Nrf2对缺氧/复氧诱导的SH-SY5Y细胞线粒体分裂的影响

目的:通过体外模拟脑缺血再灌注损伤的细胞模型,探究核转录相关因子2(nuclear factor erythroid-2 related factor 2,Nrf2)与线粒体分裂是否存在调控关系。方法:通过三气培养箱和无糖培养基模拟氧糖剥夺/复氧(Oxygen and glucose deprivation/reperfusion,OGD/R)的条件,将OGD4 h、6h、8h和10h与R0h、6h、12h、18h、24h多个时间点组合,通过CCK8试剂盒(Cell Counting Kit-8,CCK8)检测细胞的存活率,最终以细胞凋亡明显但仍有半数存活的OGD4 h/恢复R18 h为条件建立细胞OGD/R模型;用Nrf2激动剂叔丁基对苯二酚(Tert-butylhydroquinone,t BHQ)和抑制剂鸦胆子苦醇(Brusatol,Bru)对细胞进行干预处理;蛋白免疫印迹(Western blot,WB)法检测Nrf2、动力相关蛋白1(Dynamin-related protein 1,Drp1)的表达量;制备细胞沉淀的切片,通过电镜观察细胞内线粒体的形态。结果:OGD/R+t BHQ组Nrf2蛋白的表达明显高于OGD/R组,且OGD/R+t BHQ组Drp1蛋白的表达则低于OGD/R组(P0.05),而OGD/R+Bru组Nrf2蛋白的表达低于OGD/R组,且OGD/R+Bru组Drp1蛋白的表达高于OGD/R组(P0.05);电镜观察结果显示OGD/R+t BHQ组线粒体分裂的程度和比例较OGD/R组有所减少,而在OGD/R+Bru组有增多(P0.05)。结论:Nrf2对线粒体分裂有负向调控作用,可能机制是经由Drp1蛋白发挥作用。

Regulation of Nrf2 on the Mitochondrial Division of SH-SY5Y Cell under Ischemia-reperfusion Condition

ABSTRACT Objective: To investigate the relationship between Nrf2 and mitochondrial division by establishing a cell model of cerebral ischemia-reperfusion injury in vitro. Methods: A cell model of oxygen glucose deprivation/recovery was created by a three-gas incubator, sugar-free medium and normal medium. OGD4 h, 6 h, 8 h, and 10 h were matched with R0 h, 6 h, 12 h, 18 h, and 24 h to set up various time combinations. Then cell viability was determined by Cell Counting Kit-8 (CCK8 kit). Finally OGD4 h/R18 hunder which condition cellsshowed obvious apoptosis but still had half of the survival. Tert-butylhydroquinone (tBHQ) and Brusatol (Bru) were chosed as the agonist and inhibitorrespectively to deal with cells. Western blot was applid to measure the expression of proteins Nrf2 and Drp1. Meanwhile the morphology of mitochondria in the same batch of cells was observed by the electron microscopy. Results: Western blot showed that the expression of Nrf2 in OGD/R+tBHQ group was significantly higher than that in OGD/R group, and the expression of Drp1 in OGD/R+tBHQ group was lower than that in OGD/R group (P<0.05). Also the expression of Nrf2 in OGD/R+Bru group was lower than that in OGD/R group, and the expression of Drp1 in OGD/R+Bru group was higher than that in OGD/R group (P<0.05). The results of electron microscopy showed that the degree and proportion of mitochondrial division in the OGD/R+tBHQ group were decreased compared with the OGD/R group, but increased in the OGD/R+Bru group. Conclusion: Nrf2 may negatively regulated the mitochondrial division via Drp1.