Effect of Long-Snake Moxibustion on Gut Microbiota of Patients with Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a chronic and progressive arthritis disease, which mainly affects the axial bones and sacroiliac joints. Most patients show severe backache and arthrolysis [1, 2]. The progress of the advanced diseases can lead to joint deformity and limited activities, which will reduce the quality of life of patients [3, 4]. The incidence group of AS is mostly young adults aged 20–30. A recent survey found that 30 out of every 10,000 people suffer from AS, and the incidence rate is on the rise [5], which has caused a heavy burden to patients and society. At present, the clinical treatment of AS mainly depends on drug therapy, including nonsteroidal anti-inflammatory drugs, anti-rheumatic drugs, tumor necrosis factor (TNF-α) inhibitors, and biological agents. Although these drugs can improve the symptoms of patients to a certain extent, however, the course of disease cannot be controlled, and there are problems such as large side effects and high cost [6], while traditional Chinese medicine is economical and effective in treating AS, which has great advantages. Long-snake moxibustion is a traditional external treatment method of traditional Chinese medicine. Because of its definite curative effect, safety, comfort, and no side effects, it has attracted more and more attention in clinic. Our previous research found that long-snake moxibustion can significantly activate peroxisome proliferators-activated receptors, inhibit the expression of inflammatory factors such as TNF-α, IL-1β, IL-6, IL-17, and IL-23, play an anti-inflammatory role, and finally relieve AS [7]. Combined with many current studies, it has been shown that long-snake moxibustion can obviously improve the clinical symptoms of AS patients and regulate the serum inflammatory response and metabolism in vivo [8, 9]. Gut microbiota is considered as the second genome of human beings. It is found that intestinal ecological imbalance in AS patients and the species and abundance of flora have changed [10]. However, there are few studies on the regulation of intestinal microflora of AS patients by long-snake moxibustion. Therefore, this study explored the influence of long-snake moxibustion on gut microbiota of AS patients based on 16s rDNA sequencing technology and provided some experimental basis for the treatment of AS with long-snake moxibustion.

From September 2020 to June 2022, 30 AS patients (15 males and 15 females, aged 18–50 years) who were clinically diagnosed by acupuncture department of Hongdu Chinese Medicine Hospital in Nanchang City were selected. Observation indexes of long-snake moxibustion before treatment are the pretreatment group, and those after treatment are the posttreatment group. At the same time, 30 healthy volunteers (15 males and 15 females, aged 18–50 years) matched with patients’ age were selected as the health group. There was no significant difference in baseline data such as sex and age between the two groups (Table 1) (p > 0.05). This research plan was approved by the Nanchang Hongdu Hospital of Traditional Chinese Medicine Medical Ethics Committee (KY-2020-007), and all subjects volunteered to participate and signed the informed consent form.

The diagnosis of the included patients conforms to the diagnostic criteria in the Guidelines for Diagnosis and Treatment of Ankylosing Spondylitis issued by Rheumatology Branch of Chinese Medical Association in 2010: (1) low back pain lasts for at least 3 months, and the pain improves with activities, but the rest does not relieve it; (2) the lumbar spine is limited in the anteroposterior and lateral flexion directions; (3) the range of thoracic expansion is less than the normal value of the same age and gender; (4) bilateral sacroiliitis is grade II ∼ IV, or unilateral sacroiliitis is grade III ∼ IV. If the patient has (4) with any one of (1–3) attached, it can be diagnosed as AS [11]. Exclusion criteria: (1) pregnant and lactating patients; (2) patients with joint deformity, spinal rigidity, and severe dysfunction in the late stage of the disease; (3) patients with acute complications need to be treated with glucocorticoid; (4) patients with other serious primary diseases or mental diseases; (5) patients with peptic ulcer; (6) patients who have taken nonsteroidal anti-inflammatory drugs, anti-rheumatic drugs, TNF-α inhibitors, or biological agents within 3 months.

