The protective effect of Klotho on apoptosis of MC3T3-E1 osteoblasts induced by dexamethasone / 中国药理学通报

ABSTRACT

Aim To explore the effect of Klotho (KL) gene transfection on the apoptosis of MC3T3-E1 osteo-blasts induced by dexamethasone(DEX). Methods MC3T3-E1 osteoblasts were transfected by recombinant adenovirus containing KL gene(Ad-KL) and recombi-nant adenovirus containing green fluorescent protein (GFP) gene(Ad-GFP). The apoptosis model was con-structed. The transfection efficiency of Ad-KL and Ad-GFP in cells were observed 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 apoptotic rate was evaluated by flow cytom-etry. The level of mRNA and protein was analyzed by qPCR and Western blot, respectively. The level of caspase-9 protein was detected by immunofluorens-cence 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 DEX acting on the cells, the cells viability decreased and apoptotic rate increased obviously in DEX group and GFP + DEX group. The level of Bax mRNA and protein presented a upward trend in DEX group and GFP +DEX group, while the level of Bcl-2 mRNA and protein was oppo-site. But after KL transfecting MC3T3-E1 osteoblasts, the markers described above in KL group had more dramatic improvement than in DEX group and KL +DEX group. Conclusions High-dosage DEX can in-duce the apoptosis of MC3T3-E1 osteoblasts, and the pro-apoptosis effect of high-dosage DEX in MC3T3-E1 osteoblasts can be suppressed by up-regulating KL gene expression level, suggesting that the glucocorticoid-in-duced osteoporosis might be improved by up-regulating KL gene expression level, and it may be a new target for the treatment of latrogenic osteoporosis induced by high-dosage glucocorticoid in clinic.