Effects Of VRACs Inhibitor On Microglia Growth And Activation,and Mechanisms Underlying Such Effects

Part1:Effect of volume regulated anion channels (VRACs) inhibitor on proliferation of mouse microgliaObjective To study the inhibitory effect of volume regulated anion channels (VRACs) inhibitor Tamoxifen and DCPIB on mouse microglia cell line BV-2and the mechanisms underlying such effect.Methods The microglia cell line BV-2was incubated with Tamoxifen and DCPIB in different concentrations for different times. Cell inhibition ratio was assessed by MTT assay and flow cytometric analysis was performed to detect the cell cycle. Furthermore, CyclinDl expression was detected by western blot.Results MTT examination showed that BV-2cells was inhibited and cell growth curve was down-regulated obviously with concentration-dependent and time-dependent manner. Flow cytometry (FCM) showed that most BV-2cells were inhibited by Tamoxifen and DCPIB, and stagnated in G0/G1phase, while proportion of BV-2cells in G2/M phase and S phase was decreased. Western blot showed that the expression level of CyclinDl was down-regulated in BV-2cells after treated with Tamoxifen and DCPIB for72h.Conclusion The results suggest that Tamoxifen and DCPIB can markedly inhibit the proliferation of mouse microglia cell line BV-2cells. The mechanisms of inhibition may involved arresting the cell cycle in GO/Gl phase and down-regulating CyclinDl expression. Part2:Effects of volume regulated anion channels (VRACs) inhibitor on apoptosis of mouse microgliaObjective In the current study, we investigated whether Tamoxifen and DCPIB induce apoptosis in microglia cell line BV-2and whether or not such an effect is associated with the mitochondrial pathway and death receptor pathway.Methods BV-2cells were incubated with Tamoxifen and DCPIB. Cell viability was assessed by MTT assay, flow cytometric analysis was performed to detect the cell apoptosis rate and mitochondrial membrane potential, and Bax, Bcl-2, Fas, Fas-L expression was detected by western blot.Results Our results demonstrated that Tamoxifen and DCPIB decreased cell viability in a concentration-dependent and time-dependent manner, and induced apoptosis of BV-2cells in a concentration-dependent manner. When apoptosis was induced, disruption of the mitochondrial membrane potential preceded, followed by up-regulated the expression of pro-apoptotic Bax, Fas, Fas-L, down-regulated anti-apoptotic Bcl-2.Conclusion These results indicated that Tamoxifen and DCPIB induced apoptosis of BV-2cells through both mitochondria-and death receptor-mediated pathways. Part3: Inhibitory effect of volume regulated anion channels (VRACs) inhibitor on OGD-induced activation of mouse microgliaObjective To understand the inhibitory effect of volume regulated anion channels (VRACs) inhibitor (Tamoxifen and DCPIB) on Oxygen Glucose Deprivation (OGD)-induced activation of microglia, and the mechanism underlying such effect.Methods The microglia cell line BV-2were used, and divided to Control group, OGD group, DMSO group, Tamoxifen (0.luM,1uM)+OGD group and DCPIB (1uM,10uM)+OGD group. After cultured1h,6h and12h, the change of the cell form was observed by microscope. Cell viability was assessed through detecting lactate dehydrogenase (LDH) in supernatants fluid of BV-2cells. Culture supernatants were collected and IL-1β and TNF-a were detected by ELISA assay, and Nitric oxide (NO) was detected. The mRNA of iNOS, IL-1β and TNF-a were detected by RT-PCR.Results Our results demonstrated that Tamoxifen and DCPIB inhibited OGD inducing activation of BV-2cell, and decreased NO, IL-1β and TNF-a in supernatants fluid of BV-2cells(P<0.05).Conclusion These results indicated that Tamoxifen and DCPIB inhibited OGD inducing activation of BV-2cell. The mechanism underlying such effect may via blocking volume regulated anion channels...