| Background:Arrhythmia,which is manifested by tachycardia or bradycardia,is common and occurs with complex incentives.It not only affects the patient's quality of life,but also seriously threatens the patient's life.At present,there are three main causes of bradycardia:gene mutation of ion channels of the myocardial bioelectric conduction system,abnormal function of the autonomic nervous system,and drug or other diseases induced.The autonomic nervous system?ANS?plays an important regulatory role in the pathological process of bradycardia.In recent years,more and more research evidence has shown that regulating the ANS system is a safe and effective method against arrhythmia.ANS is composed of sympathetic nerves and parasympathetic?vagal?nerves,and acts on the corresponding adrenergic and cholinergic receptors by releasing neurotransmitters.The?-adrenergic effect on cardiomyocytes is the main manifestation of sympathetic regulation.By coupling the activation of G protein,the downstream pathway enzyme activity,phosphorylation process,calcium influx,etc.are affected.Sympathetic nerves suppression is easy to cause bradycardia[1].Current clinical treatments for bradycardia include western medicines such as atropine,isoproterenol,and dopamine to accelerate heart rate,as well as proprietary Chinese medicines such as Xinbao pills[2].Western medicine can be used as a quickly rescue medicine,and Chinese medicine may have advantages in long-term use.In addition,for some patients who are not suitable for placing a permanent cardiac pacemaker,the use of clinically effective Chinese medicine for intervention can reduce their treatment burden and avoid the immune response caused by implantation,which is a better choice[3].By integrating data platforms,clinical experience and the exploration of prescriptions,a macro analysis can guide the research direction.Network analysis based on a large amount of data is also the development of modern traditional Chinese medicine.The application of traditional Chinese medicine theory in the treatment of arrhythmia can be traced back to Zhang Zhongjing of the Han Dynasty.It has a long history.Shenxian Shengmai Oral Liquid is a clinically used Chinese patent medicine for treating bradycardia with heart-kidney-yang deficiency syndrome.It contents eight flavors of traditional Chinese medicine:Yinyanghuo?Hongshen?Danshen?Buguzhi?Mahuang?Xixin?Shuizhi?Gouqizi.This study aims to use big data analysis combined with isolated heart perfusion and other experimental methods to preliminary explore the mechanism of Shenxian Shengmai oral liquid on autonomic neuromodulation in treating bradycardia.Methods:The traditional Chinese medicine inheritance assistant platform was used to summarize the rules of TCM for the treatment of arrhythmia with heart-kidney-yang deficiency syndrome,and many clinical research literature was collected to evaluate the efficacy of SXSM for bradycardia.IPSC-CMs cells were cultured in vitro,and the effects of SXSM on cell beat frequency and field potential impedance were measured.Rat models in vitro and in vivo were used to test the effects of Shenxian Shengmai Oral Liquid on heart rate?HR?and heart rate variability?HRV?.The rat serum norepinephrine levels,sympathetic nerve marker protein tyrosine kinase?TH?and norepinephrine transporter NET gene expression level of myocardial tissues were measured.According to the results of the chemical identification of Shenxian Shengmai Oral Liquid in previous work,the chemical structures of all components were collected from Pubchem.The target protein action relationship,signaling pathway,and biological function prediction were performed.Based on the results,Operetta high-content imaging system was used to evaluate pathway upstream protein expression and gene level changes.Results:The analysis of the bradycardia in heart-kidney-yang deficiency medication rule revealed that the top ten used in formula were:Yinyanghuo?Shudihuang?Huangqi?Gouqizi?Danggui?Tusizi?Roucongrong?Gancao?Lurong?Renshen.Meta analysis showed that the effective rate of control group was 67.34%,while that of the Shenxian Shengmai oral liquid treatment group was 88.14%.The total effective rate was CI=7.73,I2=0.52,Z=9.47?P<0.00001?.SXSM has better clinical efficacy in bradycardia patients.Human induced pluripotent stem cells-Cardiomyocytes?i PSC-CMs?cell pulsation experiments in vitro showed that cell beat of the Shenxian Shengmai oral liquid group could be increased from 30 to 54times/min.The results of isolated heart perfusion experiments found that acetylcholine can cause significant bradycardia,the heart rate of the normal group and the model group were173±52 bpm and 67±35 bpm,respectively.The SXSM treatment group significantly increased heart rate to 168±61 bpm.The LF/HF ratio also increased significantly,with 62±15 and196±37 in the model and treatment groups,respectively.Pretreatment of SXSM could increase the release level of neurotransmitters,but it could be blocked by?-receptor antagonists.The expression level of TH in myocardial tissue in SXSM group increased,but there was no change in the expression level of the adrenaline transporter NET gene.The results of network pharmacological predictions indicate that SXSM may play a role through the?1 adrenergic receptor signaling pathway.Immunofluorescence staining results of ADRB1protein proved that high dose of Shenxian Shengmai Oral Liquid?1mg/ml?can increase the expression of this gene,and subsequent PCR results also showed the same trend.Conclusion:This study found that Shenxian Shengmai Oral Liquid can affect sympathetic nerve activity,increase the release of the neurotransmitter norepinephrine for the first time,and it can also activate the?1 adrenergic receptor signaling pathway suggesting that the neuromodulatory effect of SXSM may be one of the main mechanisms for the treatment of bradycardia.This study refers to the clinical research and the rules of medication to screen and analyze the experimental objects,highlighting the necessity of traditional Chinese medicine theory guidance for the modern pharmacology research of TCM.Cardiomyocytes pulsation,isolated heart perfusion in vitro and in vivo drug induction model were used to reveal the pharmacodynamic effect of Shenxian Shengmai Oral Liquid.Aimed at derived from clinic and used in clinic,predict and verify the mechanism of multi-target signaling pathway by network pharmacology method provides an efficient and practical method for the development of large-scale Chinese medicine varieties research. |
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