Effects of Dingjifumai Decoction on Electrocardiogram and sodium potassium pump of rats with ventricular arrhythmia

Ling Fu,Hui-Ling Liao,Yan Zhou,Fei-Hu Zou,Ling Luo

1ChongQing Traditional Chinese Medicine Hospital,ChongQing,China.2Southwest Medical University,LuZhou,SiChuan,China.3Traditional Chinese Medicine Hospital,TongLiang,ChongQing,China.

Background

Ventricular Arrhythmia(VA)is a type of the common clinical cardiovascular disease emergency,Ventricular Premature beat(VP),Ventricular Tachycardia(VT),Ventricular Fibrillation(VF)and other all belong to VA[1]the category,Common causes include various organic heartdiseases,diseasesotherthan cardiac causes,electrolyte acid-base balance disorders,etc.[2].The unpredictability and potentially catastrophic consequences of VA are forcing doctors to seek treatment to prevent and treat VA.At present,the four types of anti-arrhythmic drugs commonly used in clinical practice have significant side effects causing new arrhythmias,while traditional Chinese medicine has fewer side effects and stable efficacy,and can reduce the side effects of western medicine anti-arrhythmias[3].Therefore,it is of great significance to develop effective Chinese herbal preparations for the prevention and treatmentof ventricular arrhythmia.In the clinical diagnosis and treatment,it was found that Dingjifumai decoction(DJFM)had a better effect in anti-ventricular arrhythmia,so this experiment was designed for research.

Materials and methods

Experimental materials and instruments

Experimental animalsForty healthy male SD rats of clean grade,weighting(200±20)g(provided by Animal Experiment Center of Southwest Medical University,license number:SCXK(Chuan)2013-17).

Grouping and processingThe rats were weighed and given adaptive feeding for 3 days,and then randomly divided into 4 groups and numbered,10 rats in each group,respectively the blank group (normal saline 2ml/200g),modelgroup (normalsaline2ml/200g),Metoprolol group and DJFM group.The Metoprolol group was administered a Metoprolol suspension at a daily dose of5.2mg/kg.DJFM iscomposed of Suanzaoren(Ziziphus jujuba Mill.var.spinosa),Fushen(Poria cocosSchw.),Longgu (OsDraconis),Muli(Oyster), Guizhi (CassiaTwig),Yuanzhi (Polygala tenuifolia Willd.),Dazao(Ziziphus zizyphus),Hehuanpi(Silktree Albizia Bark),Zhigancao(Glycyrrhizae.),et al.It is purchased from Sichuan New Green Pharmaceutical TechnologyDevelopmentCo.LTD.Eachratwas gavaged once a day with a dose of 17.6 g/kg per day for a total of 14 days.

Model establishment

The rats were weighed before the last gavage on 14 days.

Ten ratsin each group were injected with 1%pentobarbital sodium at 30mg/kg and anesthetized.The anesthetized rats were placed in prone position on a wooden board.Insert the acupuncture needle into the rat,connect the electrode ang the electrocardiogram(ECG)[12].Tracing the II lead ECG,recording the normal ECG,zeroing THE baseline,waiting for the rat’s ECG to stabilize,the model group immediately injected 0.001%Aconitine 30 ug/kg solution through tail vein,and the blank group injected the volume 0.9%saline.The bolus is completed within 5 seconds,and began to record ECG for 20 minutes,record the VP,VT and VF in time.After the observation,cut the chest skin and frame with scissors,immediately open the chest,cut all blood vessels connected to the heart tissue eat the bottom of the heart and take out the whole heart,squeeze out the blood in heart cavity,and dry the residual blood on the surface of the heart gauze.After that,the left ventricle cut was excised,and a part of the cut was placed in a cryotube,stored in a-80°C refrigerator,and the heart tissue sodium potassium pump detection wasperformed according to the kit instruction.

