| Background:Atrial fibrillation(AF)is a common clinical tachyarrhythmia.It was reported that the prevalence of AF is currently 2%-4%in adults worldwide,with more than 60 million people suffering from AF.And the prevalence of AF is expected to increase exponentially in the future as people live longer,the population ages,and the diagnosis of atrial fibrillation is enhanced.AF leads to serious adverse events such as stroke,heart failure,and cognitive impairment,with high rates of disability and death,resulting in a significant medical burden.Rhythm control is an important measure in the management of AF.However,the clinical efficacy of chemical cardioversion is unsatisfactory,with arrhythmogenic and other adverse effects,and the recurrence rate after radiofrequency ablation is high.Based on many years of clinical practice,my supervisor innovatively proposed the theory of "Co-modulation of pulse and spirit " and created an effective formula for the treatment of paroxysmal atrial fibrillation,the Dingchan Formula,while the clinical efficacy of which has not yet been studied in a large sample and the mechanism of action is not yet clear.Objective:1.To further evaluate the clinical efficacy of Dingchan Formula in the treatment of paroxysmal AF(Qi Yin Liang Xu,Xin Shen Bu Ning Syndrome)with the " Co-modulation of pulse and spirit " theory.2.To initially screen the potential signaling pathways and targets for the treatment of AF through network pharmacology research.3.The animal experiments were conducted based on the results of network pharmacology and the "β1-AR/cAMP/PKA signaling pathway" to explore the potential action mechanism of Dingchan Formula in the treatment of paroxysmal AF,in order to find new approaches for the treatment of atrial fibrillation,and to lay the foundation for further research in the future.Methods:1.Clinical study:A retrospective cohort study was adopted to collect case data of outpatients with paroxysmal AF,and they were divided into exposed and non-exposed groups according to whether taking Dingchan Formula in addition to routine Western medicine treatment.The exposure time was 8 weeks,and the effects of Dingchan Formula on the times of AF episodes,duration,frequency,EHRA symptom scale,as well as the clinical efficacy and the occurrence of adverse events were observed and analyzed.2.Network pharmacology research:The TCMSP database,Herb Herbal Group,BATMAN-TCM and Shanghai Organic Chemistry database were searched to find and collect the active ingredients of the Dingchan Formula core drugs.Then these ingredients were submited to SwissTargetPrediction platform for ADME prediction.The OMIM,DrugBank,Genecards,TTD,and DisGeNET database were searched to find the relevant targets of AF,and then the screened AF-target intersection was submitted to Metascape platform for GO analysis and KEGG pathway enrichment analysis to construct the interrelationship network of "active ingredients-AF-targets ".3.Experimental study:48 SD rats of 6～8 weeks were randomly divided into blank group,model group,Dingchan Formula group and amiodarone group,and were gavaged with saline,aqueous extract of the formula and amiodarone suspension for 14 days respectively.The AF model was prepared by intraperitoneal injection of isoprenaline(ISO)combined with esophageal Burst pacing electrical stimulation.Electrophysiological indexes such as effective refractory period(ERP),AF induction rate and AF duration were observed in each group.The levels of NE and 5-HT in serum and cAMP in atrial tissues were measured by Elisa method,and the expression of β1AR,PKA,Cav1.2 and RyR2 proteins and genes in atrial tissues of rats were measured by Western-blot and Real-time PCR.Results:Clinical findings:1.Times of AF episodes:After treatment,the number of AF episodes was significantly reduced in both groups(P<0.01),and the exposed group was significantly less than that in the non-exposed group(P<0.01).2.AF duration:After treatment,the duration of AF was significantly shorter in both the exposed and non-exposed groups(P<0.01),and the exposed group was significantly less(P<0.01).3.Frequency of AF episodes:After treatment,compared to the non-exposed group,the frequency of AF episodes in the exposed group was significantly less(P<0.01).4.Clinical efficacy:The total effective rate was 77.27%in the exposed group and 53.03%in the non-exposed group after treatment,with statistically significant differences between the two groups(P<0.01).5.EHRA symptom scale:After treatment,the improvement of EHRA symptom scale score was significantly better in the exposed group than in the non-exposed group(P<0.01).6.24h Holter indexes:After treatment,the total heart rate,fastest heart rate,average heart rate and the number of premature atrial beats were significantly lower in the exposed and non-exposed groups than before(P<0.01),among which the fastest heart rate was significantly lower in the exposed group than that in the nonexposed group(P<0.01).7.TCM syndrome scores:compared to before,the scores of TCM syndrome in the two groups were significantly lower(P<0.01),and the exposed group were better(P<0.01).8.TCM symptoms efficacy:After