The Study Of Wnt1 Regulating Dexamethasone-induced Inhibition Of Osteogenic Differentiation Of Human BMSCs

Purpose:This study aimed to study the effect and mechanism of Wnt1 regulating Dex-induced inhibition of osteogenic differentiation of hBMSCs.Methods:(1)Treat hBMSCs with Dex to construct a glucocorticoid-inhibited osteogenic differentiation cell model.To explore whether Dex can inhibit the transcription of endogenous Wnt1 gene.At the cellular level,the osteogenic differentiation phenotype changes were identified by ALP staining;at the m RNA level,the transcription of Runx2,ALP,Col-1 and Sp7 osteogenic differentiation transcription factors was verified by RT-q PCR;further glucocorticoid receptor antagonism was added.RU-486 was used to investigate the transcription of endogenous Wnt1 gene.At the protein level,Western blot was used to detect the differences in the expression of Runx2 protein.(2)Co-treatment of hBMSCs with exogenous Wnt1 active protein and Dex to investigate whether Wnt1 can rescue the osteogenic differentiation of Dex-suppressed hBMSCs.At the cellular level,cell phenotypic changes are determined by ALP staining;ALP assays identify their activity.At the protein level,Western blot was used to detect the differences in the expression of Runx2 protein.(3)Co-treatment of hBMSCs with rapamycin,exogenous Wnt1 active protein and Dex to explore whether m TORC1 signaling pathway mediates the rescue effect of Wnt1 on Dex-inhibited osteogenic differentiation.At the cellular level,cell phenotypic changes are determined by ALP staining;ALP assays identify the activities.At the protein level,Western blot was used to detect the differences in the expression of Runx2 proteins,S6 and p-S6.Results:High concentrations of glucocorticoids inhibited the osteogenic differentiation of hBMSCs and inhibited the transcription of the endogenous Wnt1 gene in hBMSCs through the glucocorticoid receptor,in a dose-dependent manner.Compared with the osteogenic group,treat hBMSCs with 100 μM Dex,the transcription of Runx2,ALP and Col-1were down-regulated by 54.5%,36.9% and 21.1%,respectively,and the protein expression of Runx 2 was down-regulated by 69.5%.(2)Exogenous Wnt1 protein can partially rescue the inhibition of osteogenic differentiation of hBMSCs inhibited by glucocorticoids.Treat cells with 100ng/m L exogenous Wnt1 protein,the rescue rate of ALP activity is about 76.2%,and Runx2 The rescue rate of protein expression was 79.8%.(3)The mTORC1 signaling pathway mediates the rescue effect of exogenous Wnt1 protein on the osteogenic differentiation of hBMSCs inhibited by glucocorticoids.Compared with the OS+Dex group100ng/m L exogenous Wnt1 protein can increase the p-S6/S6 content by about 43.8%;and at this concentration,exogenous Wnt1 protein can rescue 60.6% of the down-regulated p-S6/S6 amount by Dex;20μM Rapa can partially eliminate 70.9% of Wnt1 protein rescue effect.Conclusion:This study demonstrated that high concentrations of dexamethasone inhibited the osteogenic differentiation of hBMSCs and the transcription of Wnt1 gene.Wnt1 can partially rescue the osteogenic differentiation and activity of hBMSCs inhibited by glucocorticoids,and this rescue process is partially mediated by mTORC1 signaling pathway.