Department of Emergency, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
Int J Biol Macromol. 2018 Oct 15;118(Pt A):1142-1148. doi: 10.1016/j.ijbiomac.2018.07.001. Epub 2018 Jul 3.
Oxidative stress plays an important role in myocardial ischemia-reperfusion (I/R) injury. And pNaKtide is known to inhibit Na/K-ATPase/Src/reactive oxygen species (ROS) amplification signaling. Accordingly, we aimed to investigate the effect of pNaKtide on myocardial I/R injury.
We first determine the effect of pNaKtide on hypoxia- or cobalt chloride-induced injury in embryonic heart-derived H9c2 cells via measuring lactate dehydrogenase (LDH) level and trypan blue stain assay. In addition, TUNEL stain assay and western blot analysis of cleaved-PARP and cleaved-caspase3 were performed to detect apoptosis level. Meanwhile, ROS accumulation was assessed by dichlorofluorescin diacetate (DCFH-DA) assay. Then we conducted cell counting kit-8 (CCK-8) and flow cytometry to examine cell proliferation and cell cycle respectively. We next generated rat I/R model and determined the effect of pNaKtide by measuring serum LDH and evaluating heart pathology. At last, the activities of Src and ERK1/2 were examined via western blot to clarify molecular mechanism.
In vitro, pNaKtide exposure significantly attenuated the H9c2 cells death and ROS accumulation induced by hypoxia or cobalt chloride. And no significant effect was detected on cell cycle and proliferation upon pNaKtide administration. In vivo, pNaKtide distinctly decreased serum LDH level and ameliorated I/R induced myocardial injury in the rats. Western blot analysis revealed pNaKtide decreased Src and ERK1/2 activities robustly.
The results provided evidence that pNaKtide exhibited cardioprotective effect against hypoxia-induced injury in vitro and in vivo. And pNaKtide might be a potential molecular for therapy of I/R related heart disease.
氧化应激在心肌缺血再灌注（I/R）损伤中起重要作用。已知 pNaKtide 可抑制 Na/K-ATP 酶/Src/活性氧（ROS）放大信号。因此，我们旨在研究 pNaKtide 对心肌 I/R 损伤的影响。
我们首先通过测量乳酸脱氢酶（LDH）水平和台盼蓝染色试验来确定 pNaKtide 对缺氧或氯化钴诱导的胚胎心脏衍生 H9c2 细胞损伤的影响。此外，通过 TUNEL 染色试验和 cleaved-PARP 和 cleaved-caspase3 的 Western blot 分析检测凋亡水平。同时，通过二氯荧光素二乙酸酯（DCFH-DA）试验评估 ROS 积累。然后，我们通过细胞计数试剂盒-8（CCK-8）和流式细胞术分别检测细胞增殖和细胞周期。接下来，我们生成大鼠 I/R 模型，并通过测量血清 LDH 和评估心脏病理来确定 pNaKtide 的作用。最后，通过 Western blot 检测 Src 和 ERK1/2 的活性来阐明分子机制。
在体外，pNaKtide 暴露可显著减轻缺氧或氯化钴诱导的 H9c2 细胞死亡和 ROS 积累。给予 pNaKtide 后，对细胞周期和增殖没有明显影响。在体内，pNaKtide 明显降低血清 LDH 水平并改善大鼠 I/R 诱导的心肌损伤。Western blot 分析显示，pNaKtide 可显著降低 Src 和 ERK1/2 的活性。
这些结果提供了证据表明，pNaKtide 在体外和体内均表现出对缺氧诱导损伤的心脏保护作用。pNaKtide 可能是治疗与 I/R 相关心脏病的潜在分子。
