青蒿琥酯通过铁死亡途径抑制肝星状细胞活化的作用机制

Mechanism of artesunate in inhibiting hepatic stellate cell activation via ferroptosis

目的:探究青蒿琥酯(Art)诱导肝星状细胞(HSC)发生铁死亡进而改善肝纤维化(HF)病变损伤的作用及分子机制。方法:建立CCl4致大鼠HF模型,然后腹腔注射不同剂量Art治疗4周,HE、Masson、Sirius red染色考察Art对肝组织病变损伤的影响,ELISA试剂盒检测Art对肝功能指标的影响,Real-time qPCR检测HF指标;体外培养HSC-T6细胞系,给予不同剂量Art处理24 h,Western Blot、Real-time qPCR、免疫荧光检测HSC活化指标;转染P53 siRNA,Western Blot检测P53受调控后铁死亡相关指标的影响。结果:各染色结果表明Art在体内能够改善HF病变损伤。ELISA试剂盒检测表明,与正常对照组比较,模型组ALT、AST表达水平显著升高(P<0.01),而各剂量Art较模型组能够显著降低ALT、AST表达水平(P<0.05,P<0.01),Real-time qPCR检测表明,与模型组比较,Art能够剂量依赖性地下调CollagenⅠ和α-SMA表达水平(P<0.05,P<0.01);Art可以降低HSC活化标志物CollagenⅠ、α-SMA和Fibronectin蛋白和mRNA表达水平(P<0.05),并呈现剂量依赖性,同时免疫荧光实验结果发现,CollagenⅠ以及α-SMA表达能够被Art降低(P<0.05),并呈现剂量依赖的趋势;Western Blot检测发现,Art可以显著下调HSC活化指标,而铁死亡抑制剂Fer-1显著降低了Art诱导的抑制α-SMA、CollagenⅠ表达的作用;而P53 siRNA可以抵消Art抑制α-SMA、CollagenⅠ和Fibronectin表达的影响。结论:Art通过调控P53蛋白诱导HSC铁死亡改善HF病变损伤。

Objective:To explore the molecular mechanism of artesunate(Art)in improving hepatic fibrosis(HF)by inducing HSC ferroptosis.Methods:The classic CCl4-induced HF model was established,and Art was intraperitoneally injected for 4 weeks.H&E staining,Masson staining and Sirius red staining were used to investigate the effect of Art on liver injury,and ELISA assay was used to detect the effects of Art on liver function indexes,and Realtime qPCR was used to detect the expression level of HF markers.HSC cells were treated with different doses of Art for 24 h.Western Blot,Real-time qPCR and immunofluorescence were used to detect the HSC activation markers.Transfection P53 si RNA,Western Blot was used to detect the ferroptosis inhibitors or P53 inhibitors.Results:The results of HE,Masson and Sirius red staining showed that Art could improve liver fibrosis lesion damage in vivo,and the ELISA kit showed that the expression levels of ALT and AST were significantly increased in the model group compared with the standard control group(P<0.01).In contrast,each dose of Art could significantly reduce the expression levels of ALT and AST(P<0.05,P<0.01),Real-time qPCR showed that Art could dose-dependently downregulate the expression levels of CollagenⅠandα-SMA(P<0.05,P<0.01);Art could reduce the expression levels of HSC activation markers CollagenⅠ,α-SMA and Fibronectin protein and mRNA(P<0.05),and showed dose-dependence,while the results of immunofluorescence assay revealed that the expression of CollagenⅠas well asα-SMA could be reduced by Art(P<0.05)and showed a dose-dependent trend;Western Blot assay revealed that Art could significantly down-regulate HSC activation markers,while ferrostatin-1(Fer-1),a ferroptosis inhibitor,significantly reduced artesunate-induced inhibition ofα-SMA,CollagenⅠexpression;and Art significantly downregulated HSC activation indexes,while P53 siRNA counteracted the effect of Art-induced inhibition ofα-SMA,CollagenⅠand Fibronectin expression.Conclusion:Art inhibits HSC activation by inducing P53-dependent HSC ferroptosis.