Moxibustion of Zusanli (ST36) and Shenshu (BL23) alleviates the inflammation of rheumatoid arthritis in rats through regulating macrophage migration inhibitory factor/glucocorticoids signaling

Abstract

OBJECTIVE:

To test the hypothesis that moxibustion may inhibit rheumatoid arthritis (RA) synovial inflammation by regulating the expression of macrophage migration inhibitory factor (MIF)/glucocorticoids (GCs).

METHODS:

Fifty male Sprague-Dawley rats were randomly divided into five groups (n = 10 each): blank Control (CON) group, RA Model (RA) group, Moxibustion (MOX) group, MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) group, and Moxibustion + MIF inhibitor ISO-1 (MOX + ISO-1) group. Rats in the ISO-1 group and ISO-1 + MOX group were intraperitoneally injected with the inhibitor ISO-1. The rats in the RA group, ISO-1 group, MOX group, and ISO-1 + MOX group were injected with Freund's complete adjuvant (FCA) in the right hind footpad to establish an experimental RA rat model. In the MOX group and MOX + ISO-1 group, rats were treated with Moxa. The thickness of the footpads of the rats in each group was measured at three-time points before, after modeling and after moxibustion treatment. The contents of serum MIF, corticosterone (CORT), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by enzyme-linked immunosorbent assay; and the contents of synovial MIF were detected by Western blot. Hematoxylin-eosin (HE) staining method was used to observe the pathological changes of synovial tissue under a section light microscope, and pathological scoring was performed according to the grading standard of the degree of synovial tissue disease.

RESULTS:

Moxibustion was found to reduce the level of MIF and alleviate inflammation in RA rats in this study. In addition, after inhibiting the expression of MIF, the level of CORT increased, and the level of TNF-α decreased. Treating RA rats with inhibited MIF by moxibustion, the level of CORT was almost unchanged, but the level of TNF-α further decreased. The correlation analysis data suggested that MIF was positively related to the expression of TNF-α and negatively correlated with the expression of CORT.

CONCLUSION:

Reducing MIF to increase CORT and decrease TNF-α by moxibustion treatment in RA. MIF may be a factor for moxibustion to regulate the expression of CORT, but the expression of TNF-α is due to the incomplete regulation of the MIF. This study added to the body of evidence pointing to moxibustion's anti-inflammatory mechanism in the treatment of RA.

1. INTRODUCTION

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovitis and joint damage, which leads to significant morbidity and mortality.^1,2 Macrophage migration inhibitory factor (MIF) has multiple properties as a multifunctional protein molecule, including enzymes, hormones, and cytokines. MIF has several biological effects, including participating in immune regulation, promoting inflammation, inhibiting glucocorticoid anti-inflammatory, and affecting cell apoptosis, all of which are important in the pathogenesis of inflammatory diseases.^3,⇓-5 Synovial tissue invasion of joints, macrophage infiltration, and high expression of inflammatory factors are all important inflammatory pathological features of RA. In RA, MIF is the upstream cytokine of inflammatory factors such as tumor necrosis factor-α (TNF-α), which can activate macrophages, enhance their adhesion and phagocytosis, and induce different cells to express various pro-inflammatory factors.^5,⇓,⇓-8 Neutralizing the immune response to MIF can significantly inhibit the expression of TNF-α and the accumulation of macrophages,⁹ which may improve RA synovial inflammation. Moreover, clinically, the synovial MIF concentration of RA patients is related to disease activity; the increase in MIF plasma concentration is related to more severe joint damage,^10,⇓-12 These results suggest that MIF plays a key role in the inflammatory response in RA.

Glucocorticoids (GCs) are the primary anti-inflammatory substance in the body. In chondrocytes, the endogenous GCs signaling pathway plays a role in anti-inflammatory and inhibiting cartilage destruction in joint inflammation.¹³ The physiological concentration of GCs can promote the synthesis and release of MIF, which can reversely regulate the inhibitory effect of GCs on immune cell activation and cytokine secretion.^14,15 Therefore, exogenous GCs are frequently used in clinical practice to achieve anti-inflammatory effects, but long-term use of GCs can have negative consequences. Thus, there is an urgent need to find a new treatment that is effective and safe.

