Kanae Umemoto,Tomoya Hayashi,Kaori Fukushige,Shuichi Hirai,Hayato Terayama,Kou Sakabe,Munekazu Naito

Kanae Umemoto,Graduate School of Acupuncture and Moxibustion,Meiji University of Integrative Medicine,Nantan,Kyoto 629-0392,Japan;Department of Anatomy,Division of Basic Medical Sc+ience,Tokai University School of Medicine,Isehara,Kanagawa 259-1193,Japan;Department of Anatomy,Aichi Medical University School of Medicine,Nagakute,Aichi 480-1195,Japan

Tomoya Hayashi,Graduate School of Acupuncture and Moxibustion,Meiji University of Integrative Medicine,Nantan,Kyoto,629-0392,Japan;Department of Physiology,Meiji University of Integrative Medicine,Nantan,Kyoto 629-0392,Japan

Kaori Fukushige,Munekazu Naito,Department of Anatomy,Aichi Medical University School of Medicine,Nagakute,Aichi 480-1195,Japan

Shuichi Hirai,Department of Anatomy,Aichi Medical University School of Medicine,Nagakute,Aichi 480-1195,Japan;Division of Anatomical Science,Department of Functional Morphology,Nihon University School of Medicine,Itabashi-ku,Tokyo 173-0032,Japan

Hayato Terayama,Kou Sakabe,Department of Anatomy,Division of Basic Medical Science,Tokai University School of Medicine,Isehara,Kanagawa 259-1193,Japan

Abstract OBJECTIVE:To examine whether specific stimulation of Shenshu (BL23) affects sympathetic nervous activity(SNA)-associated plasma renin concentration (PRC).METHODS:Eight healthy volunteers participated in three pattern conditions in random order:control (Cont),stimulation of Shenshu (BL23),and stimulation of sham point (Sham).All participants were initially in the supine position for >60 min,and then remained in the standing position during the experimental procedure to increase SNA.An electrocardiogram was used to calculate low frequency/high frequency (LF/HF) ratio;blood was collected to analyze PRC.RESULTS:The LF/HF ratio was significantly increased in the standing position when compared with the supine position (P < 0.01).There was no difference in LF/HF ratioduring or after stimulation of Shenshu (BL23) in the standing position when compared with before the stimulation in the supine position;however,the LF/HF ratio was significantly increased in Cont and Sham conditions (P < 0.01).There was no difference in PRC after stimulation of Shenshu (BL23) in the standing position when compared with before the stimulation in the supine position;however,there was a significant increase in PRC in the Cont and Sham conditions (Cont P < 0.05,Sham P < 0.01).CONCLUSION:Our results demonstrated that specific acupuncture stimulation of Shenshu (BL23) in the standing position decreased SNA-associated PRC,which was not observed during acupuncture stimulation of the sham point.

Keywords:Acupuncture;Point BL23 (Shenshu);Sympathetic nervous activity;Plasma renin concentration

1.INTRODUCTION

Shenshu (BL23) is a back-shu point called “Shenshu” or“Jinyu” in Chinese or Japanese traditional medicine,respectively,that is located in the lumbar region at the level of the inferior border of the second lumbar vertebral(L2) spinous process and 1.5 Bcun,which is defined as half the width of four fingers at the dorsal crease of the proximal interphalangeal joint of the middle finger,lateral to the posterior median line.1The acupuncture stimulation of Shenshu (BL23) has been reported to affect kidney function,2-4the immune system,5cerebral function,6,7and bone metabolism.8The acupuncture stimulation of Shenshu (BL23) has a diuretic action,2-4and is mediated by sympathetic nervous activity (SNA),at least in part.2,3We previously demonstrated anatomically that Shenshu (BL23) is the specific location that connects the sympathetic trunk in eight donor cadavers.9Furthermore,SNA has an indirect effect on aldosteroneviamodulation of plasma renin concentration (PRC),an enzyme produced in the kidney.10Inhibition of SNA leads indirectly to a decrease in aldosterone,which increases urine output.11

The aim of this study was to examine whether the acupuncture stimulation of Shenshu (BL23) can affect SNA-associated PRC.

