Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint inflammation driven by complex signaling pathways. Recent therapeutic approaches focus on small molecules targeting intracellular signaling to address specific physiological aspects of the disease. Previously we identified a small molecule, 2-hydroxyestradiol (2-OHE2), an inhibitor of TNF-α by an in-silico study. In the present study, our aim was to explore the efficacy of 2-OHE2 by studying the proteome profile of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) using SWATH-MS and validate its therapeutic potential in RA by in-vitro studies. Oxidative stress was assessed using various biochemical assays, and cellular bioenergetics were analyzed with the Seahorse XFe96 Analyzer. We identified 396 differential proteins by SWATH-MS, and 82 showed significant changes. PharmMapper analysis revealed the association of 2-OHE2 with HMOX1 (HO-1), confirmed by SWATH-MS data. Also, we revealed that 2-OHE2 enhanced the expression of HO-1 and lowered oxidative stress via activating the Nrf2/KEAP1/HO-1 pathway. Further, 2-OHE2 has been found to boost cellular respiration and ATP production. Our findings thus suggest that 2-OHE2 possesses therapeutic potential as an antioxidant for RA treatment, effective at low dosages.
Keywords: Oxidative stress; Rheumatoid arthritis (RA); SWATH, proteomics, HO-1/Nrf2/KEAP1; Small molecule, estrogen metabolite.
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