The Study Of Inhibition Mechanisms Of Klotho On Apoptosis Of MC3T3-E1 Osteoblasts Induced By Dexamethasone

Aim:To explore the effect of Klotho(KL)gene transfection on the apoptosis of MC3T3-E1 osteoblast induced by dexamethasone(DEX).Methods:MC3T3-E1 osteoblasts were transfected by recombinant adenovirus containing KL gene(Ad-KL)and recombinant adenovirus containing green fluorescent protein(GFP)gene(Ad-GFP).The apoptosis model was constructed.The transfection efficiency of Ad-KL and Ad-GFP in cells were observed by using inverted fluorescent microscope,and the level of KL mRNA and protein was detected by qPCR and Western blot,respectively.The cell viability after different concentrations of DEX acting on the cells and the viability of every research group were determined by cell counting kit-8(CCK-8)assay.The apoptosis rate was evaluated by flow cytometry.The level of mRNA and protein was analyzed by qPCR and Western blot,respectively.The level of caspase-9 protein was detected by immunofluorenscence assay.Results:Cells were transfected by Ad-KL and Ad-GFP successfully.KL group and KL+DEX group had higher level of KL mRNA and protein than that in other groups.The optimum concentration of DEX was 2.0 mmol·L-1.When the DEX acting on the cells,comparing with the control group,the cells viability was decreased and apoptosis rate was increased obviously in DEX group and GFP+DEX group.The level of pro-apoptotic factor Bax mRNA and protein presented a upward trend in DEX group and GFP+DEX group,while the level of anti-apoptotic factor Bcl-2 mRNA and protein was opposite.But after KL transfecting MC3T3-E1 osteoblasts,the cells viability and apoptosis rate in KL group and KL+DEX group had more dramatic improvement than DEX group.The level of Bax mRNA and protein demonstrated significantly decrease,while Bcl-2 mRNA and protein presented obvious increase in KL group and KL+DEX group.Conclusions:This study showed that the high-dosage DEX can induce the apoptosis of MC3T3-El osteoblasts,and the pro-apoptosis effect of high-dosage DEX in MC3T3-E1 osteoblasts could be suppressed by up-regulating the expression level of KL gene.It suggests that the glucocorticoid-induced osteoporosis might be improved by up-regulating the expression level of KL gene,and it could be a new target for the treatment of latrogenic osteoporosis induced by high-dosage glucocorticoid on clinic.