氧化三甲胺对血管内皮细胞线粒体功能的作用研究

Effect of trimethylamine-N-oxide on mitochondrial function of vascular endothelial cells

摘要:

目的 探讨氧化三甲胺（TMAO）对血管内皮细胞线粒体功能的影响。方法 使用不同浓度的TMAO（50、100和150 mmol/L）刺激人脐静脉血管内皮细胞（HUVEC）24 h，或使用100 mmol/L浓度的TMAO干预HUVEC不同的时间（12、24和36 h）。利用CCK-8法检测HUVEC的细胞活力；采用Annexin V-FITC/PI双染法检测细胞凋亡水平；使用酶联免疫吸附试验检测细胞上清液内皮素-1（ET-1）、白细胞介素-18（IL-18）、IL-1β、IL-6和IL-10水平；采用罗丹明-123探针检测线粒体膜电位（MMP）；采用MitoSOX Red探针检测线粒体活性氧（mtROS）；使用Calcein AM染色检测线粒体通透性转换孔（MPTP）的开放程度。结果 TMAO浓度自50 mmol/L开始，细胞活力开始降低，且呈剂量依赖性变化（P <0.05）。TMAO干预12 h后，细胞活力显著降低，且呈时间依赖性变化（P <0.05）。不同浓度TMAO组细胞凋亡率均高于对照组（P <0.05）。对照组ET-1水平低于不同浓度TMAO组（P <0.05）。对照组IL-18、IL-1β、IL-6水平均低于不同浓度TMAO组（P <0.05），IL-10水平高于不同浓度TMAO组（P <0.05）。不同浓度TMAO组罗丹明-123相对表达量均低于对照组（P <0.05）。TMAO处理24、36 h组罗丹明-123相对表达量均低于对照组（P <0.05）。100、150 mmol/L TMAO组MitoSOX相对表达量均高于对照组（P <0.05）。不同干预时间TMAO组MitoSOX相对表达量均高于对照组（P <0.05）。不同浓度TMAO组Calcein AM相对表达量均低于对照组（P <0.05）。不同干预时间TMAO组Calcein AM相对表达量均低于对照组（P <0.05）。结论 TMAO可诱导内皮细胞线粒体功能障碍。

Abstract:

Objective To explore the effect of trimethylamine-N-oxide (TMAO) on mitochondrial function of vascular endothelial cells.Methods Human umbilical vein endothelial cells (HUVEC) were either stimulated with varying concentrations of TMAO (50, 100, and 150 mmol/L) for 24 hours or treated with 100 mmol/L TMAO for 12, 24, and 36 hours. Cell viability was assessed using the CCK-8 assay. Apoptosis levels were measured by Annexin V-FITC/PI double staining. Levels of endothelin-1 (ET-1), interleukin-18 (IL-18), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) in cell culture supernatants were quantified via enzyme-linked immunosorbent assay (ELISA). Mitochondrial membrane potential (MMP) was detected using a rhodamine-123 probe. Mitochondrial reactive oxygen species (mtROS) were measured with a MitoSOX Red probe. The degree of mitochondrial permeability transition pore (MPTP) opening was evaluated by Calcein AM staining.Results Cell viability decreased in a dose-dependent manner starting at TMAO concentrations of 50 mmol/L (P < 0.05). After 12 hours of TMAO exposure, cell viability significantly decreased in a time-dependent manner (P < 0.05). The cell viability in TMAO-treated groups was higher than that in the control group across all concentrations (P <0.05). ET-1 levels in the control group were lower than those in TMAO-treated groups at different TMAO concentrations (P < 0.05). IL-18, IL-1β, and IL-6 levels in the control group were lower than those in TMAO-treated groups at different TMAO concentrations (P < 0.05), while IL-10 levels were higher in the control group than in TMAO-treated groups (P < 0.05). The relative expression of rhodamine-123 in TMAO-treated groups at different concentrations was lower than that in the control group (P < 0.05). The relative expression of rhodamine-123 in the 24- and 36-hour TMAO-treated groups was lower than that in the control group (P < 0.05). The relative expression of MitoSOX in the 100- and 150-mmol/L TMAO-treated groups was higher than that in the control group (P < 0.05). The relative expression of MitoSOX in TMAO-treated groups at different intervention times was higher than that in the control group (P < 0.05). The relative expression of Calcein AM in TMAO-treated groups at different TMAO concentrations was lower than that in the control group (P < 0.05). At different intervention times, the relative expression of Calcein AM in TMAO-treated groups was lower than that in the control group (P < 0.05).Conclusions TMAO induces mitochondrial dysfunction in vascular endothelial cells.