SH-SY5Y细胞氧糖剥夺/恢复模型中Sestrin2基因过表达对线粒体分裂的调控研究

Regulation of Sestrin2 overexpression in mitochondrial fission in glucose and oxygen deprivation/recovery SH-SY5Y cell model

目的探讨人神经母细胞瘤SH-SY5Y细胞氧糖剥夺/恢复(OGD/R)模型中应激诱导蛋白Sestrin2基因过表达对线粒体分裂的调控作用及其机制。方法(1)将SH-SY5Y细胞分为正常对照组、OGD/R组、Sestrin2过表达组及空载体组,后2组细胞预先采用慢病毒感染方法分别构建Sestrin2过表达及空载体稳转细胞株,除正常对照组细胞外均给予氧糖剥夺4 h/恢复18 h造模处理。造模完成后采用细胞计数试剂-8(CCK-8)法检测各组细胞的存活率,采用Western blotting实验检测各组细胞中Sestrin2、线粒体动力相关蛋白1(Drp1)、线粒体分裂蛋白1(Fis1)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胞浆Kelch样环氧氯丙胺相关蛋白1(Keap1)、胞核核因子E2相关因子2(Nrf2)的蛋白水平,采用透射电镜观察各组细胞中线粒体的超微结构,采用免疫荧光染色检测各组细胞中Nrf2的核移位情况。(2)将Sestrin2过表达稳转SH-SY5Y细胞株分为Sestrin2过表达组、鸦胆子苦醇+Sestrin2过表达组、二甲基亚砜(DMSO)+Sestrin2过表达组,后2组细胞在造模前分别给予Keap1/Nrf2通路抑制剂鸦胆子苦醇(终浓度100 nmol/L)或DMSO溶液(终体积分数0.1%)预处理4 h,然后各组细胞均给予氧糖剥夺4 h/恢复18 h造模处理。造模完成后采用Western blotting实验检测各组细胞中胞浆Keap1、胞核Nrf2、Drp1、Fis1的蛋白水平。结果(1)与OGD/R组相比,Sestrin2过表达组细胞的存活率明显升高(61.33%±1.15%vs.81.00%±3.00%),Bcl-2/Bax值明显升高(0.467±0.006 vs.0.880±0.010),Drp1、Fis1、胞浆Keap1蛋白水平明显降低(1.089±0.033 vs.0.865±0.014;0.829±0.009 vs.0.350±0.007;0.967±0.017 vs.0.881±0.024),胞核Nrf2蛋白水平明显升高(0.627±0.025 vs.0.957±0.015),差异均有统计学意义(P<0.05);并且,Sestrin2过表达组细胞的线粒体结构明显较OGD/R组更完整,Nrf2发生了明显的核移位。(2)与Sestrin2过表达组相比,鸦胆子苦醇+Sestrin2过表达组细胞胞核Nrf2的蛋白水平明显降低(0.920±0.013 vs.0.627±0.035),Drp1、Fis1的蛋白水平明显升高(0.994±0.020 vs.1.084±0.005;0.728±0.010 vs.0.906±0.022),差异均有统计学意义(P<0.05)。结论Sestrin2基因过表达可下调胞浆Keap1蛋白水平、促进Nrf2核移位而激活Keap1/Nrf2通路,从而抑制线粒体分裂,减少细胞凋亡,减轻SH-SY5Y细胞的OGD/R损伤。

Objective To investigate the role of Sestrin2 overexpression in regulating mitochondrial fission and its mechanism in human neuroblastoma SH-SY5Y cell model of glucose and oxygen deprivation/recovery(OGD/R).Methods(1)SH-SY5Y cells were divided into normal control group,OGD/R group,Vector group,and Sestrin2 overexpression group;Sestrin2 overexpression or empty vector stable cell lines in the Sestrin2 overexpression group and Vector group were constructed by lentivirus infection;cells in the later 3 groups were subjected to oxygen-glucose deprivation(OGD)for 4 h followed by restoration of O2 supply for 18 h.The cell survival rate was detected by cell counting kit(CCK)-8 assay.The protein levels of Sestrin2,dynamin-related protein 1(Drp1),mitochondrial fission protein 1(Fis1),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),Kelch-like ECH-related protein 1(Keap1)in the cytoplasm and nuclear factor E2-related factor(Nrf2)in the nucleus were detected by Western blotting.The mitochondria ultrastructure was observed by transmission electron microscope.The Nrf2 nuclear translocation was detected by immunofluorescence staining.(2)Cell lines with Sestrin2 overexpression were divided into Sestrin2 overexpression group,Brusatol+Sestrin2 overexpression group,and DMSO+Sestrin2 overexpression group.Cells in the Brusatol+Sestrin2 overexpression group were pretreated with normal medium containing Brusatol(Keap1/Nrf2 pathway inhibitor,final concentration:100 nmol/L)for 4 h before OGD/R;cells in the DMSO+Sestrin2 group were pretreated with normal medium containing DMSO(final volume fraction:0.1%)for 4 h before OGD/R.Cells in these groups were then subjected to OGD for 4 h followed by restoration of O2 supply for 18 h.The protein levels of Drp1,Fis1,Keap1 in the cytoplasm,and Nrf2 in the nucleus were measured by Western blotting.Results(1)As compared with those in the OGD/R group,cells in the Sestrin2 overexpression group had significantly increased survival rate(61.33%±1.15%vs.81.00%±3.00%),significantly up-regulated Bcl-2/Bax ratio(0.467±0.006 vs.0.880±0.010),significantly decreased Drp1,Fis1 and cytoplasmic Keap1 protein levels(1.089±0.033 vs.0.865±0.014;0.829±0.009 vs.0.350±0.007;0.967±0.017 vs.0.881±0.024),and significantly up-regulated nuclear Nrf2 protein level(0.627±0.025 vs.0.957±0.015,P<0.05).The mitochondrial structure in the Sestrin2 overexpression group under electron microscope was more complete than that in the OGD/R group,and obvious nuclear translocation of Nrf2 was noted.(2)As compared with the Sestrin2 overexpression group,Brusatol+Sestrin2 overexpression group had significantly decreased nuclear Nrf2 protein level(0.920±0.013 vs.0.627±0.035),and statistically increased Drp1 and Fis1 protein levels(0.994±0.020 vs.1.084±0.005;0.728±0.010 vs.0.906±0.022,P<0.05).Conclusion Sestrin2 overexpression could suppress mitochondrial fission,reduce cell apoptosis,and attenuate OGD/R injury of SH-SY5Y cells by activating Keap1/Nrf2 pathway via down-regulating cytoplasmic Keap1 protein level and promoting Nrf2 nuclear translocation.