| ObjectiveThe morbidity of alcohol induced osteonecrosis of the femoral head(ONFH) is increasing. The patients with alcohol induced ONFH are treated with conservative treatment in early stage and hip replacement in advanced stage. However, these two kinds of treatment are unsatisfactory, the former being ineffective and the latter having some complications. The etiopathogenisis and pathogenesy have been defined in recent study with the development of molecular biology. Previous studies show that alcohol may induce marrow stromal cells(MSCs) to differentiate into adipocytes, then systemic changes in metabolism follow, and osteonecrosis comes up eventually. But the study of prevention and treatment about alcohol induced ONFH has rarely been reported. Therefore, the objective of the current experiment is to isolate, obtain and cultur the SD-rat primary MSCs, into which alcohol or simvastatin were added, then to observe the inhibitive effects of simvastatin on MSCs differentiating into adipocytes induced by alcohol and the effects of enhancement of simvastatin on differentiation of MSCs into osteoblasts with use of the methods of cell and molecular biology and to further investigate the protective mechanism and the possible treatment to alcohol induced ONFH. MethodsThe 6 week-old male SD-rats offered by the experimental animal center of Henan province were used in the study. MSCs were obtained from the midshafts of femur bones, then cultured. The primary MSCs were subcultured when they got confluence. The subcultured MSCs were randomly separated into 3 groups: Group A: the cells were treated without alcohol or simvastatin as control. Group B: the cells were treated with 0.09 mol/L alcohol for 7 days and for 10 days respectively. Group C: the cells were treated with 0.09 mol/L alcohol for 7 days and 10 days respectively, then with 1×10-7mol/L simvastatin for 7 days and 11 days respectively. Morphologic features of the cells were monitored with use of a phase-contrast microscope. On the 21st day, the adipocytes induced by the alcohol in treated MSCs were stained with the SuDan III , and then the number of adipocytes(number of adipocytes per visual field , on a microscope with magnification of 100×) were counted under a microscope. The content of triglyceride in MSCs treated for 21 days and alkaline phosphatase(ALP) activity value in MSCs treated for 14 days was determined respectively with use of a biochemical assay. The content of osteocalcin(OC) which the cells secreted for 24h in the media was determined by radioimmunoassay on the 14th day. The total protein in the cell layers was measured with the use of a Coomassie brilliant blue method. The expression of genes that are indicators of adipogenesis (peroxisome proliferator-activated receptor-γ, PPARγ) and osteogenesis[OC,bone morphogenetic protein-2(BMP-2) and core binding factor-a1 (Cbfa1) ]in MSCs treated for 14 days was evaluated using reverse transcription-polymerase chain reaction(RT-PCR) assays.Results1. The number of adipocytes: The number of adipocytes in Group B increased significantly. There was statistical difference between B and the other two groups(P<0.05 ) . There was no statistical difference between Group A and Group C(P>0.05) .2. The content of TG in the MSCs : The content of TG in the cells in Group B was much higher than that in the other two Groups. There was statistical difference between Group B and the other two groups (P<0.05) . There was no statistical difference between Group A and Group C (P>0.05) .3. The content of OC in the media: The content of OC in the media in Group B was much lower than the other two Groups. There was statistical difference between Group B and the other two groups (P<0.05) . There was no statistical difference between Group A and Group C (P>0.05) .4. The ALP activity value in cells: The ALP activity value in cells in Group B was much lower than that in the other two Groups. There was statistical difference between Group B and the other two groups (P<0.05) . There was no statistical difference between Group A and Group C (P>0.05) .5. Analysis of gene expression of PPARγ: The analysis of gene expression of PPARγin Group B was higher than that in the other two Groups. There was statistical difference between Group B and the other two groups (P<0.05) . There was no statistical difference between Group A and Group C (P>0.05) .6. Analysis of gene expression of OC: The analysis of gene expression of OC in Group B was lower than that in the other two Groups. There was statistical difference between Group B and the other two groups (P<0.05) . There was no statistical difference between Group A and Group C (P>0.05) .7. Analysis of gene expression of BMP-2: The analysis of gene expression of BMP-2 in Group B was lower than that in the other two Groups. There was statistical difference between Group B and the other two groups (P<0.05) . There was no statistical difference between Group A and Group C (P>0.05) .8. Analysis of gene expression of Cbfa1: The analysis of gene expression of Cbfa1 in Group B was lower than that in the other two Groups. There was statistical difference between Group B and the other two groups (P<0.05 ) . There was no statistical difference between Group A and Group C (P>0.05) .Conclusions:1. Simvastatin may inhibit differentiation of MSCs into adipocytes induced by alcohol; 2. Simvastatin may decrease the contents of TG in MSCs induced by alcohol;3. Simvastatin may increase the contents of OC in the media of MSCs induced by alcohol;4. Simvastatin may increase the ALP activity values in MSCs induced by alcohol;5. Simvastatin may decrease the gene expression PPARγof MSCs induced by alcohol;6. Simvastatin may increase the gene expression of OC,Cbfa1 and BMP-2 of MSCs induced by alcohol.These findings above indicate that simvastatin may inhibit the differentiation of MSCs into adipocytes induced by alcohol and enhance differentiation of MSCs into osteoblasts. Simvastatin may prevent or treat alcohol induced ONFH. |
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