益气养阴通络方调控LncRNA UCA1抑制细胞焦亡机制在糖尿病肾病中的作用研究

Our previous studies found that there existed the decrease of lncRNA UCA1 and the increase of microRNA-206,inflammatory factors NLRP3, Caspase-1 as well as IL-1beta, and the mimic of microRNA-206 was stably co-transfected with lv-uca1 or si-uca1 into HK-2 cells in diabetic nephropathy.The results indicated that the mimic transfection of microRNA-206 resulted in a significant promotion of pyroptosis of HK-2 cells, while the overexpression of UCA1 brought about the reverse for the promotion of microRNA-206. Clinical researches have shown that the empirical prescription Yiqi Yangyin Tongluo recipe can reduce urinary protein, significantly improving glycometabolism and renal function in patients with early diabetic nephropathy. In the peripheral blood test of some patients with diabetic nephropathy,higher UCA1 and lower miR-206 have been displayed in the treatment group of Yiqi Yangyin Tongluo recipe, suggesting that UCA1 may inhibit renal tubules by targeting microRNA-206. Based on the basic pathogenesis of deficiency of both Qi and Yin, Blocking Collaterals by blood stasis and stagnation of collaterals in TCM, a hypothesis is proposed that Yiqi Yangyin Tongluo recipe can regulate UCA1 to inhibit pyroptosis of renal tubular epithelial cells, alleviate inflammation and delay the progress of DN.This project will study the occurrence mechanism of diabetic nephropathy from a new intervention point of lncRNA, clarifying the role and regulation mechanism of UCA1 in diabetic nephropathy from the clinical, cells and animal experimental levels and revealing the mechanism of the empirical prescription Yiqi Yangyin Tongluo recipe regulating UCA1 to inhibit pyroptosis of renal tubular epithelial cells, thus to provide new ideas for the treatment of diabetic nephropathy with Chinese medicine compound prescription.

我们前期发现，在糖尿病肾病(DN)中，lncRNAUCA1降低，miR-206、NLRP3、Caspase-1、IL-1β升高，miR-206模拟物被稳定地与lv-uca1或si-uca1共转染到HK-2细胞，结果提示模拟转染显著促进HK-2细胞焦亡，而UCA1的过度表达逆转了miR-206的促进作用，临床研究表明益气养阴通络方可以降低尿蛋白，改善早期DN患者糖代谢、肾功能，在部分患者外周血检测中，益气养阴通络方治疗组UCA1升高，miR-206降低，基于中医气阴两虚，血瘀阻络，络脉瘀滞的病机，我们提出假说：益气养阴通络方调控UCA1抑制肾小管上皮细胞焦亡，减轻炎症反应，延缓DN进展。本课题将从lncRNA新介入点探讨DN的发生机制，从临床、细胞、动物实验水平明确UCA1在DN中的作用及调控机制，阐明益气养阴通络方调控UCA1抑制肾小管上皮细胞焦亡的机制，为DN的中药复方治疗提供新思路。

糖尿病肾病（Diabetic nephropathy，DN）是终末期肾病的主因，发病率高，防治意义重大。高血糖状态下机体可生成多种危险相关分子导致肾脏炎症反应和细胞焦亡，也是防治DN的新靶点。本研究通过临床、体外细胞和体内动物三个层面，以LncRNAUCA1/miR-206和P13k/Akt调控肾小管上皮细胞焦亡和炎症反应为切入点，基于中医气阴两虚，血瘀阻络，络脉瘀滞的病机探讨益气养阴通络方(YQYY)发挥肾脏保护作用的机制。体内实验：雄性Wistar大鼠随机分为空白组、模型组、益气养阴通络方大剂量组、中剂量组、低剂量组和缬沙坦组，在造模成功给药8周后采集24h尿液和血进行肾功能指标检测，PAS、HE及AQP-2蛋白染色，qPCR检测LncRNAUCA1、miR-206以及炎性因子的表达，Westernblot检测NLRP3、Caspase-1、IL-1β、PI3K和AKT等相关蛋白表达。结果发现糖尿病肾病大鼠24hUTP、血肌酐、尿素氮、β2-MG、AQP2、PI3K、AKT、p-PI3K和p-AKT蛋白表达升高，LncRNAUCA1表达降低。体外实验：通过高糖诱导肾小管上皮细胞损伤模型，发现LncRNAUCA1表达下调，IL-1β、NLRP3和 Caspase-1蛋白炎症损伤蛋白表达上调，LncRNAUCA1过表达能够抑制高糖诱导的细胞凋亡和炎症因子表达。通过双荧光素酶报告实验、RIP和细胞回复实验证实LncRNAUCA1通过靶向调控miR-206抑制肾小管上皮细胞凋亡和炎症反应。临床水平：DN患者血清中miR-206呈表达上调。本项目从lncRNA新介入点探讨DN的发生机制，从临床、细胞、动物实验水平明确UCA1在DN中的作用及调控机制，阐明益气养阴通络方调控UCA1抑制肾小管上皮细胞焦亡的机制，为DN的中药复方治疗提供新思路。