This paper describes in detail the operation procedures and precautions to be taken during Du-moxibustion in the treatment of ankylosing spondylitis in experimental mice.
Ankylosing spondylitis (AS) is a progressively worsening and disabling form of arthritis that primarily affects the axial skeleton. This disease mainly involves the spine and the sacroiliac joint. Fusion of the spine and the sacroiliac joint may occur in the later stage of the disease, resulting in spinal stiffness and kyphosis, as well as difficulty in walking, which seriously affects the quality of work and daily living activities and imposes a heavy burden on the patient, the family, and society. Increasing attention has been paid to non-pharmacotherapy as an alternative therapy for AS. Moxibustion is an ancient therapeutic technique used in Traditional Chinese Medicine (TCM). Du-moxibustion therapy, a unique and innovative external treatment developed on the basis of ordinary moxibustion, has a definite therapeutic effect on AS. Du-moxibustion skillfully combines the compatible techniques of TCM to integrate meridians, acupoints, Chinese herbal medicine, and moxibustion. This paper describes the operation procedures and precautions to be taken during Du-moxibustion in experimental mice in detail to provide an experimental basis for the study of the mechanism of Du-moxibustion in the treatment of AS.
Ankylosing spondylitis (AS) is a progressively worsening and disabling form of arthritis that primarily affects the axial skeleton. According to the latest epidemiological survey, ~0.01%-1.8% of people in the world suffer from this condition1, most of whom are young males (male to female ratio is ~2-3:1)2. The incidence of AS in the Chinese population is 0.2%-0.4%. This disease mainly involves the spine and the sacroiliac joint. Fusion of the spine and the sacroiliac joint may occur in the later stages of the disease, resulting in spinal stiffness and kyphosis, as well as difficulty in walking, which seriously affects the quality of work and daily living activities and imposes a heavy burden on the patient, family, and society1. Currently, there is no definitive solution for AS, and the provided treatments, both pharmacological and non-pharmacological, primarily focus on alleviating pain, slowing disease progression, and enhancing the quality of life. In recent years, because pharmacotherapeutic options have been very limited3, increasing attention has been paid to non-pharmacotherapy, which has become an alternative therapy for AS. In China, patients often prefer external treatment consisting of Traditional Chinese Medicine (TCM), which has minor side effects and great convenience during the treatment.
Moxibustion is an ancient therapeutic technique used in TCM. Du-moxibustion is an external treatment technique consisting of "medicated moxibustion with foam" on the spine segment of the Du meridian. It is mainly used in the treatment of AS and has shown safety and effectiveness. Compared with ordinary moxibustion, Du-moxibustion has the characteristics of a wide moxibustion area, a large moxa cone, strong firepower, and high temperature4. A systematic review and meta-analysis suggest that moxibustion is an effective complementary treatment for AS5. A recent study confirmed that the clinical symptoms and some inflammatory factors of AS patients were improved by moxibustion6. Studies have revealed that Du-moxibustion can increase the brain content of β-endorphin and exert a central analgesic effect7, downregulate the expression of the HLA-B27 gene8, and delay the recurrence rate of AS. Du-moxibustion also has an ameliorating effect on the progression of AS by decreasing the inflammatory index ESR (ESR) and levels of C-reactive protein (CRP), C-terminal peptide of type I collagen (CTX-I), and Dickkopf1 protein (DKK1)9,10.
Another clinical study showed that Du-moxibustion can further adjust the disordered T cell subsets, Ig, and complement C3 to balance the immune mechanism11. In terms of bone metabolism, moxibustion can inhibit the rise of serum alkaline phosphatase (ALP) and serum phosphorus (S-P), increase serum calcium (S-Ca), bone mineral density (BMD), and bone strength12. Moreover, Du-moxibustion can repair spinal function at multiple points to relieve fatigue symptoms in AS patients13. Some animal studies have revealed the potential mechanism of moxibustion in treating AS. A study indicated that moxibustion significantly improved the overall health status, reduced the levels of paw thickness, and decreased the levels of IL-1β, PGE2, IL-6, and TNF-α in the ligament tissue samples of the spine. Metabolic pathway analysis linked the identified metabolites to the TCA cycle, as well as lipid, amino acid, intestinal flora, and purine metabolism14. Another study showed that moxibustion suppressed the expression of pro-inflammatory cytokines, IL-1β, TNF-α, IL-17, and IL-6; reduced the mRNA levels of RANKL, RANK, ALP, and OCN; and improved the histopathological features in AS mice15.
