miR⁃33⁃5p对糖尿病肾脏疾病大鼠肾纤维化影响机制的研究

Effect and mechanism of miR⁃33⁃5p on renal fibrosis in diabetic kidney disease rats

目的探讨miR‐33‐5p对DKD大鼠肾纤维化的影响机制。方法选取60只SD大鼠中未经任何处理的15只为正常对照(NC)组,其余45只建立DKD大鼠模型,造模成功后随机分为DKD模型组(DKD)、miR‐33‐5p拮抗剂组(miR‐33‐5p antagomir)及其阴性对照(antagomir‐NC)组,每组各15只。antagomir‐NC、miR‐33‐5p antagomir组尾静脉注射antagomir‐NC或miR‐33‐5p antagomir,每周1次,持续12周。检测各组FPG、24 hUAlb、BUN和血肌酐(Scr)含量。Masson染色观察各组肾纤维化程度。qRT‐PCR检测肾组织中miR‐33‐5p、Ⅰ型胶原蛋白(CollagenⅠ)、Ⅲ型胶原蛋白(CollagenⅢ)mRNA表达水平。Western blot检测肾组织中TGF‐β1、Smad同源物3(Smad3)、Smad2、p‐Smad3、p‐Smad2、平滑肌肌动蛋白α(α‐SMA)等蛋白表达水平。结果与DKD组比较,miR‐33‐5p antagomir组肾脏间质胶原纤维减少,24 hUAlb、FPG、BUN、Scr含量降低[(36.22±2.41)vs(19.24±1.25)mg/24 h,(29.15±1.53)vs(14.25±1.76)mmol/L,(18.33±3.16)vs(13.73±0.68)mmol/L,(60.17±2.37)vs(39.42±1.74)μmol/L,P<0.05],肾组织中miR‐33‐5p、CollagenⅠmRNA、CollagenⅢmRNA降低[(5.19±0.20)vs(1.88±0.27),(3.44±0.21)vs(1.28±0.08),(4.71±0.32)vs(2.06±0.12),P<0.05],TGF‐β1、p‐Smad3/Smad3、p‐Smad2/Smad2、α‐SMA等蛋白表达水平降低(P<0.05)。antagomir‐NC、DKD组各指标比较,差异均无统计学意义(P>0.05)。结论抑制miR‐33‐5p表达可改善DKD大鼠肾纤维化,其作用机制可能与阻断TGF‐β/Smad信号通路有关。

Objective To investigate the effect and mechanism of miR‐33‐5p on renal fibrosis in DKD rats.Methods 15 of 60 SD rats without any treatment were selected as normal control group(NC).The remaining 45 rats were established as DKD model and randomly divided into DKD model group(DKD),antagomir‐NC group and miR‐33‐5p antagomir group,with 15 rats in each group.Antagomir‐NC and miR‐33‐5p antagomir groups were injected with antagomir‐NC or miR‐33‐5p antagomir intravenously once a week for 12 consecutive weeks.The contents of FPG,24 hUAlb,urea nitrogen(BUN)and serum creatinine(Scr)were determined in each group.Masson staining was used to observe the degree of renal fibrosis.The mRNA expression levels of miR‐33‐5p,Ⅰtype collagen(CollagenⅠ)andⅢtype collagen(CollagenⅢ)in renal tissues were detected by qRT‐PCR.The protein expression levels of transforming growth factorβ1(TGF‐β1),smad homologue 3(Smad3),smad homologue 2(Smad2),p‐Smad3,p‐Smad2 and smooth muscle actinα(α‐SMA)in renal tissues were detected by Western blot.Results Compared with DKD group,miR‐33‐5p antagomir group showed decreased collagen fibers in renal interstitium,the contents of 24 hUAlb[(36.22±2.41)vs(19.24±1.25)mg/24 h],FPG[(29.15±1.53)vs(14.25±1.76)mmol/L],BUN[(18.33±3.16)vs(13.73±0.68)mmol/L]and Scr[(60.17±2.37)vs(39.42±1.74)μmol/L]were significantly decreased(P<0.05);the expression levels of miR‐33‐5p[(5.19±0.20)vs(1.88±0.27)],CollagenⅠmRNA[(3.44±0.21)vs(1.28±0.08)],CollagenⅢmRNA[(4.71±0.32)vs(2.06±0.12)]and the protein expression level of TGF‐β1,p‐Smad3/Smad3,p‐Smad2/Smad2 andα‐SMA in renal tissue was significantly decreased(P<0.05).There was no statistical differences in the above indexes between antagomir‐NC group and DKD group(P>0.05).Conclusion Inhibition of miR‐33‐5p expression can improve renal fibrosis in DKD rats,the mechanism of which may be related to blocking TGF‐β/Smad signaling pathway.