Moxibustion, as a non-specific stimulus, has been recognized as a commonly used treatment method. Studies have confirmed that it has a good effect on the treatment of RA.^16,⇓-18 Moxibustion regulate the hypothalamic-pituitary-adrenal axis and regulate mitogen-activated protein kinase and Janus kinase-signal transducer and activator of transcription intracellular signaling pathways; it inhibits the synovial inflammation of RA, and improves the abnormal secretion of synovial cells, induces synovial cell apoptosis, and reduces joint swelling. It also adjusts the body's immune function and has good anti-inflammatory, analgesic and immune regulation effects.^{19,⇓,⇓-22}

Although these studies have revealed the principles of acupuncture treatment of RA from a certain level, they have not paid enough attention to MIF, the core element of the pathogenesis of RA, and the relationship between MIF and GCs and inflammation-related factors remains unclear. Thus, in the current study, we focused on the relationship between the anti-inflammatory effect of moxibustion on MIF-GCs-inflammatory factors.

2. MATERIALS AND METHODS

2.1. Ethics statement

This study was approved by Institutional Animal Care and Use Committee (IACUC) of Chengdu University of Traditional Chinese Medicine, Chengdu, China (approval number: CUTCM-2018-11). All animal experiments were designed by the principles of the 3Rs (Replacement, Reduction and Refinement) and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.²³

2.2. Animals

Fifty male Sprague-Dawley (SD) rats [(160 ± 10) g] were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China) and dwelled in a pathogen-free environment with two animals per cage. The rats are fed in a 12-h light-dark cycle environment [temperature (20 ± 2) ℃, humidity 50%-65%], and have free access to diet and drinking water.

2.3. Experimental Design and Induction of RA

Fifty SD rats were randomly divided into five groups (n = 10) after a week of adaptive domestication: Blank Control (CON) group, RA Model (RA) group, Moxibustion (MOX) group, MIF inhibitor (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) group, and Moxibustion + MIF inhibitor ISO-1 (MOX + ISO-1) group. The rat model of RA was established as previously described.^24,25 On the third day of the experiment, the RA group, the ISO-1 group, the MOX group, and the MOX + ISO-1 group were injected with 0.1 mL Freund's complete adjuvant (FCA, Product Number: F5881, Sigma, St. Louis, MO, USA) in rats' bilateral hind foot pads. The CON group used the same method to inject sterile saline. On the 10th day of the experiment, the RA model was successful (supplementary Figure 1A).

2.4. Intraperitoneal injection with MIF inhibitor ISO-1

After reviewing the literature, in previous studies, the researchers used the inhibition of MIF before inflammation occurred.^26,⇓-28 The optimal dosing regimen and dose were determined based on the preliminary experiment's findings. ISO-1 was purchased from Selleck (Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) was purchased from Sigma (St. Louis, MO, USA) and diluted with sterile saline. On the first day of the experiment, the inhibitor ISO-1 was intraperitoneally injected at a dose of 6 mg/kg every other day in the ISO-1 group and MOX + ISO-1 group, and continued until the 30th day of the experiment with 15 injections in total.^26,⇓-28 CON, RA, and MOX groups received the same dose of sterile saline as the method group (supplementary Figure 1A).

