基于Smad7蛋白表达调控解析黄芪总皂苷与甘草酸配伍抗肝纤维化的效应机制

TGF-β1 has been long considered as a key mediator in development of fibrosis, so targeting its downstream TGF-β/Smad signaling pathway represents a specific and effective therapeutic strategy by either enhancing the expression of Smad7 or inhibiting TGF-β/Smad signaling pathway. Our research team previous findings suggest that improved expression of Smad7 protein may be a key part in astragalosides (AS) and glycyrrhiza acid (GA) synergistic treatment of liver fibrosis, but the exact regulatory mechanism is still unclear. Based on our research results, the hypothesis could be proposed that the increase of Smad7 in AS and GA synergistic action is associated with significantly activation of the IFN-γ/JAK/STAT1 signal and inhibition of the ubiquitin ligase activity. Through both Smad7-/-/DMN and Smad7-/-/CCl4 mouse model of liver fibrosis in combination with vitro experiments, the RNA interference technology and markers associated with regulation of Smad7 protein will be detect, so that the AS/GA synergy mechanism could be demonstrated, which would make a contribution to the clinical application of HuangQi decoction in anti-fibrosis.

TGF-β1是促组织、器官纤维化的关键细胞因子，有效干预其信号转导是目前抗纤维化研究的重要策略。提高Smad7蛋白表达、抑制TGF-β/Smad信号转导是抗纤维化的有效途径。课题组前期研究结果提示，提高Smad7蛋白表达可能是黄芪总皂苷与甘草酸治疗肝纤维化协同增效配伍作用的关键环节，但其确切的调控机制尚不清楚。本项目紧密围绕Smad7蛋白相关调控机制的研究进展，提出"黄芪总皂苷与甘草酸配伍上调Smad7表达的作用可能在于激活IFN-γ/JAK/STAT1信号及抑制泛素连接酶的活性"这一理论假说。通过Smad7?/?/DMN和Smad7?/?/CCl4两种肝纤维化小鼠模型与体外实验相结合，采用RNA干扰技术及调控Smad7蛋白表达相关指标的观测，以确证黄芪总皂苷/甘草酸协同增效配伍的关键环节是否在于上调Smad7的蛋白表达并解析其效应机制，为黄芪汤组分复方治疗肝纤维化的发展应用奠定科学基础。

转化生长因子（TGF-β1）是促组织、器官纤维化的关键细胞因子，有效干预其信号转导是目前抗纤维化研究的重要策略，抑制TGF-β/Smad信号转导是抗纤维化的有效途径。本项目研究表明黄芪总皂苷（AST）与甘草酸（GA）联用对人与小鼠2种肝星状细胞细胞株的活化及TGFβR1有良好协同抑制效应，证实已有的体内实验结果；发现miR744调控TGF-β1基因的作用及其在肝硬化患者血清中含量的显著降低；进一步以miR744为靶标，部分解析了黄芪总皂苷抑制TGF-β1生成的作用机制。基于既往体内实验肝组织基因芯片的分析，体外实验研究发现，睾丸孤儿样受体4（TR4）促进肝星状细胞的活化（至今尚未见文献报道），其机制与提高p-TβRⅠ、p-smad2/3蛋白表达及降低RXRα受体水平有关。黄芪总皂苷与甘草配伍抑制肝星状细胞增殖和活化具有协同作用，其机制与调控TR4→p-TβRⅠ→p-smad2/3信号通路有关。本项目同时观察了黄芪汤及其组分对胆汁淤积肝纤维化胆管上皮细胞增殖与肝纤维化的影响，发现Notch信号通路参与胆汁性肝纤维化的形成与发展，黄芪汤及其有效组分可显著抑制Notch信号通路而发挥抗纤维化的作用。本项目的完成为黄芪汤组分复方治疗肝纤维化的应用发展奠定科学基础，显现出良好的发展前景。