基于肝星状细胞中NOX4/ROS和RhoA/ROCK信号通路研究熊果酸抗肝纤维化作用的分子靶点

Inhibiting the activation of hepatic stellate cells(HSC) is the key countermeasure to prevent liver fibrosis. Recent works have suggested imperative roles of NOX4/ROS and RhoA/ROCK signaling in inducing the fibrosis: in pulmonary fibrosis,the NOX4/ROS was proved to be the upstream signal molecules to activate RhoA/ROCK signaling pathways, while the activation of RhoA/ROCK signaling pathway seemed to be a stimulus to upregulate the expressions of NOX4/ROS pathway in renal fibrosis.However, if there also exists such a functioning way similar to above-mentioned or other behaviors between these two signaling pathways in the process of liver fibrosis is still unknown. In our preliminary study, the expression of NOX4/ROS and Rac1, a member of Rho GTPase family, was upregulated in liver fibrosis rats, and Ursolic Acid(UA) seemed to reverse liver fibrosis through inhibiting the expression of NOX4/Rac1. But whether UA affects RhoA, another member of Rho GTPase family, remains unknown. Based on our previous findings, we will perform our experiments in NOX4 or RhoA gene knockout rats to investigate the roles of them to liver fibrosis in vivo,and the inhibiting effect of UA towards these two pathways.In vitro,we will deeply explore the underlying molecular mechanism of the interaction or synergistic effects between NOX4/ROS and RhoA/ROCK signaling pathway; shed futher light on the mechanism of UA to cure liver fibrosis; and eventually illuminate the NOX4/ROS and RhoA/ROCK pathway are promising molecule target for UA. This study is expected to further clarify the molecular mechanism to development of liver fibrosis,and provide novel targets for the treatment of UA to liver fibrosis.

抑制肝星状细胞（HSC）的活化是阻止肝纤维化的关键。研究发现NOX4和RhoA都在纤维化中起重要作用且两者相互调节：在肺纤维化中，NOX4/ROS为RhoA/ROCK信号通路的上游分子；在肾纤维化中，两者恰好相反。目前未见两者在肝纤维化中是否存在上述类似或其他调节机制的报道。我们前期研究发现熊果酸（UA）能通过下调HSC中NOX4/ROS的表达，抑制Rho GTP酶家族成员中Rac1的表达，逆转肝纤维化。但UA对Rho GTP酶家族另一重要成员RhoA的作用未见报道。本研究将在此基础上通过NOX4或RhoA基因敲除小鼠观察两者缺失对肝纤维化的改善作用；明确UA对两者的抑制作用；深入研究NOX4/ROS和RhoA/ROCK信号通路在肝纤维化中的交互调控机制；进一步探讨UA是否以这两条信号通路作为抗纤维化的分子靶点。研究结果有望进一步阐明肝纤维化发病的分子机制，并为UA抗肝纤维化提供新靶点。

静止型肝星状细胞增殖型肌纤维母细胞的转化是肝纤维化发病的中心事件。活化的HSC/MFB导致肝脏代谢重编程，自噬增加及实质细胞损伤加重，同时导致HSC类维生素A丢失、收缩性增强，肝脏微环境中生长因子及炎症信号因子放大，继而产生大量ECM，促使纤维化的发生与发展等。利用胶原蛋白启动子驱动的绿色荧光蛋白转基因小鼠发现，MFB主要由HSC转化而来而MFB是ECM的主要来源，因此抑制HSC的活化，或者使活化的HSC衰老、失活和凋亡是肝纤维化逆转的关键。.Rho GTP酶可以活化NOX，尤其是Rac1可以调控NOX1和NOX2的活化，我们前期研究也发现Rac1在活化的HSC中表达上调，Rac1参与NOX的活化，且熊果酸可以通过下调Rac1的表达，抑制NOX亚基的活化，逆转肝纤维化。然而RhoA/ROCK与NOX4/ROS的关系不是很清楚：在肺脏疾病的研究中，NOX4/ROS可激活RhoA/ROCK信号通路，促进肺纤维母细胞的迁移及胶原的合成，加重肺纤维化。而在肾脏疾病的研究中发现，RhoA/ROCK为NOX4/ROS的上游信号分子，RhoA/ROCK信号通路的激活可以使NOX4/ROS表达上调，促进肾肌成纤维细胞分化，加重肾纤维化。虽然RhoA/ROCK和NOX4/ROS都参与调控HSC活化和纤维化，但RhoA/ROCK和NOX4/ROS在肝纤维化中的相互调控机制未见报道。据此我们推测，NOX4/ROS与RhoA/ROCK信号通路都参与了HSC活化的调节，两者之间的具体关系可能如下：一方面，NOX4/ROS为RhoA/ROCK的上游信号分子；另一方面NOX4/ROS为RhoA/ROCK的下游信号分子；甚至RhoA/ROCK和NOX4/ROS之间可能存在反馈回路等，通过一些目前未知的机制共同促进肝纤维的发生发展。.本课题组研究发现熊果酸可以阻止HSC中Rac1的表达，抑制NOX亚基的活化，阻止NOX4产生ROS，抑制HSC激活，促进活化HSC的凋亡，逆转肝纤维化。RhoA/ROCK是除NOX4/ROS之外，熊果酸抗肝纤维化作用的又一主要分子靶点，熊果酸可能通过抑制RhoA/ROCK信号通路的激活，调控HSC细胞骨架，进而抑制HSC活化，阻止肝纤维化。因此，研究以OX4/ROS为RhoA/ROCK为作用靶点的肝纤维化机制，对熊果酸将来应用于临床治疗肝纤维化有重要意义。