Autophagy May Relieve The Inhibitory Effects Of High Glucocorticoid On Osteoblast Function

Objective:Glucocorticoid(GCs) excess decreases bone mass and strength in part by acting directly on osteoblasts and osteocytes. Because glucocorticoids have been shown to induce both osteocyte apoptosis and autophagy, we sought to determine whether osteoblast cell fate and function was influenced by autophagy in the presence of GCs by performing in vitro studies.Methods:MC3T3-E1 cells were treated with various concentration dexamethasone in α-modified essential medium at 10-8mol/L,10-6mol/L and 10-4mol/L for 24h,48h,72h and 96h. LC3 and Beclinl were measured in each group by Western blotting and GFP-LC3 punctate staining were examined by fluorescence microscope to evaluate if GCs could induce autophagy in cells and whether the autophagic process is dose dependently changed or time dependently varied. At last, flow cytometry and Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to identify the apoptosis and expression level of Alp Collal and Ocn after treated with autophagy inhibitor 3-MA and autophagy inducer rapamycin.Results:1. LC3-Ⅱ was induced in Western blot and autophagosome were formed under fluorescence microscope.2. Western blot and GFP-LC3 punctate staining showed autophagic level in a dose-dependent manner treated with Dex.3. The autophagic level in MC3T3-E1 reached the top at 48h and then descended.4. Dex induced apoptosis of MC3T3-E1 cells in a dose-dependent manner and apoptosis increased if autophagy was inhibited by 3-MA.5. The expression level of Alp Collal and Ocn gene were inhibited by Dex in a in a dose-dependent manner. Autophagy inhibitor 3-MA aggravated the inhibition and autophagy inducer rapamycin could reverse it.Conclusion:Autophagy plays a significant role in the GCs-induced bone loss, and it can reverse the apoptosis and inhibition of GCs on MC3T3-E1 cells.