Qu Jing-yang (屈敬阳), An Na (安娜), Liu Ding-ding (刘丁丁), Chen Long (陈龙), Xu Qian (许倩), Yang Zhi-xin (杨志新)

1 Hebei Province Key Laboratory of Research and Development for Chinese Medicine, Department of Traditional Chinese Medicine of Chengde Medical University, Chengde 067000, China

2 Traditional Chinese Medical College, North China University of Science and Technology, Tangshan 063000, China

3 Basic Medical Institute, Chengde Medical University, Chengde 067000, China

4 College of Traditional Chinese Medicine, Chengde Medical University, Chengde 067000, China

Complementary acupionts (CAs)[1-2], refer to the two acupoints respectively representing yin and yang,located in the head, neck, trunk, limbs, and other body parts, selected at the same time to play a synergistic effect. It’s characterized by treating yin for yang disorders and treating yang for yin disorders[3]. CAs are convenient for clinical application with good therapeutic effect[4-5]. Mast cells (MCs) are correlated with the meridian phenomenon, and play an important role in stimulation and regulation during acupuncture.Mechanical stimulation can degranulate MCs to release many active substances, such as bradykinin (BK),histamine (HIS), and prostaglandin (PG)[6-8]. The MCs stimulate multi-targeted cellular network communication by mobilizing, recruiting, degranulation and release of multiple active substances. Although CAs show satisfactory clinical efficacy, the synergistic mechanism is not clear[9].

In this study, electroacupuncture (EA) was performed to stimulate the CAs, Yinlingquan (SP 9) and Yanglingquan (GB 34). The MCs and the changes in released HIS and BK were observed to explore the basic mechanism of synergistic action of CAs, thus to provide ideas and basis for profound study and clinical application of the combination theory of CAs.

1 Materials and Methods

1.1 Laboratory animals and groups

Forty healthy female Wsitar rats, weighing 190-200 g,were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. During the experiment, the processing of animals completely conformed to theGuiding Opinions on the Treatment of Experimental Animals.

Grouping methods: Using random number table method, the rats were randomly divided into an acupuncture at Yanglingquan (GB 34) group (group A),an acupuncture at Yinlingquan (SP 9) group (group B),an acupuncture at Yanglingquan (GB 34) and Yinlingquan (SP 9) group (group AB), an acupuncture at points beside Yanglingquan (GB 34) and Yinlingquan(SP 9) group (group CD) and a normal control group(group K), 8 rats in each group.

1.2 Instruments and reagents

Thioacetylcholine (Tokyo Chemical Industry, Japan);toluidine blue O (TBO, AMRESCO, USA); cryostat slicer(Thermo Fisher Scientific, UK); Multiskan MK3 enzyme analyzer (Thermo, Finland); enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Enzyme Biotech Co., Ltd., China); G6805-Ⅱ EA instrument(Qingdao Xin Sheng Industrial Co., Ltd., China).

1.3 Experimental methods

Groups A, B, AB and CD: After the rats were fixed, the acupoints of rats were located according to theExperimental Acupuncture Science[10]. Yanglingquan(GB 34) and Yinlingquan (SP 9) were selected, and the acupuncture sites were marked with picric acid. i

Yanglingquan (GB 34): Five millimeter above and lateral to Zusanli (ST 36).

Yinlingquan (SP 9): On the medial side of the cruris,opposite to Yanglingquan (GB 34).

Group A: Acupuncture at bilateral Yanglingquan(GB 34).

Group B: Acupuncture at bilateral Yinlingquan (SP 9).Group AB: Acupuncture at bilateral Yanglingquan(GB 34) and Yinlingquan (SP 9).

Group CD: Points 3 mm away from bilateral Yanglingquan (GB 34) and Yinlingquan (SP 9) were used as control points and subjected to acupuncture[11].

Group K: Rats were fixed using the same method as rats in the other 4 groups without acupuncture stimulation.

Needles of 0.35 mm in the diameter and 40 mm in length were used for acupuncture. The handles were connected to the G6805-Ⅱ mode EA device after needling qi was obtained. Sparse-dense wave,frequency of 2 Hz/100 Hz and current of 1 mA were used to keep the needle handles slightly tremulous while the rats kept quiet. Rats were continuously stimulated by EA for 20 min each time. Experimental interventions were conducted on the 1st, 3rd, 5th and 7th days when the experiment started, for 4 times in total. Rats were housed as normal overnight after each intervention. All acupuncture interventions were conducted by trained professionals started from 3 p.m.The intervention order was group A, group B, group AB,group CD and group K.

