TWEAK负调控活化肝星状细胞衰老在肝纤维化中的作用及机制

The activation of hepatic stellate cell (HSC) is a central event of liver fibrogenesis, and it is also an important target for anti-fibrotic therapy. Previous studies have focused on promoting apoptosis of activated HSC or transforming the activation phenotype to quiescent phenotype, but recent studies have shown that senescence is also an important outcome approach of activated HSC. Our preliminary experiment has demonstrated that tumor necrosis factor-like weak inducer of apoptosis (TWEAK) inhibited the SA-β-Gal activity of activated HSC and up regulated the expression of SIRT1. Accordingly, we hypothesize: TWEAK activates SIRT1 via the receptor Fn14, and further inhibits p53 pathway to negatively regulate activated HSC senescence, subsequently promotes liver fibrosis. Firstly, we will treat cultured HSC with adenovirus vectors, the purified protein or neutralizing antibody to up/down regulate TWEAK, or with adenovirus vectors to up regulate p53 or down regulate SIRT1, to clarify the effects and mechanism of TWEAK in vitro; secondly, we will use wild-type and Sirt1-KO mouse to establish liver fibrosis model to further confirm the mechanism that TWEAK inhibits the senescence of activated HSC in vivo; finally, we will verify the association between activated HSC senescence and related gene expression in different causes of chronic liver disease. The result will demonstrate a new mechanism of liver fibrosis promoted by TWEAK via inhibiting the senescence of activated HSC and provide a new perspective for clinical treatment of liver fibrosis.

肝星状细胞（HSC）活化是肝纤维化发生的中心环节，也是抗纤维化的重要靶点。既往研究多聚焦促进活化HSC凋亡或转为静息表型，近来发现衰老也是其重要转归途径。我们的前期工作表明，肿瘤坏死因子样弱凋亡诱导因子（TWEAK）能抑制活化HSC的SA-β-Gal活性，上调SIRT1表达。据此提出假说：TWEAK通过Fn14激活SIRT1，抑制p53通路从而负调控活化HSC衰老，促进肝纤维化。本研究拟首先采用HSC体外培养，用腺病毒载体、纯化蛋白、中和抗体上/下调TWEAK，用腺病毒载体上调p53、下调SIRT1，在体外明确TWEAK的作用及机制；再采用野生型及Sirt1基因敲除小鼠肝纤维化模型，在体内进一步证实TWEAK抑制活化HSC衰老的机制；最后在人肝活检标本中验证活化HSC衰老与相关分子表达的关系。本研究将阐明TWEAK负调控活化HSC衰老而促纤维化的新机制，为临床治疗肝纤维化提供新的视角。

肝纤维化是肝硬化发生的必经阶段，其发生发展的中心环节肝星状细胞（Hepatic stellate cell，HSC）活化。因此，以活化HSC为切入点深入探讨肝纤维化的发展和逆转机制具有极其重要的意义。既往研究多聚焦于促进活化HSC凋亡或转为静息状态，而近来研究显示细胞衰老也是活化HSC的一种重要转归。我们的预实验发现肿瘤坏死因子样弱凋亡诱导因子（tumor necrosis factor-like weak inducer of apoptosis，TWEAK）能够抑制活化HSC的SA-β-Gal活性，并上调SIRT1的表达。本研究首先在体外细胞实验水平发现TWEAK通过Fn14受体发挥生物学作用，首次阐明miR-19b参与调控TWEAK对Fn14的激活，即miR-19b能够抑制Fn14的表达，而TWEAK则通过抑制miR-19b从而上调Fn14。在此基础上，我们发现TWEAK/Fn14能够促进HSC的活化，在体内能够促进肝纤维化。TWEAK对HSC的作用主要体现在抑制衰老，TWEAK主要通过上调SIRT1基因表达，从而抑制p53的乙酰化而抑制HSC的自发性衰老而促进活化。这些结果首次阐明TWEAK通过调控细胞衰老而发挥生物学作用，对肝纤维化的治疗以及细胞衰老的逆转均有潜在价值。另外，TWEAK显著促进HSC的迁移，同时较大剂量能够促进增殖。TWEAK促进LX-2细胞迁移，而同时上调MMP9的表达和活性、以及IkappaBalpha和p65。此外，TWEAK还通过激活EGFR/Src和PI3K/AKT信号通路从而促进活化HSC迁移。TWEAK能够NF-κB/STAT3通路促进活化HSC炎症因子分泌。TWEAK可以通过诱导HSC中Fn14的表达，促进IκB和JAK2磷酸化，进而激活其下游的NF-κB（p65）、STAT3磷酸化，从而促进多种炎症因子分泌，如Mcp1，IL8，IL6，RANTES。这些结果也在体内实验中得到验证。本研究通过大量实验阐明TWEAK在肝纤维化过程中作用重要，机制丰富，为临床治疗肝纤维化提供新的视角，为今后进一步探讨阻断TWEAK治疗肝纤维化的策略提供充分支撑。