从细胞自噬功能异常探讨“痰瘀”病机在NASH发病中作用及其中药干预机制

Jiang-Zhi Granule (JZG) was developed in accordance with the TCM theory, including strengthening spleen, resolving inner dampness and phlegm, and dissipating blood stasis. Our previous study showed that JZG had an anti-steatotic effect on HFD-induced rats through inhibition of SREBP-1c pathway. Steatosis in liver is regarded as the basic factor providing the setting for NAFLD. However, the mechanisms for the development from hepatic steatosis (NAFLD) to hepatic inflammation (NASH) have not been clarified completely. To date, the pathogenesis of NASH has been proposed widely as a ‘two hit concept’. Therefore, an effective early pharmacological intervention is entailed to prevent or delay the onset and progress of NASH. In the pathogenesis of NAFLD, the lipid accumulation attenuated autophagy, leading to the accumulation of pathological products, which is the material basis for the development of ‘phlegm and stasis’. After the formation of ‘phlegm and stasis’, lipid peroxidation, endoplasmic reticulum stress, release of reactive oxygen, inflammatory factors activation and other factors progressed NAFLD to NASH. In this study, we used Luciferase Assay, RNAi and many other molecular biotechnology in vitro and in vivo, to explore the effect of autophagy attenuation on the development of pathological products (‘phlegm and stasis’) and the pathogenesis of NASH. The aim of this study was to clarify the potential target for JZG treatment, and provide a novel view for the prevention and/or treatment of NASH.

青年科学基金项目研究证实，降脂颗粒应用祛湿化痰、健脾活血法治疗脂肪肝，是通过调控SREBP-1c信号通路改善肝细胞脂质代谢异常。肝细胞脂质代谢异常是NAFLD发病的基础环节，NAFLD是如何从单纯性脂肪肝进展成为脂肪性肝炎，发病机制至今尚未明确，目前普遍接受“二次打击”学说。对NASH的早期干预，是防止病变继续恶化的关键节点。课题组在前期研究的基础上，提出NAFLD发病过程中，脂质积聚后细胞自噬功能减退，细胞内病理产物堆积，成为“痰瘀”凝结的物质基础，“痰瘀”形成后，出现的脂质过氧化、内质网应激，活性氧释放、炎症因子激活等因素，使得NAFLD病情向NASH进展。本研究通过体内、体外实验，运用荧光报告素酶、RNAi等分子生物学技术，探讨肝细胞自噬功能减退，形成“痰瘀”病理产物，影响NASH发病过程的机制，阐释降脂颗粒可能的作用靶点，探索NASH防治的新思路。

非酒精性脂肪性肝病（Non-alcoholic fatty liver disease，NAFLD）可导致严重的肝脏并发症，增加死亡率，近年来受到众多学者越来越多的关注。非酒精性脂肪性肝炎（Nonalcoholic steatohepatitis，NASH）是病程演变的关键阶段，肝细胞脂质积聚后，脂质过氧化、氧化应激和炎症性因子等因素形成“二次打击”，使线粒体、内质网等细胞器的自身损伤增加。正常情况下，细胞通过自噬机制处理自身细胞内衰老的细胞器、蛋白折叠残留片段，防止其在细胞内积聚形成病理产物“痰瘀”；我们考虑肝细胞脂肪变后，细胞自噬功能减低，自我清除能力下降，肝气运行不畅，阻碍肝主疏泄功能，脏腑功能失调。降脂颗粒已获得国家食品药品监督管理局批准进行临床试验，前期研究疗效可靠。我们考虑中草药所含成分复杂，能够产生多种效应，尝试运用系统药理学方法，解释中药多组分、多靶点的协同效应。我们运用系统药理学方法：分析降脂颗粒成分-靶点-相互作用关系，筛选降脂颗粒中活性化合物的成分，关联候选基因靶点构成网络，预测分析药物作用靶点。结合前期实验数据和文献分析，自噬信号通路是药物药效机制之一，我们进一步进行实验验证。我们应用细胞实验、模式动物实验，从Lipophagy及Mitophagy两方面分析脂质过氧化和线粒体损伤时，降脂颗粒的药物作用机制。我们构建病毒转染细胞株，分析LC3-II/LC3-I表达比值及p62来确认自噬流，检测药物的有效性，确认作用时间及强度；以分子探针BODIPY、Lyso Tracker和MitoTracker复染进行细胞内定位；电镜观察自噬小体形成，多角度评估药物的干预效果；以高脂饲料和MCDD饲料喂养小鼠构建NAFLD模型，分析降脂颗粒的药效机制；运用分子生物学实验技术从基因、蛋白水平分析药效，实验证实降脂颗粒能够调控PI3K-AKT-mTOR信号通路，改善肝细胞脂质堆积，减轻肝细胞损伤。我们的研究仍然存在着诸多的不足，没有分析药物包含的复杂化合物其他靶点联合效应；没有分析其他非实质细胞的作用；我们将在后面的实验中进一步分析补充。