Fyn在肝星状细胞介导肝纤维化形成过程中的作用机制研究

The activation, and its subsequent pathological pro-fibrotic actions, of hepatic stellate cells (HSC) are the hallmarks of liver fibrosis. In response to liver damage, HSCs is activated and undergoes a phenotypic transition, and can acquires (or trans-differentiate to) a myofibroblast-like phenotype, including expression of α-smooth muscle actin (α-SMA). The activated HSCs (with myofibroblast-phenotype) show increased proliferation, unstoppable production of extracellular matrix (ECM) proteins, secretion of proinflammatory/profibrotic cytokines, and having the capacity to migrate and contract. It is important to understand the molecular mechanisms which are largely responsible for HSC activation and subsequent liver fibrosis. Understanding the major pathophysiological pathway is helpful to develop effective therapeutic interventions to limit and reverse the development of live fibrosis. Our preliminary data showed that Fyn, a Src family kinases member, plays a critical role in HSC activation and live fibrosis. Our preliminary data support that Fyn promotes the pro-fibrotic functions of HSCs, such as activation, differentiation, and migration of HSC, through the integrin/Fyn/FAK axis. Furthermore, Fyn promotes HSC to myofibroblast differentiation through a neuroal Wiskott-Aldrich Syndrome Protein (NWASP)-dependent pathway. Downregulation of Fyn (by shRNA or pharmacological inhibitor AZD0530) significantly inhibited the pro-fibrotic functions of activated HSCs. These preliminary findings lead us to hypothesize that Fyn plays a critical role in HSC activation and liver fibrosis, and Fyn is a potential therapeutic target to treat liver fibrosis. Our study seeks to define the critical role of Fyn in activation and profibrotic functions of human HSCs, to delineate the molecular mechanisms by which Fyn promotes myofibroblast differentiation and liver fibrosis in human HSCs and in animal liver fibrosis model, and to examine the effect of loss of Fyn in liver fibrosis in vivo (by using Fyn knockout mice). Our study also aims to examine the activation of Fyn in a large scale of clinical human samples to support our hypothesis. Furthermore, preliminary translational medicine experiments will be carried out to evaluate the anti-fibrotic effects of AZD0530 (a small molecule inhibitor of Fyn) in animal models of liver fibrosis. We believe that our study will greatly contribute to the understanding of profibrotic role of Fyn in liver fibrosis, and thus provide a new and feasible therapeutic target for the treatment of liver fibrosis.

肝星状细胞(Hepatic stellate cells, HSC)的活化及功能异常是肝纤维化发生、发展的核心环节。我们前期研究发现Src酪氨酸蛋白激酶家族成员Fyn可通过整合素/Fyn/FAK通路、Fyn/FAK/N-WASP通路在HSC分化和迁徙过程中发挥重要作用；shRNA和小分子Src激酶抑制剂AZD0530拮抗Fyn分子活性可抑制HSC的部分促纤维化生物学行为，提示Fyn有望成为肝纤维化治疗的新靶点。本项目拟在此基础上利用临床组织标本验证病理状态下Fyn对HSC和人肝纤维化发生、发展的影响；利用Fyn基因敲除小鼠进一步阐明Fyn在HSC介导肝纤维化形成中的作用机制；观察Fyn抑制剂AZD0530在动物模型中的抗纤维化效果，进行初步的转化医学尝试。本研究将拓展对Fyn为代表的Src激酶在肝纤维化进程中作用机制的认识，进一步诠释肝纤维化发生、发展分子机制，开发抗肝纤维化治疗的新靶点。

Src家族激酶在系统性硬化症和肺纤维化中具有重要作用。然而Src家族激酶在肝纤维化中的作用基本上是不清楚的。本项目我们研究了Src家族激酶中的Fyn在肝星状细胞活化和肝纤维化中的作用，并评估了Src蛋白激酶抑制剂AZD0530和尼达尼布抗肝纤维化的效果。我们发现临床肝纤维化组织中Fyn被显著激活，其磷酸化水平显著高于正常肝组织。体外实验显示TGF-β诱导Fyn激活。Fyn敲低能够显著抑制HSC活化、增殖和迁移。Fyn敲除小鼠能够抵抗CCl4诱导的肝纤维化。AZD0530能够抑制肝星状细胞Fyn激活和α-平滑肌肌动蛋白表达，可以抑制肝星状细胞的迁移；尼达尼布处理亦能抑制肝星状细胞中Fyn激活，下调Fyn/FAK/N-WASP信号通路，进而阻止肝星状细胞激活。尼达尼布处理能显著减弱CCl4诱导的小鼠肝纤维化严重性。我们结果证实了Fyn在肝星状细胞活化和肝纤维化进展中发挥关键作用，提示其可以作为治疗肝纤维化的潜在药物靶点。