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

刘岩; 李泱; 叶加虎; 刘昕; 王延坤; 单兆亮

doi:10.3969/j.issn.2095-5227.2021.01.015

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

摘要

摘要:
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)，H₂O₂(10 μmol/L)组(n=13)，H₂O₂(50 μmol/L)组(n=13)，H₂O₂(50 μmol/L) + APO (100 μmol/L)组(n=13)，APO (100 μmol/L)组(n=13)，应用膜片钳全细胞技术，探讨夹竹桃麻素(100 μmol/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Ca_V1.2蛋白的表达。RT-qPCR检测兔左心房中的CACNA1C mRNA表达。
结果 10 μmol/L H₂O₂组：I_Ca,L峰值从(−18.5±0.1) pA/pF减至(−10.7±0.7) pA/pF (P＜0.01)；50 μmol/L H₂O₂组：I_Ca,L峰值从(−18.5±0.1) pA/pF减至(−9.4±0.8) pA/pF(P＜0.01)；50 μmol/L H₂O₂+100 μmol/L APO组：I_Ca,L峰值为(−16.7±1.6) pA/pF，与50 μmol/L H₂O₂组比较差异有统计学意义(P ＜0.01)；100 μmol/LAPO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H₂O₂处理后缩短的APD₅₀得以恢复。与对照组比较，H₂O₂组CACNA1C mRNA、Ca_V1.2蛋白表达下降(P＜0.05)，APO+H₂O₂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。

Abstract:
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 (I_Ca,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), H₂O₂(10 μmol/L) group (n=13), H₂O₂(50 μmol/L) group (n=13), H₂O₂(50 μmol/L) + APO (100 μmol/L) group, APO (100 μmol/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 Ca_V1.2 protein levels.
Results In the H₂O₂(10 μmol/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 H₂O₂(50 μmol/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 H₂O₂(50 μmol/L) + APO (100 μmol/L) group, the peak value of I_Ca,L was (−16.7±1.6) pA/pF, significantly higher than that in the H₂O₂(50 μmol/L) group after treatment (P＜0.01); In APO (100 μmol/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 H₂O₂. Compared with H₂O₂ group, the expression of CACNA1C mRNA and the protein levels of Ca_V1.2 decreased (P＜0.05), which could be recovered by APO.
Conclusion APO have the protective effect on I_Ca,Land APD in left atrial myocytes of rabbit.

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