Abstract
To determine the active components of Eupolyphaga sinensis Walker (Tu Bie Chong) and explore the mechanisms underlying its fracture-healing ability.
A modified Einhorn method was used to develop a rat tibial fracture model. Progression of bone healing was assessed using radiological methods. Safranin O/fast green and CD31 immunohistochemical staining were performed to evaluate the growth of bone cells and angiogenesis at the fracture site. Methylthiazoletetrazolium blue and wound healing assays were used to analyze cell viability and migration. The Transwell assay was used to explore the invasion capacity of the cells. Tubule formation assays were used to assess the angiogenesis capacity of human vascular endothelial cells (HUVECs). qRT-PCR was used to evaluate the changes in gene transcription levels.
Tu Bie Chong fraction 3 (TF3) significantly shortened the fracture healing time in model rats. X-ray results showed that on day 14, fracture healing in the TF3 treatment group was significantly better than that in the control group (P =.0086). Tissue staining showed that cartilage growth and the number of H-shaped blood vessels at the fracture site of the TF3 treatment group were better than those of the control group. In vitro, TF3 significantly promoted the proliferation and wound healing of MC3T3-E1s and HUVECs (all P <.01). Transwell assays showed that TF3 promoted the migration of HUVECs, but inhibited the migration of MC3T3-E1 cells. Tubule formation experiments confirmed that TF3 markedly promoted the ability of vascular endothelial cells to form microtubules. Gene expression analysis revealed that TF3 significantly promoted the expression of VEGFA, SPOCD1, NGF, and NGFR in HUVECs. In MC3T3-E1 cells, the transcript levels of RUNX2 and COL2A1 were significantly elevated following TF3 treatment.
TF3 promotes fracture healing by promoting bone regeneration associated with the RUNX2 pathway and angiogenesis associated with the VEGFA pathway.
