Tim-3靶向CD8^(+)T细胞调控TNF-α/IL-22/STAT3通路加重自身免疫性肝炎小鼠肝损伤

Tim-3 aggravates liver injury in mice with autoimmune hepatitis via TNF-α/IL-22/STAT3 pathway by targeting CD8^(+)T cells

目的探讨T细胞免疫球蛋白和黏蛋白结构域的分子3(Tim-3)对自身免疫性肝炎的影响及其机制。方法将30只小鼠分为3组:空白组、模型组和模型+Tim-3阻断组,每组10只。空白组经尾静脉注射等量生理盐水;其他两组尾静脉注射10 mg/kg刀豆蛋白A建立自身免疫性肝炎小鼠模型,造模成功后模型+Tim-3阻断组每天1次腹腔内注射液0.1 mg/ml Tim-3,注射7 d。HE染色观察小鼠肝组织病理改变;全自动生化分析仪检测血清AST、ALT;酶联免疫吸附法检测IL-22、TNF-α水平;流式细胞分析肝组织CD8^(+)T比例;实时荧光定量PCR检测肝组织Tim-3、IL-22、TNF-α、STAT3 mRNA相对表达;免疫印迹试验检测肝组织Tim-3、IL-22、TNF-α、STAT3蛋白相对表达。结果空白组小鼠肝脏组织无变化;模型组肝脏组织大面积性肝炎;模型+Tim-3阻断组见显著淋巴细胞浸润与大量空泡。与空白组比较,模型组和模型+Tim-3阻断组ALT、AST、IL-22、TNF-α水平均升高(P<0.05);与模型组相比,模型+Tim-3阻断组ALT、AST、IL-22、TNF-α水平升高(P<0.05)。与空白组比较,模型组和模型+Tim-3阻断组CD8^(+)T细胞比例表达均升高(P<0.05);与模型组相比,模型+Tim-3阻断组CD8^(+)T细胞比例下降(P<0.05)。与空白组比较,模型组和模型+Tim-3阻断组Tim-3、IL-22、TNFα、STAT3 mRNA及蛋白表达均升高(P<0.05);与模型组相比,模型+Tim-3阻断组Tim-3 mRNA及蛋白表达下降,IL-22、TNF-α、STAT3 mRNA及蛋白相对表达升高(P<0.05)。结论阻断Tim-3可下调CD8^(+)T细胞,促进TNF-α/IL-22/STAT3炎症信号表达,加重自身免疫性肝炎小鼠肝损伤。

Objective To investigate the effect of T cell immunoglobulin and mucin domain 3(Tim-3)on autoimmune hepatitis and its mechanism.Methods Thirty mice were divided into three groups:blank group,model group and model+Tim-3 blocking group,with 10 mice in each group.The autoimmune hepatitis model was induced by injecting with 10 mg/kg cancana A through the tail vein.After the model was successfully established,the mice in model+Tim-3 blocking group were intraperitoneally injected with 0.1 mg/ml Tim-3 inhibitor once a day for 7 d.The mice in blank group were injected with the same amount of normal saline through the tail vein.HE staining was used to observe the pathological changes of liver tissue.Serum AST and ALT levels were detected by automatic biochemical analyzer.The levels of IL-22 and TNF-αwere determined by enzyme-linked immunosorbent assay.CD8^(+)T percentage in liver tissue was analyzed by flow cytometry.The relative mRNA expression levels of Tim-3,IL-22,TNF-αand STAT3 in liver tissue were detected by real-time quantitative PCR.The relative expression levels of Tim-3,IL-22,TNF-αand STAT3 in liver tissue were detected by Western blotting.Results There was extensive liver hepatitis in model group,while significant lymphocyte infiltration and numerous vacuoles were observed in model group+Tim-3 blocking group.Compared with blank group,ALT,AST,IL-22 and TNF-αlevels in model group and model+Tim-3 blocking group were increased(P<0.05).Compared with model group,ALT,AST,IL-22 and TNF-αlevels were increased in model+Tim-3 blocking group(P<0.05).Compared with blank group,the percentages of CD8^(+)T cells in model group and model+Tim-3 blocking group were increased(P<0.05).Compared with model group,the proportion of CD8^(+)T cells in model+Tim-3 blocking group was decreased(P<0.05).Compared with blank group,the mRNA and protein expression levels of Tim-3,IL-22,TNF-αand STAT3 were increased in model group and model+Tim-3 blocking group(P<0.05).Compared with model group,the mRNA and protein expression levels of Tim-3 in model+Tim-3 blocking group were decreased,while the relative mRNA and protein expression levels of IL-22,TNF-αand STAT3 were increased(P<0.05).Conclusion Tim-3 blocking can down-regulate CD8^(+)T cells and promote the expression of TNF-α/IL-22/STAT3 inflammatory signaling pathway,thus aggravating liver injury in mice with autoimmune hepatitis.