Study On The Mechanism Of Shenxian Shengmai Oral Liquid In Treating Sick Sinus Syndrome Based On Network Pharmacolog

Purpose:1.To compare the relative curative effect of Chinese patent medicines of warming yang and benefiting qi in the improvement of sinoatrial node function by means of network meta-analysis,and to provide the basis for the selection of Chinese patent medicine treatment plan.2.Predict the core mechanism of Shenxian-Shengmai Oral Liquid in the treatment of sick sinus syndrome by means of network pharmacology and molecular docking.3.Explore the therapeutic mechanism of Shenxian-Shengmai Oral Liquid on sick sinus syndrome by observing the effects of Shenxian-Shengmai Oral liquid on the expression of BMP4,TBX3 and HCN4 in HL-1 cells with siRNA targeted knockdown of Bmp4 expression level and on the beating rates and field potential duration of the human-induced pluripotent stem cell-derived atrial myocytes(hiPSC-AMs)with reduced BMP4 expression.4.Explore the therapeutic mechanism of Shenxian-Shengmai Oral Liquid on sick sinus syndrome by observing the effect of Shenxian-Shengmai Oral Liquid on the expression of KCNH2,KCNQ1 and KCNMA1 channels in HL-1 cells targeted to knock down Kcnh2,Kcnql and Kcnmal by siRNA and on the beating rates and field potential duration of hiPSC-AMs with reduced KCNH2,KCNQ1,and KCNMA1 expression.Materials and methods:This research consists of three parts:1.Network meta-analysis of warming yang and benefiting qi Chinese patent medicines in the improvement of sinoatrial node function;2.Mechanism research on the treatment of SSS with SXSM based on network pharmacology and molecular docking;3.Research on the mechanism of SXSM in the treatment of SSS based on BMP4/TBX3/HCN4 pathway and potassium channels.1 Network meta-analysis of warming yang and benefiting qi Chinese patent medicines in the improvement of sinoatrial node functionRandomized controlled trials of warming yang and benefiting qi Chinese patent medicines in the treatment on sinus bradycardia were obtained according to inclusion and exclusion criteria through computer retrieval of CNKI,Wanfang Database,VIP,CBM and PubMed.Basic information of included literature were extracted.The quality of included studies was assessed via the Cochrane risk of bias tool and data analysis was performed via R 4.0.2 software.The warming yang and benefiting qi Chinese patent medicines were sorted according to the total effective rate of symptom improvement and the improvement effect on the average heart rate of sinus bradycardia patients.2 Mechanism research on the treatment of SSS with SXSM based on network pharmacology and molecular dockingTCMSP was used to search the ingredients of SXSM in combination with literature retrieval.The targets of Shenxian-Shengmai Oral Liquid were predicted using the Swiss Target Prediction database.The targets of SSS were obtained by searching OMIM,DrugBank and DisGeNET databases.The targets of SXSM and SSS were intersected to obtain common targets with reference to the online Venn diagram program.The PPI network information of the targets was obtained via STRING database.Cytoscape 3.7.2 software was used for visual analysis,and the core targets were screened to construct the component-target network.The core targets were subjected to GO enrichment analysis and KEGG pathway enrichment analysis,and molecular docking was performed between compounds and targets via Discovery Studio 2019 software.3 Research on the mechanism of SXSM in the treatment of SSS based on BMP4/TBX3/HCN4 pathway and potassium channels3.1 HL-I cells were divided into the control group,si-NC group,model group,SXSM group and si-NC+SXSM group.The cells in the si-NC group were transfected with nonsense siRNA.The cells in the model group were transfected with the siRNA targets on Bmp4,Kcnh2,Kcnq1 and Kcnma1,respectively.At 24 h of transfection,the cells in the SXSM and si-NC+SXSM groups were added with SXSM.The concentration of SXSM was 1 mL/L.RT-qPCR and Western blot