2.METHODS

2.1.Participants

Eight healthy volunteers (seven males and one female),with a mean age of 25 years [(25 ± 3) years],provided informed consent to participate in the present study after the protocol had been approved by the Human Research Institutional Review Board at Meiji University of Integrative Medicine (reference No.25-73).Participants were excluded if they had experienced low back pain or toothache.None of the participants reported taking any medicine and acupuncture treatment,or alcohol and caffeine for at least one month or three hours,respectively,prior to the experiment.

2.2.Experimental procedure

The experimental procedure is shown in Figure 1.All participants were subjected to three pattern conditions in random order:control (Cont),stimulation of Shenshu(BL23),and stimulation of sham point (Sham:4 cm from Shenshu (BL23) in the same dermatome,45° outside and downward for 10 min;Figure 2).3Participation in each experiment occurred between 10 a.m.and 1 p.m.and was separated by a washout period of at least 7 d.

Figure 1 Experimental procedure

Figure 2 Stimulation points

The participants rested in a supine position for >60 min(SP period).Next,they moved to a standing position and remained in this position for 20 min (BS period).After the BS period,participants in the Cont condition continued to remain in the standing position for 10 min(S period).Participants in the Shenshu (BL23) and Sham conditions received stimulation for 10 min in the standing position in the S period.We used disposable sterilized acupuncture needles (0.18 mm diameter,30 mm length,Seirin Co Ltd.,Shizuoka,Japan),which are commonly used in Japan.The acupuncture needles were inserted to a depth of 15-20 mm to reach the fascia of erector spinae muscle and retained for 10 min after achieving aDe Qisensation.After the S period,all participants remained in the standing position for 20 min (AS period).Following the SP,BS,and S periods,samples of peripheral blood were self-collected into centrifuge tubes containing anticoagulant.Electro-cardiogram (ECG) was recorded using a portable electrocardiograph (CheckMyHeart,Daily Care BioMedical Inc,Taiwan,China) for 5 min prior to the end of each period,because the measurement time of the device is limited to 5 min.12The study was conducted in air-conditioned room (temperature,25-27 °C;humidity,31% ± 5%).

2.3.Evaluation of sympathetic nervous activity (SNA)using electrocardiogram (ECG)

The portable electrocardiograph was used to record the ECG in the standing position and perform off-line analysis.The electrodes of the device were attached to each participant’s right and left forearm.Data on heart rate (HR) and heart rate variability (HRV),which is variation in the beat intervals,were transferred to a computer and analyzed using HRV analysis software(CheckMyHeart v3.0,Daily Care BioMedical Inc.,Taiwan,China).The 5 min interval HRV data were used in the spectral analysis to calculate two major components,low frequency (LF:0.04-0.15 Hz) and high frequency (HF:0.15-0.40 Hz).LF was defined as an indicator of both sympathetic and parasympathetic nervous activity,whereas HF was defined as an indicator of parasympathetic nervous activity.13The ratio of LF to HF (LF/HF) ratio was used as an indicator of SNA.13

2.4.PRC

After the samples of peripheral blood were self-collected,the tubes were centrifuged immediately at room temperature.The samples were stored at－25 ℃ until PRC measurement.We conducted an enzyme-linked immunosorbent assay (ELISA) using an ELISA detection kit (RE53321,IBL International GMBH,Hamburg,Germany) with a Microplate Reader (model 550,Bio-rad Laboratories,Hercules,CA,USA) and analyzed the PRC according to the manufacturer’s instructions (RE53321,IBL international GMBH,Hamburg,Germany).

2.5.Statistical analysis

Statistical analysis was completed using the GraphPad Prism program (GraphPad Software Inc.,San Diego,CA,USA).Statistical significance of the treatment effects within each group was evaluated by a repeated measure two-way analysis of variance followed by Tukey's test.The mean HR and LF/HF in the SP period,and the mean PRC in the Post-SP period were compared between Cont,Shenshu (BL23),and Sham conditions using Dunnett’s test.Results are presented as mean ± standard deviation().The level of statistical significance was set atP <0.05.

