基于miRNA27a调控TGF-β1/Smad3信号通路探讨当归补血汤治疗糖尿病肾间质纤维化作用机制研究

Diabetic nephropathy (DN) is a chronic microvascular complication of diabetes mellitus. There is no effective intervention in clinic. Danggui Buxue Decoction (DBD) is a classical prescription of traditional Chinese medicine. Based on the theory of syndrome of "invigorating Qi and nourishing blood" in traditional Chinese medicine, our previous study found that DBD can reverse renal interstitial fibrosis in DN, but the mechanism of which remains to be elucidated. According to microRNA sequencing and other preliminary experimental results combined with domestic and foreign research reports, we believe that the therapeutic mechanism of DBD on DN renal interstitial fibrosis is related to the involvement of miRNA-27a in regulating TGF-beta1/Smad3 signaling pathway. This project firstly confirmed the correlation between the expression of miRNA-27a and DBD effect in HK-2 cell model cultured in high glucose in vitro. Secondly, reporting gene and gene transfection technology was used to further study the specific connotation mechanism of miRNA-27a regulating target gene and downstream TGF-beta 1/Smad3 signaling pathway. Finally, the animal model was used to verify the above hypothesis in vivo. The results of this project will reveal the mechanism of DBD in the treatment of DN renal interstitial fibrosis, and provide new scientific basis for clinical use of DBD in the treatment of DN.

糖尿病肾病(Diabetic Nephropathy,DN)是糖尿病慢性微血管并发重症，临床缺乏有效干预手段。当归补血汤(Danggui Buxue Decoction,DBD)是中医经典方剂，基于中医“益气养血”的证治理论，课题组前期研究发现DBD对DN肾间质纤维化有逆转作用，但机制尚待阐明。据高通量miRNA测序等预实验结果结合国内外研究报道，我们认为DBD对DN肾间质纤维化的治疗作用机制与miR-27a参与调控TGF-β1/Smad3信号通路相关。本项目拟首先在体外高糖培养的HK-2细胞模型上确证miR-27a表达变化与DBD效应之关联性，其次采用报告基因、基因转染等技术深入探究miR-27a调控下游靶基因及与TGF-β1/Smad3信号通路之内涵机制；最后利用体内动物模型验证上述假说。本课题将揭示DBD治疗DN肾间质纤维化的作用途径，为临床运用DBD治疗DN提供新的理论依据。

背景：当归补血汤(Danggui Buxue Decoction,DBD)是中医经典方剂，基于中医“益气养血”的证治理论，课题组前期研究发现DBD对DN肾间质纤维化有逆转作用，但机制尚待阐明。目的：本研究旨在探讨当归补血汤及非编码miR-27a在糖尿病肾纤维化中的作用及机制。方法：体外HK-2细胞设置对照组、高糖组、高糖+空白血清组、高糖+当归补血汤含药血清组，分别作用于细胞48h。采用MTT法检测各组细胞活力；WB检测纤维化相关蛋白的表达情况；qRT-PCR检测miR-27a及纤维化相关因子mRNA的表达情况；高糖条件下通过转染miR-27a模拟物观察细胞纤维化相关蛋白及mRNA表达的变化。将60只雄性Goto-Kakizaki（GK）大鼠采用高脂饮食喂养4周构建糖尿病肾病大鼠模型，随机分为模型组、阳性药组、当归补血汤高、中、低剂量组，10只雄性wistar大鼠作为对照组。灌胃给药8周后检测大鼠体重、血糖、血肌酐、血尿素氮等变化；WB观察纤维化相关蛋白的表达变化；qRT-PCR检测miR-27a及纤维化相关因子mRNA的表达情况；免疫组织化学和masson染色观察肾脏纤维化病变度；ELISA法检测肾脏中炎症因子的表达。结果：当归补血汤含药血清可有效改善细胞活力，对高糖条件下HK-2细胞具有保护作用；当归补血汤含药血清可明显抑制高糖诱导的HK-2细胞纤维化相关蛋白的表达，抑制高糖条件下miR27a的表达及纤维化相关因子mRNA的表达；高糖条件下转染miR-27a模拟物可促进HK-2细胞纤维化相关蛋白及mRNA的表达；体内实验表明，当归补血汤可改善糖尿病肾病大鼠肾功能；降低纤维化相关蛋白的表达；降低糖尿病肾病大鼠miR-27a及纤维化相关因子mRNA的表达；免疫组化和masson染色表明，当归补血汤明显改善肾纤维化病变程度；同时，当归补血汤还可减轻肾脏炎症因子的表达。结论：当归补血汤对高糖条件下HK-2具有保护作用，并通过抑制miR-27a/TGF-β1/Smad3 信号通路改善HK-2细胞纤维化及糖尿病肾病大鼠肾纤维化。本课题将揭示DBD治疗DN肾间质纤维化的作用途径，为临床运用DBD治疗DN提供新的理论依据。