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内质网过度应激介导低氧高二氧化碳肺动脉高压大鼠的脑损伤

项冰倩, 高慧, 陈锡文, 戴雍月, 王万铁

温州医科大学1缺血/再灌注损伤研究所；2动物实验中心，温州 325035

摘要

本文旨在研究过度内质网应激(endoplasmic reticulum stress, ERS)在低O2高CO2性肺动脉高压(hypoxia hypercapnia induced pulmonary hypertension, HHPH)大鼠脑损伤中的作用。雄性SD大鼠40只，采用随机数字表法分为4组(n = 10)：对照组、低O2高CO2组、ERS通路激动剂衣霉素(tunicamycin, TM)组、ERS通路抑制剂4-苯基丁酸(4-phenylbutyric acid, 4-PBA)组。对照组置于常氧环境中饲养，其余三组置于低O2高CO2氧舱中(8.5%~11% O2、5%~6% CO2)饲养4周。TM组和4-PBA组大鼠分别腹腔注射TM (0.08 mg/kg，一周两次)和4-PBA (每天80 mg/kg)，低O2高CO2组腹腔注射等体积的生理盐水。4周后对大鼠进行手术，记录肺动脉平均压后进行心脏灌流，结束后开颅并快速取脑组织检测脑含水量，光镜下观察脑组织形态学变化，TUNEL法检测脑细胞凋亡指数，分光光度计法检测脑组织中Caspase-3酶活性，RT-PCR和Western blot检测磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun N-terminal kinase JNK, p-JNK)、Caspase-12、CCAAT增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein, CHOP)、葡萄糖调节蛋白78 (glucose regulated protein 78 kDa, GRP78) mRNA及蛋白表达水平。结果显示：与对照组相比，其余3组肺动脉平均压、脑含水量、细胞凋亡指数、Caspase-3酶活性、p-JNK、Caspase-12、CHOP、GRP78蛋白及mRNA均有升高(P < 0.05)，组织形态学结构亦有明显的损伤性变化；与低O2高CO2组比较，TM组的肺动脉平均压、脑含水量、细胞凋亡指数、Caspase-3酶活性、p-JNK、Caspase-12、CHOP和GRP78蛋白及mRNA均有增高(P < 0.05)，脑组织结构损伤性的变化亦有明显加重，而4-PBA组以上变化均有减轻(P < 0.05)。上述结果提示，过度ERS可能参与了HHPH引起的脑组织损伤，抑制ERS有望减轻脑损伤。

关键词： 低O2高CO2; 肺动脉高压 ; 脑 ; 内质网应激 ; 细胞凋亡

分类号：R332；R363.2；R329.2

Excessive endoplasmic reticulum stress mediates brain damage in hypoxia hypercapnia induced pulmonary hypertension rats

XIANG Bing-Qian, GAO Hui, CHEN Xi-Wen, DAI Yong-Yue, WANG Wan-Tie

1Ischemia/Reperfusion Injury Research Institute； 2Animal Experiment Center, Wenzhou Medical University, Wenzhou 325035, China

Abstract

The purpose of the present study was to investigate the effect of excessive endoplasmic reticulum stress (ERS) on the brain damage in hypoxia hypercapnia induced pulmonary hypertension (HHPH) rats. Forty healthy SPF male SD rats were randomly divided into four groups (n = 10 for each): control group, hypoxia hypercapnia group, ERS pathway agonist tunicamycin (TM) group and ERS pathway inhibitor 4-phenylbutyric acid (4-PBA) group. The rats of control group lived in normal environment, while the rats of other three groups were raised for four weeks in the tank with 8.5%–11% O2 and 5%–6% CO2. TM (0.08 mg/kg, twice a week) and 4-PBA (80 mg/kg, daily) were respectively intraperitoneally injected into the rats of TM and 4-PBA groups, and the hypoxia hypercapnia group was given the same volume of normal saline. The mean pulmonary artery pressure and heart perfusion of the rats were determined and recorded after four-week raising. Then the brain tissue of the rats were quickly taken out for the brain water content measuring and morphological changes observing. The Caspase-3 activity and the apoptotic index of the brain cells were also determined. The protein and mRNA expressions of p-JNK, Caspase-12, CHOP and GRP78 in brain tissues were detected by Western blot and RT-PCR. The results showed that compared with the control group, the mean pulmonary artery pressure, brain water content and brain cells apoptotic index, Caspase-3 activity, the protein and mRNA levels of p-JNK, Caspase-12, CHOP and GRP78 were increased (P < 0.05), and the brain tissues of the rats were obviously damaged in the rats raised in the hypoxia hypercapnia environment; compared with hypoxia hypercapnia group, the mean pulmonary artery pressure, brain water content, brain apoptotic index and Caspase-3 activity, p-JNK, Caspase-12, CHOP, GRP78 protein and mRNA expression in TM group were increased (P < 0.05), and the brain tissues of the rats were obviously damaged, while all above changes were relieved in 4-PBA group (P < 0.05). These results suggest that excessive ERS may participate in the brain injury induced by HHPH in rats and inhibition of excessive ERS can relieve the brain injury in the rats with HHPH.

Key words: hypoxia hypercapnia; pulmonary hypertension ; brain ; endoplasmic reticulum stress ; cell apoptosis

收稿日期：2017-02-27　　录用日期：2017-05-31

通讯作者：王万铁　　E-mail: dyy@wmu.edu.cn

引用本文：

项冰倩, 高慧, 陈锡文, 戴雍月, 王万铁. 内质网过度应激介导低氧高二氧化碳肺动脉高压大鼠的脑损伤[J]. 生理学报 2017; 69 (4): 413-421.

XIANG Bing-Qian, GAO Hui, CHEN Xi-Wen, DAI Yong-Yue, WANG Wan-Tie. Excessive endoplasmic reticulum stress mediates brain damage in hypoxia hypercapnia induced pulmonary hypertension rats. Acta Physiol Sin 2017; 69 (4): 413-421 (in Chinese with English abstract).