当归多糖靶向肝脏激活AMPK/SIRT1正反馈环路促进自噬治疗非酒精性脂肪肝病的机制研究

Non-alcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease, yet there is not a recognized drug therapy till now. Traditional Chinese medicine polysaccharides have the pharmacological characteristics of multiple targets and multiple mechanisms, which are compatible with the etiology of multi-factors induced NAFLD, thus it is possible for the polysaccharides to treat this disease. The applicant has verified that Angelica sinensis polysaccharide (ASP) could effectively treat NAFLD, but the specific mechanism is still unclear. Our preliminary experiments of this project reveal that ASP can be specifically recognized by hepatocyte surface receptor ASGPR, then enter the hepatocyte by endocytosis of this receptor. In addition, ASP can also up-regulate the expressions of AMPK and SIRT1 in liver and promote hepatic autophagy. It has been reported that the activation of hepatic AMPK/SIRT1 positive feedback loop can promote autophagy, and the promotion of autophagy can treat NAFLD. Based on these findings, the applicant hypothesizes that ASP can treat NAFLD by specifically binding to hepatocyte ASGPR, activating hepatic AMPK/SIRT1 positive feedback loop and promoting autophagy. This project intends to treat NAFLD with ASP, combined with chemical inhibitors, RNA interference and gene knockout technologies to block the key proteins of the signaling pathway, defining the mechanism of ASP from animal, cell and molecular levels. This project will provide new ideas and theoretical basis for the drug development of NAFLD, as well as the further development and utilization of Chinese medicine angelica sinensis.

非酒精性脂肪肝病(NAFLD)已成为慢性肝病之首，尚无公认药物疗法。中药多糖多靶点多机制的特点与NAFLD多因素致病的发病机制契合，有可能治疗该病。申请人已证实当归多糖（ASP）可有效治疗NAFLD小鼠，但分子机制尚不明确。本项目前期研究发现ASP可被肝细胞表面受体ASGPR专一性识别，通过胞吞作用进入细胞，ASP还可上调肝脏AMPK和SIRT1表达量并促进肝脏自噬。文献报道，激活肝脏AMPK/SIRT1正反馈环路可促进自噬，促进自噬可治疗NAFLD。据此提出假说：ASP特异性结合ASGPR靶向肝脏激活AMPK/SIRT1正反馈环路，促进自噬缓解多重致病因子治疗NAFLD。本项目拟采用ASP治疗NAFLD模型，结合化学抑制剂、RNA干扰和基因敲除技术，分别阻断信号通路关键蛋白，从动物、细胞和分子层面明确ASP作用机制，可为NAFLD药物研发以及中药当归的进一步开发利用提供新思路和理论依据。

NAFLD影响全球四分之一的人口，可进一步发展为肝炎、肝纤维化甚至肝癌，但是尚无公认药物疗法。中药活性成分因其高效低毒的特点，在治疗非酒精性脂肪肝病(NAFLD)的药物开发中扮演重要作用。申请人长期致力于挖掘有效治疗NAFLD的活性成分及分子机制，在本项目开展的3年期间，我们团队分别从动物、细胞和分子层面明确ASP作用机制，从“确认现象→研究机制→验证机制”的思路展开探索，顺利完成既定方案：1)确认现象:建立NAFLD动物模型，ASP给药治疗后检测药效学指标，明确ASP对NAFLD的治疗作用；2)研究机制:检测AMPK/SIRT1正反馈环路和细胞自噬关键蛋白，采用免疫荧光共定位、透射电镜等方法观察肝细胞自噬的水平，探究ASP治疗NAFLD的可能分子机制；3)验证机制:阻断AMPK或者SIRT1通路，干预ASP治疗，证实ASP通过激活AMPK/自噬通路治疗NAFLD。此外，本项目还紧紧围绕中药活性成分治疗NAFLD的研究，拓展了两部分内容：1）中药活性多酚（槲皮素）通过促进AMPK介导的肝脏自噬改善NAFLD；2）中药活性多酚（木犀草素）通过调节宿主血清代谢组和肠道微生物组缓解蛋氨酸胆碱缺乏饮食引起的NAFLD。总之，本项目顺利完成了申请书既定计划，并在中药治疗NAFLD这一领域进一步延伸，取得一系列成果，为后续研究打下坚实的基础。本项目的成功实施为NAFLD药物研发以及中药活性成分的进一步开发利用提供新思路和理论依据。