Effects of moxibustion on autophagy and ferroptosis in a disease-syndrome combination rat model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation, fibroblast-like synoviocyte hyperplasia, and progressive cartilage and bone destruction. Moxibustion is widely used as a non-pharmacological adjunctive intervention for RA; however, its specific therapeutic effects and its associations with autophagy and ferroptosis remain insufficiently characterized. This protocol describes a randomized, controlled, assessor-blinded preclinical study designed to evaluate moxibustion in a disease-syndrome combination rat model of RA. Eligible rats will undergo exposure to a controlled wind-, cold-, and dampness-related environment followed by Complete Freund's Adjuvant (CFA) injection. After successful model induction has been confirmed using prespecified criteria, animals will be randomly allocated to a normal control group, an untreated model group, a heat-matched sham-moxibustion group, a Zusanli (ST36) moxibustion group, and a methotrexate active-comparator group. All animals will undergo standardized handling and restraint procedures. Moxibustion will be administered for 20 min once daily for 15 consecutive days, whereas methotrexate will be administered at the prespecified dose and frequency. The primary outcome will be the histopathological synovitis score. Secondary outcomes will include paw volume, arthritis index, joint structural damage, serum and synovial inflammatory mediators, and functional assessments. Autophagy will be evaluated using LC3-II/LC3-I, SQSTM1/p62, Beclin-1, and complementary assessments of autophagic flux. Ferroptosis-related outcomes will include GPX4, SLC7A11, ACSL4, lipid peroxidation, glutathione, reactive oxygen species, and tissue iron levels. Outcome assessment and statistical analysis will be performed by investigators blinded to group allocation. This study is expected to provide a reproducible framework for distinguishing the specific effects of moxibustion from non-specific heat, restraint, and handling effects, and to generate mechanistic evidence supporting subsequent translational and clinical investigations.