Efficacy on rabbits with arrhythmia:needling acupoint of Neiguan(PC6)at shallow or deep depth,and retaining needles for 10,20,or 30 min

GU Chunling,CHENG Kai,YANG Shouqin,RAN Shaofeng,TAI Hengyap,GAO Xiaofeng,PANG Dandan

GU Chunling,YANG Shouqin,RAN Shaofeng,TAI Hengyap,School of Acupuncture and Tuina,Beijing University of Chinese Medicine,Beijing 100029,China

CHENG Kai,School of Acupuncture and Tuina,Beijing University of Chinese Medicine,Beijing 100029,China

GAO Xiaofeng,Beijing MedEx Technology Co.,Ltd.,Beijing 100029,China

PANG Dandan,China Academy of Chinese Medical Sciences,Beijing 100700,China

Abstract OBJECTIVE:To compare the effects of Neiguan(PC6) acupuncture at different depths and retention time on arrhythmia duration,myocardial tissue morphology,mRNA expression level of L-type calcium channel α1C subunit and Ca2+-Mg2+-AtPase activity in tachirrhythmia model of rabbits.METHODS:The tachyarrhythmia model was made by intravenous injection of barium chloride into the ears of rabbits.A total of 56 healthy adult male New Zealand big-eared white rabbits,apply the random number table method,divided into normal control group (group A),model group (group B),shallow needling Neiguan (PC6) 10 min group(group C),shallow needling Neiguan (PC6) 20 min group (group D),shallow needling Neiguan (PC6)30 min group (group E),deep needling Neiguan(PC6) 10 min group (group F),deep needling Neiguan(PC6)20 min group(group G),deep needling Neiguan(PC6)30 min group(group H),7 animals in each group.Electrocardiograms were used to collect the duration of arrhythmia;hematoxylin-eosin staining method was performed on myocardial tissue,RT-PCR tested the expression of α1C subunit mRNA,and the activity of Ca2+-Mg2+-ATPase were quantified by phosphorus determination method.RESULTS:The duration of arrhythmia in each acupuncture treatment group was shortened to varying degrees.Compare to the model group,the tissue damage from barium chloride inducing was improved in the acupuncture group.Compared to the model group,except for group E,most treatment groups had varying degrees of improvement with significantly down-regulated L-type calcium channel α1C subunit mRNA expressions level and increased Ca2+-Mg2+-ATPase activity.CONCLUSIONS:The effect of acupuncture at Neiguan (PC6) with different depths and retention time can reduce the duration of arrhythmia induced by barium chloride relatively,improve the induced pathological changes,down regulate L-type calcium channel α1C subunit mRNA expressions level and increase Ca2+-Mg2+-ATPase activity.Both the shallow and deep tissues of Neiguan(PC6)may be involved in transmitting acupuncture information.There is an optimal induction period for shallow needling at Neiguan (PC6) to reach the best therapeutic effect.

Keywords:Point PC6(Neiguan);arrhythmias,cardiac;calcium channels,L-type;Ca2+-Mg2+-ATPase;needle retention time

INTRODUCTION

Neiguan (PC6) is the first choice for acupuncture and moxibustion regarding arrhythmia treatment.1There are kinds of acupuncture levels methods,such as 1/5 inch,half-inch,one inch,and penetration acupuncture through with Waiguan (SJ5).Studies have confirmed that both shallow needling (3 mm) and deep needling(5-8 mm) at Neiguan (PC6) can treat arrhythmias.2The specificity of acupoints reflects in different regulating effects of acupuncture at different levels and retention times to reach its best effect.3Most clinical studies on the relationship of the acupuncture's effect between different depths have shown that deep needling was better than shallow needling.4Previous studies proved that deep needling at Neiguan (PC6) was better than shallow needling in shortening the duration of atrial fibrillation.5Does the poor efficacy of shallow needling have something to do with the needle retention time?The needle retention time is too short to achieve the best therapeutic effect?Or is it too long that may cause tolerance and fatigue,resulting in ineffective or even inferior stimulation?6

The purpose of this experiment is to explore the acupuncture level and needle retention time to obtain the best therapeutic effect of tachyarrhythmia,in order to provide scientific basis for clinical rational use of acupoints.

MATERIALS AND METHODS

Experimental animals

A total of 56 healthy and clean adult male New Zealand white rabbits,weighing 2.0-2.5 kg.Provided by Beijing Fulong Tengfei Breeding Center,license number:SCXK (Peking) 2018-0009.They are raised in the animal room on the Liangxiang campus of Beijing University of TCM.The breeding conditions are:indoor temperature (18-22) ℃,humidity 35%-45%,natural light,food and water are provided.Rabbits were fasting 12 h before the experiment operation,shaver were used to remove the rabbit hair on the inner side of the both forearm in order to accurately locate the acupuncture points.The study was approved by the experimental animal ethics committee of Beijing University of TCM.

