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刘婉婷, 谢玉鑫, 李婧, 李朝荃, 姚慧, 付傲妮, 杨皓天, 易光辉.荷叶碱促进S1P/S1PR1/AMPK来减轻H2O2诱导的血管内皮细胞线粒体氧化应激损伤[J].湖南中医药大学学报,2025,45(9):1625-1635[点击复制] |
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| 荷叶碱促进S1P/S1PR1/AMPK来减轻H2O2诱导的血管内皮细胞线粒体氧化应激损伤 |
| 刘婉婷,谢玉鑫,李婧,李朝荃,姚慧,付傲妮,杨皓天,易光辉 |
| (心血管疾病研究所, 湖南省动脉硬化重点实验室, 湖南动脉硬化疾病国际科技合作基地, 湖南 衡阳 421001;南华大学衡阳医学院基础医学院, 湖南 衡阳 421001) |
| 摘要: |
| 目的 探究荷叶碱（NF）对H2O2诱导的人脐静脉内皮细胞（HUVECs）氧化应激引起的线粒体损伤的保护作用及其机制。方法 以H2O2诱导HUVECs氧化损伤为实验模型，将细胞分为对照组、H2O2组、H2O2+NF组、H2O2+NF+W146组、H2O2+NF+SEW2871组和H2O2+NF+Compound C组，选择S1PR1抑制剂W146（10μmol/L）、S1PR1激动剂SEW2871（20 nmol/L）和AMPK抑制剂Compound C（多索吗啡，1μmol/L）分别预处理30 min、30 min和24 h，然后加入10μmol/L NF处理12 h，最后与400μmol/L H2O2共孵育2 h。用ELISA检测S1P分泌水平；通过Western blot检测S1PR1、S1PR3、AMPK和p-AMPK的蛋白表达情况；通过二氢乙啶染色检测细胞中ROS的水平；使用透射电镜观察线粒体的超微结构；通过JC-1荧光探针和流式细胞术检测线粒体膜电位（MMP）水平；使用Calcein AM荧光探针检测线粒体通透性转换孔（MPTP）的开放程度；使用三磷酸腺苷检测试剂盒检测胞内ATP水平。结果 与对照组相比，H2O2组的细胞线粒体氧化应激损伤增加（P<0.01）。与H2O2组相比，H2O2+NF组细胞活性增加（P<0.001）,ROS水平降低、线粒体超微结构损伤和MPTP开放程度减轻，MMP水平和ATP水平增加（P<0.05）。同时，H2O2+NF组细胞S1P分泌（P<0.05）、S1PR1（P<0.05）和p-AMPK（P<0.01）蛋白表达水平增加。与H2O2+NF组相比，H2O2+NF+SEW2871组细胞p-AMPK（P<0.05）蛋白表达水平增加，线粒体氧化应激损伤减少（P<0.05）;H2O2+NF+W146组和H2O2+NF+Compound C组细胞p-AMPK（P<0.05）蛋白表达水平降低，线粒体氧化应激水平增加（P<0.05,P<0.01）。结论 NF促进S1P/S1PR1/AMPK来减轻H2O2诱导的HUVECs氧化应激引起的线粒体损伤。 |
| 关键词: 氧化应激 线粒体损伤 荷叶碱 人脐静脉内皮细胞 1-磷酸鞘氨醇/1-磷酸鞘氨醇受体1 腺苷酸活化蛋白激酶 |
| DOI：10.3969/j.issn.1674-070X.2025.09.006 |
| 投稿时间：2025-06-08 |
| 基金项目:国家自然科学基金项目(81770490); 湖南省科技计划项目(2020JJ4535)。 |
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| Nuciferine alleviating H2O2-induced mitochondrial oxidative stress damage in vascular endothelial cells by promoting S1P/S1PR1/AMPK pathway |
| LIU Wanting, XIE Yuxin, LI Jing, LI Chaoquan, YAO Hui, FU Aoni, YANG Haotian, YI Guanghui |
| (Institute of Pharmacy and Pharmacology, Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, University of South China, Hengyang, Hunan 421001, China;Institute of Cardiovascular Disease, Key Laboratory for Arteriosclerology of Hunan Province, Hunan International Scientific and Technological Cooperation Base of Arteriosclerotic Disease, Hengyang Medical School, University of South China, Hengyang, Hunan 421001, China;School of Basic Medical Sciences, Hengyang Medical College, University of South China, Hengyang, Hunan 421001, China) |
| Abstract: |
| Objective To explore the protective effects of nuciferine（NF） on mitochondrial damage caused by H2O2-induced oxidative stress in human umbilical vein endothelial cells（HUVECs） and the related mechanisms. Methods HUVECs were subjected to oxidative damage induced by H2O2as an experimental model. The cells were divided into the control group, H2O2group, H2O2+NF group, H2O2+NF+W146 group, H2O2+NF+SEW2871 group, and H2O2+NF+Compound C group. They were pretreated with the S1PR1 inhibitor W146（10 μmol/L）, S1PR1 agonist SEW2871（20 nmol/L）, and AMPK inhibitor Compound C（dorsomorphin, 1 μmol/L）for 30 min, 30 min, and 24 h, respectively. Subsequently, 10 μmol/L NF was added and incubated for 12 h, followed by coincubation with 400 μmol/L H2O2for 2 h. The secretion level of S1P was measured using ELISA. The protein expression levels of S1PR1, S1PR3, AMPK, and p-AMPK were determined by Western blot. The intracellular levels of reactive oxygen species（ROS）were measured using dihydroethidium staining. The ultrastructure of mitochondria was observed using transmission electron microscopy.The mitochondrial membrane potential（MMP） was determined using JC-1 fluorescent probes and flow cytometry. The opening degree of the mitochondrial permeability transition pore（MPTP） was examined using the Calcein AM fluorescent probe. The intracellular ATP levels were measured using an ATP assay kit. Results Compared with the control group, the H2O2group exhibited increased mitochondrial oxidative stress damage（P＜0.01）. Compared with the H2O2group, the H2O2+NF group demonstrated enhanced cell viability（P＜0.001）, reduced ROS levels, alleviated mitochondrial ultrastructural damage, and decreased MPTP opening degree, as well as elevated MMP and ATP levels（P＜0.05）. Additionally, the H2O2+NF group showed increased S1P secretion（P＜0.05） and elevated protein expression levels of S1PR1（P＜0.05） and p-AMPK（P＜0.01）. Compared with the H2O2+NF group, the H2O2+NF+SEW2871 group exhibited increased p-AMPK protein expression level（P＜0.05） and reduced mitochondrial oxidative stress damage（P＜0.05）. Conversely,the H2O2+NF+W146 and H2O2+NF+Compound C groups showed decreased p-AMPK protein expression level（P＜0.05） and elevated mitochondrial oxidative stress level（P＜0.05, P＜0.01）. Conclusion NF alleviates mitochondrial damage caused by H2O2-induced oxidative stress in HUVECs by promoting S1P/S1PR1/AMPK signaling pathway. |
| Key words: oxidative stress mitochondrial damage nuciferine human umbilical vein endothelial cell sphingosine-1-phosphate/sphingosine-1-phosphate receptor 1 adenosine 5’-monophosphate-activated protein kinase |
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