While these studies suggest that moxibustion may be effective in the treatment of AS, more research is needed to confirm these findings and determine the optimal treatment protocol for moxibustion in patients with AS. Our team created and modified the moxibustion therapy based on the basic theory and practical operation of traditional Chinese moxibustion, which has been widely carried out and applied in Chinese clinical practice for over 35 years. Although there are few high-quality studies on the treatment of AS by Du-moxibustion, its indication in treating AS has been supported by evidence. However, the mechanism of treating AS with Du-moxibustion is worthy of further study. Animal study is an important method to explore the mechanism of Du-moxibustion in the treatment of AS. The clinical operation of Du-moxibustion is relatively mature but rarely used in the study of the mechanism in animal experiments. This paper describes in detail the operation and precautions to be taken during Du-moxibustion in the treatment of AS experimental mice.
All animal experiments were approved by the Experimental Animal Welfare Ethics Review Committee of Shandong University of Traditional Chinese Medicine (No. SDUTCM20230831301).
1. Animal preparation
2. Establishment of the AS mouse model
NOTE: The AS model was induced with cartilage proteoglycan (PG), which is a classic modeling method16,17,18,19,20,21. BALB/c strain mice were repeatedly immunized with PG to induce tendinitis and spondylitis. See the Table of Materials for details related to the materials, reagents, and instruments used in this protocol.
3. Du-moxibustion
NOTE: Prepare in advance the three most important raw materials for Du-moxibustion (Figure 1).
4. Evaluation of arthritis index (AI)
5. Rotarod test
NOTE: The rotarod test is used to assess motor coordination and balance by recording the time the mice spend on the rotating drum. This experiment consisted of three trials separated by 20 min intervals. The official test data were recorded from the third trial, with the first two serving as training exercises.
6. Open field test (OFT)
NOTE: The OFT is used to evaluate the state of autonomic movement, aiming to identify pathological behavior. Four square open field arenas (50 cm x 50 cm x 40 cm) were placed together to form the apparatus. The bottom of the box was divided into nine equal squares.
7. Euthanasia and specimen collection
8. Histological analysis
9. Enzyme-linked immunosorbent assay (ELISA)
10. Statistical analysis
PG-induced model mice developed symptoms of peripheral arthritis, characterized by redness and swelling of the extremities and toes, and gradually developed axial arthritis as early as week 14, which is very similar to the manifestation of AS. Therefore, the AS mouse model was considered successful if the arthritis index (AI) was over 3 points at 14 weeks after the last injection22. When the paws of the mice appeared swollen, the Du-moxibustion treatment was carried out once a week for a total of 3x. After treatment, the mice were euthanized by cervical dislocation under isoflurane deep anesthesia, their blood was collected, and the ankles were excised.
Effects of Du-moxibustion on motor coodination and autonomic movement
Bovine PG-induced AS was adopted as a model to observe the effect of Du-moxibustion at the Du meridian on motor coordination. Initially, the joint swelling score was used to assess whether the model was successful, and motor coordination and autonomic movement were evaluated using the rotarod test and OFT.
Evaluation of AI determined the severity of peripheral arthritis. After modeling, two-way repeated-measures ANOVA revealed that the joint swelling scores in the model and Du-moxibustion groups significantly increased compared with those in the control group before the Du-moxibustion (P < 0.01; Figure 6A). Du-moxibustion treatment decreased joint swelling scores compared with those of the model group after the Du-moxibustion (P < 0.05; Figure 6A).
