丙泊酚后处理对小鼠N2a细胞氧糖剥夺/复糖复氧模型内质网IRE1-XBP1信号通路的影响

Influence of propofol postconditioning on endoplasmic reticulum inositol-requiring enzyme 1-X box binding protein 1 signaling pathway in oxygen-glucose deprivation/reperfusion model of mouse neuroblastoma cells

目的探讨丙泊酚后处理对小鼠神经母细胞瘤(N2a)细胞氧糖剥夺/复糖复氧(OGD/R)模型内质网肌醇酶1(IRE1)-X盒连接蛋白1(XBP1)信号通路的影响。方法培养小鼠N2a细胞至对数生长期,采用随机数字表法将细胞平均分为三组:对照组(C组)、OGD/R组(O组)和OGD/R+丙泊酚后处理组(P组)。C组在正常条件下培养,O组氧糖剥夺3 h后在正常条件下培养进行复糖复氧24 h,P组氧糖剥夺3 h后于复糖复氧即刻加入丙泊酚50μmol/L孵育2 h,随后在正常条件下培养22 h。三组均采用流式细胞学技术检测细胞凋亡率,CCK-8法检测细胞存活率,Western blot法检测IRE1、XBP1蛋白含量,qRT-PCR法检测IRE1、XBP1 mRNA表达量,透射电镜法观察细胞内质网形态。结果与C组比较,O组和P组细胞凋亡率明显升高(P<0.05),细胞存活率明显降低(P<0.05),IRE1、XBP1蛋白含量和mRNA表达量明显升高(P<0.05)。与O组比较,P组细胞凋亡率明显降低(P<0.05),细胞存活率明显升高(P<0.05),IRE1、XBP1蛋白含量和mRNA表达量明显降低(P<0.05)。透射电镜结果显示,C组细胞内质网形态正常、排列规则、粗面内质网清晰可见;O组细胞内质网排列不规则、水肿、扩张、断裂、呈囊池状,粗面内质网严重脱颗粒,部分与细胞膜融合;P组细胞内质网排列轻度不规则,部分内质网出现轻度水肿、扩张,粗面内质网脱颗粒现象较O组减轻。结论丙泊酚后处理可以减少小鼠N2a细胞OGD/R后细胞凋亡,提高细胞存活率,其机制可能与抑制内质网IRE1-XBP1信号通路,减轻内质网应激反应有关。

Objective To investigate the influence of propofol postconditioning on endoplasmic reticulum(ER) inositol-requiring enzyme 1(IRE1)-X box binding protein 1(XBP1) signaling pathway in oxygen-glucose deprivation/reperfusion(OGD/R) model of mouse neuroblastoma(N2 a) cells. MethodsMouse N2 a cells were cultured to logarithmic growth stage. The cells were divided into 3 groups using random number table method: sham operation group(group C), OGD/R group(group O) and OGD/R plus propofol postconditioning group(group P). Cells in group C were cultured under normal condition, in the other 2 groups were cultured with oxygen glucose deprivation for 3 hours. Then cells in group O were cultured under normal condition for reperfusion for 24 hours, those in group P were incubated with propofol 50 μmol/L for 2 hours immediately during reperfusion, followed by culturing them under normal condition for 22 hours. The apoptosis rate of cells was detected by flow cytometry, the cell survival rate was detected by cell counting kit(CCK-8), the expression level of IRE1 and XBP1 protein and mRNA were detected with Western blot and qRT-PCR, the transmission electron microscope was used to observe the morphology of endoplasmic reticulum. Results Compared with group C, the apoptosis rate was significantly increased(P < 0.05), the survival rate was significantly decreased(P < 0.05), the expression level of IRE1 and XBP1 protein and mRNA were significantly increased(P < 0.05) of cells in groups O and P. Compared with group O, the apoptosis rate was significantly decreased(P < 0.05), the survival rate was significantly increased(P < 0.05), the expression level of IRE1 and XBP1 protein and mRNA were significantly decreased(P < 0.05) of cells in group P. The result of transmission electron microscope showed that, the endoplasmic reticulum of cells in group C was normal, arranged regularly, and the rough endoplasmic reticulum was clearly visible;the endoplasmic reticulum of cells in group O was irregularly arranged, edematous, dilated, broken, and presenting cystic cistern, the rough endoplasmic reticulum was severely degranulated and partially fused with the cell membrane;the endoplasmic reticulum of cells in group P was slightly irregular, some endoplasmic reticulum showed mild edema and expansion, and the degranulation of the rough endoplasmic reticulum was less severe than that in group O. Conclusion Propofol postconditioning can reduce the apoptosis of mouse N2 a cells after OGD/R and increase the cell survival rate. Its mechanism may be related to inhibiting the ER IRE1-XBP1 signaling pathway and reducing the ER stress response.