二甲双胍基于PPARγ/p38MAPK通路抑制结核分枝杆菌感染巨噬细胞的炎症反应

Metformin inhibits the inflammatory response of Mycobacterium bovis-infected macrophages through PPARγ/p38MAPK pathway

目的基于PPARγ/p38MAPK通路探讨二甲双胍对结核分枝杆菌感染巨噬细胞炎症反应的影响。方法以牛型结核分枝杆菌减毒株(M.bovis)诱导THP-1源性巨噬细胞建立感染模型。实验分4组,即对照组、M.bovis组、M.bovis+二甲双胍(metformin,Met)组、M.bovis+Met+GW9662(PPARγ抑制剂)组。分别采用RT-PCR和Western blot检测PPARγ、磷酸化p38MAPK(p-p38MAPK)基因及蛋白表达;酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)检测各组巨噬细胞TNF-α、IL-6及IL-10水平;检测各组巨噬细胞荷菌量。结果M.bovis感染巨噬细胞中PPARγ表达较对照组细胞显著升高(P<0.05),同时M.bovis感染显著增加巨噬细胞p-p38MAPK表达及TNF-α、IL-6、IL-10水平(P<0.05);二甲双胍具有激活PPARγ的功能,与单纯M.bovis组相比,二甲双胍处理后,进一步升高了PPARγ表达,但显著抑制了M.bovis感染所致的p-p38MAPK表达,降低了TNF-α、IL-6水平,升高IL-10水平(P<0.05);特异性PPARγ拮抗剂GW9662阻断PPARγ通路后,二甲双胍的上述作用被逆转;相关性分析结果显示,二甲双胍处理后,PPARγ表达与TNF-α、IL-6呈负相关,与IL-10呈正相关(P<0.05),而p-p38MAPK表达则与TNF-α、IL-6呈正相关,与IL-10呈负相关(P<0.05)。与M.bovis组相比,M.bovis+Met及M.bovis+Met+GW9662组细菌负荷量均明显降低(P<0.05),而M.bovis+Met组和M.bovis+Met+GW9662组细菌负荷量差异无统计学意义(P>0.05)。结论二甲双胍能抑制结核分枝杆菌感染巨噬细胞炎症反应,其部分机制可能与其激活PPARγ通路,进而抑制p38MAPK活化有关。

This study was designed to investigate the effect of metformin on the inflammatory response of macrophages infected with Mycobacterium bovis(M.bovis)through PPARγ/p38MAPK pathway.Macrophage infection model was established by M.bovis.At the same time,cells were randomly divided into control group,M.bovis group,M.bovis+Met group and M.bovis+Met+GW9662(PPARγinhibitor)group.The gene and protein expressions of PPARγand phosphorylated p38MAPK were detected by RT-PCR and Western blotting,respectively.The levels of TNF-α,IL-6 and IL-10 in macrophages were detected by enzyme linked immunosorbent assay(ELISA).Bacterial load of each group was detected.Compared with control group,the expression of PPARγwas significantly increased in M.bovis group(P<0.05).Simultaneously,M.bovis infection can obviously increase the expression of p-p38MAPK and the levels of TNF-α,IL-6 and IL-10(P<0.05).Compared with M.bovis group,metformin treatment further increased the expression of PPARγ,while significantly inhibited M.bovis infection-induced p-p38MAPK expression,decreased the levels of TNF-α,IL-6 and increased the level of IL-10(P<0.05).GW9662,the specific antagonist of PPARγ,could reverse the above effects of metformin.Correlation analysis showed that after metformin treatment,PPARγexpression was negatively correlated with TNF-αand IL-6,positively correlated with IL-10(P<0.05),while p-p38MAPK expression was positively correlated with TNF-αand IL-6,and negatively correlated with IL-10(P<0.05).Compared with M.bovis group,the bacterial load of M.bovis+Met and M.bovis+Met+GW9662 groups were significantly decreased(P<0.05),but there was no significant difference in bacterial load between M.bovis+Met and M.bovis+Met+GW9662 groups(P>0.05).These results indicated that metformin can inhibit the inflammatory response of M.bovis-induced macrophages,which due partly to its active effect on PPARγpathway and inhibiting effect on p38MAPK activation.