Experiment grouping

A total of 56 rabbits applies the random number table method dividing into eight groups,seven animals in each one.There were named with the control group(group A)and the model group(group B),shallow needling Neiguan (PC6) 10 min group (Group C),shallow needling Neiguan (PC6) 20 min group (group D),shallow needling Neiguan (PC6) 30 min group (group E),deep needling Neiguan (PC6) 10 min group(group F),deep needling Neiguan (PC6) 20 min group (group G),deep needling Neiguan (PC6)30 min group(group H).

Experimental reagents and instruments

Chloral hydrate was purchased from Honghu United Chemical Industry (Beijing,China);Anhydrous Barium Chloride was purchased from Yinuokai Biology Company (Beijing,China);HiFiScript cDNA first-strand synthesis kit was purchased from Cwbiotech (Taizhou,China);SYBR FAST qPCR Kit Master Mix (2×) Universal was purchased from American KAPA Biosystems (Woburn,MA,USA);Trizol was purchased from Invitrogen (Carlsbad,California,USA);Chloroform,isopropanol was purchased from Beijing Chemical Reagent Factory (Beijing,China);Ultramicro ATP enzyme(Ca2+-Mg2+-ATP) assay kit and Total Protein assay were purchased from Beijing Rigor Bioscience(Beijing,China).

BL-420N biological signal acquisition and analysis system was purchased from Chengdutaimeng Biology Company (Chengdu,Sichuan,China);disposable sterile acupuncture needles was purchased from Zhongyantaihe Company (Beijing,China);spectrophotometer:UV-2000 was purchased from Jinghua Technology Instrument Company (Shanghai,China);real-time quantitative PCR instrument:ABI7500 was purchased from Applied Biosystems (Foster,CA,USA);fully automatic biochemical analyzer:UniCel DxC 600 Synchron was purchased from Beckman Coulter (Brea,CA,USA);Gel imager:BioSens SC 810B was purchased from Shanghai Shanfu Biotech Company(Shanghai,China).

Modeling method

After weighing,the rabbits were anesthetized by intraperitoneal injection of chloral hydrate (10%,5 mL/kg).7After completing anesthesia,the rabbit was fixed on the rabbit table in the supine position.Disposable sterile needles were used as electrodes and inserted under rabbits' skin at the right forelimb and the ends of both lower limbs.After connecting the BL-420N bio-signal acquisition and analysis system ECG electrodes to the rabbit,run the standard limb II electrocardiogram (ECG) and monitor the ECG for 5 min.In the given min,a bolus injection through the ear vein with sodium chloride solution (0.9%,1 mL/kg) was given to the control group;the other groups were injected with barium chloride solution (0.4%,1mL/kg)through the ear vein.8A successful model will be verified with the occurrence of 7 or more continuous premature ventricular contraction beats (PVC),9the PVC goes by the definition of continuous,vast,deformed,or torsion QRS waveform;with not preceded P waves and unequal R waves gap.The rate of change in heart rate after barium chloride injection versus before was greater than 0.39.

Acupuncture method

Refer to 10to locate the Neiguan (PC6) in rabbits:The lower 1/6 point of the inner forearm (approximately 1.2-1.3 cm above the wrist joint),between the flexor carpi radialis and the flexor digitorum superficialis.After the model is successfully build,prepare a disposable sterile acupuncture needle of 0.25 mm × 25 mm.Process the shallow acupuncture group needling straightly with a depth of 3 mm2the tip of the needle stops at the surface of the muscle,retain the needle for 10 min(group C),20 min (group D),and 30 min (group E).In like manner,process the deep acupuncture group needling straightly with a depth of 5-8 mm,11,12properly adjust the direction of the needle tip to touch the endings of median nerve to make the rabbit limbs twitch,retain the needles for 10 min (group F),20 min(group G),and 30 min(group H).Each group were performed with the tremorQistimulation method (that is,hold the needle handle and do a small and rapid tremor without relative displacement between the needle body and the surrounding tissues) once every 3 min with a 3 min length,this was repeated until the needle retention time was over.Group A and group B were not performed with acupuncture.

Observation indicators and detection methods

(a) Arrhythmia duration:the ECG was recorded continuously with limb lead-II standard until the sinus rhythm was restored during the experiment.The arrhythmia duration is counted from the first ventricular premature contraction beat on ECG to the beginning of the recovery of sinus rhythm.13Ventricular tachycardia disappears for more than 300 s can be diagnosed as the restoration of the rather normal heart rhythm.14

(b) Observation of myocardial morphologyviahematoxylin-eosin staining method:rabbit's left ventricular anterior wall myocardial tissue specimen was fixed in 4% paraformaldehyde,then dehydrated by gradient ethanol,transparent xylene,immersed in wax,embedded,and sectioned.The thickness of the section was about 5 μm,hematoxylin-eosin staining was performed,and the sections were sealed and observed under a microscope.

