[Artesunate enhances effect of leflunomide on inhibiting bone destruction in rheumatoid arthritis via modulating HIF-1α/ARNT signaling pathway].

Based on the hypoxia-inducible factor-1α(HIF-1α)/aryl hydrocarbon receptor nuclear translocator(ARNT) signaling pathway, this study aims to investigate the effects and potential mechanisms of artesunate in enhancing the effect of leflunomide on inhibiting bone destruction in rheumatoid arthritis(RA). By using in vitro osteoclast differentiation model induced by receptor activator of nuclear factor-κB ligand(RANKL) and a collagen-induced arthritis(CIA) mouse model, the intervention effect of combination treatment on bone destruction in RA was systematically evaluated at the cellular, histological, and molecular levels. Key mechanisms were analyzed and screened via transcriptomic sequencing, and relevant mechanisms were further validated through in vitro and in vivo experiments. The results show that artesunate can significantly enhance leflunomide's inhibitory effect on osteoclast formation, further reducing actin ring formation and bone resorption activity. In CIA mice, the combination treatment more effectively decreases the number of tartrate-resistant acid phosphatase(TRAP)-positive multinucleated osteoclasts in the joints compared with leflunomide alone, suggesting that artesunate potentiates leflunomide's ability to inhibit osteoclast formation and differentiation, thereby alleviating bone destruction in RA. Transcriptomic analysis indicates that artesunate may enhance leflunomide's inhibitory effect on bone destruction in RA by modulating the HIF-1α/ARNT signaling pathway. Further validation finds the nuclear co-localization of HIF-1α and ARNT during osteoclast differentiation. The combination treatment markedly suppresses HIF-1α/ARNT signaling and the expression of molecules related to downstream osteoclasts, while upregulating antioxidant-related proteins. The critical role of this signaling pathway in the combination treatment for inhibiting osteoclast differentiation is further supported by using an HIF-1α inhibitor. In summary, this study elucidated the critical role of the HIF-1α/ARNT signaling pathway in artesunate-enhanced leflunomide-mediated amelioration of bone destruction in RA, providing new experimental evidence and theoretical support for understanding the molecular mechanisms of bone destruction in RA and guiding combination treatment with leflunomide.