Abstract

Background Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by synovial inflammation and hyperplasia, where fibroblast-like synoviocytes (FLS) play a central role in disease progression through aberrant proliferation and cytokine secretion. Current therapies often have limited efficacy and significant side effects, highlighting the need for novel treatment strategies. Ferroptosis has emerged as a potential therapeutic target in RA. This study aimed to address whether Coumestrol, a natural phytoestrogen with known anti-inflammatory properties, can alleviate RA by inducing ferroptosis in RA-FLS. Methods The human RA-FLS cells (MH7A) were treated with Coumestrol at different concentrations (50, 100 μM). Cell viability and proliferation were assessed using CCK-8 and EdU assays. Apoptosis was evaluated via Annexin V/PI staining. cytokine levels were measured by ELISA and qPCR. Mitochondrial function was analyzed using Seahorse assays, reactive oxygen species (ROS) probes, and iron content quantification. Results Coumestrol dose-dependently suppressed RA-FLS proliferation and reduced the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β). Moreover, Coumestrol treatment induced significant apoptosis, oxidative stress, and mitochondrial dysfunction. Furthermore, Coumestrol significantly induced ferroptosis in RA-FLS, as evidenced by elevated mitochondrial ROS and iron accumulation. Mechanistically, PMAIP1 was identified as a key target responding to Coumestrol treatment. Knockdown of PMAIP1 substantially abolished Coumestrol-induced ferroptosis in RA-FLS. The mechanism by which Coumestrol upregulates PMAIP1 involves the suppression of the TRIM3-mediated ubiquitin-proteasome pathway, thereby significantly enhancing PMAIP1 protein stability. Conclusion Coumestrol mitigates RA progression by promoting PMAIP1-mediated ferroptosis in RA-FLS, leading to suppressed proliferation and inflammation. These findings suggest that Coumestrol may serve