亚低温对新生大鼠缺氧缺血性脑损伤p-ERK/p38 MAPK的影响

Effects of moderate hypothermia on p-ERK/p38 MAPK signal pathway following hypoxic-ischemic brain injury in neonatal rats

目的探讨丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPK)信号转导通路p-ERK/p38部分活化在亚低温治疗新生大鼠缺氧缺血性脑损伤(hypoxic-ischemic braindamage,HIBD)中的作用及意义。方法实验分模型组和对照组。采用7日龄Sprague-Dawley(SD)清洁级大鼠,建立新生大鼠HIBD标准模型,随机分为常温缺氧缺血组(10只、肛温=37℃)和亚低温缺氧缺血组(10只、肛温=33℃);对照组为假手术动物,也分为常温组和亚低温组(各8只,余同上)。取背侧海马冠状平面采用脱氧核糖核苷酸末端转移酶介导的原位缺口末端标记(TUNEL)结合苏木素-伊红(HE)、神经元Nissl染色和电镜观察凋亡小体检测脑细胞凋亡情况。采用Western印迹检测MAPK成分磷酸化p42/44ERK(简称p-ERK1/2)、磷酸化p-38MAPK(简称p-p38)的变化时程及其意义。结果72h亚低温缺氧缺血组脑细胞凋亡发生率(6·4±1·7)%,与常温缺氧缺血组(25·3±1·5)%比较P<0·01,差异有统计学意义。而细胞坏死发生率无明显改善。HIBD后MAPK信号转导系统活化,p-ERK1/2水平在IN组结扎侧大脑组织逐渐降低,而p-p38水平却逐渐升高,二者作用相拮抗。亚低温治疗可逆转二者的反向变化。结论亚低温治疗可通过p-ERK/p38MAPK信号转导通路抑制HIBD后的细胞凋亡。

Objective To investigate the effects of moderate hypothermia management on p-ERK/p38 MAPK signal pathway following hypoxic-ischemic brain damage （HIBD） in neonatal rats. Methods Seven-day-old SD rats were subjected to permanent unilateral carotid artery ligation and hypoxia exposure （8% oxygen in nitrogen） for 2h after which all rats were randomly divided into normothermia hypoxic group （IN group; n = 10, rectal temperature： 37℃） and moderate hypothermia hypoxic group （IH group., n= 10, rectal temperature：33℃）. Sham-operated rat pups from the same litter used as normothermia control group （CN group） and hypothermia control group （CH group） with 8 rats in each and the rectal temperature was the same as Group Ⅰand Ⅱ, respectively. In situ detection of DNA fragment was performed with TUNEL. The apoptosis and necrosis of neuronal injury was determined with adjacent Nissl-stained, hematoxylin eosin （HE）-stained, and TUNEL-labeled sections. SD rats were sacrificed at 0, 6, 24, 48 and 72 h after hypoxic exposure and Western Blot was conducted to determine the activation （phosphorylation） p-38 MAPK （p-p38） and ERK1/2 （p-ERK1/2） MAPK. Results The apoptotic neuron ratio of cerebral cells was lower in IH group than that of IN group （P〈0.05）, but no difference was found in the necrosis neuron ratio（P〉0.05）. The level of p-ERK1/2 was significantly decreased after HIBD in IN group, while the level p-p38 increased against the decreased p-ERK1/2 level. However, this effect can be reversed by hypothermia therapy. Conclusions Hypothermia therapy can inhibit the apoptosis after cerebral hypoxia-ischemia by moderate hypothermia through p-ERK1/2p-p38MAPK signal transduction pathway.