基于TLR4/NF-κB途径探讨二甲双胍对EAE小鼠Th17细胞反应及T细胞分化的作用机制

Roles of metformin in Th17 cell response and T cell differentiation of EAE mice based on the TLR4/NF-κB pathway

目的通过TLR4/NF-κB途径探讨二甲双胍对实验性自身免疫性脑脊髓炎(EAE)小鼠T辅助细胞17(T help 17cells,Th17)反应及T细胞分化的作用机制。方法通过皮下多点注射髓鞘少突胶质细胞糖蛋白(MOG)肽35-55诱导EAE模型,将30只建模成功的小鼠分为EAE组和EAE+二甲双胍组(n=15),随机选择15只健康小鼠作为对照组。二甲双胍灌胃干预,剂量为200 mg/(kg·d^(-1))。比较各组小鼠EAE病情、髓鞘完整性、脊髓Th17细胞浸润和外周血中Th17细胞比例。小鼠CD4;幼稚T细胞分为NC组、MOG组和MOG+二甲双胍组。通过CCK-8和流式细胞术检测增殖和Th17分化情况。并检测各组细胞中TLR4/NF-κB转录和翻译水平。结果EAE组的Knoz评分、髓鞘损伤程度、Th17细胞浸润情况和外周血中Th17细胞比例显著高于对照组(P<0.05)。EAE+二甲双胍组的Knoz评分、髓鞘损伤程度、Th17细胞浸润情况和外周血中Th17细胞比例显著低于EAE组(P<0.05)。对于细胞实验,3组的各项指标比较差异显著(P<0.05)。MOG组的OD值、Th17细胞比例、TLR4和NF-κB mRNA及蛋白表达水平显著高于NC组(P<0.05)。MOG+二甲双胍组的OD值、Th17细胞比例、TLR4和NF-κB mRNA及蛋白表达水平显著低于MOG组(P<0.05)。结论二甲双胍可能通过抑制TLR4/NF-κB通路抑制CD4+幼稚T细胞向Th17细胞分化并减少中枢神经系统中Th17细胞的浸润,从而缓解EAE。

The aim of this study was to explore the roles of metformin in T help 17 cells(Th17)response and T cell differentiation in experimental autoimmune encephalomyelitis(EAE)mice through the TLR4/NF-κB pathway.The EAE model was constructed by subcutaneous injection of myelin oligodendrocyte glycoprotein(MOG)peptide35-55 at multiple points.Total of 30 EAE model mice were divided into EAE group and EAE+metformin group(n 15),with 15 healthy mice as control group.Mice in EAE+metformin group were interfered with gavage intervention of metformin at a dose of 200 mg/(kg·d^(-1)).The EAE condition,myelin integrity,spinal cord Th17 cell infiltration and the proportion of Th17 cells in peripheral blood were compared in all groups.Mouse CD4;naive T cells were divided into NC group,MOG group and MOG+metformin group.The proliferation and Th17 differentiation were detected by CCK-8 and flow cytometry;TLR4/NF-κB transcription and translation levels in each group of cells were detected.Data showed that the Knoz score(2.43±0.42 points),degree of myelin damage,Th 17 cell infiltration and the proportion of Th17 cells in peripheral blood(3.06%±0.25%)in the EAE group were significantly higher than those in the control group(P<0.05).The Knoz score(1.45±0.31 points),degree of myelin damage,Th 17 cell infiltration,and the proportion of Th 17 cells in peripheral blood(1.93%±0.20%)in the EAE+metformin group were significantly lower than those in the EAE group(P<0.05).For the cell experiment,the OD value(0.71±0.07),Th17 cell ratio(25.61%±1.89%),TLR4 and NF-κB mRNA(4.61±0.41,4.40±0.39)and protein expression levels(2.98±0.25,2.72±0.24)of the MOG group were significant higher than those of the NC group(P<0.05);while the OD value(0.59±0.06),Th17 cell ratio(13.24%±1.20%),TLR4 and NF-κB mRNA(2.69±0.24,2.35±0.22)and protein expression levels(1.45±0.13,1.21±0.11)in the MOG+metformin group were significantly lower than those of the MOG group(P<0.05).In conclusion,metformin may inhibit the differentiation of CD4;naive T cells into Th17 cells by inhibiting the TLR4/NF-κB pathway to reduce the infiltration of Th17 cells in the central nervous system,thereby alleviating EAE.