基于回医“四性学说”研究回回甘松饮调控miR-192保护糖尿病肾病大鼠的作用及分子机制

Diabetic nephropathy has become one of the major diseases which threaten the Public health . The mechanism of DN has not been elucidated thoroughly yet. MiR-192 is the only miRNA which has been found in glomerular mesangial cells. Its direct target genes is SIP1 which is the important node of TGF-β1/Smad pathway . We hypothesized that: miR-192 may bind to SIP1 which can effectively control TGF-β1/Smad signal pathway, and it may be a new mechanism of DN . It was found in previous research that Hui-hui Gan-song Yin ( HGY )which is based on the "FOUR XING Theory" is effective in improving symptoms of DN patients of Hui nationality in Ningxia. And it can also reduce the level of blood glucose and blood lipid in diabetic animal model. On this basis, in order to clarify the effects of HGY on DN and its molecular mechanism, our study will observe HGY efficacy by rat DN model. Using the Molecular Biology techniques and Flow cytometry, we observe whether HGY regulates TGF-β1/Smad signaling pathway by the target point of miR-192 or involves in extracellular matrix (ECM) metabolism in the whole - cell-molecular level. This study based on the Hui nationality medicine theory, miR-192 as the starting point, elucidates the efficacy of HGY and the mechanism, providing a new thought and new target on the prevention of DN.

糖尿病肾病（DN）是严重危害人类健康的疾病,但其机制尚未彻底阐明。miR-192是唯一被发现在肾小球系膜细胞表达的miRNA,其直接靶基因SIP1是TGF-β1/Smad通路的重要节点。我们推测：miR-192与靶基因结合可有效调控TGF-β1/Smad信号通路,这可能是DN发病的一个新机制。针对宁夏回族DN发生率高的现状，前期研究发现：基于回医"四性学说"组方的回回甘松饮（HGY）有效改善宁夏回族DN患者症状；并可降低糖尿病动物模型的血糖与血脂。在此基础上，为阐明HGY对DN的药效及分子机制，本课题拟通过大鼠DN模型，观察HGY药效，并运用分子生物学等技术通过在体和离体实验观察HGY是否以miR-192为靶点，调控TGF-β1/Smad信号通路,参与细胞外基质(ECM)代谢。本课题从回族医学理论出发，以miR-192为切入点，阐明HGY对DN的药效及机制，为防治DN提供新思路和新靶点。

研究背景：前期研究发现：回回甘松饮（HGY）可有效改善糖尿病肾病（DN）患者症状；并可降低糖尿病动物模型的血糖与血脂水平。本研究旨在为寻求治疗DN有效的民族药物提供科学依据。.研究内容：（1）动物实验：选用Sprague-Dawley大鼠复制DN模型。主要检测大鼠空腹血糖、糖化血红蛋白、血脂、肾功、尿微量白蛋白；利用光镜及透射电镜观察肾脏组织病理变化。采用Western Blot、免疫组化及real-time PCR方法检测大鼠肾组织中FN、CollagenⅠ、CollagenⅥ、SIP1、TGF-β1、Smad3、Smad7蛋白及基因的表达水平（2）细胞实验：采用中药血清药理学方法制备大鼠含药血清。采用CCK-8法检测大鼠肾小球系膜细胞（MCs）增殖情况；采用流式细胞术检测细胞周期变化。采用Western Blot及real-time PCR方法检测MCs中FN、CollagenⅠ、CollagenⅥ、SIP1、TGF-β1、Smad3、Smad7蛋白及基因的表达水平。.研究结果：（1）动物实验：HGY能够调节血糖、血脂水平，降低尿量及肾指数，减轻肾脏病理变化，并能够在蛋白和基因水平上调节肾组织中FN、CollagenⅠ、CollagenⅣ、SIP1、TGF-β1、Smad3、Smad7的表达，与模型组比较具有显著性差异（P＜0.01或P＜0.05）。（2）细胞实验：HGY含药血清能够抑制高糖诱导的MCs增殖，调节细胞周期比例，并能够在蛋白和基因水平上调节MCs中FN、CollagenⅠ、CollagenⅥ、SIP1、TGF-β1、Smad3、 Smad7的表达，与高糖组比较具有显著性差异（P＜0.01或P＜0.05）。.科学意义：（1）HGY具有保护早期DN大鼠肾脏的作用。在一定范围内，HGY可以调节血糖、血脂，降低早期尿微量白蛋白水平，保护肾功能，改善DN大鼠肾脏组织的病理结构，减轻肾脏的病理损伤程度。（2）HGY含药血清可抑制高糖诱导的大鼠MCs增殖，具有增强细胞活力，调节细胞周期的药理作用。（3）HGY可能通过调控miR-192介导的TGF-β1/Smads信号通路改善肾脏组织细胞外基质堆积、抑制MCs增殖、调节细胞周期，从而保护肾脏功能，延缓DN的进程。.