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*

doi:10.3969/j.issn.1672-5069.2021.05.016

摘要/Abstract

摘要： 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。

关键词: 缺血-再灌注损伤, 西罗莫司, Toll样受体4, 自噬, 小鼠

Abstract: 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 factor α (TNF-α) and interleukin 6 (IL-6) were detected by ELISA. The expression of TLR4, nuclear factor κB (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-α and 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 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.

Key words: Ischemia-reperfusion injury, Sirolimus, Toll-like receptor 4, Autophagy, Mice

唐双意, 王希斌, 丘岳, 覃福礼, 张宏亮, 蒋霞. 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^*[J]. 实用肝脏病杂志, 2021, 24(5): 669-672.

Tang Shuangyi, Wang Xibin, Qiu Yue, et al. Effects of sirolimus on hepatic TLR4 expression and autophagy in mice with partial hepatic warm ischemia-reperfusion injury[J]. Journal of Practical Hepatology, 2021, 24(5): 669-672.

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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

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