糖克煎剂经miR-21/Tgfb1/Smad信号转导延缓糖尿病肾病肾脏纤维化

Anti-Fibrosis effect of Tangke apozem on DN rats via reglulating miR-21/Tgfb1/Smad signal

目的:探讨糖克煎剂对糖尿病肾损伤模型大鼠肾脏miR-21和Smad家族蛋白的影响。方法:选取50只雄性大鼠,随机选取10只作为空白对照组。其余大鼠采用一次性尾静脉注射链脲佐菌素(STZ)复制痰浊血瘀型糖尿病大鼠(DN)模型,并随机分为模型组、福辛普利钠0.9 mg/kg组、糖克煎剂4.5、18 g/kg剂量组灌胃给药,每日一次。观察各组大鼠的一般情况;监测血糖、体重、尿素氮(BUN)、血肌酐(Scr)及24 h尿蛋白水平;HE染色,光镜下观察肾组织形态学的改变;Western-blot法分别检测肾脏组织中Tgfb1、Smad3、Smad7、Vegf和Fn1的蛋白表达水平;实时荧光定量PCR检测肾脏组织miR-21、Smad3、Smad7、Fn1基因mRNA的表达;免疫组化法检测Smad3和Smad7蛋白在肾脏组织中分布及表达情况。结果:与模型组比较,糖克煎剂可改善DN大鼠肾指数;降低血糖、BUN、Scr及24 h尿蛋白水平;光镜下肾组织病理形态得到显著改善;并可下调Smad3、Fn1的mRNA水平和miR-21表达;下调Smad3、Vegf、Tgfb1和Fn1的蛋白表达;而Smad7的mRNA及蛋白水平则出现不同程度增加。免疫组化结果与Western-blot检测结果相一致。结论:糖克煎剂可通过调控miR-21/Smad信号转导,进而调控DN大鼠肾组织Tgfb1表达,延缓糖尿病肾病肾脏纤维化进程,起到肾脏保护作用有关。

Objective： To investigate the Anti-Fibrosis effect of Tangke apozem on DN rats by mediating microR-21/Tgfb1/Smad signal. Methods： DN rats with turbid phlegm and blood stasis syndromes were duplicated by 30 mg/kg streptozotocin injected into tail veins. Then all normal rats and DN rats was divided into the control group,DN model group,Fosinopril sodium group,Tangke apozem with 4. 5 g/kg and18 g/kg group. We analyzed the physiclal status,blood sugar,weight,Blood Urea Nitrogen（BUN）,Serum creatinine（Scr） and albuminuria. HE stain was used to observe the histomorphometric changes of kidneys. Immunocytochemistry and western blotting analyzed protein expressions of partial or all genes of Tgfb1,Smad3,Smad7,Vegf and Fn1. We also detected mRNA expressions of miR-21,Smad3,Smad7 and Fn1 by Real-time PCR. Results： Compared with DN model rats,Tangke apozem improved the kidney-weight/body-weight and reduced the blood sugar,weight,BUN,Serum creatinine（Scr） and albuminuria. Histomorphology was also improved,and mRNA expression levels of Smad3,Fn1 and microRNA-21 were down-regulated. TProtein expressions of Smad3,Vegf,Tgfb1 and Fn1 were also minimized. Simultaneously,mRNA and protein expression level of Smad7 were all up-regulated. Results of Immunocytochemistry were same with that of Western-blot. Conclusion： The renoprotective role of Tangke apozem is presented through mediating miR-21/Smad signal transduction,reducing the expression of Tgfb1 and delaying progression of renal fibrosis.