目的 观察二甲双胍对饱和脂肪酸(SFA)诱导的RAW264.7巨噬细胞炎性反应中炎性因子的影响及探讨其可能机制.方法 SFA干预RAW264.7巨噬细胞建立体外炎性反应模型;实验分为对照组、SFA干预组、二甲双胍+ SFA干预组、AMPK抑制剂Compound C+二甲双胍+SFA干预组;实时定量PCR检测TNF-α和IL-6 mRNA的表达,ELISA法检测TNF-α和IL-6蛋白的分泌,Western blot分析腺苷酸活化蛋白激酶(AMPK)的磷酸化水平.结果 与对照组比较,SFA干预组RAW264.7巨噬细胞TNF-α、IL-6 mRNA表达及蛋白分泌水平均显著升高(P<0.05);与SFA组比较,二甲双胍+SFA干预组TNF-α、IL-6 mRNA表达水平及蛋白分泌水平均降低(P<0.05),而细胞AMPK的磷酸化水平增强(P<0.05);与二甲双胍+SFA干预组比,Compound C+二甲双胍+SFA干预组TNF-α、IL-6 mRNA表达及蛋白分泌水平均升高,AMPK磷酸化水平降低(P<0.05).结论 二甲双胍激活AMPK降低饱和脂肪酸诱导的RAW264.7巨噬细胞炎性因子TNF-α、IL-6的分泌.
Objective To investigate the effect of metformin on inflammatory cytokines of saturated fatty acid (SFA)-induced RAW264.7 macrophage.Methods RAW264.7 cells were treated with SFA to establish inflammatory model in vitro.Macrophages were divided into normal control group,SFA-treated group,metformin + SFAtreated group and AMPK inhibitor Compound C + metformin + SFA-treated group.RT-PCR was used to detect mRNA expression levels of TNF-α and IL-6.ELISA was used to detect secretion levels of TNF-α and IL-6 in cell supernatants.Western blot was used to analyze phosphorylation level of AMP-activated protein kinase.Results Compared to normal control group,both mRNA expression and protein secretion of TNF-α and IL-6 increased in SFA-treated group (P < 0.05).Compared to SFA-treated group,both mRNA expression and protein secretion of TNF-α and IL-6 decreased in metformin + SFA-treated group (P <0.05),and AMPK phosphorylation level was significantly enhanced (P < 0.05).Compared to metformin + SFA treated-group,mRNA expression and protein secretion of TNF-α and IL-6 increased,and AMPK phosphorylation level deceased in Compound C + metformin +SFA-treated group (P < 0.05).Conclusions Metformin suppresses secretion of inflammatory cytokines TNF-αand IL-6 in saturated fatty acid-induced RAW264.7 macrophage via activation of AMPK.