抑制mTOR增强索拉非尼介导的HSC铁死亡

Inhibition of mTOR enhances sorafenib-induced activated HSC ferroptosis

目的:探究mTOR信号通路在索拉非尼诱导肝星状细胞(HSC)铁死亡改善肝纤维化进程中的作用及其机制。方法:用四氯化碳(CCl_(4))诱导C57BL/6雄性小鼠肝纤维化模型,随机分为正常组、模型组、索拉非尼低(2.5 mg/kg),中(5 mg/kg),高(10 mg/kg)剂量组,每组6只。除正常组外,其余4组均腹腔注射CCl_(4)建立肝纤维化模型,第4周开始给予索拉非尼灌胃。各组小鼠于造模第8周末处死。Western blot检测肝组织中mTOR和p-mTOR表达情况。体外实验中,采用PDGF-BB诱导HSC-T6细胞活化,并加入不同浓度索拉非尼(5μM,10μM,20μM)和mTOR抑制剂及激活剂,CCK8检测HSC-T6细胞活力;Western blot检测α-SMA、COL1α1、GPX4、mTOR和p-mTOR蛋白表达情况;试剂盒检测HSC-T6中iron、GSH、MDA含量;采用双氯荧光素(2′,7′-dichlorofluorescin diacetate,DCFH-DA)检测ROS水平。结果:结果显示,索拉非尼可显著降低α-SMA、COL1α1、GPX4和p-mTOR的蛋白表达,并且在HSC-T6中降低GSH的水平并提高iron、MDA、ROS的含量(P<0.05)。不同浓度索拉非尼均明显抑制PDGF-BB激活的HSC-T6的细胞活力(P<0.05)。当mTOR受到抑制时,α-SMA、COL1α1和GPX4的蛋白表达以及GSH的水平会进一步降低(P<0.05),iron、MDA、ROS的含量则升高(P<0.05)。结论:索拉非尼诱导HSC铁死亡减轻肝纤维化,抑制mTOR可进一步增强索拉非尼的作用。

Objective:To investigate the role and mechanism of mTOR in sorafenib-ameliorated liver fibrosis by inducing hepatic stellate cells(HSC)ferroptosis.Methods:The liver fibrosis models of C57BL/6 male mice were induced with carbon tetrachloride(CCl_(4))and randomly divided into normal group,model group,and sorafenib low(2.5 mg/kg),medium(5 mg/kg),and high(10 mg/kg)dose groups.Except for the normal group,the remaining four groups were treated with intraperitoneal injection of CCl_(4)for 8 weeks to establish liver fibrosis models.In addition,the corresponding concentrations of sorafenib were administered during the fourth week of modeling.Western blot was used to detect the expression of mTOR and p-mTOR protein in liver tissues.In vitro experiments,HSC-T6 cells were activated by PDGF-BB and then treated with sorafenib(5μM,10μM,20μM),an mTOR inhibitor and activator.CCK8 was used to detect HSC-T6 cell viability,Western blot detected the protein expression ofα-SMA,COL1α1,GPX4,mTOR and p-mTOR.The levels of serum iron,GSH,and MDA were measured,and the intracellular ROS level was measured by 2′,7′-dichlorofluorescein diacetate and dihydroethidium.Results:In vitro results showed that sorafenib significantly decreased the protein expression ofα-SMA,COL1α1,GPX4 and p-mTOR,and decreased the level of GSH and increased the content of iron,MDA and ROS in HSC-T6(P<0.05).Sorafenib(5μM,10μM,20μM)significantly inhibited the cell viability of PDGF-BB-enhanced HSC-T6(P<0.05).When mTOR was inhibited,the protein expressions ofα-SMA,COL1α1 and GPX4 and the level of GSH were lower(P<0.05),and the contents of iron,MDA and ROS were further increased(P<0.05).When mTOR was activated,the results were reversed.Conclusion:Sorafenib induced ferroptosis to alleviate liver fibrosis,and inhibition of mTOR further enhanced the effect of sorafenib.