WuShen Mitigates Cardiac Dysfunction And Prevents Cardiac Remodeling And Arrhythmias In Post-myocardial Infarction Rats

Part Ⅰ:Analysis of the Potential Mechanism of WuShen on Cardiovascular Disease:A Network-based Approaches in Pharmacology.PURPOSES:As the pathogenesis of cardiovascular diseases is rather multi-factorial than a single cause,the phytomedicine may offer novel treatment opportunities based on a network pharmacological effect.A systems pharmacology-based strategy was used to pinpoint the potentially active compounds and therapeutic mechanisms of WuShen prescription for Cardiovascular Diseases(CVD)treatment.Systems pharmacology is a new strategy for new drug development,especially for a complex chemical composition of TCM,through screening potential candidate compounds,predicting targets,and eventually constituting the active compounds-targets-pathways-diseases networks.Wushen Decoction is widely used in the treatment of cardiovascular diseases in clinical practice.The present study pointed out that WuShen decoction improved LV function,reduced infarction size and ventricular arrhythmias by mitigating ventricular fibrosis and Cx43 gap junction remodeling in rats with myocardial infarction.PURPOSES:The purposes of this section are to analyze the potentilly active components of WuShen,to identify the target of each active ingredient,to construct the“drug-target”,“drug-disease”network,and to predict the possible molecular mechanism of action of WuShen.Although the animal experiments of this study have been completed,the analysis of the previous research data of the potentially active ingredients of WuShen can provide us more clues for the in-depth research and future development of WuShen.METHODS&RESULTS:The potentially active ingredients of WuShen were extracted by TCM System and Network Pharmacology Database and Analysis Platform.Then,the oral bioavailability(OB)analysis,drug-likeness(DL)analysis and Half-life(HL)screening of each compound were analyzed for identifying candidate active compounds.Compounds with OB≥30%,DL T value≥0.18,and HL>4 hours were selected as the candidate active compounds.Besides,we also collected several active compounds with OB<30%,DL<0.18 and HL<4 but with bioactivity according to previously published literatures.In conclusion,total 116 compounds were collected as the candidate active compounds.The computer model is used to predict the target information of drug candidates by using the WES model calculation method.The main contents within WES calculation include the determination of the physical and chemical structural properties and pharmacological properties of the drug by CDK fingerprint matrix and Dragon descriptor matrix by using statistical methods.The work flow of WES algorithm includes,a.Recognizing the key ligand structural features highly related to the pharmacological properties in a framework of ensemble through CDK print matrix and Dragon descriptor matrix;b.Identifying the compound-target affiliations through evaluating the overall similarity of target ensemble;c.Consolidating the standardized ensemble similarities(Z score)by Bayesian network and then extracting target prediction results.To explore the potential therapeutic mechanisms of WuShen prescription on diseases,Cytoscape software was used to construct Compound-Target,Target-Pathway,Target-Disease,and the cardiovascular diseases pathway-therapeutic modules in the present work.A total of 880 compounds were collected from five herbals,including ginseng(190),salvia miltiorrhiza(495),scrophulariaceae(45),Radix glehniae(35)and Sophora flavescens(113).These compounds were subjected to computer network pharmacology.A total of 116 candidate compounds and 130 potential targets were obtained from the analysis.These 116 compounds and 130 potential targets together constitute a compound-target interaction network of 1376 sides.A target-pathway interaction network consisting of 82 targets and 24 pathways associated with cardiovascular disease was finally obtained.These targets and pathways are mainly related to cardiovascular diseases,such as atherosclerosis,myocardial infarction,myocardial reperfusion injury,hypertension,and arrhythmia.An in-depth analysis of the target-disease interaction network was obtained and suggested that,WuShen can mainly treat cardiovascular diseases(Degree=61),endocrine system diseases(Degree=31),nutritional disorders(Degree=17),neurological diseases(Degree=26)and congenital diseases(Degree=4).In turn,WuShen may play a protective role in cardiovascular system by intervening endocrine,nutritional metabolism,and nervous system.CONCLUSIONS:The network-pharmacological analysis of WuShen is