Influence of Moxibustion of Complementary Acupoints on Analgesic Effect and Mast Cell Function in CIA Rats

Haiyan LI, Hengkang ZHU, Taotao CUI, Yaying CUI, Zhixin YANG, 3*

1. College of TCM, Chengde Medical University, Chengde 067000, China; 2. Heilongjiang University of Chinese Medicine, Harbin 150040, China; 3. Hebei Key Laboratory of Nerve Injury and Repair, Chengde 067000, China

Abstract [Objectives] To explore the moxibustion effect and mechanism of "Complementary Acupoints" on rheumatoid arthritis (RA). [Methods] A total of 32 Wistar rats were randomly divided into 4 group: normal control (K), model group (M group), "the Complementary Acupoints" Yanglingquan and Yinlingquan group (AB group), Shenshu and Zusanli group (CD group), with 8 rats in each group. Moxibustion was performed using refined moxa floss (2 mg/pc) on acupoints in both sides every other day, 5 pieces for each acupoint, and implemented for 10 times. Before and after the modeling and after the intervention, the left foot sole thermal pain threshold of each group was measured. After the intervention, the rats’ left paws were examined by X-ray to detect the number of mast cells (MCs) and degranulation in the affected area of the foot. [Results] The thermal pain threshold of the model group was lower than that of the K group (P<0.05). The number of MC in the affected area and the degranulation were increased. After the moxibustion intervention, the thermal pain threshold of the AB and CD groups was higher than that of the M group (P<0.05); the number of MCs and degranulation were decreased compared with M group, and the degree of reduction was better than that of CD group. The data of foot and claw showed that the foot and paw soft tissue swelling, bone and joint deformity and bone destruction were better in AB and CD groups were more relieved than that in M group. [Conclusions] The moxibustion of Complementary Acupoints can effectively intervene with RA of rats, and its mechanism may be closely related to the MC degranulation.

Key words Complementary Acupoints, Yin and Yang assisting each other and existing together, Yinlingquan and Yanglingquan, Moxibustion, Mast cells (MCs)

1 Introduction

The Complementary Acupoints[1-2]are a kind of correspondence acupuncture point developed by Professor Yang Zhixin through summarizing the experience based on the excavation of ancient and modern documents, and in accordance with the theory of Interdependence and Interaction between Yin and Yang, TCM Yin and Yang theory, and the circulation law of meridians[3], as well as the relationship between the internal and external organs of the meridians. Complementary Acupoints combine Yin and Yang, adjust the balance between Yin and Yang, and have the characteristics of conducting Yin from Yang, and conducting Yang from Yin[4]. The advantages of this idea of Complementary Acupoints are fewer acupoints, higher pertinence, and higher reliability of efficacy[2]; after more than 20 years of clinical practice, it has achieved satisfactory efficacy[5-6].

Mast cells (MCs) are widely distributed in connective tissues throughout the body. MCs not only play an important role in protecting the body against pathogen invasion, but also regulate specific immune responses. Besides, MCs participate in inflammatory diseases, and they can be activated by non-allergic stimuli such as neuropeptides, and often produce synergistic effects. The diversity of MCs production media and the complexity of MCs functions make them the hub or node of communication between various cellular networks in the tissues[7-8], so some scholars call MCs "universal regulators"[9]. Such regulating function of MCs is produced by releasing their particles and contents. MCs activation and degranulation can produce a variety of cytokines and mediators with different effects[10].

In this experiment, through the moxibustion at Yinlingquan and Yanglingquan of Complementary Acupoints, we observed the effect of mast cell function in the affected area of rheumatoid arthritis (RA) rats, and explored the effect and mechanism of Complementary Acupoints on rheumatoid arthritis.

2 Materials and methods

2.1 MaterialsBovine Type II Collagen (Chondrex, USA, item No.20022); Freund’s complete adjuvant (Sigma, USA, item No.f5881); X-ray diagnostic system (Ysio Max, Siemens, Germany); BME-410C automatic thermal pain stimulator (Beijing KAIHUISD Technology Co., Ltd.); Toluidine Blue (American AMRESCO Company).

