mTORC1-TFEB信号通路对肝脏缺血再灌注损伤模型大鼠肝细胞线粒体结构和肝功能的影响

Effect of mTORC1-TFEB signaling pathway on mitochondrial construction and liver function in gepatic ischemia-reperfusion injury

目的探讨mTORC1-TFEB信号通路对肝脏缺血再灌注损伤模型大鼠肝细胞线粒体结构及肝功能的影响。方法36只SD雄性大鼠随机均分为对照组、模型组及药物组,药物组大鼠术前3 d注射雷帕霉素溶液[2.0 mL/(kg·d)]、对照组和模型组大鼠注射等体积生理盐水,药物组和模型组大鼠麻醉后开腹夹闭第一肝门再放开制作肝缺血再灌注损伤模型、对照组大鼠仅手术但不夹闭肝门,分别在术后24 h、72 h取大鼠肝脏组织HE染色观察肝脏组织细胞损伤程度,透射电镜分析肝细胞线粒体的超微结构变化,比色法检验肝脏组织谷草转氨酶(AST)和谷丙转氨酶(ALT)浓度水平,蛋白印迹实验(Western blot)和实时荧光PCR(RT-PCR)检测磷酸化mTORC1(p-mTORC1)和转录因子EB(TFEB)蛋白和mRNA表达。结果HE染色结果显示,对照组肝脏组织细胞在术后各个时间点未见明显异常(P>0.05),而药物组大鼠肝脏损伤程度在各个时间点均较模型组轻(P<0.05),药物组和模型组术后72 h肝损伤的程度较术后24 h减轻(P<0.05);透射电镜结果显示,在各个时间点,对照组线粒体的超微结构形态正常,药物组线粒体超微结构损伤均较模型组轻,药物组和模型组术后72 h线粒体损伤程度较术后24 h减轻;术后24 h及72 h,药物组及模型组AST、ALT表达水平高于对照组,且模型组高于药物组(P<0.05),药物组及模型组p-mTORC1的蛋白和mRNA表达水平低于对照组、而TFEB的蛋白和mRNA表达水平高于对照组,且药物组p-mTORC1的蛋白和mRNA表达水平低于模型组、而TFEB的蛋白和mRNA表达水平高于模型组(P<0.05),术后72 h药物组及模型组的AST、ALT和TFEB相关表达水平较术后24 h降低,而p-mTORC1相关表达水平较术后24 h升高(P<0.05)。结论mTORC1-TFEB信号通路对肝脏缺血再灌注损伤模型大鼠有保护作用,其机制可能与减轻线粒体损伤、改善肝功能有关。

Objective To investigate the effect of mTORC1-TFEB signaling pathway on mitochondrial construction and liver function in a rat model of hepatic ischemia-reperfusion injury.Methods Thirty-six SD male rats were randomly divided into control group,model group and drug group.The rats in the drug group were injected with rapamycin solution[2.0 mL/(kg·d)]3 d before operation,and the rats in the control group and the model group were injected with the same volume of normal saline.In the drug group and the model group,the first porta hepatis were clipped after anesthesia,and then the first porta hepatis were released.In the control group,the porta hepatis were only operated but not clipped.At 24 h and 72 h after operation,HE staining was taken from the liver tissues of rats to observe the degree of liver tissue cell damage,the ultrastructural changes of mitochondria were analyzed by transmission electron microscopy,and the concentrations of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in liver tissue were determined by colorimetry.Western blot and real-time PCR(RT-PCR)were used to detect the protein and mRNA expression of phosphorylated mTORC1(p-mTORC1)and transcription factor EB(TFEB).Results The results of HE staining showed that there was no obvious abnormality in liver tissue cells at each time point after operation in the control group(P>0.05)while the degree of liver injury in the drug group was lighter than that in the model group at each time point(P<0.05).The degree of liver injury in the drug group and the model group at 72 h after operation was lighter than that at 24 h after operation(P<0.05).The results of transmission electron microscopy showed that the morphology of mitochondrial ultrastructure in the control group was normal at each time point while the damage of mitochondrial ultrastructure in the drug group was lighter than that in the model group at each time point.The degree of mitochondrial damage in the drug group and the model group at 72 h after operation was lighter than that at 24 h after operation.At 24 h and 72 h after surgery,AST and ALT expression levels in drug group and model group were higher than those in control group,and model group was higher than drug group(P<0.05).The drug group and the model group had lower protein and mRNA expression levels of p-mTORC1 and higher protein and mRNA expression levels of TFEB than the control group,and the drug group had lower protein and mRNA expression levels of p-mTORC1 and higher protein and mRNA expression levels of TFEB than the model group(P<0.05).The expression levels of AST,ALT,and TFEB in the drug group and model group at 72 h after surgery were decreased compared with 24 h after surgery while the expression levels of p-mTORC1 were increased compared with 24 h after surgery(P<0.05).Conclusion mTORC1-TFEB signaling pathway has a protective effect on hepatic ischemia-reperfusion injury model rats,and its mechanism may be related to alleviating mitochondrial damage and improving liver function.