[Effects of exosomes derived from normoxic or hypoxic human umbilical vein endothelial cells on hypoxia-induced cardiomyocyte injury and cardiac fibroblast activation in vitro].

摘要

OBJECTIVES: To investigate the effects of exosomes derived from human umbilical venous endothelial cells (HUVECs) cultured in normoxic and hypoxic conditions (N-exo and H-exo, respectively) on hypoxia-induced injury of human cardiomyocyte AC16 cells and activation of rat cardiac fibroblasts (RCFs) in vitro. METHODS: N-exo and H-exo were extracted from HUVECs by differential centrifugation and sucrose density gradient centrifugation and characterized. AC16 cells and RCFs in normoxic culture or hypoxic culture (1% O₂ for 24 h) were treated with N-exo or H-exo, and the changes in protein and mRNA levels were examined with Western blotting and RT-qPCR. Intracellular ROS levels and apoptosis in AC16 cells were assessed by DCFH-DA staining and TUNEL staining, and the changes in proliferation of RCFs were evaluated with EdU assay. RESULTS: N-exo and H-exo with densities of 1.5×10⁹ and 7.2×10⁸ mL-1 were obtained, respectively, which showed abundant protein components and expressed the exosomal marker HSP70. In AC16 cells, hypoxic exposure significantly upregulated CK-MB mRNA and downregulated Cx43 and GPX4 mRNA levels, causing also enhanced expressions of ALP, IL-1β and cTnT proteins and increased intracellular ROS content and cell apoptosis. These changes were effectively alleviated by treatment with 10⁷ mL-1 N-exo but worsened after treatment with H-exo. In RCFs, hypoxia significantly increased the expressions of collagen I, collagen III and α‑SMA and cell proliferation, which were suppressed by N-exo treatment but further enhanced by H-exo treatment. Hypoxia exposure increased HIF-1α and VEGF expression and phosphorylation level of AKT in both AC16 cells and RCFs; N-exo obviously inhibited while H-exo further enhanced these changes. Px-478, a specific HIF-1α inhibitor, significantly inhibited hypoxia-induced AC16 cell injury and RCF activation. CONCLUSIONS: N-exo derived from HUVECs inhibits while H-exo exacerbates hypoxia-induced AC16 cell injury and RCF activation possibly via the HIF-1α pathway. 目的: 探究人脐静脉内皮细胞（HUVECs）在常氧和缺氧环境下产生的外泌体（N-exo和H-exo）对缺氧诱导的人心肌细胞AC16细胞损伤和大鼠心脏成纤维细胞 （RCFs）活化的作用和机制。方法: 缺氧培养24 h诱导AC16细胞损伤和RCFs活化。差速离心法和蔗糖密度梯度离心法提取HUVECs来源的外泌体，利用透射电子显微镜、纳米颗粒跟踪分析技术、考马斯亮蓝染色和Western blotting检测外泌体标志蛋白对外泌体进行鉴定。分别设置对照组:AC16和RCFs正常培养;Hypoxia组:1% O2培养24 h;N-exo/H-exo组:N-exo/H-exo干预24 h;Hypoxia+N-exo/H-exo组:缺氧培养同时加入外泌体干预;Western blotting法检测蛋白表达水平;RT-qPCR检测mRNA表达水平;荧光探针DCFH-DA检测AC16细胞内活性氧水平;TUNEL法检测AC16细胞凋亡;EDU法检测RCFs的增殖能力。结果: N-exo和H-exo电镜下为典型的茶托型双层囊泡状;N-exo和H-exo的浓度分别为1.5×109 mL-1和7.2×108 mL-1;考马斯亮蓝染色显示HUVEC-exo具有丰富的蛋白谱;HUVECs-exo中可检测出外泌体标志性蛋白HSP70条带;与对照组相比，Hypoxia组AC16细胞中CK-MB的mRNA表达水平上升，缝隙连接蛋白43（Cx43）和谷胱甘肽过氧化酶4（GPX4）的mRNA表达水平下降，ALP、IL-1β和cTnT的蛋白表达水平上升，细胞中活性氧含量升高，细胞凋亡增加（P<0.05）;与Hypoxia组相比，107 mL-1 N-exo组CK-MB水平降低，Cx43和GPX4水平上升，ALP、IL-1β和cTnT水平下降，细胞中活性氧含量降低，细胞凋亡减少（P<0.05）;而H-exo的作用与N-exo相反（P<0.05）。与对照组相比，Hypoxia组RCFs细胞中I型胶原蛋白（Collagen I）、III型胶原蛋白（Collagen III）和α-平滑肌肌动蛋白（α-SMA）的表达水平上升，增殖能力提高（P<0.05）;与Hypoxia组相比，107 mL-1的N-exo组上述指标均降低（P<0.05）;而H-exo的作用与N-exo相反（P<0.05）。与对照组相比，Hypoxia组的AC16和RCFs中的缺氧诱导因子1亚基α（HIF-1α）和血管内皮生长因子（VEGF）水平上升，蛋白激酶B（AKT）磷酸化水平上升（P<0.05）;而与Hypoxia组相比，N-exo组的上述指标下降（P<0.05），H-exo组的上述指标上升（P<0.05）;HIF-1α抑制剂Px-478对缺氧诱导的AC16细胞损伤和RCFs细胞活化有抑制作用（P<0.05）。结论: 常氧环境下HUVECs来源的外泌对缺氧诱导的AC16细胞损伤和RCFs活化有抑制作用，缺氧环境下HUVECs来源的外泌体则加重缺氧诱导的AC16细胞损伤和RCFs活化，该作用机制可能涉及HIF-1α信号通路。.