Department of Cardiology, The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning, China.
Central Laboratory of The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning, China.
Life Sci. 2020 Mar 15;245:117347. doi: 10.1016/j.lfs.2020.117347. Epub 2020 Jan 22.
Oxidative stress plays an important role in myocardial ischemia-reperfusion injury. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) was first identified in apoptosis induced by T cell receptor activation, and was shown to play a different role in different cell types and under different stimuli. The role and mechanism of PHLDA1 in oxidative stress-induced cardiomyocyte injury and cardiac ischemia-reperfusion were therefore determined.
Cell viability and apoptotic rate were measured by Cell Counting Kit-8 and flow cytometry, respectively. Mitochondrial membrane potential was measured using JC-1 test kit. Reactive oxygen species (ROS) production was detected using ROS kit. HE staining was used to detect histological morphology, 2,3,5-triphenyltetrazolium chloride staining to detect infarct size, terminal deoxynucleotidyl transferase dUTP nick end labeling staining to detect the apoptotic rate, and immunohistochemistry and western blot analysis to detect protein expression. The binding of PHLDA1 to Bcl-2 associated X (Bax) was detected by immunoprecipitation.
The results indicated that PHLDA1 is highly expressed in oxidative stress-induced cardiomyocyte and myocardial ischemia-reperfusion injuries. PHLDA1 overexpression in cardiomyocytes promoted oxidative stress-induced cardiomyocyte injury. At the same time, PHLDA1 knockdown improved oxidative stress-induced cardiomyocyte and myocardial ischemia-reperfusion injuries. In addition, PHLDA1 binds to Bax and the interaction is enhanced under HO stimulation.
The present results indicated that PHLDA1 interacts with Bax to participate in oxidative stress-induced cardiomyocyte injury and myocardial ischemia reperfusion injury.
氧化应激在心肌缺血再灌注损伤中起重要作用。PHLDA1 最初是在 T 细胞受体激活诱导的细胞凋亡中发现的，并且在不同的细胞类型和不同的刺激下发挥不同的作用。因此，确定了 PHLDA1 在氧化应激诱导的心肌细胞损伤和心肌缺血再灌注中的作用和机制。
通过细胞计数试剂盒-8 和流式细胞术分别测量细胞活力和细胞凋亡率。使用 JC-1 试剂盒测量线粒体膜电位。使用 ROS 试剂盒检测活性氧（ROS）的产生。通过 HE 染色检测组织形态学，通过 2,3,5-三苯基氯化四氮唑染色检测梗死面积，通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记染色检测细胞凋亡率，通过免疫组化和 Western blot 分析检测蛋白表达。通过免疫沉淀检测 PHLDA1 与 Bcl-2 相关 X（Bax）的结合。
结果表明，PHLDA1 在氧化应激诱导的心肌细胞和心肌缺血再灌注损伤中高表达。心肌细胞中 PHLDA1 的过表达促进了氧化应激诱导的心肌细胞损伤。同时，PHLDA1 的敲低改善了氧化应激诱导的心肌细胞和心肌缺血再灌注损伤。此外，PHLDA1 与 Bax 结合，并且在 HO 刺激下相互作用增强。
本研究结果表明，PHLDA1 与 Bax 相互作用，参与氧化应激诱导的心肌细胞损伤和心肌缺血再灌注损伤。
