丁苯酞对大鼠急性一氧化碳中毒后半胱氨酸蛋白酶-3和半胱氨酸蛋白酶-9表达的影响

Effects of dl-3-n-butylphthalide on expression of Caspase-3 and Caspase-9 mRNA and protein in rats with acute severe carbon monoxide poisoning

目的研究丁苯酞对大鼠急性重度CO中毒后脑组织中半胱氨酸蛋白-3(Caspase-3)和半胱氨酸蛋白酶-9(Caspase-9)表达的影响。方法通过数字表法将大鼠随机分为3组:正常组、模型组和实验组(均n=10)。用CO染毒法制作大鼠急性重度CO中毒模型。实验组大鼠给予丁苯酞80 mg·kg^(-1),灌胃1次/天,连续7 d,正常组和模型组给予等量的0.9%NaCl。在7 d后,用免疫组化染色法观测Caspase-3和Caspase-9阳性细胞数,用反转录酶-聚合酶链锁反应(RT-PCR)法及蛋白质印迹法检测Caspase-3和Caspase-9mRNA和蛋白的表达水平。结果正常组大鼠脑组织中Caspase-3和Caspase-9的阳性细胞数分别为4.98±1.34,8.65±1.89,mRNA表达水平分别为0.19±0.02,0.18±0.03,蛋白表达水平分别为0.09±0.01,0.08±0.01;模型组大鼠脑组织中Caspase-3和Caspase-9阳性细胞数分别为29.43±4.53,40.45±5.23,mRNA表达水平分别为0.47±0.05,0.75±0.05,蛋白表达水平分别为0.78±0.05,0.86±0.07。与正常组对比,模型组的Caspase-3和Caspase-9mRNA和蛋白表达水平显著增高,差异均有统计学意义(均P<0.05)。实验组大鼠脑组织中的Caspase-3和Caspase-9阳性细胞数分别为22.26±3.12,25.09±4.25,mRNA表达水平分别为0.35±0.05,0.41±0.05,蛋白表达水平分别为0.37±0.02,0.45±0.02,与模型组对比,Caspase-3和Caspase-9 mRNA和蛋白表达水平较明显下调,差异均有统计学意义(均P<0.05)。结论丁苯酞可通过下调Caspase-3和Caspase-9的表达水平来抑制神经细胞凋亡进程,从而减轻CO对大鼠脑神经的损害。

Objective To investigate the effects of dl - 3 - n - bu- tylphthalide（NBP） on the expression of Caspase - 3 and Caspase - 9 in the rat brain tissue after acute severe carbon monoxide poisoning. Methods Male Sprague- Dawley rats were randomly divided into three groups： normal group, model group and experimental group. The acute severe carbon monoxide poisoning model was made by the literature. Rats in experimental group were given 80 mg· kg^-1 NBP, and rats in normal group and model group were given same volume of saline by garage one time a day for seven days. Immunohistochemistry were used to observe the positive cells changes of Caspase- 3 and Caspase -9 after CO poisoning among the three groups. RT-PCR and Western blotting were used to detect the expression levels of Caspase -3 and Caspase -9＇s mRNA and protein. Results In normal group, the expression of Caspase - 3 and Caspase - 9 positive ceils were 4.98 ± 1.34, 8.65 ± 1.89, Caspase - 3 and Caspase - 9 of mRNA were 0. 19 ±0. 02,0. 18 ±0. 03, Caspase -3 and Caspase -9 of proteins were 0. 09 ±0. 01,0. 08 ±0. 01. In model group, Caspase - 3 and Caspase - 9 positive cells were 29. 43 ± 4. 53 ,40. 45 ± 5. 23, Caspase - 3 and Caspase - 9 of mRNA were 0.47 ±0. 05,0. 75 ± 0.05, Caspase - 3 and Caspase - 9 of proteins were 0. 78 ± 0. 05,0. 86 ± 0. 07. Compared with normal group, the expressions of Caspase -3 and Caspase -9 were increased significantly in the model group （P 〈 0.05 ） . In experimental group, Caspase - 3 and Caspase - 9 positive cells were 22.26 ± 3.12, 25.09 ±4. 25, Caspase -3 and Caspase -9 of mRNA were 0. 35 ±0. 05,0. 41 ±0.05 ,Caspasc -3 and Caspase -9 of proteins were 0.37 ± 0. 02,0. 45 ± 0. 02. Compared with model group, the Caspase - 3 and Caspase - 9 expressions were dowm regulated in rats of experimental group （P 〈 0.05 ）. Conclusion NBP seemed to play an important role in alleviation of brain damage via decreasing the expression of Caspase -3 and Caspase -9 and inhibiting the apotosis of ceils.