Department of Cardiac Surgery, the Affiliated Hospital of Guizhou Medical University, Guiyang, China.
Pharmacogenet Genomics. 2023 Aug 1;33(6):117-125. doi: 10.1097/FPC.0000000000000499. Epub 2023 Jun 8.
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.
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.
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.
BMSC-derived exosomes carrying ITCH suppressed cardiomyoblast apoptosis, promoted cardiomyoblast viability, and improved myocardial injury in AMI by mediating ASK1 ubiquitination.
骨髓间充质干细胞（BMSC）衍生的外泌体已被证实可在治疗急性心肌梗死（MI）中发挥有效作用。在此，我们旨在研究携带瘙痒 E3 泛素连接酶（ITCH）的 BMSC 衍生外泌体在 MI 中的作用及其涉及的潜在机制。
从大鼠骨髓中分离 BMSCs，使用超速离心法提取外泌体。通过 PKH-67 染色确定外泌体被心肌细胞摄取的情况。体外模型中，用缺氧刺激大鼠心肌细胞系 H9C2。用流式细胞术测定 H9C2 细胞凋亡情况。通过细胞计数试剂盒-8 测定细胞活力。用 Western blot 测定 ITCH、凋亡信号调节激酶 1（ASK1）和凋亡相关蛋白 cleaved-caspase 3 和 Bcl-2 的表达。采用泛素化测定法检测 ASK1 的泛素化水平。
BMSC 衍生的外泌体被 H9C2 心肌细胞内吞。BMSC-Exo 下调缺氧处理下 cleaved-caspase 3 的表达，上调 Bcl-2 的表达，进一步抑制 H9C2 细胞凋亡，同时下调 ASK1 的表达，在 BMSC 培养上清（BMSC-S）中也观察到类似的作用。然而，这些作用被外泌体抑制剂 GW4869 逆转。BMSC 来源的外泌体增强了 ASK1 的泛素化和降解。机制上，ITCH 敲低 BMSC 的外泌体促进 H9C2 细胞凋亡，上调 ASK1 的表达。过表达 ITCH 增强了 ASK1 的泛素化和降解。进一步，ASK1 和 cleaved-caspase 3 的蛋白表达上调，Bcl-2 蛋白表达下调。ITCH 敲低 BMSC 外泌体增加心肌细胞凋亡。
携带 ITCH 的 BMSC 衍生外泌体通过调节 ASK1 泛素化抑制心肌细胞凋亡，促进心肌细胞活力，改善 AMI 中的心肌损伤。
