To investigate the mechanism by which hypoxia-reoxygenation (HR) mediates macrophage polarization to the M1 phenotype and then mediates cardiomyocyte (CM) pyroptosis through exosome release. Mouse bone marrow macrophages and CMs were cultured in vitro under hypoxia for 12 h and reoxygenation for 6 h to establish an HR cell model. qPCR was used to detect the M1 or M2 macrophage markers IL-1β, TNF-α, MR, and Arg, and a macrophage and CM coculture system was then established. Macrophages were transfected with an exosome-CD63-red fluorescent protein (RFP) lentivirus, allowing secretion of exosomes expressing RFP, and GW4869 was used to inhibit exosome release by macrophages. qPCR detected miR-29 expression in macrophage-derived exosomes, and macrophages were transfected with miR-29a inhibitors to obtain exosomes with low miR-29a expression (siR-exos). Pyroptosis indicators were detected by Western blot and ELISA. Importantly, LPS induced bone marrow macrophage polarization to the M1 type as a positive control to further verify that these exosomes (LPS-exos) regulated CM pyroptosis by delivering miR29a. Dual luciferase reporter and Western blot assays were adopted to analyze the miR-29a and MCL-1 target relationship. In addition, MCL-1 overexpression was used as a rescue experiment to determine whether miR-29a regulates pyroptosis in CM by targeting MCL-1. Macrophages expressed the M1 macrophage markers IL-1β and TNF-α after HR exposure. After CM coculture, RFP expression was significantly higher in the HR group than in the normal (Nor) group but significantly reduced in the GW4869 group. Immunofluorescence showed that caspase-1 mRNA and protein expression in the HR group was significantly higher than that in the Nor group (P < 0.05). Caspase-1 expression was significantly decreased in the GW4869 group compared with the HR group (P < 0.05). Western blotting showed that the pyrolysis-related NLRP3 and ASC protein expression levels were significantly upregulated in the HR group compared with the control (Ctr) and Nor groups (P < 0.05). However, GW4869 effectively inhibited pyroptosis-related protein expression (P < 0.05). In addition, ELISA showed that the expression of the inflammation indicators IL-1β and IL-18 was significantly increased in the HR group compared to the Ctr group (P < 0.05) but decreased in the GW4869 group (P < 0.05). qPCR showed that miR-29a was upregulated in the HR group compared to the Nor group. Moreover, HR-induced exosomes (HR-exos) from macrophages exacerbated HR-induced CM pyroptosis, while inhibition of miR-29a in exosomes partially offset CM pyroptosis induction. LPS-exos promoted pyroptosis-related protein expression, as the IL-1β and IL-18 concentrations were increased in the LPS-exos group. However, pyroptosis-related proteins were observably decreased, and IL-1β and IL-18 were also significantly decreased after miR-29a inhibition when compared with that in the HR-exos and LPS-exos groups. Mcl-1 overexpression reversed miR-29a-mediated CM pyroptosis in an HR environment. HR treatment induced macrophage polarization towards the M1 phenotype, which mediated CM pyroptosis through exosomal miR-29a transfer by targeting MCL-1.

研究缺氧-复氧 (HR) 介导巨噬细胞极化为 M1 表型，然后通过外泌体释放介导心肌细胞 (CM) 焦亡的机制。小鼠骨髓巨噬细胞和CMs在体外缺氧12 h、复氧6 h建立HR细胞模型。采用qPCR检测M1或M2巨噬细胞标志物IL-1β、TNF-α、MR、Arg，建立巨噬细胞与CM共培养体系。巨噬细胞用外泌体-CD63-红色荧光蛋白（RFP）慢病毒转染，允许分泌表达RFP的外泌体，GW4869用于抑制巨噬细胞释放外泌体。qPCR检测巨噬细胞来源的外泌体中miR-29的表达，将巨噬细胞转染miR-29a抑制剂，获得miR-29a低表达的外泌体（siR-exos）。通过Western印迹和ELISA检测细胞焦亡指标。重要的是，LPS 诱导骨髓巨噬细胞极化为 M1 型作为阳性对照，以进一步验证这些外泌体（LPS-exos）通过传递 miR29a 调节 CM 细胞焦亡。采用双荧光素酶报告基因和蛋白质印迹分析来分析 miR-29a 和 MCL-1 靶标关系。此外，MCL-1 过表达被用作拯救实验，以确定 miR-29a 是否通过靶向 MCL-1 调节 CM 中的细胞焦亡。巨噬细胞在暴露于 HR 后表达 M1 巨噬细胞标志物 IL-1β 和 TNF-α。CM共培养后，HR组的RFP表达显着高于正常（Nor）组，但GW4869组的RFP表达显着降低。P  < 0.05)。与HR组相比，GW4869组Caspase-1表达显着降低（P  < 0.05）。蛋白质印迹显示，与对照（Ctr）和Nor组相比，HR组中热解相关的NLRP3和ASC蛋白表达水平显着上调（P  <0.05）。然而，GW4869 有效抑制焦亡相关蛋白的表达（P  < 0.05）。此外，ELISA 显示，与 Ctr 组相比，HR 组炎症指标 IL-1β 和 IL-18 的表达显着升高（P  < 0.05），而在 GW4869 组中则降低（P < 0.05)。qPCR 显示，与 Nor 组相比，HR 组中的 miR-29a 上调。此外，来自巨噬细胞的 HR 诱导的外泌体（HR-exos）加剧了 HR 诱导的 CM 焦亡，而外泌体中 miR-29a 的抑制部分抵消了 CM 焦亡的诱导。LPS-exos 促进了细胞焦亡相关蛋白的表达，因为 LPS-exos 组中 IL-1β 和 IL-18 浓度增加。然而，与 HR-exos 和 LPS-exos 组相比，在 miR-29a 抑制后，焦亡相关蛋白显着降低，IL-1β 和 IL-18 也显着降低。Mcl-1 过表达逆转了 HR 环境中 miR-29a 介导的 CM 焦亡。HR 治疗诱导巨噬细胞向 M1 表型极化，M1 表型通过靶向 MCL-1 的外泌体 miR-29a 转移介导 CM 焦亡。