Results

Model establishment

In thisstudy,aconitine wasnotused to induce arrhythmias in the blank group,so VP,VT,VF and other VA did not occur in the blank group,so the pair-based comparison of experimental results did not involve the blank group.After intravenous injection of Aconitine,the incidence of VP,VT and VF in the model group was 100%,indicating that the VA rat model was successfully constructed.(Figure 1)

The effects of DJFM on ECG of rats with VA

①The incidence of VP:Compared with the model group,the time of VP was significantly delayed in the DJFM group and the metoprolol group,and the difference was statistically significant(P<0.05),while the difference between the Metoprolol group and DJFM group was not statistically significant(P>0.05).②Onset time of VT:Compared with the model group,the onset time of VT was significantly delayed in the DJFM group and the Metoprolol group,and the difference was statistically significant(P<0.05),while the difference between the Metoprolol group and DJFM group was not statistically significant(P>0.05).③ Time of occurrence of VF:Compared with the model group,the time of occurrence of VF was significantly delayed in the DJFM group and the Metoprolol group,and the difference was statistically significant(P<0.05),while the difference between the metoprolol group and DJFM group was not statistically significant(P>0.05).(Table 1)

Figure 1 electrocardiogram of ventricular arrhythmia rats in aconitine model

Table 1 Effect of DJFM on electrocardiogram results of rats with ventricular arrhythmia induced by aconitine model(x ±S，n=10)

Effect of DJFM on sodium potassium pump in VA heart tissue of rats

Compared with the blank group,the sodium potassium pump value in the model group was significantly decreased,and the difference was statistically significant(P<0.05).Compared with the model group,the sodium potassium pump value of the tissues in the Metoprolol group and the DJFM group increased,and the difference was statistically significant(P<0.05).There was no significant difference in sodium potassium pump between the metoprolol group and the dDJFM group(P>0.05).(Table 2,Figure 2).

Table 2 Effects of DJFM on sodium potassium pump content in heart tissues of rats with ventricular arrhythmia(x ±S，n=10)

Figure 2 Effects of DJFM on sodium potassium pump content in heart tissues of rats with ventricular arrhythmiaS，n=10)

Discussion

The Animal models of VA are mainly made by rabbits,guinea pigs,rats,mice,cats,dogs or goats,et al.Rats are easy to obtain and widely used,with physiological and anatomical structures similar to humans,so they are widely used in VA models[4].The establishment of VA modelhasseveralmethodssuch asdrug-induced,electrical stimulation induced,coronary artery ligation and stress induced VA.The drug-induced method is easier operation,controllable condition,stability,relatively short building period and high success rate.So this experiment method induced by drugs.

Aconitine is a well-known alkaloid causing arrhythmia.Its mechanism of action to induce ventricular arrhythmia is able to make the myocardial cell membrane of the high of voltage-gated sodium channels be trigger activation.After activation of Na+channel,it is an open state,and the influx of Na+in myocardial cell increases,which accelerates the depolarization of the cell membrane,thus increase the self-discipline of pacemaker,induce ectopic rhythm point[4-6].Aconitine can also increase Ca2+in myocardial cells by increasing the expression of ICa-L,induced intracellular Ca2+elevation triggering activity,delayed depolarization[8].It can improve the self-regulation of fast-responding cells,thus forming one source ormultiple sourcesofheterotopic rhythm,shortening the refractory period of myocardium and leading to arrhythmia.

Myocardialcells almostallrely on aerobic metabolism.Sodium potassium pump is a biofilm enzyme system ubiquitous on the cell membrane of the body[8-10].Its main function is to transport Na+and K+across the membrane.It can at the resist electrochemical gradient on either side of the cell membrane while hydrolyzing ATP,and keep the ion concentration difference inside and outside the membrane stable,keeping the balance of electrolyte and fluid,maintaining the activity of Sodium potassium pump is beneficial to normalphysiologicalfunction ofcells.Myocardial membrane Sodium potassium pump is also a cardiac glycoside receptor,which plays a regulatory role in the metabolism and function of the heart,and improves the ability of anti-arrhythmia from multiple perspectives.Sodium potassium pump can hydrolyze ATP to release energy and realize the continuous activity of sodium potassium pump through reverse electrochemical gradient.Only maintaining the activity of sodium potassium pump can maintain the difference in ion concentration and ensure the normal electrical activity of tissue cells.When myocardial cell sodium potassium pump is inhibited,a series of changes in ion flow will occur,making extracellular Na+flow into the cell,and intracellular Na+increase indirectly activates Na+-Ca2+exchange protein,making intracellular Ca2+excessive and causing Ca2+overload to damage myocardial contractility[11],thus resulting in ventricular arrhythmia.Therefore,the content of sodium potassium pump in cardiac myocytes is an importantindicatorto monitorthe occurrence of ventricular arrhythmias.

