基于ChREBP和PPARα正负反馈级联调控探讨葛根芩连汤干预肝糖脂代谢网络改善胰岛素抵抗的作用及机制研究

葛根芩连汤干预胰岛素抵抗(IR)相关的湿热型糖尿病，临床降糖疗效确切但作用机制不明。前期大鼠肝转录干预研究显示：负责脂氧化的过氧化物酶体增殖物激活受体α(PPARα)和脂合成的葡萄糖反应元件结合蛋白(ChREBP)同时被激活。体外拮抗肝PPARα上调ChREBP表达。两者均调控大量糖脂代谢靶基因表达，推测存在正负反馈进行震荡调节。鉴于肝糖输出对血糖贡献最大，因而提出假说“葛根芩连汤干预ChREBP和PPARα正负反馈级联调控肝糖脂代谢网络改善IR”。本课题拟采用多重分子药理手段，以葛根芩连汤-主成分-极低浓度入血成分混合物为主线，体内外多层次多水平研究其干预肝ChREBP和PPARα表达、活性、相互调控、其它核转录因子调节、上游磷酸化激酶修饰调控，结合下游糖脂代谢效应基因集研究其正负反馈级联网络多重调控肝降糖通路改善IR，为方剂临床治疗糖尿病提供理论依据。

Gegen Qinlian Decoction (GQD) was recently found favorable curative effects on the clinical treatment of “damp-heat” T2DM related with insulin resistance(IR). However, so far, the precise anti-diabetic mechanism of GQD remains has not been fully understood. Liver transcriptomics data showed that many genes involving in the pathways of lipid synthesis and transport were activated as well as the pathway of lipid decomposition in the GQD-treated diabetic rats. Glucose-stimulated ChREBP directly regulates glycolysis and glucose transport genes, as well as de novo lipogenesis genes such as ACC and FASn on the transcriptional level, whereas PPARα directly regulated CPT-1α, the rate-limiting enzyme of fatty acid β-oxidation. Thus, concomitantly activated hepatic PPARα and ChREBP genes in the GQD-treated diabetic rats were predicted as key nuclear transcript factors for recovering the dynamic balance of glucose homeostasis and lipid metabolism. GW6471, a specific PPARα antagonist elevated Chrebp mRNA expression of IR-HepG2 with GQD-containing serum treatment. The results indicated that it maybe exists a negative feedback looping between PPARα and ChREBP for the regulatory network of GQD intervention in vitro. Considering hepatic glycemic output contributes most to blood glucose, hence the hypothesis is putting forward that the crosstalk between ChREBP and PPARα based on the positive and negative feedback cascade regulation is favorable for hepatic glucose and lipid homeostasis to alleviate insulin resistance (IR) for the treatment of GQD. In this study, the integrated regulatory networks of hepatic glucose and lipid metabolism will be performed in the multilevel manners, including whole animal, tissue, cell and molecular levels with GQD - main active components – compounds including low blood concentration of active components. The intervened pathways of hepatic glucose and lipid metabolism based on the coordinate regulation of ChREBP and PPARα would be defined by molecular pharmacological methods in vivo and in vitro. The transcription, translation and post-translation regulation of ChREBP and PPARα expression, their interaction as well as interaction with other transcription factors would be performed in this study. Moreover, upstream phosphorylated kinase modification would be performed in this study. The loss of function test (antagonist and/or RNAi) would be used to detect the expression of downstream target genes with transcript profiles to define ChREBP and PPARα function based on the positive and negative feedback cascade as these regulation nodes of hepatic glucose and lipid metabolism. This study would provide theoretical basis for clinical treatment and medication guide intervened with GQD.