Objectives: Trimethylamine oxide (TMAO) is a metabolite of intestinal flora and is known to promote the progression of atherosclerotic plaques. However, how TMAO works, including its effect on vascular endothelial cells, is not fully understood. This study aims to explore the biological role of TMAO in human umbilical vein endothelial cells (HUVECs) and the underlying mechanism.
Methods: Cell pyroptosis and the loss of plasma membrane integrity were induced under TMAO stimulation in HUVECs. The plasma membrane integrity of the cells was measured by Hoechst 33342/propidium iodide (PI) staining and lactate dehydrogenase leakage assay, and the changes in cell morphology were observed by atomic force microscope. The expression of proteins related to pyroptosis was determined by Western blotting or immunofluorescence. Mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) activity in HUVECs was measured by the ALDH2 activity assay kit, and the level of reactive oxygen species (ROS) was detected by fluorescent probe DCFH-DA.
Results: TMAO induced pyroptotic cell death, manifesting by the presence of propidium iodide-positive cells, the leakage of lactate dehydrogenase, the production of N-terminal gasdermin D (GSDMD-N), and the formation of plasma membrane pores. Moreover, TMAO induced elevated expression of inflammasome components, nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1 in cells. TMAO significantly inhibited ALDH2 activity and increased intracellular ROS production. However, the activation of ALDH2 by pharmacological manipulation attenuated TMAO-induced inflammasome activation and GSDMD-N production.
Conclusions: TMAO induces pyroptosis of vascular endothelial cells through the ALDH2/ROS/NLRP3/GSDMD signaling pathway, which may be a potential therapeutic target for improving the treatment of atherosclerosis.
目的 : 氧化三甲胺(trimethylamine oxide，TMAO)是肠道菌群的代谢产物，可促进动脉粥样硬化斑块的发展。然而，TMAO对血管内皮细胞的作用尚不清楚。本研究旨在探讨TMAO对人脐静脉内皮细胞的生物学作用及其机制。 方法 : 以TMAO刺激人脐静脉内皮细胞诱导细胞焦亡和质膜完整性丧失。采用Hoechst 33342/PI染色法和乳酸脱氢酶释放实验测定细胞质膜完整性，原子力显微镜观察细胞形态变化。通过蛋白质印迹法或免疫荧光法测定细胞焦亡相关蛋白质的表达。用乙醛脱氢酶2(acetaldehyde dehydrogenase 2，ALDH2)活性测定试剂盒测定人脐静脉内皮细胞中ALDH2的活性，用荧光探针DCFH-DA检测活性氧(reactive oxygen species，ROS)水平。 结果 : TMAO可诱导内皮细胞焦亡，表现为细胞死亡增加、乳酸脱氢酶外渗、成孔蛋白GSDMD-N段产生和质膜孔形成。同时，TMAO诱导细胞中炎性体成分核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3，NLRP3)、凋亡相关微粒蛋白(apoptosis-associated speck-like protein containing a caspase activation and recruitment domain，ASC)和半胱氨酸蛋白酶-1(caspase-1)蛋白质的表达升高。此外，TMAO显著抑制线粒体ALDH2活性并增加细胞内ROS的产生，ALDH2的激活还可减弱TMAO诱导的炎症小体活化和 GSDMD-N生成。 结论 : TMAO通过ALDH2/ROS/NLRP3/GSDMD信号通路诱导血管内皮细胞焦亡，这可能是治疗动脉粥样硬化的潜在靶点。.
目的: 氧化三甲胺(trimethylamine oxide，TMAO)是肠道菌群的代谢产物，可促进动脉粥样硬化斑块的发展。然而，TMAO对血管内皮细胞的作用尚不清楚。本研究旨在探讨TMAO对人脐静脉内皮细胞的生物学作用及其机制。
方法: 以TMAO刺激人脐静脉内皮细胞诱导细胞焦亡和质膜完整性丧失。采用Hoechst 33342/PI染色法和乳酸脱氢酶释放实验测定细胞质膜完整性，原子力显微镜观察细胞形态变化。通过蛋白质印迹法或免疫荧光法测定细胞焦亡相关蛋白质的表达。用乙醛脱氢酶2(acetaldehyde dehydrogenase 2，ALDH2)活性测定试剂盒测定人脐静脉内皮细胞中ALDH2的活性，用荧光探针DCFH-DA检测活性氧(reactive oxygen species，ROS)水平。
结果: TMAO可诱导内皮细胞焦亡，表现为细胞死亡增加、乳酸脱氢酶外渗、成孔蛋白GSDMD-N段产生和质膜孔形成。同时，TMAO诱导细胞中炎性体成分核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3，NLRP3)、凋亡相关微粒蛋白(apoptosis-associated speck-like protein containing a caspase activation and recruitment domain，ASC)和半胱氨酸蛋白酶-1(caspase-1)蛋白质的表达升高。此外，TMAO显著抑制线粒体ALDH2活性并增加细胞内ROS的产生，ALDH2的激活还可减弱TMAO诱导的炎症小体活化和 GSDMD-N生成。
结论: TMAO通过ALDH2/ROS/NLRP3/GSDMD信号通路诱导血管内皮细胞焦亡，这可能是治疗动脉粥样硬化的潜在靶点。
Keywords: atherosclerosis; endothelial cell pyroptosis; endothelial dysfunction; trimethylamine oxide.