DJFM consists of ten kinds of Chinese herbal medicines such as Suanzaoren,Fushen,Chuanxiong,Longgu,Muli,Guizhi,Yuanzhi,Dazao,Hehuanpi,Zhigancao.This prescription mainly uses methods of enriching blood,nourishing blood,tranquilization and nourishing Yin.In this prescription,Suanzaoren plays a role of the monarch in traditional Chinese medicine formulation and has flavours of sweet and sour.It acts on the heart meridian and liver meridian,and has functions of nourishing heart yin,supplementing liver blood and tranquilizing the mind.Fushen plays a role of the minister in traditional Chinese medicine formulation and has nature of calm and flavours of sweet and tasteless.It acts on the heart meridian and spleen meridian,and has functions ofsupplementing heartand spleen and tranquilizing the mind.Chuangxiong plays a role of the assistant in traditional Chinese medicine formulation and has functions of tonifying Qi and activating blood.Guizhi plays a role of the assistant in traditional Chinese medicine formulation and has functions of warm the heart-Yang,promoting heart-blood circulation,warming spleen and stomach and improving symptoms such as palpitation.The functions of compatibility of medicines between Chuangxiong,Guizhiand Suanzaoren are nourishing blood,activating blood and improving the ability of integration of enriching blood and promoting blood circulation through combine of acrid taste for dispersing,pungent-warm and sour for astringbg.Zhiyuan plays a role of the assistant in traditional Chinese medicine formulation and has functions of tranquilizing the mind and eliminating phlegm.Hehuanpi plays a role of the assistant in traditional Chinese medicine formulation and has functions of tranquilizing the mind and harmonizing qi and blood.Longgu and Muli play a role of the assistant in traditional Chinese medicine formulation and have functions of tranquilizing,invigorating Yin and conwerhencing dissipation of heart Qi.Separately,Longgu has effect of tranquillizing on liver meridian and Muli has effect of anchoring mind on lung meridian.Zhigancao and Suanzaoren play a role of the guide in traditional Chinese medicine formulation.Baked Licoric has effect of Yin-enriching,Qi-boosting,dredging Yang and recovering the pulse.Fructus Ziziphi Jujubae has effect of invigorating spleen to replenish Qi,nourishing blood and tranquilizing mind.

This prescription combines Suanzaoren with Longgu,Muli,et al.for the purpose of increasing synergy and curative effect,avoiding the disadvantages of ineffective efficacy and unstable efficacy ofsinglemedicinal materials.This prescription combines these medicines has effect of enriching blood,nourishing blood,tranquilizing mind and nourishing Yin.Clinical studies suggest that Suanzaoren’s nature is calm,flavours are sour and sweet,and the main components of Wild jujube are saponins,flavonoids,alkaloids and the like.Related studies have shown that Suanzaoren has sedative,anticonvulsant,anti-arrhythmia, anti-atherosclerosis, improved microcirculation,anti-ischemia and immune enhancement,and has a positive cardiovascular pharmacological effect[12].Huang Yisheng et al.confirmed that jujube saponin A can inhibit the decrease of Bcl2 and Bax expression in rat myocardial tissue after reperfusion injury,and can resist arrhythmia by significantly reducing serum LDH and myocardialMDA content[13-14],increasing myocardial SOD activity and the ability to resist oxygen free radicals.The Longgu and Muli have good sedative,hypnotic and anticonvulsant effects[15].Oxygen free radicals may play an important role in the induction of potentially fatal arrhythmias,and Muli aqueous extracts can enhance the activity of SOD and reduce the content of MDA[16],thus playing a role in the prevention and treatment of ventricular arrhythmias.Fushen has a variety of biological active ingredients such as lycium polysaccharides,tetracyclic triterpenoids,et al.It has the functions of calming hypnosis,nourishing the heart,calming the nerves,resisting tumors,enhancing immunity and lowering blood glucose.Lin Xiaoming etal.confirmed that Fuling can enhance SOD activity[17],inhibitMDA production,and have the effectof scavenging oxygen free radicals.The main chemical components of Guizhi are volatile oils,phenols,organic acids,et al.Guizhi has the functions of dilating blood vessels,anti-oxidation,lowering blood lipid,lowering blood pressure, promoting blood circulation and anti-platelet aggregation, anti-inflammatory and anti-allergic effects[18].For example,cinnamaldehyde can dilate blood vessels,thereby enhance blood circulation [19-20],increase coronary blood flow,enhance myocardial contractility,and slow heart rate abnormalities,thereby maintaining the normal function of the heart;such as Guizhi liquid extracted by hydroalcohol method can reduce blood lipids[21],improve myocardial ischemia and reduce the incidence of arrhythmia in rats with coronary heart disease.Licorice mainly contains triterpenoids,flavonoids,alkaloids,organic acids,licorice polysaccharides and other chemical components.The most important active substances are triterpenoid saponins and flavonoids.Licorice has many effects such as anti-oxidation, lowering blood lipids and anti-atherosclerosis, anti-arrhythmia, anti-myocardial ischemia and immune regulation[22-23].Many studies have confirmed that Zhigancao can exert anti-arrhythmia effects through various ways.Xie Shirong et al.found that glycyrrhetinic acid can inhibit aconitine and coronary artery ligation-induced reperfusion ventricular arrhythmia[23].Total flavonoids from licorice can delay the occurrence of arrhythmia in aconitine model rats and reduce the incidence ofventricularfibrillation in chloroform model rats, indicating that it has anti-ventricular arrhythmia effect[25].

