Xia Linzhen, Wang Zuo, Chen Xiangyu
Molecular Pathology Laboratory, Department of Pathology, Changsha Hospital for Maternal and Child Health Care, Hunan Normal University, Changsha, Hunan, People's Republic of China.
Institute of Cardiovascular Disease, Key Laboratory for Arteriosclerology of Hunan Province, Hunan International Scientific and Technological Cooperation Base of Arteriosclerotic Disease, Department of Bioinformatics and Medical Big Data, Hengyang Medical School, University of South China, Hengyang, Hunan, People's Republic of China.
J Inflamm Res. 2025 Jul 2;18:8719-8733. doi: 10.2147/JIR.S527437. eCollection 2025.
PURPOSE: The study was aimed at identifying that cytochrome b (CYTB) expression regulation by trimethylamine N-oxide (TMAO) can induce mitochondria reactive oxygen species (ROS) and promote vascular endothelial cells (VECs) pyroptosis. METHODS: VECs were transfected with TET methylcytosine dioxygenase 2 (TET2)/CYTB overexpression lentivirus, CYTB siRNA, TET2 shRNA, or NC. ROS levels were measured using MitoSOX Red fluorescence, and pyroptosis was evaluated via Hoechst 33342/PI staining. Western blot was used to measure TET2, the NOD-like receptor thermal protein domain associated protein 3 (NLRP3), proteolytic cleavage of gasdermin D (GSDMD), CYTB, and Caspase-1 expression. Interleukin (IL)-1β was quantified by ELISA. The mRNA expression of IL-1β, CYTB, ND2, and TET2 was measured by qRT-PCR. Cellular ultrastructure was examined by electron microscope, and calcium flux was monitored with Fluo-4AM. methylation was detected using Targeted Bisulfite Sequencing. RESULTS: This study showed that TMAO can down-regulate the expression of CYTB inVECs, cause VECs pyroptosis and mitochondrial dysfunction (MDF). CYTB overexpression antagonized the effect of TMAO. Further, silencing CYTB promoted mtROS production, and MitoTEMPO, a ROS scavenger, inhibited VECs pyroptosis caused by CYTB silencing. In addition, TET2 had demethylation activity. The expression of CYTB was positively regulated by TET2. TMAO was able to inhibit the expression of TET2 and promote the methylation level of the gene promoter. CONCLUSION: TMAO promotes the methylation level of the gene promoter and down-regulates the expression of CYTB by inhibiting the expression of TET2. The decreased expression level of CYTB induces ROS, promoting VECs pyroptosis.
目的：本研究旨在确定氧化三甲胺（TMAO）对细胞色素b（CYTB）表达的调控作用是否能诱导线粒体活性氧（ROS）生成并促进血管内皮细胞（VECs）焦亡。 方法：用TET甲基胞嘧啶双加氧酶2（TET2）/CYTB过表达慢病毒、CYTB小干扰RNA（siRNA）、TET2短发夹RNA（shRNA）或阴性对照（NC）转染VECs。使用MitoSOX Red荧光法检测ROS水平，通过Hoechst 33342/碘化丙啶（PI）染色评估焦亡情况。采用蛋白质免疫印迹法检测TET2、NOD样受体热蛋白结构域相关蛋白3（NLRP3）、gasdermin D（GSDMD）的蛋白水解切割、CYTB和半胱天冬酶-1（Caspase-1）的表达。通过酶联免疫吸附测定（ELISA）定量白细胞介素（IL）-1β。采用实时荧光定量聚合酶链反应（qRT-PCR）检测IL-1β、CYTB、ND2和TET2的mRNA表达。用电子显微镜检查细胞超微结构，并用Fluo-4AM监测钙流。使用靶向亚硫酸氢盐测序检测甲基化。 结果：本研究表明，TMAO可下调VECs中CYTB的表达，导致VECs焦亡和线粒体功能障碍（MDF）。CYTB过表达可拮抗TMAO的作用。此外，沉默CYTB可促进线粒体ROS生成，而ROS清除剂MitoTEMPO可抑制CYTB沉默引起的VECs焦亡。另外，TET2具有去甲基化活性。CYTB的表达受TET2的正向调控。TMAO能够抑制TET2的表达并提高该基因启动子的甲基化水平。 结论：TMAO通过抑制TET2的表达促进该基因启动子的甲基化水平并下调CYTB的表达。CYTB表达水平降低诱导ROS生成，促进VECs焦亡。
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