夹竹桃麻素对兔心房肌细胞氧化应激损伤L型钙电流及动作电位时程的影响

Effect of apocynin on L-type calcium current and action potential duration in rabbit atrial myocytes under oxidative stress in vitro

背景房颤的发生、发展机制复杂。大量研究表明,氧化应激与房颤关系密切。夹竹桃麻素(apocynin,APO)是氧化应激损伤过程中重要的氧化酶抑制剂,其能够对抗氧化应激的损伤作用。目的探讨夹竹桃麻素对体外兔左心房肌细胞L型钙电流(L-type calcium current,I_(Ca,L))及动作电位时程(action potential duration,APD)氧化应激损伤的保护作用。方法酶解法分离得到单个兔左心房肌细胞,并将其分为5组:对照组(n=13),H_(2)O_(2)(10μmol/L)组(n=13),H_(2)O_(2)(50μmol/L)组(n=13),H_(2)O_(2)(50μmol/L)+APO(100μmol/L)组(n=13),APO(100μmol/L)组(n=13),应用膜片钳全细胞技术,探讨夹竹桃麻素(100μmol/L)对兔左心房肌细胞I_(Ca,L)及APD氧化损伤的保护作用。Western blot检测各组兔左心房中CaV1.2蛋白的表达。RT-qPCR检测兔左心房中的CACNA1C mRNA表达。结果10μmol/L H_(2)O_(2)组:I_(Ca,L)峰值从(−18.5±0.1)pA/pF减至(−10.7±0.7)pA/pF(P<0.01);50μmol/L H_(2)O_(2)组:I_(Ca,L)峰值从(−18.5±0.1)pA/pF减至(−9.4±0.8)pA/pF(P<0.01);50μmol/L H_(2)O_(2)+100μmol/L APO组:I_(Ca,L)峰值为(−16.7±1.6)pA/pF,与50μmol/L H_(2)O_(2)组比较差异有统计学意义(P<0.01);100μmol/L APO组:I_(Ca,L)峰值为(−17.2±1.3)pA/pF,与对照组比较差异无统计学意义。门控机制研究表明,APO的作用主要是通过使稳态激活曲线向负方向移动、稳态失活曲线向正方向移动实现电流恢复,而与电流失活后恢复过程关系不大。进一步,APO使H_(2)O_(2)处理后缩短的APD50得以恢复。与对照组比较,H_(2)O_(2)组CACNA1C mRNA、CaV1.2蛋白表达下降(P<0.05),APO+H_(2)O_(2)组可以使表达恢复(P<0.05)。结论APO对兔左心房肌细胞I_(Ca,L)和APD具有保护作用。

Background The occurrence and development of atrial fibrillation is complex.A large number of studies have shown that oxidative stress is closely related to atrial fibrillation.Apocynin(APO)is an important oxidase inhibitor in the process of oxidative stress injury,which can resist oxidative stress injury.Objective To study the protective effect of apocynin on L-type calcium current(ICa,L)and action potential duration(APD)in rabbit left atrial myocytes under oxidative stress injury.Methods We used enzyme to get single left atrial myocytes of rabbit,and they were divided into control group(n=13),H_(2)O_(2)(10μmol/L)group(n=13),H_(2)O_(2)(50μmol/L)group(n=13),H_(2)O_(2)(50μmol/L)+APO(100μmol/L)group,APO(100μmol/L)group(n=13).The whole cell patch clamp technique was used to study the protective effect of APO on I_(Ca,L)and APD under oxidative stress injury.Real-time qPCR and Western blot were used to detect the expression of CACNA1C mRNA and CaV1.2 protein levels.Results In the H_(2)O_(2)(10μmol/L)group,the peak value of I_(Ca,L)declined from(−18.5±0.1)pA/pF to(−10.7±0.7)pA/pF(P<0.01);In the H_(2)O_(2)(50μmol/L)group,the peak value of I_(Ca,L)declined from(−18.5±0.1)pA/pF to(−9.4±0.8)pA/pF(P<0.01);In H_(2)O_(2)(50μmol/L)+APO(100μmol/L)group,the peak value of I_(Ca,L)was(−16.7±1.6)pA/pF,significantly higher than that in the H_(2)O_(2)(50μmol/L)group after treatment(P<0.01);In APO(100μmol/L)group,the peak of I_(Ca,L)was(−17.2±1.3)pA/pF,similar to the control group(P>0.01).APO also recovered the shortened APD50 induced by H_(2)O_(2).Compared with H_(2)O_(2)group,the expression of CACNA1C mRNA and the protein levels of CaV1.2 decreased(P<0.05),which could be recovered by APO.Conclusion APO have the protective effect on ICa,Land APD in left atrial myocytes of rabbit.