Isoimperatorin improves osteoporosis by increasing YBX1 expression to promote BGLAP m5C modification.

Osteoporosis is a chronic metabolic bone disease that is prone to fractures. Isoimperatorin (ISO) has been shown to alleviate the bone loss in ovariectomized (OVX) rats. The aim of this study was to investigate the effect and the mechanism of ISO on osteoporosis using animal study and cell experiments. Osteogenic differentiation was assessed by alkaline phosphatase activity detection, and alizarin red S staining. The expression of osteogenic differentiation-related genes and m5C regulators was measured using quantitative real-time PCR. Hematoxylin eosin (H&E) staining and microCT were performed to evaluate osteoporosis in vivo. The m5C levels in mice were measured by dot blot assay, and the binding between ISO and YBX1 was assessed by biolayer interferometry (BLI) analysis and molecular docking. Methylated RNA immunoprecipitation was performed to identify the target gene of YBX1. The interaction between YBX1 and BGLAP was assessed using RIP and luciferase reporter assay. Results suggested that ISO significantly promoted osteogenic differentiation of MC3T3 cells and alleviated osteoporosis in OVX mice. Moreover, ISO increased m5C level and YBX1 expression in OVX mice, while YBX1 knockdown inhibited osteogenic differentiation in ISO-treated MC3T3 cells, and restored osteoporosis in OVX mice ameliorated by ISO. Additionally, YBX1 knockdown inhibited the m5C level of BGLAP through inhibiting its mRNA stability. In conclusion, we demonstrated that ISO improved osteoporosis through increasing YBX1 expression thereby upregulating the m5C modification of BGLAP. These results may provide a novel theoretical basis for ISO treatment of osteoporosis.