乌帕替尼对氧糖剥夺/复氧后BV2细胞极化的影响

Effects of upadacitinib on BV2 cells after oxygen–glucose deprivation/recovery

目的研究乌帕替尼对氧糖剥夺再复氧(OGD/R)后BV2小胶质细胞极化及炎症的影响,并探讨其作用机制。方法实验分对照组、OGD组和乌帕替尼组3组。BV2细胞经OGD/R处理后,噻唑蓝试剂(MTT)检测细胞生存率,划痕实验观察细胞迁移能力,实时荧光定量多聚核苷酸链式反应(qPCR)检测BV2细胞M1型极化标志物(CD11b、CD32、iNOS)和M2型极化标志物(Arg-1、IL-10、CD206)的mRNA水平,酶联免疫吸附测定(ELISA)检测培养基中IL-1β、IL-6、TNF-α含量,蛋白质印迹法(Western Blot)检测JAK1/STAT6通路相关蛋白表达水平。结果乌帕替尼能增加OGD/R后BV2细胞的生存率(P<0.05),能减少BV2细胞向M1型极化(P<0.05)。乌帕替尼可明显降低OGD/R诱导的BV2细胞的迁移能力(P<0.05),减少OGD/R诱导BV2细胞分泌的炎症因子:IL-1β、IL-6、TNF-α(P<0.05)。乌帕替尼提高共培养PC12细胞的生存率(P<0.05)。乌帕替尼可显著抑制由OGD/R诱导激活BV2细胞p-JAK1和p-STAT6蛋白的表达水平(P<0.05)。结论乌帕替尼可减少OGD/R诱导BV2细胞向M1型极化和减少炎症反应,与JAK1/STAT6通路有关。

Objective To investigate the effects of upadacitinib on the polarization and inflammation of BV2 microglia after oxygen glucose deprivation/recovery(OGD/R)and to explore its mechanism of action.Methods The experiment was divided into 3 groups:control group,OGD group and upadacitinib treatment group.After BV2 cells were treated with OGD/R,MTT was used to detect cell survival rate.Wound scratch assay was used to detect the cell migration ability.qPCR was used to detect mRNA levels of M1-type polarization markers(CD11 b,CD32,iNOS)and M2-type polarization markers(Arg-1,IL-10,CD206)of BV2 cells.ELISA was used to detect the levels of IL-1β,IL-6,and TNF-αin the culture medium.Western blot was used to detect the expression levels of JAK1/STAT6 pathway-related proteins.Results Upadacitinib increased the survival of BV2 cells after OGD/R(P<0.05),reduced the polarization of BV2 cells to M1 type(P<0.05).Upadacitinib significantly decreased the migration ability of BV2 cells induced by OGD/R(P<0.05),reduced the inflammatory factors secreted by BV2 cells induced by OGD/R:IL-1β,IL-6,TNF-α(P<0.05).Upadacitinib increased the survival rate of co-cultured PC12 cells(P<0.05).Upadacitinib significantly inhibited the expression levels of p-JAK1 and p-STAT6 proteins in BV2 cells activated by OGD/R induction(P<0.05).Conclusion Upadacitinib decreases polarization of BV2 induced by OGD/R to M1 type and reduces inflammation,which is related to JAK1/STAT6 pathway.