expected to screen out the active ingredients,to determine the drug-target,drug-disease network,to predict the possible molecular mechanism of action of Wuhen.These information can provide more clues for the in-depth research and development of WuShen in the future and help create more changes for drug discovery and disease treatment.Part Ⅱ:Development and Validation of U-HPLC-LTQ-Orbitrap MS Method for Chinese Ancient Medical Prescription WuShen.PURPOSES:WuShen decoction has been clinically used in the treatment of CVD such as coronary heart disease,heart failure and arrhythmias,etc.,and has gained much attention because of its well health benefits.Our present study showed WuShen decoction could significantly improve cardiac function and inhibited inducible ventricular arrhythmias and the inducibility and duration of atrial fibrillation.It is particularly important to detect and identify the material basis of WuShen.The purpose of this study is therefore to initially identify the potential active ingredients of WuShen and establish the quality standard of WuShen in this research system.The significance of this session includes:a.Preliminary establishment of the fingerprint of WuShen decoction used in this research system;b.Establishing quality standards for this study which is conducive to the repeatability of the experiment;c.Exploring the active ingredients of the compound to facilitate a further development of the prescriptions;d.Preliminary determination of the approximate content of the main active ingredients is also conducive to the exploration and estimation of the dosage in future clinical trials.METHODS&RESULTS:Sample of WuShen decoction was prepared by water-extraction and alcohol-precipitation method.Radix Ginseng Rubra,Salvia Miltiorrhiza Bunge,Radix Scrophulariae,Radix Glehniae,Radix Sophorae Flavescentis,and Wushen(the prescription),were decocted and condensed into an extractum(concentration lg/mL)respectively.The extractum was cooled down and mixed in 70%alcohol for removing impurities(alcohol solutions were collected).Then,the solutions were removal of alcohol by Rotary evaporator and condensed into a concentration of lg/mL.Ultra-performance liquid chromatography-mass spectrometer(UPLC-MS)was used to identify the key active constituents of Wushen decoction.Total twelve standard products of Chinese medicine were used as reference standards.Chromatographic separation was performed on an AccelaTM U-HPLC system as previously reported.The LC conditions 1 was as follows:Column:Inertsil ODS-3 C18,100 X 2.1 mm,3.0 μm particle size(Shimadzu,Japan);Mobile phase:(A)MeOH;(B)Water with 0.1%formic acid;Flow rate:300 μL/min;Injection volume:3 μL;LC Gradient for positive ESI-MS:5-25%A(0-21 min),25-58%A(21-30 min),58-73%A(30-65 min),100%A(75-80 min).LC Gradient for negative ESI-MS:25%A(0-21 min),25-58%A(21-30 min),58-73%A(30-65 min),73-100%A(65-75 min),100%A(75-80 min).The MS analysis was operated in both positive and negative electrospray mode,a mass range of 125-1500 with resolution set at 30,000.The data-dependent MS/MS events were performed on the most intense ions detected in full scan MS,and the normalized collision energy was 35%.Nitrogen was used as sheath gas and helium served as the collision gas.The key electrospray parameters were as follows:sheath gas(nitrogen):45 L/min,auxiliary gas flow:5 L/min,capillary temperature;325.0℃.The source voltage:4.0 kV(Positive ion mode)/3.5 kV(Negative ion mode).Data was collected and analyzed with Xcalibur 2.07(Thermo Scientific).The Orbitrap mass analyzer was calibrated according to the manufacturer’ s guidance using a commercial calibration fluid.A total of 12 active ingredients of 5 herbals were identified,including:matrine,oxymatrine,Salvianolic acid B,Ginsenoside Rd,Ginsenoside Rg1,Ginsenoside Re,Angoroside C,Harpagoside,Scopoletin,Tanshinone ⅡA and Cryptonshinone.CONCLUSIONS:A total of 12 active ingredients of 5 herbals were identified,including:matrine,oxymatrine,Salvianolic acid B,Ginsenoside Rd,Ginsenoside Rg1,Ginsenoside Re,Angoroside C,Harpagoside,Scopoletin,Tanshinone ⅡA and Cryptonshinone.These active ingredients are important material basis for the efficacy of WuShen.Part Ⅲ:WuShen Decoction Improves Ventricular Function and Prevents Ventricular Remodeling and Arrhythmias in post-MI rats.PURPOSES:Traditional Chinese Medicine(TCM)has long been used as major therapy for the treatment of cardiovascular diseases(CVD)in China since thousands of years ago.There are tremendous numbers of classics of TCM documenting the physiology and pathology of CVD and the principles and measures of prevention and treatment of CVD.The prescription“WuShen”was jot down firstly in