CARDIOMYOCYTE AND VASCULAR ENDOTHELIAL CELL SIGNALING BY CHOLESTEROL OZONATION

胆固醇臭氧化作用下的心肌​​细胞和血管内皮细胞信号转导

• 批准号: 7959463
• 负责人: RAO M UPPU
• 金额: $ 6.62万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959463
• 关键词: Acetylcysteine; Apoptosis; Apoptotic; Attenuated; Biochemical; Biological Assay; Biomedical Research; Cardiac Myocytes; Cell Line; Cells; Cessation of life; Cholesterol; Computer Retrieval of Information on Scientific Projects Database; Environment; Funding; Gene Expression; Generations; Glutathione; Grant; Institution; Ketones; Louisiana; Membrane Potentials; Mitochondria; Ozone; Pathway interactions; Proteins; Reaction; Reactive Oxygen Species; Reporting; Research; Research Personnel; Resources; Signal Transduction; Source; Time; Trolox; United States National Institutes of Health; Vascular Endothelial Cell; Western Blotting; alanylaspartic acid; apoptotic protease-activating factor 1; aqueous; base; caspase-3; caspase-8; caspase-9; cytochrome c; cytotoxicity; inhibitor/antagonist; overexpression; receptor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have previously reported that cholesterol secoaldehyde (ChSeco), a putative product of ozone reaction with cholesterol in aqueous environments, induces apoptosis in H9c2 cell line. The present study further investigated the involvement of apoptotic-related proteins and gene expression using quantitative real-time (RT) PCR and western blot analyses, and appropriate biochemical assays. The RT-PCR analysis revealed that ChSeco activates the expression of genes involved in the death receptor (extrinsic) pathway. The significance of this pathway was also evident based on increased activity of caspase-8. The overexpression of Apaf-1, loss of mitochondrial transmembrane potential and subsequent release of cytochrome c, and increased activity of caspase-9, further provide strong evidence for the involvement of mitochondrial (intrinsic) pathway. Thus, it appears that a combined activation of the intrinsic and extrinsic pathways resulted in the activation of effector caspase-3/7. Prior treatment of H9c2 cells with caspase-8- and -9-specific inhibitors decreased the activity of caspase-3 and the extent of cytotoxicity was significantly lowered by Z-Val-Ala-Asp-fluoromethyl ketone, a pancaspase inhibitor (PCI). Western blot analysis showed that, in H9c2 cells exposed to ChSeco, the expression of neither Bcl-2 or Bax was altered, although there was a decrease in the phosphorylated Bcl-2. A time-course analysis of the ChSeco-treated H9c2 cells showed an upstream rapid increase in the generation of reactive oxygen species (ROS) and the associated decrease in intracellular glutathione. N-Acetyl-L-cysteine and Trolox significantly attenuated the ChSeco-induced ROS formation and cytotoxicity and also down-regulated the expression of genes of all the players of either pathway. This study clearly demonstrated that ChSeco induces apoptosis in H9c2 cells through ROS generation and activation of both the intrinsic and extrinsic pathways.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们之前曾报道过胆固醇仲乙醛 (ChSeco) 是水环境中臭氧与胆固醇反应的推定产物，可诱导 H9c2 细胞系凋亡。本研究使用定量实时 (RT) PCR 和蛋白质印迹分析以及适当的生化测定进一步研究了凋亡相关蛋白和基因表达的参与。 RT-PCR 分析显示 ChSeco 激活死亡受体（外在）途径相关基因的表达。基于 caspase-8 活性的增加，该途径的重要性也很明显。 Apaf-1 的过度表达、线粒体跨膜电位的丧失和随后细胞色素 c 的释放以及 caspase-9 活性的增加，进一步为线粒体（内在）途径的参与提供了有力的证据。因此，内在和外在途径的联合激活似乎导致了效应器 caspase-3/7 的激活。先前用 caspase-8 和 -9 特异性抑制剂处理 H9c2 细胞会降低 caspase-3 的活性，并且 Z-Val-Ala-Asp-氟甲基酮（一种 pancaspase 抑制剂 (PCI)）显着降低细胞毒性程度。 Western blot 分析显示，在暴露于 ChSeco 的 H9c2 细胞中，尽管磷酸化的 Bcl-2 有所减少，但 Bcl-2 或 Bax 的表达均没有改变。对 ChSeco 处理的 H9c2 细胞的时程分析显示，上游活性氧 (ROS) 的产生迅速增加，并且细胞内谷胱甘肽相应减少。 N-乙酰-L-半胱氨酸和 Trolox 显着减弱 ChSeco 诱导的 ROS 形成和细胞毒性，并下调任一途径所有参与者的基因表达。这项研究清楚地表明，ChSeco 通过 ROS 的产生和内在和外在途径的激活来诱导 H9c2 细胞凋亡。