新疆特色植物锁阳中笼状碳苷黄烷Songarin A对肝纤维化大鼠的作用机制研究

Previous researches have found that Cynomorium songaricum Rupr., which is the characteristic and traditional Chinese medicine in Xin jiang, having significant protective effect for acute and chronic hepatic injury animal model caused by CCl4. Active tracking filter has found the active ingredients , flavanol compounds, Songarin A, being a cage structure of carbon glycosides. This topic proposes around the activation , proliferation and apoptosis of HSC, and the process of collagen production, studying the effect and mechanism of liver hepatic fibrosis. .First of all, on the basis of large-scale preparation Songarin A , utilizing liver hepatic fibrosis in rats model, we identify the role of Songarin A and its function characteristics in liver hapatic fiborsis. Secondly, using HSC T6 cell lines model to detect the proliferation and apoptosis mechanism of HSC by Scanning electron microscopy , flow cytometry and TUNEL. Finally, detecting the signal transduction molecules of HSC Smad3, Smad7 and ERK expression with Western blot and RT-PCR; detecting the rat HSC I and type III collagen mRNA expression levels in rat with Songarin A medication before and after by RT-PCR and Iimmunohistochemistry methods. Through the related research in vivo and in vitro, to clear the mechanism of action and targets of Songarin A in rat model with liver fibrosis.

前期研究发现：新疆特色植物锁阳对四氯化碳所致肝损伤动物模型具有显著的保护作用。活性跟踪筛选发现其活性成分为笼状碳苷结构黄烷类化合物-Songarin A。本课题拟围绕肝星状细胞（hepatic stellate cells，HSC）激活、增殖、凋亡和胶原产生的过程，研究其对肝纤维化大鼠的作用机理。.首先，大量制备Songarin A，复制大鼠肝纤维化模型，明确Songarin A对肝纤维化大鼠的作用和作用特点；其次，利用HSC-T6细胞株,用电镜、流式细胞仪及TUNEL法检测HSC凋亡及增殖；Western blot 和RT-PCR法检测HSC中的Smad3、Smad7以及ERK等信号转导分子；用RT-PCR法和免疫组化技术检测Songarin A用药前后大鼠HSC中I、III型胶原mRNA表达水平。通过体内外相关研究，明确Songarin A对肝纤维化大鼠的作用机制和作用靶点。

1.从锁阳属中首次分离得到两个化合物1,2,4-trihydroxybenzene、3,4,5-trihydroxybenzoic acid ethyl ester，通过研究锁阳对CCl4诱导的肝纤维化小鼠的治疗作用，发现锁阳具有抗肝纤维化的作用，并可以通过调控TGF-β1/Smad信号通路及ERK1/2信号通路来治疗肝纤维化。.2.锁阳能保护大鼠肝功能，明显降低大鼠肝纤维化程度,减缓大鼠肝纤维化的发展，其机理可能与抑制TGF-β1、PDGF、TNF-α的改变有关。.3.锁阳对小鼠肝纤维化及其对Hedgehog信号通路干预作用的初步探讨.（1）对雄性C57小鼠一周两次腹腔注射CC14，第三周时可成功复制肝纤维化模型。在正常状态时，Hedgehog信号通路在小鼠体内是被抑制的；当受到CCl4损伤和刺激时，Hedgehog通路被激活，从而使α-SMA和Gli1在肝脏中的表达增加。所以，Hedgehog信号通路可能与肝纤维化的发生关系密切。.（2）锁阳用药对小鼠肝纤维化有改善作用，并且锁阳高剂量组用药效果显著，其作用机制可能与Hedgehog信号通路的抑制有关，可能成为治疗肝纤维化新的药物。.4.自噬对肝星状细胞中Hedgehog信号通路的影响.（1）雄性小鼠通过腹腔注射CCl4和橄榄油（1:3的比例）混合物，可以成功复制小鼠肝纤维化模型。.（2）在正常小鼠肝脏中较少发生自噬，而在肝纤维化发展中自噬活动增强。.（3）在炎性微环境下，自噬可以通过调节原代M-HSC中Hedgehog信号通路中Gli1、Gli2的表达，从而促进HSC的活化以及肝纤维化的形成。