novel antagonist of serotonin receptor 5-HT2B, prevents monocrotaline-induced pulmonary arterial hypertension in rats by David A. Zopf; Liomar A.A. das Neves; Kristen J. Nikula; Jinbao Huang; Peter B. Senese; Michael R. Gralinski (195-203).
Pulmonary arterial hypertension (PAH) is a chronic disease characterized by sustained elevation of pulmonary arterial pressure that leads to right ventricle failure and death. Pulmonary resistance arterioles in PAH undergo progressive narrowing and/or occlusion. Currently approved therapies for PAH are directed primarily at relief of symptoms by interfering with vasoconstrictive signals, but do not halt the microvascular cytoproliferative process. In this study we show that C-122 (2-amino-N-(2-{4-[3-(2-trifluoromethyl-phenothiazin-10-yl)-propyl]-piperazin-1-yl}-ethyl)-acetamide trihydrochloride, a novel antagonist of serotonin receptor 5-HT2B (Ki = 5.2 nM, IC50  = 6.9 nM), when administered to rats for three weeks in daily oral 10 mg/kg doses, prevents not only monocrotaline (MCT)-induced elevations in pressure in the pulmonary arterial circuit (19 ± 0.9 mm Hg vs. 28 ± 2 mm Hg in MCT-vehicle group, P < 0.05) and hypertrophy of the right ventricle (right ventricular wt./body wt. ratio 0.52 ± 0.02 vs. 0.64 ± 0.04 in MCT-vehicle group, P < 0.05), but also muscularization of pulmonary arterioles (23% vs. 56% fully muscularized in MCT-vehicle group, P < 0.05), and perivascular fibrosis in the lung. C-122 is orally absorbed in the rat, and partitions strongly into multiple tissues, including heart and lung. C-122 has significant off-target antagonist activity for histamine H-1 and several dopamine receptors, but shows no evidence of crossing the blood–brain barrier after a single 10 mg/kg oral dose in rats. We conclude that C-122 can prevent microvascular remodeling and associated elevated pressures in the rat MCT model for PAH, and offers promise as a new therapeutic entity to suppress vascular smooth muscle cell proliferation in PAH patients.
Keywords: Pulmonary arterial hypertension; Rat monocrotaline model; Serotonin antagonist; Antiproliferative; Vascular remodeling; Hemodynamics;

In this communication we show that the same principle that underlies the use of the isobolograph for assessing agonist interactions also leads to a method for analyzing the opposing effects of a single agonist. This is the principle of dose equivalence whose application is illustrated here and applied to the endothelium-dependent relaxing component of two putative vasoconstrictor peptides. These studies, employing angiotensin II and endothelin-1, were conducted with isolated preparations of rat aorta that were measured for agonist-induced isometric tension development in both endothelial-denuded and -intact vessels. The dose–effect relation of the relaxing component of each agonist, which should not be calculated from simple effect subtraction, was derived by the method described here.
Keywords: Isobole; Dose equivalence; Additivity; Endothelium; Angiotensin II; Endothelin-1;

Proximal tubules and podocytes are toxicity targets of bucillamine in a mouse model of drug-induced kidney injury by Yoko Fujiwara; Hiroyoshi Tsuchiya; Nobuya Sakai; Katsushi Shibata; Akio Fujimura; Taka-aki Koshimizu (208-215).
Effective detection of potential nephrotoxicity is crucial for pre-clinical drug development. We evaluated a sensitive animal model for drug-induced kidney injury, which includes hemi-nephrectomy of mice. Although bucillamine and d-penicillamine are used for the treatment of rheumatoid arthritis in Japan, drug-related adverse effects on the kidney can limit their therapeutic utilities. When bucillamine (1000 or 2000 mg/kg/day) or d-penicillamine (2000 mg/kg/day) were orally administered to hemi-nephrectomised BALB/c mice, the urinary protein levels of bucillamine-treated mice, but not of those treated with d-penicillamine, the vehicle, or in bucillamine treated unnephrectomized mice, were significantly increased and remained high during the 4-week drug-loading period. Membranous glomerulonephropathy occasionally seen in bucillamine/d-penicillamine-treated arthritis patients was not reproduced in mice. Instead, our mouse model showed proximal tubular injury and podocyte foot process effacement in the bucillamine-treated kidneys. These two cell types are also the primary targets of the experimental Heymann membranous glomerulonephropathy. Gene expression profiling of the bucillamine-treated mice identified lipocalin 2 as a significantly up-regulated transcript together with cytochrome P450 CYP4a14, a group-specific component, and proprotein convertase subtilisin/kexin type 9. Moreover, large amounts of lipocalin 2 were detected in the urine of the bucillamine-treated mice, but not in the hemi-nephrectomised control mice. These results indicate that hemi-nephrectomy effectively promotes acute kidney injury by bucillamine, which is accompanied by up-regulation of the urinary biomarker lipocalin 2. Our mouse model with initial stage of kidney injury should be useful to analyse the pathogenesis of drug-induced glomerular and tubular injuries.
Keywords: NGAL (Neutrophil gelatinase-associated lipocalin); Bucillamine; d-Penicillamine; Nephrotoxicity; Animal model; DNA microarray;