(c) RT-PCR technique was used to observe the mRNA expression level of LTCC α1C subunit:extract total RNA was extracted from the tissue with TRIzol reagent,reverse transcription kit was used to synthesize cDNA by reverse transcription.The primers and cDNA were added to the SYBR FAST qPCR Kit Master Mix (2×) Universal PCR system,respectively,then the experiment was performed in the real-time fluorescence quantitative PCR (plus stepone,ABI),with a final volume of 20 μL.GAPDH was used for references,and the 2－△△Ctmethod was used for relative quantitative analysis of the data.All experiments were repeated at least three times(Tables 1,2).

Table 1 Primer sequence of detection gene

Table 2 Reaction conditions

(d) Determination of Ca2+-Mg2+-ATPase activity via phosphorus determination method:After collecting rabbit's cardiac muscle tissue,prepare normal saline tissue homogenate,centrifuge at 3000-4000 r/min for 10 min,and then extract 100 μL of supernatant.After enzymatic reaction,comparison tube and measuring tube,extracted the supernatant 300 μL phosphonium,Ca2+-Mg2+-ATPase activity value detection,according to the kit instructions,enzyme activity was determined by the enzymatic reaction and phospho-assay method.

Statistical analysis

All data were analyzed using SAS (vers 9.4,SAS Institute,Chicago,IL,USA) software.Measurement data were expressed as mean ± standard deviation (±s).When comparing between the groups,the data of each group was conformed to normality and homogeneity of variance using two independent sample t-tests or analysis of variance.There were statistical differences in the analysis of variance,and the least significant difference test was used for pairwise comparison between groups.The interaction was analyzed by factorial (2*3)analysis designed of variance.P <0.05 was used as the standard for statistically significant differences.

RESULTS

Comparison of intervention effects on the duration of tachyarrhythmia in rabbits of each group

Compared with the model group [(4387 ± 728) s],the duration of arrhythmia in each acupuncture treatment group was shortened to varying degrees (P <0.05).In terms of the duration of arrhythmia,shallow acupuncture groups showed that group D [(1636 ± 196) s]

Comparison of the morphology of rabbit ventricular myocytes in each group

In Figure 1,the rabbit's left ventricle anterior wall of the cardiac muscle was observed.The cardiac muscle tissue structure of the control group was typical;the cell nucleus was intact and elliptical with narrow blankspace surrounding it;both longitudinal and transverse sections showing that the cytoplasm was relatively uniform and had no abnormal changes.The ventricular wall fibers were densely arranged near the center in sheets-like formations;the tissue near the heart cavity(papillary muscles)was loose and in fascicle order.

Figure 1 Hematoxylin-eosin staining results of rabbit myocardium(cross section,×200 times)

In the model group,there were noticeable structural changes:pronounced edema of the cardiomyocytes forming it into granular shape,vacuoles changes in myocardial cells;some of the nuclei have a sign of lysis,shrinkage and wrinkled around the edge forming into irregular shapes;widened perinuclear space;pale stained cytoplasm;disordered fiber arrangement and distinctive wave-like degeneration.

In the control group and model groups,capillaries could be seen between the myocardial cells.There were different degrees of myocardial cell edema,cell vacuolation,myocardial fiber arrangement disorder,wrinkled nucleus,widened perinuclear space,pale stained cytoplasm,and wave-like degeneration in each acupuncture treatment group.Capillary dilation with new red liquid in between was seen in all acupuncture treatment groups.

Comparison mRNA expression of L-type calcium channel α1C subunit in myocardial tissue of rabbits in different groups

Compared with the control group (0.25 ± 0.04),the L-type calcium channel α1C subunit mRNA expression of the model group (1.49 ± 0.29) is significantly higher (P <0.01).The expression of majority treatment group noticeably decreased(P<0.05),except for group E (P >0.05).Comparing the expression level of each group showing a result of — shallow acupuncture groups,group D (0.65 ± 0.25)

Comparison Ca2+-Mg2+-ATPase activity in myocardial tissue of rabbits in various groups

Compared with the control group [(8.4 ± 0.6) μmol·mg－1·h－1],the activity of Ca2+-Mg2+-ATPase was decreased in the model group[(7.3±0.5)μmol·mg－1·h－1]and the difference was statistically significant (P <0.01).Except for the group E (P >0.05),compared with the model group,the activities of Ca2+-Mg2+-ATPase in other acupuncture treatment groups were increased in varying degrees,and the difference was statistically significant(P<0.05).In the activity of Ca2+-Mg2+-ATPase,shallow acupuncture groups showed that group E [(7.3 ± 0.4) μmol·mg－1·h－1]

DISCUSSION

The modeling method in this experiment is to inject barium chloride solutionviathe ear vein to induce tachyarrhythmia in rabbits.After the induction,the incidence rate of ventricular tachycardia is 100%,as well as the survival rate,proving that the induction method is successful.The sinus rhythm of the model group's rabbit usually needs 60-70 min to recover after the induction of barium chloride,but in comparison to the model group,each acupuncture treatment group it shortened the duration of arrhythmia to varying degrees.

