Department of Cardiac Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China.
Department of Cardiac Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China.
J Biochem Mol Toxicol. 2022 Nov;36(11):e23199. doi: 10.1002/jbt.23199. Epub 2022 Aug 17.
Ischemic preconditioning (IPC), and ischemic postconditioning (IPost) have a significant protective effect on myocardial ischemia/reperfusion (MI/R) injury by alleviating oxidative stress and mitochondrial disturbances, although the underlying molecular mechanisms are unclear. The study was to demonstrate that cardioprotection against anoxia/reoxygenation (A/R) injury is transduced via the Notch1/Hes1/VDAC1 signaling pathway. Using mass spectrometry and tandem affinity purification (TAP), to screen for differentially expressed proteins associated with Hes1, followed by standard bioinformatics analysis. The co-immunoprecipitation (Co-IP) assay confirmed an interaction between Hes1 and VDAC1 proteins. H9c2 cells were transfected with Hes1 adenoviral N-terminal TAP vector (AD-NTAP/Hes1) and Hes1-short hairpin RNA adenoviral vector (AD-Hes1-shRNA) to establish A/R injury, IPC, and IPost models, respectively. The expression of Hes1 and VDAC1 proteins were measured by western blot analysis, while the levels of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and apoptosis were evaluated by flow cytometry. AD-NTAP/Hes1 can activate the exogenous protein expression of Hes1, thus decreasing creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) activity and promoting cell viability. The study found that VDAC1 was a potential target protein for Hes1 and the overexpression of Hes1 protein expression downregulated protein expression levels of VDAC1, reduced ROS production, stabilized ΔΨm, and inhibited apoptosis in H9c2 cells. Additionally, downregulation of Hes1 protein expression also upregulated VDAC1 protein expression, increased ROS production, imbalanced ΔΨm, promoted cell apoptosis, and attenuated the cardioprotection afforded by IPC and IPost. The Notch1/Hes1 signaling pathway activated by IPC/IPost can directly downregulate the protein expression of VDAC1 and consequently relieve A/R injury.
缺血预处理（IPC）和缺血后处理（IPost）通过减轻氧化应激和线粒体紊乱对心肌缺血/再灌注（MI/R）损伤具有显著的保护作用，尽管其潜在的分子机制尚不清楚。本研究旨在证明缺氧/复氧（A/R）损伤的心脏保护作用是通过 Notch1/Hes1/VDAC1 信号通路转导的。使用质谱和串联亲和纯化（TAP）筛选与 Hes1 相关的差异表达蛋白，然后进行标准的生物信息学分析。共免疫沉淀（Co-IP）测定证实 Hes1 与 VDAC1 蛋白之间存在相互作用。用 Hes1 腺病毒 N 端 TAP 载体（AD-NTAP/Hes1）和 Hes1-shRNA 腺病毒载体（AD-Hes1-shRNA）转染 H9c2 细胞，分别建立 A/R 损伤、IPC 和 IPost 模型。通过 Western blot 分析测定 Hes1 和 VDAC1 蛋白的表达水平，通过流式细胞术评估活性氧（ROS）、线粒体膜电位（ΔΨm）和细胞凋亡水平。AD-NTAP/Hes1 可激活外源性 Hes1 蛋白表达，从而降低肌酸磷酸激酶（CPK）和乳酸脱氢酶（LDH）活性，促进细胞活力。研究发现，VDAC1 是 Hes1 的潜在靶蛋白，Hes1 蛋白表达上调可下调 VDAC1 蛋白表达水平，减少 ROS 生成，稳定 ΔΨm，抑制 H9c2 细胞凋亡。此外，下调 Hes1 蛋白表达也可上调 VDAC1 蛋白表达，增加 ROS 生成，破坏 ΔΨm 平衡，促进细胞凋亡，并减弱 IPC 和 IPost 提供的心脏保护作用。IPC/IPost 激活的 Notch1/Hes1 信号通路可直接下调 VDAC1 蛋白表达，从而减轻 A/R 损伤。
