西罗莫司对肝脏热缺血-再灌注损伤小鼠肝组织TLR4表达和细胞自噬功能的影响

Effects of sirolimus on hepatic TLR4 expression and autophagy in mice with partial hepatic warm ischemia-reperfusion injury

目的探讨西罗莫司对肝脏热缺血-再灌注(I/R)损伤小鼠肝组织Toll样受体4(TLR4)表达和细胞自噬功能的影响。方法将40只Balb/c小鼠随机分为对照组、模型组、小剂量西罗莫司干预组(1 mg.kg-1)和大剂量西罗莫司干预组(3 mg.kg-1),每组10只,采用手术夹闭小鼠Glison鞘左支和中支1 h构建部分肝脏I/R模型,其中干预组在造模前2 d腹腔注射西罗莫司。采用ELISA法检测血清肿瘤坏死因子α(TNF-α)和白介素6(IL-6)含量,采用Western blot法检测肝组织TLR4、核因子κB(P65)、磷酸化核因子κB(p-P65)、微管相关蛋白1轻链3B-Ⅱ(LC3B-Ⅱ)和P62蛋白表达,使用透视电镜检测肝组织自噬小体数量。结果在恢复灌注6 h后,模型组小鼠血清ALT、AST、TNF-α和IL-6水平分别为(1370.6±245.7)U/L、(1584.3±321.5)U/L、(69.4±8.8)pg/mL和(154.1±22.7)pg/mL,显著高于对照组【分别为(34.31±4.3)U/L、(72.8±13.9)U/L、(10.7±3.4)pg/mL和(36.2±6.9)pg/mL,P<0.05】,而小剂量和大剂量西罗莫司干预组血清ALT和AST水平均显著低于模型组(P<0.05);模型组小鼠肝组织TLR4蛋白表达和p-p65/p65比值均较对照组显著升高(P<0.05),小剂量和大剂量西罗莫司干预组小鼠均较模型组显著降低(P<0.05),而LC3B-Ⅱ、P62蛋白表达和自噬小体数量均较模型组显著增加(P<0.05)。结论西罗莫司可能通过抑制TLR4/NF-κB信号通路激活抑制炎症反应,同时通过诱导肝细胞自噬,有效减轻小鼠部分肝脏热缺血-再灌注损伤。

Objective The aim of this study was to explore the effects of sirolimus on hepatic Toll-like receptor 4(TLR4)expression and autophagy in mice with partial hepatic warm ischemia-reperfusion(I/R)injury.Methods Forty Balb/c mice were randomly divided into control,model,low-dose(1 mg.kg-1)and high-dose(3 mg.kg-1)of sirolimus group,with 10 mice in each group.The left and middle branches of Glison sheath were surgically clamped to construct partial hepatic warm I/R model.The mice in the intervention group were intraperitoneally injected with sirolimus 2 days before modeling.After 6 hours of reperfusion,serum tumor necrosis factorα(TNF-α)and interleukin 6(IL-6)were detected by ELISA.The expression of TLR4,nuclear factorκB(P65),phosphorylated P65(p-P65),microtubule-associated protein 1 light chain 3B-II(LC3B-II)and P62 protein in liver tissues was detected by Western blot.The autophagosomes were detected by fluoroscopic electron microscope.Results After 6 h of reperfusion,serum ALT,AST,TNF-αand IL-6 levels in the model group were(1,370.6±245.7)U/L,(1,584.3±321.5)U/L,(69.4±8.8)pg/mL and(154.1±22.7)pg/mL,significantly higher than[(34.31±4.3)U/L,(72.8±13.9)U/L,(10.7±3.4)pg/mL,(36.2±6.9)pg/mL,respectively,P<0.05]in the control group,while serum ALT and AST levels in low-dose and high-dose of sirolimus-intervened groups were significantly lower than those in the model group(P<0.05);the hepatic expression of TLR4 protein and p-p65/p65 ratio in the model group were significantly stronger than those in the control group(P<0.05),while the expression of TLR4 protein and p-p65/p65 ratio in the low-dose and high-dose of sirolimus-intervened groups were significantly weaker than those in the model group(P<0.05),the expression of LC3B-II and P62 proteins,and the number of autophagosomes were significantly stronger or higher than those in the model group(P<0.05).Conclusion Sirolimus might reduce inflammatory response by inhibiting the activation of TLR4/NF-κB signaling pathway,which could effectively protect partial hepatic warm I/R injury in mice by inducing autophagy of hepatocytes.