熊果酸抗大鼠肝纤维化的作用靶点及安全性研究：对HSC、肝细胞及枯否细胞的NOX的影响及生物学效应。

Hepatic stellate cell(HSC) activation and proliferation represents a central event in liver fibrosis, apoptosis of activated HSC have been recognized as orchestrated programs during resolution of liver fibrosis. Recent studies provided evidence for a central role of NADPH oxidase(NOX) in the regulation of HSC activation, proliferation and liver fibrosis, demonstrate it as a novel targets for antifibrotic therapies. Our previously studies discovered that UA selectively induction of activated HSC apoptosis but promoted hepatocytes proliferation for the first time. Recently we observed that UA can down-regulated the expression of NOX subunits , inhibited NOX activation to generate ROS and NOX-mediated signal transduction in HSC. In order to clarify the hypothesis that UA may induce activated HSC apoptosis and promote hepatocytes proliferation by target of NOX in vivo, the present study was designed to investigate the effects of UA on progression of hepatic fibrosis, HSC activation and apoptosis, the expression of NOX subunits, ROS generation and signal transduction in CCl4-induced rats liver fibrosis ; equally observe its effect on the liver injury, hepatocytes proliferation and apoptosis, NOX subunits expression and biological function in hepatocytes. Finally inspect that if UA have adverse effect in vivo by observe its effects on the expression of NOX subunit, superoxide procudion and host defense function in Kupffer cells. The present study examined the therapeutic target of UA in whole - celluar - molecular level, and provided evidence for clinical applications to treatment of hepatic fibrosis.

肝星状细胞（HSC）的激活、增殖是肝纤维化的中心事件，其凋亡是肝纤维化逆转程序。近年研究表明NADPH 氧化酶（NOX）在HSC的激活、增殖及肝纤维化发病中起关键作用、是抗肝纤维化的新靶点。本团队首次在体外发现熊果酸选择性诱导活化型HSC凋亡、促进肝细胞增殖；近期发现它在体外能下调HSC的NOX亚基表达、抑制NOX激活产生ROS及其调控的信号转导。本研究拟在大鼠肝纤维化模型及组织细胞上观察熊果酸对肝纤维化发展、HSC活化与凋亡、NOX亚基表达、ROS产生及其调控信号通路的影响；观察它对肝细胞损伤、增殖、凋亡，对肝细胞NOX亚基表达及上述生物学功能的影响，以论证熊果酸在体内以NOX为作用靶点诱导活化型HSC凋亡、促进肝细胞增殖的假说；观察它对枯否细胞NOX亚基表达、超氧化物及宿主防御功能的影响以评估其安全性。该研究将在整体-细胞-分子水平阐明熊果酸作用靶点，为其用于肝纤维化治疗提供理论依据。

肝纤维化是各种病因（如病毒、血吸虫、酒精、非酒精性脂肪性肝炎、胆汁淤积、自身免疫性肝病等）引起肝损伤后细胞外基质(ECM) 沉积或瘢痕形成的过，肝纤维化的持续发展最终可发展为肝硬化，甚至肝癌，严重危害人类健康。因此基于肝纤维化的发病机制研发抗肝纤维化药物具有重要意义。 . 本课题组前期体外实验发现熊果酸具有独特的抗肝纤维化作用：（1）在细胞水平上明确了熊果酸能抑制肝星状细胞增殖、诱导其凋亡，并具有抑制Ⅰ型胶原和TIMP-1表达,促进MMP-1表达的作用。（2）在细胞水平上揭示了熊果酸能抑制肝细胞（HCs）上的NOX产生ROS，促进肝细胞增殖，抑制肝细胞凋亡。（3）在细胞水平发现了熊果酸可以抑制肝星状细胞（HSCs）上的NOX产生ROS，并观察到熊果酸对瘦素诱导的NOX的亚基NOX2、p22phox、p67phox、Rac1的表达均有不同程度的抑制作用。. 从文献报道可知HSCs、HCs和枯否细胞（KCs）三者表达的NOX亚基及其产生的ROS在肝纤维化中起重要作用，由体外实验可知熊果酸能抑制NOX的活化并减少ROS的表达，延续前期研究，我们建立ccl4大鼠肝纤维化模型，体内探究熊果酸抗肝纤维化作用及其机制，初步研究结果表明：（1）熊果酸能减轻或逆转肝纤维化；（2）熊果酸能抑制原代HSC、肝细胞和枯否细胞表面NOX1、NOX2、NOX4、p22phox、p47phox、p67Phox及rac1 mRNA的活化，减少三种细胞中ROS的产生；（3）熊果酸能调控HSCs及HCs凋亡相关蛋白Bcl2、XIAP、Gadd45β、Bax、CD95L 、CD95、Caspase3 和Caspase 9 的蛋白水平，进而促进HSCs凋亡，抑制HSCs增殖，保护HCs，抑制HCs凋亡；（4）熊果酸不影响正常及肝纤维化大鼠单核巨噬细胞的吞噬功能。.这些结果表明熊果酸体内抗肝纤维化的主要机制为以HSC的NOX为作用靶点选择性抑制HSC活化，诱导活化型HSC凋亡，同时，熊果酸还可通过下调HCs中NOX的表达和ROS的产生进而保护肝细胞损伤、促进肝细胞增殖，最后，虽然熊果酸可下调枯否细胞NOX的表达，但其对宿主的防御功能无明显影响。因此，研究以NOX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。