Combined Effects Of Tumor Necrosis Factor-alpha And Cyclic Stretch On The Osteogenic Differentiation In MC3T3-E1 Cells

Objective:The aim of this study was to investigate the combined effects of cyclic stretch and tumor necrosis factor-alpha(TNF-α)on osteogenic differentiation of MC3T3-E1 cells and the role of the nuclear factor-kappa B(NF-κB)signaling pathway in this process.Methods:MC3T3-E1 cells were treated with 0.5 or 10 ng/m L TNF-α and cyclically stretched using the Flexcell tension system 4000 with 12 % elongation for 12 h.Furthermore,to explore which cytokines might be regulated by the NF-κB signaling pathway in osteogenic differentiation,the cells were pre-treated with NF-κB pathway inhibitor,pyrrolidine dithiocarbamate(PDTC),and then cyclically stretched for 12 h in the presence of 10 ng/m L of TNF-α.Western blot and RT-PCR were used to detect the expression of osteocalcin(OPG),type I collagen(COL1),alkaline phosphatase(ALP),runt-related transcription factor2(Runx2),osteocalcin(OCN),receptor activator of nuclear factor-kappa B ligand(RANKL),NF-κB and phosphorylated NF-κB(p-NF-κB)at protein and gene levels.Results:Cyclic stretch alone increased the expression of OPG,COL1,Runx2,ALP,and OCN,decreasing the expression of RANKL and the RANKL/OPG ratio.TNF-α alone reduced the expression of OPG,COL1,Runx2,ALP,and OCN while increased RANKL and RANKL/OPG ratio.The upregulation or downregulation induced by cyclic stretch were restrained in the presence of TNF-α.The p-NF-κB/NF-κB ratio increased at any stimulation.Inhibition of NF-κB signaling pathway restrained the expression variations of COL1,OCN,ALP,and OPG induced by TNF-α combined with cyclic stretch.Conclusion:The results indicated that TNF-α inhibited osteogenic differentiation of MC3T3-E1 cells,while cyclic stretch promoted the osteogenic differentiation.In addition,TNF-αinhibited the osteogenic differentiation of MC3T3-E1 cells induced by cyclic stretch and NF-κB signaling pathway may participate in this process by regulating the expression of OPG,COL1,ALP and OCN.