The rotarod test was employed to evaluate motor coordination and balance. Two-way repeated-measures ANOVA revealed significant differences over time (F = 13.928, P = 0.000 < 0.05) and interaction effects of time and group (F = 12.583, P = 0.000 < 0.05). After modeling, the latency time of the control group was not different from that before modeling. However, after modeling, the latency times of the model and Du-moxibustion groups significantly decreased compared with those in the control group (P < 0.01; Figure 6B), and there were no significant differences between the model group and the Du-moxibustion group (P > 0.05; Figure 6B). After treatment, the latency time of the Du-moxibustion group significantly increased compared to that of the model group (P < 0.05; Figure 6B). These results indicated that Du-moxibustion was effective in improving motor coordination in AS mice.
OFT was performed to detect autonomic movement. The parameters were collected and analyzed automatically using the software (Figure 6C–E); the total distance and average speed were mainly observed in this study. There was no significant difference in the total distance and average speed among the groups before modeling (P > 0.05; Figure 6C,D). After modeling, the total distance and average speed significantly reduced compared with the control group (P < 0.05; Figure 6C,D). After treatment, the total distance and average speed of the Du-moxibustion group significantly increased compared to those of the model group (P < 0.05; Figure 6C,D). These results indicated that Du-moxibustion was effective in improving autonomic movement in AS mice.
Effects of Du-moxibustion on ankle joint pathology
HE staining was used to observe the pathological changes in the ankle joint. In the control group, there was no damage to the synovium structure, no congestion in the synovium, and no obvious inflammatory cell infiltration and necrosis. However, in the model group, synovium hyperplasia and infiltration were observed in the joint cavity, in addition to inflammatory cell infiltration and tissue edema. Combined with AI evaluation, it was proved that the AS model was successfully established. Compared with the model group, the synovium of mice in the Du-moxibustion group was more intact, interstitial edema was reduced, and the articular cartilage was relatively intact (Figure 7).
Effects of Du-moxibustion on plasma IL-17 and TNF-α levels
ELISA was performed to measure IL-17 and TNF-α in the plasma. The plasma IL-17 and TNF-α levels of the model group were significantly increased compared with those of the control group (P < 0.01; Figure 6F,G). Du-moxibustion decreased plasma IL-17 and TNF-α levels (P < 0.05; Figure 6F,G).
Figure 1: The raw materials needed for Du-moxibustion. Please click here to view a larger version of this figure.
Figure 2: Making a moxa cone. Please click here to view a larger version of this figure.
Figure 3: Prepare ginger water and make a trapezoidal ginger column. Please click here to view a larger version of this figure.
Figure 4: Location of GV14 and GV2 in mouse. (A) GV14 located at the subspinous process of the seventh cervical vertebra; GV2 located in the hiatus sacral canal. (B) Schematic diagram of Du moxibustion on the mouse. (C) Stimulated area of Du moxibustion. Please click here to view a larger version of this figure.
Figure 5: Operation procedure of Du-moxibustion in mouse. (A) Shave the hair and smear the ginger juice on the mouse in the prone position. (B,C) Sprinkle TCM powder evenly. (D) Cover the powder with mulberry paper. (E) Fix the ginger column on the mulberry paper firmly. (F) Put a moxa cone on the ginger column; then, light it. Please click here to view a larger version of this figure.
Figure 6: Effects of Du-moxibustion on motor coordination, autonomic movement, and IL-17 and TNF-α levels. (A) The joint swelling scores in mice. (B) The latency time in the rotarod test. (C–E) Total distance, average speed, and track diagram of OFT. (F) IL-17 levels in plasma. (G) TNF-α levels in plasma. Values are expressed as the mean ± SEM (n = 6). ##P < 0.01, #P < 0.05 versus the control group; $$P < 0.01, $P < 0.05 versus the model group. Abbreviations: IL = interleukin; TNF = tumor necrosis factor; OFT = open field test. Please click here to view a larger version of this figure.
Figure 7: HE staining of the ankle joint. (A) The joint cavity was smooth without inflammatory infiltration in the control group. (B) In the model group, synovium hyperplasia and infiltration were observed in the joint cavity. (C) In Du-moxibustion group, the joint cavity of mice was smooth and edema was alleviated. Scale bars = 200 µm. Abbreviation: HE = hematoxylin-eosin. Please click here to view a larger version of this figure.
