肾消方对自发性糖尿病肾脏疾病db/db小鼠肾小球系膜细胞RUNX3表达和分布的影响

The degree of glomerular and renal tubule fibrosis is always an important symbol of diabetic kidney disease (DKD) progress, which is mediated by TGF-β1. Our previous studies showed that compared to the patients with diabetes, DKD patients' RUNX3 have shown that on the TGF-βpathway, the transcription factor (RUNX) related with RUNT which interactses with the receptor regulates Smad proteins.But the role of DKD has not yet been reported. The project explores Shenxiao Formula therapy by which TGF-β-RUNX3-SMAD protein pathway is intervened so that the occurrence and development of the epigenetic mechanisms DKD are delayed. The research includes (1) putting forward DKD "Qi Deficiency" and "the triple warmer unfavorable" pathogenesis hypothesis, exploring the "invigorating qi" and "the triple warmer reorganization" treatment of Shenxiao Formula, working out the way to regulate the glomerular endothelial cells which influence the development of DKD through the DKD animal models TGF-beta-RUNX3-SMAD,detecting the distribution and molecules expression of RUNX3 in glomerular mesangial cells.(2) Making it clear that RUNX3 plays an important role on glomerular endothelial cells and mesangial cell growth, migration, adhesion and cell factor synthesis ; Defining the synthesis influence of the RUNX3 on functional protein.The location and interaction of RUNX3 and smad3 /β-catenin.

TGF-β1介导的肾小球或肾小管纤维化是糖尿病肾脏疾病（DKD）进展的重要标志。我们前期的研究显示，DKD患者相较于糖尿病患者RUNX3甲基化升高，RUNX3蛋白表达下降。有研究表明部分疾病TGF-β通路上，RUNX与受体相互作用调控Smad蛋白，但是在DKD中的作用尚未见报道。本项目探讨肾消方通过干预TGF-β1—RUNX3—SMAD蛋白途径起到延缓DKD发生发展的表观遗传机制，内容包括：（1）提出DKD的“中气不足”、“三焦不利”病机假说，探讨采用“补益中气”和“疏理三焦”治法的肾消方，通过DKD动物模型TGF-β1-RUNX3-SMAD途径调控肾小球内皮细胞来影响DKD的发生发展，以及RUNX3在肾小球系膜细胞表达、分布情况。（2）明确RUNX3对人肾小球内皮细胞和系膜细胞生长、迁移粘附、细胞因子合成和相关功能蛋白的合成的影响，以及与smad3/β-catenin位置和相互作用。

本研究通过观察肾消方对糖尿病肾脏疾病（Diabetic Kidney Disease，DKD）db/db小鼠的肾脏治疗作用，检测肾脏纤维化指标、RUNX3、Sirt1、Smad3蛋白水平及磷酸化改变，探讨其对TGF-β1—RUNX3—Smad3蛋白途径的影响，提示肾消方能改善肾脏病理程度，肾消方可能通过TGF-β1—RUNX3—Smad3蛋白途径从而延缓DKD的发生；使用生物信息学分析鉴定与DKD相关的小鼠系膜细胞中潜在的治疗靶基因，鉴定肾小球系膜细胞（MES-13）中TGF-β1，高葡萄糖和葡萄糖胺（GlcN）作用下的基因变化；此外，本研究初步探讨了circ_0000491通过靶向miR-101b抑制TGFβRI，加重了DKD肾小球系膜细胞的细胞外基质；本研究还通过网络药理学研究提示ALB，IL6，VEGFA，JUN，TNF，AKT1，TP53，EGFR，IL1B，IGF1，PTGS2，MMP9和TGFB1被确定为主要结节，在这些基因中，糖尿病并发症中7/13个主要结节主要集中在AGE-RAGE信号通路中；研究结果还系统地阐明了Smad2/3和RUNX3在TGF-β信号通路中的机制，而RUNX3可能与糖尿病并发症中的ECM产生有关。