曲格列汀改善高糖条件下人脐静脉内皮细胞氧化应激水平

Trelagliptin alleviates oxidative stress in human umbilical vein endothelial cells exposed to high glucose

目的:观察曲格列汀在高糖条件下对人脐静脉内皮细胞氧化应激水平的影响,并初步探讨其分子机制。方法:在正常葡萄糖浓度(5 mmol/L)及高糖(25 mmol/L)条件下培养人脐静脉内皮细胞(HUVECs),并在此期间分别加入1、5、10μmol/L曲格列汀作用24 h。随后CCK-8实验测定细胞增殖情况,荧光探针检测细胞内活性氧(ROS)变化,RT-qPCR以及Western blot分别检测NAD(P) H氧化酶和Nrf2及相关基因HMOX和NQO1的mRNA或蛋白表达水平以及内质网应激相关分子Bip1/Grp78、PDI、Ero1-Lα、Calnexin、IRE-1α和p-eIF2α表达的变化。结果:1、5和10μmol/L曲格列汀作用细胞24 h并不能降低其活性,但可有效降低高糖调剂下细胞内的ROS水平。曲格列汀处理也能明显抑制NAD(P) H氧化酶p22phox亚基mRNA的表达,并能抑制细胞核内核转录因子Nrf2的水平以及上调氧化应激相关基因HMOX和NQO1的表达。此外,高糖条件下内质网应激相关分子Bip1/Grp78、PDI、Ero1-Lα、Calnexin、IRE-1α和p-eIF2α表达水平明显增高,给予曲格列汀处理后,这些分子表达水平明显减少。结论:曲格列汀能增加高糖条件下人脐静脉内皮细胞Nrf2相关基因的表达并减轻内质网应激水平。

Objective: To verify a potential protective action of Trelagliptin in endothelial cells exposed to high glucose.Methods: Human umbilical vein endothelial cells(HUVECs) were cultured under normal(5 mmol/L) or high glucose(25 mmol/L) with different concentrations of Trelagliptin(1,5 and 10 μmol/L) for 24 h.CCK-8 assay was used to test cell proliferation.Changes of reactive oxygen(ROS) were determined using fluorescent probes.RT-qPCR and Western blot were used to detect mRNA and protein expression level of NAD(P) H oxidase,Nrf2 and Nrf2 related genes HMOX and NQO1,respectively.Expression levels of endoplasmic reticulum stress-related molecules Bip1/Grp78,PDI,Ero1-Lα,Calnexin,IRE-1α and p-eIF2α were determined by Western blot.Results: Cell treated with different concentrations of Trelagliptin(1,5 and 10 μmol/L) for 24 h could not decrease the activity of ROS,but could significantly reduce the level of ROS in the cells.Trelagliptin could also significantly inhibit the mRNA expression of NAD(P) H oxidase p22^phox subunit,the level of nuclear transcription factor Nrf2 and up-regulate the expression of oxidative stress-related genes HMOX and NQO1.Moreover,the expression levels of endoplasmic reticulum stress-related molecules Bip1/Grp78,PDI,Ero1-Lα,Calnexin,IRE-1α and p-eIF2α were significantly increased under high glucose conditions.However,the expression levels of these molecules were significantly reduced when cells were treated with Trelagliptin.Conclusion: Trigliptin could increase the expression of Nrf2-related genes in human umbilical vein endothelial cells under high glucose conditions and reduce endoplasmic reticulum stress.