Objectives To investigate the effects of β-ecdysterone on fracture healing and the underlying mechanism. Methods MTT 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. Results The 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. Conclusion β-ecdysterone might facilitate fracture healing by activating autophagy through suppressing PI3K/AKT/mTOR signal pathway.

This study uses the knee Osteoarthritis (OA) rat (constructed by the anterior cruciate ligament transection) and the inflammatory chondrocyte (constructed by 20 ng mL−1 tumor necrosis factor‐α (TNF‐α)) to study the improvement effect of injectable β‐ecdysterone (β‐Ec) hydrogel consisting of poly (D, L‐lactic‐co‐glycolic acid)‐hyaluronate (HA‐β‐Ec‐Gel) on cartilage damage. The results show that the HA‐β‐Ec‐Gel improves the three‐dimensional of bone structure. The HA‐β‐Ec‐Gel treatment improves cartilage tissue injury of OA rats. It advances the levels of autophagic factor Beclin1 (Beclin1) and microtubule–associated protein 1 light chain 3 (LC3) in cartilage tissue. The lysyl oxidase like 3 (LOXL3), ras homologue enriched (Rheb), phosphorylated (p)‐V–akt murine thymoma viral oncogene homolog (AKT)/AKT, and p‐mechanistic target of rapamycin (mTOR)/mTOR signal expression are inhibited by HA‐β‐Ec‐Gel intervention. In inflammatory chondrocytes, β‐Ec improves the cell viability, autophagosome numbers, and Beclin1 and LC3 II/I levels, also inhibits apoptotic rate, LOXL3 and Rheb expression levels, and the AKT/mTOR pathway. The 3‐methyladenine addition reverses these effects of β‐Ec. The Rheb or LOXL3 knockdown chondrocytes aren't sensitive to TNF–α‐induced damage. Their autophagy level is high. This study reveals the improvement effects of injectable HA‐β‐Ec‐Gel on OA, which provides a scientific basis and new direction for the development of OA treatment strategies.

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Objectives: To investigate the effects of β-ecdysterone on fracture healing and the underlying mechanism.Methods: MTT 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.Results: The 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.Conclusion: β-ecdysterone might facilitate fracture healing by activating autophagy through suppressing PI3K/AKT/mTOR signal pathway.