基于PPARγ节点双向调控探寻葛根芩连汤改善脂肪胰岛素抵抗的分子网络机制

Diabetes and obesity have become a major threat to human health. The vast majority is type 2 diabetes mellitus (T2DM). The pathogenesis of T2DM is mainly insulin resistance （IR）and β-cell defects. Ge Gen Qin Lian Decoction (GGQLD) is now widely and effectively used to improve IR effect in clinical treatments of traditional Chinese medicines (TCM). It was reported that these active components of GGQLD such as berberine，bailcain,puerarin and glycyrrhizic acid influence the PPARγ expression level. Since the IR effect is found in adipose tissue in the earliest stage of T2DM, high expression of PPARγ from adipose tissue may be the target protein of GGQLD intervention. It is worthwhile for us to determine if PPARγ mediates lipid and glucose metabolic pathways upon GGQLD intervention by molecular biology, cell biology, biochemical and metabolic methods. Firstly, we will examine if mRNA and protein levels of PPARγ are significantly changed in adipose tissue isolated from in vivo and in vitro models; secondly, we will investigate the influence of PPARγ on the expression level of downstream target genes aP2/FABP4, LDL, CD36/ FATP-1, FASN，GK，GLUT4 and adipocytokines such as TNF-α, IL-6, leptin, resistin, visfatin and ADPN by ELISA ,Western blot and fluorescence quantitative RT-PCR. Furthermore, miRNAs regulation based on PPARγ mediated metabolic pathway from adipocyte cell will be studied in this proposal. mRNA and miRNA expression array will be employed in IR Rat model to obtain the expression profiles based on PPARγ regulation. This study will not only provide a theoretical basis for GGQLD treatment of T2DM but also provide important theoretical and realistic significance for the corresponding development of TCM preparation.

2型糖尿病发病机制主要是胰岛素抵抗（IR）。中医认为IR与过食肥甘厚腻导致脾胃虚损相关，滋生痰湿与内热所诱发。葛根芩莲汤复方改善IR治疗糖尿病，临床疗效确切但干预机制不明确。研究表明小檗碱、黄芩苷、葛根素和甘草黄酮等影响与胰岛素增敏密切相关的PPARγ基因表达。这提示葛根芩连汤可能作用于代谢调控节点PPARγ来改善脂肪IR。本课题拟利用体内外IR模型，采用ELISA、Western blot、EMSA、荧光定量PCR和数字PCR等多种手段，重点研究究PPARγ调控的下游靶基因与脂肪分泌因子的分子调控网络。引入转录组芯片技术研究葛根芩莲汤干预糖尿病大鼠脂肪转录表达谱差异构建转录调控网络。结合代谢组学研究结果，系统阐明葛根芩连汤基于PPARγ双向调控通路改善脂肪IR的分子网络机制，为葛根芩连汤治疗糖尿病提供理论参考和科学依据，并为清热燥湿中药复方治疗代谢性疾病的多靶点网络作用机制研究提供新思路

2型糖尿病(T2DM)的主要病理基础为胰岛素抵抗（IR），脂肪组织是率先发生IR的外周组织，其引起糖脂代谢紊乱易诱发全身系统性IR导致糖尿病的形成。葛根芩连汤是苦寒燥湿的经典名方，对肥甘厚腻饮食诱导的T2DM临床疗效确切。在临床结果的基础上，我们采用体内外动物模型分析葛根芩连汤改善脂肪IR的信号转导机制，取得如下成果：（1）葛根芩连汤可显著降低T2DM大鼠空腹血糖、胰岛素、胰岛素抵抗指数、甘油三酯、总胆固醇和低密度胆固醇含量，具有抗糖尿病效应；（2）确认葛根芩连汤干预白色脂肪组织中PPARγ为节点的糖脂代谢网络来改善脂肪IR的效应机制；（3）葛根芩连汤和主药葛根含药血清可稳定升高IR-3T3-L1脂肪细胞模型葡萄糖消耗量，增加脂联素分泌量和减低甘油三酯含量，减少大脂滴形成来增强胰岛素敏感性；（4）确认葛根芩连汤及主药葛根干预IR-3T3-L1脂肪细胞中PPARγ为节点的复杂糖脂代谢网络来协调调控糖脂稳态，多靶点干预胰岛素降糖相关通路改善脂肪IR；（5）葛根芩连汤下调靶向PPARγ的miR-107-3p和miR-103-3p表达；（6）分子对接预测葛根芩连汤中36个成分结合PPARγ，主药葛根中有17个成分结合PPARγ；（7）确认葛根芩连汤核心成分葛根素和入血成分混合物A干预IR-3T3-L1细胞中PPARγ为节点的糖脂代谢网络来改善脂肪IR，具有明显降糖效应，揭示葛根芩连汤复方中低浓度多成分之间存在协同增效；（8）重要活性成份药根碱多重调控GLUTs改善脂肪IR。此外，小檗碱及异甘草素在IR-3T3-L1细胞降糖效应及干预胰岛素通路改善脂肪IR的分子机制；（9）主要活性成份葛根素、小檗碱、黄芩苷等可差异化影响GLUTs增加葡萄糖摄取，增加糖原合成来加快葡萄糖消耗改善肝IR状态。通过本项目研究，可为葛根芩连汤临床应用提供理论依据和用药指导，为葛根芩连汤复方及组分合理配伍提供优化方向。