沙格列汀通过miR-15a-5p/INSR轴缓解糖尿病大鼠胰岛素抵抗的机制研究

The mechanism of saxagliptin alleviates insulin resistance in diabetic rats through miR-15a-5p/INSR axis

目的探讨沙格列汀通过miR-15a-5p/胰岛素受体(INSR)轴缓解2型糖尿病大鼠胰岛素抵抗的机制。方法以INSR为常用转录本ENST00000341500为输入,分别获取TargetScan、miRDB和miRWalk数据库中可能靶向INSR的miRNA并取交集。随后荧光素酶实验验证靶向关系。构建胰岛素抵抗2型糖尿病(IR-T2DM)模型。随后对IR-T2DM大鼠进行阴性对照、miR-15a-5p mimic、miR-15a-5p inhibitor转染或沙格列汀处理,8周后处死大鼠获取股四头肌及外周血。对各组大鼠血压、血糖、脂代谢相关指标进行测定,同时测定稳态模型评估(HOMA)胰岛素抵抗指数(IRI)和胰岛素敏感指数(ISI)。定量-PCR检测各组大鼠骨骼肌中交集miRNA、INSR、IRS-1和GLUT4 mRNA的表达。Western blot检测各组大鼠骨骼肌中INSR、p-IRS-1和GLUT4蛋白表达。结果miR-15a-5p被选定为INSR的靶向miRNA。与阴性对照组相比,IR-T2DM大鼠经miR-15a-5p inhibitor或沙格列汀处理后骨骼肌中INSR、IRS-1和GLUT4表达、外周血中HDL-C浓度、BW、ISI升高(均P<0.05),但外周血中糖化血红蛋白(HbA1c)、三酰甘油(TG)和低密度脂蛋白胆固醇(LDL-C)浓度、收缩压(SP)、舒张压(DP)和IRI在上述组中降低(均P<0.05)。上述指标的趋势在miR-15a-5p mimic组中与miR-15a-5p inhibitor及沙格列汀组完全相反(均P<0.05)。此外,与沙格列汀或miR-15a-5p inhibitor单独处理比较,两者联合处理能更有效地缓解糖尿病大鼠胰岛素抵抗(均P<0.05)。结论沙格列汀能通过下调miR-15a-5p来上调INSR表达从而缓解糖尿病大鼠胰岛素抵抗。

Objective To study the mechanism of saxagliptin in alleviating insulin resistance in diabetic rats by miR-15a-5p/insulin receptor(INSR)axis.Methods Firstly the intersection of miRNA that might target INSR was sellected from Targetscan,miRDB and miRWalk by inputing the transcript of INSR ENST00000341500.Then dual luciferase reporter assay was used to verify the targeting relationship.Subsequently the insulin resistance-type 2 diabetes mellitus(IR-T2DM)models were constructed.Then,IR-T2DM rats were treated with miR-15a-5p mimic,miR-15a-5p inhibitor or saxagliptin.Rats were sacrificed after 8 weeks to obtain quadriceps femoris muscles and peripheral blood.Blood pressure,blood glucose,lipid metabolic parameters,homeostasis model assessment(HOMA)insulin resistance index(IRI)as well as insulin sensitivity index(ISI)were measured in each group,respectively.Quantitative PCR was used to detect the expression of intersection miRNA and IRS-1,IRS1,GLUT4 mRNA while Western blot was adopted to test the protein expression of INSR,p-IRS-1 and GLUT4 in skeletal muscle of rats in each group.Results miR-15a-5p was selected as the targeting miRNA of INSR.Compared with negative control group,the expression of INSR,IRS-1 and GLUT4 in skeletal muscle as well as the concentration of HDL-C,BW and ISI in peripheral blood of IR-T2DM rats treated with miR-15a-5p inhibitor or saxagliptin markedly increased(P<0.5),but the concentration of hemoglobin A1c(HbA1c),triglyceride(TG)and low-density liPoprotein cholesterol(LDL-C),systolic blood pressure(SP),diastolic blood pressure(DP)and IRI in peripheral blood significantly increased in the above two groups(P<0.05).The trend of the above indexes in miR-15a-5p mimic group was completely opposite to that in miR-15a-5p inhibitor and saxagliptin group(P<0.05).In addition,saxagliptin combined with miR-15a-5p inhibitor had more effictive capacity in relieving insulin resistance than treating the diabetic rats with saxagliptin or miR-15a-5p inhibitor alone(P<0.05).Conclusion Based on the results observed during this study,the paper shows that saxagliptin has distinct effect on relieving insulin resistance by down regulating miR-15a-5p to up regulate INSR expression in diabetic rats.