Astragaloside Ⅳ Prevents Glucocorticoid-induced Osteonecrosis Of The Femoral Head Via Akt Pathway

Purpose:This study intends to investigate whether V-type proton ATPase subunit H(ATP6V1H)can promote osteogenic differentiation by acting on the Akt pathway,and the relationship between astragaloside IV(AS-IV)and Akt pathway,and to explore the feasibility and mechanism of AS-IV for preventing glucocorticoid-induced osteonecrosis of the femoral head.Methods:Models of glucocorticoid-induced osteonecrosis of the femoral head with AS-IV intervention were established,and the degree of angiogenesis and bone loss was evaluated by histological staining,micro-CT,and three-dimensional microangiography.Western blot,immunohistochemistry and immunofluorescence staining were used to evaluate the expression of osteogenesis,angiogenesis,apoptosis,oxidative stress-related proteins and Akt pathway proteins.In vitro,the effects of dexamethasone and AS-IV on HUVECs and BMSCs were studied by CCK-8,transwell assay,cell scratch assay,tube formation assay and ALP staining.After blocking the PI3K/Akt signaling pathway with LY294002,the role of AS-IV was evaluated again in vivo and in vitro.To explore the effect of ATP6V1H on Akt/GSK3β pathway and osteogenic differentiation,OM(osteogenic medium)and HG(high glucose and free fatty acids)were used to induce the MC3T3-E1 cells into osteogenic differentiation;CCK-8 and alizarin red staining were used to detect cell function.After ATP6V1H overexpression and knockout,we detected protein levels such as Akt pathway by qPCR and Western blot.Results:Western blot,immunofluorescence,and immunohistochemical staining confirmed that AS-IV can simultaneously activate HIF-1α/VEGF pathway,Bad/Bcl-2 pathway,and Nrf2/HO-1 pathway through Akt,preventing bone loss and increasing blood vessel density.After blocking the PI3K/Akt pathway with LY294002,the effect of AS-IV was inhibited.In vitro,CCK-8,transwell assay,cell scratch assay,and tube formation assay confirmed that AS-IV can promote HUVECs proliferation,migration,and tube formation ability.CCK-8 and ALP staining confirmed that AS-IV can promote the proliferation and osteogenic differentiation of BMSCs.ATP6V1H expression decreased in OM+HG-MC3T3-E1 cells.Overexpression of ATP6V1H inhibited Akt/GSK3β signaling pathway and promoted osteogenic differentiation.Conclusion:AS-IV can activate the PI3K/Akt pathway which can simultaneously act on the HIF-1α/VEGF pathway,the Bad/Bcl-2 pathway,and the Nrf2/HO-1 pathway.It can regulate vascular / apoptotic / oxidative stress coupling to promote bone and angiogenesis,inhibit apoptosis and oxidative stress,and thus prevents glucocorticoid-induced ONFH in rats.ATP6V1H can facilitate osteogenic differentiation in MC3T3-E1 cells via Akt/GSK3β signaling pathway.