test were used to detect mRNA and protein expression levels of related genes in HL-1 cells.3.2 The hiPSC-AMs were divided into the control group,si-NC group,model group and SXSM group.The cells in the si-NC group were transfected with nonsense siRNA.The cells in the model group were transfected with the siRNA targets on BMP4,KCNH2,KCNQ1 and KCNMA1,respectively.At 24 h of transfection,the cells in the SXSM group were added with SXSM.The concentration of SXSM was 1 mL/L.The beating rates and field potential duration of each group of the hiPSC-AMs were systematically analyzed via CardioExcyte 96.Result:1 Network meta-analysis of warming yang and benefiting qi Chinese patent medicines in the improvement of sinoatrial node function1.1 A total of 17 studies and 1206 patients with sinus bradycardia were included in this research,involving 3 kinds of warming yang and benefiting qi Chinese patent medicines,including Shenxian-Shengmai Oral Liquid,Xinbao Pill and Shenfu Injection.Among them,16 studies have reported the total effective rate of symptom improvement of sinus bradycardia patients,and 10 studies have reported the average heart rate of sinus bradycardia patients.12 studies have reported the information of adverse reactions.1.2 In terms of the total effective rate of symptom improvement,the order of therapeutic efficiency of warming yang and benefiting qi Chinese patent medicines is:Shenxian-Shengmai Oral Liquid>Shenfu Injection>Xinbao Pill.1.3 In the improvement of the average heart rate of patients,the order of efficiency of warming yang and benefiting qi Chinese patent medicines is:Shenxian-Shengmai Oral Liquid>Xinbao Pill.2 Mechanism research on the treatment of SSS with SXSM based on network pharmacology and molecular docking2.1 In conjunction with literature search,170 active ingredients of Shenxian-Shengmai Oral Liquid were screened and obtained via TCMSP database,including squalene,sugiol,danshenol A,etc.;947 potential targets were obtained,including PPARG,MTOR,CACNA1C,KCNH2,etc.;883 targets of SSS were obtained,including BPM4,KCNQ1,CACNA2D1,etc.;and 55 common targets were obtained.2.2 62 BP items were obtained through GO enrichment analysis,mainly including the regulation of heart development and cardiac conduction system;15 CC items,mainly including membrane protein complex,voltage-gated potassium channel complex,etc.;8 MF items,mainly including scaffold protein binding,voltage-gated calcium channel activity,etc.11 signaling pathways were obtained through KEGG pathway enrichment analysis,mainly including adrenergic signaling pathway,calcium signaling pathway,and renin secretion.2.3 According to the results of molecular docking analysis,16 compounds such as bavachin and erythrinin A can spontaneously bind to BMP4,KCNH2,KCNQ1 and KCNMA1.Among them,corylidin and KCNH2 can form the most stable molecular docking model.3 Research on the mechanism of SXSM in the treatment of SSS based on the BMP4/TBX3/HCN4 pathway3.1 RT-qPCR results showed that compared with the si-NC group,the mRNA levels of Bmp4,Tbx3 and Hcn4 in the si-Bmp4 group were significantly decreased(P<0.01);Compared with the si-Bmp4 group,the mRNA levels of Bmp4,Tbx3 and Hcn4 in the SXSM group were significantly increased(P<0.01);Compared with the si-Bmp4 group,the mRNA levels of Bmp4,Tbx3 and Hcn4 in the si-NC+SXSM group were significantly increased(P<0.01);There was no significant difference in the expression of Bmp4,Tbx3 and Hcn4 between the control group and the si-NC group.3.2 According to results of Western blot test,compared with the si-NC group,the protein levels of BMP4 and HCN4 in the si-Bmp4 group were significantly decreased(P<0.01);Compared with the si-Bmp4 group,the protein levels of BMP4 and HCN4 in the SXSM group were significantly increased(P<0.01);Compared with the si-Bmp4 group,the protein levels of BMP4 and HCN4 in the si-NC+SXSM group were significantly increased(P<0.01).3.3 According to the detection results of the beating rates of hiPSC-AMs,compared with the