the Volume 12th of book Qianjin Yifang by Sun Simiao,used for the treatment of CVD and gastrointestinal dysfunction initially.Since the reform and opening up of China,Wushen decoction has been clinically used in the treatment of CVD such as coronary heart disease,heart failure and arrhythmias,etc.However,little is known about its specific mechanism.The present study aimed to investigate the therapeutic effect of WuShen decoction in cardiac dysfunction and to demonstrate whether WuShen decoction could reduce the inducibility of ventricular arrhythmias induced by programmed electrical stimulation(PES)in a rat model of myocardial infarction with left anterior descending ligation.METHODS:Three days after surgery,post-MI rats were randomly treated with low-dose,middle-dose,and high dose of WuShen decoction(WS-L,WS-M and WS-H,n=21,21 and 21 respectively)or vehicle(n=25 for 5 weeks.Sham-operated rats were treated with the same dose vehicle(n=15).Echocardiography was used to assess cardiac function.PES test was used to induce ventricular arrhythmias which were then valued by a scoring system.Sirius red staining was used to assess infarction size and collagen deposition.Immunohistochemistry or western blot were conducted to investigate proteins expression involved in TGF β 1/Smads and TXNIP/NLRP3/Caspasel/IL1 β/IL18 mediated fibrosis and Racl/NADPH oxidase/CTGF/Cx43 mediated gap junction remodeling.RESULTS:WuShen decoction significantly improved cardiac function and inhibited the severity and inducibility of ventricular arrhythmias.WuShen decoction significantly reduced cardiac index and fibrosis and ventricular enlargement,improved the protein expression and distribution of Cx43 gap junction.Besides,WuShen decoction significantly inhibited TGF β 1/Smads and TXNIP/NLRP3/Caspase1/IL1 β/IL18 mediated fibrosis.CONCLUSIONS:WuShen decoction improved LV function,reduced infarction size and ventricular arrhythmias by mitigating ventricular fibrosis and Cx43 gap junction remodeling,potentially via inhibiting TGF β 1/Smads and TXNIP/NLRP3/Caspase1/IL1 β/IL18 pathways.Part Ⅳ:WuShen Decoction Reduces the Susceptibility to Atrial Fibrillation by Inhibiting Atrial Structural Remodeling in post-MI rats.PURPOSES:Atrial fibrillation(AF),the most common sustained cardiac arrhythmia,is now becoming increasingly more and more widespread.Atrial interstitial fibrosis and atrial enlargement are the main aspect of atrial structural remodeling and play an important role in the occurrence and maintenance of AF.Therapies toward inhibiting atrial structural remodeling are regarded the upstream therapy for AF.WuShen decoction is used in the treatment of cardiovascular diseases in China.However,its applications in the prevention of atrial structural remodeling and AF are not largely revealed.METHODS:Three days after surgery,post-MI rats were randomly treated with low-dose,middle-dose,and high dose of WuShen decoction(WS-L,WS-M and WS-H,n=21 in each group)or vehicle(n=25)for 5 weeks followed with the route in research section 1.Sham-operated rats were treated with the same dose vehicle(n=15).After 5 weeks of treatment,echocardiography was used to assess atrial inner diameter.The morphologies of electrocardiogram including P-waves duration,PR intervals and P to PR ratio were also analyzed to assess atrial remodeling partly.Transesophageal atrial burst pacing was implemented to test the vulnerability to AF.Pacing was repeated three times to acquire AF inducibility and AF duration.After all of these measurements,Hearts were then moved out,dewatered,embedded in paraffin,cut(3 μm thickness),and stained with Sirius red/fast-green counter stain to assess atrial interstitial fibrosis.RESULTS:Results from echocardiographic assessment showed that MI induced a significant enlargement of the left atrium,however,WuShen decoction significantly inhibited left atrial diameter(all P<0.01,MI compared with WS-L,WS-M and WS-H).Besides,WuShen decoction also reduced significantly the P-waves duration(all P<0.001,MI compared with WS-L,WS-M and WS-H)and P to PR ratio(all P<0.001,MI compared with WS-L,WS-M and WS-H),and decreased both AF inducibility(all P=0.000,MI compared with WS-L,WS-M and WS-H)and duration(all P=0.000,MI compared with WS-L,WS-M and WS-H).Sirius-red staining showed that WuShen decoction significantly inhibited atrial fibrosis including both left atrial septum and left atrial appendage.CONCLUSIONS:WuShen decoction significantly inhibited left atrial diameter,P to PR ratio,decreased both AF inducibility and duration,mitigated atrial fibrosis,suggesting WuShen decoction can constitute an upstream therapy for AF by suppressing atrial enlargement and fibrosis.