2.2 Methods

2.2.1Experiment grouping and treatment. A total of 32 healthy male clean-grade Wsitar rats, weighing (140±15) g were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. [(license: SCXK (Beijing) 2016-0006], and raised in a barrier environment animal laboratory [license: SYSK (Hebei) 2017-001]. They were randomly divided into 4 groups: normal control group (K), model group (M group), "the Complementary Acupoints" Yanglingquan and Yinlingquan group (AB group), Shenshu and Zusanli group (CD group), with 8 rats in each group. In the experiment, the treatment of animals was in full compliance with the provisions on the treatment of laboratory animals in theRegulationofthePeople’sRepublicofChinaontheAdministrationofLaboratoryAnimal.

CIA model making: the operation was performed under aseptic conditions: the bovine type II collagen (2 mg/mL) and CFA were mixed in equal volume, and the rubber head dropper was used to repeatedly suck and mix (in an ice bath) to make it fully mixed, and emulsified (the mixture was a milky white viscous liquid and not dispersed after dripping into the water). After intraperitoneally anesthetized rats with 4 g/100 mL chloral hydrate at a dose of 0.8 mL/100g, with a well-prepared emulsion, the rats were injected intracutaneously at a dose of 0.2 mL/piece. Multi-point injection was performed on the back and tail root (the injection angle was almost flat, and the angle between the injection angle and the rat’s skin was not greater than 15 degrees to prevent injection into the subcutaneous tissue. According to the hand feel, the needle should move freely when swinging the needle from side to side. When you feel obstruction, it means the intradermal injection site is appropriate. After the injection, the skin present round and bulging. 7 d later, performed the operation again with the same injection method as above[11]. The arthritis index[12](AI ≥ 9) was used as the criterion for successful modeling.

Rats were shaved at the acupoints on both sides, using 2 mg of refined moxa floss, 5 pieces for each acupoint, and implemented for 10 times. The acupoints of the model group and normal group were shaved in the same way, fixed in the same way, without any intervention.

2.2.2Detection indicators and methods. We used the BME-410C automatic thermal pain stimulator to detect the thermal pain threshold. First, placed the rat in the isolation frame, and after stabilized, focused on the middle of the left hind foot sole with a heat source. When the rat licks its feet and shakes feet quickly, it is considered to be positive, set 20 S as the upper limit of the thermal pain threshold to prevent thermal burns; selected the value between 6-20 S as the effective value, and monitored continuously, with an interval of 5-10 min each time, and selected the average of the two effective values as the thermal pain threshold.

After anesthesia with chloral hydrate, rats were fixed and placed on an X-ray diagnostic system. X-rays were taken to observe the soft tissue and bone damage of the left foot and paw.

Rats were killed after anesthesia, then samples were taken, routinely fixed, dehydrated, and embedded, and a modified toluidine blue staining method[13]was used to stain the affected part of the foot. Under an optical microscope × 400 magnification, 4 slices were observed for each rat, 3 fields of view were selected for each slice, and the average value was taken to observe the number and degranulation of MCs in each group.

3 Results and analysis

3.1 Thermal pain thresholdThere was no difference in heat pain threshold of rats in each group before modeling (P>0.05), and after modeling, they were all lower than group K (P<0.05); after moxibustion intervention, the thermal pain threshold of group AB and CD was higher than group M (P<0.05), indicating that moxibustion has a therapeutic effect on CIA rats, and the group AB is better than the CD group, as shown in Fig.1.

Note: compared with the group K, *P<0.05; compared with the group M, #P<0.05.

3.2 X-ray images of left foot and pawThe paw soft tissue, bone growth, and bone and joint structure of the group K were normal; the paw soft tissue of the group M was severely swollen, the foot bones and joints were severely deformed, and the heel bone was severely damaged; in the AB and CD groups, the paw soft tissue was swollen, the deformity of the foot bones and joints and the degree of bone destruction were all reduced compared with the M group, and the reduction degree of the AB group was better than that of the CD group, as shown in Fig.2.

