基于“miR34a/Nampt-NAD+-TAC”通路研究痰瘀同治法调控糖脂代谢的作用机制

Dan-gua Fang,which reflects the tanyu-tongzhi method (the method of treating phlegm simultaneously with smoothing blood stasis,痰瘀同治法), has been used in our hospital for more than 10 years and has obtained the national invention patent.In the previous series of research projects on national natural science foundation, we found that Dan-gua Fang regulation of glycolipid metabolism was relatedand with increasing the metabolic flux of TAC to promote energy expenditure, and meanwhile up-regulation of Nampt gene expression.The miR-34a / Nampt-NAD + -TAC pathway is a key physiological mechanism for the regulation of glycolipid metabolism.Therefore,the hypothesis has been established that the tanyu-tongzhi method may improve glucolipid metabolism by regulating miR-34a / Nampt-NAD + -TAC pathway to promote energy consumption. There are four cell experiments, in which the pathway and its related factors in upstream and downstream will be studied multdimensionally from genes, protein expression, key enzymes and intermediates to effects by qRT-PCR, Western blot, immunohistochemistry, ELISA, flow cytometry, high performance liquid chromatography etc., combined with micoRNA blocking and simulation techniques.And then the results of above experiments will still be verified through the experimental five in vivo experiments. The results of this study will clarify the mechanism on tanyu-tongzhi method improving gluolipid metabolism disorder which characterized by the excessive energy storage, and the relationship between the mechanism and the pathway-mediated metabolic flux regulation. These will provide references to improve the clinical use of tanyu-tongzhi method to interfere with the obese glycolipid metabolic disease efficacy.

体现痰瘀同治法的丹瓜方为我院院内制剂已使用10余年并获国家发明专利,降脂降糖降体重疗效好。前期系列国自然研究中，发现丹瓜方调控糖脂代谢与增加三羧酸循环（TAC)代谢通量、促进能量消耗有关，且上调Nampt基因表达。miR-34a/Nampt-NAD+-TAC通路是糖脂代谢调控的关键生理机制。故提出假说：痰瘀同治法可能通过调控miR-34a/Nampt-NAD+-TAC通路促进能量消耗以改善糖脂代谢。本项目通过4个细胞实验，以qRT-PCR、免疫印迹、免疫组化、ELISA、流式细胞术、高效液相色谱法等，结合micoRNA阻断与模拟技术，对该通路及上、下游相关因素，从基因、蛋白表达、关键酶及中间产物、效应等多维度进行研究，且通过实验五在体实验进行系统验证。以期探明痰瘀同治法改善糖脂代谢紊乱的机制，及其与该通路介导的代谢通量调节的关系，为提高临床用痰瘀同治法干预肥胖的糖脂代谢疾病疗效提供借鉴。

从整体观角度来看，糖脂代谢疾病虽然涉及面、涉及病种较广，但其本质只有一个，那就是热量超载。大部分化学药物不但不能解决热量超载这个根本问题，有的药反而抑制分解代谢、增加体重。本项目从整体观看问题，抓住热量超载这个基本点，用痰瘀同治中药方化痰活血，促进分解代谢，以促进人体过剩热量贮备的消除。本项目通过4个细胞实验，以qRT-PCR、免疫印迹、免疫组化、ELISA、细胞线粒体压力测试等，结合miRNA阻断与模拟技术，对该通路及上、下游相关因素，从基因、蛋白表达、关键酶及中间产物、效应等多维度进行研究，且通过实验五在体实验进行系统验证。系统细胞实验后，动物实验验证研究显示各组FBG、PBG及HbA1c比较与正常组比较，模型组FBG、PBG及HbA1c水平升高（P<0.01）。与模型组比较，丹瓜方组FBG和HbA1c水平降低（P<0.05，P<0.01）。与丹瓜方组比较，丹抑组、抑制组FBG及HbA1c均显著增高（P<0.01）。基线时各组间的体重比较无显著差异。模型组第2、4、6、8、10周体重明显低于正常组（P<0.01），在4、6、8周时差异有统计学意义（P<0.05）。在第2周，抑制剂组的体重显著高于丹瓜组（P<0.05）。与正常组比较，模型组肝组织Nampt蛋白及miR-34a-mRNA表达升高（P<0.01）；Nampt-mRNA则 降低（P<0.05）。与模型组比较，丹瓜组肝组织Nampt蛋白水平及mRNA表达升高（P<0.01，P<0.05），肝miR-34a-mRNA表达明显降低而血miR-34a-mRNA明显升高（P<0.05）。与丹瓜组比较，丹抑组肝组织Nampt-mRNA表达、抑制组肝Nampt蛋白及血 miR-34a-mRNA 表达下降（P<0.05，P<0.01）。肝组织染色（HE、Masson、油红O）结果均表明丹瓜方干预组显著的改善糖脂代谢疗效。