The Effect Of Glucocorticoid-induced Oxidative Stress On The Expression Of Cbfa1

Objective To study the effect of glucocorticoid (GC)-induced oxidative stress on the expression of Cbfal, to investigate the intervention effect of N-acetyl cysteine and to explore the molecular mechanisms in glucocorticoid-induced osteoporosis.Methods 1. The calvarial osteoblasts Of the neonatal SD rat were isolated and digested with trypsin and colla genase repeatedly, which were then cultured and observed the morphologic character both in the light and phase-contrast microscopy. Osteoblasts were identified by alkaline phosphatase (ALP) and mineralization nodules staining.2. The second passage osteoblasts which were in the logarithmic phase were cultured under a-MEM to 80% confluence, and growth-arrest in serum-free a-MEM for 24 hours to synchronize the cell growth. After this time the medium was changed to the following groups. The cultured cells were randomly divided into four groups:the normal control group, various concentrations of dexamethasone (0.01-10 umol/L, DEX) groups, N-acetyl cysteine (2 mmol/L, NAC) group, and N-acetyl cysteine plus dexamethasone (2 mmol/L NAC+10 umol/L DEX) group.3. Osteoblasts proliferation ability within 24,48,72 h was detected by MTT colorimetry. After osteoblasts being induced-cultured for 3 d and 7 d, the activity of ALP was assayed. The content of bone gla protein (BGP) in the culture supernatant was determined by ELISA on cells being induced-cultured for 3 d and 14 d. Using Peroxide-sensitive fluorescent probe-DHE analyze intracellular reactive oxygen species (ROS) by flow cytometry within 2,4,8,16,24 h. The content of malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in the cell culture supernatant were detected after cultured 24 h. Collecting osteoblasts and the corresponding supernatant after cultured 8,16,24,48,72 h, the expression of Cbfal was observed using RT-PCR.Results 1. In all groups the OD values of MTT showed an increase trend with the incubation time. Compared with the control group, the cell proliferation of NAC group was no significant change; 0.01 umol/L DEX could promote the proliferation of osteoblasts (P＜0.05, P＜0.01); DEX (0.1,1,10 umol/L) inhibited the proliferation of osteoblasts, OD values were significantly lower (P＜0.01). The OD values of NAC+DEX group compared with the 10 umol/L DEX group were increased (P<0.01).2. Compared with the control group, no significant change being found in NAC group, DEX at the concentration of 0.01 umol/L could promote osteoblasts differentiation, the activity of ALP and the concentration of BGP showing increases (P<0.05, P<0.01), but DEX (0.1,1,10 umol/L) inhibited the differentiation of osteoblasts, the activity of ALP and the content of BGP showing decreases (P＜0.05, P＜0.01). The activity of ALP and the content of BGP showed increases in the NAC+DEX group, compared with the 10 umol/L DEX group (P<0.05, P<0.01).3. Compared with the control group, the level of intracellular ROS and the content of MDA were markedly increased and the ability of T-AOC was reduced in DEX (0.1, 1,10 umol/L) groups (P＜0.05, P<0.01). The level of intracellular ROS and the content of MDA were decreased, but the ability of T-AOC was increased in NAC+DEX group, compared with the DEX (10 umol/L) (P<0.01).4. Compared with the control group, no significant change being found in NAC group, DEX increased the expression of Cbfal mRNA at the concentration of 0.01 umol/L (P＜0.01); but DEX at the concentrations of (0.1,1,10 umol/L) inhibited the expression of Cbfal (P<0.01). The expression of Cbfal mRNA was significantly up-regulated in NAC+DEX group, compared with the DEX (10 umol/L) (P＜0.01).Conclusion DEX (0.1,1,10 umol/L) decreases the expression of Cbfal and inhibits differentiation and function of osteoblasts by inducing oxidative stress. The antioxidant NAC can mitigate the oxidative stress damaging effect of DEX.