TNF-α induces PIP3-mediated necroptosis in MLO-Y4 cells / 中国病理生理杂志

ABSTRACT

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.