二甲双胍对缺氧状态下高血糖诱导N9小胶质细胞释放炎性因子的调节作用

摘要

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（siRNA）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（qRT-PCR）和蛋白质印迹法检测AMPKα1的mRNA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-α mRNA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。%ObjectiveTo investigate the role of metformin in the hyperglycemia induced proinlfammatory cytokine secretion in N9 microglia under hypoxic condition.MethodN9 cells cultured in vitro were divided into euglycemia group, euglycemia+metformin group, hyperglycemia group and hyperglycemia+metformin group. 2 mmol/L metformin was added into euglycemia+metformin group and hyperglycemia+metformin group for 12 hours. Then, four groups were changed into hypoxia conditions (3% O2) for 24 hours. AMPKα1 siRNA were used to knockout AMPKα1 expression. Quantitative real-time PCR and Western blotting were applied to detect the mRNA and protein level of AMPKα1. ELISA were used to determine secretion levels of IL-6、IL-1β and TNF-α among four groups.ResultMetformin had an inhibitory effect on protein secretion of the inlfammatory cytokines IL-6, IL-1β and TNF-α, and the inhibition effect gradually increased with the increase of metformin dose. As compared with euglycemia group, the protein secretion levels of IL-6, IL-1β and TNF-α signiifcantly increased in hyperglycemia group (P<0.05). As compared with hyperglycemia group, the secretion levels of IL-6, IL-1β and TNF-α signiifcantly decreased in hyperglycemia+metformin group (P<0.05). There were no differences in the protein secretion levels of IL-6, IL-1β and TNF-α between euglycemia group and euglycemia+metformin group (P>0.05). Silencing of AMPKα1 significantly upregulated IL-6, IL-1β and TNF-α secretion levels under hyperglycemia+metformin condition, as compared with the si-control transfected cells.ConclusionMetformin can inhibit hyperglycemia induced proinflammatory cytokine secretion in microglia under hypoxic condition, and AMPKα1 plays a vital role in the metformin induced effect.