摘要
目的 研究miR-155-5p靶向沉默信息调节因子1(SIRT1)调节TGF-β/Sma和Mad相关蛋白(TGF-β/Smad)信号通路,对DKD大鼠肾纤维化的影响机制.方法 48 只大鼠随机分为正常对照(NC)组、模型(Mod)组、抑制剂阴性对照(anti-NC)组、miR-155-5p抑制剂(anti-miR-155-5p)组、miR-155-5p抑制剂+小分子干扰RNA阴性对照(anti-miR-155-5p+si-NC)组、miR-155-5p抑制剂+SIRT1 小分子干扰RNA(anti-miR-155-5p+si-SIRT1)组,每组 8 只.检测各组FPG、24 h UAlb、BUN、血肌酐(Scr).HE、Masson 染色比较各组肾组织病理变化,计算胶原容积分数(CVF).RT-qRCR检测miR-155-5p、SIRT1 mRNA表达,Western blot法检测各组SIRT1、TGF-β1、Smad3、Smad7、结缔组织生长因子(CTGF)、Ⅰ型胶原蛋白(COLⅠ)蛋白表达.结果 与NC组比较,Mod、anti-NC组FPG、24 hUAlb、BUN、Scr、CVF、miR-155-5p表达及TGF-β1、Smad3、CTGF、COLⅠ蛋白表达升高,SIRT1 mRNA和蛋白表达、Smad7 蛋白表达降低(P<0.05).与anti-NC组比较,anti-miR-155-5p、anti-miR-155-5p+si-NC组SIRT1 mRNA和蛋白表达、Smad7蛋白表达升高(P<0.05),FPG、24 hUAlb、BUN、Scr、CVF、miR-155-5p表达及TGF-β1、Smad3、CTGF、COLⅠ蛋白表达降低(P<0.05).与anti-miR-155-5p、anti-miR-155-5p+si-NC组比较,anti-miR-155-5p+si-SIRT1 组FPG、24 hUAlb、BUN、Scr、CVF、miR-155-5p 表达及 TGF-β1、Smad3、CTGF、COLⅠ蛋白表达升高(P<0.05),SIRT1 mRNA和蛋白表达、Smad7蛋白表达降低(P<0.05).SIRT1 mRNA的3'UTR含有miR-155-5p序列保守碱基.结论 DKD大鼠中升高miR-155-5p可靶向调控SIRT1,改善肾纤维化.
Abstract
Objective To explore the influence of miR-155-5p on renal fibrosis in diabetic kidney disease(DKD)rats by targeting silent information regulator 1(SIRT1)/transforming growth factor-beta/Sma-and Mad-related protein(TGF-β/Smad)signaling pathway.Methods Forty-eight rats were randomly divided into normal control(NC)group,model(Mod)group,inhibitor negative control(anti-NC)group,miR-155-5p inhibitor(anti-miR-155-5p)group,miR-155-5p inhibitor+small interfering RNA negative control(anti-miR-155-5p+si-NC)group,and miR-155-5p inhibitor+SIRT1 small interfering RNA(anti-miR-155-5p+si-SIRT1)group,with 8 rats in each group.FPG,24 h UAlb,BUN and serum creatinine(Scr)were detected in each group.HE and Masson staining were used to compare the pathological changes of renal tissue in each group,and the collagen volume fraction(CVF)was calculated.The mRNA expressions of miR-155-5p and SIRT1 were detected by real-time fluorescence quantitative PCR(RT-qRCR),and the protein expressions of SIRT1,TGF-β1,Smad3,Smad7,connective tissue growth factor(CTGF)and collagen type Ⅰ(COLⅠ)were detected by Western blot.Results Compared with NC group,the expressions of FPG,24 hUAlb,BUN,Scr,CVF,miR-155-5p,TGF-β1,Smad3,CTGF and COLⅠincreased(P<0.05),while the expressions of SIRT1 mRNA and protein and Smad7 protein decreased in Mod and anti-NC groups(P<0.05).Compared with the anti-NC group,in the anti-miR-155-5p,anti-miR-155-5p+si-NC groups,the expression of SIRT1 mRNA and protein,Smad7 protein increased(P<0.05),and FPG,24 hUAlb,BUN,Scr,CVF,miR-155-5p,TGF-β1,Smad3,CTGF and COLⅠprotein were expressed(P<0.05).Compared with the anti-miR-155-5p and anti-miR-155-5p+si-NC groups,the anti-miR-155-5p+si-SIRT1 group showed the expression of FPG,24 hUAlb,BUN,Scr,CVF,miR-155-5p,TGF-β1,Smad3、CTGF、and COLⅠprotein increased(P<0.05),the expression of SIRT1 mRNA and protein,Smad7 protein decreased(P<0.05).The 3'UTR of SIRT1 mRNA contains the conserved base of miR-155-5p sequence.Conclusions Elevated miR-155-5p in DKD rats can target and regulate SIRT1 to alleviate the process of renal fibrosis.
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