The results of this study suggest that compared with the model group,DJFM group and metoprolol group can significantly delay the occurrence of VP,VT and VF,the difference is statistically significant(P<0.05).The DJFM group and the metoprolol group had the same effect in delaying the appearance of VP,VT and VF,and there was no significant difference(P>0.05).It is indicated that DJFM can prolong the occurrence of ventricular arrhythmias such as VP,VT and VF in aconitine model rats.The mechanism may be related to the inward rectifier potassium channel(IK+),which can prevent arrhythmia by promoting K+efflux to reduce intracellular positive potential and reduce self-discipline.On the other hand,it may also be related to fast Na+channels,which may reduce theself-discipline of fast-reacting cells by inhibiting Na+influx,thereby achieving the effect of preventing and treating arrhythmia.It is indicated that DJFM may have the function of preventing and treating ventricular arrhythmia models of different ion channels.

The experimentalso found thatthe sodium potassium pump in the tissues of the model group was significantly lower than theblank group,and the difference was statistically significant(P<0.05).The metoprolol group and DJFM group can increase the sodium potassium pump in the tissues of rats with ventriculararrhythmia(P < 0.05).Therewasno significant difference in the sodium potassium pump between the DJFM group and the metoprolol group(P>0.05).It was concluded that DJFM can protect sodium potassium pump.The mechanism may be to reduce intracellular Ca2+concentration indirectly by reducing intracellular Na+concentration,thereby preventing myocardial contractility damage caused by Ca2+concentration overload,and thus preventing ventricular arrhythmia.

In summary,the intravenous injection of aconitine can successfully prepare a rat model of ventricular arrhythmia.DJFM is similar to metoprolol in preventing and treating arrhythmia.The mechanism may be related to the protection of myocardial tissue sodium potassium pump,and the inhibition of Na+influx reduces the self-discipline of fast-reacting cells,thereby protecting the cardiomyocytes and improving myocardial metabolism.However,there are few side effects of traditional Chinese medicine.If the syndrome type is consistent,there is no obvious contraindication.Therefore,we should promote the use of traditional Chinese medicine in clinical practice and provide treatment services for more patients with arrhythmia.

1.Cao KJ,Chen ML,Jiang H,et al.Chinese expert consensus on ventricular arrhythmias.Chin J Card Pacing Electrophysiol,2016.30:283-325.

2.Chen X.Huang wan clinical electrocardiogram.6th ed.Beijing:People's Medical Publishing House,2016:185-186.

3.Xu HM,Ren FY,Jiang XG.Discussion on the treatment of arrhythmia with traditional Chinese medicine.Proceedings of the 12th annual meeting of the heart disease branch of the Chinese society of traditional Chinese medicine,2010:268-272.

