抗纤益心方通过调节Drp1活性抑制去甲肾上腺素诱导大鼠H9c2细胞肥大

Kangxian Yixin Decoction(抗纤益心方)Inhibits Norepinephrine-Induced Hypertrophy of Rat H9c2 Cells by Regulating DRP1 Activity

目的:探讨抗纤益心方对去甲肾上腺素(NE)诱导大鼠H9c2细胞肥大的影响及作用机制。方法:H9c2细胞培养24 h,用NE诱导建立H9c2细胞肥大模型,将细胞分为空白对照组、模型对照组、抗纤益心方250 mg/mL组、线粒体动力分裂蛋白1抑制剂1×10^(-5)mol/L组、线粒体动力分裂蛋白1抑制剂1×10^(-5)mol/L+抗纤益心方250 mg/mL组,药物作用24 h后,显微镜下观察细胞形态及检测单个心肌细胞表面积;RT-PCR法检测细胞肥大因子心房钠尿肽(Anp)、脑钠肽(Bnp)mRNA表达;电镜检测细胞线粒体超微结构;JC-1检测细胞线粒体膜电位(MMP)水平;Western blot法检测细胞ANP、BNP、发动蛋白相关蛋白1(DRP1)及其p-DRP1(s616)蛋白表达。结果:与空白对照组比较,模型对照组细胞相对表面积增大,Anp、Bnp mRNA表达均上调,线粒体结构损伤,MMP水平明显下调(P<0.05),ANP、BNP、p-DRP1蛋白表达上调(P<0.05);与模型对照组比较,抗纤益心方组及抗纤益心方+线粒体动力分裂蛋白1抑制剂组,细胞相对表面积减小、Anp、Bnp mRNA表达下调(P<0.05),线粒体结构损伤改善,MMP水平上调(P<0.05),ANP、BNP及p-DRP1蛋白表达下调(P<0.05或P<0.01)。结论:抗纤益心方能够抑制NE诱导H9c2细胞肥大,其机制可能与改善线粒体结构与功能,下调DRP1去磷酸化表达有关。

Objective:To investigate the mechanism of Kangxian Yixin Decoction(抗纤益心方)(KYD)against hypertrophy of H9 c2 cardiomyocytes induced by norepinephrine(NE).Methods:H9 c2 cardiomyocytes were cultured for 24 h,and NE was used to induce the hypertrophy.The cells were classified into blank control group,model group,250 mg/mL KYD group,1×10^(-5)mol/L mitochondrial division inhibitor-1(Mdivi-1)group,and combination group(250 mg/mL KYD+1×10^(-5)mol/L Mdivi-1).After the treatment for 24 h,cell morphology was observed and surface area of individual cardiomyocytes was measured under the microscope.The mRNA expression of cell hypertrophy markers atrial natriuretic peptide(Anp)and brain natriuretic peptide(Bnp)was detected by RT-PCR.Cell mitochondrial structure was detected by electron microscopy.Mitochondrial membrane potential(MMP)was assessed by JC-1.The protein expression of ANP,BNP,dynamin-related protein 1(DRP1),and phosphorylated(p)-DRP1(S616)was detected by Western blotting.Results:Compared with blank control group,the model group showed large relative surface area of cells,high mRNA expression of Anp and Bnp,damaged mitochondrion structure,low protein expression of MMP(P<0.05),and high protein expression of ANP,BNP,and p-DRP1(P<0.05).Compared with the model group,KYD group and combination group had small relative surface area of cells,low mRNA expression of Anp and Bnp(P<0.05),improvement of mitochondrion structure injury,high protein expression of MMP(P<0.05),and low protein expression of ANP,BNP,and p-DRP1(P<0.05 or P<0.01).Conclusion:KYD can inhibit NE-induced hypertrophy of H9 c2 cells and the mechanism is that it improves mitochondrial structure and function and down-regulates p-DRP1.