2＋浓度、膜电位、二氢乳清酸脱氢酶(DHODH)表达以及氧化型辅酶Q10(CoQ10)的影响；荧光探针法观察DHODH抑制剂以及氧化型CoQ10对血管内皮细胞ROS含量、Ca2＋浓度的影响。 [结果]DS处理后,血管内皮细胞线粒体固缩、体积变小、双层膜电子密度增加、嵴数量减少,嵴稳态相关蛋白MIC60表达下调23%,ROS含量升高,Ca2＋浓度增加,线粒体膜电位降低(P＜0.05或P＜0.01)；GPR91激动剂处理后,线粒体嵴稳态相关蛋白MIC60表达下调31%,ROS含量升高27%,钙离子浓度升高36%,线粒体膜电位降低(P＜0.05或P＜0.01)；GPR91抑制剂处理后,线粒体嵴稳态相关蛋白MIC60上调22%,ATP5I上调40%,ROS含量降低41%,Ca2＋浓度降低67%,线粒体膜电位恢复正常(P＜0.05或P＜0.01)。DS处理后,DHODH的表达下调43%,氧化型CoQ10的含量增加120%(P＜0.05或P＜0.01)；GPR91激动剂处理后,DHODH的表达下调22%,氧化型CoQ10的含量增加36%(P＜0.05或P＜0.01)；GPR91抑制剂处理后,DHODH的表达上调40%,氧化型CoQ10的含量降低39%(P＜0.01)；DHODH抑制剂处理后,ROS含量增加20%,Ca2＋浓度增加28%,线粒体膜电位降低(P＜0.05或P＜0.01)；外源加入氧化型CoQ10处理后,ROS含量降低30%,Ca2＋浓度降低20%(P＜0.05或P＜0.01)。 [结论]琥珀酸/GPR91可能通过影响线粒体嵴稳态下调DHODH的表达进而抑制氧化型CoQ10还原,导致线粒体损伤。"/>

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琥珀酸/GPR91通过DHODH/CoQ10促血管内皮细胞线粒体损伤
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作者单位:

(1.南华大学心血管疾病研究所 动脉硬化学湖南省重点实验室 湖南省动脉硬化性疾病国际科技创新合作基地,湖南省衡阳市 421001;2.江门国际旅行卫生保健中心,广东省江门市 529030)

作者简介:

秦文华,硕士研究生,研究方向为动脉粥样硬化发病机制及其防治,E-mail:2315780198@qq.com。

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基金项目:

国家自然科学基金项目(31870937)


Succinate/GPR91 promotes mitochondrial damage in vascular endothelial cells through DHODH/CoQ10
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Affiliation:

1.Institute of Cardiovascular Disease, University of South China & Key Laboratory for Arteriosclerology of Hunan Province & Hunan International Scientific and Technological Cooperation Base of Arteriosclerotic Disease, Hengyang, Hunan 421001, China;2.Jiangmen International Travel Healthcare Center, Jiangmen, Guangdong 529030, China)

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    摘要:

    目的]探讨琥珀酸/G蛋白偶联受体91(GPR91)对血管内皮细胞线粒体的影响及其调节机制。 [方法]采用透射电镜、Western blot、荧光显微镜观察琥珀酸类似物琥珀酸二乙酯(DS)、GPR91激动剂及抑制剂对血管内皮细胞线粒体形态、嵴、嵴稳态相关蛋白、活性氧(ROS)含量、Ca2＋浓度、膜电位、二氢乳清酸脱氢酶(DHODH)表达以及氧化型辅酶Q10(CoQ10)的影响；荧光探针法观察DHODH抑制剂以及氧化型CoQ10对血管内皮细胞ROS含量、Ca2＋浓度的影响。 [结果]DS处理后,血管内皮细胞线粒体固缩、体积变小、双层膜电子密度增加、嵴数量减少,嵴稳态相关蛋白MIC60表达下调23%,ROS含量升高,Ca2＋浓度增加,线粒体膜电位降低(P＜0.05或P＜0.01)；GPR91激动剂处理后,线粒体嵴稳态相关蛋白MIC60表达下调31%,ROS含量升高27%,钙离子浓度升高36%,线粒体膜电位降低(P＜0.05或P＜0.01)；GPR91抑制剂处理后,线粒体嵴稳态相关蛋白MIC60上调22%,ATP5I上调40%,ROS含量降低41%,Ca2＋浓度降低67%,线粒体膜电位恢复正常(P＜0.05或P＜0.01)。DS处理后,DHODH的表达下调43%,氧化型CoQ10的含量增加120%(P＜0.05或P＜0.01)；GPR91激动剂处理后,DHODH的表达下调22%,氧化型CoQ10的含量增加36%(P＜0.05或P＜0.01)；GPR91抑制剂处理后,DHODH的表达上调40%,氧化型CoQ10的含量降低39%(P＜0.01)；DHODH抑制剂处理后,ROS含量增加20%,Ca2＋浓度增加28%,线粒体膜电位降低(P＜0.05或P＜0.01)；外源加入氧化型CoQ10处理后,ROS含量降低30%,Ca2＋浓度降低20%(P＜0.05或P＜0.01)。 [结论]琥珀酸/GPR91可能通过影响线粒体嵴稳态下调DHODH的表达进而抑制氧化型CoQ10还原,导致线粒体损伤。

