Fn14通过抑制SIRT4调控肝星状细胞谷氨酰胺代谢促进肝纤维化的研究

The activation of hepatic stellate cells (HSCs) is a central event of liver fibrogenesis. Our previous studies focused on Fn14 pathway in the regulation of HSCs activation, and the gene expression profiling revealed that Fn14 inhibited the expression of SIRT4, however, the role of SIRT4 in liver fibrosis is still unclear. Our preliminary experiment showed that SIRT4 inhibited GDH activity and glutamine metabolism of HSCs, while Fn14 had the opposite effect. Accordingly, we hypothesize: Fn14 inhibits SIRT4 to induce GDH activity, and further enhances glutamine metabolism to promote the activation and proliferation of HSCs, subsequently mediates liver fibrosis. Firstly, we will treat cultured HSCs with adenovirus vectors to up / down regulate Fn14, or with overexpression plasmid to up regulate SIRT4, or with EGCG to inhibit GDH activity to clarify the effects and mechanism of Fn14 and SIRT4 in vitro; secondly, we will use the wild-type and Sirt4-KO mice to establish liver fibrosis model to further confirm the mechanism in vivo; finally, we will verify the association between liver fibrosis and the related gene expression in human liver biopsy specimens. The results will demonstrate a new mechanism of liver fibrosis and provide a new idea for clinical treatment of liver fibrosis.

肝星状细胞（HSCs）活化是肝纤维化的中心环节。我们的前期工作聚焦于Fn14通路调控HSCs活化，表达谱芯片分析显示Fn14抑制SIRT4的表达，而SIRT4在肝纤维化中的作用尚不清楚。预实验表明：SIRT4能够抑制HSCs的GDH活性和谷氨酰胺代谢，而Fn14作用相反。我们据此提出假说：Fn14通过抑制SIRT4而诱导GDH活性，增强谷氨酰胺代谢，从而促进HSCs的活化和增殖，最终介导肝纤维化。本研究拟首先通过体外培养HSCs，用腺病毒载体上/下调Fn14、过表达质粒上调SIRT4、EGCG抑制GDH活性，在体外明确Fn14、SIRT4的作用及机制；再采用野生型及Sirt4基因敲除小鼠肝纤维化模型，在体内进一步验证；最后在人肝活检标本中验证相关分子表达与肝纤维化的关系。本研究将阐明肝纤维化的新机制，为临床治疗肝纤维化提供新思路。

肝纤维化是肝硬化的必经阶段，而肝硬化及其并发症后果严重。肝星状细胞（HSCs）活化是肝纤维化的中心环节。我们的前期工作聚焦于Fn14通路调控HSCs活化，表达谱芯片分析显示Fn14抑制SIRT4的表达，而SIRT4在肝纤维化中的作用尚不清楚。我们的预实验表明：SIRT4能够抑制HSCs的GDH活性和谷氨酰胺代谢，而Fn14作用相反。据此提出本研究的假说：Fn14通过抑制SIRT4而诱导GDH活性，增强谷氨酰胺代谢，从而促进HSCs的活化和增殖，最终介导肝纤维化。为此，本研究首先在细胞实验、动物模型及人体标本水平，证明在肝纤维化过程中，SIRT4的表达降低，而过表达SIRT4则抑制HSCs的增殖、活化，从而抑制肝纤维化；并进一步证实上调的谷氨酰胺代谢对维持HSCs活化及纤维化水平具有关键作用，而过表达SIRT4则能够通过显著抑制谷氨酰胺代谢而发挥抗纤维化作用；进而发现TWEAK/Fn14能够通过抑制SIRT4而促进肝纤维化，抑制Fn14则能够上调SIRT4而逆转肝纤维化。最后，在人肝活检标本及外周血中验证了相关分子表达的关系。在上述结果的基础上，进一步的拓展研究还发现：抑制Fn14通路能够通过抑制活化HSCs内质网应激而逆转肝纤维化；Mn2+通过激活cGAS-STING通路诱导HSCs衰老而抑制肝纤维化。这些研究结果首次揭示Fn14/SIRT4调控谷氨酰胺代谢在肝纤维化发生发展和逆转中的重要作用及机制，有助于从新的视角探索肝纤维化逆转的新方法，为临床治疗肝纤维化提供新的理论和实验依据。