3.RESULTS

3.1.Evaluation of sympathetic nervous activity (SNA)using spectral analysis of electrocardiogram (ECG)

There was no significant difference between the mean HR in the Cont,Shenshu (BL23),and Sham conditions in the SP period (Dunnett’s testP >0.05).The mean HR in the BS,S,and AS periods was significantly increased when compared with that in the SP period in all three pattern conditions (P <0.01,Figure 3B).The mean HR in the Cont and Sham conditions in the AS period was significantly increased when compared with that in the BS period (P <0.05,Figure 3B);however,there was no significant difference in the mean HR between the BS and AS periods in the Shenshu (BL23) condition (Figure 3B).

There was no significant difference between the mean LF/HF in Cont,Shenshu (BL23),and Sham conditions in the SP period (Dunnett’s testP >0.05).The mean LF/HF ratio in the BS period was significantly increased when compared with that in the SP period in all three pattern conditions (P <0.01,Figure 4).Moreover,the LF/HF ratio in the S and AS periods was significantly increased when compared with that in the SP period in Cont and Sham conditions (P <0.01,Figure 4);however,there was no significant difference in the LF/HF ratio in the S or AS period when compared with that in the SP period in the Shenshu (BL23) condition (Figure 4).

3.2.PRC

There was no significant difference between the mean PRC in Cont,Shenshu (BL23),and Sham conditions in the Post-SP period (Dunnett’s testsP >0.05).The mean PRC in Cont and Sham conditions was significantly increased in the Post-S period when compared with that in the Post-SP period (Cont:P <0.05;Sham:P <0.01,Figure 5).There were no significant differences in PRC between the Post-SP and Post-S periods in the Shenshu(BL23) condition (Figure 5).

Figure 3 HR in the Cont,Shenshu (BL23),and Sham conditions

Figure 4 LF/HF ratio during each period

Figure 5 PRC during each period

4.DISCUSSION

In this study,we demonstrated that specific acupuncture stimulation of Shenshu (BL23) decreased SNAassociated PRC in the participants with increased SNA.To the best of our knowledge,this is the first study to indicate that Shenshu (BL23) is the specific location that can affect SNA-associated PRC.

Clinically,acupuncture stimulation has been used to modulate SNA.The effects of SNA-induced acupuncture stimulation have been examined in a variety of ways,such as HR,salivary α-amylase and plasma norepinephrine concentration.Imai14reported that acupuncture stimulation of Hegu (LI4),Zusanli (ST36),Taichong (LR3),Liangmen (ST21),Jizhong (CV6),Shenzhu (CV12),and sham points significantly decreased HR.Akimotoet al15have reported that acupuncture stimulation of Hegu (LI4),Jiache (ST6),Zusanli (ST36),and Kongzui (LU6) can significantly decrease salivary α-amylase concentration.Furthermore,Sakumaet al16have demonstrated that acupuncture stimulation of Pishu (BL20),Shenshu (BL23),Dachangshu (BL25),and other points can decrease plasma norepinephrine concentration.

Several studies have reported that acupuncture stimulation decreases SNA.In this study,we used the LF/HF ratio as an indicator for SNA,which has been used in previous studies.13Liet al17have reported that acupuncture stimulation of Hegu (LI4) and Neiguan(PC6) significantly decreases the LF/HF ratio.In addition,Liu18reported that electroacupuncture stimulation of ST36 and PC6 can significantly decrease the LF/HF ratio.Our results revealed that HR and LF/HF ratio were significantly increased when participants remained in a standing position (Figure 3B,4).In the Shenshu (BL23) condition,there was no corresponding increase in the LF/HF ratio during or after stimulation,although the LF/HF ratio in the control and sham groups was significantly increased (Figure 4).Our results indicated that specific acupuncture stimulation of the Shenshu (BL23),and not the sham point,decreased the SNA in these participants.

It has been reported that acupuncture stimulation of Hegu(LI4),Zusanli (ST36),Taichong (LR3),Liangmen(ST21),Jizhong (CV6),Shenzhu (CV12),and sham points significantly decreases HR,although there was no significant difference in HR between these points.14However,the previous study was conducted in the supine position;therefore,SNA may have been affected by each individual’s condition.14In this study,the participants remained in the standing position in order to increase SNA.Our findings demonstrated that specific acupuncture stimulation of Shenshu (BL23) decreased SNA in the participants in the standing position.Furthermore,acupuncture stimulation of the sham point had no effect on SNA.This suggests that the increase in SNA by the standing position must contribute to the effect by acupuncture stimulation of Shenshu (BL23).

