PDF(Pubmed)
Abstract：
The present study was designed to explore the role of M2 macrophage‑derived exosomes (M2‑exos) on the KCa3.1 channel in a cellular atrial fibrillation (AF) model using rapidly paced HL‑1 myocytes. M2 macrophages and M2‑exos were isolated and identified. MicroRNA (miR)‑146a‑5p levels in M2 macrophages and M2‑exos were quantified using reverse transcription‑quantitative PCR (RT‑qPCR). HL‑1 myocytes were randomly divided into six groups: Control group, pacing group, pacing + coculture group (pacing HL‑1 cells cocultured with M2‑exos), pacing + mimic‑miR‑146a‑5p group, pacing + NC‑miR‑146a‑5p group and pacing + pyrrolidine dithiocarbamate (PDTC; a special blocker of the NF‑κB signaling pathway) group. Transmission electron microscopy, nanoparticle tracking analysis, western blotting, RT‑qPCR and immunohistochemistry were performed in the present study. A whole‑cell clamp was also applied to record the current density of KCa3.1 and action potential duration (APD) in each group. The results revealed that miR‑146a‑5p was highly expressed in both M2 macrophages and M2‑exos. Pacing HL‑1 cells led to a shorter APD, an increased KCa3.1 current density and higher protein levels of KCa3.1, phosphorylated (p‑)NF‑κB p65, p‑STAT3 and IL‑1β compared with the control group. M2‑exos, miR‑146a‑5p‑mimic and PDTC both reduced the protein expression of KCa3.1, p‑NF‑κB p65, p‑STAT3 and IL‑1β and the current density of KCa3.1, resulting in a longer APD in the pacing HL‑1 cells. In conclusion, M2‑exos and their cargo, which comprised miR‑146a‑5p, decreased KCa3.1 expression and IL‑1β secretion in pacing HL‑1 cells via the NF‑κB/STAT3 signaling pathway, limiting the shorter APD caused by rapid pacing.
摘要：
本研究旨在探索M2巨噬细胞衍生的外泌体（M2-exos）在使用快速起搏的HL-1心肌细胞的细胞心房颤动（AF）模型中对KCa3.1通道的作用。分离并鉴定了M2巨噬细胞和M2-exos。使用逆转录定量PCR（RT-qPCR）对M2巨噬细胞和M2-exos中的MicroRNA（miR）-146a-5p水平进行定量。HL-1心肌细胞随机分为6组：对照组，起搏组,起搏+共培养组（起搏HL‑1细胞与M2‑exos共培养），起搏+模拟-miR‑146a‑5p组，起搏+NC‑miR‑146a‑5p组和起搏+吡咯烷二硫代氨基甲酸酯（PDTC；NF‑κB信号通路的特殊阻断剂）组。透射电子显微镜，纳米粒子跟踪分析，西方印迹,在本研究中进行了RT-qPCR和免疫组织化学。还应用全细胞钳记录每组中KCa3.1的电流密度和动作电位持续时间（APD）。结果显示，miR‑146a‑5p在M2巨噬细胞和M2‑exos中均高表达。起搏HL‑1细胞导致APD变短，与对照组相比，KCa3.1电流密度增加，KCa3.1蛋白水平升高，磷酸化（p-）NF-κBp65，p-STAT3和IL-1β。M2-exos，miR‑146a‑5p‑mimic和PDTC均降低了KCa3.1，p‑NF‑κBp65，p‑STAT3和IL‑1β的蛋白表达以及KCa3.1的电流密度，导致起搏HL‑1细胞的APD更长。总之,M2-exos及其货物，其中包括miR-146a-5p，通过NF‑κB/STAT3信号通路降低起搏HL‑1细胞中KCa3.1的表达和IL‑1β的分泌，限制快速起搏引起的较短APD。