2.5. Moxibustion treatment

On the 10th day of the experiment, moxibustion was performed as described previously.²⁵ The rats were placed on a rat table with their heads covered in breathable gloves and their back and hind limbs exposed (shaved at the treatment point). Acupoints of “Shenshu” (BL23, the second lumbar spinous process adjacent to open 1.5 inch) and “Zusanli” (ST36, distal to the head of the tibia in a depression between the muscles of the cranial tibia and the long digital extensor) were selected for intervention (supplementary Figure 1B). The two acupoints' locations were determined using the Chinese government's Channel and Points Standard GB12346-90, “Experimental Acupuncture” and “The Veterinary Acupuncture of China.” In the MOX group and MOX + ISO-1 group, rats were treated with Moxa (5 mg/column), purchased from Nanyang Yilejia (Nanyang, China), which was ignited and placed on the acupuncture point (Supplementary Figure 1C). To avoid scalding the rat, experimenter would remove one moxa pillar after it was burned to about two-thirds and replace it with a new one. Moxibustion was performed in each acupoint of the rats with five columns at a time, once a day, alternately on both sides. A six-day course of treatment consists of three courses of treatment, with one day off between courses. The rats in the CON, RA, and ISO-1 groups were all fixed using the same method without moxibustion.^25,29

Figure 1. Measurement of MIF expression in synovium by Weston blot .

A: Western blot results showing expression level of MIF protein; B: Western blot analysis. CON: blank control group, rats did not undergo any treatment; RA: RA Model group, rats were injected with 0.1 mL FCA in rats' bilateral hind foot pads on the third day of the experiment, MOX: moxibustion group, Moxibustion was performed to rats on the 10th day of the experiment, a six-day course of treatment consists of three courses of treatment, with one day off between courses. MIF: macrophage migration inhibitory factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; FCA: Freund's complete adjuvant. Data are presented as mean ± standard deviation (n = 10). ^aP < 0.05, compared with the CON group; ^bP < 0.05, compared with the RA group.

2.6. Changes in swelling degree in rats’ hind limb

Observe the rats' overall condition and bipedal lesions daily. Use a waterproof marker to mark the middle of the hind paw of the rat, which is the position of each measurement. Before modeling, after successful modeling, and after treating (corresponding to the 3rd, 10th, and 31st days in the experiment), the thickness of the foot pads were measured with a vernier caliper.

2.7. Hematoxylin and eosin (HE) staining

The ankle joint was fixed in 4% Paraformaldehyde fixative (Servicebio, Wuhan, China). After fixation, each tissue sample was processed routinely and embedded in paraffin. Then, 5 μm sections were taken from the tissue blocks and stained with HE. For histopathological examination, these sections were photographed under a light microscope (Nikon, Tokyo Met, Japan). The slides were graded according to inflammatory changes,²⁰ by a single investigator blinded to the group assignment. Rats synovial pathology score includes synovial tissue hyperplasia, inflammatory cell infiltration and macrophage hyperplasia (Table 1).

Table 1.

Rat synovial pathology scoring standard

Pathological changes Score	Synovial tissue hyperplasia	Inflammatory cell infiltration	Macrophage proliferation
0	None	None	None
1	Mild	Few	Few
2	Moderate	Dense	Dense
3	Massive	Numerous	Numerous

2.8. Western blot

The synovial tissue was washed 2-3 times with cold phosphate buffer saline to remove blood stains, cut into small pieces and placed in a homogenizer. Add protease inhibitor, then add 10 times the volume of this reagent to homogenize thoroughly on ice. Transfer the homogenate to a 1.5 mL centrifuge tube and shake. Pipette repeatedly in the ice bath for 30 min to ensure complete cell lysis. Centrifuge at 12000 ×g for 10 min, and collect the supernatant to obtain the total protein solution. The protein concentration was measured by the BCA method. Proteins were extracted and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gel. A polyethylene difluoride film was used to transfer the band. After blocking with 5% non-fat dry milk in Tris-buffered saline for 1 h at room temperature, membranes were incubated with rabbit anti-MIF (Abcam, Cambridge, UK) overnight at 4 ℃. After extensive washing, the membranes were incubated with a secondary antibody conjugated with HRP at 37 ℃ for 2 h. The images were collected under the chemiluminescence imaging system (Servicebio, Wuhan, China), and the ChemiScope analysis software (Servicebio, Wuhan, China) of the system was used for grayscale analysis.

