The Network Target Prediction And Experimental Verification Of Wenxin Granule In Preventing And Treating Atrial Fibrillation

Background:Atrial fibrillation(AF)is a common arrhythmia in clinical practice,which can significantly increase the cardiovascular morbidity and mortality.At present,there are some limitations in western medicine and surgical treatment.The treatment of traditional Chinese medicine(TCM)has a unique advantage in the prevention and treatment of AF.Wenxin Keli(WXKL)is composed of Codonopsis Radix?Notoginseng Radix?Polygonati Rhizoma?Nardostachyos Radix Et Rhizoma?Ambrum.It has the functions of nourishing qi and Yin,promoting blood circulation and removing blood stasis,which is widely used in the treatment of AF.Because sy stematic research of whole animal model of WXKL for the prevention and treatment of AF is still lacking,the mechanism of its clinical efficacy is not clear,so further research is needed.Objectives:1.To predict the targets and signal pathway of WXKL in the prevention and treatment of AF and construct the WXKL-AF biological network by using the method of network pharmacology,so as to reveal the complex mechanism of multi-component,multi-target and multi-channel of WXKL in the prevention and treatment of AF.2.To observe the effect of WXKL on AF inducibility and AF duration in SAF rats and verify the predicted network targets preliminarily so as to lay a foundation for further exploring the mechanism of WXKL in the prevention and treatment of SAF.Methods:1.In the study of WXKL in the prevention and treatment of AF with the method of network targets prediction.TCMSP and TCM-mesh databases were searched to obtain the chemical components and targets of WXKL.CTD,OMIM and TTD databases were searched to obtain the related genes of AF,and the PPI(protein-protein interaction)network was integrated through STRING database.Cytoscape was used to construct the WXKL PPI network,AF PPI network.Through the analysis of network topology,the key targets of WXKL and AF network were obtained.Overlaping the intersection of the two networks to construct the WXKL-AF PPI network.Using the plug-in MOCDE to cluster the WXKL-AF PPI network.so as to identify the core cluster module of the PPI network.Finally,the David database was used to analyze the targets of core modules by GO and KEGG pathway analysis,and summarize the results.2.To establish the model of SAF by isoproterenol(ISO)combined with transesophageal atrial burst pacing.WXKL was used as the observation drug and amiodarone as the positive control drug.The two drugs were intervened two weeks in advance,and the burst group and ISO combined burst group(model group)were set up for comparison.The changes of heart rate,mortality,AF inducibility and AF duration were monitored by electrophysiological instrument.3.Through the experimental methods of Quantitative real-time PCR to detect the relative gene expression of PI3K?AKT1?NOS3?RYR2 in each group of rats.Results:1.The prediction and analysis of network targets(1)236 active components and 699 targets of WXKL were obtained from TCMSP and TCM-mesh databases.695 targets were obtained by mapping the targets to the STRNG database.The WXKL PPI network was constructed,and 113 core nodes of the network were obtained through network topology analysis.Most of these nodes were related to cell proliferation,growth,metabolism,differentiation,apoptosis,and neurotransmitters and endocrine hormone,inflammation,intravascular environment,and so on.(2)324 genes related to AF were obtained from CTD,OMIM and TTD databases.285 disease targets were obtained by mapping the genes to STRING database to construct the AF PPI network.The network topology analysis showed 110 core nodes of the network,most of which were related to ion channels,cell proliferation and apoptosis,inflammation,intravascular environment,etc.(3)The WXKL-AF PPI network contains 80 targets*The WXKL PPI network regulates 84.38%nodes in the AF PPI network with degree value greater than 50,After cluster analysis,5 modules were obtained,and 2 core modules with score greater than 5.It mainly includes the targets of AKT1,NOS3,ADRB1,RyR2,KCNH2.SCN5A and VEGFA.(4)Through the analysis of the gene ontology(GO)enrichment analysis and the Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis,it was found that WXKL mainly regulates the development of AF through protein synthesis and transport,cell proliferation and apoptosis,cardiac conduction and heart rate regulation,hormone endocrine,intravascular environment,oxidative stress and inflammatory response,and may play a role in AF through PI3K-Akt signaling pathway,adrenergic signaling in cardiomyocytes,TNF signaling pathway,MAPK signaling pathway,HIF-1 signaling pathway.2.The exploration of the pharmacodynamic of animal experiment(1)Mortality:compared with the control group,the mortality of rats in the model group(P<0.01),(low dose of WXKL group)WXKL-L group(P<0.05),amiodarone group(P<0.05)was increased;compared with the burst group,the mortality of rats in the model group was increased(P<0.05);compared with the model group,the mortality of rats in the(high dose of WXKL group)WXKL-H group was decreased(P<0.05).(2)Heart rate:compared with the control and burst group,the heart rate of rats in the amiodarone group(P<0.001)and the model group(P<0.01)decreased significantly after two weeks of intervention;compared with the amiodarone group,the initial heart rate of rats in the WXKL-L and WXKL-H group was faster(P<0.01),and the heart rate remained more stable.After the injection of ISO,the heart rate of rats in each group generally increased by about 20%compared with the initial heart rate.After transesophageal atrial burst pacing,the heart rate of rats in each group was generally reduced by 10-20 bpm.(3)AF inducibility:compared with the control group,other groups' AF inducibility significantly increased(P<0.01),which indicates that the model was stable;compared with the burst group,the AF inducibility of the model group increased,but there was no statistical difference;compared with the model group,the AF inducibility in the WXKL-H group decreased,but there was no statistical difference.(4)AF duration:compared with the control group.the duration of AF in other groups was significantly longer(P<0.05);compared with the burst group(12.7±5.2s),the injection of ISO could significantly increase the duration of AF(421.50±130.1 s,P<0.01);compared with model group,WXKL-H group(24.7±13.1,P<0.01)and amiodarone group(19.8±12.2,P<0.01)significantly shortened the duration of AF.3.The validation of network targetsThe expression of PI3K?AKT1?NOS3?RYR2 in model group was significantly decreased compared with control group.The expression ofA KT1?NOS3?RYR2 in WXKL-H group and amiodarone group was significantly increased compared with model group.The expression of PI3K also increased,but there was no statistical difference.Conclusion:In this study,through the method of network target prediction,it concluded that the network targets mainly include AKT1?NOS3?ADRB1?RYR2?KCNH2?SCN5A?VEGFA,etc.The biological functions of WXKL in the prevention and treatment of AF were preliminarily explained which mainly reflected in protein synthesis and transport,cell proliferation and apoptosis,cardiac conduction and heart rate regulation,intravascular environment,hormone endocrine,oxidative stress and inflammatory response and so on.WXKL may plays a role in the prevention and treatment of atrial fibrillation through PI3K-Akt signaling pathway,adrenergic signaling in cardiomyocytes,TNF signaling pathway.MAPK signaling pathway and HIF-1 signaling pathway.The results of animal experiments showed that WXKL could shorten the AF duration of rats induced by ISO combined with transesophageal atrial burst pacing,and to a certain extent reduce the AF inducibility,reduce the mortality of rats.The experimental validation of network targets suggests that WXKL may inhibit the development of AF by regulating PI3K?AKT1?NOS3?RYR2 to inhibit apoptosis,improve endothelial function,and maintain myocardial calcium homeostasis.The establishment of the animal model and the preliminary verification of its mechanism laid a foundation for further exploration of the mechanism of WXKL in the prevention and treatment of SAF.