缺氧/复氧诱导心肌细胞分泌高表达miR-208b的外泌体而调控心肌成纤维细胞活化、迁移和铁死亡

目的　探讨缺氧/复氧诱导心肌细胞所分泌的外泌体能否通过miR-208b调控心肌成纤维细胞的生物学功能。方法　心肌细胞进行缺氧/复氧处理后，收集所分泌外泌体与心肌成纤维细胞进行共培养，然后用荧光定量PCR、Western blot或酶联免疫吸附试验（ELISA）检测miR-208b、α-平滑肌肌动蛋白（α-SMA）、Collagen Ⅰ、Collagen Ⅲ和谷胱甘肽过氧化物酶4（GPX4）的表达，细胞计数试剂盒-8（CCK-8）检测细胞存活力，Transwell检测细胞迁移，商用试剂盒检测活性氧（ROS）、丙二醛（MDA）和Fe²⁺的累积。采用单因素方差分析（ANOVA）。结果　缺氧/复氧诱导心肌细胞和其分泌的外泌体高表达miR-208b，将此类外泌体加入到心肌成纤维细胞进行共培养时发现，心肌成纤维细胞可以摄入外泌体，从而上调自身miR-208b的表达，进而促进心肌成纤维细胞的存活力和迁移，增强α-SMA、Collagen Ⅰ和Collagen Ⅲ的表达，不过miR-208b的抑制物能显著减弱上述外泌体对心肌成纤维细胞生物学功能的调控作用。同时，缺氧/复氧心肌细胞源性外泌体能进一步增强铁死亡主要指标ROS、MDA和Fe²⁺的累积，抑制铁死亡关键调控因子GPX4的表达，不过miR-208b的抑制物能明显减弱Erastin和缺氧/复氧心肌细胞源性外泌体对铁死亡的影响作用。结论　缺氧/复氧心肌细胞源性外泌体能通过高表达的miR-208b而调控心肌成纤维细胞的生物学功能，表明miR-208b是介导心肌细胞和心肌成纤维细胞间通讯的关键分子。

Hypoxia/reoxygenation inducing cardiomyocytes to secrete exosomes with high expression of miR-208b to regulate activation,migration, and ferroptosis of cardiac fibroblasts

Objective　To explore whether exosomes secreted by hypoxia/reoxygenation-induced cardiomyocytes can regulate the biological function of cardiac fibroblasts through miR-208b. Methods　After cardiomyocytes were treated with hypoxia/reoxygenation, the secreted exosomes were collected and co-cultured with cardiac fibroblasts, and then the expressions of miR-208b, alpha-smooth muscle actin (α-SMA), Collagen Ⅰ, Collagen Ⅲ, and glutathione peroxidase 4 (GPX4) were detected by fluorescence quantitative PCR, Western blot, or enzyme linked immunosorbent assay (ELISA), the cell viability was detected by Cell Counting Kit -8 (CCK-8), the cell migration was detected by Transwell assay, and the accumulation of reactive oxygen species (ROS), malonaldehyde (MDA), and Fe²⁺ was detected by commercial kits. One-way analysis of variance (ANOVA) was used. Results　Hypoxia/reoxygenation induced high expression of miR-208b in cardiomyocytes and their secreted exosomes. When these exosomes were added to cardiac fibroblasts for co-culture, it was found that cardiac fibroblasts could take up exosomes, thereby upregulating the expression of miR-208b, promoting the viability and migration of cardiac fibroblasts, and enhancing the expressions of α-SMA, Collagen Ⅰ, and Collagen Ⅲ, but miR-208b inhibitors significantly reduced the effects of these exosomes on cardiac fibroblasts. At the same time, hypoxic/reoxygenated cardiomyocyte-derived exosomes could further enhance the accumulation of ROS, MDA, and Fe²⁺, the main indicators of ferroptosis, and inhibit the expression of GPX4, a key regulator of ferroptosis. However, miR-208b inhibitors significantly attenuated the effects of Erastin and hypoxic/reoxygenated cardiomyocyte-derived exosomes on ferroptosis. Conclusion　Hypoxic/reoxygenated cardiomyocyte-derived exosomes can regulate the biological function of cardiac fibroblasts through highly expressed miR-208b, indicating that miR-208b is a key molecule mediating the communication between cardiomyocytes and cardiac fibroblasts.