Regulatory Effect of Moxibustion on Macrophage Polarization and Migration in RA Model Rats and the Role of TIM-3

Summary

This study examines the regulatory effects of moxibustion on macrophage polarization and migration in rat models of rheumatoid arthritis, with a focus on the role of T cell immunoglobulin and mucin domain–containing protein 3 (TIM-3), to elucidate the therapeutic mechanisms of moxibustion in this disease.

Abstract

This study aimed to examine the regulatory effects of moxibustion on macrophage polarization and migration in a rat model of rheumatoid arthritis, as well as the role of T cell immunoglobulin and mucin domain–containing protein 3 (TIM-3) in this process. On day 0 of the experiment, Sprague–Dawley (SD) rats were divided into five groups. Complete Freund’s adjuvant (CFA) was injected into the hind footpads to establish the rheumatoid arthritis model. Additionally, lentivirus-mediated Havcr2 RNA interference (LV-Havcr2-RNAi) was injected into the hind footpads to inhibit TIM-3 expression. Starting on day 7, rats underwent moxibustion treatment in cycles comprising six days of treatment and one day of rest, for a total of three complete cycles. After completion of moxibustion therapy, the inflammatory status was assessed through histological analysis and measurement of paw thickness. Phenotypic markers of M1 and M2 macrophages were assessed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. TIM-3 expression was detected via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Furthermore, peritoneal exudate macrophages (PEMs) were isolated from additional SD rats, and macrophage migration was examined using a Transwell assay. The study found that after CFA injection, rats exhibited significant paw redness and swelling, along with an imbalance in macrophage phenotype expression. Additionally, Transwell migration assays revealed that CFA-induced model rats showed increased macrophage migration relative to control specimens. However, moxibustion treatment attenuated these changes. Results also demonstrated that moxibustion increased TIM-3 expression, and after lentiviral intervention, TIM-3 levels were markedly reduced, leading to exacerbation of inflammatory manifestations and macrophage imbalance. These results indicate that TIM-3 serves as a key mediator in moxibustion regulation of macrophage polarization and migration.

Introduction

Rheumatoid arthritis (RA) is a prevalent systemic autoimmune disorder that clinically presents with symmetrical inflammatory arthritis. The inflammation primarily targets the synovial membrane, cartilage, and bone, resulting in tissue destruction and culminating in irreversible joint deformity¹. Current pharmacological management of RA primarily relies on disease-modifying anti-rheumatic drugs (DMARDs)², which often require lifelong administration. However, these therapeutic agents are associated with various adverse effects. Consequently, a significant number of patients seek safe and effective complementary therapies to alleviate clinical symptoms and potentially reduce their reliance on conventional drug regimens. As a key component of traditional Chinese medicine, moxibustion may serve as a promising complementary therapy for RA. It exerts its therapeutic effects through systemic anti-inflammatory and immunomodulatory mechanisms³. Evidence indicates that moxibustion can alleviate symptoms and protect joint structures⁴. Furthermore, compared with the potential hepatorenal toxicity, infection risks, and high costs associated with DMARDs, moxibustion offers superior safety and lower cost. However, moxibustion is not a panacea and cannot wholly replace DMARDs for managing RA. Moxibustion typically requires repeated, long-term treatment sessions to achieve its effects. During acute exacerbations of RA, it may not provide rapid symptom control. Furthermore, the therapeutic outcome of moxibustion is highly dependent on the practitioner’s skill and experience, introducing variability. Consequently, integrating moxibustion with DMARDs should be considered to achieve a synergistic “enhanced efficacy with reduced toxicity” effect, making it a viable strategy for the long-term management of RA⁵.

Although numerous studies have confirmed the therapeutic efficacy of moxibustion in RA^6,7,8, its precise mechanisms of action remain unclear. This study investigates the effects of moxibustion on macrophage polarization and migration in RA, using T cell immunoglobulin and mucin domain–containing protein 3 (TIM-3) as a mechanistic entry point. This approach is designed to elucidate a potential immunomodulatory pathway through which moxibustion exerts its anti-arthritic effects.

As pivotal immune cells, macrophages play a central role in the pathogenesis and progression of RA^9,10. Upon the onset of inflammation, tissue-resident macrophages release a variety of cytokines and chemokines. These signaling molecules act on vascular endothelial cells, promoting the differentiation of circulating monocytes into macrophages and their subsequent recruitment to inflamed tissue^11,12. These infiltrating macrophages further amplify the inflammatory response by producing additional mediators, which in turn recruit more macrophages and exacerbate tissue infiltration, thereby establishing a vicious cycle of inflammation and tissue infiltration.