• Moxibustion treatment area: from Dazhui point (in the spinal region, in the subspinous process depression of the seventh cervical vertebra, on the posterior median line) of governor vessel to Yaoshu point (in the sacral region, just opposite the sacral canal hiatus, on the posterior median line).

• Operation method: the patient lies prone on the treatment bed, with a comfortable posture and bare back. The performer routinely disinfects the spinal region of the patient for three times, smears ginger juice along the spinal column and spreads 2–3 layers of gauze, spreads ginger paste with a width of 4–5 cm and a height of 1–2 cm on the gauze, heap moxa (about 200 g) on ginger paste to form a long serpentine moxa cone with a bottom width of 2–3 cm, a height of 2 cm, a lower width and a top tip. Ignite the head, body, and tail of the moxa cone to make it burn naturally, which is one moxa cone. After burning completely, replace moxa and continue moxibustion for three times. After the treatment, remove the moxa ash and ginger paste, wipe the patient's back with a warm towel, drink a proper amount of warm water after dressing, and rest for 10 min.

• Treatment cycle: once a week, with 4 weeks as a course of treatment, and continuous treatment for 3 courses.

• Matters needing attention: keep the ambient temperature moderate; before moxibustion, patients should avoid overeating, starvation, or strenuous exercise; pay attention to ask the patient’s temperature when applying moxibustion, and adjust the moxibustion in time to avoid burns; if burns and scalds occur, they should be disinfected and smeared with scald ointment, and the severe cases should be treated surgically; after moxibustion, ask the patient to take shelter from the wind and cold, and take a bath after 5–6 h.

Visual analogue scale (VAS) [12], Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), and Bath Ankylosing Spondylitis Function Index (BASFI) were used to score AS patients (0–10 points) [13, 14]. The higher the score, the higher the disease activity.

In the morning, 1 g morning stool samples of healthy volunteers and AS patients were collected before and after treatment and transported to the refrigerator at −80°C on ice within 30 min for storage. 16S rDNA sequencing analysis was carried out on the collected specimens, including fecal DNA extraction and detection, PCR amplification, product purification, library construction, and quality inspection. The library was sequenced by Illumina Miseq/HiSeq2500 platform, and the sequence was clustered into OTUs according to the consistency of ninety-seven percent, and the species annotation was carried out with Silva database. According to the species annotation, the flora structure and diversity were calculated, the differences between groups were compared, and the different characteristics of sample community structure were analyzed.

Statistical analysis was carried out by SPSS 26.0 statistical software, and the measurement data were expressed by mean standard deviation. If the data followed a normal distribution, a t test was used; if it did not follow a normal distribution, a rank-sum test was employed. A difference was considered statistically significant if p< 0.05.

After long-snake moxibustion treatment, the scores of VAS, BASDAI, and BASFI of AS patients were significantly lower than those before treatment (p < 0.05), as shown in Table 2.

As shown in Figures 1 and 21,585 OTUs were obtained from the three groups, including 14,694 OTUs in the health group, 15,415 OTUs in the pretreatment group and 14,293 OTUs in the posttreatment group, 10,657 OTUs in the health group and the pretreatment group, 10,912 OTUs in the pretreatment group and the posttreatment group, and 10,090 OTUs in the health group and the posttreatment group.

The alpha diversity index of the three groups of samples is shown in Table 3. Before treatment, the Shannon and Simpson indexes of the group were not statistically different from those of the health group (p > 0.05), while the Ace and Chao1 indexes were higher than those of the health group (p < 0.05). There was no significant difference in Shannon, Simpson, Ace, and Chao1 between the pretreatment group and the posttreatment group (p > 0.05). It is suggested that the richness of intestinal flora in AS patients is higher than that in healthy people, but the treatment of long-snake moxibustion has no obvious effect on it.

Based on weighted_unifrac algorithm, the results of PCoA analysis are plotted (Fig. 2a and b). The closer the samples are on the graph, the greater the similarity. As shown in the figure, there is a slight clustering phenomenon between the health group and the pretreatment group, but it has not reached a significant level, and there is no significant difference between the pretreatment group and the posttreatment group.