1.4 Observation items and testing methods

Specimens from rats in each group were collected 2 h after the 7th day. Hair around the CAs was shaved,firstly with an electric hair clipper, and then with an electric shaver. The skin (about 1 cm × 1 cm) was marked with the acupoint as the center. The anesthetized rats using intraperitoneal injection of 3.5%chloral hydrate [10 mL/(kg·bw)] were sacrificed by dislocation after blood collection via abdominal aorta.

1.4.1 Serum HIS and BK levels

The abdominal aorta blood was harvested and centrifuged at 3 500 r/min immediately after anesthesia.The serum was collected and the levels of HIS and BK were determined by ELISA according to the kit instruction manual.

1.4.2 The number and the degranulation rate of MCs in tissues of the acupoint regions

Tissues (including the skin, subcutaneous tissue and a small amount of muscle, about 1 cm × 1 cm × 0.4 cm) of the acupoint regions were collected. The skin and subcutaneous tissues of the acupoint regions were collected in group A and group B; the skin and subcutaneous tissues of Yinlingquan (SP 9),Yanglingquan (GB 34) and those between the two acupoints were collected in group K, group AB and group CD. The collected tissues were wrapped with foil,put into the labeled frozen tube, and stored at –80 ℃refrigerator. Sections (thickness of 15 μm) were sliced in a constant cold box for immune-histochemistry staining against acetylcholinesterase with TBO counterstaining[12-13], followed by a gradient of alcohol dehydration, xylene transparent and neutral gum seal.Four sections were randomly selected for each rat, and 3 fields were observed for each section under optical microscope (× 100). The number of MCs and number of degranulated MCs were recorded in each field.

Statistics of the MCs numbers and degranulated MCs numbers: The MCs number for each slice was the total number of MCs in 3 fields per slice, and the average MCs number of the 4 slices was designated as the MCs number of each rat.

Degranulation rate = Number of degranulated MCs ÷MCs number × 100%.

1.5 Statistical method

Data were analyzed using SPSS 19.0 version statistical analysis software. Measurement data were presented as mean ± standard deviation (x±s), and the differences between groups were compared using one-way analysis of variance. If the variance was homogeneous, the least significant difference (LSD)method was used for comparison; if the variance was heterogeneous, then the Tamhane method was used for comparison.P＜0.05 indicated that the difference was statistically significant.

2 Results

2.1 General conditions of the rats

Before the intervention, the rat’s body weight in each group was 190-200 g and showed no statistically significant difference among groups (P=0.41), indicating the comparability. After the intervention, the rat’s body weight in each group was 200-220 g and showed no statistically significant difference among groups (P=0.89),(Table 1).

Table 1. Changes in rat’s body weight of each group (x±s, g)

2.2 Number and degranulation of MCs in tissues of the acupoints

The morphological observation of tissue sections in this experiment showed that different sizes and different numbers of MCs were distributed in dermis,subcutaneous tissue and muscle of the acupoints in each group (Figure 1). The shape of MCs was regular,mostly round or oval; the cell membrane was integral;the cytoplasm of MCs was filled with heterogenous particles; rare cells around showed scattered blue particles; degranulation was not obvious, around acupoints in rats of group K and group CD. In group A, B,and AB, the numbers and the volumes of MCs were increased; the cell edges were irregular; the cell membrane became thinner and ruptured; and a large number of blue particles were distributed around the cells with obvious degranulation.

Data statistics: Compared with group K, the MCs numbers in group AB, group A, group B and group CD were significantly increased (allP＜0.05), which was highest in group AB and statistically significantly different from the other groups (allP＜0.05). MCs numbers showed no statistically significant difference between group A and group B (P＞0.05). Compared to group K, the MCs degranulation rates of the other 4 groups all increased (allP＜0.05). MCs degranulation rate of group AB was the highest, there were statistical differences between group AB and the other 3 groups(group A, group B and group CD); MCs degranulation rate was not significantly different between group A and group B (P＞0.05), nor between group CD and group K(Figure 2).

Figure 1. Morphological structure and distribution of MCs in the tissues of the acupoints in each group (TBO staining, × 100)

Figure 2. MCs number and degranulation rate in tissues of the acupoints in each group

2.3 Comparison of histamine and bradykinin levels

The order from high to low of histamine level in rats’serum: group AB ＞ group B ＞ group A ＞ group CD ＞group K; the bradykinin level in group AB was significantly higher than those in the other groups (allP＜0.05); the difference in bradykinin level between group A and group B was not statistically significant(P＞0.05); the difference in bradykinin level between group CD and group K was not statistically significant(P＞0.05), (Table 2).

