沉默微小RNA-218表达保护STZ诱导的糖尿病大鼠肾组织

目的:探讨沉默微小RNA-218(microRNA-218,miR-218)表达对链脲佐菌素(streptozotocin,STZ)诱导的糖尿病肾病大鼠肾脏组织的保护作用及其可能机制。方法:采用单次腹腔注射STZ(50 mg/kg)方法制备糖尿病大鼠模型并构建miR-218短发夹RNA(short hairpin RNA,shRNA)慢病毒载体。SD大鼠被随机分为健康对照组、糖尿病模型组、空载慢病毒组及miR-218-shRNA组。于自动生化仪上检测不同时点(4、8和12周)大鼠血糖、24h尿蛋白量、血清肌酐(serum creatinine,SCr)及血尿素氮(blood urea nitrogen,BUN)含量。实时荧光定量PCR(RTqPCR)检测肾脏组织miR-218的表达。RT-qPCR和Western blot检测血红素氧合酶1(heme oxygenase-1,HO-1)、肾病蛋白(nephrin)和p38丝裂原激活的蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)的mRNA及蛋白表达水平。Caspase-3活性检测试剂盒检测caspase-3活性。末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling,TUNEL)法检测肾脏组织细胞凋亡。结果:与健康对照组相比,STZ处理后大鼠miR-218表达水平显著升高。同时模型大鼠的血糖、24 h尿蛋白量、SCr及BUN含量显著升高(P0.05);模型大鼠肾脏组织中HO-1和nephrin的mRNA和蛋白表达水平显著降低,而p38 MAPK蛋白的磷酸化水平显著升高;另外,模型大鼠肾脏组织中的caspase-3活性也显著升高。模型大鼠感染miR-218-shRNA后,miR-218表达水平显著下降并可以显著逆转上述效应。miR-218-shRNA组肾脏组织细胞的凋亡水平显著低于糖尿病模型组及空载慢病毒组。结论:miR-218参与了糖尿病大鼠的肾脏损伤,慢病毒载体沉默其表达能有效抑制肾脏组织细胞的凋亡,提示miR-218可以作为糖尿病肾病的基因治疗靶点。

Protective effect of microRNA-218 silencing on kidney tissue in STZ-induced diabetic rats

AIM:To investigate the protective effect of microRNA-218(miR-218) silencing on kidney tissue of streptozotocin (STZ)-induced diabetic nephropathy rats and the potential mechanism.METHODS:The diabetic rat model was established by a single intraperitoneal injection of STZ (50 mg/kg).Meanwhile,the miR-218 short hairpin RNA (shRNA) lentiviral vector was constructed.The Sprague-Dawley rats were randomly divided into 4 groups:healthy control group,diabetes group,empty vector group and miR-218-shRNA group.The blood glucose,24 h urinary protein,serum creatinine (SCr) and blood urea nitrogen (BUN) in the rats at different time points (4,8 and 12 weeks) were measured by an automated analyzer.The expression of miR-218 was detected by RT-qPCR,while the expression of heme oxygenase-1(HO-1),nephrin and p38 mitogen-activated protein kinase (p38 MAPK) at mRNA and protein levels in the kidney tissues was determined by RT-qPCR and Western blot.The caspase-3 activity was detected by caspase-3 activity assay kit,and the cell apoptosis of the kidney tissues was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).RESULTS:Compared with healthy control group,the expression of miR-218 was significantly increased in STZ-treated rats.Meanwhile,the concentrations of blood glucose,24 h urinary protein,SCr and BUN were significantly increased in STZ-treated rats (P<0.05).The mRNA and protein expression of HO-1 and nephrin was significantly decreased,while the level of phosphorylated p38 MAPK was significantly increased in STZ-treated rats.In addition,the activity of caspase-3 was also significantly increased in STZ-treated rats.When the model rats were infected with miR-218-shRNA,the expression of miR-218 was significantly decreased and the above effects were markedly reversed.Furthermore,TUNEL results showed that compared with diabetic group and empty vector group,miR-218 silencing significantly attenuated the cell apoptosis in the kidney tissues in miR-218-shRNA group.CONCLUSION:miR-218 is involved in the kidney injury in diabetic rats,and silencing of miR-218 by lentiviral vector-mediated miR-218-shRNA transfection effectively inhibits kidney cell apoptosis,suggesting that miR-218 is a potential target for the treatment of diabetic nephropathy.