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目的探讨二甲双胍对缺氧状态下高血糖诱导小胶质细胞释放炎性因子的调节作用。方法将体外培养的N9小胶质细胞分为四组:正常血糖组、正常血糖+二甲双胍组、高血糖组、高血糖+二甲双胍组。正常血糖+二甲双胍组与高血糖+二甲双胍组细胞均加入2 mmol/L二甲双胍处理12小时,四组细胞均进行缺氧处理24小时(3%O2)。采用小干扰RNA(si RNA)靶向沉默腺苷酸活化蛋白激酶α1(AMPKα1),采用实时定量聚合酶链式反应(q RT-PCR)和蛋白质印迹法检测AMPKα1的m RNA和蛋白水平,并采用酶联免疫吸附测定(ELISA)检测各组细胞白细胞介素(IL)-6、IL-1β、肿瘤坏死因子-α(TNF-α)分泌水平。结果二甲双胍对缺氧状态下高血糖诱导的炎性因子(IL-6、IL-1β、TNF-α)蛋白分泌具有抑制作用,并随着浓度的增加,抑制作用增强。高血糖组IL-6、IL-1β、TNF-αm RNA及蛋白分泌水平高于正常血糖组(P<0.05);高血糖+二甲双胍组IL-6、IL-1β、TNF-α蛋白分泌水平低于高血糖组(P<0.05);正常血糖组与正常血糖+二甲双胍组IL-6、IL-1β、TNF-α蛋白分泌水平比较差异无显著性(P>0.05)。而沉默AMPKα1能够上调高血糖+二甲双胍状态下IL-6、IL-1β、TNF-α蛋白分泌水平,与仅转染si-control细胞比较差异具有显著性(P<0.05)。结论二甲双胍能够抑制缺氧状态下高血糖诱导小胶质细胞炎性因子的释放,而AMPKα1在其中发挥关键作用。
Objective To investigate the regulatory effect of metformin on hyperglycemia-induced inflammatory factor release from microglia in hypoxia. Methods N9 microglial cells cultured in vitro were divided into four groups: normal blood glucose group, normal blood glucose + metformin group, hyperglycemia group, hyperglycemia + metformin group. The cells in normal blood glucose + metformin group and hyperglycemia + metformin group were treated with 2 mmol / L metformin for 12 hours, and all four groups were subjected to hypoxia for 24 hours (3% O2). Small interfering RNA (siRNA) was used to target AMPKα1, and m RNA and protein levels of AMPKα1 were detected by real-time quantitative polymerase chain reaction (q RT-PCR) and Western blotting The levels of interleukin (IL) -6, IL-1β and tumor necrosis factor-α (TNF-α) were measured by enzyme linked immunosorbent assay (ELISA). Results Metformin inhibited the secretion of inflammatory cytokines (IL-6, IL-1β, TNF-α) induced by hyperglycemia in hypoxia condition. With the increase of concentration, metformin enhanced the inhibitory effect. The levels of IL-6, IL-1β, TNF-αmRNA and protein in hyperglycemia group were higher than those in normal blood glucose group (P <0.05). The levels of IL-6, IL-1β and TNF- (P <0.05). The levels of IL-6, IL-1β and TNF-α in normal blood glucose group and normal blood glucose + metformin group had no significant difference (P> 0.05). Silencing AMPKα1 could up-regulate the secretion of IL-6, IL-1β and TNF-α in hyperglycemia and metformin, which was significantly different from that of si-control alone (P <0.05). Conclusion Metformin can inhibit hyperglycemia-induced release of microglial inflammatory cytokines under hypoxia, whereas AMPKα1 plays a key role.