IGFBPrP1在致肝纤维化形成中对肝星状细胞自噬的调节机制

Transforming growth factor β1 (TGFβ1) is known to be one of the strongest cytokines that induces hepatic fibrosis. Recently, it was reported that TGFβ1 can affect autophagy activation of hepatic stellate cells (HSC). Furthermore, we previously found that insulin-like growth factor binding protein related protein-1 (IGFBPrP1) was a novel factor involved in hepatic fibrogenesis, and that reciprocal causation existed between IGFBPrP1 and TGFβ1. However, the relationship between IGFBPrP1 and autophagy of HSC is unclear. Here, we aim to explore the involvement of HSC autophagy in the pathogenesis of IGFBPrP1-induced liver fibrosis. SiAtg5 or siAtg7 was used to knockdown Atg5 or Atg7 genes, respectively, both in the JS1 cell line and in C57BL/6 mice post transfection with Ad-IGFBPrP1. In addition, the cells and mice will be treated with an autophagy inhibitor or its signal pathway specific inhibitor. Finally, the expression of α-SMA of HSC activation and numerous key autophagy markers such as LC3B, Atg5, Atg7 or mTOR of signal pathway of autophagy and ECM of hepatic fibrosis markers will be detected by the following methods: Real-time PCR, western blot, flow cytometry, immunofluorescence, MDC staining and transmission electron microscopy. The results from this study may provide a new therapeutic strategy to determine the progression of liver fibrosis by specifically inhibiting activated HSC.

TGFβ1是目前公认的最强的致肝纤维化因子，近期有学者发现其可引起肝星状细胞(HSC)自噬活性发生改变。申请者前期研究发现IGFBPrP1是致肝纤维化新因子，且与TGFβ1可互为因果关系。但IGFBPrP1与HSC自噬的关系目前一无所知。故本项目拟以HSC自噬为切入点，采用沉默自噬相关基因或给予自噬或其相关信号通路的特异性抑制剂作用于经腺病毒IGFBPrP1转染的HSC(原代分离及小鼠HSC株)或C57BL/6野生型小鼠，经实时荧光定量PCR、western blot、流式细胞术、免疫荧光、MDC染色及透射电镜等方法测定反映HSC活化的α-SMA、反映自噬的LC3B、自噬相关基因Atg5、Atg7、自噬信号的“阀门分子”mTOR、反映肝纤维化程度的ECM等动态变化，从细胞及整体动物水平探究IGFBPrP1在致肝纤维化形成中对HSC自噬的调节机制，为有效靶向HSC的抗肝纤维化治疗提供新策略。

目前抗肝纤维化的临床治疗方案远远不能满足医学要求，抑制肝星状细胞（HSC）活化是逆转肝纤维化的重要方法。本项目是以肝星状细胞自噬为切入点，从体外细胞与在体动物水平研究了IGFBPrP1致肝纤维化形成的机制。在实施过程中成功分离、鉴定及培养了SD大鼠原代肝星状细胞，通过胆总管结扎术制备C57BL/6野生型小鼠肝纤维化模型，体外培养了小鼠肝星状细胞株JS-1，分别给予自噬抑制剂3MA或自噬相关信号通路特异性抑制剂雷帕霉素或LY294002，采用了实时荧光定量PCR、Western blot、透射电镜、MDC染色、细胞免疫荧光、免疫组织化学染色、双荧光素酶报告基因检测等方法进行了研究。在C57BL/6野生型小鼠被胆总管结扎或AdIGFBPrP1基因转染后，形成小鼠肝纤维化的在体动物实验中，肝组织自噬指标LC3、SQSTM1/p62、Beclin1的表达均增高，IGFBPrP1可通过自噬诱导小鼠肝纤维化形成（肝组织HE染色、天狼星红染色、免疫组化CollagenI染色等发生改变）；且在该过程中Atg9a、lncRNANEAT1、lncRNAH19的表达是增高的，lncRNAMEG3和MiR-29b表达是减少的，Akt与mTOR磷酸化水平是被抑制的。在SD大鼠原代HSC和小鼠HSC株JS-1的体外细胞实验中，IGFBPrP1能诱导HSC发生自噬、促进其活化，导致引起肝纤维化形成的细胞外基质（ECM）含量增多；且该过程中IGFBPrP1作为上游调控因素增加lncRNANEAT1、lncRNAH19和Atg9a的表达，减少lncRNAMEG3、MiR-29b的表达，抑制Akt与mTOR磷酸化水平。本项目阐明了IGFBPrP1在致肝纤维化形成中对肝星状细胞自噬活性的调节机制，为IGFBPrP1成为临床逆转肝纤维化新靶标及未来推进具有抗肝纤维化特性的药物开发提供了重要依据。