DataSheet1_β-Ecdysterone Enhanced Bone Regeneration Through the BMP-2/SMAD/RUNX2/Osterix Signaling Pathway.docx

Bone defects are a global public health problem. However, the available methods for inducing bone regeneration are limited. The application of traditional Chinese herbs for bone regeneration has gained popularity in recent years. β-ecdysterone is a plant sterol similar to estrogen, that promotes protein synthesis in cells; however, its function in bone regeneration remains unclear. In this study, we investigated the function of β-ecdysterone on osteoblast differentiation and bone regeneration in vitro and in vivo. MC3T3-E1 cells were used to test the function of β-ecdysterone on osteoblast differentiation and bone regeneration in vitro. The results of the Cell Counting Kit-8 assay suggested that the proliferation of MC3T3-E1 cells was promoted by β-ecdysterone. Furthermore, β-ecdysterone influenced the expression of osteogenesis-related genes, and the bone regeneration capacity of MC3T3-E1 cells was detected by polymerase chain reaction, the alkaline phosphatase (ALP) test, and the alizarin red test. β-ecdysterone could upregulate the expression of osteoblastic-related genes, and promoted ALP activity and the formation of calcium nodules. We also determined that β-ecdysterone increased the mRNA and protein levels of components of the BMP-2/Smad/Runx2/Osterix pathway. DNA sequencing further confirmed these target effects. β-ecdysterone promoted bone formation by enhancing gene expression of the BMP-2/Smad/Runx2/Osterix signaling pathway and by enrichment biological processes. For in vivo experiments, a femoral condyle defect model was constructed by drilling a bone defect measuring 3 mm in diameter and 4 mm in depth in the femoral condyle of 8-week-old Sprague Dawley male rats. This model was used to further assess the bone regenerative functions of β-ecdysterone. The results of micro-computed tomography showed that β-ecdysterone could accelerate bone regeneration, exhibiting higher bone volume, bone surface, and bone mineral density at each observation time point. Immunohistochemistry confirmed that the β-ecdysterone also increased the expression of collagen, osteocalcin, and bone morphogenetic protein-2 in the experiment group at 4 and 8 weeks. In conclusion, β-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 newly discovered function of β-ecdysterone has revealed a new direction of osteogenic differentiation and has provided novel therapeutic strategies for treating bone defects.