miR-214靶向PTEN介导PI3KAKT信号通路对糖尿病肾病大鼠的保护机制

Protective mechanism of miR-214 targeting PTEN mediated PI3K/Akt signaling pathway in diabetic nephropathy rats

目的探讨微小RNA(miR-214)靶向磷酸酶与张力蛋白同源物(PTEN)介导磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路减轻糖尿病肾病(DN)大鼠的氧化应激和肾间质纤维化的作用和机制。方法将40只SD雄性大鼠分为对照组(control组)、模型组(DN组)、miR-214阴性对照组(DN+miR-NC组)和miR-214模拟物组(DN+miR-214 mimics组),每组10只;qRT-PCR检测各组大鼠肾组织中miR-214的表达;电子天平称量各组大鼠体质量和肾质量,计算肾指数;血糖仪测定各组大鼠的空腹血糖(FPG);双缩脲法测定各组大鼠24 h尿蛋白(UP)水平;全自动生化分析仪检测各组大鼠血清肌酐(Scr)和血清尿素氮(BUN)的水平;HE染色观察各组大鼠肾组织病理变化;Masson染色观察各组大鼠肾组织间质纤维化;ELISA检测各组大鼠血清中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-PX)的表达;免疫组织化学法检测各组大鼠肾组织中转化生长因子β1(TGF-β1)的表达;生物信息学工具预测靶基因;双荧光素酶报告基因检测基因或蛋白之间的相互作用;Western blot检测各组大鼠肾组织中PTEN、p-PI3K、p-AKT和磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)的表达。结果miR-214在DN大鼠肾组织中呈低表达,在DN+miR-214 mimics组大鼠肾组织中表达显著上调(均P<0.001);miR-214 mimics使大鼠的体质量升高,肾指数、FPG、UP、Scr及BUN水平下调(均P<0.05);DN+miR-214 mimics组大鼠肾组织中肾间质中的炎性细胞浸润减少,胶原纤维沉积减少(P<0.01);miR-214 mimics使大鼠血清中MDA的水平降低,SOD、GSH、GSH-PX的水平升高(均P<0.05),大鼠肾组织中TGF-β1的表达水平下降(P<0.05);miR-214与PTEN存在靶向关系;miR-214 mimics使大鼠肾组织中PTEN蛋白表达下调,p-PI3K、p-AKT和p-mTOR蛋白表达上调(均P<0.05)。结论miR-214在DN大鼠肾组织中低表达,过表达miR-214对DN大鼠的肾组织有保护作用,可能与降低肾组织的氧化应激水平、TGF-β1蛋白表达和抑制PI3K/AKT通路激活有关。

Objective To investigate the role and mechanism of miR-214 targeting PTEN-mediated PI3K/AKT signaling pathway in reducing oxidative stress and renal interstitial fibrosis in diabetic nephropathy in rats.Methods SD rats were divided into control group(control group),model group(DN group),miR-214 negative control group(DN+miR-NC group)and miR-214 mimic group(DN+miR-214 mimics group).qRT-PCR was used to detect the expression of miR-214 in kidney tissue of each group of rats.The electronic balance was used to weigh the body weight and kidney weight of each group of rats,and calculates the renal index.Blood glucose meter was used to measure fasting blood glucose(FPG)of rats in each group.Biuret method was used to determine the 24-hour urine protein(UP)level of rats in each group.Automatic biochemical analyzer to detect serum creatinine(Scr)and serum urea nitrogen(BUN)levels in each group of rats.HE staining was used to observe the pathological changes of kidney tissue in each group of rats.Masson staining was used to observe the renal interstitial fibrosis in each group of rats.ELISA was used to detect the expression of malondialdehyde(MDA),superoxide dismutase(SOD),glutathione(GSH),and glutathione peroxidase(GSH-PX)in the serum of each group of rats;Immunohistochemical method was used to detect the expression of TGF-β1 in kidney tissue of rats in each group.Bioinformatics tools was used to predict target genes.Dual luciferase reporters was used to detect interactions between genes or proteins;Western blot was used to detect the expression of PTEN,p-PI3K,p-AKT and p-mTOR proteins in the kidney tissues of rats in each group.Results miR-214 in renal tissue of DN rats was low expressed(P<0.001),and significant up-regulated in renal tissue of DN+miR-214 mimics group rats(P<0.001).miR-214 mimics increased body weight in rats,decreased renal index,FPG,UP levels,Scr levels,and the BUN level(all P<0.05).In the DN+miR-214 mimics group,the infiltration of inflammatory cells in the renal interstitial tissue and the deposition of collagen fibers in the renal interstitial tissue were reduced.miR-214 mimics reduced MDA levels in rat serum,elevated SOD,GSH,GSH-PX levels(all P<0.05),and TGF-β1 expression in rat kidney tissues was decreased(P<0.05).There is a targeting relationship between miR-214 and PTEN.miR-214 mimics down-regulated PTEN protein expression in rat kidney tissues,and up-regulated p-PI3K,p-AKT,and p-mTOR protein expressions(all P<0.05).Conclusion miR-214 is under-expressed in the kidney tissues of DN rats,but over-expression of miR-214 has a protective effect on the kidney tissues of DN rats,which may be related to reducing the level of oxidative stress in kidney tissues,the expression of TGF-β1 protein,and inhibiting PI3K/AKT pathway activation is involved.