DCPIB, A Potent Volume-regulated Anion Channel Antagonist,Attenuates Microglia-mediated Inflammatory Response And Neuronal Injury Following Focal Cerebral Ischemia

Part1:DCPIB attenuates oxygen-glucose deprivation-induced proliferation of microglia and secretion of pro-inflammation cytokinesObjective To explore the influence of DCPIB on the oxygen-glucose deprivation-induced proliferation of microglia and secretion of pro-inflammation cytokines.Methods The microglial cell line BV-2was purchased from the Cell Center of Chinese Academy of Medical Science.Frozen BV-2was cultured in poly-lysine coated24-well plastic culture plates. Microglial cells were randomly grouped into control group, OGD group and OGD+DCPIB group. The culture medium was replaced with glucose-and serum-free medium.And BV-2cells were cultured in an oxygen-deprivation (93%N2/5%CO2/2%O2) incubator at37℃for10min. To terminate OGD and start reperfusion, the cells were returned to normoxia (95%O2/5%CO2) for3h and the medium was replaced with high glucose and serum-free one. After being fixed in4%paraformaldehyde, permeabilized with0.25%Triton X-100for10min and blocked in10%normal goat serum for1h, microglia were co-incubated with relative primary antibodies (OX-42and Ki67、TNF-α、IL-1β respectively)overnight at4℃.Primary antibodies were detected with corresponding fluorescence-conjugated secondary antibodies.. Slides were observed with the help of a fluorescence microscope。All rates of Ki67-,TNF-a-and IL-Iβ-positive microglia were calculated.SPSS19.0was applied to data analysis. Results Ki67was located in nucleus. More significant up-regulated expression of ki-67was shown in OGD group compared to that of in vehicle group (P<0.01, n=6); and expression of ki-67in microglia in OGD+DCPIB (10μM) group indicated obvious down-regulation compared to that of in OGD group (P<0.01, n=6).The positive rates of TNF-αand IL-Iβere elevated in10min OGD group compared to that of in vehicle group. The administration of DCPIB in microglia significantly suppressed the expression of TNF-αand IL-Iβin OGD+DCPIB group compared to that of in OGD group.Conclusion Transient OGD induces microglia proliferation and activation. Proliferation of microglia and secretion of microglal pro-inflammation cytokines are effectively suppressed. Part2:DCPIB suppresses OGD-induced migration of microgliaObjective To explore the effects of DCPIB on OGD-induced migration of microgliaMethods BV-2cells line were cultured in6-well plastic plates. The microglia were randomly grouped into control group, OGD group and OGD+DCPIB group upon the coverage of BV-2cells reaching80%area. Cell monolayers were injured with a sterile micropipette tip and then subjected to10min of OGD in37℃,2%O2,5%CO2incubator with serum-and glucose-free medium in the absence or presence of DCPIB (10μg/mL).The control group cells were incubated in serum-free high glucose medium in37℃,5%CO2incubator.Then, the cells of OGD+DCPIB group were returned to normoxia environment with DCPIB treatmentPhotographs were taken at different times (3,6,12,24, and48h) after transient OGD and reperfusion injury using a phase contrast microscope. The size of migration was analyzed by calculating the denuded area using NIH Image J software, and the SPSS software, version19.0. was used to analyze all data.Results The scratched area was significantly reduced compared to that of in control group (P<0.01, n=6); Such a DCPIB induced depression of microglia migration was significantly enhanced compared to that of in OGD group (P<0.01, n=6).Conclusion Transient OGD stimulates microglial migration, and this change is depressed in the presence of DCPIB. Part3:DCPIB attenuates microglial activation-mediated neuronal injury under ischemic conditions in vitro and in vivoObjective To study the neuroprotective of DCPIB under ischemic conditions in vitro and in vivoMethods Microglia/neuron co-culture system, BV-2cells were subcultured onto24-well Transwell collagen-coated membrane