以NADPH氧化酶信号网络为靶点探讨大黄蛰虫丸抗肝纤维化作用的分子机制

As an ancient herbal formula that has functions of promoting blood circulation to remove blood stasis and removing damp-heat, Dahuang Zecong Pill(DHZCP) has been proved to possess ability of anti- fibrosis and -cirrhosis of the liver by current clinic. However, the mechanism of above ability is still unclear now. Recent studies have shown that NADH oxidase (NOX) and its signal network played a key role in the pathogenesis of liver fibrosis and were viewed as one of the important new targets for anti-liver fibrosis research. In our previous study, we found that DHZCP could reduce the NOX activity of the hepatic stellate cells (HSC) and liver fibrosis in rat liver tissue and suppress the expression of NOX key subunits and the collagen type Ⅰ. Thus, it is important and valuable to understand that how DHZCP inhibit NOX signal network to achieve the effect of anti-fibrosis. In this project, we proposed to use RT-PCR, western blot and flow cytometry, confocal laser surgery, signaling pathway block technique and other modern molecular biology techniques to investigate the effect of DHZCP to HSC, liver cells, liver fibrosis models of NOX and more importantly, the signal network. It is expected to clarify the functions of DHZCP in anti-liver fibrosis and the regulation of HSC in biological characteristic target processing from cellular and protecting the liver cells, molecular and the integral levels, and thus, to demonstrate the mechanism of anti-liver fibrosis and to provide theoretical basis for TCM approved internationalization and modernization of DHZCP.

作为活血化瘀、清泻湿热的古方大黄蛰虫丸，目前临床已证实对肝纤维化肝硬化具有肯定的疗效，但具体作用机制不明。新近研究表明，NADPH氧化酶（NOX）及其调控的信号网络在肝纤维化的发病中起关键作用，是抗肝纤维化的重要新靶点。我们前期研究发现，大黄蛰虫丸能下调肝星状细胞（HSC）和肝纤维化大鼠肝组织中NOX的活性、抑制NOX重要亚基及Ⅰ型胶原的表达，那么大黄蛰虫丸是否通过抑制NOX信号网络达到抗纤维化作用呢?具有很强的探索性。本课题拟在前期研究基础上，通过采用RT-PCR、免疫印迹、激光共聚焦术、流式细胞术、信号通路阻断等现代分子生物学技术，分别观察大黄蛰虫丸对HSC、肝细胞、肝纤维化模型NOX及其调控信号网络的影响，从细胞、分子、整体水平来阐明大黄蛰虫丸调控HSC、保护肝细胞及抗纤维化的作用靶点，为揭示大黄蛰虫丸抗肝纤维化机制提供实验基础；为古方大黄蛰虫丸获得国际认可及中药现代化提供理论依据。

大黄蛰虫丸是中医经典方，应用于临床抗肝纤维化，然而分子机制尚不清楚。因此，本研究以大鼠及HSC-T6细胞为研究对象，从体内外探讨大黄蛰虫丸抗肝纤维化的作用、对胶原形成的改变、对HSC-T6细胞生物学特性的影响，并揭示其分子机制。研究结果表明：1、大黄蛰虫丸能清除H2O2诱导的细胞内ROS、促进HSC损伤后修复，抑制低氧环境诱导的HSC的增殖,并呈明显的剂量-效应反应关系；2、大黄蛰虫丸能显著缓解CCl4诱导的大鼠肝纤维化，改善肝功能、减少血清中炎症细胞因子的产生；3、大黄蛰虫丸能显著减少胶原生成，可能是通过调节MMPs和PI3K信号网络；4、二代测序及生物信息显示大黄蛰虫丸可能通过ECM受体, 粘附激酶和PI3K–Akt等信号网络参与抗纤维化进程；5、大黄蛰虫丸可通过lncRNA gas5抑制p-ERK和p38等信号通路的激活，发挥抗肝纤维化作用。本研究从细胞、分子、整体水平证明了大黄蛰虫丸对肝纤维化的治疗效应、作用环节及其机制，为临床肝纤维化的防治提供新方法，也为拓宽大黄蛰虫丸临床应用范围提供科学依据。