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

Abstract

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.

Keywords: AKT; Autophagy; Fracture healing; Rapamycin; mTOR; β-Ecdysterone.

Fig. 1

The cell viability at of osteoblasts treated with different strategies was evaluated by MTT assay

Fig. 2

The osteogenesis differentiation in fractured osteoblasts was facilitated by β-ecdysterone. A The ALP activity in each group was measured by a commercial kit. B The gene expression level of RunX2 was detected by qRT-PCR assay. C The expression level of RunX2 was determined by Western botting assay (*p < 0.05 vs. control, **p < 0.01 vs. control)

Fig. 3

The apoptotic state of fractured osteoblasts was significantly alleviated by β-ecdysterone. A The apoptotic state of treated osteoblasts was visualized by AO/PI staining. B The apoptotic state of treated osteoblasts was quantified by flow cytometry assay

Fig. 4

The autophagy in fractured osteoblasts was activated by β-ecdysterone. A The gene expression level of ATG7 and LC3 was evaluated by qRT-PCR assay. B The protein expression level of ATG7 and LC3 was determined by Western botting assay (*p < 0.05 vs. control, **p < 0.01 vs. control)

Fig. 5

The PI3K/AKT/mTOR signal pathway in fractured osteoblasts was inhibited by β-ecdysterone. The expression level of PI3K, p-AKT1, AKT1, p-mTOR, mTOR, p-p70S6K, and p70S6K was detected by Western botting assay (*p < 0.05 vs. control, **p < 0.01 vs. control)

Fig. 6

The pathological state in fractured femurs was dramatically alleviated by β-ecdysterone. A X-ray assay was performed on fractured femurs isolated from each group. B The pathological state in fractured femurs was visualized by HE staining assay

Fig. 7

The autophagy in fractured femurs was activated by β-ecdysterone. A The expression level of Beclin-1 in the fractured femurs was detected by immunohistochemical assay. B The expression level of LC3 in the fractured femurs was determined by immunofluorescence assay. C Western blotting assay was utilized to measure the expression level of ATG7 and LC3-II/LC3-I in the fractured femurs (*p < 0.05 vs. control, **p < 0.01 vs. control)

Fig. 8

The PI3K/AKT/mTOR signal pathway in fractured femurs was inhibited by β-ecdysterone. The expression level of PI3K, p-AKT1, AKT1, p-mTOR, mTOR, p-p70S6K, and p70S6K was detected by Western botting assay (*p < 0.05 vs. control, **p < 0.01 vs. control)