Research Of Vitamin K₂ On Prevention Of Glucocorticoid Induced ONFH

OBJECTIVE:The current research aimed to study the effect of VK₂ on glucocorticoid treated BMSC,osteoblast and vascular endothelial cell,and to study the effect of VK₂ on glucocorticoid-induce ONFH in vivo.METHODS:The first part:BMSC extracted from SD rats were treated with 5*10^-5methylprednisolone?MP?and MP plus with gradient concentrations of VK₂(10^-7M-10^-5M),and then the proliferation,the viability,the expression of osteogenesis related proteins including Runx2,ALP,OCN and the mineralization of BMSC were detected.The second part:MC3T3-E1 was treated with 10^-5M DEX with or without gradient concentrations of VK₂(10^-7M-10^-5M),then the proliferation,the apoptosis under FBS-free condition,the expression of osteogenesis related proteins including Runx2,ALP,OCN and the mineralization of MC3T3-E1 were detected.The third part:EAhy926 and MG63 were treated with 0^-5M DEX,10^-6M VK₂ and DEX plus 10^-6M VK₂ separately,then the proliferation,apoptosis,in vitro tube formation and the migration of EAhy926 were detected,furthermore,the expression of angiogenesis related proteins in EAhy926 and MG63 was also detected.The fourth part:First of all,we constructed the glucocorticoid-induced ONFH model with MP injection in rats,then the rats were pretreated with VK₂ followed by MP injection,the OCN level in serum,the rate of ONFH,the Runx2,OCN expression in femoral heads and the blood supply of femoral heads in these rats were evaluated.The fifth part:hBMSC were treated with gradient concentrations of VK₂(10^-7M-10^-5M),the expression of miR-133a and osteogenesis related proteins including Runx2,ALP and OCN were detected.Moreover,we constructed hBMSC with miR-133a overexpression with lentivirus transfection method,and cultured them with gradient concentrations of VK₂(10^-7M-10^-5M)to detect the expression of miR-133a and osteogenesis related proteins.RESULTS:The first part:MP significantly inhibited the proliferation,the viability and the expression of osteogenesis related proteins,while VK₂ promoted the proliferation,the viability,the expression of osteogenesis related proteins and the mineralization ability of MP-treated BMSC.The second part:DEX obviously inhibited the proliferation,the expression of osteogenesis related proteins,the mineralization and promoted the apoptosis of MC3T3-E1,while with VK₂cotreatment,the proliferation,the expression of osteogenesis related proteins and the mineralization of MC3T3-E1 were promoted significantly and the apoptosis was inhibited significantly versus DEX treatment solely.The third part:No obvious influence of DEX and VK₂ on the proliferation of EAhy926 was observed in this study,however,DEX significantly inhibited the tube formation and the expression of angiogenesis related proteins,while when cotreated with VK₂,the tube formation ability and expression of angiogenesis related proteins were strongly upregulated.The fourth part:VK₂ strongly enhanced the level of carboxylated OCN and uncarboxylated OCN in serum of rats with MP injection,simultaneously,VK₂ promoted the carboxylation of OCN.Furthermore,VK₂ decreased the incidence of ONFH in MP treated rats and promoted the expression of Runx2,OCN in femoral heads.The blood supply of femoral heads in MP-treated rats was also ameliorated by VK₂.The fifth part:Overexpression of miR-133a obviously inhibited the expression of osteogenesis related proteins of hBMSC,while VK₂ simultaneously downregulated miR-133a expression in hBMSC with and without miR-133a overexpression and promoted their osteogenesis.CONCLUSIONS:VK₂ has the ability to protect BMSC,osteoblast and vascular endothelial cell from damage by glucocorticoids and prevent the incidence of glucocorticoid-induced ONFH.Furthermore,VK₂ may promote osteogenesis of BMSC through the inhibition of miR-133a expression.