PERK通路对大鼠脑出血继发性脑损伤及神经细胞凋亡的作用

PERK pathway on secondary brain injury and neuronal cell apoptosis after cerebral hemorrhage in rats

目的:探讨PERK通路对大鼠脑出血继发性脑损伤及神经细胞凋亡的作用。方法:将48只大鼠随机分为6组,构建脑出血(ICH)模型。取其中1组作为对照组ICHsham,其余5组按时间进程编为ICH6h、ICH12h、ICH24h、ICH48h、ICH72h。将48只大鼠随机分为6组,取其原代海马神经元,在体外使用氧血红蛋白(OxyHb)处理。取其中1组作为对照组control,其余5组按时间进程编为OxyHb6h、OxyHb12h、OxyHb24h、OxyHb48h、OxyHb72h。将36只大鼠随机分为4组,按处理方式编为sham、ICH、GSK2606414、salubrinal。采用免疫印迹、免疫荧光和TUNEL染色等方法,观察上述各组大鼠PERK通路相关蛋白的表达及神经元细胞的凋亡情况。结果:①ICH模型组的p-eIF2α和ATF-4表达水平较ICHsham组显著性升高,并在ICH 48 h达到峰值,其差异具有统计学意义(P<0.05);ICH模型组的ATF-6和XBP-1表达增加较ICHsham组亦有显著性差异。②OxyHb处理组的p-eIF2α和ATF-4表达水平显著性升高,并在OxyHb处理48 h达到峰值;OxyHb处理组的ATF-6和XBP-1表达增加较control组亦有显著性差异。③与ICH模型组相比,GSK2606414组的p-eIF2α和ATF-4蛋白表达减少;与GSK2606414组相比,Salubrinal组的p-eIF2α和ATF-4蛋白含量增加。表明GSK2606414处理可以显著逆转ICH诱导的CHOP和cleaved caspase-12蛋白水平的升高。与sham组相比,ICH后TUNEL和ATF-4双阳性细胞数量增加,但GSK2606414处理可以消除了这种效应。④与OxyHb处理相比,GSK2606414组的p-eIF2α和ATF-4的蛋白质含量明显下降,salubrinal组的p-eIF2α和ATF-4的蛋白质含量明显增加,GSK2606414组的CHOP和cleaved-caspase-12蛋白水平均显著降低,salubrinal组的CHOP和cleaved-caspase-12蛋白水平均显著升高;TUNEL染色结果表明,GSK2606414组原代神经元凋亡数量较OxyHb组显著性减少,Salubrinal组原代神经元凋亡数量显著性增加(P<0.05)。结论:PERK信号通路在ICH诱导的细胞凋亡中发挥重要作用,阻断其激活,具有神经保护作用,减轻继发性脑损伤(SBI),提示靶向该通路可能是改善ICH患者预后的一种有前途的治疗策略。

Objective:To investigate the effect of PERK pathway on secondary brain injury and neuronal apoptosis after cerebral hemorrhage in rats.Methods:48 rats were randomly divided into 6 groups to construct ICH model.One group was used as the control group as ICHsham,and five groups were labeled as ICH6 h,ICH12 h,ICH24 h,ICH48 h and ICH72 h by time course.Other 48 rats were randomly divided into 6 groups,and their primary hippocampal neurons were treated with oxyhemoglobin(OxyHb)in vitro.One group was used as the control group,and five groups were labeled as OxyHb6 h,OxyHb12 h,OxyHb24 h,OxyHb48 h and OxyHb72 h by time course.In addition,36 rats were randomly divided into 4 groups,and labeled as sham,ICH,GSK2606414 and salubrinal according the processing method.Western blot,immunofluorescence and TUNEL staining were used to observe the expression of PERK pathwayrelated proteins and the apoptosis of neurons in the above experimental groups.Results:①The expression levels of PeIF2αand ATF-4 of ICH model groups were significantly higher than those of ICHsham group,and reached the peak at48 h after ICH,the difference was statistically significant(P<0.05);The expression of ATF-6 and XBP-1 of ICH model groups increased significantly compared with ICHsham group.②The expression levels of P-eIF2αand ATF-4 of OxyHb treatment groups were significantly higher than those of control group,and reached the peak at 48 h;The expression of ATF-6 and XBP-1 of OxyHb treatment groups also increased significantly compared with the control group.③Compared with ICH group,the expression of p-eIF2αand ATF-4 was decreased in GSK2606414 group.Co mpared with the GSK2606414 group,the protein content of p-eIF2αand atf-4 was increased in Salubrinal group.In addition,the increased levels of CHOP and cleaved caspase-12 protein induced by ICH were significantly reversed by GSK2606414 treatment.Compared with sham group,the number of TUNEL and ATF-4 double positive cells increased after ICH treatment,but this effect could be eliminated by GSK2606414 treatment;④Compared with OxyHb treatment,the protein contents of p-eIF2αand ATF-4 in GSK2606414 treatment group were significantly decreased,the protein contents of p-eIF2αand ATF-4 in Salubrinal treatment group were significantly increased,the levels of CHOP and cleaved caspase-12 protein in GSK2606414 treatment group were significantly lower,the levels of CHOP and cleaved caspase-12 protein in Salubrinal treatment group were significantly higher.Tunel staining results showed that compared with OxyHb treatment,the number of primary neurons in GSK2606414 treatment group decreased,while the number of primary neurons in salubrinal treatment group increased.Conclusion:PERK signaling pathway plays an important role in ICH induced apoptosis,which blocks its activation and has neuroprotective effect to reduce secondary brain injury(SBI),suggesting that targeting this pathway may be a promising therapeutic strategy to improve the prognosis of ICH patients.