Du-Moxibustion in a Mouse Model of Ankylosing Spondylitis

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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

1. Determine the experimental groups and the number of animals per group in advance.
2. Choose male BALb/c mice, house them at a constant temperature of 22 ± 1 °C, constant humidity of 60 ± 5%, and 12 h diurnal cycle light environment.

2. Establishment of the AS mouse model

NOTE: The AS model was induced with cartilage proteoglycan (PG), which is a classic modeling method^{16,17,18,19,20,21}. 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.

1. Use a 200 µL pipette to draw 200 µL of PG and CFA (or IFA) into a 1.5 mL centrifuge tube. Shaking gently to mix the liquid in the centrifuge tube.
2. Place the centrifuge tube in the tissue grinder for homogenization and then incubate on ice for 4 min. Repeat 5x. Set the following parameters in the tissue grinder: 60 Hz, grinding 30 s, interval 10 s for a total of 6 cycles.
3. Extract 0.2 mL of emulsified PG protein from the centrifuge tube with a 1 mL syringe.
4. Grab and fix the mouse with the left hand. Lay the mouse in a supine position, keeping the head lower than the tail to prevent damage to the abdominal organs when the syringe is inserted.
5. Sterilize the abdomen with alcohol swabs using the right hand.
6. With the syringe in the right hand, insert the needle subcutaneously slightly to the left or right of the linea alba ventralis in the lower half of the abdomen.
7. Push the needle 3-5 mm subcutaneously parallel to the midabdominal line and then push the needle into the abdominal cavity at a 45° angle to the skin.
8. Penetrate the peritoneum, withdraw the needle plug, and slowly inject the emulsified PG protein.

3. Du-moxibustion

NOTE: Prepare in advance the three most important raw materials for Du-moxibustion (Figure 1).

1. Prepare moxa cones. Take the velvet in the hand, with two palms facing opposite directions exerting a twisting force to rub the velvet into a spindle shape (Figure 2)
2. Cut the ginger into small pieces and puree it in a juicer (Figure 3). Filter the ginger puree through cotton gauze. Separate the ginger juice and ginger mud from the ginger puree to prepare for the next step.
3. Prepare 10 g of ginger mud and 20 mL of ginger juice and place them in paper cups for later use.
4. Weigh 0.1 g of TCM powder (a mixture of cinnamon powder, clove powder, notopterygium powder, antharidin powder, and artificial musk)²² and grind it in a mortar.
5. Place the mice in the anesthesia induction box of the animal anesthesia machine and anesthetize them with a mixture of 2-3% isoflurane and oxygen at a flow rate of 2 L/min.
6. Assess the depth of anesthesia by observing the absence of a startle reflex initially and verify the level of surgical anesthesia by checking for the absence of a pedal reflex in response to a gentle toe pinch.
7. After the mouse has reached surgical plane anesthesia, take it out of the box and fix it on the operating table in a prone position. Align the mouse's mouth and nose with the outlet of the anesthetic.
8. Administer anesthesia via a mask attached to the anesthetic machine, through which isoflurane and oxygen are delivered via a non-rebreathing circuit. Maintain the anesthesia and keep the mouse on a 37 °C heating pad during the entire operational procedure. Apply lubricant to the eyes to prevent desiccation.
9. Locate the acupoints and identify the stimulation area (Figure 4).
10. Shave the hair on the back of the mouse until the flesh-colored skin on the back of the mouse is seen. Ensure the width of hair removal is ~1.5 cm and the length is from the seventh cervical vertebra to the tail vertebra (Figure 5A).
11. Apply the prepared ginger juice with a cotton gauze to the shaved area (Figure 3).
12. Sprinkle the TCM powder evenly with a writing brush on the median line of the back of the mouse and cover it with mulberry paper (Figure 5B-D).
13. Make a trapezoidal ginger column 6 cm long, 1.5 cm wide, and 3 mm thick with ginger mud. Fix the ginger column on the mulberry paper (Figure 5E).
14. Make a groove above the midline of the ginger column (Figure 3), and place the moxa cone on the ginger column.
15. Light the moxa cone, placing a new moxa cone after the previous one burns out. Check the firmness of the ginger column to prevent burning the mouse, burning a total of three cones (Figure 5F).
16. After the burning of the moxa cones, wipe off the ginger and the TCM powder, and clean the back of the mouse. Discontinue the anesthesia at this point.
17. Allow the mouse to regain consciousness in a new cage with unrestricted access to food and water. Place the cage on a heated pad to aid in the recovery process. Monitor the animal until it regains sufficient consciousness.
18. Once the mouse has fully recovered, confirm the presence of the righting reflex before returning it to its regular housing.
    ​NOTE: During the operation, it is necessary to monitor the mental state of the mice to avoid weak breathing. Prevent scalding, but keep the mice warm, and avoid bruising of the claws and tail.