Table 2. Comparison of histamine and bradykinin levels in rat’s serum of each group (x±s, ng/mL)

3 Discussion

Human body consists of yin and yang, thus the pathogenesis is nothing more than imbalance between yin and yang (excess or deficiency). ‘Complementary acupiont’ is a new theory of piont combination based on the meridian pathways, the relationship between meridians and points, meridian theory, especially the yin and yang theory[3]. Yinlingquan (SP 9) and Yanglingquan (GB 34) are relative in yin and yang, and a pair of CAs widely used clinically[14]. Therefore,Yanglingquan (GB 34) and Yinlingquan (SP 9) were selected to study.

It has been proven that MCs are involved in the initiation and conduction of acupuncture effect information[15], thus are one of the key factors in acupuncture signal cascade amplification. After acupuncture stimulation, the MCs in the tissues ofthe acupoints are activated and degranulated to release various active substances such as HIS, BK,5-hydroxytryptamine (5-HT), PG and substance P (SP),which transform the physical stimulation of acupuncture into biological information to produce acupuncture effect, through the conduction and integration of the complex systems of nerve, endocrine,immune network, thus to play a regulatory role in target organs[16]. Therefore, the MCs number and degranulation rate, and the levels of histamine and bradykinin released by MCs in tissues of the acupoints were selected to observe the changes of body after acupuncture at the CAs.

HIS is a vasoactive amine compound in the tissues of human body. It exists widely in the tissues of the human body and is highest in MCs. HIS is also present in the tissues of skin, peripheral nerves, lungs, liver and gastric mucosa. In most cases, HIS is released by MCs after trauma or external specific antigen stimulation. Studies have shown that, HIS in the peripheral nerve is a pain-inducing substance that can cause physical pain and antagonist of acupuncture analgesia; in the central nervous system, it is an alleviation that plays a role in analgesic effect and synergetic acupuncture analgesia[17].Previous studies have shown that the 5-HT or HIS was positive in basophilic granules released from MCs in tissues of the acupoints after stimulation by acupuncture and moxibustion[18].

BK is an essential inflammatory mediator, converted from prokinin under the action of kallikrein after external irritant. The role of BK in the body is extremely broad. On the one hand, it can sensitize the sensory neurons and trigger the body's inflammatory response;on the other hand, it also involves in glandular secretion,smooth muscle contraction and other physiological activities. BK has played an integral role in the production and conduction of pain, and acupuncture analgesic process[19].

The results of this experiment showed that acupuncture could increase local MCs recruitment and degranulation rate, which was most significant after acupuncture at CAs of Yinlingquan (SP 9) and Yanglingquan (GB 34), and thus confirmed the‘amplifier’ of acupuncture effect[20]. These results were in line with the previous results and experimental hypotheses. Acupuncture at Yanglingquan (GB 34) and Yinlingquan (SP 9) group was significantly higher than acupuncture at Yanglingquan (GB 34) group,acupuncture at Yinlingquan (SP 9) group, and acupuncture at points beside Yanglingquan (GB 34) and Yinlingquan (SP 9) group, indicating that the acupuncture effect in acupuncture at CAs group was better than acupuncture at single acupoint and non-acupoints. Therefore, this experiment basically concluded that the increase of MCs recruitment and degranulation rate was one of the possible factors for the synergism of CAs combination. The statistical results showed that the serum levels of HIS and BK in rats after acupuncture were increased, which showed a positive correlation between them; and the serum histamine level reached the highest after acupuncture at CAs of Yinlingquan (SP 9) and Yanglingquan (GB 34). It is basically considered that the increase of serum HIS and BK may be one of the reasons for the synergy of CAs.

In this experiment, we found that the changes in morphology and recruitment trends of MCs, HIS and BK levels were related to the synergistic features of CAs.The synergism of acupuncture at CAs was confirmed via experimental study, which provided a new idea and basis for further exploring the mechanism of synergism,and the wider clinical application of CAs.

At the same time, it is suggested that the changes in MCs recruitment and degranulation in tissues of the acupoints and the changes in serum HIS and BK levels can be used as reference indicators to quantify the acupuncture effect, and provide some experimental and theoretical basis for the clinical evaluation of acupuncture efficacy.

Conflict of Interest

The authors declared that there was no potential conflict of interest in this article.

This work was supported by Project of Natural Science Foundation of Hebei Province (河北省自然科学基金项目, No. H2014406047); Key Program of Science and Technology of Hebei Higher Education (河北省高等学校科学技术研究重点项目, No. ZD20131001).

Statement of Human and Animal Rights

The treatment of animals conformed to the ethical criteria in this experiment.

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