3.3 Number and degranulation of MCsUnder the microscope, cells with smooth cell membranes and round and oval shapes are normal MCs; cells with ruptured cell membranes, irregular shapes, and blue-stained particles from the periphery are degranulated MCs. The number and degranulation of MCs were increased in the M group compared with the K group; the AB and CD groups were increased compared with the K group, and the degranulation increased; compared with the M group, the number of MCs was decreased, and the AB group was less than that of the CD group, the difference was statistically significant (P<0.05), as shown in Fig.3 and Table 1.

Note: Arrows denote pathological abnormality.

Fig.3 Number and degranulation of mast cells in the affected foot of each group of experimental rats (400×)

Table 1 Comparison of the number of foot mast cells and degranulation rate between groups n=8)

4 Discussions

Rheumatoid arthritis (RA) is a chronic autoimmune system disease with joint synovial lesions as the main clinical manifestations. The incidence rate of RA in China is 0.2%-0.9%[14], and both the incidence and disability are high, it seriously affect the quality of life of patients. So far there is no clinically effective method to cure RA, and the main purpose of treatment is to relieve pain symptoms and improve quality of life of patients. In animal experimental studies, Shenshu and Zusanli are commonly used acupoints for intervention in RA[15].

Moxibustion not only has anti-inflammatory effects, but also adjusts, catalyzes, and activates immune function, thereby improving the patient’s immune response level and enhancing the body’s immune and anti-inflammatory ability. In the theory of TCM, RA belongs to the category of impediment disease. Studies[14-16]have shown that moxibustion has a good effect of warming meridians, dredging collaterals, reducing inflammation and pain, and can significantly improve the clinical efficacy of RA patients.

Complementary acupoints[1-2]refer to the two compatiable acupoints on the head, neck, trunk, limbs and body parts with Yin and Yang opposite to each other, which can play a synergistic effect through Yin and Yang assisting each other and existing together. Its advantage is high pertinence and exact curative effect.

Complementary acupoints Yinlingquan and Yanglingquan, located on both sides of the knee joint, are commonly used acupoints for clinical treatment of joint diseases[17]. Collagen-induced arthritis (CIA) model has many similar characteristics with RA in terms of clinical manifestations, pathology, immunological changes and pathological mechanisms, and is an ideal animal model for RA[18].

In the 1970s, Chinese scholars discovered that the distribution of MC at the meridian points is specific[19]. The number of MCs in some acupoints in the human body is larger than other non-acupoint areas. The number of MCs in the deep tissues of the meridians is more than that of the superficial ones, and MCs usually run along the meridians. MCs are usually distributed in the direction of small blood vessels and nerve vesicles along the meridian direction, which is related to the meridian phenomenon[19-20]. The specific distribution of MCs on the meridian lines is conducive to the reaction of MCs and their released mediators such as histamine and substance P with local nerves and blood vessels, and this reaction affects the local and overall functional state.

Studies[15]have confirmed that moxibustion on Shenshu and Zusanli can significantly alleviate the infiltration of inflammatory cells on the synovial membrane in RA model rats and improve the

proliferative response of synovial cells. This study also shows that moxibustion on Complementary acupoints Yinlingquan and Yanglingquan of RA rats, has a good effect on the soft tissue swelling, foot joint deformity, and bone destruction in the affected area of RA rats, indicating the effectiveness of moxibustion on Complementary Acupoints Yinlingquan and Yanglingquan on intervention of RA rats. In addition, moxibustion on complementary acupoints Yinlingquan and Yanglingquan can reduce the number and degranulation of MCs and in the affected area of the foot, which may be closely related to the mechanism of the moxibustion effect of complementary acupoints on rheumatoid arthritis. Furthermore, the improvement degree of Complementary acupoints Yinlingquan and Yanglingquan are better than Shenshu and Zusanli. The reasons may be the action of Yin and Yang assisting each other and existing together, and the selected two points being close to the joints, indicating that meridians and acupoints have relatively specificity in regulating this function of target organs.