Abstract
Background: OP(Osteoporosis) is a common bone metabolic disorder in the elderly characterized by loss of bone mass and a tendency to fracture. The mammalian target of rapamycin (mTOR) pathway in autophagy plays an indispensable role in maintaining the stability of the intracellular environment and ensuring the normal physiological functions of cells. Methods: In this study, different concentrations(20, 40, 60, 80, 100, 120, 140, 160, 180 and 200nM) of rapamycin were used to act on MC3T3-E1 osteoblasts for different time lengths(6, 12, 24, 36 and 48 hours). CCK8 was used to detect the proliferative activity of cells and screen suitable rapamycin concentration for subsequent experiments. Western blot and real-time quantitative PCR were used to detect the expression changes of phosphorylated mTOR, upstream and downstream mTOR pathway, autophagy and osteogenic differentiation markers. The expression of LC3 was observed by immunofluorescence. The differentiation ability of osteoblasts was observed by alizarin red and alkaline phosphatase staining.Results: The results showed that the induction of proliferation activity of osteoblasts from 20 nM to 200 nM presented a parabolic feature. After the action time of 50 μM rapamycin exceeded 12 hours, the proportion of S stage cells was significantly increased. The results of gene and protein analysis showed that rapamycin significantly inhibited the phosphorylation of mTOR, and the phosphorylation of the downstream factors of mTOR, 4E-BP1(eIF4E-binding protein 1) and S6K1(p70 ribosomal S6 kinase 1) also decreased. Rapamycin significantly increased the expression of LC3 II (microtubule associated protein 1 light chain 3-α), significantly increased the ratio of LC3II/LC3I, and significantly decreased the expression of p62(sequestosome-1). Rapamycin significantly induced the expression of ALP(Alkaline phosphatase), Runx2(Runt-related transcription factor 2) and osterix. Conclusions: This study confirmed that rapamycin stimulates the autophagy of osteoblasts by inhibiting mTOR and promotes their proliferation and differentiation, suggesting that mTOR may be a potential therapeutic target for osteoporosis.
Cell-autonomous requirement for mammalian target of rapamycin (Mtor) in spermatogonial proliferation and differentiation in the mouse†
