Name: Author Spotlight: Evaluating Traditional Chinese Therapy for Ankylosing Spondylitis in Mice
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Description: Watch this Scientific Journal Video about Evaluating Traditional Chinese Therapy for Ankylosing Spondylitis in Mice at JoVE.com

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.

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
<ol>
	<li>Determine the experimental groups and the number of animals per group in advance.</li>
	<li>Choose male BALb/c mice, house them at a constant temperature of 22 &#177; 1 &#176;C, constant humidity of 60 &#177; 5%, and 12 h diurnal cycle light environment.</li>
</ol>
<p cl...

Exploring the Therapeutic Potential of Du-Moxibustion in Ankylosing Spondylitis

<ol>
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Y., Qu, J., Li, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+thunder-fire+moxibustion+in+treating+ankylosing+spondylitis+of+kidney+deficiency+and+governor+meridian+cold+and+its+influence+on+TNF-&#945;+and+RANKL:+study+protocol+for+a+prospective,+nonblinded,+single-center,+randomized+controlled+trial.">Efficacy of thunder-fire moxibustion in treating ankylosing spondylitis of kidney deficiency and governor meridian cold and its influence on TNF-&#945; and RANKL: study protocol for a prospective, nonblinded, single-center, randomized controlled trial.</a> Trials. 23 (1), 344 (2022).</li><li>Chong, G. Q., Ma, Y., Sun, H. S., Zhang, T., Peng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+clinical+and+analgesic+mechanism+of+moxibustion+in+the+treatment+of+ankylosing+spondylitis.">Study on clinical and analgesic mechanism of moxibustion in the treatment of ankylosing spondylitis.</a> J Clin Acupunct Moxib. (06), 48-49 (1999).</li><li>Liu, W. Q., Lu, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+observation+of+Melilotus+extract+tablets+combined+with+Dujiu+for+treating+ankylosing+spondylitis.">Clinical observation of Melilotus extract tablets combined with Dujiu for treating ankylosing spondylitis.</a> Pharm Res. 35 (03), 182-184 (2016).</li><li>Zhang, Y. L., Lin, J. H., Zhou, Y. Y., Zhao, G. Q., He, Y. 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Y., Li, F., Yan, J., Wan, C., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+study+of+Du-moxibustion+combined+with+needling+Huatuo+Jiaji+acupoint+in+the+treatment+of+AS+of+kidney-Yang+deficiency.">Clinical study of Du-moxibustion combined with needling Huatuo Jiaji acupoint in the treatment of AS of kidney-Yang deficiency.</a> J Clin Acupunct Moxib. 35 (06), 41-44 (2019).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+analysis+of+biochemical+changes+in+the+tissue+and+urine+of+proteoglycan-induced+spondylitis+in+mice+after+treatment+with+moxibustion.">Metabolomic analysis of biochemical changes in the tissue and urine of proteoglycan-induced spondylitis in mice after treatment with moxibustion.</a> Integr Med Res. 10 (1), 100428 (2021).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moxibustion+attenuates+inflammation+and+alleviates+axial+spondyloarthritis+in+mice:+Possible+role+of+APOE+in+the+inhibition+of+the+Wnt+pathway.">Moxibustion attenuates inflammation and alleviates axial spondyloarthritis in mice: Possible role of APOE in the inhibition of the Wnt pathway.</a> J Tradit Complement Med. 12 (5), 518-528 (2022).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+intensity-dependent+expression+of+osteoinductive+wnt+proteins+is+critical+for+ectopic+new+bone+formation+in+ankylosing+spondylitis.">Inflammation intensity-dependent expression of osteoinductive wnt proteins is critical for ectopic new bone formation in ankylosing spondylitis.</a> Arthritis Rheum. 70, 1056-1070 (2018).</li><li>Cui, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCL12/CXCR4-Rac1-mediated+migration+of+osteogenic+precursor+cells+contributes+to+pathological+new+bone+formation+in+ankylosing+spondylitis.">CXCL12/CXCR4-Rac1-mediated migration of osteogenic precursor cells contributes to pathological new bone formation in ankylosing spondylitis.</a> Sci Adv. 8 (14), eabl8054 (2022).</li><li>Braem, K., Lories, R. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+to+cartilage+proteoglycans+in+BALB/c+mice+with+progressive+polyarthritis+and+ankylosing+spondylitis+induced+by+injection+of+human+cartilage+proteoglycan.">Immunity to cartilage proteoglycans in BALB/c mice with progressive polyarthritis and ankylosing spondylitis induced by injection of human cartilage proteoglycan.</a> Arthritis Rheum. 30, 306-318 (1987).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenascin-C-mediated+suppression+of+extracellular+matrix+adhesion+force+promotes+entheseal+new+bone+formation+through+activation+of+Hippo+signalling+in+ankylosing+spondylitis.">Tenascin-C-mediated suppression of extracellular matrix adhesion force promotes entheseal new bone formation through activation of Hippo signalling in ankylosing spondylitis.</a> Ann Rheum Dis. 80 (7), 891-902 (2021).</li><li>Glant, T. 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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...

