一氧化碳中毒大鼠脑红蛋白表达变化及对半胱氨酸天冬氨酸蛋白酶3表达的影响

Change of the expression of neuroglobin in rats after acute carbon monoxide poisoning and the effect on cysteiny1 aspartate-specific proteinases-3 expression

目的:观察急性一氧化碳中毒后脑红蛋白和半胱氨酸天冬氨酸蛋白酶3表达的变化及氯化血红素对上述两者表达变化的影响。方法:实验于2005-01/08在华北煤炭医学院解剖学实验室完成。150只雄性SD大鼠随机分成3组,每组50只。①一氧化碳中毒组:大鼠放入染毒罐,静式吸入一氧化碳与空气的混合气60min。造模后分别于1,3,5,7和14d处死,每个时间点10只大鼠。②氯化血红素治疗组:除给予一氧化碳中毒组同样的处理外,于造模后立即注射氯化血红素(30mol/kg,3次/d)直至相应时间点处死,每个时间点10只大鼠。③对照组:不作任何处理,在相应时间点处死。各组大鼠分别行免疫组化染色和免疫蛋白印迹法,观察大鼠脑内的脑红蛋白和半胱氨酸天冬氨酸蛋白酶3表达的变化。结果:150只大鼠均进入结果分析。①免疫组化阳性神经细胞数目:一氧化碳中毒组大鼠海马脑红蛋白阳性神经元数目持续下降,至中毒后14d最低,与对照组相比差异有统计学意义(P<0.01);同时半胱氨酸天冬氨酸蛋白酶3阳性细胞逐渐增多,至中毒后5d达高峰,与对照组相比差异有统计学意义(P<0.01)。经氯化血红素治疗后,与中毒组相比脑红蛋白的表达明显增多,与中毒组相比差异有统计学意义(P<0.01);同时半胱氨酸天冬氨酸蛋白酶3的阳性细胞数明显下降,与中毒组相比差异有统计学意义(P<0.05)。②免疫蛋白印迹灰度分析:一氧化碳中毒组脑红蛋白的表达持续下降,半胱氨酸天冬氨酸蛋白酶3的表达上升,与对照组相比差异有统计学意义(P<0.01);氯化血红素治疗组与中毒组相比,脑红蛋白表达量上升(P<0.01);半胱氨酸天冬氨酸蛋白酶3表达下降,治疗5d最显著(P<0.01)。结论:一氧化碳中毒后大鼠脑红蛋白表达呈持续下降;刺激脑红蛋白表达可抑制急性一氧化碳中毒后大鼠脑半胱氨酸天冬氨酸蛋白酶3的表达。

AIM： To observe the changes of the expression of neuroglobin and cysteinyl aspartate-specific proteinases-3 （Caspases-3） in rats after acute carbon monoxide （CO） poisoning and the influence of hemin on the expressions of neuroglobin and Caspases-3. METHODS： The experiment was carried out in the laboratory of the Department of Anatomy, North China Coal Medical College from January to August 2005. Totally 150 male Sprague-Dawley rats were randomly divided into 3 groups with 50 rats in each group. ① CO-exposed group： The rats were fixed in a poisoning box （50 cm in diameter, 75 cm in height）. The chemical carbon monoxide （0.998） was admitted into the poisoning box. Rats were exposed to the mixture of carbon monoxide and air for 60 minutes. After making model, the rats were killed at corresponding time points with 10 rats at each time respectively. ② Hemin-treated group： After the rats were treated with CO as those in the CO-exposed group, they were treated with hemin （30 mol/kg）, three times a day, till killed at corresponding time points with 10 rats at each time respectively. ③ Control group： Do nothing to the rats till they were killed at 1, 3, 5, 7 and 14 days with 10 rats at each time respectively, lmmunohistoehemistry stain and Western blotting were used to observe the expressions of neuroglobin and Caspase-3 in the brain of rats. RESULTS： All the 150 rats were involved in the analysis of results. ① The number of positive neuronal cells of immunohistochemical staining： The number of the neuroglobin-positive cells in hippocampus was decreased continuously, and the expression was the lowest at 14 days after CO exposure, it was lower in the CO-exposed group than in the normal control group （P 〈 0.01）; While the number of Caspase-3-posiiive cells in hippocampus was increased sharply, it reached the maximum at 5 days after carbon monoxide exposure, it was higher in the CO-exposed group than in the normal control group （P 〈 0.01）. The immunohistochemistry showed that the expression of neuroglobin in the heroin-treated group was higher than that in the CO-exposed group （P 〈 0.01）, and the expression of Caspase-3 in the hemin-treated group was lower than that in the COexposed group （P 〈 0.05）. ② The Western blotting results： The expression of neuroglobin in the CO-exposed group was decreased, and it was lower than that in the normal control group （P 〈 0.01）. The expression of Caspase-3 was increased, and reached its peak at 5 days after CO exposure, and it was higher than that in the normal control group （P 〈 0.01）. The expression of neuroglobin in the hemin-treated group was higher than that in the CO-exposed group （P 〈 0.01）. The expression of Caspase-3 in the hemin-treated group was decreased, and reached the minimum at 5 days （P 〈 0.01）. CONCLUSION： The expression of neuroglobin in rats after acute carbon monoxide poisoning decreased steadily, and rising expression of neuroglobin inhibited the expression of Caspase-3 in rats after acute carbon monoxide poisoning.