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bioRxiv · 10.1101/2021.05.28.446186

Regulation of osteoblast autophagy based on PI3K/AKT/mTOR signaling pathway study on the effect of β-ecdysterone on fracture healing

Tang, Y.·Mo, Y.·Xin, D.·Xiong, Z.·Zeng, L.·Luo, G.
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Abstract

PurposeTo investigate the effects of {beta}-ecdysterone on fracture healing and the underlying mechanism. MethodsMTT assay was used to detect the cell viability and alkaline phosphatase (ALP) activity was measured using a commercial kit. AO/PI and flow cytometry assays were used to determine the state of apoptosis of osteoblasts. 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 to evaluate the pathological state. 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 expression of RunX2 in fractured osteoblasts were significantly suppressed by 3-methyladenine and elevated by rapamycin, 60, and 80 M {beta}-ecdysterone. The apoptotic state of fractured osteoblasts was enhanced by 3-methyladenine and alleviated by rapamycin, 60, and 80 M {beta}-ecdysterone. The state of autophagy both in fractured osteoblasts and femurs was inhibited by 3-methyladenine and facilitated by rapamycin and {beta}-ecdysterone. Compared to control, Garrett score in 3-methyladenine group was significantly decreased and promoted in rapamycin and {beta}-ecdysterone groups, accompanied by ameliorated pathological state. Lastly, the PI3K/AKT/mTOR pathway both in fractured osteoblasts and femurs was activated by 3-methyladenine and inhibited by rapamycin and {beta}-ecdysterone. Conclusion{beta}-ecdysterone might facilitate fracture healing by activating autophagy through suppressing PI3K/AKT/mTOR signal pathway.

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Tang, Y., Mo, Y., Xin, D., Xiong, Z., Zeng, L., Luo, G.. 2021-05-28. Regulation of osteoblast autophagy based on PI3K/AKT/mTOR signaling pathway study on the effect of β-ecdysterone on fracture healing. https://doi.org/10.1101/2021.05.28.446186

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