Department of Internal Medicine I, Division of Cardiology, University Hospital Jena, 07747 Jena, Germany.
Department of Anesthesiology and Intensive Care Medicine, University Hospital Jena, 07745 Jena, Germany.
Int J Mol Sci. 2021 Nov 1;22(21):11852. doi: 10.3390/ijms222111852.
Doxorubicin (Dox) is a chemotherapeutic agent with cardiotoxicity associated with profibrotic effects. Dox increases ceramide levels with pro-inflammatory effects, cell death, and fibrosis. The purpose of our study was to identify the underlying ceramide signaling pathways. We aimed to characterize the downstream effects on cell survival, metabolism, and fibrosis. Human fibroblasts (hFSF) were treated with 0.7 µM of Dox or transgenically overexpressed ceramide synthase 2 (FLAG-CerS2). Furthermore, cells were pre-treated with MitoTempo (MT) (2 h, 20 µM) or Fumonisin B1 (FuB) (4 h, 100 µM). Protein expression was measured by Western blot or immunofluorescence (IF). Ceramide levels were determined with mass spectroscopy (MS). Visualizations were conducted using laser scanning microscopy (LSM) or electron microscopy. Mitochondrial activity was measured using seahorse analysis. Dox and CerS2 overexpression increased CerS2 protein expression. Coherently, ceramides were elevated with the highest peak for C24:0. Ceramide- induced mitochondrial ROS production was reduced with MT or FuB preincubation. Mitochondrial homeostasis was reduced and accompanied by reduced ATP production. Our data show that the increase in pro-inflammatory ceramides is an essential contributor to Dox side-effects. The accumulation of ceramides resulted in a lipotoxic shift and subsequently mitochondrial structural and functional damage, which was partially reversible following inhibition of ceramide synthesis.
阿霉素（Dox）是一种具有心脏毒性的化疗药物，与成纤维作用有关。Dox 增加神经酰胺水平，具有促炎作用、细胞死亡和纤维化。我们研究的目的是确定潜在的神经酰胺信号通路。我们旨在描述对细胞存活、代谢和纤维化的下游影响。用人成纤维细胞（hFSF）用 0.7µM 的 Dox 或过表达神经酰胺合酶 2（FLAG-CerS2）处理。此外，细胞用 MitoTempo（MT）（2 小时，20µM）或 Fumonisin B1（FuB）（4 小时，100µM）预处理。通过 Western blot 或免疫荧光（IF）测定蛋白表达。用质谱（MS）测定神经酰胺水平。使用激光扫描显微镜（LSM）或电子显微镜进行可视化。使用 Seahorse 分析测量线粒体活性。Dox 和 CerS2 过表达增加了 CerS2 蛋白表达。一致地，神经酰胺升高，C24:0 达到峰值。用 MT 或 FuB 预孵育可减少神经酰胺诱导的线粒体 ROS 产生。线粒体稳态降低，并伴有 ATP 生成减少。我们的数据表明，促炎神经酰胺的增加是 Dox 副作用的一个重要因素。神经酰胺的积累导致脂毒性转移，随后线粒体结构和功能受损，用神经酰胺合成抑制剂抑制后部分可逆。
