基于GLP-1受体相关信号通路的京尼平苷与绿原酸组合治疗非酒精性脂肪肝效应机制研究

Basing on the preliminary work of the applicant, the optimized prescription composed of Geniposide and Chlorogenic acid (the "GC" prescription) has an ideal effect on anti-experimental liver steatosis found by the uniform designed research, which has a great advantage compared with every single components of the prescription, combined our preliminary results of the study of gene chip and the progress of pharmacological research with the recent new knowledge ,that the GLP-1-associated-receptor signaling pathways play roles on the new therapeutic targets to NAFLD, we raised the hypothesis that the GC prescription may play a key role on the GLP-1-associated-receptor signaling pathways, through which it regulates the liver lipid metabolism on the treatment of fatty liver diseases with compatibility synergy effects. We plant to use two kinds of fatty liver models in rats(lack of congenital leptin receptor (db/db mice), high-fat diet induced ones) and two kinds of cellular models (FFA induced hepatic steatosis, siRNA silences HuH7 hepatocellular Glp-1 receptor) which are compared with the Glp-1 agonists and antagonists. Arounding the key downstream components of the relevant GLP-1-receptor signaling pathways (cAMP-PKA, PI3K-Akt, SIRT1-AMPK) and the hepatic lipid metabolism, we will from both positive and negative aspects to test weather the "GC" prescription has the pharmacological effects on these signaling pathways, especially on the individual components, and to reveal the target position and the main mechanism and of "GC" prescription on the treatment of fatty liver disease. It will provide scientific bases for our further development of new drugs.

本项目在前期运用均匀设计筛选发现京尼平苷与绿原酸特定配比组合（GC方）有理想的抗实验性脂肪肝效应，显著优于单一成分，且基因芯片观察发现该方对GLP-1受体下游信号通路的AMPK、PKA有重要影响的基础上，结合 "肝脏GLP-1受体信号通路是非酒精性脂肪性肝病（NAFLD）治疗靶点"的新认识，提出"GC方影响肝GLP-1受体相关信号通路调节肝脏脂肪代谢"的假说。拟采用两种整体模型（高脂饮食诱导、瘦素受体基因缺失db/db小鼠）、两种细胞模型（FFA诱导肝细胞脂肪变性、siRNA沉默HuH7肝细胞GLP-1受体），设GLP-1受体激动剂与拮抗剂对照，围绕肝脏GLP-1受体下游cAMP-PKA、PI3K-Akt、SIRT1-AMPK信号通路及肝脂肪代谢关键环节，正反两方面验证GC方是否作用于肝细胞GLP-1受体调节相关信号通路，以阐释该方治疗NAFLD的关键靶位和机制，为今后研发新药奠定基础。

课题围绕“京尼平苷与绿原酸组合（GC方）影响肝脏GLP-1受体相关信号通路调节肝脏脂肪代谢”假说，采用高脂饮食体内外动物及细胞模型，围绕肝脏GLP-1受体下游SIRT1-AMPK、cAMP-PKA信号通路及肝脂肪代谢关键环节，探讨该方治疗NAFLD的关键靶位和机制。.课题实施进展顺利，完成了任务书计划中高脂饮食诱导的小鼠脂肪肝模型、先天性瘦素受体基因缺乏db/db小鼠模型体内实验及肝细胞脂肪变性体外模型实验。获得以下主要研究结果：①与模型组相比， GC方治疗后非酒精性脂肪肝小鼠肝脏脂肪含量显著下降，肝脏炎症减轻，血糖血脂显著降低； GC方药物血清或直接添加均可显著降低FFA诱导的肝细胞脂肪含量、改善肝细胞脂肪变性；②GC方可显著升高NAFLD模型小鼠肝组织或脂肪变性肝细胞GLP-1R蛋白及基因表达，并升高脂肪肝小鼠血清GLP-1含量；③GC方可显著升高肝组织或肝细胞GLP-1R下游SIRT1、LKB1、p-AMPK、p-ACC蛋白及基因表达水平，升高肝组织或肝细胞cAMP含量及PKA蛋白含量。.综合体内外实验结果，研究获得以下主要结论：①京尼平苷与绿原酸组合（GC方）可显著改善高脂饮食诱导脂肪肝模型小鼠肝脏脂肪变性及糖脂代谢，显著减轻肝脏炎症。②升高血清GLP-1含量，上调肝组织GLP-1R表达，并激活GLP-1R下游 SIRT1-AMPK及cAMP-PKA信号通路是GC方有效防治实验性脂肪肝的主要作用机制。(详见研究技术报告).项目研究工作顺利完成，达到预期目标。已发表标注论文16篇（其中SCI收录4篇），国际会议口头汇报交流论文1篇，美肝会壁报交流论文1篇；获专利授权2项；获上海市科技进步三等奖1项，中国中西医结合学会科技进步二等奖1项；新获国家自然基金面上项目资助1项；培养研究生3名。研究为探索成分明确、并可进一步提高疗效的抗脂肪肝中药成分复方制剂提供坚实理论依据。