基于自噬相关PI3K/Akt/mTOR信号通路探索五味子总木脂素降肝脂作用及作用机制

Fatty liver is the most prevalent liver desease. The pathogenesis of fatty liver remains unclear, and lack of effective therapeutic methods. Recent studies reported that autophagy is one of the hepatic lipid metabolism pathways, and drug-induced autophagy can promote hepatic lipid elimination. Schisandra Chinensis lignans (SCL) is the most effective part for liver protection. It has the potential ability to inhibit hepatic steatosis. Previous study in our group found that SCL could decrease the hepatic lipids and induce autophagy at the same dosage in vitro. The underlying mechanism of SCL-induced autophagy related the regulative effect of PI3K/Akt/mTOR signaling pathway. Based on these results, we hypothesize that PI3K/Akt/mTOR pathway is involved in the lowering hepatic lipids effect of SCL. To address this hypothesis, we propose to use RNA silence and overexpression approaches to establish Atg5 gene knockdown and Akt gene activation of cells. We also use the corresponding autophagic inhibitors to systematic analysis whether regulates these moleculars can weaken the lowering hepatic lipids action of SCL, and identify the underlying mechanism of SCL-induced autophagy and related signaling pathway in hepatic lipid metabolism. We also confirm the role of PI3K/Akt/mTOR signaling pathway in the lowering hepatic lipids effect of SCL in vivo. This project explores the lipid lowering mechanism of SCL, and provides new ideas to investigate the lipid lowering effect of Traditional Chinese Medicine.

脂肪肝是最普遍的肝脏疾病，目前发病机制尚未明确且缺乏有效干预手段。最新研究表明自噬是肝脂代谢最主要的途径之一，通过药物诱导肝细胞自噬，可以促进肝脂消除。五味子总木脂素是五味子中保肝活性最强的有效部位，具有明确的降肝脂作用。本课题研究证实，五味子总木脂素在降低脂肪肝细胞模型脂质含量的同时能诱导肝细胞自噬，其诱导自噬机制与调控PI3K/Akt/mTOR信号通路有关。基于此，我们提出以下假说：五味子总木脂素是通过调控PI3K/Akt/mTOR信号通路诱导自噬发挥降肝脂作用。本研究拟采用RNA基因沉默及过表达技术，分别构建Atg5基因抑制和Akt基因过表达脂肪肝细胞模型，并采用相应自噬抑制剂，系统分析调控上述分子后五味子总木脂素降肝脂作用的差异，明确该药物诱导的自噬及相关信号通路在调节肝脂代谢中的机制，再在体内进行验证。本项目的实施可阐明五味子总木脂素降肝脂作用机制，为中医药防治脂肪肝提供新思路。

本课题围绕“五味子总木脂素（SCL）是通过调控Akt/mTOR/PI3KC3信号通路诱导自噬，影响脂质代谢，从而发挥其降肝脂作用”假说，采用RNA基因沉默及过表达技术，分别构建ATG5基因抑制和Akt基因过表达脂肪肝细胞模型，并采用相应自噬抑制剂，系统分析调控上述分子后五味子总木脂素降肝脂作用及相关自噬和脂质代谢通路的差异，探讨SCL调控的自噬相关Akt/mTOR/PI3KC3信号通路在其降肝脂的作用及机制。课题实施进展顺利，获得以下主要研究结果：（1）SCL可以抑制游离脂肪酸（FFA，油酸：棕榈酸=2：1）诱导的HepG2和肝原代脂肪肝细胞Akt/mTOR/PI3KC3信号通路，从而激活自噬；（2）抑制肝细胞自噬后（3-MA处理和siATG5转染）能降低SCL降肝脂能力，且SCL上调肝细胞β氧化相关蛋白的能力减弱；（3）SCL能激活高脂饲料喂养的脂肪肝小鼠模型肝细胞自噬，3-MA处理后SCL降肝脂及激活肝细胞β氧化能力变弱；（4）Akt激活（IGF-1处理和Akt1 ORF表达质粒转染）后SCL诱导的脂肪肝细胞自噬和降肝脂能力降低，且上调脂肪肝细胞β氧化相关蛋白的能力减弱。（5）Akt激活（Akt1 ORF表达质粒尾静脉注射）后SCL诱导的脂肪肝小鼠模型肝细胞自噬和降肝脂能力降低，且激活肝细胞β氧化能力降低。综合体内外实验结果，研究获得以下主要结论：（1）SCL是通过诱导肝细胞自噬发挥其降肝脂作用，相关机制与其激活肝细胞β氧化有关；（2）SCL是通过调控自噬Akt/mTOR/PI3KC3信号通路发挥其降肝脂作用。项目研究工作顺利完成，达到预期目标。已发表标注论文5篇（其中SCI收录5篇），正培养研究生2名。研究从自噬及相关信号通路角度，揭示SCL降肝脂作用的内在机制，为中医药防治脂肪肝提供新的思路。