基于miRNA层面的肝脾肾同调法保护胰岛β细胞的机制研究

The theory of “simultaneous regulation of liver, spleen and kidney for treating Type 2 diabetes”is grounded in the idea of zang and fu relativity and combination of traditional Chinese and western medicine. Under the guidance of the theory, the recipe of jiangtangxiaoke has been prescribed. So far, clinical study and experimental research had demonstrated that the recipe is good for treating Type 2 diabetes. And the miRNA expression profiles are different in pre-experiment. In order to discuss the theory of “simultaneous regulation of liver, spleen and kidney for treating Type 2 diabetes”in detail, we are going to study on the mechanism based on miRNA network and carry out four studying aspects. ①“simultaneous regulation of liver, spleen and kidney” can protect effectively the pancreatic islet B cells..②The miRNA expression profile are found out which exhibit higher differential expression level by gene chip technique. According to the results, the potential target genes and control network are analyzed by the bioinformatics software.③In order to validate this conclusion, some miRNAs are going to be suppressed. At the same time, the relationship of protecting B cells, miRNA network and FoxO1 will be analyzed. ④The expression of FoxO1 in B cells will be tested. The result will be used to validate the relationship. In final, we may certify that “simultaneous regulation of liver, spleen and kidney” can protect effectively B cells. And the mechanism may be that the miRNA network disrupts normal expression of FoxO1 by affecting many signaling pathways.

肝脾肾同调法以中医脏腑相关为特点，融合中西医基本观点辨治2型糖尿病。在此理论指导下所组降糖消渴方已经证实临床疗效显著，实验研究亦显示其具有较好的调节糖脂代谢和胰腺保护作用。预实验也发现胰腺miRNA表达谱有变化。基于此，本项目拟从miRNA层面揭示肝脾肾同调法治疗T2DM的表观遗传学机制，为此开展如下研究：①肝脾肾同调法保护糖尿病大鼠胰岛β细胞的数目及功能。②肝脾肾同调法对糖尿病大鼠胰腺miRNA表达谱的影响，筛选差异表达的miRNA，利用生物信息学推测可能的靶基因及调控网络。③抑制有意义的胰岛β细胞特异性miRNA的表达，以验证其功能，同时分析miRNA调控网络对细胞数目及功能和FoxO1表达的影响。④肝脾肾同调法对FoxO1表达的影响，印证生物信息学推测结果。据此以证实“肝脾肾同调法可保护胰岛β细胞，其机制可能与调节胰腺特异性miRNA的表达，多途径影响FoxO1的表达相关”这一假说。

2型糖尿病（T2DM）是胰岛功能减退、胰岛素抵抗等而引发的糖、蛋白质、脂肪、水和电解质等一系列代谢紊乱综合征。近年来，T2DM具有显著的持续增多和年轻化趋势。中医药以其独特的疗效优势，目前已经成为防治T2DM及其并发症不可或缺的重要部分。.肝脾肾同调法以中医脏腑相关为特点，融合中西医基本观点辨治2型糖尿病，在临床中取得了一定的疗效。降糖消渴颗粒是在该大法指导下所组系列方剂中的代表方剂之一。前期实验研究显示该方具有较好的调节糖脂代谢和胰腺保护作用，为进一步研究药效机制，本项目拟从miRNA层面揭示肝脾肾同调法治疗T2DM的表观遗传学机制，分别设立动物实验和细胞实验。.项目主要研究内容包括：1、肝脾肾同调法对糖尿病大鼠胰腺的保护作用，以及对miRNA表达谱的影响，筛选差异表达的miRNA，利用生物信息学推测可能的靶基因及调控网络。2、肝脾肾同调法对胰岛β细胞的保护作用，包括对细胞数目和细胞功能的影响。3、肝脾肾同调法保护胰岛β细胞的机制及对FoxO1表达的影响，印证生物信息学推测结果。4、抑制miR-489和miR-760表达，分析对FoxO1表达的影响，验证其功能。.动物实验发现，肝脾肾同调法能够很好地改善糖尿病鼠糖脂代谢水平，对糖尿病鼠胰腺具有保护作用，其作用机制与影响胰腺miRNA表达谱，通过miRNA-mRNA调控网络，潜在影响多条信号通路，尤其与PI3K/AKT/FOXO1通路关系密切。经细胞实验证实，降糖消渴颗粒可提高AKT的表达，促进FoxO1的磷酸化出核，抑制FoxO1的核转录，经PI3K/AKT/FOXO1通路，实现抑制细胞凋亡，促进细胞增殖，改善胰岛素分泌功能，以保护胰岛β细胞的作用。.以上研究结论为肝脾肾同调法治疗糖尿病提供了实验室依据，为降糖消渴颗粒的临床开发及应用提供了研究基础，也为从miRNA层面探讨中医药治疗糖尿病的机制研究提供了思路。