The therapeutic potential of cannabidiol, the major non-psychotropic Cannabis constituent, was investigated in rats exposed to ischemia/reperfusion liver injury. Ischemia was induced by clamping the pedicle of the left hepatic lobe for 30 min, and cannabidiol (5 mg/kg, i.v.) was given 1 h following the procedure and every 24 h thereafter for 2 days. Ischemia/reperfusion caused significant elevations of serum alanine aminotransferase and hepatic malondialdehyde, tumor necrosis factor-α and nitric oxide levels, associated with significant decrease in hepatic reduced glutathione. Cannabidiol significantly attenuated the deterioration in the measured biochemical parameters mediated by ischemia/reperfusion. Histopathological examination showed that cannabidiol ameliorated ischemia/reperfusion-induced liver damage. Immunohistochemical analysis revealed that cannabidiol significantly reduced the expression of inducible nitric oxide synthase, cyclooxygenase-2, nuclear factor-κB, Fas ligand and caspase-3, and increased the expression of survivin protein in ischemic/reperfused liver tissue. These results emphasize that cannabidiol represents a potential therapeutic option to protect the liver against hypoxia–reoxygenation injury.
Keywords: Cannabidiol; Ischemia/reperfusion; Oxidative stress; Inflammation; Liver;

Antidepressants, including selective serotonin reuptake inhibitors (SSRIs), cause erectile dysfunction; however, the mechanism by which they cause erectile function is unclear. We investigated the reactivity of the corpus cavernosum after chronic fluoxetine treatment in rabbits. Twelve rabbits were randomly divided into two groups: control (n = 6) or 20 mg/kg/day of fluoxetine delivered i.p. (n = 6). The reactivity of the corpus cavernosum tissue from the fluoxetine-treated and control groups was studied in organ chambers after 21 days of fluoxetine injection. In the fluoxetine-treated group, endothelium-dependent relaxation of the corpus cavernosum in response to acetylcholine was significantly decreased compared to the control group. However, the sensitivity (i.e., pD2) of the fluoxetine-treated cavernosal tissue strips to acetylcholine was not changed with respect to controls. Electrical field stimulation (EFS)-induced neurogenic relaxation was also significantly reduced in the fluoxetine-treated group. Relaxation in response to the nitric oxide (NO) donor sodium nitroprusside was similar between the cavernosal tissues from the two groups. There was also no change in agonist potency between the two groups. Additionally, chronic fluoxetine treatment had no effect on KCl-induced contractile responses. When tissue contraction was produced with phenylephrine to study relaxation in response to various stimuli, the tension induced was similar between the fluoxetine-treated and control groups. This study suggests that chronic fluoxetine treatment causes significant functional changes to the penile erectile tissue of rabbits, and these changes may contribute to the development of impotence.
Keywords: Penis; Corpus cavernosum; Nitric oxide; Fluoxetine; Rabbit; Erectile dysfunction;