2.9. Enzyme-linked immunosorbent assay (ELISA)

After the rats were anesthetized, 5 mL of abdominal artery blood was taken, centrifuged at 3000 r/min for 10 min, and the supernatant was stored at －80 ℃ for future use. Before the ELISA test, all serum samples were taken out of the refrigerator 20 min ahead of time and brought to room temperature. Follow the instructions of the ELISA kit (Elabscience, Wuhan, China). The test procedures were as follows: standard dilution, sample addition, washing, color development, reaction termination, then the absorbance of each well was measured, and finally calculated the concentration of MIF, corticosterone (CORT), and TNF-α by drawing a standard curve.

2.10. Statistical analysis

All data were presented as mean ± standard deviation ($\bar{x}±s$). The data were analyzed using SPSS 26.0 (IBM, Armonk, NY, USA). Analysis of variance was used to compare the groups after the data for each group was checked for normality and homogeneity of variance. When the variances are uniform, the least significant difference test was used, and when they are not uniform, the Tamhane's T2 test was used. Spearman bivariate correlation was used to determine the relationship between the MIF expression and the expression of CORT and TNF-α. P < 0.05 was considered significant.

3. RESULTS

3.1. MIF expression in synovium increased in RA rats, but decreased after moxibustion

Compared with the CON group, the expression of MIF in the synovium of RA rats increased (P < 0.05). After moxibustion treatment, the MIF in synovium decreased (P < 0.05, Figure 1A, 1B). The above results showed that the expression of MIF was significantly increased in synovium of RA rats and that moxibustion on Shenshu (BL23) and Zusanli (ST36) can reduce its expression.

3.2. ISO-1 can inhibit the expression of MIF in RA rats

Use MIF inhibitor ISO-1 to down-regulate the expression of MIF in RA rats. Compared with the RA group, the expression of MIF in the synovium (P < 0.05, Figure 2A, 2B) and serum (P < 0.01, Figure 2C) in the ISO-1 group were reduced, indicating that MIF was successfully inhibited. When MIF was inhibited, compared with the RA group, the serum COTR level increased (P < 0.01, Figure 2D) and the TNF-α level decreased in the ISO-1 group (P < 0.05, Figure 2E). MIF inhibits the expression of CORT and promotes the secretion of the inflammatory factor TNF-α.

Figure 2. ISO-1 can inhibit the expression of MIF in RA rats.

A: Weston blot was used to detect the expression of MIF in the synovium of rats ankle joint; B: Western blot analysis; C: ELISA was used to detect the levels of MIF in rats serum; D: ELISA was used to detect the levels of CORT in rats serum; E: ELISA was used to detect the levels of TNF-α in rats serum. RA: RA Model group, rats were injected with 0.1 mL FCA in rats' bilateral hind foot pads on the third day of the experiment, ISO-1: MIF inhibitor ISO-1 group, rats were intraperitoneally injected ISO-1 every other day on the first day of the experiment with 15 injections in total, and be injected with 0.1 mL FCA in rats' bilateral hind foot pads on the third day of the experiment. MIF: Macrophage migration inhibitory factor; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; ISO-1: (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester; CORT: corticosterone; TNF-α: tumor necrosis factor-α; FCA: Freund's complete adjuvant; ELISA: Enzyme-linked immunosorbent assay. Data are mean ± standard deviation (n = 10). ^aP < 0.05, ^bP < 0.01, compared with the RA group.

3.3. Moxibustion alleviated the symptoms of arthritis in RA model rats

There was no difference in the basal footpad thickness of SD rats in each group before injecting FCA (P > 0.05). On the 7th day after the FCA injection, compared with the CON group, the foot pads of the RA group and MOX group were significantly swollen (P < 0. 01) (Table 2). Several inflammatory infiltrate were found in synovial tissue, as well as an increase in macrophage numbers, synovium hyperplasia, and thickening [Figure 3 (A-I)], synovial pathological score increased (Figure 3J), and the level of TNF-α in serum increased in RA group (P < 0.05, Figure 3L); But the CORT had no significant change (Figure 3K). Compared with the RA group, the swelling of the foot pad, inflammatory infiltration and synovial hyperplasia and thickening in synovial tissue were alleviated in the MOX group, the synovial pathological score decreased, the levels of TNF-α and MIF in serum decreased, and the level of CORT was increased (Table 2, Figure 3). Moxibustion can relieve the symptoms of joint inflammation in RA model rats, according to the findings.

