基于miR-192/TGF-β/Smad信号通路研究消渴平合剂治疗早期糖尿病肾病的分子机制

Diabetic kidney disease was the most serious microvascular complications of diabetes. The expression of miR-192 ,which is mediated by TGF-β pathway, has played an important role in the pathogenesis of DKD process. Xiaokeping mixture is our hospital preparations (approval number：20100002), which has the function of promoting blood circulation and remove blood stasis, Yiqiyangyin. Our previous studies have confirmed that xiaokeping can reducing proteinuria significantly of DKD patients , and inhibit the expression of TGF-β of DKD rats ,but the intracellular signal transduction process and the imolecular mechanism is not clear.Combining traditional Chinese and western medicine understanding of DKD, We hypothesized that: xiaokeping may delay the process of DKD by cutting the expression of miR-192 ,so it inhibit the activation of TGF-β. This study will be carried out around the gene regulation of miR-192, we will use methods such as molecular biology, cell transfection, serum pharmacology by the early DKD rat model induced by STZ, our study will observe whether xiaokeping regulates TGF-β/Smad signaling pathway by the target point of miR-192 and delay the process of DKD .This study put miR-192 as the breakthrough point, try to clarify the molecular mechanism of xiaokeping to treat DKD through experiments in vivo and in vitro respectively,so we can provide powerful evidence for xiaokeping to use in the treatment of DKD.

糖尿病肾病（DKD）是糖尿病最严重的微血管并发症，由TGF-β通路介导的miR-192表达在DKD的发病过程中起重要作用。消渴平是医院制剂（浙药制字20100002），具有活血化瘀、养阴通络等功效，前期研究证实其可显著减少DKD患者蛋白尿、抑制DKD大鼠TGF-β1表达，但消渴平对TGF-β1胞内信号传导的过程及具体分子机制还不明确。结合中西医对DKD的认识，我们提出了消渴平通过下调miR-192表达从而抑制TGF-β激活延缓DKD进程的假说。本课题拟围绕miR-192的基因调控开展研究，通过建立STZ诱导的早期DKD大鼠模型，运用分子生物学、细胞转染、血清药理学等方法深入观察消渴平是否以miR-192为靶点调控TGF-β/Smad通路，继而延缓DKD进程。本项目以miR-192为切入点，分别通过体内外实验阐明消渴平治疗早期DKD的分子机制，为消渴平应用于DKD的治疗提供有力的佐证。

糖尿病肾病是糖尿病常见且严重的微血管并发症之一，已成为导致糖尿病死亡率增加的主要原因。消渴平是医院制剂（浙药制字20100002），具有活血化瘀、养阴通络等功效。本项目在前期研究基础上，围绕miR-192的基因调控开展研究，运用分子生物学、细胞转染、血清药理学等方法深入观察消渴平是否以miR-192为靶点调控TGF-β/Smad通路，继而延缓糖尿病肾病进程。实验结果：（1）构建miR-192过表达慢病毒，miR-192 mimics转染大鼠肾小球系膜细胞后，细胞活性显著下降。消渴平对高糖诱导的肾小球系膜细胞具有调节细胞周期作用，可明显抑制高糖诱导的系膜细胞增殖。（2）消渴平可显著降低高糖诱导的系膜细胞miR-192、TGF-β1、Collagen1和Smad3的表达，明显升高SIPI和Smad7的表达。（3）消渴平可显著抑制高糖诱导的系膜细胞凋亡，抑制系膜细胞cleaved caspase-3和Bax、Cyt-C表达，增加Bcl-2表达，与厄贝沙坦组相比无明显差异。（4）消渴平能明显降低db/db小鼠Scr、BUN、CysC、糖化血清蛋白GSP、24h尿蛋白水平，可调节血脂，改善肾脏病理学结构改变，具有一定的肾功能保护作用，实验发现消渴平可明显降低模型组小鼠TGF-β1及 p-Smad3蛋白表达,升高Smad7和S1P1 蛋白表达，进一步Western blot 及荧光定量PCR实验表明消渴平可上调足细胞Nephrin、Podocin、CD2AP的表达，降低P38MAPK、NF-κB p65、IL-6的表达。综上研究证实，消渴平合剂具有一定的肾功能保护作用，其机制可能与调控miR-192/TGF-β/Smad信号通路相关，研究为祖国传统中医药预防和干预糖尿病及其并发症提供了理论与实践依据，可推动中药制剂的进一步开发利用。