基于AMPK/ROS/JNK信号通路研究健肝消脂方调控胰岛素抵抗改善非酒精性脂肪性肝病的机制

Our previous studies conformed that insulin resistance (IR) could be improved in patients with non-alcoholic fatty liver disease (NAFLD) and lipid accumulation was decreased significantly in liver on experimental model by treatment of Jiangan Xiaozhi recipe. IR is considered as common pathological basis of metabolic diseases and plays a critical role in the occurrence of NAFLD. Current studies demonstrate the adenosine monophosphate activated protein kinase (AMPK)/reactive oxygenspecies (ROS)/ c-Jun N terminal kinase (JNK) pathway plays an outstanding role in addition to the close relation of hepatic lipid accumulation with the expression of AMPK and JNK. Therefore, we propose that treatment of Jiangan Xiaozhi recipe improves IR and decreases hepatic lipid accumulation by modulating AMPK/ROS/JNK pathway in the occurrence of NAFLD. On the basis of the previous studies, we are going to investigate the molecular mechanism underlying the therapeutic effects of Jiangan Xiaozhi Recipe on NAFLD in vivo and in vitro models by molecular biology methods. The effect of Jiangan Xiaozhi recipe for IR will be evaluated by using hyperinsulinemic-euglycemic clamp and glucose consumption assay which are approved internationally. In addition, the expression of proteins associated with the signal pathway will be detected by Western Blot and the activity of ROS will be estimated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. This study will elucidate the molecular mechanism and target of Jiangan Xiaozhi recipe treating NAFLD, and it will also contribute to the clinical use of the recipe to realize the prevention and treatment of NAFLD.

前期研究证实健肝消脂方能减少实验模型肝脏脂质沉积，还可改善非酒精性脂肪性肝病（NAFLD）病人的胰岛素抵抗（IR）。IR被认为是代谢性疾病共同的病理基础，在NAFLD发病中具有核心作用。研究显示AMPK/ROS/JNK信号通路在IR发生中有重要作用，且AMPK、JNK表达与肝脏脂质沉积密切相关。因此，我们提出假说：健肝消脂方可能通过AMPK/ROS/JNK信号通路调控IR进而改善NAFLD。本课题拟在前期研究基础上通过体内外实验模型及分子生物学研究方法进一步研究健肝消脂方改善NAFLD的分子机制。以国际公认高胰岛素正葡萄糖钳夹实验和细胞葡萄糖消耗量评价该方对IR的作用，用Western blot、ELISA和流式细胞术等技术对模型通路相关蛋白及ROS活性进行检测。本研究将证实健肝消脂方改善NAFLD确切的分子机制及作用靶点，将极大促进该方在临床上应用，对NAFLD的防治具有重要现实意义。

为揭示健肝消脂方治疗非酒精性脂肪性肝病（NAFLD）的分子机制，同时为健肝消脂方临床应用及其进一步开发奠定理论基础，本项目主要探讨了健肝消脂方对非酒精性脂肪性肝病大鼠模型的作用及其AMPK/ROS/JNK 通路干预机制、健肝消脂方含药血清对3T3-L1前脂肪细胞胰岛素抵抗细胞模型的作用及其对AMPK/ROS/JNK信号通路的干预机制、健肝消脂方对NAFLD模型大鼠肝脏相关mRNA表达的影响、健肝消脂方含药血清及该方医院制剂的相关问题。现已证实健肝消脂方治疗NAFLD的分子机制与提高AMPK活性密切相关，同时在不同前提下对ROS/JNK通路活性也有不同影响，健肝消脂方还影响了NAFLD模型大鼠肝脏中的多种分子通路，目前我们已初步探明了健肝消脂方含药血清中的有效成分，并已制定了健肝消脂方医院制剂的初步质量标准。概述如下。1.分子机制方面：在动物实验及细胞实验两个层面，健肝消脂方均能够改善胰岛素抵抗，并能改善脂质代谢与脂质沉积，其分子机制可能与提高AMPKα磷酸化水平有关。在动物实验中我们还发现，健肝消 脂方可以降低NAFLD模型大鼠肝脏组织中ROS含量、JNK磷酸化水平；而在细胞实验中，我们则发现健肝消脂方能够适度提高ROS水平、并可上调p-Akt（S473）、p-Akt（T308）、LKB1、PI3K、p-JNK活性、p-IRS-1活性。动物实验与细胞实验中健肝消脂方对于ROS、JNK的不同作用，可能是造模方式不同而引起，并可能与“二次打击”学说有关；而另一方面，健肝消脂方可能还影响了NAFLD模型大鼠肝脏中的血管内皮生长因子（VEGF）信号通路、丝裂原活化蛋白激酶（MAPK）信号通路、Toll样受体信号通路等分子机制。2.健肝消脂方药物方面：使用液相色谱串联质谱（LC-MS/MS）方法可测定健肝消脂方含药血清中三七皂苷R1、人参皂苷Rg1、芍药苷、人参皂苷Rb1、黄芪甲苷、丹参酮Ⅰ的含量；对于健肝消脂方医院制剂，采用薄层色谱法可定性鉴别该医院制剂中的三七、赤芍、青皮、黄芪，采用高效液相色谱法定量可对该医院制剂中丹酚酸B含量进行测定，其线性范围在0.101µg-2.525µg之间，该方法可初步用于控制该制剂的质量。本项目现已发表论文6篇，培养研究生4人。本项目研究成果丰富了健肝消脂方防治NAFLD的理论依据，并奠定了该方深入开发的基础，具有重要的理论意义及临床实用价值