Bufalin inhibits the osteogenic differentiation of renal interstitial fibroblasts through the OCT4/BMP2/Smads signaling axis

This study aimed to investigate the role of bufalin in inhibiting the osteogenic differentiation of human renal interstitial fibroblasts (hRIFs) and explore its potential mechanism through the OCT4/BMP2/Smads signaling axis. Osteogenic differentiation of hRIFs was induced via osteogenic medium (OM), and this osteogenic differentiation was evaluated via alizarin red staining for calcium nodule formation, cobalt chloride staining for alkaline phosphatase activity, and Western blotting (WB) analysis for the expression of key osteogenic protein. Then, differentiated osteoblasts were treated with various concentrations of bufalin, and cell viability was assessed via the MTT assay to determine the optimal concentration. To investigate whether the inhibitory effect of bufalin on osteogenic differentiation involves the OCT4/BMP2/Smads signaling pathway, Western blot analysis, OCT4 overexpression, and BMP2-targeting short hairpin RNA (sh-BMP2) were used. OM successfully induced osteogenic differentiation in hRIFs and this differentiation was inhibited by bufalin. Mechanistically, bufalin suppressed OCT4 expression and consequently inhibited BMP2/Smads pathway activation. Notably, OCT4 upregulation reversed the inhibitory effects of bufalin on hRIF growth and osteoblast differentiation. Conversely, BMP2 downregulation counteracted the pro-osteogenic effects induced by OCT4 upregulation in bufalin-treated cells. Bufalin inhibits the osteogenic differentiation in hRIFs, via a mechanism involving the downregulation the OCT4/BMP2/Smads pathway. These findings reveal that bufalin plays an important regulatory role in the osteogenic differentiation of hRIFs, which may provide a theoretical foundation for future research into the treatment of Randall's plaque formation.