si-NC group,the beating rates of hiPSC-AMs in the si-BMP4 group were significantly decreased(P<0.01);Compared with si-BMP4 group,the beating rates of hiPSC-AMs in the SXSM group were significantly increased(P<0.05);There were no significant difference in the beating rates of cells between the control group and the si-NC group.According to the detection results of the field potential duration of hiPSC-AMs,compared with the si-NC group,the field potential duration of hiPSC-AMs in the si-BMP4 group was significantly longer(P<0.01);Compared with si-BMP4 group,the field potential duration of hiPSC-AMs in the SXSM group was significantly shortened(P<0.05).There was no significant difference in the field potential duration of hiPSC-AMs between the control group and the si-NC group.4 Research on the mechanism of SXSM in the treatment of SSS based on KCNH2,KCHQ1 and KCNMA14.1 According to RT-qPCR results,compared with the si-NC group,the mRNA levels of Kcnh2,Kcnq1 and Kcnmal in the si-Kcnh2 group,si-Kcnq1 group and si-Kcnma1 group were significantly decreased(P<0.01);Compared with the si-Kcnh2 group,si-Kcnq1 group and si-Kcnmal group,the mRNA levels of Kcnh2,Kcnq1 and Kcnmal in the SXSM group were significantly increased(P<0.01 or P<0.01);Compared with the si-Kcnh2 group,si-Kcnql group and si-Kcnmal group,the mRNA levels of Kcnh2,Kcnq1 and Kcnmal in the si-NC+SXSM group were significantly increased(P<0.01);There was no significant difference in mRNA expression between the control group and the si-NC group.4.2 According to the results of Western blot test,compared with the si-NC group,the protein levels of KCNH2,KCNQ1 and KCNMA1 in the si-Kcnh2 group,si-Kcnq1 group and si-Kcnmal group were significantly decreased(P<0.01);Compared with the model group,the protein expression levels of KCNH2,KCNQ1 and KCNMA1 in the SXSM group were significantly increased(P<0.01);Compared with the model group,the protein expression levels of KCNH2,KCNQ1 and KCNMA1 in the si-NC+SXSM group were significantly increased(P<0.01).4.3 According to the detection results of the beating rates of hiPSC-AMs,compared with the si-NC group,the beating rates of hiPSC-AMs in the si-KCNH2 group,si-KCNQ1 group and si-KCNMA1 group were significantly decreased(P<0.01);Compared with the si-KCNH2 group,si-KCNQ1 group and si-KCNMA1 group,the beating rates of hiPSC-AMs in the SXSM group were significantly increased(P<0.05 or P<0.01);There were no significant difference in the beating rates of hiPSC-AMs between the control group and the si-NC group.According to the detection results of the field potential duration of hiPSC-AMs,compared with the si-NC group,the field potential duration of hiPSC-AMs in the si-KCNH2 group,si-KCNQ1 group and si-KCNMA1 group was significantly longer(P<0.01);Compared with the si-KCNH2 group,si-KCNQ1 group and si-KCNM41 group,the field potential duration of hiPSC-AMs in the SXSM group was significantly shortened(P<0.01).There was no significant difference in the field potential duration of hiPSC-AMs between the control group and the si-NC group.Conclusion:1.In terms of the improvement of symptoms and heart rate of sinus bradycardia patients,among 3 kinds of warming yang and benefiting qi Chinese patent medicines,Shenxian-Shengmai Oral Liquid can be the best treatment plan for SSS for non-emergency.Warming yang and benefiting qi Chinese patent medicines in the treatment of sinus bradycardia has a small incidence of adverse reactions and high safety compared with western medicine.2.Shenxian-Shengmai Oral Liquid is a multi-component and multi-target therapy for SSS.Its therapeutic mechanism may be related to the differentiation of pacemaker cells,the regulation of cardiac conduction system,the transmembrane transport of calcium and potassium ions,as well as the cascade regulation of mitogen-activated protein kinase.3.The effect of SXSM in the treatment of SSS may be related to the up-regulation of BMP4/TBX3/HCN4 pathway and the expression of KCNH2,KCNQ1,KCNMA1 potassium channels.