SNA plays an important role in the control of PRCviathe renal nerves.10It is considered that acupuncture stimulation of Shenshu (BL23) decreases PRC;however,these effects have been reported in only three studies,which have not been replicated recently.2,3,18Nakamuraet al2have shown that PRC was not significantly altered by acupuncture stimulation of Shenshu (BL23) in 7 healthy males,with a mean age of 23 years.In addition,Suzuki3have reported that PRC did not differ between acupuncture stimulation of Shenshu (BL23) and a sham point in 9 healthy males,ranging in age from 20 to 27 years.Leeet al18investigated the effect of acupuncture stimulation on PRC in 36 healthy males,with a mean age of 22 years.Participants were divided into 3 groups:Shenshu (BL23) (n=12),Dachangshu (BL25) (n=12),and non-stimulation group (n=12).They showed that acupuncture stimulation of Shenshu (BL23) significantly decreased PRC;18however,the experimental design was insufficient in these previous reports because some did not include non-stimulation and some do not include a stimulation of a sham point.Moreover,the PRC in previous reports may have been affected by each individual’s condition because these studies were conducted in a prone position.2,3,18In this study,because PRC is influenced by a diurnal rhythm,participation in each experiment occurred between 10 a.m.and 1 p.m.according to previous report.19There was no significant difference between the mean PRC in the Cont,Shenshu(BL23),and Sham conditions in the Post-SP period(Dunnett’s testP >0.05).The participants remained in the standing position in order to increase the PRC,as previously reported.19The PRC in the Cont and Sham conditions was significantly increased during the Post-S periods when compared with the Post-SP period (ContP<0.05;ShamP <0.01) (Figure 5).However,there was no significant difference in mean PRC during the Post-SP and Post-S periods in the Shenshu (BL23) condition(Figure 5).The ages and genders of participants and the diameter of acupuncture needle in previous studies were approximately the same as that in this study.2,3,18

Therefore,our results demonstrated that the acupuncture stimulation of Shenshu (BL23) specifically decreased PRC in participants in the standing position.Inhibition of SNA-associated PRC leads indirectly to a decrease in aldosterone,which increases urine output.It was reported that acupuncture stimulation of Shenshu (BL23)increased urine output compared to Weishu (BL21),Dachangshu (BL25),or Zhishi (BL52).4Leeet al18demonstrated that acupuncture stimulation of Shenshu(BL23) decreased PRC while that of Xinshu (BL15) did not change.

We have shown that acupuncture needles inserted at Shenshu (BL23) reached the region supplied by the intermediate and lateral branches of the posterior ramus of the L2 spinal nerve,which is connected to the muscle branch that supplies the erector spinae muscles.9The lateral branch stimulates the cutaneous branch,which innervates the skin on the back.9In this study,we revealed that acupuncture stimulation of Shenshu (BL23)can decrease SNA when compared with a sham point at the same dermatome as Shenshu (BL23),during increased SNA caused by the standing position.This decrease may be mediated by the muscle branch of the intermediate and lateral branches of the posterior ramus of the L2 spinal nerve.We suggest that Shenshu (BL23)is the specific location that can affect SNA-associated PRC.

Although this study has shown that acupuncture stimulation of Shenshu (BL23) decreased SNAassociated PRC,there are some limitations.The participants in this study were healthy volunteers as in the case with previous studies.2,3,18Several studies demonstrated acupuncture has clinical efficacy on various autonomic nerve-related disorders,20,21while it is not clear whether specific an acupuncture point regulates autonomic responses.If patients had participated in this study,it might have been demonstrated that acupuncture stimulation of Shenshu (BL23) treated autonomic nerverelated diseases through decreasing SNA-associated PRC.Although the participants in this study were healthy volunteers,our findings demonstrated that acupuncture stimulation of Shenshu (BL23) decreased SNAassociated PRC.Such knowledge may further lead to the development of effective treatment for autonomic nerverelated diseases.

5.ACKNOWLEDGEMENTS

The authors would like to thank Hiroe Kataoka,M.S.and Xiyao Shan,Ph.D.The authors also gratefully thank the students of Meiji University of Integrative Medicine for their cooperation.