益心解毒方对Nox2和Nox4亚基过表达的心肌细胞NADPH氧化酶活性的影响

Effects of Yixin Jiedu Formula on NADPH oxidase activity in H9c2 myocardial cells with overexpressed Nox2 and Nox4 subunits

目的通过观察益心解毒方含药血清对Nox2、Nox4亚基过表达的H9c2心肌细胞NADPH氧化酶活性的影响,揭示其作用环节是否与干预相应亚型的NADPH氧化酶调控环节有关。方法采用PCR方法扩增Nox2、Nox4基因全长序列,经双酶切、连接载体和转化后提取重组质粒,经酶切鉴定的阳性质粒进行DNA测序,测序正确后按照lipofectamine 2000试剂的说明书瞬时转染H9c2细胞,转染后的心肌细胞分组给予不同的药物干预,24 h后检测NADPH氧化酶活性。结果 1含有Nox2/Nox4亚基的重组质粒载体通过测序鉴定,结果与Gen Bank报道的完全一致。2通过荧光显微镜下观察转染72 h后的H9c2细胞,可见大量发绿色荧光的细胞,流式细胞术计数结果显示转染率均在60%左右,符合实验要求。3空质粒载体组、模型组、益心解毒方组的NADPH氧化酶活性表达明显高于正常组(P<0.01,P<0.05);模型组和益心解毒方组的NADPH氧化酶活性表达高于空载体组(P<0.01);模型组NADPH氧化酶活性表达均明显高于其他各组,而益心解毒方各组的NADPH氧化酶活性表达均明显低于模型组(P<0.01,P<0.05)。结论成功构建大鼠心肌细胞Nox2、Nox4亚基的重组质粒载体,该重组质粒载体可以在心肌细胞中过表达,益心解毒方可以有效地降低NADPH氧化酶活性的表达。提示此复方治疗心衰的机制可能是抑制了Nox2和Nox4型NADPH氧化酶的表达。

Objective To observe the effect of Yixin Jiedu Formula drug serum on NADPH oxidase activity in H9c2 myocardial cells with overexpressed Nox2 and Nox4 subunits,and reveal whether it could influence the regulation and control process of corresponding subtypes ＇NADPH oxidase.Methods Nox2 and Nox4 genes ＇ total sequence were amplified by PCR,recombinant plasmid was extracted after cut of double enzyme,connection of carrier and conversion. DNA sequencing was carried out for positive plasmid after enzyme digestion identification. After that, H9c2 cells got transient transfection following the instruction of lipofectamine 2000,and different drug intervention were given to the cells. After 24 h,NADPH oxidase activity was detected. Results 1 Sequencing identification of recombinant plasmid carrier with Nox2 / Nox4 subunits was completely accord with the reported resultsfrom Gen Bank. 2 Under fluorescence microscope,the transfected H9c2 cells were observed after 72 h,a great deal of green fluorescent cells were observed. And the transfected rate was about 60% according to flow cytometry count result,which met the requirements of the experiment. 3 Positive expression of NADPH oxidase in empty plasmid vector group,model group and Yixin Jiedu Formula group were significantly higher than the control group,the differences were significant（ P〈0. 01,P〈0. 05）; Positive expression of NADPH oxidase in model group and Yixin Jiedu Formula group were higher than the empty plasmid vector group,the differences were significant（ P〈0. 01）; Positive expression of NADPH oxidase in model group was significantly higher than other groups（ P〈0. 01）,and positive expression of NADPH oxidase in different Yixin Jiedu Formula groups were significantly lower than the model group（ P〈0. 05）. Conclusions Recombinant plasmid carrier of Nox2 and Nox4 subtypes in rats＇ myocardial cells is constructed successfully,which has an overexpression in myocardial cells. Yixin Jiedu Formula could effectively decrease the positive expression of NADPH oxidase,which indicates the possible mechanism of this formula in treating heart failure is restraining the expression of Nox2 and Nox4 NADPH oxidase.