N-(2-Hydroxyphenyl)acetamide: a Novel Suppressor of RANK/RANKL Pathway in Collagen-Induced Arthritis Model in Rats.

RANKL and RANK are potential contributors of inflammatory cascade in human and animal model of arthritis. The current study aims to investigate the effect of N-(2-hydroxyphenyl)acetamide (NA-2) on regulation of RANKL pathway in collagen-induced arthritis (CIA) model in rats. CIA was induced using bovine type II collagen in female Wistar rats. The clinical parameters, level of pro-inflammatory and oxidative stress markers were measured to determine the progression of the disease. The mRNA level of RANKL and RANK and downstream mediators of inflammation i.e. c-fos, c-jun, NF-κB and Akt were analysed in spleen tissue using real-time PCR. Immunohistochemical analysis of iNOS, pAkt and c-Fos was also done in spleen tissue. Treatment with NA-2 and indomethacin showed increase in body weight and significant reduction in paw volume and arthritic score (p < 0.0001). Marked reduction in the level of oxidative stress markers, NO, PO and GSH (p < 0.0001), and pro-inflammatory markers, IL-1β (p < 0.0001) and TNF-α (p < 0.01), was also observed. Likewise, NA-2 and indomethacin treatment also significantly suppressed the mRNA expression of RANKL, RANK, c-fos, c-jun, NF-κB (p < 0.0001) and Akt (p < 0.01) and protein expression of iNOS, pAkt and c-Fos (p < 0.0001) compared to the arthritic control group. Our findings suggest that NA-2 is an antiarthritic agent acting in a pleiotropic manner in CIA rats by not only reducing the clinical signs of arthritis, inflammatory cytokines and free radical production but also attenuating the RANK/RANKL signaling pathway.