Department of Cardiovascular Medicine, Chongqing University Center Hospital (Chongqing Emergence Medical Center), No. 1, Jiankang Road, Yuzhong District, Chongqing, 400014, China.
Department of Nursing, Chongqing Gaoxin District People's Hospital, Chongqing, 400039, China.
Biomed Eng Online. 2021 Aug 31;20(1):88. doi: 10.1186/s12938-021-00923-2.
Myocarditis, an inflammatory disease of the myocardium, is a serious hazard to human life due to the expansion of inflammatory lesions in the myocardium. The aim of this study was to investigate the role of hypoxia-inducible transcription factor (HIF)-1α and its inhibitor topotecan in the pathogenesis of myocarditis.
H9c2 cardiomyoblasts was stimulated with lipopolysaccharide (LPS) to simulate myocarditis model in vitro. The levels of myocardial damage markers were determined using commercially available kits. Western blotting was used to evaluate HIF-1α expression after LPS challenge. Then, after HIF-1α silencing, the contents of inflammatory factors were determined with enzyme-linked immunosorbent assay (ELISA). Cell viability was tested by means of a cell counting kit-8 (CCK-8) assay. Cell apoptosis was assessed by flow cytometry, and the expression of apoptotic proteins was examined using western blot analysis. Subsequently, HIF-1α was overexpressed and topotecan was employed to treat H9c2 cells under LPS exposure condition. The biological functions were detected again.
LPS significantly elevated the levels of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) and cardiac troponin-I (cTn-I) in supernatant of H9c2 cell lysates. Additionally, LPS led to the notably upregulated expression of HIF-1α. HIF-1α-knockdown markedly decreased the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 compared with the LPS-induced group. Moreover, the cell viability was conspicuously enhanced and cell apoptotic ratio was remarkably reduced, accompanied by downregulated expression of Bax, Bim, caspase 3 and caspase 9 after HIF-1α silencing. Consistently, HIF-1α gain-of-function significantly promoted the production of inflammatory cytokines and cell apoptosis, which was partially counteracted by topotecan administration.
To conclude, these findings demonstrated that HIF-1α inhibition by topotecan ameliorates LPS-induced myocarditis in vitro, providing a new approach in the treatment of myocarditis.
心肌炎是一种心肌炎症性疾病，由于心肌炎症病变的扩大，对人类生命构成严重威胁。本研究旨在探讨缺氧诱导因子-1α（HIF-1α）及其抑制剂拓扑替康在心肌炎发病机制中的作用。
用脂多糖（LPS）刺激 H9c2 心肌细胞，在体外模拟心肌炎模型。使用商业试剂盒测定心肌损伤标志物水平。用 Western blot 检测 LPS 刺激后 HIF-1α的表达。然后，沉默 HIF-1α后，用酶联免疫吸附试验（ELISA）测定炎症因子的含量。用细胞计数试剂盒-8（CCK-8）测定细胞活力。用流式细胞术评估细胞凋亡，用 Western blot 分析检测凋亡蛋白的表达。随后，在 LPS 暴露条件下，过表达 HIF-1α并使用拓扑替康处理 H9c2 细胞，再次检测其生物学功能。
LPS 显著增加了 H9c2 细胞裂解物上清液中乳酸脱氢酶（LDH）、肌酸激酶-MB（CK-MB）和心肌肌钙蛋白-I（cTn-I）的水平。此外，LPS 导致 HIF-1α的表达明显上调。与 LPS 诱导组相比，HIF-1α 敲低组肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6 和 IL-8 的浓度明显降低。此外，HIF-1α 沉默后，细胞活力明显增强，细胞凋亡率明显降低，Bax、Bim、caspase 3 和 caspase 9 的表达下调。同样，HIF-1α 功能获得显著促进了炎症细胞因子和细胞凋亡的产生，而拓扑替康的给药部分逆转了这一作用。
综上所述，这些发现表明，拓扑替康抑制 HIF-1α可改善 LPS 诱导的体外心肌炎，为心肌炎的治疗提供了一种新方法。
