Conclusions
4EBP1-eIF4E pathway is activated in the RIP3/MLKL mediated-necroptosis. 目的: 程序性坏死是一种由受体相互作用蛋白3(receptor interacting protein 3，RIP3)和混合谱系激酶域样蛋白(mixed lineage kinase domain-like protein，MLKL)介导的细胞死亡形式，有研究指出哺乳动物雷帕霉素靶蛋白通路可能参与程序性坏死的调控，真核细胞翻译起始因子4E结合蛋白1(eukaryotic translation initiation factor 4E-binding protein 1，4EBP1)-真核起始因子4E(eukaryotic initiation factor 4E，eIF4E)通路是哺乳动物雷帕霉素靶蛋白最重要的下游分子通路之一，然而此通路是否参与程序性坏死的发生，目前尚无相关研究。本研究旨在探索4EBP1-eIF4E通路在程序性坏死中是否发生改变。方法: 首先向小鼠上皮样成纤维细胞系L929细胞中加入程序性坏死诱导剂TSZ(TNF-α/SM-164/Z-VAD-FMK)，在光学显微镜下观察细胞坏死情况；进一步向敲除RIP3和MLKL基因的L929细胞中加入TSZ，采用碘化丙啶(propidium iodide，PI)染色观察细胞坏死情况，实时荧光定量聚合酶链反应和蛋白质印迹法检测4EBP1和eIF4E的mRNA和蛋白质表达水平。结果: 野生型L929细胞用TSZ处理后，坏死细胞增加，4EBP1的mRNA和蛋白质表达水平明显下调且磷酸化的4EBP1(phosphorylated 4EBP1，p-4EBP1)/4EBP1值增加(P<0.05或P<0.01)，eIF4E的mRNA表达水平明显上调且磷酸化的eIF4E(phosphorylated eIF4E，p-eIF4E)/eIF4E值增加(均P<0.01)；在敲除L929细胞中的RIP3和MLKL基因后，PI阳性坏死细胞明显减少，4EBP1的mRNA和蛋白质表达水平明显上调且p-4EBP1/4EBP1值下降(P<0.05或P<0.01)，eIF4E的mRNA表达水平明显下调且p-eIF4E/eIF4E值下降(均P<0.01)。结论: 4EBP1-eIF4E通路在RIP3/MLKL介导的程序性坏死中发生活化。.
Methods
TNF-α/SM-164/Z-VAD-FMK (TSZ), a necroptosis inducer, was used to induce necroptosis in murine fibroblastoid cell line L929. Cell necrosis was observed under an optical microscope. Then, TSZ was added to L929 cells with RIP3 and MLKL gene knockout. Propidium iodide (PI) staining was used to observe cell necrosis. Real-time fluorescence quantitative PCR and Western blotting were used to determine the mRNA and protein expression of 4EBP1 and eIF4E, respectively.
Results
After treating L929 cells with TSZ, the number of necrotic cells was increased, the mRNA and protein expression levels of 4EBP1 were significantly downregulated, and the ratio of phosphorylated 4EBP1 (p-4EBP1) to 4EBP1 was increased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly upregulated, and the ratio of phosphorylated eIF4E (p-eIF4E) to eIF4E was increased (both P<0.01). After knocking out RIP3 and MLKL in L929 cells, PI positive necrotic cells were significantly reduced, the mRNA and protein expression levels of 4EBP1 were significantly upregulated, and the ratio of p-4EBP1 to 4EBP1 was decreased (P<0.05 or P<0.01); the mRNA expression level of eIF4E was significantly downregulated, and the ratio of p-eIF4E to eIF4E was decreased (both P<0.01). Conclusions: 4EBP1-eIF4E pathway is activated in the RIP3/MLKL mediated-necroptosis. 目的: 程序性坏死是一种由受体相互作用蛋白3(receptor interacting protein 3，RIP3)和混合谱系激酶域样蛋白(mixed lineage kinase domain-like protein，MLKL)介导的细胞死亡形式，有研究指出哺乳动物雷帕霉素靶蛋白通路可能参与程序性坏死的调控，真核细胞翻译起始因子4E结合蛋白1(eukaryotic translation initiation factor 4E-binding protein 1，4EBP1)-真核起始因子4E(eukaryotic initiation factor 4E，eIF4E)通路是哺乳动物雷帕霉素靶蛋白最重要的下游分子通路之一，然而此通路是否参与程序性坏死的发生，目前尚无相关研究。本研究旨在探索4EBP1-eIF4E通路在程序性坏死中是否发生改变。方法: 首先向小鼠上皮样成纤维细胞系L929细胞中加入程序性坏死诱导剂TSZ(TNF-α/SM-164/Z-VAD-FMK)，在光学显微镜下观察细胞坏死情况；进一步向敲除RIP3和MLKL基因的L929细胞中加入TSZ，采用碘化丙啶(propidium iodide，PI)染色观察细胞坏死情况，实时荧光定量聚合酶链反应和蛋白质印迹法检测4EBP1和eIF4E的mRNA和蛋白质表达水平。结果: 野生型L929细胞用TSZ处理后，坏死细胞增加，4EBP1的mRNA和蛋白质表达水平明显下调且磷酸化的4EBP1(phosphorylated 4EBP1，p-4EBP1)/4EBP1值增加(P<0.05或P<0.01)，eIF4E的mRNA表达水平明显上调且磷酸化的eIF4E(phosphorylated eIF4E，p-eIF4E)/eIF4E值增加(均P<0.01)；在敲除L929细胞中的RIP3和MLKL基因后，PI阳性坏死细胞明显减少，4EBP1的mRNA和蛋白质表达水平明显上调且p-4EBP1/4EBP1值下降(P<0.05或P<0.01)，eIF4E的mRNA表达水平明显下调且p-eIF4E/eIF4E值下降(均P<0.01)。结论: 4EBP1-eIF4E通路在RIP3/MLKL介导的程序性坏死中发生活化。.