Ulcerative colitis is a nonspecific inflammatory disorder characterized by oxidative and nitrosative stress, leucocyte infiltration and up-regulation of pro-inflammatory cytokines. The aim of this study is to evaluate the effect of taurohyodeoxycholic acid (THDCA) isolated from Pulvis Fellis Suis on acute ulcerative colitis model induced by trinitrobenzene sulfonic acid (TNBS) in mice. The efficacy of THDCA was studied by macroscopical and histological scoring systems as well as myeloperoxidase (MPO) activity. Serum levels, including tumor necrosis factor (TNF)-α and interleukin (IL)-6 were assayed by enzyme-linked immunoassay. The expression of cyclooxygenase (COX)-2 in the colons was assessed by immunohistochemical analysis. Treatment with THDCA in doses of 25, 50 and 100 mg/kg/day and sulfasalazine in a dose of 500 mg/kg/day used as reference for 7 consecutive days after the induction of colitis, significantly decreased colonic MPO activity, TNF-α, IL-6 serum levels and the expression of COX-2 in colon compared with TNBS induced ulcerative colitis model group. Moreover, THDCA attenuated the macroscopic colonic damage and the histopathological changes induced by TNBS. All the effects of these parameters were comparable to that of the standard sulfasalazine, especially at the highest dose level. The results suggested that THDCA from Pulvis Fellis Suis has a protective effect in TNBS-induced ulcerative colitis which might be due to its anti-inflammatory activities, and that it may have therapeutic value in the setting of inflammatory bowel disease.
Keywords: Ulcerative colitis; Taurohyodeoxycholic acid; MPO; TNF-α; IL-6; COX-2;

Muscarinic receptor subtypes involved in regulation of colonic motility in mice: Functional studies using muscarinic receptor-deficient mice by Takaji Kondo; Miwa Nakajima; Hiroki Teraoka; Toshihiro Unno; Sei-ichi Komori; Masahisa Yamada; Takio Kitazawa (236-243).
Although muscarinic M2 and M3 receptors are known to be important for regulation of gastric and small intestinal motility, muscarinic receptor subtypes regulating colonic function remain to be investigated. The aim of this study was to characterize muscarinic receptors involved in regulation of colonic contractility. M2 and/or M3 receptor knockout (KO) and wild-type mice were used in in vivo (defecation, colonic propulsion) and in vitro (contraction) experiments. Amount of feces was significantly decreased in M3R-KO and M2/M3R-KO mice but not in M2R-KO mice. Ranking of colonic propulsion was wild-type = M2R-KO > M3R-KO > M2/M3R-KO. In vitro, the amplitude of migrating motor complexes in M2R-KO, M3R-KO and M2/M3R-KO mice was significantly lower than that in wild-type mice. Carbachol caused concentration-dependent contraction of the proximal colon and distal colon from wild-type mice. In M2R-KO mice, the concentration-contraction curves shifted to the right and downward. In contrast, carbachol caused non-sustained contraction and relaxation in M3R-KO mice depending on its concentration. Carbachol did not cause contraction but instead caused relaxation of colonic strips from M2/M3R-KO mice. 4-[[[(3-chlorophenyl)amino]carbonyl]oxy]-N,N,N-trimethyl-2-butyn-1-aminium chloride (McN-A-343) caused a non-sustained contraction of colonic strips from wild-type mice, and this contraction was changed to a sustained contraction by tetrodotoxin, pirenzepine and L-nitroarginine methylester (L-NAME). In the colon of M2/M3R-KO mice, McN-A-343 caused only relaxation, which was decreased by tetrodotoxin, pirenzepine and L-NAME. In conclusion, M1, M2 and M3 receptors regulate colonic motility of the mouse. M2 and M3 receptors mediate cholinergic contraction, but M1 receptors on inhibitory nitrergic nerves counteract muscarinic contraction.
Keywords: Mouse colon; Muscarinic receptor; Knockout mouse; Nitrergic nerve;

