DDAH1/ADMA促高糖诱导血管内皮细胞线粒体功能障碍  

DDAH1/ADMA promotes high glucose-induced mitochondrialdysfunction in vascular endothelial cells

作　　者：陈素雅 陈慧丽 彭锦鸿 李年生 江俊麟 CHEN Su-ya;CHEN Hui-li;PENG Jin-hong;LI Nian-sheng;JIANG Jun-lin(Dept of Pharmacology,Xiangya School of Pharmaceutical Sciences,Central South University,Changsha 410078,China;Rehabilitation Therapy,School of Medicine in Hunan Normal University,Changsha 410013,China)

机构地区：[1]中南大学湘雅药学院药理学系,湖南长沙410078 [2]湖南师范大学医学院康复治疗学专业,湖南长沙410013

出　　处：《中国药理学通报》2025年第2期258-267,共10页Chinese Pharmacological Bulletin

基　　金：湖南省自然科学基金资助项目(No 2022JJ30776,2022JJ30777)。

摘　　要：目的探讨二甲基精氨酸二甲胺水解酶1(dimethylarginine dimethylamino hydrolase 1,DDAH1)对高糖诱导的血管内皮细胞线粒体功能障碍及自噬的影响。方法采用JC-1染色检测线粒体膜电位;DCFH-DA荧光探针检测活性氧水平;Hoechst法检测细胞凋亡;Real-time PCR法检测DDAH1 mRNA水平;Western blot检测DDAH1、LC3-I和LC3-II蛋白表达;线粒体探针Mitotracker与LC3细胞免疫荧光共定位法检测线粒体自噬水平;高效液相法检测细胞上清液中非对称二甲基精氨酸(asymmetric dimethylarginine,ADMA)水平。结果高糖处理人脐静脉内皮细胞(HUVECs)48 h明显降低线粒体膜电位,增加活性氧产生,促进细胞凋亡;高糖下调LC3-Ⅱ/LC3-Ⅰ蛋白表达,减少Mitotracker与LC3共定位,抑制线粒体自噬。自噬抑制剂3-MA或CQ加重高糖诱导的HUVECs线粒体损伤和细胞凋亡;自噬激动剂RAPA减轻高糖诱导的HUVECs线粒体损伤和凋亡。高糖下调HUVECs DDAH1蛋白表达,增加ADMA水平。DDAH1 siRNA抑制线粒体自噬,降低线粒体膜电位,促进细胞凋亡,而DDAH1过表达则促进线粒体自噬,减轻高糖诱导的HUVECs凋亡。结论高糖诱发内皮细胞线粒体功能损伤与其抑制DDAH1表达,增加ADMA水平,降低线粒体自噬有关。Aim To investigate the effects of dimethylarginine dimethylamino hydrolase 1(DDAH1)on high glucose-induced mitochondrial dysfunction and mitophagy in vascular endothelial cells.Methods JC-1 staining was used to detect mitochondrial membrane potential.DCFH-DA fluorescent probe was employed to measure reactive oxygen species(ROS)levels.Hoechst staining was used to assess cell apoptosis.Real-time PCR was conducted to detect DDAH1 mRNA levels.Western blot was performed to analyze the expression of DDAH1,LC3-I and LC3-II proteins.Mitochondrial probe Mitotracker and autophagosome marker protein LC3 were used in cell immunofluorescence co-localization to assess mitochondrial autophagy,and high-performance liquid chromatography was utilized to measure the levels of asymmetric dimethylarginine(ADMA)in cell supernatant.Results High glucose treatment for 48 h significantly reduced mitochondrial membrane potential,increased ROS production,and promoted apoptosis in human umbilical vein endothelial cells(HUVECs).High glucose downregulated the expression of LC3-II/LC3-I proteins,reduced the co-localization of Mitotracker and LC3,and inhibited mitochondrial autophagy.Autophagy inhibitors 3-MA or CQ exacerbated high glucose-induced mitochondrial damage and apoptosis in HUVECs,while autophagy activator RAPA alleviated these effects.High glucose significantly downregulated DDAH1 protein expression in HUVECs and increased ADMA levels in cell supernatant.DDAH1 siRNA inhibited mitochondrial autophagy,reduced mitochondrial membrane potential,and promoted apoptosis,whereas DDAH1 overexpression enhanced mitochondrial autophagy and alleviated high glucose-induced apoptosis in HUVECs.Conclusion High glucose-induced endothelial mitochondrial dysfunction is associated with the suppression of DDAH1 expression,the increase of ADMA levels,and thereduction of mitochondrial autophagy.

关 键 词：二甲基精氨酸二甲胺水解酶1 非对称二甲基精氨酸 线粒体自噬 线粒体功能障碍 血管内皮细胞 凋亡 

分 类 号：R322.123[医药卫生—人体解剖和组织胚胎学] R329.24[医药卫生—基础医学] R329.25R341.7R392