Effect of Moxibustion on Inflammatory Cytokines in Animals with Rheumatoid Arthritis: A Systematic Review and Meta-Analysis

Background This study aims to systematically evaluate the effect of moxibustion on the level of inflammatory cytokines in animal models with rheumatoid arthritis (RA) and to provide evidence for the clinical application of moxibustion to the treatment of RA and related basic researches. Methods The databases employed in this study include PubMed, EMBASE, Web of Science, China National Knowledge Infrastructure (CNKI), Chinese Science and Technology Periodical Database (VIP), SinoMed, and Wanfang Data Information Site. The retrieval time was from the establishment of these databases to March 2020. The reviewers made use of the CAMARADES 10-item checklist to evaluate the quality of each included study. The inflammatory cytokines were considered as the outcome measure. The Revman 5.3 software was used to conduct meta-analysis on the outcome indicators of the studies included. Results A total of 648 articles were retrieved and 18 animal experiments were included in this study. The quality scores of the studies ranged from two to eight with a mean of 5.8. Compared with the effect of the control group, moxibustion reduced the expression of TNF-α (SMD 2.95, 95% CI: 1.99–3.92, P < 0.00001), IL-1β (SMD 4.10, 95% CI: 2.37–5.84, P < 0.00001), IFN-γ (MD 25, 95% CI: 16.17–33.82, P < 0.00001), IL-6 (MD 11.83, 95% CI: 6.22–17.44, P < 0.0001), and IL-17 (MD 99.3, 95% CI: 86.83–111.76, P < 0.00001). At the same time, the level of IL-2 (SMD 8.89, 95% CI: 0.93–16.86, P=0.03), IL-4 (MD 1.79, 95% CI: 0.26–3.32, P=0.02), and IL-10 (MD 5.93, 95% CI: 1.37–10.49, P=0.01) increased after moxibustion treatment. Asymmetric funnel plots indicated that there was publication bias. Conclusion The findings of the present review indicate that moxibustion can protect the synovium of joint in animal models with RA by upregulation of the level of anti-inflammatory cytokines and downregulation of the level of proinflammatory cytokines. Moxibustion has the potential to relieve inflammation of RA.


Introduction
Rheumatoid arthritis (RA) is an autoimmune disease that starts with the inflammation of synovium. e main symptoms of RA include pain, swelling, and stiffness of the joint. With the progression of the disease, cartilage destruction and bone erosion may occur, eventually leading to severe disability. e occurrence of RA is closely related to autoimmune disorders. Previous studies have revealed that many factors including gene, environment, hormone, and infection are involved in the persistent immunological response [1]. In addition, people with RA have an increased risk of cardiovascular disease, fragility fracture, and venous thromboembolism [2][3][4]. e incidence of RA varies from country to country and region to region, ranging from 0.5% to 1% in adults [5]. It is estimated that there are over 19 million prevalent cases of RA globally, which increased by 7.4% from 1990 to 2017 [6]. Global Burden of Diseases 2017 found that the crude disability adjusted life years rate increased by 25% during 1990-2017 [7]. e pain and joint dysfunction caused by RA urgently need an effective treatment to alleviate the inflammatory reaction and delay the progression of the disease. Among the RA therapeutic strategies, drug treatment is the most prominent one. However, both anti-inflammatory and biological agents have side effects (such as cardiovascular and gastrointestinal lineages complications) and patients treated with them may develop drug resistance [8,9]. erefore, it is of great value to seek a treatment that has significant clinical effect and low recurrence rate without side effects.
Moxibustion is an external therapy based on the theory of traditional Chinese medicine (TCM). As a complementary therapy, its effective mechanism may be associated with the thermal effects, radiation effects, and pharmacological actions produced by the burning of moxa and the influence of meridians and acupoints. e theory of TCM holds that moxibustion can invigorate Qi and stimulate blood circulation, warm meridians, and regulate Yin and Yang [10]. Accumulate studies demonstrate that moxibustion plays an important role in anti-inflammatory and pain relief, especially for the treatment of musculoskeletal diseases, such as primary osteoporosis [11], cervical spondylosis [12], and knee osteoarthritis [13]. Studies found that the therapeutic effects of moxibustion seem to be related to anti-inflammatory and immune regulation. In recent years, a large number of studies, especially animal experiment studies, have illustrated the anti-inflammatory effects of moxibustion for RA.
Reviews based on animal research will be beneficial to the design of animal experiment in the future and provide a basis for clinical studies. Furthermore, animal experiment provides important data and theoretical support for the potential mechanism of moxibustion therapy for RA. erefore, the current study conducted a systematic review and meta-analysis to analyze the effect of moxibustion on inflammatory cytokines in animal models of RA to provide guidance for future animal experiments and clinical trials.