Long-acting fluticasone furoate has a superior pharmacological profile to fluticasone propionate in human respiratory cells by Christos Rossios; Yasuo To; Masako To; Misako Ito; Peter J. Barnes; Ian M. Adcock; Malcolm Johnson; Kazuhiro Ito (244-251).
Currently available glucocorticoids are relatively short acting and may be less effective in patients with chronic obstructive pulmonary disease (COPD) where high levels of oxidative stress are seen. Here we show that a novel glucocorticoid, fluticasone furoate (FF), has a longer duration of action in several cell systems compared with fluticasone propionate (FP) and budesonide, and unlike FP, FF is resistant to oxidative stress. FF had similar or slightly higher potency to FP and was 2–9 fold more potent than budesonide, when assessed at 4 h, in inhibiting inflammatory cytokine production in epithelial cell lines (BEAS2B, A549), primary bronchial epithelial cells and a monocytic cell line (U937). The potency of FF was sustained beyond 16 h with or without washout compared with FP or budesonide, such that it showed a greater duration of action in this range of cellular assays. The activated YFP-conjugated glucocorticoid receptor was detectable in nuclei of FF treated BEAS2B cells for at least for 30 h, and FF had a longer duration of action than FP in inhibiting activation of transcription factors such as NF-κB and AP-1. In addition, FF showed superior effects to FP in peripheral blood mononuclear cells from patients with COPD and also in U937 cells or primary bronchial epithelial cells under conditions of oxidative stress. The longer duration of action and oxidative stress insensitivity of FF compared with FP has potential clinical implications for the control of inflammation in respiratory diseases, such as COPD.
Keywords: Chronic obstructive pulmonary disease (COPD); Steroid insensitivity; Fluticasone furoate; Glucocorticoid receptor; Long acting;

Large conductance voltage- and Ca2+-activated K+ (BK) channels are important in regulating detrusor smooth muscle (DSM) function. Here, we examined systematically how the BK channel pharmacological activation modulates DSM contractility. NS1619, a potent BK channel activator, was utilized as a pharmacological tool to investigate the effect of BK channel activation on rat DSM contractility. Isometric tension recordings of DSM strips isolated from rat urinary bladder were performed systematically under various experimental conditions. NS1619 (30 μM) substantially diminished DSM spontaneous contraction amplitude, muscle force integral, frequency, duration and muscle tone. This effect was blocked by iberiotoxin, a BK channel selective inhibitor. NS1619 inhibited the phasic and tonic contractions in DSM strips pre-contracted with either the cholinergic agonist, carbachol (0.1 μM), or the depolarizing agent, KCl (20 mM). In the presence of elevated KCl (60 mM KCl), the inhibitory effect of NS1619 was significantly reduced, indicating that BK channel activation is the underlying mechanism of NS1619 action. BK channel activation with NS1619 dramatically decreased the amplitude of electrical field stimulation (EFS)-induced contractions under a range of stimulation frequencies (0.5–50 Hz). In the presence of specific neurotransmitter inhibitors, BK channel activation with NS1619 significantly decreased both cholinergic and purinergic components of EFS-induced contractions. We conclude that BK channel activation with NS1619 significantly inhibited spontaneous, pharmacologically induced and nerve-evoked DSM contractions. Targeting the BK channel with selective openers may offer a unique opportunity to control DSM contractile activity, including pathophysiological conditions such as overactive bladder and detrusor overactivity, regardless of the underlying cause.
Keywords: Urinary bladder; K+ channel opener; Iberiotoxin; Detrusor contractility;

Effects of age and hypertension on α1-adrenoceptors in the major source arteries of the rat bladder and penis by Makoto Yono; Takanori Tanaka; Shigeki Tsuji; Shin Irie; Yukikuni Sakata; Masayuki Otani; Masaki Yoshida; Jamshid Latifpour (260-265).
α1-Adrenoceptors regulate blood pressure, regional vascular resistance and tissue blood flow. As aging and hypertension may impact pelvic arterial blood flow resulting in bladder and penile dysfunction, we investigated effects of age and hypertension on α1-adrenoceptors in the major source arteries of the rat bladder and penis. Using radioligand receptor binding, real-time reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent microsphere infusion techniques, we compared 3 and 22-month-old male Fischer rats, and male normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Twenty-two-month-old rats and SHRs had significantly higher total α1-adrenoceptor density in the internal iliac artery and lower blood flow to the bladder and penis than 3-month-old and WKY rats, respectively. RT-PCR data showed an age and hypertension related increase in the expression of α1B-adrenoceptor mRNA in the internal iliac, vesical and internal pudendal arteries and a switch from α1A predominance in 3-month-old and WKY rats to α1B  > α1A in 22-month-old rats and SHRs. Our data indicate the presence of age and hypertension related alterations in vascular α1-adrenoceptor subtype distribution and in blood flow to the rat bladder and penis. These findings suggest that pharmacological blockade of the vascular α1B-adrenoceptor, which could increase pelvic blood flow, may contribute to the improvement of bladder and penile dysfunctions in animal models for aging and hypertension.
Keywords: Aging; Hypertension; Vascular α1-adrenoceptor; Bladder; Penis; Rat;

