miR⁃210激动剂减轻糖尿病肾脏病大鼠肾脏炎性反应和纤维化

miR⁃210 agonist alleviates renal inflammatory response and fibrosis in diabetic kidney disease rats

目的探讨微小RNA⁃210激动剂(agomiR⁃210)对糖尿病肾脏病(DKD)大鼠的肾脏保护作用及其机制。方法36只5周龄雄性SD大鼠被分为正常对照(NC)组、agomiR⁃NC对照组、agomiR⁃210对照组、DKD模型组、DKD+agomiR⁃NC组和DKD+agomiR⁃210组,每组6只。采用高脂饮食联合腹腔注射链脲菌素(STZ)法构建糖尿病大鼠模型,持续喂养12周后建立DKD大鼠模型。于持续喂养期的第2周至第4周给予DKD+agomiR⁃210组大鼠尾静脉注射agomiR⁃210(20 nmol/kg),每周2次。定期检测空腹血糖、24 h尿白蛋白(Alb)和尿微量白蛋白(MAU)水平。实验第12周末处死大鼠,留取肾脏组织。HE、PAS和Masson染色法评估肾组织病理学改变;实时荧光定量PCR(RT⁃qPCR)和Western印迹法检测肾组织中肿瘤坏死因子α(TNF⁃α)、白细胞介素(IL)⁃1β和IL⁃6的mRNA和蛋白表达;免疫组化法检测肾组织中α平滑肌肌动蛋白(α⁃SMA)、Ⅰ型胶原(Col⁃Ⅰ)、Ⅳ型胶原(Col⁃Ⅳ)和纤连蛋白(FN)的分布与表达;Western印迹和免疫组化法检测肾组织中磷酸化(p)⁃Smad3和p⁃核因子κB p65(p⁃NF⁃κB p65)蛋白的表达。结果与DKD模型组比较,DKD+agomiR⁃210组大鼠肾组织病理学改变明显改善,血糖水平、糖原沉积和胶原蓄积均显著降低(均P<0.05),尿Alb与MAU排泄均明显减少(均P<0.01);肾组织TNF⁃α、IL⁃1β、IL⁃6的mRNA和蛋白表达均显著降低,α⁃SMA、Col⁃Ⅰ、Col⁃Ⅳ、FN、p⁃Smad3和p⁃NF⁃κB p65蛋白表达均明显降低(均P<0.01)。结论agomiR⁃210可减轻大鼠DKD肾脏病理改变和减少尿Alb、MAU排泄,作用机制可能与其抑制Smad3和NF⁃κB活性有关。

Objective To investigate the protective effect and mechanism of microRNA⁃210 agonist(agomiR⁃210)on kidney in diabetic kidney disease(DKD)rats.Methods Thirty⁃six 5⁃week⁃old male SD rats were divided into normal control(NC)group,agomiR⁃NC control group,agomiR⁃210 control group,DKD model group,DKD+agomiR⁃NC group and DKD+agomiR⁃210 group,with 6 rats in each group.Diabetic rats were established by a high⁃fat diet combined with intraperitoneal injection of streptozotocin(STZ),then were fed for 12 consecutive weeks to construct DKD model rats.During 2nd⁃4th week of continuous feeding,the rats in DKD+agomiR⁃210 group were injected with 20 nmol/kg agomiR⁃210 via tail vein twice a week.Blood glucose levels,24 h urine albumin(Alb)and 24 h urine microalbumin(MAU)contents were measured regularly.At the end of the 12th week,the rats were sacrificed,and renal tissues were collected.The renal histopathological changes were assessed by HE,PAS and Masson staining methods.The mRNA and protein expression levels of tumor necrosis factor⁃α(TNF⁃α),interleukin(IL)⁃1βand IL⁃6 in renal tissues were detected by RT⁃qPCR and Western blot.The distributions and expressions ofα⁃smooth muscle actin(α⁃SMA),typeⅠcollagen(Col⁃Ⅰ),typeⅣcollagen(Col⁃Ⅳ)and fibronectin(FN)in renal tissues were detected by immunohistochemical method.The protein expression levels of phospho(p)⁃Smad3 and p⁃NF⁃κB p65 in renal tissues were detected by Western blot and immunohistochemical methods.Results Compared with DKD model group,the renal pathological damages in DKD+agomiR⁃210 group were improved,the blood glucose level,glycogen deposition and collagen accumulation were significantly decreased(all P<0.05),the urinary excretions of Alb and MAU were significantly reduced(all P<0.01),and the expressions of TNF⁃α,IL⁃1β,IL⁃6,α⁃SMA,Col⁃Ⅰ,Col⁃Ⅳ,FN,p⁃Smad3 and p⁃NF⁃κB p65 in renal tissues were significantly decreased(all P<0.01).Conclusion AgomiR⁃210 can alleviate renal pathological changes and urinary Alb and MAU excretion in rats with DKD,which may be related to its inhibition of Smad3 and NF⁃κB activity.