Registration.
Following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines, the manuscript has been registered in PROSPERO (No. CRD 42020171320).

Search Strategy.
Four Chinese databases (CNKI, SinoMed, VIP, and Wanfang) and three English databases (PubMed, EMBASE, and Web of Science) were retrieved. e retrieval time was from the establishment of these databases to March 2020. e languages used in the studies were limited to Chinese and English. Search terms consisted of two aspects: subject (rheumatoid arthritis) and intervention (moxibustion and other related terms).

Exclusion Criteria
(1) Studies conducted in vitro or in humans (2) Moxibustion therapies combined with other therapies such as Chinese herbal medicine or Western medicine (3) e control group not treated with purified water injection, saline injection, or with no treatment (4) Studies without a separate control group (5) Duplicated publications, reviews, meta-analysis, or case reports; studies with abstract only; studies without extractable data 2.5. Study Selection and Data Extraction. One researcher (YMZ) retrieved the relevant databases according to the search strategy and made a list of all the records. Two evaluators (YNS and WTL) independently assessed the articles based on the inclusion and exclusion criteria. Firstly, the evaluators screened the title and abstract of the articles to exclude the obviously irrelevant literature. Secondly, the reviewers scanned the full text of these selected articles that met the previously mentioned criteria. If there was a disagreement, it would be resolved with negotiation between two researchers (YNS and WTL) or confirmed by the third researcher (LLZ). Two researchers (YNS and WTL) extracted data from the selected studies. e contents of the data extraction mainly included the following: (1) general information: the first author, the year of publication, and nationality; (2) rat information: the number of rats in the experimental group and control group, gender, species, weight of rats, and modeling method; (3) treatment information: types of moxibustion, acupoint selection, duration, and period of treatment; (4) results: TNF-α, IFN-c, IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-17. When the data in the selected studies was presented as graph, the researchers would try to contact the author to obtain detailed data. If the author had no response after three times of e-mail contact, the study would be excluded since it lacked key information. e third reviewer would make a final decision on any divergence regarding the outcome data.
2.6. Quality Assessment. CAMARADES 10-item checklist was used to assess the methodological quality of the studies included [14]. e checklist included (1) peer-reviewed publication; (2) control of temperature; (3) random allocation to treatment or control; (4) blinded induction of model; (5) blinded assessment of outcome; (6) use of anesthetic without significant intrinsic neuroprotective activity; (7) appropriate animal model; (8) sample size   2 Evidence-Based Complementary and Alternative Medicine calculation; (9) compliance with animal welfare regulations; (10) statement of potential conflict of interests.
Each study was given a comprehensive quality score out of a possible total of 10 points and the group median was calculated. Two reviewers (YNS and WTL) independently extracted data and assessed the quality of each study. Discrepancies were resolved by the third reviewer.

Statistical Analysis.
Meta-analysis and statistical calculations were performed with the assistance of RevMan 5.3 software.
e data of inflammatory cytokines were considered as continuous data. Mean difference (MD) would be employed to estimate the combined effect sizes of the indicators when the outcomes among different studies were tested with the same way. If the measurement unit of the outcome indicator was different, the standardized mean difference (SMD) would be given to estimate the combined effect sizes. e confidence interval (CI) was established at 95%. e I 2 statistic was given to assess the heterogeneity among these studies. If I 2 < 50%, it indicated that the heterogeneity among the studies included was low and a fixed effect model would be used. Otherwise, a random effect model would be performed. When significant heterogeneity existed, the sensitivity analyses and subgroup analysis would be conducted according to the moxibustion methods, animal species, animal weight, animal gender, and modeling methods. Funnel plot was used to evaluate the publication bias. When P value was less than 0.05, the outcome would be statistically significant.

Control Interventions.
To better evaluate the anti-inflammatory effects of moxibustion, the current study excluded studies using therapies including western medicine, traditional Chinese medicine, or acupuncture for the control group and chose studies employing therapies like purified water injection, saline injection, or no treatment for the control group. All literature included set no treatment as control interventions.