AS is clinically manifested as back, lumbar, and joint swelling pain, and spinal deformity and joint rigidity may occur in severe cases26. Early diagnosis and treatment can improve the quality of life, reduce disability rates, and improve the prognoses of patients with AS. According to traditional Chinese medicine (TCM), AS is believed to be caused by a deficiency of Yang in the kidney and the Du meridian, as well as an accumulation of cold evil internally. TCM holds the view that moxibustion, based on the concept of meridians, can generate Qi that warms the body's surface. This Qi then travels downwards along the meridians and acupoints. In The Inner Canon of Huangdi (a very classic and important TCM masterwork), it is recorded that "the diseased Du meridian is treated." As AS is located in the Du meridian, stimulating the Du meridian is used as a therapeutic point. The Du meridian is considered to be the ocean of Yang channels where Yang Qi reaches its peak. Moxibustion on the Du meridian can supplement Yang qi, activate the effect of the Du meridian, and adjust the warming effect of kidney Yang, that is, enhance the immune regulation function of Western medicine.
From the perspective of Western medicine, the spinal cord plays an important role in transmitting neural information. Du moxibustion, which acts on the entrance and exit of spinal nerve roots, can affect the function and regulatory mechanism of the nervous system through the regulation of neurotransmitters. In the 1990s, it was demonstrated that Du-moxibustion increased the content of β-endorphins in the hypothalamus of rats, thus having a central analgesic effect7. In addition, enthesitis is a common complication of AS, characterized by persistent pain and muscle stiffness, which can affect joint and body movement when severe. As a TCM treatment method that has been clinically validated, Du-moxibustion can effectively alleviate inflammation, promote tissue repair, and improve enthesitis in treating AS.
Specifically, the effects of acupuncture and moxibustion are mainly reflected in the following aspects. First, they promote blood circulation and the absorption of inflammation. Du-moxibustion stimulates blood circulation and metabolism in the tissues surrounding the spine through the warming effect and promotes the absorption of inflammation and tissue repair, thereby reducing the degree of inflammation and pain of tendon attachment inflammation. Second, they improve muscle and ligament tension. The stimulation effect of Du-moxibustion can improve the muscle tension around the spine, relax the hardened soft tissues, promote the activity of spinal joints, and reduce the pain and stiffness caused by muscle tension and ligament stretching. In summary, Du-moxibustion, as an adjunctive therapy, can comprehensively regulate the activities of the body's nervous, immune, and circulatory systems by acting on the spinal region, making the body more balanced as a whole.
Inflammation is the main manifestation of AS27. TNF-α is a multi-effector cytokine and inflammatory factor produced under the stimulation of mononuclear macrophages28. The activity of ALP and collagen synthesis can be inhibited by TNF-α, resulting in bone loss and cartilage destruction29. In recent years, studies have found that IL-17 plays an important role in the pathogenesis of AS, which has been fully demonstrated in a review30. IL-17 can directly act on osteoblasts, promote the expression of nuclear transcription factor-κB (NF-κB) receptor activator ligand (RANKL), and promote the robust generation of osteoclasts. At the same time, IL-17 also affects mesenchymal stem cells (MSCs), inducing their proliferation and promoting their differentiation into osteoblasts31. Kenna32 et al. found that the level of IL-17 in the peripheral blood of AS patients increased by almost two-fold compared with healthy controls. The study suggests that IL-17 antagonists may be a new option for AS patients who do not respond well to TNF-α antagonists33. A clinical study showed that a dosing regimen of IL-17 antagonists was superior to a placebo for improving radiographic axial spondyloarthritis signs and symptoms in patients34. The results of this study showed that the plasma levels of TNF-α and IL-17 in the model group were significantly higher than those in the control group, and the plasma levels of TNF-α and IL-17 were significantly decreased after Du-moxibustion treatment.
