Isopsoralen suppresses receptor activator of nuclear factor kappa-β ligand-induced osteoclastogenesis by inhibiting the NF-κB signaling

Abstract

Osteoporosis is a serious systemic metabolic bone system disease.This study aimed to identify the target genes of isopsoralen and the signaling pathways involved in the differential expression of the genes involved in osteoclast differentiation. We hypothesized that isopsoralen may inhibit osteoclast differentiation by blocking the nuclear factor kappa-B (NF-κB) signaling pathway and verified our hypothesis through basic experiments. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to detect the effect of isopsoralen on the proliferation and viability of primary mouse bone marrow monocytes (BMMCs). The effect of isopsoralen on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation was determined by using tartrate-resistant acid phosphatase (TRAP) staining. Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression of the related genes and proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of isopsoralen target genes were obtained through comprehensive analysis using the STITCH database, Cytoscape 3.8.2, and R-Studio software. Differentially expressed genes (DEGs) were found in osteoclasts induced by RANKL before and after 3 days using R-Studio, following which KEGG analysis was performed. Next, enrichment analysis was performed on the KEGG pathway shared by the target genes of isopsoralen and the differentially expressed genes during osteoclast differentiation to predict the signaling pathway underlying the inhibition of osteoclast differentiation by isopsoralen. Finally, Western blot was used to detect the effect of isopsoralen on the activation of signaling pathways to verify the results of our bioinformatics analysis. Based on the enrichment analysis of isopsoralen target genes and differentially expressed genes during osteoclastogenesis, we believe that isopsoralen can inhibit RANKL-induced osteoclastogenesis by inhibiting the NF-κB signaling pathway.