Roles Of FoxO1 In Osteoclast Differentiation Of RAW264.7 Cells

Objective: Research the role of FoxO1 in osteoclast differentiation of RAW264.7 CellsMethod: 1.Three pieces of FoxO1-siRNA were established,then used to silenced FoxO1 gene in RAW264.7 cells,according RNA interference effece.The best piece of FoxO1-siRNA was picked out for the later experiment.2.The RAW264.7 cells were divided into the following groups randomly: control group,S group（FoxO1-siRNA transfection）group,H group（100μmol/l H₂O₂）,N group（si-Scrambled transfection）,However,it should be noted that in all H groups,flask was added 100μmol/l H₂O₂ for an hour and the medium inside was changed into RANKL induced medium later.TRAP staining was used to authenticate osteoclast,when all groups were induced culture for 5d.3.The mRNA expression level of osteoclast marker genes（TRAP,BMMP-9,CathepsinK）were detected by RT-PCR assays.4.Apoptosis was detected by a flow cytometry.The intracellular ROS was detectedby a flow cytometry,and the MDA,SOD and GSH-PXwere detected by the detection kits.Results: 1.Selection of FoxO1-siRNA: RT-PCR results showed that the FoxO1 mRNA expression level were reduced in all FoxO1-siRNAtransfection groups（P<0.05）.The most obviously silenced group was FoxO1-siRNA-2,which reached 78%（P<0.05）.At the same time,Western-blot assay showed that FoxO1 protein expression level was lowest（P<0.05）too.The FoxO1-siRNA-2 was selected for FoxO1 gene silencing of RAW264.7 cells.2.TRAP staining and the RT-PCR results showed: RAW264.7 cells could be induced osteoclast differentiation successfully by 100ng/mlRANKL.Some cells can be found 3-5 nucleus,and the cytoplasm stained into wine red,when observed under inverted microscope.3.All RANKL induced groups had higher mRNA expression level ofTRAP,CathepsinK,BMMP-9（P<0.05）.The mRNA expression level of osteoclast marker genes was promoted by FoxO1 genesilencing,as well as H₂O₂.It was worth noting that H₂O₂ had more obvious effect in osteoclast differentiation than FoxO1.3.Apoptosis results showed: FoxO1 genesilencing and H₂O₂ could promote apoptosis,and the FoxO1 genesilenced had a stronger effect in apoptosis（P<0.05）.4.Oxidative stress: ROS and MDA were elevated in FoxO1 genesilencing group and H₂O₂ group（P<0.05）,at the same time,SOD and GSH-PX were reduced in these groups.（P<0.05）.Conclusion: FoxO1 has inhibiting effect in apoptosis and osteoclast differentiation.The effect of FoxO1 in osteoclast differentiation is related to regulate the level of antioxidant enzymes and inhibited apoptosis.