当归挥发油对血管紧张素Ⅱ所致肥大心肌细胞CaN信号通路和T型钙通道的影响

Effects of Angelicanaphtha on CaN signal pathways and T-type calcium channel of angiotensin Ⅱ-induced hypertrophy cardiomyocyte

目的观察当归挥发油对血管紧张素Ⅱ诱导的肥大心肌细胞内Ca^(2+)浓度、钙调神经磷酸酶(CaN)信号通路及T型钙通道相关基因Cav3.1、Cav3.2表达的影响,探讨当归挥发油抑制血管紧张素Ⅱ诱导心肌细胞肥大的作用机制。方法选择H9c2心肌细胞株,实验分为5组:正常组采用无血清培养液培养;血管紧张素Ⅱ组加入终浓度为10^(-4)mg/L的血管紧张素Ⅱ培养建立心肌细胞肥大模型;当归挥发油低、中、高剂量组均先加入终浓度为10^(-4)mg/L血管紧张素Ⅱ孵育2 h,后分别加入终浓度为5 mg/L、10 mg/L、20 mg/L的当归挥发油培养;各组均培养24 h。激光扫描共聚焦显微镜观察各组心肌细胞内Ca^(2+)表达情况(以荧光强度表示其浓度),Western blot法检测心肌细胞中CaN、Cav3.1、Cav3.2蛋白表达情况。结果血管紧张素Ⅱ组心肌细胞存活率、心肌细胞内Ca^(2+)荧光强度和心肌细胞中CaN、Cav3.1、Cav3.2蛋白相对表达量均明显高于正常组(P均<0.05);当归挥发油各组心肌细胞存活率、心肌细胞内Ca^(2+)荧光强度和当归挥发油高、中剂量组细胞中CaN、Cav3.1、Cav3.2蛋白相对表达量均明显低于血管紧张素Ⅱ组(P均<0.05)。结论Ca^(2+)超载和CaN、Cav3.1、Cav3.2蛋白的过度表达与血管紧张素Ⅱ引起的心肌细胞肥大相关;当归挥发油可能通过拮抗血管紧张素Ⅱ引起的Ca^(2+)超载,下调CaN、Cav3.1、Cav3.2蛋白表达,抑制心肌细胞肥大。

Objective It is to observe the effects of Angelicanaphtha(AN)on Ca^(2+)concentration,calcineurin(CaN)signaling pathway and the expression of T-type calcium channel-related genes Cav3.1 and Cav3.2 in angiotensinⅡ-induced hypertrophic cardiomyocytes,and to explore the mechanism of the inhibition of angiotensinⅡ-induced cardiomyocyte hypertrophy by AN.Methods The H9c2 cardiomyocyte cell line was selected,and the experiment was divided into 5 groups:the normal group was cultured with serum-free culture medium;the angiotensinⅡgroup was cultured with angiotensinⅡat a final concentration of 10-4 mg/L to establish cardiomyocyte hypertrophy models;and the AN low-,medium-,and high-dose groups were all firstly incubated for 2 h by angiotensinⅡat a final concentration of 10-4 mg/L,then cultured with AN at a final concentration of 5 mg/L,10 mg/L,and 20 mg/L,respectively.All the groups were cultured for 24 h.The expression of Ca^(2+)in cardiomyocytes of each group(its concentration was expressed as fluorescence intensity)was observed by Laser scanning confocal microscopy,and the expression of CaN,Cav3.1,and Cav3.2 protein in cardiomyocytes were detected by Western blot method.Results The cardiomyocyte survival rate,Ca^(2+)fluorescence intensity in cardiomyocytes,and the relative expressions of CaN,Cav3.1,and Cav3.2 proteins in cardiomyocytes in the angiotensinⅡgroup were significantly higher than those in the normal group(all P<0.05);the cardiomyocyte survival rate,Ca^(2+)fluorescence intensity in cardiomyocytes in each AN groups,and and the relative expressions of CaN,Cav3.1,and Cav3.2 proteins in cardiomyocytes in the AN medium-,and high-dose groups were significantly lower than those in the angiotensinⅡgroup(all P<0.05).Conclusions Ca^(2+)overloading,the over-protein expression of Cav3.1,Cav3.2 are involved in the AngⅡ-induced cardiac myocyte hypertrophy.AN can reverse the AngⅡ-induced cardiac myocyte hypertrophy,possibly by reducing the calcium concentration induced by AngⅡ,down-regulating the protein expression of CaN,Cav3.1,Cav3.2.