黄芩苷对脑出血后小胶质细胞炎症反应的影响

Effects of baicalin on inflammation and biological behavior of microglia cells after intracerebral hemorrhage

目的探究黄芩苷对脂多糖(LPS)诱导的小鼠小胶质细胞HMC3炎症反应及酪氨酸蛋白激酶2(JAK2)/信号转导和转录启动因子3(STAT3)信号通路的调控作用。方法体外培养小鼠小胶质HMC3细胞,分为对照组(不做干预)、LPS组(1μg·mL^(-1)LPS)、实验组(1μg·mL^(-1)LPS+5、10、20、40μmol·L^(-1)黄芩苷)、抑制剂组(1μg·mL^(-1)LPS+20μmol·L^(-1)JAK2/STAT3通路抑制剂AG490)、黄芩苷+Y组(1μg·mL^(-1)LPS+10μmol·L^(-1)黄芩苷+20μmol·L^(-1)AG490)和黄芩苷+A组(1μg·mL^(-1)LPS+10μmol·L^(-1)黄芩苷+0.5μmol·L^(-1)JAK2/STAT3通路激活剂colivelin),干预24 h。用酶联免疫吸附试验(ELISA)检测炎症因子白细胞介素(IL)-10、IL-1β和肿瘤坏死因子(TNF)-α的表达水平;细胞计数试剂盒-8(CCK-8)测定细胞活力;用Hoechst 33258染色法检测细胞的凋亡率;Transwell小室测定细胞的迁移能力;蛋白印迹(Wb)法测定上皮间质转化(EMT)及JAK2/STAT3通路相关蛋白表达水平。结果与对照组相比,LPS组IL-1β、TNF-α表达水平显著升高(P<0.05),IL-10表达水平与细胞活力显著降低(P<0.05);与LPS组相比,除LPS+5组外,其余实验组IL-1β和TNF-α表达水平均显著降低(P<0.05),IL-10表达显著升高(P<0.05),而细胞活力只有LPS+5和LPS+10组显著升高(P<0.05),因此选择细胞活力提升更高且抑炎效果综合更佳的LPS+10组进行后续实验。与对照组相比,LPS组细胞E-钙粘蛋白(E-cadherin)蛋白表达水平显著降低(P<0.05),细胞凋亡率、迁移数、N-钙粘蛋白(N-cadherin)、波形蛋白(Vimentin)、纤维粘连蛋白(FN)以及p-JAK2和p-STAT3蛋白表达显著升高(P<0.05);与LPS组相比,黄芩苷组和抑制剂组显著扭转了上述指标的变化(P<0.05);与黄芩苷组相比,黄芩苷+Y组细胞E-cadherin蛋白表达水平显著升高(P<0.05),细胞凋亡率、迁移数、N-cadherin、Vimentin、FN以及p-JAK2和p-STAT3蛋白表达显著降低(P<0.05),黄芩苷+A组则显著扭转了上述指标的变化(P<0.05)。结论黄芩苷可显著抑制LPS诱导的小鼠小胶质HMC3细胞的炎症、凋亡、迁移及EMT进程,其作用机制可能与抑制JAK2/STAT3通路的信号转导相关。

Objective To explore the regulatory effects of baicalin on HMC3 inflammation and tyrosine protein kinase 2(JAK2)/signal transduction and transcription promoter 3(STAT3)signaling pathways in mouse microglia cells induced by lipopolysaccharide(LPS).Methods Mouse microglia HMC3 cells were cultured in vitro.They were divided into control group(no intervention),LPS group(1μg·mL^(-1)LPS),experimental group(1μg·mL^(-1)LPS+5,10,20,40μmol·L^(-1)baicalin),inhibitor group(1μg·mL^(-1)LPS+20μmol·L^(-1)AG490),Baicalin+Y group(1μL g·mL^(-1)LPS+10μmol·L^(-1)Baicalin+20μmol·L^(-1)JAK2/STAT3 pathway inhibitor AG490)and Baicalin+A group(1μg·mL^(-1)LPS+10μmol·L^(-1)baicalin+0.5μmol·L^(-1)JAK2/STAT3 pathway activator colivelin),was treated for 24 h.The expression levels of inflammatory factors interleukin(IL)-10,IL-1βand tumor necrosis factor(TNF)-αwere detected by enzyme-linked immunosorbent assay(ELISA);cell counting kit 8(CCK-8)was used to measure cell viability;hoechst 33258 staining was used to detect the apoptosis rate;transwell chamber was used to measure the migration ability of cells;the protein expression levels of EMT and JAK2/STAT3 pathway were determined by Western blotting(Wb).Results Compared with the control group,the expression levels of IL-1βand TNF-αin LPS group were significantly increased(P<0.05),IL-10 expression level and cell viability were significantly decreased(P<0.05);compared with LPS group,the expression levels of IL-1βand TNF-αwere significantly decreased in all experimental groups except LPS+5 group(P<0.05),IL-10 expression was significantly increased(P<0.05),and cell viability was significantly increased only in LPS+5 and LPS+10 groups(P<0.05),the cell viability of the other two groups had no significant difference and was less than 50%.Therefore,LPS+10 group with higher cell viability and better anti-inflammatory effect was selected to add JAK2/STAT3 signaling pathway inhibitor AG490 and activator colivelin for JAK2/STAT3 pathway validation experiment.Compared with the control group,the expression level of E-cadherin protein in LPS group was significantly decreased(P<0.05),the apoptosis rate,migration number,N-cadherin,Vimentin,FN and the protein expressions of p-JAK2 and p-STAT3 were significantly increased(P<0.05);compared with LPS group,Baicalin goup and inhibitor group significantly reversed the changes of the above indexes(P<0.05);compared with Baicalin group,the expression level of E-cadherin protein in Baicalin+Y group was significantly increased(P<0.05),the apoptosis rate,migration number,N-cadherin,Vimentin,FN and the protein expressions of p-JAK2 and p-STAT3 were significantly decreased(P<0.05),Baicalin+A group significantly reversed the changes of the above indexes(P<0.05).Conclusion Baicalin can significantly inhibit the inflammation,apoptosis,migration and EMT process of mouse microglia HMC3 cells induced by LPS,and its mechanism may be related to the inhibition of JAK2/STAT3 pathway signal transduction.