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 t....

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 mon....

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