绿原酸-栀子苷组合抗非酒精性脂肪性肝病的剂量配比优化及对上皮间质转化的作用机制研究

In view of nonalcoholic fatty liver disease (NAFLD) as the critical factor for the development of hepatic fibrosis, cirrhosis and liver cancer, it is very important to prevent the progression of NAFLD by exploring effective drugs. Previous studies have showed that chlorogenic acid and geniposide have function to reduce TG content in HepG2 cell induced by FFA, and also mechanism of action of capillary extract for NAFLD is related to inhibiting expression of p-IKB, TNF alpha. On that basis, we put forward the scientific hypothesis "chlorogenic acid and geniposide,as the main active ingredients of wormwood and gardenia for NAFLD, are interacted to each other, and have significant intervention effect on liver lipid, inflammation, epithelial-mesenchymal transition signaling pathway". For this, rat induced by high-fat diet and HepG2 cell induced by FFA, taken as the model of NAFLD, will be given, with uniform design method, chlorogenic acid and geniposide. At the end of the experiment, check differernt indexes about lipid inflammatory damage, epithelial-mesenchymal transition, to reveal the ratio optimization between Chlorogenic Acid and Geniposide, and to make clear the pharmacological effect,their interactions, of chlorogenic acid and geniposide for NAFLD, together with the signalling pathways of epithelial-mesenchymal transition, which is to date still rarely involved in the field of NAFLD.

鉴于非酒精性脂肪性肝病（NAFLD）是肝纤维化、肝硬化、肝癌发生、发展的重要因素，探索抗NAFLD的药物及其作用机制是防止慢性肝病进展的重要措施。在前期研究发现绿原酸-栀子苷组合具有降低FFA诱导HepG2细胞TG含量作用及茵陈提取物抗NAFLD作用机理与抑制p-IκB、TNF-α表达有关基础上，提出"绿原酸、栀子苷作为茵陈、栀子抗非酒精性脂肪性肝病的重要有效成分，存在交互作用特点，对肝脂毒性炎症损伤、上皮间质转化相关信号转导通路有显著的干预作用"的科学假说。为证实该假说，拟采用高脂饮食诱导大鼠NAFLD模型及FFA诱导HepG2细胞脂肪变性模型，阐明NAFLD肝脂毒性炎症损伤、上皮间质转化的发病机制，以均匀设计法优化绿原酸-栀子苷组合的剂量配比，明确其抗NAFLD的药理效应、交互作用特点，并围绕肝脂毒性炎症损伤及目前抗NAFLD研究领域尚极少涉及的上皮间质转化信号通路，探讨药物作用机制。

研究背景：鉴于非酒精性脂肪性肝病（NAFLD）是肝纤维化、肝硬化、肝癌发生、发展的重要因素，探索抗NAFLD的药物及其作用机制是防止慢性肝病进展的重要措施。在前期研究发现绿原酸-栀子苷组合具有降低FFA诱导HepG2细胞TG含量作用，提出“绿原酸、栀子苷作为茵陈、栀子抗非酒精性脂肪性肝病的重要有效成分，存在交互作用特点，对肝脂毒性炎症损伤、上皮间质转化相关信号转导通路有显著的干预作用”的科学假说。.研究内容：采用高脂饮食诱导大鼠NAFLD模型及FFA诱导HepG2细胞脂肪变性模型，阐明NAFLD肝脂毒性炎症损伤、上皮间质转化的发病机制，以均匀设计法优化绿原酸-栀子苷组合的剂量配比，明确其抗NAFLD的药理效应，并围绕肝脂毒性炎症损伤及上皮间质转化信号通路，探讨药物作用机制。.研究结果及意义：（1）采用高脂饮食诱导的大鼠脂肪肝模型，运用数学模型“均匀设计法"观察肝组织TG、FFA含量、ALT、AST活性、MDA、SOD含量变化，优选绿原酸-栀子苷组合治疗NAFLD的最优剂量配比为90 mg绿原酸与90 mg栀子苷。（2）以p-IκB、TNF-α、Snail、E-cadherin蛋白和mRNA表达为指标，证实绿原酸-栀子苷组合显著改善大鼠NAFLD药理效应与其影响肝脂毒性炎症损伤以及上皮间质转化信号转导通路密切相关。（3）观察大鼠体重、肝湿重、脂肪重量以及TG、FFA、FAS、AMPK、ACCase、Malonyl-CoA、血清CHO、LDL-C含量及血清AST、ALT活性变化，证实栀子苷显著改善高脂饮食诱导的大鼠NAFLD药理效应与其改善游离脂肪酸代谢“AMPK-ACCase-Malonyl-CoA-FFA”轴密切相关。（4）观察栀子与栀子苷对高脂饮食诱导的金黄地鼠NAFLD模型脂质代谢、肝组织病理及炎症损伤的影响，证实栀子苷作为栀子的主要有效成分，其对NAFLD的药效优于栀子。（5）在前期研究证实10-5M绿原酸、栀子苷对HepG2细胞无毒性剂量前提下，证实栀子苷及绿原酸-栀子苷组合显著降低FFA诱导HepG2细胞肝脂毒性炎症损伤与抑制TG含量、PKR、p-IκB、TNF-α、Snail、E-cadherin蛋白及 mRNA表达有关。（6）研究表明栀子苷具有显著抑制细胞内NF-κB活性、细胞上清TNF-α, IL-6表达的作用特点，与肝脂毒性炎症损伤信号通路有关。