4.Zhou GX,Gao C,Xu P.Replication Methodology of AnimalModelsforHumanDisease.Shanghai:Shanghai Scientific and Technological Literature Press,2008:13-15.

5.Wang GB,Fu Q,Lin XM,et al.Experimental Study on Antiarrhythmic Effects of Xinlu Tai Capsule.Tradi Chin Drug Res Clin Pharmacol,2003.5:105-107.

6.Li HW.Experimental study on the mechanism of yiqi fumaimixture in the treatmentofpremature ventricular contraction. Beijing university of traditional Chinese medicine,2013.

7.Zhou YH,Piao XM,Liu X,et al.Arrhythmogenesis toxicity of aconitine is related to intracellular Ca2+signals.Int J Med Sci.2013.10:1242-1249.

8.Hu X,Wang DS.Effect of Dingxin decoction on arrhythmia in rat model.Chin J Mod Med,2016.26:18-21.

9.Schemer Bobis G.The sodium pump.Its molecular properties and mechanics of ion transport.Eu r J Biochem,2002.269:2424 2433.

10.Zang GY.Na+,K+-ATPase,Health and Sports.J Shenyang Sport Univ,2006.6:59-62.

11.Noble D,Noble PJ.Late sodium current in the pathophysiology of cardiovascular disease:consequences of sodium-calcium overload.Heart.2006.4:iv1-iv5.

12.Geng X,Li TL.Progress on Chemical Composition and Pharmacological Activities of Semen Zizyphi Spinosae.ActaChinMedPharmacol,2016.44:84-86.

13.Huang YS,Mi XY,Xiong YH.Effect of Mechanism of Zizyphi Spinosaeand on arrhythmia of myocardial ischemia reperfusion injury in rat.Nei Mongol J Tradi Chin Med,2013.29:45-46.

14.Huang YS,Jia YH,Sun XG,et al.Effects of jujube saponins A on arrhythmia and bcl-2 and BAX expression in ischemia-reperfusion injury rats.Tradi Chin Drug Res Clin Pharmacol,2011.13:51-54.

15.Zhang H,Zhang L,Liu Y.Studies on chemical components and pharmacological activities of Os Draconis(Longgu)and Ostreae Concha.Chin J Chin Mater Med,2011.36:1839-1840.

16.Feng L,Zhao WJ,Chang WZ.Research progress on pharmacological action and clinical application of oyster.Inf Tradi Chin Med,2011.28:114-116.

17.Lin XM,Feng JY,Long Z,et al.Effect of tremella,poria and gynostemma on murine immunological and scavenging free radicals function.J Beijing Med Univ,1995.37:455-457+473.

18.Hou JY.Pharmacology ofTraditionalChinese Medicine.Beijing:China Press ofTraditional Chinese Medicine,2002:30-31.

19.Liu P,Zhang LP.Studies on Chemical Constituents in Dried TenderStem ofCinnamomiCassia.Liaoning J Tradi Chin Med,2012.39:1926-1927.

20.Chen JY,Ya QK,Lu WJ.Overview of research on cinnamon.Guangxi Med J,2009.31:872-874.

21.Jiang B,Jiang HY,Ren LL,et al.Regulatory effect of Guizhi on the blood lipo level of Mouse model of Coronary heart disease.Shenzhen J Integr Tradi Chin Western Med,2003.3:234-235.

22.Zhang YL,Wang MY,Ynag JY,et al.Research progress of chemical constituents and pharmacological effects of backed licorice.Acad J Shanghai Univ Tradi Chin Med,2015.29:99-102.

23.Wang B,Wang YX,Zhao HY,et al.Research progress of the major components and the pharmacological effect of Glycyrrhiza uralensis fisch.J Jilin Med Coll,2013.34:215-218.

24.Xie SR,Huang CY,Huang SY,et al.Antiarrhythmic effect of glycyrrhetinic acid.Herald Med,2004.19:140-142.

25.Hu XY,Peng GP,Chen RY.Effects of glycyrrhiza flavonoids on arrhythmia.Chin Tradi Herb Drugs,1996.27:733-735.