Effects Of Statins On TNF-a-induced Growth Inhibition And Apoptosis In MC3T3-E1 Osteoblastic Cells

[AIM]Tumor necrosis factor-alpha(TNF-α) plays a key role in the regulation of bone metabolism.It is believed that an increase in the TNF-αlevel and a decrease in the number of osteoblasts might be responsible for bone loss in vivo.Statins stimulate in vivo bone formation in rodents and increase new bone volume in cultures from mouse calvaria.Statins stimulate the expression of bone anabolic factors such as vascular endothelial growth factor(VEGF) and bone morphogenetic protein-2(BMP-2). Up to now,no experiments were performed to determine if simvastatin could attenuate TNF-α-induced apoptosis in osteoblastic cells.This study further examined the cellular mechanisms how statins inhibit TNF-a-mediated apoptosis in osteoblastic MC3T3-E1 cells.[METHODS]Murine osteoblastic MC3T3-E1 cells were cultured in DMEM essential medium supplemented with 10%fetal bovine serum and antibiotics. 3-(4,5-Dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide(MTT) was used to evaluate cell viability.Hoechst staining of cells treated with TNF-a showed typical pyknotic fragmented nuclei and apoptotic bodies,cells were examined under a microscope equipped for epifluorescence illumination.Apoptosis-mediated death of MC3T3-E1 cells was examined by Flow cytometric analyses after Annexin V/7-AAD double staining.[RESULTS]1.MC3T3-E1 cells were exposed to various concentrations(10-100 ng/ml) of TNF-a decreased the viability in time and dose-dependent manners,such that a 72-h incubation of the cells with 100 ng/ml TNF-a resulted in 36%viability compared with that of the untreated cells.Therefore,the dose(100 ng/ml) and time(72 h) conditions of the TNF-a treatment were used as a model condition to determine the effects of quercetin on TNF-a-induced growth inhibition and cytotoxicity.2.MC3T3-E1 cells were exposed to various concentrations(10^-10-10^-7 mol/L) of statins,such as simvastatin,rosuvastatin,fluvastatin and atorvastatin increase growth and viability.In contrast,high concentrations of statins(10^-5-10^-3 mol/L) decrease growth and viability of MC3T3-E1 cells,and showed a significant cytotoxicity.3.MC3T3-E1 cells treated with statins in the presence of TNF-αshowed a markedly increased level of growth and viability that of untreated control cells.TNF-α-induced decrease in cell viability were inhibited by the statins treatment in a dose-dependent manner.4.Experiments were performed to determine if simvastatin could attenuate TNF-α-induced apoptosis in murine osteoblasts.DNA fragmentation was assessed by Hoechst staining method.MC3T3-E1 cells treated with TNF-αshowed typical pyknotic fragmented nuclei and apoptotic bodies.TNF-α-induced increase of apoptosis was attenuated by the addition of simvastatin treatment in a dose-dependent manner.5.Annexin V/7-AAD double staining was carried out in order to determine if TNF-α-induced cell death and its inhibition by simvastatin in MC3T3-E1 cells were mediated by apoptosis or necrosis.When MC3T3-E1 cells were treated with 10 ng/ml TNF-α,the proportion of apoptotic cells was measured to be 29.2%of total cell population.The apoptotic cell populations were decreased by simvastatin treatment in a dose-dependent manner such that when the cells were treated with simvastatin (10^-10-10^-7mol/L) in the presence of 10 ng/ml TNF-α,approximately 31.34%,48.5%, 52.11%and 65.73%of inhibitory rate.[CONCLUSION]Our results demonstrate that statins can increase growth and viability of osteoblastic cells and decrease TNF-a induced growth inhibition and apoptosis in murine osteoblastic MC3T3-E1 cells.