Study Quality Assessment.
e quality score of the included studies ranged from two to eight. More specifically, one study scored two, two studies scored three, five studies scored four, four studies scored seven, and six studies scored eight. Fourteen studies were published in peer-reviewed journal [16-21, 23-29, 32], but four studies were not because they were master's or doctoral thesis [15,22,30,31]. Eleven studies elaborated the control of temperature [17, 18, 20, 23-26, 28-30, 32]. All studies randomly allocated rats to the control group and the treatment group and adopted appropriate animal models. Blinded induction of model and blinded assessment of outcome were stated, respectively, in four [17,18,23,24] and five studies [20,26,28,31,32].
3.9. Assessment of Bias. Funnel plot was applied to assess the publication bias. e asymmetry funnel plot indicated that there was publication bias (Figure 6).

Principle Findings.
e current study represents the first meta-analysis to evaluate the effect of moxibustion for the treatment of RA in animal experiments with the results of inflammatory cytokines as outcome indicators. is meta-    inflammatory and analgesic effects of conventional medicine and downregulate HIF-1α/VEGF contents to inhibit angiogenesis [33]. e conclusion is similar to the previous clinical meta-analysis [34].

4.2.
Possible erapeutic Mechanism of the TCM. " e Yellow Emperor's Internal Classic," an ancient medical canon of TCM in China, holds that the occurrence of RA is closely related to the invasion of three perverse trends of wind, cold, and dampness. e primary pathogenesis is the deficiency of healthy Qi in viscera and the meridian is blocked by accumulated dampness, which has close relationship with spleen, stomach, and kidney. erefore, warming the meridian and removing dampness through invigorating spleen and kidney are the basic principles of treatment [35]. Almost all the selected acupoints belong to the Stomach Meridian and Kidney Meridian. It is consistent with the basic pathogenesis of the Qi deficiency and pathogenic stagnation, which plays a key role in ensuring effectiveness by warming the meridian and eliminating dampness through invigorating spleen and kidney, dredging channel of Qi and blood, and harmonizing Yin and Yang. e theory of TCM holds that spleen and stomach provide the material basis of the acquired constitution. ST 36, the he-sea point of Stomach Meridian of Foot-Yangming, can strengthen the function of spleen and stomach and eliminate dampness. e results of previous studies indicated that acupuncture at ST 36 can promote gastrointestinal motility and improve gastrointestinal function [36,37]. e research found that electroacupuncture at ST 36 might activate AMPK, produce stable ULK1/AMPK compound, and increase the mitochondrial autophagy, which could regulate spleen-stomach and treat spleen deficiency [38]. Meanwhile, acupuncture at ST 36 reduced the lipopolysaccharide-(LPS-) induced serum levels of TNF, IL-6, and INF-c [39] and could promote eosinophils apoptosis to inhibit the development of inflammatory reaction [40]. BL23 is the back-shu point of kidney. Moxibustion at BL23 for the treatment of RA can not only nourish kidney and benefit   [16] Reduce TNF-α, IL-1β, IL-17 Wang et al. 2017 [17] Regulated the immune function Wu et al. 2017 [18] Decreased TNF-α, IL-1β Su et al. 2015 [19] Reduced T cell activation Regulated the immune function Wu et al. 2018 [20] Regulated the circadian rhythm of TNF-α Fan et al. 2017 [21] Decreased TNF-α, IL-6, NO, TNF-α mRNA Cui 2019 [22] Reduced TNF-α, BK Huo et al. 2020 [23] Decrease TNF-α, IFN-c, IL-1β, IL-4 Restore the balance between 1/ 2 Yin et al. 2017 [24] Decrease TNF-α, IL-17 Luo et al. 2011 [25] Increased IL-2 Decreased TNF-α, IL-1β Wu et al. 2018 [26] Rhythmic anti-inflammatory Gao et al. 2011 [27] Increased IL-4 Decreased IL-1β, MT Restore the balance between 1/ 2 Zhang et al. 2014 [28] Increased IL-2, Bcl-2 Decreased IL-1, caspase- 3 Bao 2007 [29] Regulated the immune function  Evidence-Based Complementary and Alternative Medicine essence, but also dispel cold and relieve pain, so as to achieve the effect of treating both symptoms and root causes.

