大鼠急性一氧化碳中毒后迟发性脑病模型海马区缺氧诱导因子-1α及血红素加氧酶-1的表达

Hippocampus hypoxia-inducible factor-1 alpha and heme oxygenase-1 expression in the delayed encephalopathy after acute carbon monoxide poisoning rat model

目的研究急性CO中毒后迟发性脑病（DEACMP）大鼠模型海马区缺氧诱导因子-1α（HIF-1α）及血红素加氧酶-1（HO-1）的表达及相关作用。方法将156只大鼠按随机数字表法抽样并分成急性CO中毒后迟发性脑病组（一氧化碳组）60只、空气对照组60只及空白对照组36只，再按随机数字表法均分成染毒后1、3、7、14、21、28 d 6个亚组。采用蛋白免疫印迹法及免疫组织化学法检测大鼠海马区HIF-1α及HO-1的表达，用普鲁士蓝染色检测铁离子的表达，原位末端转移酶标记染色检测细胞凋亡情况，Morris水迷宫试验分析大鼠行为学变化，观察海马区细胞形态学变化并监测动脉血一氧化碳血红蛋白（ HbCO）浓度。结果一氧化碳组大鼠海马区HIF-1α、HO-1及铁离子的表达均呈现染毒后先增高[3 d达峰值（HIF-1α：7.4±1.0，HO-1：9.8±0.9，铁离子：44.60±1.77）]，再降低的趋势，细胞凋亡的峰值延迟至染毒后7～14 d（7 d：19.4±1.1，14 d：17.8±1.0），与空气对照组（HIF-1α：1.7±0.7， HO-1：2.1±0.4，细胞凋亡：4.2±1.0，由于空气对照组及空白对照组的铁离子含量极少，无法进行统计，铁离子未进行组间比较，仅在CO组内进行比较）及空白对照组（ HIF-1α：2.0±0.5， HO-1：2.1±0.4，细胞凋亡：3.9±0.6）相同时间点比较差异具有统计学意义（F＝77.86、228.77、258.54，均P＜0.05）。各峰值与组内其他时间点比较差异具有统计学意义。水迷宫试验发现大鼠于染毒后14 d出现认知功能下降及行为学改变。形态学发现海马区锥体细胞变性、坏死。染毒过程中大鼠动脉血HbCO浓度＞50％维持超过16 h。结论急性重度CO中毒时，大鼠海马区HIF-1α在缺氧应激下增加导致下游靶基因HO-1增加，两者及代谢产物的过度表达共同参与促进海马区细胞迟发性凋亡可能是导致DEACMP的一种机制。

Objective To research the expression of hypoxia-inducible factor-1 alpha （ HIF-1α） and heme oxygenase-1 （ HO-1 ） in hippocampus of rats with delayed encephalopathy after acute carbon monoxide poisoning （ DEACMP） and its functions.Methods One hundred and fifty-six rats were selected and randomly divided into several groups：group CO of 60 rats, group air control （ AC） of 60 rats and group blank control （ BC） of 36 rats according to the random number table method , and then randomly divided into six subgroups after exposure , 1, 3, 7, 14, 21 and 28 d.The expression of HIF-1αand HO-1 in rat hippocampus was detected by Western blotting and immunohistochemistry method , the expression of iron ion was detected by the blue staining , the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay staining was used to detect the apoptosis of cells , the Morris water maze was used to analyze the behavioral changes of rats , the changes of cell morphology in the hippocampus were observed and the carboxyhemoglobin （HbCO） concentration in arterial blood was monitored.Results The expression of HIF-1α, HO-1 and iron in hippocampus of rats in group CO increased and reached a peak at the third day （ HIF-1α：7.4 ±1.0, HO-1：9.8 ±0.9, iron：44.60 ±1.77）, and then decreased, with the peak of apoptosis delayed at the 7th to the 14th day（7 d：19.4 ±1.0;14 d：17.8 ±1.0）.The difference of the same time points between the group AC （HIF-1α：1.7 ±0.7, HO-1：2.1 ±0.4, apoptosis：4.2 ±1.0） and the group BC （HIF-1α： 2.0 ±0.5, HO-1： 2.1 ±0.4, apoptosis： 3.9 ±0.6） was statistically significant （ F=77.86, 228.77, 258.54,all P〈0.05）.The difference between each peak value and that at other time points in the same group was statistically significant.The water maze test showed that the memory cognitive function and behavioral changes of rats declined in the 14th day.The pyramidal cells in the hippocampus were found degenerated and necrotic through morphological observation.The concentration of HbCO in rats′aorta was above 50% for 16 hours throughout the exposure.Conclusions The increase of HIF-1αin the rat′s hippocampus resulted in the increase of the downstream target gene HO-1 under hypoxia stress with acute severe CO toxicity.The excessive expression of the both and metabolic products promoting the delayed apoptosis of pyramidal cells in the hippocampus may be a mechanism of DEACMP.