Table 2.

Thickness of foot pad in the experiment ($\bar{x}±s$)

Group	n	Foot pad thickness (mm)
		3rd		10th 31st
Control	10	3.85±0.13	3.80±0.13		3.76±0.21
Model	10	3.87±0.34	6.68±0.89a		7.34±0.60b
Moxibustion	10	3.72±0.20	6.64±0.64a		5.72±0.43c

Notes: there was no difference in the basal footpad thickness of rats in each group on the third day of the experiment. CON: Blank Control group, rats did not undergo any treatment; RA: RA Model group, rats were injected with 0.1 mL FCA in rats' bilateral hind foot pads on the third day of the experiment, MOX: Moxibustion group, rats were injected with 0.1 mL FCA in rats' bilateral hind foot pads on the third day of the experiment, and moxibustion was performed to rats on the 10th day of the experiment, a six-day course of treatment consists of three courses of treatment, with one day off between courses. On the 10th day of the experiment, ^aP < 0.01, compared with the CON group; on the 31st day of the experiment; ^bP < 0.01, compared with the CON group, ^cP < 0.01, compared with the RA group.

Figure 3. Moxibustion alleviate inflammation and hyperplasia of ankle synovium in RA rats.

A: photo of footpads of rats in the CON group; B: HE stained image of ankle synovial tissue of CON groups, magnification:× 25 and scale bars represent 200 μm; C: HE staining magnified image of ankle joint synovial tissue in the CON group, magnification: × 100 and scale bars represent 50 μm; D: photo of footpads of rats in the RA group; E: HE stained image of ankle synovial tissue of RA groups, magnification: × 25 and scale bars represent 200 μm; F: HE staining magnified image of ankle joint synovial tissue in the RA group, magnification: × 100 and scale bars represent 50 μm. G: photo of footpads of rats in the MOX group; H: HE stained image of ankle synovial tissue of MOX groups, magnification: × 25 and scale bars represent 200 μm; I: HE staining magnified image of ankle joint synovial tissue in the MOX group, magnification: × 100 and scale bars represent 50 μm. J: rat synovial pathology scores of different groups. K: expression of CORT in serum in in different groups. L: expression of TNF-α in serum in in different groups. CON: Blank Control group, rats did not undergo any treatment; RA: RA Model group, rats were injected with 0.1 mL FCA in rats' bilateral hind foot pads on the third day of the experiment, MOX: Moxibustion group, rats were injected with 0.1 mL FCA in rats' bilateral hind foot pads on the third day of the experiment, and moxibustion was performed to rats on the 10th day of the experiment, a six-day course of treatment consists of three courses of treatment, with one day off between courses. CORT: corticosterone; TNF-α: tumor necrosis factor-α; HE: hematoxylin and eosin; FCA: Freund's complete adjuvant. Data are mean ± standard deviation (n = 10). ^aP < 0.01, compared with the CON group; ^bP < 0.01, compared with the RA group.

3.4. Moxibustion treatment of RA rats may exert anti-inflammatory effects through MIF/CORT

After three weeks of moxibustion treatment, compared with the RA group, serum MIF and TNF-α in the MOX + ISO-1 group decreased (P < 0.01) (Figure 4A, 4C), CORT increased (P < 0.01) (Figure 4B), and the ankle synovial inflammation alleviated (Figure 4D-4I). Compared with the ISO-1 group, the level of serum TNF-α in the MOX + ISO-1 group continued to decrease, but there were no differences in synovial inflammation, serum MIF and serum CORT expression (P > 0.05) (Figure 4).

Figure 4. Moxibustion treatment of RA rats may exert anti-inflammatory effects through MIF/CORT.