Depending on environmental stimuli, macrophages display significant plasticity by polarizing into distinct functional phenotypes, mainly M1 (classically activated) and M2 (alternatively activated) states¹³. Under physiological conditions, macrophage polarization maintains a dynamic equilibrium between M1 and M2 phenotypes. However, in RA, this equilibrium is disrupted, characterized by enhanced M1 polarization with increased release of pro-inflammatory mediators, including interferon (IFN)-γ and inducible nitric oxide synthase (iNOS), which exacerbates inflammatory responses. Concurrently, M2 polarization is weakened, accompanied by reduced release of anti-inflammatory factors, including interleukin (IL)-4 and CD206, impairing their capacity to inhibit T-cell activation and proliferation, thereby aggravating disease progression¹⁴. A number of investigations have revealed that modulating the M1/M2 macrophage balance may mitigate RA progression^15,16.

TIM-3, identified as a T cell surface glycoprotein, is widely expressed on a spectrum of immune cells, including T cells, macrophages, and natural killer cells, where it binds to specific ligands to exert immunoregulatory functions¹⁷. TIM-3 signaling exerts a definitive influence on macrophage inflammatory cytokine expression and polarization. Notably, TIM-3 is highly expressed on M2 macrophages, where it promotes the M2 phenotype while suppressing the M1 phenotype. Conversely, research indicates that TIM-3 inhibition produces the opposite effect, driving M1 polarization and attenuating M2 polarization^18,19,20. Additionally, evidence highlights TIM-3 as a key regulator of macrophage migratory behavior^21,22. Therefore, therapeutic intervention targeting TIM-3 to control macrophage polarization and migration dynamics shows considerable promise as an RA treatment strategy.

Based on the aforementioned rationale, this study was conducted to contribute to the growing body of knowledge regarding the mechanisms by which moxibustion exerts its therapeutic effects in RA.

Protocol

Ethical Statement and Safety Precautions
All animal experiments have been approved by the Ethics Committee of Chengdu University of Traditional Chinese Medicine (2024029). During all injection procedures, including the administration of CFA and lentivirus, operators must wear personal protective equipment (PPE) consisting of disposable gloves, goggles, and laboratory coats. These procedures must be conducted within a biosafety cabinet or a well-ventilated environment. All sharps that have come into contact with reagents must be immediately discarded into designated sharps containers. Throughout the moxibustion treatment, the operator closely monitored the animal's response. The moxa cone was promptly replaced to prevent thermal injury to the skin. Furthermore, flammable materials should be kept clear of the burning moxa to eliminate fire hazards. 

1. Establishment of RA model

Thirty male SD rats (weight: 200 ± 20 g), aged six weeks (license number: SCXK 2020-030), were randomly divided into five groups (n = 6): control group, RA model group (RA group), RA + moxibustion treatment group (Mox group), RA + TIM-3 lentivirus interference group (RA + TIM-3– group), and RA + TIM-3 lentivirus interference + moxibustion treatment group (RA + TIM-3– + Mox group). Before the experiment, rats were acclimatized for seven days in a controlled environment with a temperature of 22–24 °C, 20% humidity, and a natural light/dark cycle, with free access to food and water. On day 23 of the experiment, an additional five three-week-old male SD rats were purchased from the same company. These rats were acclimatized for five days under identical housing conditions before being used for PEM extraction. A schematic diagram summarizing the experimental procedure and representative procedural photographs are shown in Figure 1.

On day 0 of the experiment, 0.5 mL/kg of CFA was injected into the hind paw pads of all rats except those in the control group to induce RA. The same volume of saline was administered at the same location in control group rats. Paw thickness was used as the criterion to evaluate successful establishment of the RA model²³.

2. Lentivirus injection

Rats in the RA + TIM-3^- and RA + TIM-3^- + Mox groups received injection of LV-Havcr2-RNAi (94435-1) into the hind paw pads (3.5 × 10⁷ TU/mL; 10 µL per pad) on day 1 of the experiment. All other rats received identical injections of normal saline at the same anatomical site²⁴.

3. Moxibustion

On day 7 of the experiment, rats in the Mox and RA + TIM-3^- + Mox groups received moxibustion treatment. To facilitate the procedure, hair within an approximately 1 × 1 cm area around the BL23 and ST36 acupoints was shaved (this procedure was also performed for rats in the remaining groups). During moxibustion, each rat was gently secured to a wooden block slightly larger than its body size using two elastic bands to fully expose the treatment areas (rats in other groups were similarly restrained). Subsequently, grain-shaped moxa cones were applied to both acupoints. To ensure consistency in thermal stimulation across individuals, each moxa cone (diameter: 2 mm; height: 5 mm; moxa wool content: 60%) was prepared from a standardized 5 mg portion of moxa wool. Each day, five cones were applied to the two acupoints on one side of the body, with treatment alternating to the contralateral side on the following day. This alternating treatment protocol was maintained for three weeks, with a one-day rest period every seventh day.

