小窝蛋白-3/RISK 介导HBO-PC 对 心肌的保护作用研究

Hyperbaric oxygen preconditioning exerts myocardial protection through caveolin-3/RISK pathway

摘要:

目的 探究高压氧预处理（HBO-PC）减轻心肌缺血再灌注（I/R）损伤的具体分子机制。 方法 对心肌细胞予以HBO-PC 处理，并复制缺氧复氧（H/R）模型，用试剂盒测定丙二醛（MDA）、超 氧化物歧化酶（SOD）、乳酸脱氢酶（LDH）及一氧化氮NO 的含量，Western blot 检测促/ 抗凋亡蛋白、 蛋白激酶Cε（PKCε）、小窝蛋白-3（Cav-3）、再灌注损伤挽救激酶（RISK）通路中蛋白及其磷酸化水 平的变化，免疫共沉淀和免疫荧光染色检测PKCε 与Cav-3 的结合；同时使用PKC 抑制剂Chelerythrine （CHE）和RISK 通路抑制剂进一步验证HBO-PC 对心肌细胞I/R 损伤的作用机制。结果 H/R 模型模 拟I/R 损伤过程。HBO-PC 降低MDA 和LDH 含量、Caspase-3 活性和Bax 的表达，增加SOD 和NO 含 量、Bcl-2 表达，抑制心肌细胞凋亡，减轻I/R 损伤。同时，HPO-PC 促进PKCε 活化及其与Cav-3 的结 合，增加Cav-3 蛋白表达，以及RISK 通路中胞外信号调节激酶（ERK）1/2、蛋白激酶B（Akt）、磷脂酰 肌醇3- 羟激酶（PI3K）和下游效应激酶糖原合酶激酶（GSK）3β 的磷酸化水平；CHE 能够抑制HPOPC 的作用。RISK 通路抑制剂增加Caspase-3 活性和Bax 表达，减少Bcl-2 表达，阻断HBO-PC 对心肌细 胞凋亡的拮抗作用。结论 HBO-PC 可能通过调控PKCε/Cav-3/RISK 通路，抑制心肌细胞凋亡，抵抗 心肌I/R 损伤，发挥心肌保护作用。

Abstract:

Objective To investigate the specific molecular mechanism of hyperbaric oxygen preconditioning (HBO-PC) on myocardial ischemia-reperfusion (IR) injury. Methods HBO-PC and hypoxia-reoxygenation (HR) models were performed in cardiomyocytes. Commercial kits were used to analyze the content of MDA, SOD, LDH, and NO. Western blot was used to detect the expressions of pro-/anti-apoptotic proteins, and the levels of protein kinase C ε (PKCε), caveolin (Cav)-3 and reperfusion injury salvage kinase (RISK) pathway related proteins. The combination of PKCε and Cav-3 was determined by co-immunoprecipitation and immunofluorescence staining. At the same time, cardiomyocytes were treated with the inhibitors of PKC (Chelerythrine, CHE) and RISK pathway to confirm the mechanism of HBO-PC on myocardial IR injury. Results The HR model mimicked the process of IR injury. HBO-PC decreased the levels of MDA and LDH, caspase-3 activity and Bax expression, and increased the levels of SOD, NO and Bcl-2, which suggested that HBO-PC dampened myocardial cell apoptosis and IR injury. In addition, HPO-PC promoted the binding of PKCε to Cav-3 and up-regulated the levels of Cav-3, p-ERK1/2, p-Akt, p-PI3K and p-GSK3β; while CHE inhibited the effect of HPO-PC. RISK pathway inhibitors increased caspase-3 activity and Bax expression, reduced Bcl-2 expression, which showed RISK inhibitors attenuated the effect of HBO-PC on myocardial cell apoptosis. Conclusions HBO-PC may protect the heart from myocardial IR injury through regulating PKCε/Cav-3/RISK pathway and subsequently inhibiting myocardial cell apoptosis.