Abstract
ObjectivesTo investigate the effects of β-ecdysterone on fracture healing and the underlying mechanism.MethodsMTT assay was used to detect the cell viability. AO/PI and flow cytometry assays were used to determine the apoptotic rate. The expression level of RunX2, ATG7 and LC3 was evaluated by qRT-PCR and Western blot assays. X-ray and HE staining were conducted on the fractured femur. Immunohistochemical assay was used to detect the expression level of Beclin-1 and immunofluorescence assay was used to measure the expression level of LC3 in the fractured femurs. Western blot was utilized to determine the expression level of PI3K, p-AKT1, AKT1, p-mTOR, mTOR, p-p70S6K, and p70S6K.ResultsThe ALP activity and the expression of RunX2 in fractured osteoblasts were significantly elevated, the apoptotic rate was suppressed by rapamycin, 60, and 80 μM β-ecdysterone. The state of autophagy both in fractured osteoblasts and femurs was facilitated by rapamycin and β-ecdysterone. Compared to control, Garrett score was significantly promoted in rapamycin and β-ecdysterone groups, accompanied by ameliorated pathological state. Lastly, the PI3K/AKT/mTOR pathway both in fractured osteoblasts and femurs was inhibited by rapamycin and β-ecdysterone.Conclusionβ-ecdysterone might facilitate fracture healing by activating autophagy through suppressing PI3K/AKT/mTOR signal pathway.
Highlights
Fracture healing is a complicated and well-organized regulatory progress induced by series of histological and biochemical changes
The osteogenic differentiation in osteoblasts and the fracture healing of fracture rats were significantly facilitated by the autophagy inducer rapamycin, which was consistent with the reports claimed previously [15, 16]
The osteogenic differentiation in osteoblasts and the fracture healing of fracture rats were dramatically repressed by the autophagy inhibitor 3-methyladenine, which further identified the involvement of autophagy in the repair of fracture healing
Summary
Fracture healing is a complicated and well-organized regulatory progress induced by series of histological and biochemical changes. Autophagy is an important cellular mechanism maintaining the balance between cellular survival and cell death under stress state [4]. Researches explored the impact of autophagy on the activity of osteoblasts by establishing animal models. By high throughput screening technology, rapamycin, an inducer of autophagy, is found to facilitate the differentiation and growth of osteoblasts [8]. These reports indicate that autophagy might exert an important role in the progress of bone growth and mineralization of bone tissues, which provides a potential therapeutic idea for the treatment of clinical delayed fracture union or nonunion
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