After three cycles of moxibustion treatment, the rats were anesthetized using isoflurane (4% induction, 2% maintenance, inhalation), and 5 mL of arterial blood was collected from the abdominal aorta. After centrifugation (2,000 × g, 15 min, 4 °C), the supernatant serum was transferred into microcentrifuge tubes and stored at −20 °C. Following blood sampling, the rats were euthanized by cervical dislocation. The ankle joints were carefully dissected and fixed in 4% paraformaldehyde. Synovial tissues and spleens were rapidly frozen using liquid nitrogen and stored at −80 °C.

4. Measurement of rat paw thickness

The midpoints of the rats’ hind paws were marked with a waterproof marker to ensure consistent measurement positioning. Paw thickness was measured using vernier calipers before modeling, after successful modeling, and between each treatment course (on days 0, 7, 14, 21, and 28 of the experiment).

5. Histological analysis

Following fixation in 4% paraformaldehyde, rat ankle joints were placed in centrifuge tubes containing an ample volume of decalcification solution (10% formic acid). The tubes were mounted on a shaker and subjected to gentle agitation at room temperature for 30 h. During this period, the decalcification solution was replaced at regular intervals, and tissue condition was monitored to prevent over-decalcification. Subsequently, tissues were transferred to a 5% sodium thiosulfate solution for neutralization over 6 h, followed by a thorough 4 h rinse under running tap water²⁵. The tissues were then dehydrated and embedded in paraffin blocks. Sections were transversely cut at a thickness of 5 µm. After deparaffinization and hydration, sections were stained with hematoxylin for 3 min, rinsed with water, differentiated briefly (5 s), rinsed again, and blued for 5 s, followed by a thorough rinse under running tap water. Subsequently, sections were dehydrated in 95% ethanol for 1 min and counterstained with eosin for 15 s. Finally, sections were dehydrated, cleared, and mounted with a coverslip for observation under a light microscope. With this staining, nuclei appeared blue, whereas cytoplasm, collagen fibers, and red blood cells were visualized in varying shades of pink; the keratinized layer typically stained bright red.

6. ELISA

Following the manufacturer’s protocol, a 100 µL aliquot of each serum sample (pre-diluted 1:2) was added to the designated wells. Blank and standard wells were included in parallel. After sealing, the plate was incubated at 37 °C for 90 min. Following a wash step, the detection antibody was added and incubated at 37 °C for 60 min. After another wash, streptavidin–HRP conjugate was added. Following thorough washing, the substrate solution was added for color development, which was stopped by the addition of stop solution. Absorbance was measured at 450 nm (OD₄₅₀). Sample concentrations of IL-4 and IFN-γ were determined by interpolation from the standard curve, and final concentrations were calculated by multiplying by the respective dilution factor.

7. qRT-PCR

Total RNA was extracted from serum samples using TRIzol reagent according to a modified protocol. Briefly, 100 µL of serum was homogenized with 1 mL of TRIzol reagent by vigorous pipetting. Subsequently, 100 µL of chloroform was added, and the mixture was vigorously shaken for 15 s, followed by incubation at room temperature for 3 min. The sample was then centrifuged at 12,000 × g for 15 min at 4 °C to achieve phase separation. The upper aqueous phase was carefully transferred to a new tube, mixed with 0.5 mL of isopropanol, and incubated at room temperature for 10 min. Subsequently, total RNA was pelleted by centrifugation at 12,000 × g for 10 min at 4 °C. The resulting RNA pellet was washed once with 1 mL of 75% ethanol (centrifugation at 8,000 × g for 5 min at 4 °C) and air-dried before final dissolution. The obtained RNA underwent reverse transcription using a cDNA synthesis kit. Gene expression analysis was performed by qRT-PCR using real-time quantitative PCR system software. β-actin was used as the housekeeping gene for normalization. The relative expression of TIM-3 mRNA was calculated using the 2^−ΔΔCt method. The details of primers are added in Table 1.

8. Western blot

The extracted spleen was washed three times with precooled phosphate-buffered saline (PBS), cut into small pieces, and lysed by adding lysis buffer at a volume tenfold greater than that of the tissue. Homogenization was performed using a homogenizer tube. The homogenate was placed on ice for 30 min for lysis, with five to six rounds of vortexing to ensure complete tissue disruption. The samples were centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was collected as the total protein extract. Target protein concentrations were measured using the bicinchoninic acid (BCA) assay according to the kit protocol.

