Effects And Mechanisms Of FoxO1/β-catenin Signal Pathway In Osteoblast Oxidative Stress

Objective Constructing FoxO1-siRNA to silence the expression of FoxO1in MC3T3-E1in vitro. Using MC3T3-E1cells interfered by H2O2to construct a model for osteoblast oxidative stress, and investigate the effects and mechanisms of FoxO1/β-catenin signal pathway in osteoblast oxidative stress.Methods1. Build3groups of FoxO1-siRNA, transfected MC3T3-E1cells, using thiazole blue method (MTT) to detect cell proliferation after culturing for12h,24h,48h and72h, and choose the best proliferation period for further experiments. Using RT-PCR and Western-blot method to detect FoxO1mRNA and protein expression respectively, screening the best silence effected siRNA for further experiments.2. Inoculated cells in good growth condition into6hole plate, and incubated it at37℃, in a humidified atmosphere of5%CO2. Waiting for the cells grow to about80%, and then divided them into normal control group (group NG), FoxO1-siRNA transfection group (transfected by FoxO1-siRNA, group F), H2O2interfering group (0.3mmol/L H2O2interfered for4hours, group.H), Scrambld-siRNA transfection and H2O2interfering group (transfected by Scrambld-siRNA,0.3mmol/L H2O2interfered for4hours, group S+H), FoxO1-siRNA transfection and H2O2interfering group (transfected by FoxO1-siRNA,0.3mmol/L H2O2interfered for4hours, group F+H). To all those groups, after culturing for12h,24h,48h,72h, interfered it by0.3mmol/L H2O2for4hours according to its group requirement respectively, and detected its cells proliferation by MTT colorimetry, then selecting the best growth period for further experiments. Then, after each group cultured for48hours, detected its ALP, SOD, MDA levels; cell apoptosis was detected by flow cytometry rate; collected the total RNA of each group, using RT-PCR method to detect the expression of osteoblast marker gene BMP2, Runx2, OPG mRNA, and the FoxO1/β-catenin signaling pathway downstream genes β-catenin, Gadd45, Bim, PPAR-ymRNA.Results1. The cell proliferation ability of MC3T3-E1for each group had the same trend along with time, which reached a maximum value at48h (P＜0.05), and decreased at72h (P＜0.05); the cell proliferation ability of each group transfected by FoxO1-siRNA was lower than the group NG (P＜0.05), specially, the group si-FoxO-2cell proliferation activity decreased most obviously at48hour (P＜0.05); RT-PCR results displayed that the FoxO1mRNA was silenced most obviously in the group si-FoxO-2(P＜0.05), which reached79%; at the same time, Western-blot assay showed that si-FoxO-2group of FoxO1protein expression was the lowest,(P＜0.05). Si-FoxO-2on MC3T3-E1cells with FoxO1gene silencing has the best effect.2.①The cell proliferation ability for each group had the same trend along with time, the proliferation ability of each interfered group was lower than that in group NG, among them, the decreasing for group F+H was significant at48hours (P＜0.05);②ALP and SOD level in every interfered group was lower then that in group NG, respectively (P＜0.05), in which group F+H was lower then that in group F and group H (P＜0.05); MDA level in every interfered group was higher then that in group NG respectively (P＜0.05), in which group F+H was higher then that in group F and group H (P＜0.05);③The apoptosis rate of osteoblast in each interfered group was higher then that in group NG, in which group F+H was the highest(P＜0.05).④The expression of FoxO1mRNA in group H was significantly higher than that in group NG (P＜0.05);⑤The expression of BMP2, Runx2, OPGmRNA in each interfered group was lower than that in group NG (P＜0.05), and the expression for group F and group H were close (P＞0.05), group F+H was lower than that in group H and group F (P＜0.05).⑥Expression of (3-catenin, Gadd45mRNA in group F was lower than group NG (P>0.05), group H was higher than group NG (P＜0.05), group F+H was lower than that in group H and group F (P＜0.05); compared with group NG, the expression of Bim, PPAR-ymRNA in every interfered group was increased, respectively, the expression of group F+H was higher than that in group F (P＜0.05).Conclusion1. It can be implemented by siRNA interference to silence the expression of FoxO1in MC3T3-E1cells in vitro.2. The effects of FoxO1/β-catenin signaling pathway in proliferation and differentiation of osteoblasts may achieved through its regulation on the balance of oxidation antioxidation.3. The effects of FoxO1/β-catenin signal pathway in osteoblast oxidative stress may ralected to up regulation the level of antioxidant enzymes and the expression of NDA damage repair related genes Gadd45, and inhibit the expression of apoptosis related gene Bim and osteoblast inhibition gene PPAR-y.