Du-moxibustion therapy skillfully uses the compatible techniques of TCM to integrate meridians, acupoints, Chinese herbal medicine, and moxibustion. The raw materials of Du-moxibustion therapy mainly included moxa velvet, ginger, and TCM powder. From the perspective of TCM, moxa velvet, a processed product of mugwort, has the function of activating collaterals, dispelling cold, and relieving pain. Ginger plays a role in relieving external symptoms and dispelling cold. Modern research also shows that ginger has anti-inflammatory and analgesic properties35. Wiping the skin with ginger juice before treatment helps open the pores and promote transdermal permeation36, in addition to helping the TCM powder adhere to the skin. The trapezoidal ginger column is firmly fixed on the back to provide a solid base for the moxa cone. After moxa cone burning, the effects of firepower, ginger, and TCM powder work together on the Du meridian. In view of the pathogenesis of Yang deficiency in this disease, Du-moxibustion directly acts on the site of the disease, warming the meridians, dispersing cold, supplementing Yang Qi and essence, promoting blood circulation, and balancing Yin and Yang to treat the root of disease.
When performing Du-moxibustion, several details should be considered. First, the operation site of Du-moxibustion is fixed, from the Dazhui point (GV14) to the Yaoshu point (GV2) (anatomically from the 7th cervical spine spinous process to the tail vertebra, including the entire spine), which is consistent with the incidence site of AS (Figure 4). Second, the humidity and thickness of ginger are important. The excess ginger juice must be squeezed out after the ginger is processed into ginger mud so that the ginger mud has no granular feeling and its thickness is appropriate to be made into the trapezoidal ginger column. High humidity and moisture in the ginger mud can easily hinder the penetration of moxibustion fire and reduce the amount of moxibustion stimulation. If the ginger raw material is too dry and the particles are too large, it is difficult to make the trapezoidal ginger column, leading to rapid heat penetration and scalding of the skin. Third, the size and firmness of the moxa cone should be considered. Moxa cones used for moxibustion in mice are ~4 cm long and 0.5 cm in diameter and should be tight at the time of preparation. A test for the tightness is when the cone does not unravel even after being dropped on a surface. If the moxa cone is not compact enough, it will lead to a reduction in the amount of moxa velvet, a shorter burning time, and a reduction in firepower. Fourth, when igniting the moxa cone, the head, middle, and tail of the moxa cone should be lit with fine incense to make the moxa cone burn slowly to achieve the nourishing effect of ebb and flow. Fifth, musk is an important ingredient in the medicinal powder of moxibustion. The second day after moxibustion, there should be crystal clear blisters like small pearls on the Du meridian.
A large number of clinical studies have confirmed the definite efficacy of Du-moxibustion in the treatment of AS4,5,6. However, research on its mechanism of action in animal experiments progresses slowly. Through research and innovation, years of experience in the operation of Du-moxibustion have led us to investigate its use in mice experiments. This paper describes in detail the operation procedures and precautions of Du-moxibustion in the treatment of AS in experimental mice. In addition, the effect of moxibustion on motor coordination and the plasma levels of TNF-α and IL-17 in AS mice were observed. This study provided the most basic experimental basis for the study of the mechanism of Du-moxibustion in the treatment of AS. There are still some limitations to this study. For example, in future studies, we will pay attention to the evaluation of different parameters such as placebo moxibustion, dose, range, and treatment time to obtain the most effective combination schemes. In later studies, imaging methods can be used to evaluate the treatment results in a more intuitive way.
The application prospect of Du-moxibustion therapy is promising. The Du meridian, known as the Governor Vessel, plays a crucial role in regulating the flow of Qi and blood throughout the body. Moxibustion, a traditional Chinese therapy, involves the burning of mugwort on specific acupoints to stimulate the meridians and promote the body's self-healing mechanism. Compared with acupuncture, Du-moxibustion is a combination of various techniques that act on the body surface, which has the characteristics of comfort and a strong and wide range of action. Moreover, Du-moxibustion therapy focuses on the back and spinal area, targeting the Du meridian's pathway. It can enhance the body's immune system, improve blood circulation, and alleviate various physical and mental conditions. The therapy has demonstrated its effectiveness in treating diseases such as musculoskeletal disorders, neurological conditions, gynecological disorders, and mental health issues. With advances in research and technology, the application of Du-moxibustion therapy is expected to expand further. Future studies may explore its potential in areas such as pain management, stress reduction, digestive disorders, and respiratory conditions. The combination of traditional knowledge and modern scientific understanding could lead to better integration of Du-moxibustion therapy into mainstream healthcare practices. In conclusion, Du-moxibustion therapy holds great promise in its application and integration into various healthcare fields. Further research, clinical trials, and collaboration between traditional Chinese medicine practitioners and modern healthcare professionals are essential to unlock its full potential.
