4,4′-二异硫氰基芪-2,2′-二磺酸对血管紧张素Ⅱ诱导心肌细胞肥大的作用

Effect of chloride channel blocker DIDS on angiotensinⅡ-induced cardiomyocyte hypertrophy

目的探讨氯通道阻滞剂4,4′-二异硫氰基芪-2,2′-二磺酸(DIDS)对血管紧张素Ⅱ(AngⅡ)诱导H9C2心肌细胞肥大的作用及其可能机制。方法应用AngⅡ1μmol/L刺激心肌H9C2细胞株,构建心肌细胞肥大模型,观察12、24、36、48和72h各项表达,另给予50、100、200μmol/L DIDS预处理H9C2细胞,细胞分组:空白组、AngⅡ组、DIDS组、AngⅡ+DIDS组;免疫荧光染色测量细胞表面积,BCA法检测蛋白含量,实时定量PCR法检测心肌肥厚基因心房钠尿肽(ANP)和肌球蛋白重链(β-MHC)mRNA表达,二氯荧光素二乙酸检测活性氧水平。结果48h细胞表面积最大(50 166±2697)μm2和总蛋白含量最大(1.68±0.06)mg/107个,36hANP mRNA为4.08±0.38和β-MHC mRNA为2.80±0.18,表达量均最高。与空白对照组比较,AngⅡ组心肌细胞表面积、总蛋白含量、ANP、β-MHC mRNA表达量和细胞内活性氧水平明显增加(P<0.05),DIDS组无显著差异(P>0.05);与AngⅡ组比较,DIDS+AngⅡ组的心肌细胞表面积明显减小(P<0.05)、总蛋白含量明显减少(P<0.05)、ANP mRNA和β-MHC mRNA表达量明显减低(P<0.05)、细胞内活性氧水平明显下降(P<0.05)。结论 DIDS可以有效抑制AngⅡ诱导的H9C2心肌细胞肥大,其机制可能与降低活性氧的水平有关。

Objective To study the effect of chloride channel blocker DIDS on Ang Ⅱ-induced car- diomyocyte hypertrophy and its mechanism. Methods A cardiomyocyte hypertrophy model was established by stimulating H9C2 with Ang Ⅱ. The H9C2 were preconditioned with DIDS at the concentration of 50,100 and 200μmol/L respectively,and were divided into control group,Ang Ⅱ treatment group,DIDS treatment group and Ang Ⅱ ＋DIDS treatment group. The surface area of H9C2 was measured with immunofluorescence staining, the protein level was assayed by BCA, the expressions of ANP and β-MHC mRNA were detected by RT-qPCR,the reactive oxygen species （ROS） level was measured by DCFH-DA. Results The largest surface area of H9C2 was 50,166±2,697μm^2 , the highest total protein level was 1.68±0.06 mg/10^7 , the highest ANP and β-MHC mRNA expression levels were 4.08±0.38 and 2.80±0.18 respectively,which were significantly higher in Ang 11 treatment group than in control group （P（0.05） with no significant difference in DIDS treatment group （P〉0.05） ,and were significantly lower in Ang Ⅱ＋DIDS treatment group than in Ang Ⅱ treatment group （P〈0.05）. Conclusion DIDS can effectively inhibit Ang Ⅱ-induced cardiomyocyte hypertrophy by reducing the ROS level.