Kaempferol suppresses fibroblast-like synoviocyte proliferation in rheumatoid arthritis by targeting glutamate-cysteine ligase modifier subunit to inhibit ferroptosis resistance

Aims: Kaempferol has demonstrated promising therapeutic potential in the treatment of rheumatoid arthritis (RA), yet its underlying mechanisms remain to be fully elucidated. This study aimed to investigate the role of kaempferol in modulating Ferroptosis resistance in tumour necrosis factor-alpha (TNF-α)-induced fibroblast-like synoviocytes (RA-FLS), and to explore its molecular targets and signalling pathways involved in RA progression.

Methods: Proteomic and transcriptomic analyses were employed to identify the key pathways regulated by kaempferol in TNF-α-induced RA-FLS. The effects of kaempferol on oxidative stress, Ferroptosis, and cellular proliferation in RA-FLS were evaluated using flow cytometry, measurements of malondialdehyde (MDA) and superoxide dismutase (SOD) activity, FerroOrange staining, transmission electron microscopy (TEM), cell counting kit (CCK-8) assay, and 5-ethynyl-2-deoxyuridine (EdU) assay. Protein and messenger RNA (mRNA) expression levels were validated using Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, a TNF-α-induced collagen-induced arthritis (CIA) rat model was established to examine the anti-inflammatory and antiproliferative effects of kaempferol in vivo. The functional role of the glutamate-cysteine Ligase modifier (GCLM) subunit was further investigated using GCLM-overexpressing RA-FLS models.

Results: Kaempferol significantly modulated Oxidative Phosphorylation and glutathione metabolism pathways, reducing Reactive Oxygen Species (ROS) and lipid peroxidation levels in RA-FLS. It suppressed TNF-α-induced RA-FLS proliferation by promoting ROS accumulation and inducing Ferroptosis. In the CIA model, kaempferol effectively alleviated joint swelling and reduced inflammatory cytokine levels. Mechanistically, kaempferol downregulated GCLM expression in RA-FLS, resulting in decreased p65 nuclear translocation and inhibition of nuclear factor kappa B (NF-κB) activation. This regulatory effect restored intracellular ROS, thereby overcoming Ferroptosis resistance and inhibiting synoviocyte hyperproliferation.

Conclusion: Kaempferol exerts anti-inflammatory and antiproliferative effects in RA by targeting the GCLM/NF-κB signalling axis. By restoring ROS and iron homeostasis and overcoming Ferroptosis resistance, kaempferol inhibits RA-FLS proliferation and mitigates synovial inflammation, suggesting its potential as a novel therapeutic agent for RA treatment.