Abstract：
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.
摘要：
缺血预处理（IPC），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)损伤有明显的保护作用,尽管潜在的分子机制尚不清楚。该研究表明，通过Notch1/Hes1/VDAC1信号通路转导了针对缺氧/复氧（A/R）损伤的心脏保护。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)测定证实了Hes1和VDAC1蛋白之间的相互作用。用Hes1腺病毒N端TAP载体(AD-NTAP/Hes1)和Hes1短发夹RNA腺病毒载体(AD-Hes1-shRNA)转染H9c2细胞,建立A/R损伤,IPC,和IPost模型，分别。蛋白质印迹分析检测Hes1和VDAC1蛋白的表达,而活性氧（ROS）的水平，线粒体膜电位(ΔkW)，用流式细胞术评价细胞凋亡。AD-NTAP/Hes1可以激活Hes1的外源蛋白表达，从而降低肌酸磷酸激酶（CPK）和乳酸脱氢酶（LDH）的活性并促进细胞活力。研究发现，VDAC1是Hes1的潜在靶蛋白，过表达Hes1的蛋白表达下调了VDAC1的蛋白表达水平，减少了ROS的产生，稳定的ΔkW,抑制H9c2细胞凋亡。此外,下调Hes1蛋白表达也上调VDAC1蛋白表达，增加ROS产量，不平衡ΔkW,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蛋白的表达，从而减轻A/R损伤。
