{"title":"Bone marrow mesenchymal stem cell-derived exosomes carrying E3 ubiquitin ligase ITCH attenuated cardiomyocyte apoptosis by mediating apoptosis signal-regulated kinase-1.","authors":"Xuejun Li, Xuanyi Hu, Qiansu Chen, Tian Jiang","doi":"10.1097/FPC.0000000000000499","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have been verified to perform an effective role in treating acute myocardial infarction (MI). Herein, we aimed to investigate the role of BMSC-derived exosomes carrying itchy E3 ubiquitin ligase (ITCH) in MI and the underlying mechanism involved.</p><p><strong>Methods: </strong>BMSCs were isolated from rat bone marrow and exosomes were extracted using ultra-high speed centrifugation. Exosomes uptake by cardiomyoblasts was determined by PKH-67 staining. Rat cardiomyoblast cell line H9C2 was stimulated by hypoxia, as in vitro model. H9C2 cell apoptosis was determined by flow cytometry. Cell viability was examined by cell counting kit-8 assay. Western blotting was performed to determine the expression of ITCH, apoptosis signal-regulated kinase-1 (ASK1), and apoptotic-related protein cleaved-caspase 3 and Bcl-2. Ubiquitination assay was employed to measure the levels of ASK1 ubiquitination.</p><p><strong>Results: </strong>Exosomes derived from BMSCs were endocytosed by H9C2 cardiomyoblasts. BMSC-Exo downregulated cleaved-caspase 3 expression, upregulated Bcl-2 expression, further suppressed H9C2 cell apoptosis under hypoxia treatment, meanwhile the expression of ASK1 was downregulated, and similar effects were observed in BMSC-cultured supernatant (BMSC-S). However, these effects were reversed by exosome inhibitor GW4869. BMSC-derived exosomes enhanced ASK1 ubiquitination and degradation. Mechanically, exosomes of ITCH-knockdown BMSCs promoted H9C2 cell apoptosis and upregulated ASK1 expression. Overexpression of ITCH enhanced ASK1 ubiquitination and degradation. Further, the protein expression of ASK1 and cleaved-caspase 3 was upregulated and Bcl-2 protein expression was downregulated. ITCH-knockdown BMSC exosomes increased cardiomyoblast apoptosis.</p><p><strong>Conclusion: </strong>BMSC-derived exosomes carrying ITCH suppressed cardiomyoblast apoptosis, promoted cardiomyoblast viability, and improved myocardial injury in AMI by mediating ASK1 ubiquitination.</p>","PeriodicalId":19763,"journal":{"name":"Pharmacogenetics and genomics","volume":"33 6","pages":"117-125"},"PeriodicalIF":1.7000,"publicationDate":"2023-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Pharmacogenetics and genomics","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1097/FPC.0000000000000499","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Background: Bone marrow mesenchymal stem cell (BMSC)-derived exosomes have been verified to perform an effective role in treating acute myocardial infarction (MI). Herein, we aimed to investigate the role of BMSC-derived exosomes carrying itchy E3 ubiquitin ligase (ITCH) in MI and the underlying mechanism involved.
Methods: BMSCs were isolated from rat bone marrow and exosomes were extracted using ultra-high speed centrifugation. Exosomes uptake by cardiomyoblasts was determined by PKH-67 staining. Rat cardiomyoblast cell line H9C2 was stimulated by hypoxia, as in vitro model. H9C2 cell apoptosis was determined by flow cytometry. Cell viability was examined by cell counting kit-8 assay. Western blotting was performed to determine the expression of ITCH, apoptosis signal-regulated kinase-1 (ASK1), and apoptotic-related protein cleaved-caspase 3 and Bcl-2. Ubiquitination assay was employed to measure the levels of ASK1 ubiquitination.
Results: Exosomes derived from BMSCs were endocytosed by H9C2 cardiomyoblasts. BMSC-Exo downregulated cleaved-caspase 3 expression, upregulated Bcl-2 expression, further suppressed H9C2 cell apoptosis under hypoxia treatment, meanwhile the expression of ASK1 was downregulated, and similar effects were observed in BMSC-cultured supernatant (BMSC-S). However, these effects were reversed by exosome inhibitor GW4869. BMSC-derived exosomes enhanced ASK1 ubiquitination and degradation. Mechanically, exosomes of ITCH-knockdown BMSCs promoted H9C2 cell apoptosis and upregulated ASK1 expression. Overexpression of ITCH enhanced ASK1 ubiquitination and degradation. Further, the protein expression of ASK1 and cleaved-caspase 3 was upregulated and Bcl-2 protein expression was downregulated. ITCH-knockdown BMSC exosomes increased cardiomyoblast apoptosis.
Conclusion: BMSC-derived exosomes carrying ITCH suppressed cardiomyoblast apoptosis, promoted cardiomyoblast viability, and improved myocardial injury in AMI by mediating ASK1 ubiquitination.
期刊介绍：
Pharmacogenetics and Genomics is devoted to the rapid publication of research papers, brief review articles and short communications on genetic determinants in response to drugs and other chemicals in humans and animals. The Journal brings together papers from the entire spectrum of biomedical research and science, including biochemistry, bioinformatics, clinical pharmacology, clinical pharmacy, epidemiology, genetics, genomics, molecular biology, pharmacology, pharmaceutical sciences, and toxicology. Under a single cover, the Journal provides a forum for all aspects of the genetics and genomics of host response to exogenous chemicals: from the gene to the clinic.
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