基于TGF-β1/smad与JAK/STAT多信号转导通路的舒肝和络醒脾法抗肝纤维化作用机制研究

Hepatic fibrosis (HF) is involved in the pathophysiological process of a multi factor. Based on the theory of “the long illness into collaterals”，the “extracellular matrix deposition (liver damage)-liver fibrosis characteristic of change (fibrosis) - a large number of false lobule formation (cirrhosis)” is amount to “ collateral stasis-small ZhengJi disease- ZhengJi formation”in Chinese Medicine theory. If we can block the formation of tiny, eliminate disease products help to reverse HB. The research group proposed "The Anti-hepatic Fibrosis mechanism of the therapy of regulate the liver，harmonize the collaterals, and enlivening the spleen therapy by the method of eliminating micro ZhengJi，was related to the TGF-β1/smad and JAK/STAT signal transduction pathway" hypothesis. Intends to use molecular biology and cytology observation: First, the impact of the therapy of regulate the liver，harmonize the collaterals, and enlivening the spleen to HB model rat liver tissue TGF β1/ Smad, JAK / STAT signaling pathway related factors and TIMPs/MMPs, ILS and other cytokines. Second，the effective components of the compound, inhibits the proliferation of HSC-T6 cells with exogenous TGF-β1 induced cell apoptosis, cell cycle and TGF-β1/Smad, JAK/STAT signaling pathway. The purpose is to clarify the anti-HB blocking mechanism through signal transduction pathway by the therapy of regulate the liver，harmonize the collaterals, and enlivening the spleen, laying the foundation for screening anti-HB's healing method.

肝纤维化（HF）是一个多因素参与的病理生理过程。基于“久病入络”理论，其“细胞外基质沉积（肝损伤）-肝纤维化特征性改变（纤维化）-大量假小叶形成（肝硬化）”过程相当于中医学“络脉瘀滞-微小癥积-癥积形成”。若能消除“微癥积”证素，则有助于逆转HF。课题组提出“舒肝和络醒脾法通过消除微癥积抗HF，其作用机制与TGF-β1/smad、JAK/STAT等多信号转导通路有关”的假说。拟运用分子生物学和细胞学技术观察：1、舒肝和络醒脾法复方对FB模型大鼠肝组织TGF-β1/Smad、JAK/STAT信号通路相关因子及TIMPs/MMPs，ILs等细胞因子的影响；2、复方有效成分对外源性TGF-β1刺激后的HSC-T6细胞增殖抑制、细胞凋亡、细胞周期以及TGF-β1/Smad、JAK/STAT信号传导通路的影响，目的是阐明舒肝和络醒脾法抗HF的多信号转导通路阻断机制，为防治HF提供思路与方法。

本研究应用四氯化碳诱导的肝纤维化大鼠模型，应用ELISA、HE、MASSON、免疫组化、WB、PR-PCR等多种分子生物学方法，通过体内外实验，从形态和功能学角度验证了舒肝和络醒脾方的抗肝纤维作用及机制，将西医中“细胞外基质沉积（肝损伤）-肝纤维化特征性改变（纤维化）-大量假小叶形成（肝硬化）”的病理过程，与中医学久病入络的“络脉瘀滞-微小癥积-癥积形成”的理论相结合，阐明了舒肝和络醒脾法可通过消除“微癥积”从而逆转肝纤维化的分子机理。进而证实了我们提出“舒肝和络醒脾法通过消除微癥积抗HF，其作用机制与TGF-β1/smad、JAK/STAT等多信号转导通路有关”假说的科学性。.研究发现，舒肝和络醒脾方可从分子和蛋白水平，阻断TGF-β1/smad、JAK/STAT等多条信号转导通路中TGF-β1、smad2、JAK2、STAT3、TIMP-1、MMP-2、IL-8、TNF、COLⅢ、α-SMA等关键分子和蛋白的表达，从而发挥防治肝纤维的作用。其与阳性对照西药相比，其抗肝纤维效果更为明显，且在抗纤维化的过程中还对肝功能有一定的保护作用。同时筛选出舒肝和络醒脾方的疗效“小组方”，黄芪当归按3:1比例混合，体外抗纤维化效果最佳。.本实验研究结果为舒肝和络醒脾方在临床上的开发应用提供了实验依据和理论支撑，对中医药服务区域卫生健康和地方经济具有一定的推动作用。