基于线粒体VDAC1/mPTP通路探讨番泻苷A干预非酒精性脂肪性肝病肝损伤的作用机制

Traditional Chinese Medicine (TCM) considers that the interior retention of damp-heat is one of the major causes of NAFLD. Correspondingly, eliminating dampness and heat is an important method to treat NAFLD. Rheum officinale is a representative TCM under the guidance of this method for eliminating dampness and heat. However, the intervention mechanism in NAFLD of sennoside A (SA), the main laxative active ingredient of Rheum officinale, has not been reported. Mitochondrial dysfunction leads to hepatic oxidative injury, which plays an important role in the pathogenesis of NAFLD. Thus, the improvement of mitochondrial function is of great value in the treatment of NAFLD. Our previous studies have found that SA could down-regulate the expression of hepatic VDAC1 protein, improve mitochondrial structure and function of liver, and then delay the progression of NAFLD, but the mechanism has not been clarified. In view of the key role of the VDAC1/mPTP pathway in hepatic injury of NAFLD, this project intends to explore new potential targets of SA in the intervention of hepatic injury in NAFLD from a new perspective of regulating mitochondrial pathway using the models of normal, VDAC1 silent, VDAC1 overexpressed hepatocytes and VDAC1 knockout mice, thereby elucidating the effect of VDAC1 on the development and progression of NAFLD and the mechanism of SA in improving mitochondrial function by targeting the VDAC1/mPTP pathway, so as to provide a new idea for the treatment of NAFLD with TCM.

中医学认为湿热内蕴是非酒精性脂肪性肝病(NAFLD)的主要病机之一，清热利湿是治疗NAFLD的重要方法。大黄是该方法指导下的代表性中药，但其主要泻下有效成分番泻苷A(SA)干预NAFLD的机制尚不清楚。线粒体功能失调导致肝氧化损伤，在NAFLD的发病机制中起到重要的作用，因此改善线粒体功能对治疗NAFLD具有重要价值。我们前期研究发现SA可下调肝脏VDAC1表达，改善肝脏线粒体结构及功能，延缓NAFLD进展，但作用机制尚未阐明。鉴于VDAC1/mPTP通路在NAFLD肝损伤中的关键作用，本项目拟从调节线粒体通路的新视角，利用正常、沉默及过表达VDAC1的肝细胞和肝脏VDAC1敲除小鼠，在动物及细胞分子水平探讨番泻苷A干预NAFLD肝损伤的潜在新靶点，从而阐明VDAC1对NAFLD发生发展的影响以及番泻苷A基于VDAC1/mPTP通路改善线粒体功能的作用机制，为中药治疗NAFLD提供新思路。

线粒体功能失调导致的肝氧化损伤，在非酒精性脂肪性肝病(NAFLD)的发病机制中起到重要的作用。前期研究发现中药大黄的主要泻下有效成分番泻苷A(SA)可下调肝脏线粒体膜转运蛋白VDAC1表达，改善肝脏线粒体结构及功能，延缓NAFLD进展，但具体作用机制尚未阐明。SA可能通过调节线粒体膜VDAC1/mPTP通路改善线粒体功能，从而缓解NAFLD肝损伤。本项目体外采用正常、沉默及过表达VDAC1的肝细胞模型，分别设置空白对照组(CON)、脂肪变性模型组(OPA)、SA干预组(OPA+SA)，利用油酸和软脂酸诱导细胞脂肪变性进行实验。结果发现SA可通过抑制VDAC1有效改善脂肪变性肝细胞凋亡、脂肪变性、脂肪沉积、线粒体形态异常及氧化应激损伤，其机制与下调VDAC1表达从而抑制线粒体膜转运通路及其介导的线粒体凋亡通路有关。另外，体内通过高脂诱导NAFLD小鼠模型，分成6组：SA干预(HFD-SA)组、腺相关病毒干预对照(HFD-AAV-NC)组、VDAC1敲低干预(HFD-KO)组、VDAC1敲低+SA干预(HFD-KO-SA)组、VDAC1过表达干预(HFD-OE)组、VDAC1过表达+SA干预(HFD-OE-SA)组，并设正常(NCD)组和模型对照(HFD)组，总共8组小鼠进行实验。结果发现SA可通过抑制肝脏VDAC1有效改善高脂诱导的肥胖、肝肿大、肝脂肪堆积、肝脂肪变性、肝功能损伤、肝脂质代谢紊乱以及肝氧化损伤，其机制与下调肝脏VDAC1表达从而抑制NAFLD小鼠肝脏线粒体膜转运通路及其介导的线粒体凋亡通路有关。因此，本项目明确了VDAC1是SA缓解NAFLD肝氧化损伤的重要作用靶点之一，为中药大黄干预NAFLD肝损伤的现代机制研究提供新思路，丰富中医清热治则的科学内涵，为中药大黄的临床应用提供药理学基础。