inserts and incubated in basal medium for24h. Then, the Transwell inserts were placed in wells containing primary hippocampal neurons. BV-2cells were cultured in Transwell chamber (in lower chamber). Primary hippocampal neurons were cultured in24-well plastic plates (in upper chamber). the microglia/neuronal cultures system were randomized to control group, OGD group and OGD+DCPIB group, medium was replaced with glucose-and serum-free medium.and then subjected to10min of OGD in37℃,2%O2,5%CO2incubator in the absence or presence of DCPIB (10μg/mL).and then the control group cells were incubated in serum-free high glucose medium in37℃,5%CO2incubator for3h.Then, the cells of OGD+DCPIB group were returned to normoxia environment with DCPIB treatment. Randomly, SD rats were assigned to3groups:sham-operated group, rMCAO group, and rMCAO+DCPIB group. At the onset of MCA occlusion,10μL of DCPEB(1mM in dimethyl sulfoxide (DMSO)) or DMSO alone was administered manually for20s by intracerebroventricularin fusion. Immunofluorescence was applied to stain hippocampal neurons.Neuronal viability was analyzed in vitro and in vivo. All data were performed with the SPSS software, version19.0.Results10min OGD caused a considerable loss of expression of neun-positive neurons. compared to that of in control group (P<0.01, n=6). In the presence of10μM DCPIB in co-culture medium improved the survival of neurons significantly (P<0.01, n=6)..Pyramidal neurons in the CA1region were significantly damaged after rMCAO. The VRAC inhibitor DCPIB diminished this injury. Conclusion DCPIB inhibits neuronal death in vivo and in vitro state after microglia—mediated ischemia/reperfusion. Part4:Suppression of microglia activation by DCPIB involves MAPK signal pathwayObjectiveTo explore the effect of DCPIB on signal pathway in the process of LPS or OGD-induced microglial activation.Methods Microglial BV-2cells were cultured in glass flasks. and then were randomly grouped into control group, LPS group and LPS+DCPIB, LPS+PD98059group, LPS+SB203580group, PS+SP600125group (n=4). The LPS group and LPS+DCPIB group medium was replaced with serum-free medium and high glucose plus LPS (10μg/ml). while the LPS+DCPIB group, LPS+PD98059group, LPS+SB203580group and LPS+SP600125group were given DCPIB, PD98059, SB203580and SP600125(10μM). Each group collected and extracted microglia total protein Western blot was applied to obtain the expression of p-ERK1/2, p-JNK and p-p38. Similarly, BV-2microglial cells line were cultured in the control group, OGD group and OGD+DCPIB group randomly. And BV-2cells were cultured in an oxygen-deprivation (93%N2/5%CO2/2%O2) incubator at37℃for10min. To terminate OGD and start reperfusion, the cells were returned to normoxia (95%O2/5%CO2) for3h and the medium was replaced with high glucose and serum-free one. after being cultured,in normoxia for0,0.5h,1h,3h and6h, total protein was extracted at different time points in each group for the purpose of obtaining p-ERK1/2, p-JNK and p-p38protein. Statistical software was used for semi-quantitatve analysis, and and the SPSS software, version19.0. was used to analyze all data.Results LPS or OGD increased the expression of p-ERK1/2, p-JNK and p-p38in MAPK pathway (P<0.01, n=4) and proliferation of microglia Compared to that of in control group, the expression of p-ERK1/2, p-JNK and p-p38significantly decreased (P<0.05, n=4) in LPS+DCPIB group; p-ERK1/2expression decreased significantly in the presence of PD98059(P<0.05, n=4);the effect of SB203580on expression of p-p38and SP600125on p-JNK were also the same state (P<0.05, n=4).rapid phosphorylation of p38, ERK, and JNK reach the peak levels of at30,15, and30min after oxygen-glucose deprivation respectively. DCPIB significantly suppressed the phosphorylation of all three kinases at the peak of their activation in OGD+DCPIB group compared with that of in OGD group.Conclusion DCPIB could have an inhibitory effect on microglial activation by MAPKs signaling pathway.