白藜三醇通过下调microRNA-21减轻快速电刺激所致心房肌细胞电重构

目的:研究白藜三醇(resveratrol,RSV)对快速电刺激(rapid electrical stimulation,RES)导致乳鼠心房肌细胞电重构时微小RNA-21(microRNA-21,miR-21)表达的影响,探讨RSV通过miR-21参与电重构的可能机制。方法:采用胰酶、Ⅰ型胶原酶双酶法及差速贴壁法分离培养乳鼠心房肌细胞。通过RES建立乳鼠心房肌细胞房颤模型,心房肌细胞随机分为4组:空白对照(control)组、RSV组、RES组和RSV+RES组。为了证实RSV是否通过调控miR-21的表达参与电重构,除了上述4组,另增加过表达和沉默miR-21组:RES+阴性对照组(RES+NC组)、RES+miR-21 mimics组、RES+miR-21 mimics+RSV组、RES+miR-21 inhibitor组和RES+miR-21 inhibitor+RSV组。CCK-8法检测心房肌细胞活性以确定RSV最佳作用浓度及时间,q PCR法检测各组细胞内miR-21及L型钙离子通道CACNA1C、CACNB2 mRNA的表达水平,Western blot检测L型钙离子通道Cav1.2和Cavβ_2 的蛋白表达水平。结果:与control组相比,RES组miR-21表达明显上调(P0.05),加入RSV预处理后miR-21表达下调(P0.05)。与RES+miR-21 mimics组相比,RES+miR-21 mimics+RSV组miR-21表达下调(P0.05),而CACNA1C和CACNB2 mRNA及Cav1.2和Cavβ_2 蛋白表达量增加(P0.05)。与RES组比,RES+miR-21 inhibitor和RES+miR-21 inhibitor+RSV组的miR-21表达下调(P0.05),CACNA1C和CACNB2 mRNA及Cav1.2和Cavβ_2 蛋白表达量增加,但RES+miR-21 inhibitor组与RSV+RES组比,miR-21表达、CACNA1C和CACNB2 mRNA及Cav1.2和Cavβ_2 蛋白表达量的差异无统计学显著性。结论:在快速电刺激乳鼠心房肌细胞模拟房颤模型中,RSV干预可能通过下调miR-21表达而调控其下游靶基因这一途径来减轻心房肌细胞电重构。

Resveratrol reduces electrical remodeling in atrial fibrillation by down-regulating microRNA-21 in neonatal rat atrial myocytes

AIM: To detect the effects of resveratrol (RSV) on the expression of microRNA-21 (miR-21) in primarily cultured neonatal rat atrial myocytes with electric remodeling induced by rapid electrical stimulation (RES). Furthermore, to find out the possible mechanism of miR-21 regulating electrical remodeling. METHODS: The neonatal rat atrial myocytes were isolated by double-enzyme (trypsin and collagenase I) digestion and differential adhesion method. The atrial fibrillation (AF) model was induced by RES. Atrial myocytes were randomly divided into 4 groups:control group, RSV group, RES group, and RSV+RES group. To further detect whether RSV regulated electric remodeling by miR-21, except the 4 groups, we add miR-21 over-expression group and miR-21 inhibitor group:RES+negative control (NC) group, RES+miR-21 mimics group, RES+miR-21 mimics+RSV group, RES+miR-21 inhibitor group, and RES+miR-21 inhibitor+RSV group. The optimal concentration and pretreatment time of resveratrol were determined by CCK-8 assay. The expression of miR-21 and the mRNA expression of L-type calcium channels CACNA1C and CACNB2 in atrial myocytes were detected by qPCR. The protein expression of L-type calcium channels Cav1.2 and Cavβ₂ in the atrial myocytes was analyzed by Western blot. RESULTS: The expression of miR-21 in RES group was significantly increased compared with control group, while preconditioning with RSV decreased the expression of miR-21. Compared with RES+miR-21 mimics group, the expression of miR-21 in RES+miR-21 mimics+RSV group was significantly decreased. Meanwhile, the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ₂ were increased (P<0.05). Compared with RES group, the expression of miR-21 in RES+miR-21 inhibitor group and RES+miR-21 inhibitor+RSV group was decreased, while the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ₂ were increased. However, no difference of the expression of miR-21, the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ₂ among RSV+RES, RES+miR-21 inhibitor and RES+miR-21 inhibitor+RSV groups was observed (P<0.05).CONCLUSION: In AF model induced by RES, RSV may reduce electric remodeling by inhibiting the expression of miR-21 and regulating the downstream target genes.