Evaluating the role of 2-hydroxyestradiol in modulating TNF-α signaling and its implications in rheumatoid arthritis

Estrogen metabolites, a subset of female sex hormones, are known to play a role in autoimmunity and the inflammatory response associated with rheumatoid arthritis (RA). Current therapeutic approaches have focused on the development of small molecules that can easily alter intracellular components within a signaling pathway, thereby selectively targeting the disease physiology. In the present study, small molecule 2-hydroxyestradiol (2-OHE2) was utilized to investigate the potential anti-inflammatory effects by in vitro studies using primary fibroblast-like synoviocytes (RA-FLS) and in vivo studies using collagen-induced arthritis (CIA) rat model. A biophysical interaction study between 2-OHE2 and TNF-α was conducted using tyrosine quenching and thermal shift assay, and the maximum fluorescence quenching of the tyrosine residue was observed at a ligand concentration of 1 mM. Further, the impact of 2-OHE2 on the TNF-α signaling pathway was evaluated in RA-FLS, focusing on the inhibition of inflammation, cell proliferation, and apoptosis activation, using Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), caspase 3/7 activity, scratch assays, and immunocytochemistry. We found that 2-OHE2 effectively down-regulates inflammatory mediators, adhesion markers, and metalloproteinases and regulates apoptotic markers in RA-FLS. 2-OHE2 also exhibited anti-inflammatory effects in the CIA rat model. Our findings indicate that 2-OHE2 may be a promising potential anti-inflammatory compound. It targets TNF-α signaling, exhibiting pro-apoptotic and anti-proliferative effects, suggesting that 2-OHE2 could have therapeutic implications in the pathophysiology of RA.