Kinetics of relaxation by cGMP/cGKI signaling in fundus smooth muscle by Claudia Ertl; Robert Lukowski; Katja Sigl; Jens Schlossmann; Franz Hofmann; Jörg W. Wegener (266-271).
cGMP-dependent kinase I (cGKI) is a major mediator of smooth muscle relaxation and exists in two isoforms, α and β. Both isoforms are supposed to mediate their effects via different intracellular signaling pathways. To verify this concept, the kinetics of relaxation mediated by either isoform was analyzed in gastric fundus smooth muscle from mice. Muscles from mice that express selectively the Iα or Iβ isoform of cGKI in smooth muscle (sm-cGKIα or sm-cGKIβ mice) were compared to muscles from conventional cGKI−/− mice. Fundus muscles were contracted by carbachol and then relaxed by 8-Br-cGMP or by electrical field stimulation (EFS). The time course of relaxation by 8-Br-cGMP was not different between muscles from sm-cGKIα and sm-cGKIβ mice. EFS induced a fast transient relaxation in muscles from sm-cGKIα and sm-cGKIβ mice that was blocked by the NO synthase inhibitor L-NAME. Recovery from this relaxation was about 4-times slower in muscles from sm-cGKIα mice than in muscles from sm-cGKIβ mice. The different kinetic of recovery from relaxation after EFS in sm-cGKIα and sm-cGKIβ mice suggests that different signaling pathways exist for each cGKI isoform in vivo in fundus muscles.
Keywords: Fundus; Relaxation; NANC; Nitric oxide; cGMP;

Thalidomide modulates Mycobacterium leprae-induced NF-κB pathway and lower cytokine response by Maristela de Oliveira Hernandez; Tatiana de Oliveira Fulco; Roberta Olmo Pinheiro; Renata de Meirelles Santos Pereira; Paulo Redner; Euzenir Nunes Sarno; Ulisses Gazos Lopes; Elizabeth Pereira Sampaio (272-279).
It is widely accepted that tumor necrosis factor alpha (TNF-α) plays a critical role in the development of tissue and nerve damage in leprosy and during the reactional episodes of acute inflammation. Thalidomide (N-α-phthalimidoglutarimide), a drug used to treat leprosy reaction, modulates immune response, inhibits inflammation and NF-κB activity. Here we investigated whether thalidomide inhibits NF-κB activation induced by Mycobacterium leprae, p38 and ERK1/2 MAPK activation. EMSA and supershift assays were performed to investigate NF-κB activation in response to M. leprae and its modulation following in vitro treatment with thalidomide. Luciferase assay was assayed in transfected THP-1 cells to determine NF-κB transcriptional activity. Flow cytometry and immunofluorescence were used to investigate p65 accumulation in the nucleus. Immunoblotting was used to investigate p38 and ERK1/2 phosphorylation. Following activation of PBMC and monocytes with M. leprae, the formation and nuclear localization of NF-κB complexes composed mainly of p65/p50 and p50/p50 dimers was observed. Induction of NF-κB activation and DNA binding activity was inhibited by thalidomide. The drug also reduced M. leprae-induced TNF-α production and inhibited p38 and ERK1/2 activation. Definition of the activation mechanisms in cells stimulated with M. leprae can lead to the development of new therapy applications to modulate NF-κB activation and to control the inflammatory manifestations due to enhanced TNF-α response as observed in leprosy and in leprosy reactions.
Keywords: Thalidomide; NF-κB; TNF-α; M. leprae; Leprosy;

