参仙升脉口服液对病态窦房结综合征小鼠ROS表达的影响和对HCN4离子通道的调控作用

Effect of Shenxianshengmai Oral Liquid on ROS expression in mice with sick sinus syndrome and its regulation on HCN4 ion channel

目的:探讨参仙升脉口服液对病态窦房结综合征(SSS)小鼠窦房结线粒体活性氧(ROS)释放的影响及其对环化核苷酸调控阳离子通道蛋白亚型4(HCN4)离子通道的调控作用,探讨参仙升脉口服液改善SSS的作用机制。方法:30只C57B6小鼠随机分为假手术组、SSS组和参仙升脉口服液治疗组(SXSM组),每组10只。通过Alzet渗透压泵泵入血管紧张素Ⅱ(ANGⅡ)建立SSS小鼠模型,SXSM组小鼠给予0.2 mL·d-1参仙升脉口服液灌胃,持续28 d。观察各组小鼠心电图并分析心率,HE和Masson染色观察各组小鼠窦房结组织病理形态表现,免疫荧光法检测各组小鼠窦房结组织中ROS和HCN4水平,实时荧光定量PCR(RT-qPCR)法检测各组小鼠窦房结组织中电压门控Na+通道α亚单位SCN5A、L-型电压依赖钙离子通道α1C亚基(CACNα1C)蛋白和电压依赖性钙通道α1G亚基蛋白(CACNα1G)mRNA表达水平,Western blotting法检测各组小鼠窦房结组织中SCN5A、CACNα1C、CACNα1G和HCN4蛋白表达水平。结果:与假手术组比较,SSS组小鼠心率明显降低(P<0.05),窦房结组织中细胞出现溶解坏死,未见细胞核,结缔纤维组织大量增生,窦房结组织中ROS水平升高(P<0.05),HCN4水平降低(P<0.05),SCN5A mRNA和蛋白表达水平明显降低(P<0.05),CACNα1C和CACNα1G mRNA和蛋白表达水平明显升高(P<0.05),HCN4蛋白表达水平明显降低(P<0.05);与SSS组比较,SXSM组小鼠心率明显升高(P<0.05),窦房结组织中结缔纤维组织少量增生,细胞部分坏死溶解,ROS水平降低(P<0.05),HCN4水平升高(P<0.05),SCN5A mRNA和蛋白表达水平升高(P<0.05),CACNα1C和CACNα1G mRNA和蛋白表达水平明显降低(P<0.05),HCN4蛋白表达水平明显升高(P<0.05)。结论:参仙升脉口服液能够提高心率,抑制SSS小鼠窦房结组织中ROS的释放,抑制窦房结纤维化,其机制与调控HCN4离子通道有关。

Objective:To investigate the effect of Shenxianshengmai Oral Liquid on the release of reactive oxygen species(ROS)in sinoatrial node tissue of the sinoatrial node syndrome(SSS)mice and its regulation on cyclic nucleotide regulated cation channel protein subtype 4(HCN4)ion channel,and to explore the mechanism of Shenxianshengmai Oral Liquid in improving the SSS.Methods:A total of 30 C57B6 mice were randomly divided into sham operation group,SSS group,and Shenxianshengmai Oral Liquid treatment(SXSM)group,and there were 10 mice in each group.The SSS mice model was established by pumping angiotensinⅡ(ANGⅡ)with the Alzet osmotic pressure pump.The mice in SXSM group were given 0.2 mL of Shenxianshengmai Oral Liquid per day for 28 d.The electrocardiogram of the mice in various groups was observed and the heart rates were analyzed;HE and Masson staining methods were used to observe the pathomorphology of sinoatrial node tissue of the mice in various groups,immunofluorescence method was uesd to detect the levels of ROS and HCN4 in sinoatrial node tissue of the mice in various groups;Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of voltage-gated Na+αsubunit(SCN5 A),L-type voltage dependent calcium channelsα1C subunit protein(CACNα1 C)and voltage dependent calcium channelα1G subunit protein(CACNα1G)mRNA in sinoatrial node tissue of the mice in various groups;Western blotting method was used to detect the expression levels of SCN5A,CACNα1C,CACNα1G,and HCN4 proteins in sinoatrial node tissue of the mice in various groups.Results:Compared with sham operation group,the heart rate of the mice in SSS group was decreased significantly(P<0.05),the cells were dissolved and necrotic,there was no nucleus,and there was a large number of connective fiber tissue;the ROS level in sinoatrial node tissue was increased(P<0.05),the HCN4 level in sinoatrial node tissue was decreased(P<0.05),the expression levels of SCN5A mRNA and protein in sinoatrial node tissue were decreased significantly(P<0.05),the expression levels of CACNα1C and CACNα1G mRNA and proteins in sinoatrial node tissue were increased significantly(P<0.05),and the expression level of HCN4 protein in sinoatrial node tissue was decreased significantly(P<0.05);compared with SSS group,the heart rate of the mice in SXSM group was increased significantly(P<0.05),a small amount of connective fiber tissue showed hyperplasia,partial of the cells showed necrosis and dissolution,the ROS level in sinoatrial node tissue was decreased(P<0.05),the HCN4 level in sinoatrial node tissue was increased(P<0.05),the expression levels of SCN5A mRNA and protein in sinoatrial node tissue were increased(P<0.05),the expression levels of CACNα1C and CACNα1G mRNA and proteins in sinoatrial node tissue were decreased significantly(P<0.05),and the expression level of HCN4 protein in sinoatrial node tissue was increased significantly(P<0.05).Conclusion:Shenxianshengmai Oral Liquid can increase the heart rate,inhibit the release of ROS in sinoatrial node tissue of the SSS mice,and inhibit the fibrosis of the sinoatrial nodes.The mechanism is related to the regulation of HCN4 ion channels.