4. Evaluation of arthritis index (AI)

1. Evaluate the AI score three times: before modeling, after modeling, and after treatment. The assessment standards are as follows: 0, no redness or swelling; 1, slight redness or swelling in a few toes; 2, redness and swelling in most toe joints and toes; 3, serious redness and swelling in feet and below ankle joint; and 4, redness and swelling in feet and ankle joint²³.
2. Calculate the AI scores of all four paws, with a maximum value of 16.

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.

1. Place the mice on rotating drums that rotate under continuous acceleration from 4 to 40 rpm over 300 s²⁴.
2. Measure the latency time (the time until the mouse falls off the rod). Provide padding to prevent injury when it falls off.

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.

1. Control the background noise of the laboratory to below 65 dB.
2. Wipe the entire apparatus with 75% ethanol before each trial.
3. Place all mice in the testing room 1 h before the test to allow them to adapt to their environment.
4. Put the mice in the square open field arenas and allow them to explore for 10 min.
5. Record the total distance and movement speed for 10 min.

7. Euthanasia and specimen collection

1. Place the mouse in the anesthesia induction box and anesthetize it with a mixture of 2-3% isoflurane and oxygen at a flow rate of 2 L/min. Once the mouse enters a state of deep anesthesia confirmed by a lack of pedal reflex, transfer it to the operating table for maintenance anesthesia.
2. Grab and fix the mouse with the non-dominant hand.
3. Lightly press on the eye area to make the eyeballs congested and protrude.
4. Use scissors to trim the whiskers of the mouse to prevent hemolysis caused by hairs.
5. Confirm the depth of anesthesia by checking for the absence of a pedal reflex in response to a toe pinch. Use forceps to grasp the eyeball and quickly remove it, allowing the blood to flow into a microcentrifuge tube from the eye socket.
   NOTE: Alternative blood collection methods, such as cardiac puncture, can also be used.
6. When the blood dripping rate slows down, gently press on the mouse's heart area to speed up the blood pumping and obtain more blood. Then, euthanize the mouse using cervical dislocation.
7. Hold the leg just above the ankle joint using forceps in the non-dominant hand. Then, cut the leg just above the ankle joint using scissors.

8. Histological analysis

1. Immerse the ankle specimens in 4% paraformaldehyde for more than 24 h. Decalcify in 10% EDTA (pH = 7.4) for 1 month.
2. Soak the specimens in 4% paraformaldehyde for 2 h.
3. Then, soak the specimens in a gradient of increasing ethanol concentrations (70%, 80%, 90%, 100%, 100%, 100%). Soak each ethanol concentration for 1 h.
4. Soak the specimens in xylene for 3 h.
5. Immerse the ankle specimens in paraffin for 7 h. Set aside paraffin blocks in a 4 °C refrigerator.
6. Slice paraffin blocks to a thickness of 6 µm using a semi-automatic paraffin sectioning machine.
7. Soak the slices for 4 x 5 min in xylene to complete dewaxing.
8. Soak the slices in a gradient of decreasing ethanol concentrations (100%, 100%, 95%, 75%) for 2 min each. Soak in distilled water for 2 min.
9. Stain the slices with hematoxylin for 10 min and rinse with distilled water for 5 s.
10. Soak the slices for 30 s in the differentiation fluid and rinse with distilled water for 5 s.
11. Stain the slices with eosin for 1 min and rinse with distilled water for 3 min.
12. Soak the slices in a gradient of increasing ethanol concentrations (80%, 95%, 100%, 100%) for 1 min each. Soak in xylene for 3 x 3 min and seal the slices²⁵.

9. Enzyme-linked immunosorbent assay (ELISA)

1. Measure the IL-17 and TNF-α concentrations in plasma using the respective kits according to the manufacturers' instructions.

10. Statistical analysis

1. Express the data as mean ± standard error of the mean (SEM).
2. Determine the significance through one-way analysis of variance (ANOVA) or two-way repeated measures ANOVA, followed by Bonferroni or Tamhane T2's post hoc tests.
3. Consider P < 0.05 to be statistically significant.

Results

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 ...

Discussion

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, bas...

Materials

Name	Company	Catalog Number	Comments
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