人参皂苷Re激活AMPK调控非酒精性脂肪性肝炎炎性反应的研究

Nonalcoholic steatohepatitis(NASH)is a disease which is not associated with excessive alcoholic intake, characterized by inflammation of the liver with concurrent fat accumulation in liver. NASH is also associated with metabolic syndrome such as obesity, diabetes and hyperlipidemia. The applicant confirmed for the first time that ginsenoside Re improved nonalcoholic simple fatty liver disease to activated AMP-activated protein kinase(AMPK)-a major cellular energy regulator. Based on this research, we will discuss the mechanism of gisenoside Re on NASH deeply. Using RAW264.7cells, primary hepatocyte, Kupper cells to induce the inflammatory cell model, we investigated the effect of ginsenoside Re on inflammatory signaling pathways and AMPK signaling pathways by using some techniques such as ELISA, western blot, real-time PCR and immunofluorescence staining. Next, we examine the correlation between AMPK and inflammatory signaling pathway by using AMPK wild type and AMPK dominant negative HepG2 cells. Finally, we use high fat high cholesterol (HFHC) diet to induce NASH animal models and AMPK-/- NASH models to confirm the in vitro results. The aim of this study is to investigated the effect of ginsenoside Re activated AMPK to down regulation of inflammatory factors and provides theoretical basis for the pathogenesis of NASH. In addition, assuming that AMPK is an important drug target in this research, looking for AMPK agonist has an important significance for the treatment of nonalcoholic fatty liver disease, diabetes and metabolic syndrome.

非酒精性脂肪性肝炎（NASH）是一种无过量饮酒，以肝脏炎症和脂肪堆积为特征的疾病。我们首次证实了人参皂苷Re通过激活单磷酸腺苷活化蛋白激酶(AMPK)-一种细胞能量调节器,而改善非酒精性单纯性脂肪肝，本课题将进一步探讨Re对NASH作用机制。用RAW264.7巨噬细胞、大鼠原代肝细胞、肝巨噬细胞等诱导炎症模型后，经各种分子生物学实验探讨Re对炎症信号和AMPK上下游信号的影响，以及用AMPK wild type和AMPK dominant negative HepG2细胞阐明AMPK与炎症信号的关系。小鼠给予高脂高胆固醇饲料建立NASH动物模型、AMPK-/-的NASH动物模型，证实体外实验。本课题旨探讨Re通过激活AMPK而调节炎症因子的表达，对NASH的发病机制提供研究线索及理论基础，将AMPK作为重要药物设计靶点，寻找AMPK激动剂，对非酒精性脂肪肝及代谢综合征具有重要意义。

非酒精性脂肪性肝炎是以脂肪浸润、肝细胞损害、炎症和纤维化为特征的肝脏疾病。人参皂苷是一种主要存在于人参属药材中的甾类化合物，其中人参皂苷Re活性高，因此有较高的研究价值。本课题使用脂多糖处理的RAW264.7细胞、大鼠原代肝细胞、kupffer细胞探讨人参皂苷Re的抗炎作用。首先，在RAW264.7细胞中， 用Griess法观察到Re显著抑制了NO的生成；用Elisa法观察到Re抑制TNF-α和IL-6水平；用western blot法和RT-PCR法观察到Re抑制了iNOS与COX2的蛋白表达和mRNA表达。大鼠原代肝细胞中，Re抑制了NO和PGE2的生成；同样也降低了TNF-α和IL-6水平；iNOS和COX2的蛋白和mRNA表达量在Re组显著降低；RT-PCR法观察到Re显著降低IL-1β、IL-6和TNF-α的mRNA水平；western blot法观察到Re在高浓度显著抑制p-IkBa的表达的同时还抑制LPS诱导细胞核中p65的表达、Re可以调控MAPK蛋白磷酸化表达、降低TLR4、MYD88、p-TAK的蛋白水平、增加Akt和AMPK的磷酸化、降低pmTOR和pS6K的蛋白表达，共同处理AMPK抑制剂compound C和Re后逆转了pAKT-pAMPK-pmTOR-pS6的结果。在枯否细胞中，得到了与大鼠原代肝细胞相同的结果。同样，观察到了Re抑制培养液中TNF-α和IL-6水平；降低iNOS、COX2、TLR4、MYD88、MAPK等蛋白表达。利用MCD+60%HFD饲料诱导NASH动物模型，明确观察到治疗9周后，Re组小鼠的体质量明显得到改善；肝脏颜色渐接近于健康的暗红色；与NASH组比较，Re组的肝质量显著增加，肝质量与体质量的比值显著增加；Re的LDL、GOT、GPT水平显著降低，HDL值则显著增高，与NASH组比较人Re组TG和CHO偏低，但均无统计学意义；Re抑制血清中TNF-α和IL-6水平；HE 染色结果显示，Re组脂肪变性减轻，与NASH比较有少量炎性细胞浸润。利用Raybiotech QAM-CYT-5 蛋白芯片技术，观察到Re可能影响细胞因子细胞因子受体相互作用，TLR信号通路，TNF信号通路等，JAK-STAT信号通路。通过上述分析将进一步为人参皂苷Re的炎症作用提供一个新的思路，为防治NASH奠定基础。