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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 我们先前已经报道说，胆固醇塞多尔醛（Chseco）是在水性环境中与胆固醇反应的推定产物，可在H9C2细胞系中诱导凋亡。本研究进一步研究了使用定量实时（RT）PCR和Western Blot分析以及适当的生化分析的凋亡相关蛋白和基因表达的参与。 RT-PCR分析表明，Chseco激活了与死亡受体（外部）途径有关的基因的表达。基于caspase-8的活性增加，该途径的重要性也很明显。 APAF-1的过表达，线粒体跨膜电位的丧失以及随后释放细胞色素C以及CASPASE-9的活性增加，进一步为线粒体（内在）途径的参与提供了有力的证据。因此，似乎固有和外在途径的联合激活导致效应子caspase-3/7的激活。用caspase-8-和-9特异性抑制剂对H9C2细胞的先前处理可降低caspase-3的活性，而Z-VAL-Ala-Ala-Appas-asp-氟甲基酮（一种pancaspase抑制剂（PCI））显着降低了caspase-3的活性。 Western印迹分析表明，在暴露于Chseco的H9C2细胞中，尽管磷酸化的Bcl-2降低了Bcl-2或BAX的表达。对CHSECO处理的H9C2细胞进行的时间课程分析显示，活性氧（ROS）的产生迅速增加，而细胞内谷胱甘肽的相关降低。 N-乙酰基L-半胱氨酸和Trolox显着减弱了CHSECO诱导的ROS形成和细胞毒性，并且也下调了所有这两种途径的所有参与者的基因的表达。这项研究清楚地表明，CHSECO通过ROS的产生和固有和外在途径的激活诱导H9C2细胞的凋亡。