基于自噬途径探讨肝纤维化细胞上皮间质转化现象的分子机制及扶脾柔肝方的干预研究

HHepatic fibrosis (HF) is a necessary intermediate link in the process which chronic hepatopathy evolves to liver cirrhosis, and the inducing factor of HF lies in hepatic stellate cell(HSC) activation. Autophagy is a vacuolar process of cytoplasmic degradation by lysosome.) which can yield the energy for the activation of HSC and accelerate the occurrence of HF. In other words, the activation of HSC, the fibroblast phenotype from the epithelial mesenchymal transition(EMT) was gradually transformed into the myofibroblasts (MFB), and finally caused the occurrence and development of HF. This research is based on theory of "nourishing spleen", which means that "when partial liver disease occurs, it will spread to the whole liver;therefore we should nourish the spleen before treating the liver "; moreover. It is from "the autophagy of HSC -and EMT-energy metabolism" link that so much are learned about the pathophysiological mechanism of HF and the action or effects of traditional Chinese medicine Tonifying Spleen and Liver(TSL) on HF , Through the establishment of HF animal and EMT cell model ,and the regulation of autophagy pathway, expression of key signal transduction molecules of Akt, mTOR, Beclin 1, LC3- Ⅱ, TGF- β 1, Smad3, Snail, E-cadherin, a-SMA, AMPK in PI3K/Akt/mTOR pathway and EMT TGF- β /Smads/Snail pathway,and the probe on synergistic effect of autophagy and EMT, it is aimed to provide experimental basis for Traditional Chinese Medicine (TCM)theory of "Nourishing Spleen Method" and the effective mechanism of TSL powder in the treatment of HF.

肝纤维化(HF)是慢性肝病向肝硬化发展的必经中间环节，其中心环节在于肝星状细胞(HSC)的激活。自噬通过降解脂滴为HSC活化提供能量，促进HF的发生。HSC活化通过上皮间质转化（EMT）获得成纤维细胞的表型,转变为肌成纤维细胞(MFB),促进HF的发生与发展。本研究基于"见肝之病，知肝传脾，当先实脾"的"实脾"理论，从"自噬-能量代谢-EMT"的环节探讨HF的分子机制及中药扶脾柔肝方对HF干预的效应机制。通过建立HF动物模型及EMT细胞模型，对自噬通路的调控，检测自噬PI3K/Akt/mTOR通路、EMT TGF-β/Smads/Snail通路中关键的信号传导分子Akt、mTOR、Beclin 1、LC3-Ⅱ、TGF-β1、Smad3、Snail、E-cadherin、a-SMA、AMPK的表达情况，探讨自噬与EMT的协同作用，为揭示中医"实脾"理论及扶脾柔肝方治疗HF的作用机制提供依据。

肝纤维化是是由损伤因素所引起的细胞外基质在肝脏内大量沉积为主要病理特征的疾病。“肝纤维化-肝硬化-肝癌”这一链条被认为是各种慢性肝病导致严重后果的共同途径。.本研究从“HSC自噬-能量代谢-EMT”关键环节探讨HF的病理生理机制及中药扶脾柔肝方对HF模型大鼠及HSC的效应机制，通过建立HF动物模型及EMT细胞模型，对自噬通路的调控，检测自噬PI3K/Akt/mTOR通路、EMT、TGF-β/Smads通路中关键的信号传导分子的表达情况，探讨自噬与EMT的协同作用，为揭示中医肝脾相关理论及扶脾柔肝方治疗HF的作用机制提供依据。.动物实验结果显示，在大鼠肝纤维化模型中，扶脾柔肝方具有抗肝纤维化，降低肝纤维化血清标志物，对肝纤维化自噬PI3K/Akt/mTOR通路、EMT、TGF-β/Smads通路具有调节作用。与正常组比较，模型组HA、LN、IV-C、PCⅢNP模型升高；与模型组比较，扶脾柔肝方能降低血清HA、LN、IV-C、PCⅢNP含量。与正常组比较，模型组ALT、AST、γ-GGT、TBIL含量显著增高，ALB含量显著降低；与模型组比较，扶脾柔肝方能降低血清ALT、AST、γ-GGT、TBIL含量，升高ALB含量。与正常组比较，模型组Beclin 1、Akt、mTOR、P70S6K、TGF-β1、Smad3、Snail、a-SMA、AMPK mRNA及蛋白表达升高，E-cadherin mRNA及蛋白表达下降；与模型组比较，扶脾柔肝方能下调Beclin 1、Akt、mTOR、P70S6K、TGF-β1、Smad3、Snail、a-SMA、AMPK mRNA及蛋白表达，上调E-cadherin mRNA及蛋白表达。.体外细胞实验结果显示，扶脾柔肝方对HF“HSC自噬-能量代谢-EMT”轴具有调节作用。与空白组比较，模型组Beclin 1、LC3-Ⅱ、Akt、mTOR、P70S6K、Smad3、Snail、a-SMA、AMPK、TIMP mRNA及蛋白表达升高，E-cadherin、MMP2表达降低，HSC脂质含量下降；与模型组比较，扶脾柔肝方含药血清能降低Beclin 1、LC3-Ⅱ、Akt、mTOR、P70S6K、Smad3、Snail、a-SMA、AMPK、TIMP mRNA及蛋白表达，升高E-cadherin、MMP2表达，增加HSC脂质含量。