Prdx1基因敲减促进氧糖剥夺/复氧诱导的小鼠神经母细胞瘤N2a细胞凋亡

Knockdown of Prdx1 gene promotes apoptosis of mouse neuroblastoma N2a cells induced by oxygen-glucose deprivation/reoxygenation

目的:观察过氧化还原蛋白1(Prdx1)基因敲减对氧糖剥夺/复氧(OGD/R)诱导的小鼠神经母细胞瘤N2a细胞凋亡的作用,并进一步探讨其可能机制。方法:体外培养小鼠神经母细胞瘤N2a细胞,构建OGD/R诱导的N2a细胞损伤模型。将对数生长期的N2a细胞随机分为正常对照组、OGD/R组、OGD/R+c-Jun氨基末端激酶(JNK)抑制剂(SP600125)组、OGD/R+阴性对照序列(siNC)组、OGD/R+siPrdx1组和OGD/R+siPrdx1+SP600125组。用siRNA转染细胞;CCK-8比色法检测细胞活力;TUNEL染色法检测细胞凋亡;RT-qPCR检测Prdx1 mRNA的表达;Westernblot检测Prdx1、JNK、磷酸化JNK(p-JNK)及凋亡执行蛋白cleavedcaspase-3的蛋白水平。结果:体外培养的小鼠神经母细胞瘤N2a细胞经OGD/R处理后出现明显损伤,表现为细胞活力显著降低(P<0.05),细胞凋亡率显著增高(P<0.05);此外,Prdx1蛋白表达水平亦增高(P<0.05)。Prdx1基因敲减显著促进了OGD/R诱导的JNK和caspase-3激活(P<0.05),细胞活力显著降低(P<0.05),同时显著加剧细胞凋亡(P<0.05)。SP600125可显著抑制OGD/R诱导的JNK和caspase-3激活(P<0.05),减少细胞凋亡(P<0.05),逆转OGD/R条件下Prdx1基因敲减对N2a细胞的损伤作用。结论:Prdx1基因敲减促进OGD/R诱导的小鼠神经母细胞瘤N2a细胞凋亡,其机制与进一步激活JNK/caspase-3信号通路有关。

AIM:To investigate the effect of peroxiredoxin 1(Prdx1)gene knockdown on the apoptosis of mouse neuroblastoma N2a cells induced by oxygen-glucose deprivation/reoxygenation(OGD/R)in vitro,and to further explore the mechanism.METHODS:Mouse neuroblastoma N2a cells were cultured in vitro,and OGD/R model was established to simulate cerebral ischemia-reperfusion injury.The N2a cells were randomly divided into normal control group,OGD/R group,OGD/R+c-Jun N-terminal kinase(JNK)inhibitor(SP600125)group,OGD/R+siNC group,OGD/R+siPrdx1 group and OGD/R+siPrdx1+SP600125 group.The cell viability was measured by CCK-8 assay.Apoptosis was detected by TUNEL staining.The mRNA expression of Prdx1 was detected by RT-qPCR.The protein levels of Prdx1,JNK,phosphorylated JNK(p-JNK)and cleaved caspase-3 were determined by Western blot.RESULTS:After OGD/R treatment,N2a cells showed significant damage,their viability was decreased significantly(P<0.05),and the apoptotic rate and the expression level of Prdx1 were increased significantly(P<0.05).Knockdown of Prdx1 gene significantly promoted the activation of JNK and caspase-3 induced by OGD/R(P<0.05),further decreased the cell viability(P<0.05),and significantly aggravated the apoptosis(P<0.05).SP600125 significantly inhibited the activation of JNK and caspase-3 induced by OGD/R(P<0.05),reduced the apoptosis(P<0.05),and reversed the damage by Prdx1 gene knockdown under OGD/R condition.CONCLUSION:Knockdown of Prdx1 gene promotes OGD/R-induced apoptosis of mouse neuroblastoma N2a cells in vitro,which may be related to further activation of JNK/caspase-3 signaling pathway.