扶正化瘀方重要组分丹参酚酸B抑制肝星状细胞自噬的作用机制

This subject is a study follow-up already concluding subject of Natural Science Foundation of China. As recently discovered in abroad, autophagy is an important stimulator for activity of hepatic stellate cells (HSC) leading to hepatic fibrosis. Fuzheng Huayu recipe is created by our research group and already used in clinic to treat hepatic fibrosis effectively. Fuzheng Huayu recipe at present is used to treat chronic hepatitis C on clinic trial phase II in United States. It was demonstrated that Fuzheng Huayu recipe and its effective component, Salvianolic Acid B (SA-B), can inhibit activity of HSC. We guess that the mechanism is also related to inhibit autophagy in HSC. The relationship between transforming growth factor ?1 and autophagy in activation of HSC for forming hepatic fibrosis will be innovatively studied by protein chip, RNAi and other technique in vitro. The effect and regulatory mechanism of SA-B will be studied too on inhibition of autophagy in mice HSC. The inhibition of SA-B and Fuzheng Huayu recipe for autophagy will be tested and verified during anti-fibrosis in the liver in vivo. This subject will follow the new hot point on research of hepatic fibrosis in the world. The aim is to uncover the effect and mechanism of Fuzheng Huayu recipe and SA-B through inhibiting autophagy to suppress activation of HSC. It will deepen in research frontier that the study of herb medicine on anti-fibrosis if this subject will be approved. The study will help the traditional Chinese medicine to be accepted by academe of hepatology and help to create more effective Chinese materia medica preparation for treatment of hepatic fibrosis.

本项目是已结题国家自然科学基金项目的后续研究。 国外新近发现，自噬是激活肝星状细胞（HSC）致肝纤维化形成的重要环节。本课题组研制并已上市的抗肝纤维化临床疗效确切的扶正化瘀方目前正在美国开展治疗慢性丙型肝炎的Ⅱ期临床试验。以往研究已证实该方及其有效组分丹参酚酸的部分作用机制在于能抑制HSC活化，推测可能也与抑制HSC的自噬有关。本项目体外实验应用蛋白芯片、RNA干扰等技术，创新研究转化生长因子与自噬在激活HSC导致纤维化中的关系、丹参酚酸抑制小鼠肝星状细胞株自噬的作用及其调控机制；体内实验验证丹参酚酸和扶正化瘀方抗肝纤维化的同时对自噬的抑制作用。本项目跟踪国际肝纤维化研究新热点，揭示扶正化瘀方重要组分丹参酚酸通过干预HSC自噬而抑制其活化的效应及作用机制。在科研前沿深化中药抗肝纤维化的研究，将有助于肝病学术界接受中医药，并有利于开发更高效的抗肝纤维化中药制剂。

本课题组以往对扶正化瘀复方中君药丹参的组分丹参酚酸B（Salvianolic Acid B，SA-B）作了大量的研究，证实其有确切的抗肝纤维化作用。SA-B能通过抑制转化生长因子（transforming growth factor-β1 ,TGF-β1）在肝星状细胞（hepatic stellate cells，HSC）内的MAPK的ERK和p38信号通路，抑制HSC活化、收缩和活化HSC的迁移。本项目在此基础上，在肝组织病理学、肝组织及肝星状细胞超微结构、肝星状细胞活化、自噬及凋亡等重要环节，从整体、细胞和分子水平，进一步揭示SA-B通过干预HSC自噬而抑制HSC活化的效应及作用机制。.研究工作进展顺利，达到预期目标。已在国内核心期刊发表论文4篇，培养研究生4名。.体内研究结果表明：1、在四氯化碳诱导的肝纤维化动物模型中，观察到HSC的自噬。2. 雷帕霉素能促进肝细胞和HSC的自噬,与模型组相比，LC3BII的表达明显增加（P<0.05）。3. 扶正化瘀方和SA-B均能对抗雷帕霉素的促自噬作用。体外研究结果表明：1.从急性肝损伤模型小鼠分离培养的原代HSC的活化伴有自噬的增加。 2.TGF-β1可以通过诱导自噬（P<0.01）从而激活HSC（P<0.05）。3. SA-B能够通过抑制自噬（P<0.01）；从而抑制TGF-β1激活HSC（P<0.05）。4、通过基因转染使HSC过表达Atg 5，可使Col.1表达的灰度值明显增加（P<0.05）， 这种增加可被SA-B明显抑制（P<0.05）；而自噬相关基因Atg7被干扰后能够阻断TGF-β1诱导的HSC活化，同时SA-B抑制HSC自噬和活化的作用更加明显。5、蛋白芯片检测显示，SA-B可下调HSC表达AKT、HSP27、JNK、MKK3、P38等激酶蛋白。6、验证实验证实SA-B通过下调MAPK通路的ERK（P<0.05）、p38（P<0.05）和JNK（P<0.05）通路抑制自噬从而抑制TGF-β1诱导的HSC活化。.本研究得出的结论是：“扶正化瘀方”重要组分SA-B通过下调TGF-ββ1的MAPK通路抑制自噬从而抑制HSC活化。