treatment,the total efficacy of TCM symptoms in the exposed group was 84.85%,while the total efficacy of TCM symptoms in the non-exposed group was 42.42%,and the exposed group was significantly better than that in the non-exposed group(P<0.01).9.Western medicine reduction or discontinuation:After treatment,the reduction or discontinuation rate of anti-arrhythmic Western medicine in the exposed group was 30.3%,and that in the non-exposed group was 7.58%,and the exposed group was significantly better than that in the non-exposed group(P<0.01).Network pharmacology research results:1.Drug and disease target screening:996 targets were obtained from the 7 core drugs of the Dingchan Formula,955 targets were obtained for AF disease,and 168 targets were finally obtained for AF disease-Dingchan Formula intersection.2.GO analysis results:1953 biological processes were involved,mainly including blood circulation,ion transport,hormone regulation,transmembrane transport,ion transmembrane transport regulation,etc;199 molecular functions were involved,mainly including adrenergic receptor activity,voltage-gated ion channel activity,protein kinase activity,cation channel activity,catecholamine binding,etc.;120 cellular components were involved,mainly including membrane rafts,cation channel complexes,potassium channel complexes,etc.3.KEGG pathway enrichment analysis results:197 signaling pathways were involved,mainly including 5-hydroxytryptaminergic synapses,cAMP signaling pathway,adrenergic signals in cardiac myocytes,etc.;4.The results of active ingredient-target-pathway network construction of Dingchan Formula:the active ingredients of Dingchan Formula for AF treatment mainly included kaempferol,quercetin,leucovorin,leucovorin.hippurine,glycyrrhizin B,naringenin,glycyrrhizin,DL-hojenine,alanine,date palm cyclopeptide,daidzein and so on.The core targets mainly include MMP9,MMP2,ADORA1,HTR1 A,ADRB 1,ADRA2C,ADRA2A,TADRB2,CACNA1C,HTR1B and so on.Animal experimental results:Study one:1.Heart rate comparison:after intraperitoneal injection of ISO,compared with the control group,the heart rate of rats in the model group,Dingchan Formula group and amiodarone group increased significantly(P<0.01,P<0.05);compared with the pre-injection,the heart rate of rats in the model group,Dingchan Formula group and amiodarone group increased more significantly(P<0.01),and no statistical difference among each group was found(P>0.05).2.Atrial ERP comparison:compared with the model group,the ERP was prolonged in both the Dingchan Formula group and the amiodarone group,while the difference was not significant(P>0.05).3.Times of AF induced comparison:The times of AF induced in the Dingchan Formula group and the amiodarone group was significantly reduced compared to the model group(P<0.01),and the difference between the Dingchan Formula group and the amiodarone group was not statistically significant(P>0.05).4.Comparison of the AF duration:the duration of AF was significantly shorter in the amiodarone group than in the Dingchan Formula group(P<0.05);5.Ventricular fibrillation(VF)/ventricular tachycardia(VT)induction:the amiodarone group had the most ventricular fibrillation induction times and the longest duration.Study two:1.Comparison of serum NE and 5-HT levels:The serum levels of 5-HT and NE were significantly higher in the model group rats compared to the control group(P<0.01).The serum levels of 5-HT were increased in the Dingchan Formula and amiodarone groups(P>0.05),and the levels of NE were significantly lower compared to the model group(P<0.05).2.Comparison of cAMP levels in atrial tissues:There was no significant difference in cAMP levels among different groups in rat atrial tissues(P>0.05).3.Expression results of ADRB1,PKA,Cav1.2,and RyR2 proteins and genes in atrial tissues:Compared with the control group,the expression levels of ADRB1 protein,ADRB1 gene,PKA gene and RyR2 gene in the atrial tissues of model group were significantly increased(P<0.01),and the levels of Cav1.2 protein and gene were significantly decreased(P<0.01).The expression levels of ADRB1 protein,PKA protein,ADRB1 gene,PKA gene and RyR2 gene in the Dingchan Formula group and amiodarone group were significantly decreased compared with the model group(P<0.01,P<0.05),while the expression levels of Cav1.2 protein and gene were significantly increased(P<0.01).No statistical difference was found in PKA or RyR2 protein expression among each group(P>0.05).Conclusions:Dingchan Formula guided by the theory of "Co-modulation of pulse and spirit "can significantly decrease the times,duration,and frequency of paroxysmal AF episodes,lower the heart rate,reduce atrial ectopic beats,alleviate the discomfort symptoms,reduce the use and dose of anti-arrhythmic western drugs,and effectively inhibit the occurrence and maintenance of AF.The potential scientific mechanism of "Co-modulation of pulse and spirit " for the treatment of AF is related to the β1-AR and Cav1.2 in myocardial cells,which may play an antagonistic role in paroxysmal AF by inhibiting sympathetic nerve activity and calcium overload. |
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