According to the histogram of relative abundance of intestinal flora (Fig. 3a, b), at the phylum level, the top five relative abundance distributions of the three groups of intestinal flora are Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Tenericutes; compared with the health group, before treatment, the relative abundance of Firmicutes and Proteobacteria decreased, while the relative abundance of Bacteroides and Actinomycetes increased. Compared with the pretreatment group, the relative abundance of Firmicutes increased and the relative abundance of Actinomycetes decreased in the posttreatment group, but there was no significant statistical difference between the above changes combined with the data in Table 4 (p > 0.05). At the genus level, the top five relative abundance distributions of the three groups of intestinal flora are Subdoligranulum, Bacteroides, Faecalibacterium, Romboutsia, and Blaut. According to the data in Table 5, compared with the health group, the relative abundance of Micrococcus rariorum increased (p < 0.05), and the relative abundance of Bifidobacterium and Streptococcus decreased (p < 0.05) in the pretreatment group. Compared to the pretreatment group, the relative abundance of Roma Boots increased significantly after treatment (p < 0.05), while the relative abundance of Eubacterium huaxiensis decreased.

The AS belongs to the categories of “arthralgia syndrome” and “rheumatism” in Chinese medicine, and its basic pathogenesis is deficiency of kidney essence and cold-dampness stagnation, which leads to obstruction of meridians, obstruction of qi and blood, malnutrition of bones and muscles, and arthralgia for a long time. This disease is closely related to governor vessel, for example, “Plain question bone theoretical” records that “The pathological changes of governor vessel will cause the symptoms of stiff spine, and low back pain can’t be shaken,” and another example is “The Union of Heaven and Human Beings,” which says that “Yang qi can’t be opened or closed, and cold comes from it, which leads to great rolls,” and the governor vessel is just the sea of Yang meridian, so the treatment often takes both the symptoms and root causes into consideration, mainly focusing on tonifying kidney and strengthening bones, regulating the governor, and regulating qi, warming yang, and dispelling cold. Long-snake moxibustion is a traditional Chinese medicine external treatment method. Because it is moxibustion with ginger as a spacer on governor vessel, it is also called “Du moxibustion” or “Spreading Moxibustion.” This therapy combines the warming effect of ginger with the warming effect of moxibustion and penetrates into the governor vessel, which can play a therapeutic role in strengthening the true elements, warming the governor vessel, and dredging qi and blood for AS patients.

Gut microbiota is the most diverse and dense microflora in human body [15, 16]. Its main function is to protect the host from pathogens and exert benefits through various mechanisms, which is closely related to the occurrence of various host diseases. Studies have shown that intestinal microflora plays a key role in the development and function of intestinal innate and adaptive immune systems, and dysbacteriosis may lead to the occurrence and progress of autoimmune diseases [17]. The pathogenesis of AS is closely related to inflammatory bowel disease. More than 70% of AS patients have subclinical intestinal inflammation. The inefficient separation of the intestinal cavity from subepithelial space is related to the abnormal activation of immune cells residing in the mucosa. These immune cells are recycled, which may transfer inflammation to joints and establish pathogenic intestinal-articular axis [18]. AS early as 2002, there was the first assessment of the intestinal microflora in AS patients [19], and since then, many studies have been carried out, indicating that the intestinal microflora in AS patients is obviously different from that in healthy people. For example, Costello and others found that compared with healthy control groups, the species abundance of intestinal microflora in AS patients increased, including Lachnospiraceae, Ruminococcaceae, Rikenellaceae, Porphyromonadaceae, and Bacteroidaceae. Species richness of Veillonellaceae and Prevotellaceae decreased [20]. However, Lin et al. [21] observed that the abundance of Prevotellaceae increased and that of Rikenellaceae decreased in HLA-B27 transgenic rats. Although the flora structure is influenced by many factors, there is no unified conclusion on the species of different bacteria at present, and it is certain that intestinal microflora plays a key role in the pathogenesis of AS. At present, there are many targeted intestinal flora therapies to prevent or treat AS, including using antibiotics to eliminate harmful bacteria in the intestine, taking probiotics and prebiotics or increasing beneficial bacteria through fecal microflora transplantation, and using biological agents or Chinese herbal medicines to improve intestinal microecology [22].