Antinociceptive and anti-inflammatory properties of 7-epiclusianone, a prenylated benzophenone from Garcinia brasiliensis by Flávia V. Santa-Cecília; Lissara A.S. Freitas; Fabiana C. Vilela; Clarice de C. Veloso; Cláudia Q. da Rocha; Maria E.C. Moreira; Danielle F. Dias; Alexandre Giusti-Paiva; Marcelo H. dos Santos (280-285).
7-Epiclusianone, a natural prenylated benzophenone, was extracted from Garcinia brasiliensis Planch. & Triana (Clusiaceae), a native plant commonly known as bacupari and used in traditional Brazilian medicine for the treatment of inflammatory diseases. As a result of the wide spectrum of biological activities attributed to polyisoprenylated benzophenones, the aim of this study was to evaluate the analgesic and anti-inflammatory effects of 7-epiclusianone using two animal models. Carrageenan-induced paw oedema and peritonitis were used to investigate the anti-inflammatory activity of 7-epiclusianone in rats. The acetic acid-induced writhing, formalin and hot-plate tests were used to investigate its antinociceptive activity in mice. At test doses of 5, 10 and 15 mg/kg p.o., 7-epiclusianone had an anti-inflammatory effect as demonstrated by the reduction of paw oedema induced by carrageenan and the inhibition of leukocyte recruitment into the peritoneal cavity. At the same doses, 7-epiclusianone inhibited nociception induced by an intraperitoneal injection of acetic acid, observed by the decrease in the number of writhing episodes. Additionally, 7-epiclusianone decreased licking time caused by a subplantar injection of formalin. Moreover, the hot plate test produced a significant increase in latency reaction, demonstrating an antinociceptive effect. The experimental data demonstrated that the polyisoprenylated benzophenone 7-epiclusianone has remarkable anti-inflammatory and antinociceptive activities.Display Omitted
Keywords: 7-Epiclusianone; Garcinia brasiliensis; Anti-inflammatory; Antinociceptive;

Regulatory effect of daphnetin, a coumarin extracted from Daphne odora, on the balance of Treg and Th17 in collagen-induced arthritis by Rongfeng Yao; Yingyuan Fu; Sha Li; Lina Tu; Xiaoping Zeng; Nanzhen Kuang (286-294).
Daphnetin extracted from Daphne odora Var. marginata contains coumarin compounds, which possess properties of analgesic and anti-inflammatory effects. In this study, we investigated the therapeutic effect of daphnetin on anti-arthritis and its role on the balance of Tregs and Th17, using a collagen-induced arthritis rat model. Collagen-induced arthritis rats were treated with daphnetin for 21 days. The therapeutic effects of daphnetin were evaluated by clinical symptoms and histopathology. The levels of Th17-, Treg-, Th2-, Th1-type cytokines in serum were determined by ELISA. The expression levels of related receptors RORγt, NF-κB, Foxp3 and CD77 in joint tissues were detected by immunohistochemistry. Our results showed that administration of daphnetin significantly alleviated the severity of the arthritis, as evidenced by the reduction of arthritis scores, suppression of the infiltration of inflammatory cells and prevention of synovial hyperplasia, thereby resulting in the joint destruction in the arthritis rats. Additionally, daphnetin treatment reduced the serum level of Th17-, Th2- and Th1-type in collagen-induced arthritis rats. Correspondingly, the expression of RORγt, NF-κB and CD77 in joint tissue of collagen-induced arthritis rats was remarkably decreased, while the expression of Foxp3 and IL-10 was remarkably increased after being administered with daphnetin. Collectively, this study demonstrated that administration of daphnetin attenuated the clinical symptoms and pathological destruction of arthritis joints. The therapeutic effects were associated with the up-regulation of Tregs, down-regulation of Th17-, Th2- and Th1-type cell responses. The results provide novel evidence that daphnetin has therapeutic effects on autoimmune arthritis through modulating the balance of Tregs and Th17.
Keywords: Daphnetin; Rheumatoid arthritis; Collagen-induced arthritis; Th17; Treg; RORγt; Foxp3; TGF-β; IL-17;