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 &#34;medicated moxibustion with foam&#34; 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 &#946;-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-1&#946;, PGE2, IL-6, and TNF-&#945; 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-1&#946;, TNF-&#945;, 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% EDTA</td>
			<td>Shanghai Macklin Biochemical Co., Ltd.,Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alcohol pads</td>
			<td>HYNAUT, Qingdao Hainuo Biological Engineering Co., LTD, Qingdao, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anesthesia machine</td>
			<td>Medical Supplies &#38; Services INT. LTD, Keighley, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge tubes</td>
			<td>Axygen, Corning, NewYork, UAS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>complete Frech&#39;s adjuvant (CFA)</td>
			<td>aladdin,Shanghai, China</td>
			<td>F393378</td>
			<td></td>
		</tr>
		<tr>
			<td>cotton ball</td>
			<td>Henan RUIKE MEDICAL Equipment Co., Ltd.,Xinxiang, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cotton gauze</td>
			<td>Henan RUIKE MEDICAL Equipment Co., Ltd.,Xinxiang, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cutting board</td>
			<td>self-preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>decorin from bovine articular cartilage</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>D8428</td>
			<td></td>
		</tr>
		<tr>
			<td>depilatory cream</td>
			<td>Veet, Reckitt Benckiser (China) Investment Co. LTD, Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>electronic scale</td>
			<td>Shanghai Yajin Electronic Technology Co., Ltd.,Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tube</td>
			<td>Axygen, Corning, NewYork, UAS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>eye lubricant</td>
			<td>Beijing Shuangji Pharmaceutical Co., LTD., Beijing, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ginger</td>
			<td>self-preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 7 software</td>
			<td>GraphPad Software,Boston, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>hair clipper</td>
			<td>Super human Group CO LTD, Jinhua, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>heating pads</td>
			<td>Shenzhen Leshuo Tech Co., Ltd.,Shenzhen, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>incomplete Freund&#8217;s adjuvant (IFA)</td>
			<td>aladdin,Shanghai, China</td>
			<td>F393371</td>
			<td></td>
		</tr>
		<tr>
			<td>injection&#160;syringe</td>
			<td>Shandong Xinhua Ande Medical Supplies Co., LTD, Zibo, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>Shenzhen Rayward Life Technology Co., LTD, Shenzhen, China</td>
			<td>R510-22-10</td>
			<td></td>
		</tr>
		<tr>
			<td>joss stick</td>
			<td>Shijiazhuang Lidu Fragrant Industry Co., LTD.,Shijiazhuang, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>juicer</td>
			<td>Braun (Shanghai) Co., Ltd.,Shanghai, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>knife</td>
			<td>self-preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>lighter</td>
			<td>Zhejiang tiger-lighter Co. LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mortar</td>
			<td>Luoyang Yinai Ceramic Technology Co., LTD.,Luoyang, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-17 ELISA Kit</td>
			<td>absin, Shanghai, China</td>
			<td>abs520009-96T</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse&#160;TNF-&#945; ELISA Kit</td>
			<td>Wuhan&#160;Sanying, Wuhan, China</td>
			<td>KE10002</td>
			<td></td>
		</tr>
		<tr>
			<td>mulberry paper</td>
			<td>Yishui County Mulinsang paper Co., LTD, Linyi, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OFT</td>
			<td>Xinruan,Shanghai, China</td>
			<td>XR-XZ301</td>
			<td></td>
		</tr>
		<tr>
			<td>paper cup</td>
			<td>self-preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pipettes</td>
			<td>OXFORD BIO INSTRUMENTS INC.,Oxford, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>refined moxa velvet</td>
			<td>self-preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rotarod</td>
			<td>Xinruan,Shanghai, China</td>
			<td>XR-6C</td>
			<td></td>
		</tr>
		<tr>
			<td>scientz-48L cryogenic high throughput tissue grinder</td>
			<td>Ningbo Xinzhi Biotechnology Co., LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>Shandong Jiaren Medical Supplies Co., Ltd., Zibo, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>semi-automatic paraffin sectioning machine</td>
			<td>Leica Camera AG, Watznach, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS 25.0 software</td>
			<td>International Business Machines Corporation, NewYork, UAS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TCM powder</td>
			<td>self-preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tips</td>
			<td>Biosharp, Labgic, Beijing, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>writing brush</td>
			<td>Yishui County Mulinsang paper Co., LTD, Linyi, China</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

du-moxibustion in a mouse model of ankylosing spondylitis

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.

Author Spotlight: Evaluating Traditional Chinese Therapy for Ankylosing Spondylitis in Mice 

Du-Moxibustion in a Mouse Model of Ankylosing Spondylitis

The scope of our research focuses on the treatment of ankylosing spondylitis using du-moxibustion, the traditional Chinese external therapy. This paper describes in detail the operating procedures and precautions to be taken during du-moxibustion in treating ankylosing spondylitis in experimental mice. Our study found that du-moxibustion could improve motor coordination and autonomic movement, reduce ankle swelling, and alleviate inflammation in mice with AS.The traditional Chinese medicine external treatment of du-moxibustion was introduced for the first time in detail and more experiments, laying the foundation for future treatment of AS mechanism research.As a external therapeutic technique used in traditional Chinese medicine, du-moxibustion skillfully combines the compatible technicals of TCM to integrate meridians, acupoints, Chinese herbal medicine, and moxibustion, which directly acts on the spine area and makes overall adjustment to related site. 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.

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 ...

Watch this Scientific Journal Video about Evaluating Traditional Chinese Therapy for Ankylosing Spondylitis in Mice at JoVE.com

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 ...