The results of this study show that after long-snake moxibustion treatment, the symptom score of AS patients is significantly lower than before, suggesting that long-snake moxibustion can improve the clinical symptoms of AS. Romboutsia has the potential to produce short-chain fatty acids (SCFAs) [23, 24], and SCFA is a beneficial metabolite in the intestine which is very important to maintain the stable balance of mammalian immune system. SCFA, including acetic acid, propionic acid, and butyric acid, is formed in the microbial fermentation process of carbohydrate from diet or host, which provides energy for the host, but also mediates the interaction with the immune system [25]. The latter can not only improve the intestinal mucosal barrier function but also regulate the intestinal energy homeostasis, enhance the intestinal immune cell function, inhibit the production of pro-inflammatory cytokines in the intestine, and promote the expression of anti-inflammatory factors, and has anti-inflammatory activity. Studies have shown that the species producing butyrate in fecal samples of inflammatory bowel disease patients decreased and the level of SCFA decreased. In addition, SCFA can also induce the proliferation and differentiation of Treg cells, and the accumulation of Treg cells in inflammatory joints contributes to the spontaneous regression and relief of peripheral spinal arthritis, and its functional defect can promote the occurrence and development of AS [18, 22]. The above evidence shows that when the abundance and level of SCFA-producing flora in the intestine increase, it is beneficial to the prognosis of AS diseases. Our experiment found that the Romboutsia of intestinal flora in patients with AS showed a downward trend compared with the healthy group. After long-snake moxibustion treatment, the relative abundance of the genus was significantly higher than that before treatment, suggesting that long-snake moxibustion may promote the formation of SCFA by improving the relative abundance level of species in the intestine, thus playing a role in treating AS.

The research design is highly pragmatic, considering various real-life factors, and features rigorous data analysis and presentation of results. Therefore, in terms of generalizability to the “real-world” population, this study demonstrates significant external validity.

One of the limitations of our study is the small sample size. Although the study included 30 AS patients and 30 healthy volunteers, the sample size remains limited for the complex analysis of the microbiome. Additionally, the male-to-female ratio in Chinese AS patients is typically 2–4:1, whereas in our study, it was 1:1. This disparity may affect the reliability and generalizability of certain statistical analyses. Another shortcoming is the lack of future research directions. While the article mentions that moxibustion may improve AS symptoms by modulating the gut microbiome, it does not delve into future research directions, such as gaining a deeper understanding of treatment mechanisms or conducting larger scale clinical trials.

Our research shows that long-snake moxibustion improves the clinical symptoms of patients with ankylosing spondylitis by regulating the abundance of intestinal microorganisms, increasing beneficial bacteria, and maintaining the steady state of intestinal microorganisms, which provides a new theoretical basis for clinical use of long-snake moxibustion to treat ankylosing spondylitis.

This research plan was approved by the Nanchang Hongdu Hospital of Traditional Chinese Medicine Medical Ethics Committee (KY-2022–007). Written informed consent to participate in the study has been obtained from the participants.

The authors have no conflicts of interest to declare.

This study was supported by the General Project of Key R&D Plan of Jiangxi Science and Technology Department (No. 20202BBGL73002); Medical and Health Science and Technology Support Project of Nanchang Science and Technology Bureau (No. Hongkezi [2021] 129-12); Nanchang High-level Technological Innovation Talent “Double Hundred Plan” Project (No. Hongkezi [2022] 312-37).

Y.X. and H.X. conceived the study. Y.X. and J.M. performed the study and drafted the manuscript. H.H., Q.F., G.L., G.R., and Z.X. designed the study and participated in data acquisition. All authors read and approved the final version of the manuscript accepted for publication.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.