内质网应激在高氧导致新生小鼠脑白质损伤中的调控作用

Endoplasmic reticulum stress in hyperoxia-induced white matter damage of newborn mice

目的研究内质网应激（ERS）GRP78-PERK-CHOP通路分子在不同高氧暴露时间下参与高氧所致新生鼠脑白质凋亡的情况。方法新生6日龄C57BL/6小鼠共14窝,每窝8～10只,随机分为高氧组（80%O2）及对照组（21%O2）各7窝。分别于高氧6、12、24、48 h以及高氧48 h后4、7、22天（P12、P15、P30）断头取脑,并与对照组比较。采用RT-PCR和蛋白质印迹法检测高氧后GRP78、PERK、CHOP mRNA和蛋白表达变化,TUNEL法检测小鼠脑白质细胞凋亡情况,透射电镜观察小鼠脑白质髓鞘化改变。结果与对照组相比,高氧6 h后,GRP78 mRNA和蛋白表达增高（P=0.001,0.004）,12、24、48 h差异均无统计学意义（P〉0.05）。高氧6 h后,PERK mRNA和蛋白表达高于对照组（P=0.001,0.001）,余时间点组间比较,差异均无统计学意义（P〉0.05）。高氧12 h后,CHOP mRNA和蛋白表达高于对照组（P=0.011,0.003）,余时间点组间比较差异均无统计学意义（P〉0.05）。与对照组比较,P15高氧组未见成熟的髓鞘结构,P30高氧组髓鞘平均厚度和轴突直径明显降低,出现异常的未折叠的髓鞘和异常的环形结构。P12、P15高氧组凋亡指数明显高于对照组（P=0.001,0.030）。结论高氧暴露可导致新生小鼠脑白质损伤,GRP78-PERK-CHOP通路启动的凋亡途径可能参与其中。

Objective To study the effects of the GRP78-PERK-CHOP signaling pathway which indicated endoplasmic reticulum stress（ ERS） in the hyperoxia-induced white matter damage of newborn mice. Methods Total of 14 groups C57 BL /6 mice were randomly assigned into hyperoxia group（ exposed in 80% oxygen） and control group（ inhaled room air） started on day 6 after birth（ P6）. The pups were sacrificed at 6 h,12 h,24 h and 48 h after exposing to 80% oxygen and on P12,P15 and P30. Transmission electron microscope（ TEM） was used to detect the changes of cerebral white matter myelination. The white matter apoptosis was measured using TUNEL method. RT- PCR and Western Blot were used to detect the expression of mRNA and protein of GRP78,PERK,CHOP. Results The expressions of GRP78 and PERK mRNA and protein after 6 h hyperoxia exposure were significantly higher than the control group（ P〈0. 05）. The expression of CHOP mRNA and protein after 12 h hyperoxia exposure was significantly higher than the control group（ P〈0. 05）. Mice exposed to neonatal hyperoxia exhibited a significantly decrease in mean myelin thickness at P15. In the process of analyzing myelin ultra-structure,an abnormal number of myelin outfoldings and extra loops were observed in mice exposed to neonatal hyperoxia at P30. The apoptosis indexs in hyperoxia group were significantly higher than control group at P12 and P15. Conclusions High oxygen exposure induce white matter damage in newborn mice. The activation of the GRP78-PERK-CHOP signaling pathway initiated apoptosis may be invloved in the process of hyperoxia-induced white matter damage.