miR-34a/ATG4B对db/db小鼠肾脏损伤中自噬和氧化应激的影响

Effect of miR-34a/ATG4B Pathway on Autophagy and Oxidative Stress in Diabetic Kidney Disease in db/db Mice

目的探讨miR-34a/自噬相关基因(ATG)4B对2型糖尿病(db/db)小鼠肾脏损伤中自噬和氧化应激的影响。方法将30只4周龄健康雄性db/db小鼠适应性喂养2周,后采用随机数字表法将其分为5组:模型组(无任何干预)、miR-34a激动剂组(尾静脉注射miR-34a激动剂,首次20 nmol,维持剂量每3天5 nmol)、miR-34a激动剂对照组(尾静脉注射miR-34a激动剂阴性对照物,方法剂量同激动剂)、miR-34a拮抗剂组(尾静脉注射miR-34a拮抗剂,首次200 nmol,维持剂量每3天50 nmol)、miR-34a拮抗剂对照组(尾静脉注射miR-34a拮抗剂阴性对照物,方法剂量同拮抗剂),每组6只;同时选取6只6周龄健康雄性db/m小鼠作为正常对照组(无任何干预)。4周后检测空腹血糖、血脂、尿白蛋白、肾脏组织中ATG4B、自噬标记蛋白[微管相关蛋白1轻链3(LC3)Ⅱ和p62]和氧化应激水平[超氧化物歧化酶(SOD)、丙二醛(MDA)和活性氧类(ROS)]。结果10周龄时,模型组小鼠的体重、空腹血糖、糖化血红蛋白、总胆固醇、三酰甘油水平及尿蛋白/尿肌酐比值以及肾脏miR-34a、p62蛋白、MDA和ROS水平均高于对照组,但ATG4B、LC3Ⅱ、SOD水平低于对照组(P<0.01)。miR-34a激动剂组、miR-34a拮抗剂组与模型组小鼠血糖和糖化血红蛋白水平比较差异无统计学意义(P>0.05),但miR-34a激动剂组尿白蛋白/尿肌酐比值明显高于模型组,而miR-34a拮抗剂组尿白蛋白/尿肌酐比值明显低于模型组(P<0.01)。miR-34a激动剂组小鼠肾脏ATG4B、LC3Ⅱ、SOD水平低于激动剂阴性对照组,而p62、MDA和ROS水平高于激动剂阴性对照组(P<0.01);miR-34a拮抗剂组小鼠肾脏ATG4B、LC3Ⅱ、SOD水平高于拮抗剂阴性对照组,而p62蛋白表达水平低于拮抗剂阴性对照组(P<0.01)。结论miR-34a通过下调糖尿病小鼠肾脏中ATG4B蛋白表达抑制自噬和诱导氧化应激,导致肾脏损伤。

Objective To explore the effect of miR-34 a/ATG4 B on autophagy and oxidative stress in diabetic kidney disease in db/db mouse.Methods Thirty 4-week-old healthy male db/db mice were adaptively fed for 2 weeks,and they were randomly divided into five groups:a model group(without any intervention),a miR-34 a agonist group(tail vein injection of miR-34 a agonist,20 nmol for the first time,maintenance dose of 5 nmol every 3 days),a miR-34 a agonist control group(tail vein injection of miR-34 a agonist negative control,method and dose same as the agonist),a miR-34 a antagonist group(tail vein injection of miR-34 a antagonist,200 nmol for the first time,maintenance dose of 50 nmol every 3 days),a miR-34 a antagonist control group(tail vein injection of miR-34 a antagonist negative control,method and dose same as the antagonist),6 mice in each group;at the same time,six 6-week-old healthy male db/m mice were selected as a normal control group(without any intervention).Four weeks later,the fasting blood glucose,blood lipid,urinary albumin,ATG4 B,autophagy labeled proteins[microtubule associated protein 1 light chain 3(LC3)Ⅱand p62]and oxidative stress levels[superoxide dismutase(SOD),malondialdehyde(MDA)and reactive oxygen species(ROS)]were detected.Results At the age of 10 weeks,the body weight,fasting blood glucose,glycosylated hemoglobin,total cholesterol,triglyceride levels,urinary protein/creatinine ratio and renal miR-34 a,p62 protein,MDA and ROS levels of the model group were higher than those of the control group,but the levels of ATG4 B,LC3Ⅱand SOD were lower than those of the control group(P<0.01).There was no significant difference in blood glucose and glycosylated hemoglobin between the miR-34 a agonist group,miR-34 a antagonist group and model group(P>0.05),but the ratio of urinary albumin/creatinine in the miR-34 a agonist group was significantly higher than that in the model group,while the ratio of urinary albumin/creatinine in the miR-34 a antagonist group was significantly lower than that in the model group(P<0.01).The levels of ATG4 B,LC3Ⅱand SOD in the miR-34 a agonist group were lower than those in the negative control group,while the levels of p62,MDA and ROS in the miR-34 a agonist group were higher than those in the negative control group(P<0.01);the levels of ATG4 B,LC3Ⅱand SOD in the miR-34 a antagonist group were higher than those in the antagonist negative control group,while the expression level of p62 protein in the miR-34 a antagonist group was lower than that in the antagonist negative control group(P<0.01).Conclusion In diabetic mice model,miR-34 a contributes to diabetic kidney injury byinhibiting autophagy and oxidative stress through down-regulating ATG4 B.