基于AMPK/SIRT1通路探讨降脂颗粒改善非酒精性脂肪性肝病脂质代谢紊乱的调控机制

The prevalence of NAFLD(nonalcoholic fatty liver disease)is increasing, however, effective medicine for its treatment still lacks. Among the complex pathogenesis, lipid metabolism disorders play an important role in the pathogenesis of NAFLD. In the recent years, studies have shown that AMPK/SIRT1 pathway is important in regulating liver lipid metabolism which is closely related to the development of NAFLD. The traditional Chinese medicine, Jiang Zhi Granules(JZG),has proved to be safe and effective in long-term clinical practice for NAFLD patients. And previous study has shown its role of promoting AMPK activation. Therefore, through in vitro and in vivo experiments, this project intends to clarify the role of AMPK/SIRT1 pathway in the development of NAFLD and explore the relative mechanism, which might provide a new potential target for NAFLD. In addition, according to the pathogenesis of NAFLD in Chinese medicine"spleen deficiency as root and phlegm stasis and damp heat as manifestation", the decoction for"replenishing spleen and activating blood circulation, clearing heat and expelling dampness", JZG, will be employed to treat the NAFLD models. Using biological techniques, we will study the effect of JZG and protopanaxadiol (PPD, one compound from the herb of JZG), on NAFLD, and try to reveal its mechanism of improving lipid dysfunction in NAFLD through regulating AMPK/SIRT1 pathway to inhibit lipid synthesis and promote lipid degradation. The results will provide scientific proof for the treatment of NAFLD with traditional Chinese medicine and establish experimental basis for developing new medicine for this disease.

非酒精性脂肪性肝病(NAFLD)的发病率逐年升高，目前仍缺乏有效治疗药物。脂质代谢紊乱在NAFLD发病机制中起重要作用。近年来研究显示，AMPK/SIRT1是调节肝脏脂质代谢的重要通路，与NAFLD发生发展密切相关。中药复方降脂颗粒，在长期临床实践中治疗NAFLD安全有效。前期动物实验显示该复方可促进AMPK活化。因此本项目拟通过体内、外实验，明确AMPK/SIRT1通路在NAFLD发生发展中的作用，进一步阐述其相关机制，为NAFLD提供新的诊疗靶标。同时针对NAFLD “以脾虚为本，痰瘀、湿热为标”的中医病机，治以“健脾活血、清热利湿”方剂降脂颗粒。应用生物学技术研究降脂颗粒及有效成分原人参II醇对NAFLD的药效作用，并基于AMPK/SIRT1通路调控脂质合成与分解，揭示其改善NAFLD中脂质代谢紊乱的机制，为应用推广中医药治疗NAFLD提供科学依据，并为新型药物的研发建立实验基础。

非酒精性脂肪性肝病(NAFLD)的发病率逐年升高，目前仍缺乏有效治疗药物。发病机制尚未完全清楚，脂质代谢紊乱在NAFLD发病机制中起重要作用。近年来研究显示，AMPK/SIRT1是调节肝脏脂质代谢的重要通路，与NAFLD发生发展密切相关。中药复方降脂颗粒，在长期临床实践中治疗NAFLD安全有效。前期动物实验显示该复方可促进AMPK活化。项目通过体内、外实验，分别建立NAFLD小鼠动物模型和人肝癌细胞株HepG2细胞和小鼠正常肝细胞AML2细胞肝细胞脂肪变模型，研究中药复方降脂颗粒及其有效成分原人参II醇通过调控AMPK/SIRT1信号通路影响NAFLD脂质代谢的药效机制。运用RT-PCR及Western-Blot对AMPK/SIRT1信号通路相关分子基因及蛋白表达水平进行检测，采用DAPI+Lipi-Red双染法进行染色，并通过高内涵细胞成像分析系统分析各组细胞情况，发现降脂颗粒及其有效成分原人参Ⅱ醇均能减轻NAFLD小鼠体重、肝重，降低小鼠血清AST、ALT、LDH、TNF-α水平。还可降低肝组织TG、TC含量，改善肝细胞脂肪变，上调AMPK的磷酸化水平和SIRT1的表达水平下调SUV39H2和SIRT1乙酰化水平。还可上调PPARα、ACC的磷酸化水平、PGC-1α、CPT-1、ACOX1等脂质氧化基因的表达，下调ACC、FAS、SREBP-1等脂质合成基因的表达，且降脂颗粒的高剂量组和原人参Ⅱ醇的中剂量组的效果最佳。原人参Ⅱ醇的高、中、低剂量均降低细胞内TG的水平，且具有剂量依赖性。在使用AMPK\SIRT1抑制剂后，原人参Ⅱ醇对脂质代谢的调控作用被显著抑制。进一步明确了AMPK/SIRT1通路在NAFLD发生发展中的作用。因此，降脂颗粒及其有效成分原人参Ⅱ醇能改善体外肝细胞和NAFLD小鼠肝脏脂肪变，主要是降低甘油三酯含量，其作用机制包括促进AMPK调控和SIRT1的表达，激活AMPK/SIRT1信号通路，抑制脂肪酸合成，促进脂肪酸氧化，从而为NAFLD提供新的诊疗靶标，揭示其改善NAFLD中脂质代谢紊乱的机制，为应用推广中医药治疗NAFLD提供科学依据，并为新型药物的研发建立实验基础。