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

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。

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 Cε(PKCε),caveolin(Cav)-3 and reperfusion injury salvage kinase(RISK)pathway related proteins.The combination of PKCεand 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 PKCεto 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 HBOPC 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.