摘要目的：探讨积雪草酸（AA）对异丙肾上腺素（ISO）诱导心肌细胞肥大的影响。方法体外培养大鼠心肌细胞H9c2，分为对照组、ISO组和ISO+AA组。ISO组使用10 mmol/L ISO处理H9c2细胞诱导心肌细胞肥大，ISO+AA组加入不同浓度AA和10 mmol/L ISO处理H9c2细胞，对照组加入0.5%二甲基亚砜（DMSO）作为溶媒对照。实时定量PCR检测心房钠尿肽（ANP）、β?肌球蛋白重链（β?MHC）、谷胱甘肽过氧化物酶（GSH?Px）、超氧化物歧化酶（SOD）、血红素加氧酶1（HO?1）基因的表达，免疫荧光染色测量心肌细胞横截面积，免疫印迹法检测心肌肥大相关信号通路改变，荧光探针DCFH?DA检测细胞内活性氧簇（ROS）水平。结果实时定量PCR显示，与对照组比较，ISO处理48 h后H9c2细胞心肌肥大标志物ANP和β?MHC mRNA表达上调（均P<0.05），而5、10、20μmol/L的AA干预48 h可显著降低ISO诱导的ANP和β?MHC mRNA表达上调（均P<0.05），且具有浓度依赖性；20μmol/L AA处理H9c2细胞12、24和48 h，均能抑制ISO诱导的ANP和β?MHC mRNA表达上调（均P<0.05）。免疫荧光染色测量各组心肌细胞横截面积显示，对照组、ISO组和ISO（10 mmol/L）+AA（20μmol/L）组心肌细胞横截面积分别为（1946±49）μm2、（2952±111）μm2、（2548±59）μm2。与对照组比较，ISO组心肌细胞横截面积增加，而20μmol/L AA能显著降低ISO导致的心肌细胞肥大（均P<0.05）。免疫印迹显示ISO可导致蛋白激酶B（AKT）?糖原合成酶激酶3β（GSK3β）和细胞外信号调节激酶（ERK）?c?Jun氨基末端激酶（JNK）等心肌肥大相关信号通路活化，而20μmol/L AA能够抑制上述信号通路的活化。荧光探针DCFH?DA检测显示，20μmol/L AA能够明显抑制ISO诱导的细胞内ROS水平增加。实时定量PCR检测显示，20μmol/L AA能够明显抑制ISO诱导的GSH?Px、SOD和HO?1 mRNA表达上调。结论 AA可通过抑制AKT?GSK3β和ERK?JNK信号通路减弱氧化应激，从而抑制ISO所致的心肌细胞肥大。

abstractsObjective To investigate the effects of asiatic acid (AA) on isoproterenol (ISO) induced myocardial hypertrophy. Methods In?vitro cultured rat myocardial cells H9c2 were divided into the control group,ISO group and ISO+AA group. The H9c2 cells in the ISO group were treated with 10 mmol/L ISO to induce cardiomyocyte hypertrophy,those in ISO+AA group with different concentrations of AA and 10 mmol/L ISO,and those in the control group with 0.5%dimethyl sulfoxide(DMSO)as a vehicle control. Real?time quantitative PCR was performed to detect the expressions of atrial natriuretic peptide(ANP),β?myosin heavy chain(β?MHC),glutathione peroxidase(GSH?Px),superoxide dismutase(SOD)and hemoglobin oxygenase 1(HO?1)genes. Immunofluorescence staining was used to measure cross?sectional area of the&nbsp;cardiomyocytes. Western blot was used to detect the changes in signaling pathway related to myocardial hypertrophy. DCFH?DA fluorescent probe was used to detect the intracellular levels of reactive oxygen species(ROS). Results As shown by real?time quantitative PCR,mRNA expression of cardiac hypertrophy markers ANP andβ?MHC in H9c2 cells was up?regulated after 48 h treatment with ISO compared with the control group(both P<0.05),while 48 h intervention with 5,10 or 20μmol/L AA significantly reduced the ISO?induced up?regulation of ANP andβ?MHC mRNA expression(all P<0.05)in a concentration?dependent manner;and treatment with 20μmol/L AA for 12,24 or 48 h was shown to inhibit ISO?induced ANP andβ?MHC mRNA up?regulation in H9c2 cells(all P<0.05). Cross?sectional area of cardiomyocytes as measured by immunofluorescence staining was(1 946 ± 49)μm2 in the control group,(2 952 ± 111)μm2 in the ISO group and(2 548 ± 59)μm2 in the ISO(10 mmol/L)+AA(20 μmol/L)group. Compared with the control group,cross?sectional area of cardiomyocytes increased in ISO group,while 20μmol/L AA could significantly reduce the ISO?induced myocardial hypertrophy(both P<0.05). Western blotting indicated that ISO could lead to activation of AKT/GSK3β and ERK/JNK related to cardiac hypertrophy,while 20 μmol/L AA could inhibit the activation of these signaling pathways. DCFH?DA fluorescent probe assay showed that 20μmol/L AA could significantly inhibit the ISO induced increase in intracellular ROS levels. Real?time quantitative PCR showed that 20μmol/L AA could inhibit ISO?induced up?regulation of GSH?Px,SOD and HO?1 mRNA expression. Conclusion AA may attenuate oxidative stress by blocking the AKT/GSK3β and ERK/JNK signaling pathways,thereby inhibiting the ISO?induced myocardial hypertrophy.