A: levels of MIF in serum of different groups; B: levels of CORT in serum of different groups; C: levels of TNF-α in serum of different groups; D: photo of footpads of rats in the RA group; E: HE stained image of ankle synovial tissue of RA groups, magnification: × 25 and scale bars represent 200 μm; F: HE staining magnified image of ankle joint synovial tissue in the RA group, magnification: × 100 and scale bars represent 50 μm; G: photo of footpads of rats in the MOX + ISO-1 group; H: HE stained image of ankle synovial tissue of MOX + ISO-1 groups, magnification: × 25 and scale bars represent 200 μm; I: HE staining magnified image of ankle joint synovial tissue in the MOX + ISO-1 group, magnification: × 100 and scale bars represent 50 μm. CON: Blank Control group, rats did not undergo any treatment; RA: RA Model group,rats were injected with 0.1 mL FCA in rats' bilateral hind foot pads on the third day of the experiment; ISO-1: MIF inhibitor ISO-1 group, rats were intraperitoneally injected ISO-1 every other day on the first day of the experiment with 15 injections in total and be injected with 0.1mL FCA in rats' bilateral hind foot pads on the third day of the experiment; MOX + ISO-1: Moxibustion + MIF inhibitor ISO-1 group, rats were intraperitoneally injected ISO-1 every other day on the first day of the experiment, be injected with 0.1 mL FCA in rats' bilateral hind foot pads on the third day of the experiment, and moxibustion was performed to rats on the 10th day of the experiment. MIF: macrophage migration inhibitory factor; CORT: corticosterone; TNF-α: tumor necrosis factor - α; HE: hematoxylin and eosin; ISO-1: (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester; FCA: Freund's complete adjuvant. Data are mean ± standard deviation (n = 10). ^aP < 0.01, compared with the RA group; ^bP < 0.05, compared with the ISO-1 group.

3.5. Correlation between TNF-α/CORT and MIF Expression in serum

A spearman bivariate correlation analysis was performed to determine the relationship between synovium MIF and serum TNF-α and CORT expression. Analysis showed that the expression of serum MIF was positively correlated with the expression of TNF- α (r = 0.722, P < 0.001, supplementary Figure 2A) and negatively correlated with the expression of CORT (r = -0.434, P < 0.01, supplementary Figure 2 B). These data collectively suggested that MIF was positively related to the expression of TNF-α and negatively correlated with the expression of CORT.

4. DISCUSSION

In the clinical treatment of RA, non-steroidal anti-inflammatory drugs (NSAIDs), GCs, disease-modifying antirheumatic drugs (DMARDs) and biological agents are often used. They have good advantages in analgesia and anti-inflammatory effects, but they also have some limitations.³⁰ NSAIDs influence renal blood circulation and may lead to cardiovascular problems.³¹ Long-term use of GCs can have some adverse effects, such as osteoporosis, disturbed glucose tolerance, hypertension, elevated intraocular pressure and a higher risk of infections.³² DMARDs have a good benefit-to-risk profile, and the primary risk is infections.³³ Biological agents are alternative treatment options; however, it entails a high cost.³⁴ Therefore, it needs to find a complementary and alternative medicine (CAM) that is both effective and safe. Moxibustion has a long history of being used to treat RA in Traditional Chinese Medicine. Moxibustion treatment is effective in treating RA in both clinical trials and animal experiments,^{29,35,⇓,⇓,⇓-39} which inhibits the release of pro-inflammatory factors and increases the synthesis of anti-inflammatory factors. This is in line with the findings of this study, and it could be an important mechanism for moxibustion's resistance to RA inflammation.

