参芪益心方抑制阿霉素诱导大鼠H9c2细胞凋亡的机制研究

Study on Mechanism of Shenqi Yixin Formula in Inhibiting Adriamycin Induced Apoptosis of Rats H9c2 Cells

目的基于心肌细胞内质网应激观察参芪益心方对大鼠H9c2心肌细胞内Ca^(2+)及半胱氨酸天冬氨酸酶(Caspase)-3、Caspase-12、葡萄糖调节蛋白-78(GRP78)、增强型蛋白同源蛋白(CHOP)、肌质网钙离子ATP酶(SERCa2a)蛋白和mRNA表达的影响。方法SD大鼠制备参芪益心方含药血清。体外培养H9c2心肌细胞,用阿霉素诱导凋亡建立模型,将细胞分为模型组(8%空白血清)、高剂量组(8%含药血清)、中剂量组(4%含药血清+4%空白血清)、低剂量组(2%含药血清+6%空白血清)和卡托普利(1×10^(-5)mol/L)组,另设空白组(8%空白血清)。Real-time PCR及Western Blot分别检测Caspase-3、Caspase-12、GRP78、CHOP、SERCa2a mRNA及蛋白的表达,Fluo-3AM流式细胞仪检测H9c2细胞内Ca^(2+)荧光强度。结果与空白组比较,模型组Caspase-3、Caspase-12、GRP78、CHOP的蛋白、mRNA表达及Ca^(2+)荧光强度增加,SERCa2a蛋白及mRNA表达下降(均P<0.01)。与模型组比较,各干预组Caspase-3、Caspase-12、GRP78、CHOP蛋白及mRNA表达降低,Ca^(2+)荧光强度降低,SERCa2a蛋白及mRNA表达增加(P<0.01)。与卡托普利组比较,中药高剂量组SERCa2a蛋白及mRNA表达升高,Ca^(2+)荧光强度升高(P<0.01)。结论参芪益心方可通过降低Ca^(2+)浓度,下调Caspase-3、Caspase-12、GRP78、CHOP的表达,上调SERCa2a的表达,减轻内质网的过度应激,抑制心肌细胞凋亡。

Objective Based on the endoplasmic reticulum stress in cardiomyocytes,to observe the effect of Shenqi Yixin Formula(SQYXF)on the fluorescence intensity of Ca^(2+),protein and mRNA expression of Caspase-3,Caspase-12,glucose-regulated protein-78(GRP78),enhancer-binding protein homologous protein(CHOP),sacro/endoplasmic reticulum Ca^(2+)-ATPase(SERCa2a)in rats-derived H9c2 cardiomyocytes.Methods SQYSF Drug-containing serum was prepared in SD rats.H9c2 cardiomyocytes were cultured in vitro,and apoptotic cell model were established by adriamycin.The apoptotic cells were divided into model group(8%blank serum),high dose group(8%drug-containing serum),medium dose group(4%drug-containing serum),low dose group(2%drug-containing serum)and captopril group(1×10^(-5) mol/L).In addition,a blank group was set up(H9c2 cardiomyocytes treated with 8%blank serum).Real-time PCR and Western Blot were used to detect Caspase-3,Caspase-12,GRP78,CHOP,SERCa2a protein and mRNA expression.Fluorescence intensity of Ca^(2+)in H9c2 cells was measured by fluo-3 AM flow cytometry.Results Compared with blank group,Caspase-3,Caspase-12,GRP78,CHOP protein and mRNA expression and fluorescence intensity of Ca^(2+)increased,SERCa2a protein and mRNA expression decreased in model group(all P<0.01).Compared with model group,Caspase-3,Caspase-12,GRP78,CHOP protein and mRNA expression and fluorescence intensity of Ca^(2+)decreased,and SERCa2a protein and mRNA increased in intervention groups(P<0.01).Compared with captopril group,SERCa2a protein and mRNA expression and fluorescence intensity of Ca^(2+)increased in the high dose group(P<0.01).Conclusion SQYXF can alleviate the excessive stress of endoplasmic reticulum and inhibit cardiomyocyte apoptosis through reducing Ca^(2+)concentration,down-regulating the expression of Caspase-3,Caspase-12,GRP78,CHOP,and up-regulating the expression of SERCa2a.