Study on the mechanism of moxibustion regulating ferroptosis-lipid metabolism pathway to improve synovitis inflammatory injury in rheumatoid arthritis rats

Abstract

To explore the mechanism of moxibustion in improving synovitis inflammatory injury in rheumatoid arthritis (RA) model rats based on the ferroptosis-lipid metabolism pathway. Forty-eight SD rats were randomly divided into normal, model, moxibustion and moxibustion + long-chain acyl-CoA synthetase 4 (ACSL4) inhibitor groups, with 12 rats in each group. The RA model was replicated using environmental factors of wind, cold, and dampness combined with Freund's complete adjuvant injection. The moxibustion group received moxibustion at "Shenshu" (BL23) and "Zusanli" (ST36) for 20 minutes per acupoint each time, once daily at a single acupoint (both sides) with alternating acupoints over 15 consecutive days. The moxibustion + ACSL4 inhibitor group received intraperitoneal injection of the ACSL4 inhibitor rosiglitazone (0.4 mg/kg) after moxibustion, once daily for 15 consecutive days. Histopathological changes in synovial tissue were observed using HE staining；serum contents of glutathione (GSH) and malondialdehyde (MDA) were detected using biochemical methods；reactive oxygen species (ROS) levels in synovial tissues were detected using flow cytometry；the expressions of glutathione peroxidase 4 (GPX4), ACSL4, and lysophosphatidylcholine acyltransferase 3 (LPCAT3) proteins in synovial tissues were detected using Western blot；and serum contents of interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were detected using ELISA. Compared with the normal group, the serum contents of GSH decreased (P<0.01) while MDA, IL-6, and TNF-α contents increased (P<0.01)；the ROS levels, ACSL4 and LPCAT3 protein expressions increased (P<0.01) while GPX4 protein expression decreased (P<0.01) in synovial tissue in the model group. Compared with the model group, the serum contents of GSH increased (P<0.01) while MDA, IL-6, and TNF-α contents decreased (P<0.01)；the ROS levels, ACSL4 and LPCAT3 protein expressions decreased (P<0.01) while GPX4 protein expression increased (P<0.01) in synovial tissue in the moxibustion group and moxibustion + ACSL4 inhibitor group. Compared with the moxibustion group, the serum contents of GSH increased (P<0.01) while MDA, IL-6, and TNF-α contents decreased (P<0.01)；the ROS levels, ACSL4 and LPCAT3 protein expressions decreased (P<0.01, P<0.05) while GPX4 protein expression increased (P<0.05) in synovial tissue in the moxibustion + ACSL4 inhibitor group. HE staining showed that the model group had significantly increased synovial hyperplasia and inflammatory cell infiltration；the moxibustion group and moxibustion + ACSL4 inhibitor group showed varying degrees of alleviation in inflammatory cell infiltration and hyperplasia in synovial tissue；compared with the moxibustion group, the moxibustion + ACSL4 inhibitor group showed more significant improvements in inflammatory infiltration and hyperplasia of synovial cells, reduced layers of synovium. Moxibustion at BL23 and ST36 can alleviate synovial inflammatory injury, and its mechanism may be related to reducing lipid peroxidation and ROS levels, and inhibiting the occurrence of ferroptosis.