摘要
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(P<0.05);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。
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 as-say 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 signifi-cantly 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 silen
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2017年第7期1251-1257,共7页
Chinese Journal of Pathophysiology
