小泛素样修饰蛋白特异性蛋白酶1介导糖尿病性血管内皮细胞损伤修复的研究

Repair of SUMO specific protease 1-induced injury in diabetic vascular endothelial cells

目的利用小泛素相关修饰体(SUMO)特异性蛋白酶1(SENP1)解离过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)的小泛素样修饰蛋白1(SUMO1)修饰。方法人脐静脉内皮细胞培养传代后分对照组、高糖组及SENP1组;Western blot法检测SENP1、SUMO1、PGC-1α和半胱氨酸天冬氨酸蛋白酶3(Caspase-3)水平;实时荧光定量PCR检测线粒体转录因子A(TFAM)、核呼吸因子2α(NRF-2α)和雌激素受体相关受体α(ERRα)的mRNA表达;ELISA测定乳酸脱氢酶(LDH);细胞划痕愈合方法、Transwell方法和体外血管模拟形成实验分别检测细胞自愈、迁移和形成血管拟态的能力。结果与对照组比较,高糖组共价SUMO1、PGC-1α和活化Caspase-3蛋白表达明显升高,SENP1蛋白表达明显降低,TFAM、NRF-2α和ERRα的mRNA表达明显降低,细胞划痕修复能力及模拟血管形成能力下降(P<0.05,P<0.01)。与高糖组比较,SENP1组SENP1蛋白表达明显升高,共价SUMO1、PGC-1α和活化Caspase-3蛋白表达明显降低,TFAM、NRF-2α和ERRα的mRNA表达明显升高,细胞划痕修复和迁移能力及模拟血管形成能力明显改善(P<0.05,P<0.01)。对照组、高糖组和SENP1组细胞上清液中LDH水平比较,差异有统计学意义[(24.66±6.39)ng/ml vs(302.45±30.54)ng/ml vs(174.08±21.03)ng/ml,P<0.01]。结论SENP1能够诱导PGC-1α发生去SUMO修饰,解除其对PGC-1α下游转录因子的抑制作用,改善线粒体功能,抑制高糖诱导的血管内皮细胞功能损伤作用。

Objective To dissociate the SUMO1 from peroxisome proliferators-activated receptorγcoactivator-1α(PGC-1α)using SUMO specific protease 1(SENP1).Methods HUVEC were divided into control group,high glucose group and high glucose+SENP1 group.The expressions of SENP1,SUMO1,PGC-1αand Caspase-3 proteins were detected by Western blot while those of TFAM,NRF-2αand ERR-αmRNA were detected by RT-PCR.The activity of lactate dehydrogenase(LDH)was measured by ELISA.The self-healing,migration and angiogenesis of HUVEC were examined by scratch test,transwell test and simulated angiogenesis test respectively.ResultsThe expression levels of SUMO1,PGC-1αand Caspase-3 proteins were significantly higher while those of SENP1 protein and TFAM,NRF-2α,ERR-αmRNA were significantly lower in high glucose group than in control group(P<0.05,P<0.01).Scratch test showed that the repair and angiogenesis of HUVEC were significantly poorer in high glucose group than in control group(P<0.05,P<0.01).The expression levels of SENP1 and TFAM,NRF-2αand ERR-αmRNA were significantly higher while those of SUMO1 and PGC-1αand activated Caspase-3 proteins were significantly lower in SENP1 group than in high glucose group(P<0.05,P<0.01).Scratch test showed that the migration and angiogenesis were significantly better in SENP1 group than in high glucose group(P<0.05,P<0.01).A significant difference in serum LDH level was detected in control group,high glucose group and SENP1 group(24.66±6.39 ng/ml vs 302.45±30.54 ng/ml vs 174.08±21.03 ng/ml,P<0.01).Conclusion SENP1 can induce the deSUMOylation of PGC-1α,relieve the inhibitory effect of PGC-1αdownstream transcription factors,improve the mitochondrial function,and inhibit the high glucose-induced injury of HUVEC.