Department of Anesthesiology, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, China.
Oxid Med Cell Longev. 2018 Jun 26;2018:3091424. doi: 10.1155/2018/3091424. eCollection 2018.
The aim of this study was to identify the role of the precursor of the brain-derived neurotrophic factor (pro-BDNF) in myocardial hypoxia/reoxygenation injury (H/R) and to address the underlying mechanisms. For this purpose, myocardial microvascular endothelial cells (MMECs) exposed to a high concentration of glucose (30 mM) for 48 h were subjected to 4 h of hypoxia followed by 2 h of reoxygenation. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining and flow-cytometric analysis were performed to detect apoptosis. Cell scratch and capillary-like-structure formation assays were employed to evaluate cell function. The levels of apoptosis-related proteins were evaluated by Western blotting and immunofluorescence assays. Our results showed that H/R resulted in MMEC injury, as indicated by significant increases in TUNEL-positive cell numbers and a reduction in MMEC migration and in capillary-like-structure formation coupled with increased pro-BDNF protein expression. In addition, overexpression of pro-BDNF in MMECs via a viral vector led to increased pro-BDNF expression, and this upregulation induced apoptosis. Mechanistic experiments revealed that H/R did not influence BDNF, JNK, and caspase 3 expression, but upregulated pro-BDNF, p75, sortilin, phospho-JNK, and cleaved caspase 3 protein levels. In contrast, neutralization of endogenous pro-BDNF with an antibody significantly attenuated H/R-induced upregulation of pro-BDNF, p75, sortilin, p-JNK, and cleaved caspase 3 protein levels, indicating that p75-sortilin signaling and activation of JNK and caspase 3 may be involved in these effects. In conclusion, H/R-induced injury may be mediated by pro-BDNF, at least in part through the regulation of p75-sortilin signaling and activation of JNK and caspase 3.
本研究旨在探讨脑源性神经营养因子（BDNF）前体（pro-BDNF）在心肌缺氧/复氧（H/R）损伤中的作用，并探讨其潜在机制。为此，将暴露于高浓度葡萄糖（30mM）中的心肌微血管内皮细胞（MMECs）孵育 48 小时，随后进行 4 小时缺氧和 2 小时复氧。采用末端脱氧核苷酸转移酶（TdT）dUTP 缺口末端标记（TUNEL）染色和流式细胞术分析检测细胞凋亡。采用细胞划痕和毛细血管样结构形成实验评估细胞功能。通过 Western blot 和免疫荧光实验检测凋亡相关蛋白水平。结果显示，H/R 导致 MMEC 损伤，表现为 TUNEL 阳性细胞数明显增加，MMEC 迁移和毛细血管样结构形成减少，同时 pro-BDNF 蛋白表达增加。此外，通过病毒载体过表达 MMECs 中的 pro-BDNF 导致 pro-BDNF 表达增加，而上调 pro-BDNF 诱导细胞凋亡。机制实验表明，H/R 不影响 BDNF、JNK 和 caspase 3 的表达，但上调 pro-BDNF、p75、sortilin、磷酸化 JNK 和 cleaved caspase 3 蛋白水平。相反，用抗体中和内源性 pro-BDNF 可显著抑制 H/R 诱导的 pro-BDNF、p75、sortilin、p-JNK 和 cleaved caspase 3 蛋白水平上调，表明 p75-sortilin 信号通路和 JNK 和 caspase 3 的激活可能参与这些效应。综上所述，H/R 诱导的损伤可能至少部分通过调节 p75-sortilin 信号通路和激活 JNK 和 caspase 3 来介导。
