Division of Metabolic and Cardiovascular Sciences, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida.
Am J Physiol Heart Circ Physiol. 2019 Aug 1;317(2):H460-H471. doi: 10.1152/ajpheart.00056.2019. Epub 2019 Jun 7.
Doxorubicin (Dox)-induced cardiac side effects are regulated through increased oxidative stress and apoptosis. However, it remains unknown whether Dox induces the specific inflammatory-mediated form of cell death called pyroptosis. The current study is undertaken to determine whether Dox induces pyroptosis in an in vitro model and to test the potential of exosomes derived from embryonic stem cells (ES-Exos) in inhibiting pyroptosis. H9c2 cells were exposed to Dox to generate pyroptosis and then subsequently treated with exosomes to investigate the protective effects of ES-Exos. Mouse embryonic fibroblast-exosomes (MEF-Exos) were used as a cell line control. We confirmed pyroptosis by analyzing the presence of Toll-like receptor 4 (TLR4)-pyrin domain containing-3 (NLRP3) inflammasome that initiates pyroptosis, which was further confirmed with pyroptotic markers caspase-1, IL-1β, caspase-11, and gasdermin-D. The presence of inflammation was confirmed for proinflammatory cytokines, TNF-α, and IL-6. Our data show that Dox exposure significantly ( < 0.05) increases expression of TLR4, NLRP3, pyroptotic markers (caspase-1, IL-1β, caspase-11, and gasdermin-D), and proinflammatory cytokines (TNF-α and IL-6) in H9c2 cells. The increased expression of inflammasome, pyroptosis, and inflammation was significantly ( < 0.05) inhibited by ES-Exos. Interestingly, our cell line control, MEF-Exos, did not show any protective effects. Furthermore, our cytokine array data suggest increased anti-inflammatory (IL-4, IL-9, and IL-13) and decreased proinflammatory cytokines (Fas ligand, IL-12, and TNF-α) in ES-Exos, suggesting that anti-inflammatory cytokines might be mediating the protective effects of ES-Exos. In conclusion, our data show that Dox induces pyroptotic cell death in the H9c2 cell culture model and is attenuated via treatment with ES-Exos. Doxorubicin (Dox)-induced cardiotoxicity is mediated through increased oxidative stress, apoptosis, and necrosis. We report for the first time as per the best of our knowledge that Dox initiates Toll-like receptor 4 and pyrin domain containing-3 inflammasome formation and induces caspase-1-mediated inflammatory pyroptotic cell death in H9c2 cells. Moreover, we establish that inflammation and pyroptosis is inhibited by embryonic stem cell-derived exosomes that could be used as a future therapeutic option to treat Dox-induced cardiotoxicity.
多柔比星（Dox）引起的心脏副作用是通过增加氧化应激和细胞凋亡来调节的。然而，目前尚不清楚 Dox 是否会引起特定的炎症介导的细胞死亡形式，即细胞焦亡。本研究旨在确定 Dox 是否会在体外模型中诱导细胞焦亡，并测试胚胎干细胞（ES-Exos）衍生的外泌体是否能抑制细胞焦亡。将 H9c2 细胞暴露于 Dox 中以产生细胞焦亡，然后用外泌体处理以研究 ES-Exos 的保护作用。使用小鼠胚胎成纤维细胞衍生的外泌体（MEF-Exos）作为细胞系对照。我们通过分析起始细胞焦亡的 Toll 样受体 4（TLR4）-pyrin 结构域包含 3（NLRP3）炎性小体的存在来确认细胞焦亡，并用细胞焦亡标志物 caspase-1、IL-1β、caspase-11 和 gasdermin-D 进一步确认。炎症的存在通过促炎细胞因子 TNF-α 和 IL-6 得到证实。我们的数据表明，Dox 暴露显著（<0.05）增加了 H9c2 细胞中 TLR4、NLRP3、细胞焦亡标志物（caspase-1、IL-1β、caspase-11 和 gasdermin-D）和促炎细胞因子（TNF-α 和 IL-6）的表达。ES-Exos 显著（<0.05）抑制了炎性小体、细胞焦亡和炎症的表达增加。有趣的是，我们的细胞系对照 MEF-Exos 没有显示出任何保护作用。此外，我们的细胞因子阵列数据表明，ES-Exos 中抗炎细胞因子（IL-4、IL-9 和 IL-13）增加，促炎细胞因子（Fas 配体、IL-12 和 TNF-α）减少，表明抗炎细胞因子可能介导 ES-Exos 的保护作用。总之，我们的数据表明，Dox 在 H9c2 细胞培养模型中诱导细胞焦亡，并通过 ES-Exos 治疗减弱。多柔比星（Dox）诱导的心脏毒性是通过增加氧化应激、细胞凋亡和坏死介导的。我们首次报道，据我们所知，Dox 引发了 Toll 样受体 4 和 pyrin 结构域包含 3 炎性小体的形成，并在 H9c2 细胞中诱导了 caspase-1 介导的炎症性细胞焦亡。此外，我们还确定，炎症和细胞焦亡被胚胎干细胞衍生的外泌体抑制，这可能被用作治疗 Dox 诱导的心脏毒性的未来治疗选择。