MIF functions as an immunomodulatory factor in the innate and adaptive immune systems. It leads to persistent inflammation and bone degradation of RA by promoting angiogenesis, pro-inflammatory cytokines and matrix metalloproteinase. Studies have shown that the level of MIF in synovial fluid and synovium of patients with RA significantly increases, which may be closely related to the expression of VEGF and PLA2.⁴⁰ In this study, it was also confirmed that the levels of MIF in synovium and serum of RA rats increased. GCs are an effective regulator of RA inflammation, and endogenous GCs also play an immunosuppressive and immunomodulatory role in the anti-inflammatory process of RA. The physiological concentration of GCs can promote MIF synthesis and release, which can reversely regulate glucocorticoids' inhibitory effect on immune cell activation and cytokine secretion.^14,15 In experimental arthritis, for example, adrenalectomy results in synovial reduced MIF expression, indicating that MIF is under the control of endogenous GCs.⁴¹ In vitro, MIF is secreted in response to GCs in monocytes and T cells, neuronal cells and synovial fibroblasts.^8,15,42 These studies suggest that MIF is a physiological counter-regulator of the anti-inflammatory effects of GCs.^14,42 Thus, MIF functions acts as a physiological counter-regulator of the anti-inflammatory effects of GCs. MIF and GCs can regulate each other in the inflammatory response. Many inflammatory factors, such as TNF-α, are regulated by MIF in inflammatory diseases. After receiving MIF inhibitor treatment, the mRNA levels of IL-1β, and TNF-α in the blood vessels of Uric Acid (UA) mice decreased. MIF inhibitors significantly reduce the severe inflammatory response induced by UA, which includes vascular macrophage infiltration and upregulation of pro-inflammatory mediators in blood vessels.⁴³ Therefore, in the strategy of treating inflammatory diseases, MIF is the key entry point to reduce the level of inflammation.

As a CAM for the treatment of RA, moxibustion has been proved to reduce the symptoms of joint inflammation, alleviate synovial inflammation and slow down the process of joint bone destruction, with few adverse reactions after treatment, which is conducive to the improvement of patients' quality of life.³⁵ Some studies have shown that moxibustion can increase the level of CORT in RA rats.⁴⁴ The anti-inflammatory effect of GCs may down-regulate the level of PGE2, TNF-α and IL-1β by regulating the expression of glucocorticoid receptors (GR)-related signal molecules such as NF-κB and AP-1.²² Our previous experimental studies have also confirmed that moxibustion can effectively regulate the expression of GCs and GR by regulating the function of HPAA axis in RA model, so as to effectively exert its anti-inflammatory effect.^45,46 In this study, the results of WB and ELISA showed that the expression of MIF in synovium and serum decreased and the level of serum CORT significantly increased in RA rats treated with moxibustion.

Moxibustion may regulate MIF to regulate GCs to achieve the anti-inflammatory effect, MIF is an important upstream regulator of synovitis in RA. Therefore, moxibustion to inhibit MIF activity may be a more effective measure to treat RA. Previous studies only showed that moxibustion can regulate the MIF or GCs. However, the overall relationship between moxibustion-treated RA and MIF/GCs was not considered. In this study, after injecting MIF inhibitor ISO-1, to inhibit the expression of MIF in RA rats, it can be observed that TNF-α significantly decreased and CORT increased in moxibustion RA rats. Through Pearson correlation analysis, there was a positive correlation between TNF-α and MIF expression in RA, and a negative correlation between CORT and MIF. This study investigated the anti-inflammatory mechanism of moxibustion on FCA-induced RA model rats. The results show that moxibustion of Shenshu (BL23) and Zusanli (ST36) can effectively increase the level of CORT and decrease the level of pro-inflammatory factor TNF-α by inhibiting the expression of MIF, resulting in an anti-inflammatory effect. But when MIF is inhibited, moxibustion can further reduce the expression of TNF-α, indicating that moxibustion regulates TNF-α not only through the MIF and CORT pathways, but also through other pathways. This gives us a better understanding of the mechanism of action of moxibustion in the treatment of RA. However, the specific mechanisms by which moxibustion affects RA remain to be elucidated.

In conclusion, Moxibustion of Zusanli (ST36) and Shenshu (BL23) acupoints alleviated the synovial inflammation in the RA model of rats. And the effects of moxibustion on RA may be associated with regulation of MIF/GCs signaling. Moreover, this also gives us ideas for future research into other moxibustion mechanisms in the treatment of RA.

5. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.cn.