    Abstract:

    Aim To explore the effect of succinate/G protein coupled receptor 91 (GPR91) on mitochondria in vascular endothelial cells and its regulatory mechanisms. Methods Transmission electron microscopy, Western blot and fluorescence microscopy were used to observe the effects of succinate analogues diethyl succinate (DS), GPR91 agonist and inhibitor on the mitochondrial morphology, cristae, cristate homeostasis related proteins reactive oxygen species (ROS) content, Ca2＋ concentration, mitochondrial membrane potential, the expression of dihydroorotate dehydrogenase (DHODH) and oxidized coenzyme Q10 (CoQ10). Fluorescence probes were used to observe the effect of DHODH inhibitor and CoQ10 on ROS level and Ca2＋concentration of endothelial cells. Results After DS treatment, the mitochondria showed pyknosis and mitochondrial volume significantly decreased, electron density of the mitochondrial membrane increased, and the number of cristae decreased in endothelial cells; the expression of cristae homeostasis related proteins MIC60 decreased by 23%, while cellular ROS level and Ca2＋ concentration increased; mitochondrial membrane potential decreased (P＜0.05 or P＜0.01). After GPR91 agonist treatment, the expression of cristae homeostasis related proteins MIC60 decreased by 31%, meanwhile, cellular ROS level increased by 27% and Ca2＋ concentration increased by 36%; mitochondrial membrane potential decreased (P＜0.05 or P＜0.01). After GPR91 inhibitor treatment, the expression of cristae homeostasis related proteins MIC60 increased by 22% and ATP5I increased by 40%; the levels of ROS decreased by 41% and Ca2＋ concentration decreased by 67%; and the mitochondrial membrane potential was restored to normal (P＜0.05 or P＜0.01). After DS treatment, the expression of DHODH decreased by 43% and the level of oxidized CoQ10 increased by 120% (P＜0.05 or P＜0.01). After GPR91 agonist treatment, the expression of DHODH decreased by 22% and the level of oxidized CoQ10 increased by 36% (P＜0.05 or P＜0.01). After GPR91 inhibitor treatment, the expression of DHODH increased by 40% and the level of oxidized CoQ10 decreased by 39% (P＜0.01). After DHODH inhibitor treatment, the ROS level increased by 20% and Ca2＋ concentration increased by 28%, and mitochondrial membrane potential reduced at same time (P＜0.05 or P＜0.01). Exogenous oxidized CoQ10 inhibited ROS production by 30% and decreased Ca2＋ concentration by 20% (P＜0.05 or P＜0.01). Conclusion Succinate/GPR91 promotes mitochondrial damage in endothelial cells, and its mechanism may relate to down-regulating the expression of DHODH and inhibiting the reduction of CoQ10 by affecting the mitochondrial cristae homeostasis.

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秦文华,袁楚楚,孙玉慧,余博,危当恒.琥珀酸/GPR91通过DHODH/CoQ10促血管内皮细胞线粒体损伤[J].中国动脉硬化杂志,2024,32(6):466~472.

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  • 收稿日期:2024-01-20
  • 最后修改日期:2024-04-07
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  • 在线发布日期: 2024-07-04
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