过表达脑红蛋白通过激活PI3K／Akt信号通路防治AD的体外研究

摘要

AIM: To study the neuroprotective roles of neuroglobin (NGB) over-expression in the SH-SY5Y cells transfected with pAPPswe.METHODS:The plasmid pEGFP-NGB was successfully constructed and transfected into the SH-SY5Y cells, which were pretreated with pAPPswe.MTT assay was applied to detect the effect of NGB over-expres-sion on the cell survival rates.JC-1 staining was used to detect the level of mitochondrial transmembrane potential.The cell apoptosis was analyzed by flow cytometry.The effects of NGB over-expression on the protein level of p-Akt, Akt and caspase-3/9 were determined by Western blotting.The generation of Aβ42 in the cells was measured by ELISA.RE-SULTS:The cell survival rate was remarkably increased after transfection with NGB compared with control group and emp-ty plasmid group (P<0.05).The over-expression of NGB significantly inhibited the decrease in mitochondrial membrane potential induced by pAPPswe.The over-expression of NGB inhibited the apoptosis of the cells.Furthermore, over-expres-sion of NGB not only inhibited the expression of caspase-3 and caspase-9, but also induced the production of p-Akt, which was prevented by LY294002, an inhibitor of PI3K/Akt.The generation of Aβ42 was inhibited in the cells with the over-ex-pression of NGB.CONCLUSION: Over-expression of NGB significantly inhibits the SH-SY5Y cell injuries induced by pAPPswe and inhibits the expression of caspase-3/9, which is tightly related with cell apoptosis.Furthermore, the neuro-protective roles of NGB may be via activating PI3K/Akt signaling pathway.%目的：探索过表达脑红蛋白（neuroglobin，NGB）对转染了pAPPswe的SH-SY5Y细胞的神经保护作用及机制。方法：成功构建过表达NGB的质粒pEGFP-NGB并转染入已预先转染了pAPPswe的SH-SY5Y细胞， MTT法检测过表达NGB对该细胞存活率的影响；JC-1法检测其对细胞线粒体膜电位的影响；流式细胞术检测过表达NGB对细胞凋亡的影响；Western blotting法检测其对细胞中p-Akt、 Akt和caspase-3／9表达的影响；ELISA法检测其对细胞内Aβ42生成的影响。结果：MTT结果显示，与对照组和空质粒组比较，转染NGB后，pAPPswe-SH-SY5Y细胞的存活率明显提高，差异有统计学意义（P＜0.05）。 JC-1染色结果显示过表达NGB能够明显抑制转染pAPP-swe对SH-SY5Y细胞线粒体膜电位的降低作用（ P＜0.05）。流式细胞术结果显示过表达NGB能够抑制早、晚期细胞的凋亡。而Western blotting显示过表达NGB不仅能抑制细胞内caspase-3和caspase-9蛋白水平的表达，而且还能够促进细胞内p-Akt蛋白的表达，而这种促进作用能够被PI3K／Akt的抑制剂LY294002所抑制。 ELISA结果显示过表达NGB能够明显抑制细胞内Aβ42的生成。结论：过表达NGB能够显著抑制pAPPswe诱导的细胞损伤，而且还抑制与细胞凋亡密切相关的caspase-3和caspase-9等蛋白表达。 NGB的神经保护作用可能是通过激活PI3K／Akt信号通路来实现的。