Following separation by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), the extracted protein samples were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked at room temperature for 1 h with Tris-buffered saline containing Tween 20 (TBST) supplemented with 5% skim milk, followed by overnight incubation at 4 °C with primary antibodies. After thorough washing, the membrane was incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies at 37 °C for 2 h. Finally, images were captured using a chemiluminescence imaging system and analyzed for grayscale intensity using ChemiScope analysis software. Details of the primary and secondary antibodies are listed in Table 1.

9. Immunofluorescence detection

The extracted synovial tissue was immersed in 4% paraformaldehyde, dehydrated, and embedded in paraffin before being sectioned into 5 µm-thick slices. The sections were sequentially dewaxed in xylene I, xylene II, and xylene III (10 min each), followed by three washes in absolute ethanol (5 min each) and a final rinse with distilled water.

After antigen retrieval in ethylenediaminetetraacetic acid (EDTA), the slides were washed three times in phosphate-buffered saline (PBS) (5 min each) with gentle agitation. The sections were then blocked by incubation with 3% bovine serum albumin (BSA) for 30 min at room temperature. Following blocking, primary antibodies, including anti-inducible nitric oxide synthase (iNOS) (1:500) and anti-CD206 (1:400), were applied and incubated at 4 °C for 24 h.

After three 5 min PBS washes, the sections were incubated for 50 min at 37 °C in the dark with a secondary antibody (Cy3-conjugated goat anti-rabbit IgG, 1:300). Nuclear staining was performed by washing the slides three times with PBS (5 min per wash), followed by treatment with 4′,6-diamidino-2-phenylindole (DAPI) staining solution and 10 min incubation at room temperature in the dark. The images were examined under a fluorescence microscope.

10. PEMs isolation

Following euthanasia by cervical dislocation, rats were disinfected in 75% ethanol for 10 min and transferred to a sterile biosafety cabinet. The abdominal skin was incised with sterile scissors, followed by intraperitoneal injection of 15 mL phosphate-buffered saline (PBS) using a syringe. The abdomen was gently massaged before the peritoneal fluid was aspirated back into the syringe and transferred to centrifuge tubes to obtain macrophage suspensions.

The cell suspension was washed three times with PBS before being seeded in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Cells were cultured for 24 h in a humidified atmosphere of 5% CO₂ at 37 °C. The adherent cells obtained after this incubation period were identified as macrophages.

11. Transwell

The migration assay was conducted using 24-well plates. After 24 h of serum starvation, 4 × 10⁴ macrophages in 300 µL of DMEM supplemented with 1% fetal bovine serum (FBS) and 1% antibiotics (50 U/mL penicillin, 50 µg/mL streptomycin) were added to the upper chamber, while 700 µL of DMEM containing 10% FBS and 1% antibiotics was introduced into the lower chamber. The cells were pretreated with serum from different rat groups for 24 h. Thereafter, the plates were incubated at 37 °C in a 5% CO₂ environment for 24 h. After crystal violet staining, the upper chamber was swabbed to remove non-migrated cells. Migrating cells were quantified by manual counting across four randomly selected fields of view under an optical microscope.

12. Statistical analysis

Statistical analyses were performed using SPSS version 25.0. Datasets were first tested for normality and homogeneity of variances. When these parametric assumptions were met, intergroup comparisons were conducted using one-way analysis of variance (ANOVA) followed by least significant difference (LSD) post hoc analysis. For data that violated these assumptions, the nonparametric Kruskal–Wallis test was applied, followed by Mann–Whitney U tests with appropriate correction for multiple comparisons. All values were expressed as mean ± standard deviation, and P < 0.05 was considered statistically significant.

Results

Data availability
The raw data generated during the current study are available in the Figshare repository (https://doi.org/10.6084/m9.figshare.31550197)

Articular pathology and paw thickness in rats after moxibustion treatment
Rats injected with CFA developed obvious paw swelling and redness. Hematoxylin and eosin staining demonstrated that the control group exhibited intact joint architecture with smo...

Discussion

Current management of RA relies predominantly on DMARDs. However, these pharmacological agents are often costly and associated with non-negligible adverse effects, including hepatorenal impairment and severe infections², imposing a substantial burden on individuals and healthcare systems. In contrast, as an external treatment, moxibustion exhibits a favorable safety profile with minimal reported risk of systemic toxicity. Its technique is relatively straightforward, allowing for potential self-adm...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Complete Freund’s Adjuvant (CFA)	Sigma-Aldrich	F5881	Used for inducing rheumatoid arthritis (RA) model in rats
Lentivirus (TIM-3 RNAi)	GenChem Biotechnology, Shanghai	3.5×107 TU/mL	Used to silence TIM-3 gene expression
Moxa Cones (Wheat-sized)	Traditional Chinese Medicine Supplier	N/A	Used for moxibustion at BL23 and ST36 acupoints