The authors have nothing to disclose.
This work was supported by the General Program of the National Natural Science Foundation of China (No.82174491) and the Qilu School of Traditional Chinese Medicine Academic School Inheritance Project (No. Lu Weihan [2020]132). Thanks to the Laboratory Animal Center of Shandong University of Traditional Chinese Medicine for providing us with experimental conditions.
10% EDTA | Shanghai Macklin Biochemical Co., Ltd.,Shanghai, China | ||
alcohol pads | HYNAUT, Qingdao Hainuo Biological Engineering Co., LTD, Qingdao, China | ||
anesthesia machine | Medical Supplies & Services INT. LTD, Keighley, UK | ||
centrifuge tubes | Axygen, Corning, NewYork, UAS | ||
complete Frech's adjuvant (CFA) | aladdin,Shanghai, China | F393378 | |
cotton ball | Henan RUIKE MEDICAL Equipment Co., Ltd.,Xinxiang, China | ||
cotton gauze | Henan RUIKE MEDICAL Equipment Co., Ltd.,Xinxiang, China | ||
cutting board | self-preparation | ||
decorin from bovine articular cartilage | Sigma-Aldrich, MO, USA | D8428 | |
depilatory cream | Veet, Reckitt Benckiser (China) Investment Co. LTD, Shanghai, China | ||
electronic scale | Shanghai Yajin Electronic Technology Co., Ltd.,Shanghai, China | ||
Eppendorf tube | Axygen, Corning, NewYork, UAS | ||
eye lubricant | Beijing Shuangji Pharmaceutical Co., LTD., Beijing, China | ||
ginger | self-preparation | ||
GraphPad Prism 7 software | GraphPad Software,Boston, USA | ||
hair clipper | Super human Group CO LTD, Jinhua, China | ||
heating pads | Shenzhen Leshuo Tech Co., Ltd.,Shenzhen, China | ||
incomplete Freund’s adjuvant (IFA) | aladdin,Shanghai, China | F393371 | |
injection syringe | Shandong Xinhua Ande Medical Supplies Co., LTD, Zibo, China | ||
isoflurane | Shenzhen Rayward Life Technology Co., LTD, Shenzhen, China | R510-22-10 | |
joss stick | Shijiazhuang Lidu Fragrant Industry Co., LTD.,Shijiazhuang, China | ||
juicer | Braun (Shanghai) Co., Ltd.,Shanghai, China | ||
knife | self-preparation | ||
lighter | Zhejiang tiger-lighter Co. LTD | ||
mortar | Luoyang Yinai Ceramic Technology Co., LTD.,Luoyang, China | ||
Mouse IL-17 ELISA Kit | absin, Shanghai, China | abs520009-96T | |
Mouse TNF-α ELISA Kit | Wuhan Sanying, Wuhan, China | KE10002 | |
mulberry paper | Yishui County Mulinsang paper Co., LTD, Linyi, China | ||
OFT | Xinruan,Shanghai, China | XR-XZ301 | |
paper cup | self-preparation | ||
pipettes | OXFORD BIO INSTRUMENTS INC.,Oxford, UK | ||
refined moxa velvet | self-preparation | ||
rotarod | Xinruan,Shanghai, China | XR-6C | |
scientz-48L cryogenic high throughput tissue grinder | Ningbo Xinzhi Biotechnology Co., LTD | ||
scissors | Shandong Jiaren Medical Supplies Co., Ltd., Zibo, China | ||
semi-automatic paraffin sectioning machine | Leica Camera AG, Watznach, Germany | ||
SPSS 25.0 software | International Business Machines Corporation, NewYork, UAS | ||
TCM powder | self-preparation | ||
tips | Biosharp, Labgic, Beijing, China | ||
writing brush | Yishui County Mulinsang paper Co., LTD, Linyi, China |