Possible Modern Biological Mechanism.
e combination of the previously mentioned analysis and numerous research results indicates that the biological mechanism of moxibustion in the treatment of RA involves several aspects. Researchers have conducted many studies from the perspective of anti-inflammatory, immune regulation, regulation of circadian rhythm, and bone protection [41][42][43][44]. Among them, there are studies concentrating on anti-inflammatory effect as the most important aspect because RA is characterized by synovitis, which may result in cartilage and bone destruction. Previous studies explored the mechanisms of moxibustion in treating RA from several aspects, including restoring the balance between 1 and 2 by regulating the levels of inflammatory cytokines [23,27], modulating the pathological circadian rhythm of TNF-α, IL-1β, and IL-6 [20,25,31], regulating the function of HPAA [31], alleviating cartilage degradation through RANKL/OPG signaling pathway [44], and strengthening the negative regulation of PD-1/PD-L1 signaling pathway [30,32].
is study suggested that moxibustion can relieve synovitis of the joint with RA mainly by inhibiting the secretion of proinflammatory cytokines and enhancing the secretion of anti-inflammatory cytokines to restore the equilibrium between 1 and 2. Moxibustion is effective in treating RA, but its mechanism of action involves many aspects, and its antiinflammatory effect is just one of them. erefore, it needs to be further explored in future studies.

e Value of Animal Studies and Systematic
Review of the Effectiveness of Moxibustion for RA in Animal Model. As a complementary therapy, moxibustion is safe and effective for people suffering from RA to some extent. However, there is also a controversial view that moxibustion has no therapeutic effect but only a warming effect. Revealing the mechanisms of moxibustion for RA and making sure that the effect is not just warming are significant for the development of moxibustion. To dispel these doubts, it is necessary to objectively investigate the effects of moxibustion on synovium and related tissues, cells, and signal pathways. As is known to all, it is unethical and forbidden to obtain tissues or cells from human body. For this reason, animal researches of moxibustion for RA are of great value and cannot be replaced completely by clinical studies. Meta-analysis is a kind of statistical method that can comprehensively analyze the results of many studies and help researchers promote the methodological quality of preclinical animal researches. erefore, we conducted this systematic review and metaanalysis to assess the effectiveness of moxibustion in the treatment of RA in animal models and found that moxibustion has a definite effect to relieve inflammation in animal models with RA.

Subgroup Analysis.
e high heterogeneity among the studies based on inflammation cytokines cannot be completely explained. e reviewers carried out subgroup analysis on the included studies based on moxibustion methods, modeling methods, animal species, animal gender, and animal weight, and the researchers also conducted sensitivity analysis, but no evident cause was found. However, in subgroup analysis, it was found that suspended moxibustion was significantly better than seed-sized moxa cone in improving the level of inflammatory cytokines. e reviewers also found that 13 of the 18 studies used ST36 and BL23 points, and the remaining five studies involved acupoints including CV4, GV4, GB34, SP9, and behind ST36 or a combination of the previously mentioned points. e acupoints used were inconsistent. Furthermore, the sample size also covered a wide scope ranging from 16 to 48 and no study mentioned sample size calculation. ese findings suggested that, in addition to the differences in moxibustion method, modeling methods and animal species, acupoint combinations, and sample sizes may be another source of heterogeneity among the studies.

Limitations and Strengths.
e present review has several limitations. First, it is the language. e researchers limited the languages of all studies to English and Chinese. Of all the included studies, only one was written in English, which may cause potential publication bias. Second, the scope of sample size still needs to be expanded. Although the researchers conducted a comprehensive literature search and traced the references of the included literature to supplement the relevant literature, no literatures met the inclusion criteria. ird, the quality of the included studies is unsatisfactory and the differences in the selection of acupoints and operation methods may greatly affect the outcome of the meta-analysis. Finally, the blind method was not performed well. Although it is difficult to blind moxibustion for operators, it should be strictly carried out in the process of molding and the assessment of outcome.
According to the above limitations, more databases with different languages should be searched to increase the  diversity of the included literature. Meanwhile, in terms of experimental design, especially the random allocation of treatment and control, blind building of model and blind assessment of outcome should be strictly carried out in future animal researches. In addition, a unified standard of moxibustion therapeutic schedule should be seriously performed because the details of moxibustion manipulation are closely related to the efficacy. e choice of moxibustion methods and moxibustion acupoints, duration of each moxibustion, and course of treatment are necessarily to be standardized.

Conclusions
e findings of the present review indicate that moxibustion can protect the synovium of joint in animal models with RA by enhancing the level of anti-inflammatory cytokines and decreasing the level of proinflammatory cytokines. Moxibustion has the potential to relieve inflammation in animal models with RA. However, the specific mechanism still needs to be further explored and the quality of studies should also be improved in the future. erefore, future researches need to be more standardized in terms of experimental design, especially the design of blind method, selection of inflammatory cytokines, and moxibustion methods so as to provide a reliable theoretical basis for the application of basic research achievements to clinical practice.

Conflicts of Interest
e authors declare that they have no conflicts of interest.