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BACKGROUND: Tumor necrosis factor α (TNF-α) is a pro-inflammatory factor that can induce osteoblast apoptosis and enhance osteoclast function, resulting in inflammatory bone destruction. However, the specific mechanism is unclear. OBJECTIVE: To investigate the effect of TNF-α on the proliferation and apoptosis of MLO-Y4 cells and the possible mechanism. METHODS: MLO-Y4 cells were divided into control group, TNF-α group and ERK1/2 inhibitor group, followed by incubation with α-MEM complete medium containing nothing, 50 μg/L TNF-α, and 50 μmol/L PD98059 for 24 hours, respectively. Cell proliferation was detected by MTT method, and cell apoptosis were detected by flow cytometry. To assess the level of oxidative stress, malondialdehyde, superoxide dismutase, and glutathione peroxidase levels were detected. The protein levels of PCNA, cleaved Caspase-3, p-ERK1/2 and ERK1/2 were measured by western blot. RESULTS AND CONCLUSION: Compared with the control group, treatment with 50 μg/L TNF-α for 24 hours reduced the cell proliferation ability and increased the apoptosis rate increased; levels of lipid peroxidase and malondialdehyde increased significantly, whereas the activities of superoxide dismutase and glutathione peroxidase decreased significantly. Compared with the control group, significantly decreased PCNA and p-ERK1/2 as well as significantly up-regulated cleaved caspase-3 were observed in the TNF-α group; however, the expression of total protein ERK1/2 remained unchanged. There was no significant difference between the ERK1/2 inhibitor group and TNF-α group. To conclude, 50 μg/L TNF-α can decrease the proliferation and increase the apoptosis of MLO-Y4 cells. The mechanism may be related to the inhibition of ERK1/2 signaling pathway.
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AIM: To explore whether tumor necrosis factor-α (TNF-α) induces necroptosis in murine long bone osteocyte-like cell line MLO-Y4 and the possible mechanism.METHODS: The MLO-Y4 cells were divided into control group, TNF-α group, TNF-α+necrostatin-1 (Nec-1) group, TNF-α+Z-VAD group and TNF-α+receptor-interacting protein 3 (RIP3)-siRNA group.The death rate of MLO-Y4 cells was assessed by flow cytometry with Annexin V-FITC/PI staining.The morphological features of the cells were observed under transmission electron microscope (TEM).The protein levels of RIP1, RIP3 and cleaved caspase-3 were determined by Western blot.Finally, the numbers of total cells and RIP1-RIP3-positive cells were observed under laser scanning confocal microscope.The production of reactive oxygen species (ROS) in the cells was measured by DCFH-DA staining.RESULTS: Compared with control group, the apoptotic or necroptotic rate of the cells induced by TNF-α was increased significantly (P<0.01).The increased apoptotic or necroptotic rate was dramatically reduced by treating with Nec-1, Z-VAD or RIP3-siRNA transfection (P<0.01).In TNF-α group and TNF-α+Z-VAD group, a lot of MLO-Y4 cells with typical necroptotic morphological features were observed under TEM.However, obvious necroptotic cells were not found in Nec-1 or RIP3-siRNA treatment group.The protein level of RIP1 in the cells treated with Nec-1 was sharply lower than that in TNF-α group (P<0.01).However, Z-VAD did not reduce the elevated levels of RIP1 and RIP3.RIP3-siRNA effectively down-regulated the protein level of RIP3 compared with TNF-α group (P<0.01).Nec-1 effectively down-regulated the protein levels of RIP1 colocalized with RIP3 compared with TNF-α group (P<0.01).However, Z-VAD did not reduce the levels of RIP1 colocalized with RIP3.Nec-1, Z-VAD and RIP3 siRNA significantly decreased the ROS levels (P<0.01).CONCLUSION: TNF-α induces the necroptosis of MLO-Y4 cells.RIP3 play vital roles in the cell necroptotic signal pathway.ROS may be the executor of necroptosis of MLO-Y4 cells.