Compared to the control group,the tissue from the model group,which was damaged by the injection of barium chloride,shows noticeable structural changes.Damage such as nucleus shrinkage,lysis,disordered fiber arrangement,and substantial wave-like degeneration were seen widely.On the other hand,the pathological changes in the treatment group were lessen to varying degrees,dilated capillaries filled with red liquid were also seen.We speculate that acupuncture treatments may increase the myocardial blood flow,which triggers this phenomenon.For groups G and H,the myocardial morphology tends to be normal,reflecting that acupuncture Neiguan (PC6) has a certain degree of therapeutic protection against myocardial damage.

Mechanism of arrhythmia induced by barium chloride may be related to delayed depolarization and triggering activity induced by increased Na+/Ca2+influx in myocardial working cells.15,16In the plateau phase of the myocardial action potential,extracellular Ca2+enters into the cell through the L-type calcium ion channel,which increased the intracytoplasmic Ca2+concentration and triggered the release of large amounts of Ca2+from the sarcoplasmic reticulum.17-19Ca2+-Mg2+-ATPase can pump a small amount of Ca2+out of the cells or into the sarcoplasmic reticulum when the intracellular Ca2+was increased to a certain extent,thereby reducing the intracellular Ca2+concentration.20

The density and distribution of LTCC joined a crucial role in the occurrence and maintenance of arrhythmia,21,22α1C is the main subunit that constitutes in ventricular L-type calcium channels,abnormal changes of α1C protein expression at right ventricular outflow tract trigger arrhythmia;23,24the inactivation of α1C and down-regulated mRNA expression promoted electrical remodeling of cardiomyocytes which induct and maintain the event of arrhythmia.25-27Comparing with the control group,the expression of LTCC was up-regulated in the inducted groups,resonating with the cited article above.LTCC expression in other acupuncture treatment groups excluding group E decreased to varying degrees compared with the model group.It shows that acupuncture of different depth at Neiguan (PC6) had a certain degree of effectiveness of down regulating the expression of α1C mRNA.This result is substantiated with a previous study — Neiguan(PC6)regulatory effect on the expression of calcium ion transmembrane channels in cardiomyocytes.28Decreased Ca2+-Mg2+-ATPase activity will cause intracellular calcium overload,damage mitochondria's function,increase the concentration of oxygen radicals,and therefore causes arrhythmia.29-31Compared to the model group with the control group,Ca2+-Mg2+-ATPase activity decreased after the successful modeling in this experiment,which substantiates the theory of the cited article above.Furthermore,compared to the model group,except for group E;other treatment groups had various increases in Ca2+-Mg2+-ATPase activity,indicating that acupuncture at Neiguan (PC6) can increase the activity of Ca2+-Mg2+-ATPase,consequently reduces the overload of intracellular calcium and the degree of cardiomyocytes' damage,showing a corresponding result with the cited article.32

In this experiment,the statistical results of each index proved that the curative effects of acupuncture comparison for both the deep needling and shallow needling at Neiguan (PC6) were statistically significant (P <0.05).Thus,it suggested that both the shallow and deep tissues of Neiguan (PC6) may participate in the transmission of acupuncture information.

The effect trend of each index showed that the effect of superficial level's curative from large to small was acupunctured 20,10,30 min.The therapeutic effect of shallow needling for 20 min was better than that of shallow needling for 30 min (P <0.05),which suggest that there are an optimal induction period for shallow needling at Neiguan(PC6).

The degree of deep needling curative effect ranges from large to small was acupunctured 30,20,and 10 min.With the prolongation of the needle retaining time,the acupuncture effect of deep needling at Neiguan (PC6) was gradually enhanced,but whether the acupuncture effect will continue strengthening or gradually decreased or produces negative feedback with the further prolongation needle retaining time remains to be proved by further research.

The stimulation effect of shallow needling was more sensitive to the response of needle retention time,the optimal induction period was shorter,and it was easier to produce invalid stimulation due to long needle retention time,suggesting that shallow needling should be paying more attention to the relationship of time and effect.