Anti-inflammatory effects of lindenenyl acetate via heme oxygenase-1 and AMPK in human periodontal ligament cells by Gil-Saeng Jeong; Dong-Sung Lee; Bin Li; Jong-Jin Kim; Eun-Cheol Kim; Youn-Chul Kim (295-303).
The molecular basis for the anti-inflammatory effects of lindenenyl acetate (LA) was investigated in the lipopolysaccharide (LPS)-stimulated human periodontal ligament (HPDL) cell model. LA concentration-dependently inhibited LPS-induced inducible nitric oxide synthase (iNOS) derived nitric oxide (NO) and cyclooxygenase-2 (COX-2) derived prostaglandin E2 (PGE2) production in HPDL cells. LA also attenuated the production of LPS-induced tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-12. LA stimulated heme oxygenase-1 (HO-1) protein expression and enzyme activity of HPDL cells in a dose-dependent manner. Pretreatment with the HO-1 inhibitor, tin protoporphyrin (SnPP), attenuated the inhibitory activities of LA on LPS-induced inflammatory NO, PGE2, IL-1β, TNF-α, IL-6 and IL-12 production. LA induced translocation of Nrf-2. Furthermore, an inhibitor of JNK MAPK abolished LA-induced HO-1 expression. LA exposure up-regulated the levels of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and its upstream kinase activators, including LKB1 and Ca2+/calmodulin-dependent protein kinase kinase-II. Furthermore, compound C, a specific AMPK inhibitor, partially blocked the LA-induced anti-inflammatory effect. Taken together, these results indicate that LA has anti-inflammatory activity in HPDL cells that might be mediated by the HO-1, AMPK, JNK MAPK, and Nrf-2 pathways. Thus, LA may serve as a potential therapeutic agent in periodontal disease.
Keywords: Lindenenyl acetate; Inflammation; Periodontal ligament cell; Heme oxygenase-1; AMP-activated protein kinase;

Inhibiting effects of Leflunomide metabolite on overexpression of CD147, MMP-2 and MMP-9 in PMA differentiated THP-1 cells by Jian-lin Huang; Shi-yao Wu; Xu-jing Xie; Ming-xia Wang; Shangling Zhu; Jie-ruo Gu (304-310).
Recent studies have reported elevated expression of cluster of differentiation (CD) 147 on CD14+ monocytes of the peripheral blood of patients with active rheumatoid arthritis and a correlation of CD147 expression with Disease Activity Score. Thus, CD147 may be a new target for treatment of rheumatoid arthritis. Leflunomide is a disease-modifying antirheumatic drug that is commonly used to treat rheumatoid arthritis. The effect of leflunomide in blocking the up-regulation of CD147 and in blocking the down-regulation of metalloproteinases (MMP)-2 and MMP-9 in active macrophages has not yet been established. In this study we investigated the effect of A771726, the active metabolite of leflunomide, on expression of CD147 and on the gelatinolytic activity of MMP-2 and MMP-9 in phorbol myristate acetate (PMA) differentiated THP-1 cells. The expression of CD147, MMP-2, and MMP-9 mRNAs were determined by real-time quantitative reverse transcription PCR, the levels of cellular surface expression of CD147 were determined by flow cytometry, and the gelatinolytic activity of MMP-2 and MMP-9 were determined by zymography. Our results showed that A771726 significantly inhibited the expression of CD147 on the cell surface of activated THP-1 cells in a dose-dependent manner (P  < 0.01), inhibited the expression of MMP-2 and MMP-9 mRNAs in a dose-dependent manner (P <  0.01), and inhibited the gelatinolytic activity of MMP-2 and MMP-9 at concentration of 15 μg/ml and 45 μg/ml (P <  0.01). Our results indicate that A771726, the active metabolite of leflunomide, inhibited CD147 expression at the protein level and inhibited gelatinolytic activity of MMP-2 and MMP-9 in PMA-differentiated THP-1 cells.
Keywords: Leflunomide metabolite A771726; Cluster of differentiation 147; Matrix metalloproteinase; Human monocytic cell line THP-1;

Pro-inflammatory cytokine-mediated pancreatic β-cell dysfunction is a key pathological event in type 1 diabetes mellitus. There are few studies about the protection of epigallocatechin-3-gallate (EGCG) against pro-inflammatory cytokine-induced β-cell apoptosis. To examine the direct effects of EGCG on β-cells, insulin-producing RINm5F cells were exposed to a combination of recombinant interleukin-1beta (IL-1β), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ), with or without EGCG pretreatment for 24 h. Cell death was monitored by the MTT assay. Glucose-stimulated insulin release was measured using radio immunoassay. Intracellular reactive oxygen species was examined with dichlorofluorescein (DCF) fluorescence by flow cytometry. To evaluate RINm5F cells mitochondrial function, change in mitochondrial membrane potential, intracellular ATP levels, and nitric oxide was assessed. The expression of cytochrome c, Bax, Bcl-2, and iNOS proteins was measured by western blotting. In the present study, EGCG pretreatment protected against cytokines inducing cell death and restored glucose stimulated-insulin secretion in RINm5F cells. EGCG reduced the cytokine-induced generation of reactive oxygen species, the loss of mitochondrial membrane potential (Δψm), the release of cytochrome c from the mitochondria, and translocation of Bax protein to the mitochondria from the cytosol. EGCG pretreatment prevented cytokine-induced iNOS overexpression and NO generation. In summary, pro-inflammatory cytokines lead to a reduction of glucose-induced insulin secretion, mitochondrial activity and viability in RINm5F cells. The pro-inflammatory cytokine-induced effects can be prevented by EGCG pretreatment via the mitochondrial pathway.
Keywords: Epigallocatechin-3-gallate; Pro-inflammatory cytokine; Insulin-producing cell; Mitochondrial pathway;

