ß-Ecdysterone Enhanced Bone Regeneration Through The BMP-2/SMADs/RUNX2/Osterix Signaling Pathway

Bone defects are a global public health problem. However, the available methods for bone regeneration are limited. In recent years, the application of traditional Chinese herbs for bone regeneration has attracted increasing attention. ß-ecdysterone is a herbal extract similar to estrogen that promotes the metabolism of cell protein synthesis; however, its function on bone regeneration is not yet clear. In this study, we investigated the function of ß-ecdysterone on osteoblast differentiation and bone regeneration in vitro and in vivo. MC3T3-E1 cells were used to test the function of ß-ecdysterone on osteoblast differentiation and bone regeneration in vitro. The results of the CCK-8 assay suggested that the proliferation of MC3T3-E1 cells was promoted by ß-ecdysterone. Furthermore, ß-ecdysterone influenced the expression of osteogenesis-related genes, and the bone regeneration capacity of MC3T3-E1 cells was detected by PCR, the alkaline phosphatase (ALP) test, and the Alizarin Red test. ß-ecdysterone could up-regulate the expression of osteoblastic-related genes, and promoted ALP activity and the formation of calcium nodules. Moreover, we also determined that ß-ecdysterone increased the mRNA and protein levels of components of the BMP-2/Smad/Runx2/Osterix pathway. DNA sequencing further confirmed these target effects. ß-ecdysterone promoted bone formation by enhancing gene expression of the BMP-2/Smad/Runx2/Osterix signaling pathway and by the enrichment biological processes. For in vivo experiments, the femur condyle defection model was constructed by drilling a critical-sized (3×3 mm) defect in 12-week-old SD male rats to further assess the bone regeneration functions of ß-ecdysterone. The results of micro-CT showed that ß-ecdysterone could accelerate bone regeneration and exhibited higher bone volume, bone surface, bone mineral density at each observation time point. Immunohistochemistry confirmed that the ß-ecdysterone also increased the expression of collagen, osteocalcin, and BMP2 in the experiment group at 8 weeks. In conclusion, discovered ß-ecdysterone is a new bone regeneration regulator that can stimulate MC3T3-E1 cell proliferation and induce bone regeneration through the BMP-2/Smad/Runx2/Osterix pathway. This new function of ß-ecdysterone has revealed a new direction of osteogenic differentiation and has provided novel therapeutic strategies for treating bone defection. Overall design: To observe and compare gene expression in MC3T3-E1 cells treated with ß-ecdysterone, we performed RNA sequencing of the samples.