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.%目的: 探讨肿瘤坏死因子α(TNF-α)能否诱导小鼠长骨骨样细胞株MLO-Y4发生程序性坏死及其发生机制.方法: 将MLO-Y4细胞分为正常对照(control)组、TNF-α处理组、TNF-α+necrostatin-1 (Nec-1)处理组、TNF-α+Z-VAD处理组和TNF-α+受体相互作用蛋白3(RIP3)-siRNA组.用流式细胞术检测各组细胞凋亡或坏死率,透射电镜鉴定细胞形态学变化,用Western blot法测定RIP1、RIP3和cleaved caspase-3的蛋白水平,应用激光共聚焦显微镜观察RIP1和RIP3蛋白的共表达,应用荧光标记法检测各组细胞活性氧(ROS)水平.结果: TNF-α诱导MLO-Y4细胞24 h,凋亡和坏死率明显高于control组(P<0.01).与TNF-α组相比,Nec-1、Z-VAD和RIP3-siRNA均能降低细胞的凋亡或坏死率(P<0.01).在TNF-α组可见大量坏死样细胞,在Z-VAD组仍可见到坏死样细胞,而在Nec-1和RIP3-siRNA组未见到坏死样细胞.Western blot实验结果显示Nec-1可有效抑制RIP1蛋白表达,而Z-VAD对RIP1和RIP3蛋白表达无影响,RIP3-siRNA可有效降低RIP3蛋白表达(P<0.01).与TNF-α组比较,Nec-1可有效降低RIP1-RIP3蛋白共表达阳性细胞百分率(P<0.01),而Z-VAD对其无影响.与control组相比,TNF-α组的ROS水平明显增高(P<0.01);与TNF-α组相比,Nec-1、Z-VAD及RIP3-siRNA均能有效抑制ROS水平(P<0.01).结论: TNF-α能诱导MLO-Y4细胞发生RIP3介导的程序性坏死;ROS可能是MLO-Y4细胞程序性坏死的执行者.
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