Oral administration of quercetin inhibits bone loss in rat model of diabetic osteopenia by Wei Liang; Zhonghua Luo; Shuhua Ge; Mo Li; Junjie Du; Min Yang; Ming Yan; Zhengxu Ye; Zhuojing Luo (317-324).
Diabetic osteopenia can result in an increased incidence of bone fracture and a delay in fracture healing. Quercetin, one of the most widely distributed flavonoids in plants, possesses antioxidant property and beneficial effect on osteoporosis in ovariectomized mice. All these properties make quercetin a potential candidate for controlling the development of diabetic osteopenia. Therefore, the present study was designed to investigate the putative beneficial effect of quercetin on diabetic osteopenia in rats. Diabetes mellitus was induced by streptozotocin. The diabetic rats received daily oral administration of quercetin (5 mg/kg, 30 mg/kg and 50 mg/kg) for 8 weeks, which was started at 4 weeks after streptozotocin injection. Quercetin at 5 mg/kg showed little effect on diabetic osteopenia, while quercetin at 30 mg/kg and 50 mg/kg could increase the decreased serum osteocalcin, serum alkaline phosphatase activity, and urinary deoxypyridinoline in diabetic rats. In addition, quercetin (30 mg/kg and 50 mg/kg) could partially reverse the decreased biomechanical quality and the impaired micro-architecture of the femurs in diabetic rats. Histomorphometric analysis showed that both decreased bone formation and resorption were observed in diabetic rats, which was partially restored by quercetin (30 mg/kg and 50 mg/kg). Further investigations showed that quercetin significantly lowered the oxidative DNA damage level, up-regulated the total serum antioxidant capability and the activity of serum antioxidants in diabetic rats. All those findings indicate the beneficial effect of quercetin on diabetic osteopenia in rats, and raise the possibility of developing quercetin as potential drugs or an ingredient in diet for controlling diabetic osteopenia.
Keywords: Diabetic osteopenia; Quercetin; Bone loss; Antioxidant; Mechanical strength;

Considerable evidence shows that chronic hyperglycemia can cause pancreatic beta-cell dysfunction, which contributes to progressive deterioration of glucose homeostasis and overt diabetes. In the present study, we found that kaempferol, a flavonol compound present in various Chinese medicinal herbs, has cytoprotective effects on cultured clonal beta-cells and pancreatic human islets. Kaempferol treatment dose-dependently promoted viability, inhibited cellular apoptosis, and reduced caspase-3 activity in beta-cells and human islets exposed to chronic high glucose, with 10 μM kaempferol exerting the maximum effect. In addition, kaempferol treatment improved the expression of anti-apoptotic proteins Akt and Bcl-2 that was significantly reduced in beta-cells and human islets chronically exposed to hyperglycemia. Furthermore, exposure of beta-cells and human islets to kaempferol restored high glucose-attenuated intracellular cAMP and ATP production. Inhibition of protein kinase A or Akt activation ablated the anti-apoptotic effect of kaempferol. These cytoprotective effects of kaempferol were associated with improved insulin secretory function and synthesis in beta-cells and human islets. These findings provide evidence that kaempferol may be a naturally occurring anti-diabetic compound by protecting pancreatic beta-cell survival and function in a hostile environment that would otherwise lead to type 2 diabetes.
Keywords: Kaempferol; Beta-cell viability; Insulin secretion; Hyperglycemia;