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目的 探讨丁苯酞(NBP)对急性一氧化碳(CO)中毒大鼠血脑屏障(BBB)超微结构损伤的干预作用及其对紧密连接相关蛋白ZO-1、claudin-5表达的影响.方法 按随机数字表法将144只健康雄性SD大鼠分为正常对照组、CO中毒组和NBP干预组,每组48只.采用高压氧舱吸入法建立急性CO中毒动物模型并给予高压氧舱治疗,正常对照组自由呼吸新鲜空气.NBP干预组大鼠于中毒2 h后灌胃NBP 60 mg/kg、每日2次,直至处死;正常对照组和CO中毒组灌胃等量纯橄榄油.各组分别于制模后1、3、7、14 d取4只大鼠,透射电镜下观察BBB超微结构改变,用免疫荧光标记及蛋白质免疫印迹试验(Western Blot)检测ZO-1和claudin-5的蛋白表达,用免疫荧光双标染色观察两种蛋白在脑组织中的定位关系,用线性回归分析两种蛋白的相关性.结果 正常对照组大鼠BBB超微结构无明显异常,可见部分ZO-1和大量claudin-5阳性细胞.CO中毒组BBB超微结构严重损坏;脑组织ZO-1和claudin-5阳性细胞明显减少,中毒后1 d蛋白表达即较正常对照组明显降低(ZO-1蛋白:3.38±0.30比24.50±5.62,claudin-5蛋白:11.38±0.93比46.35±6.88,均P<0.05), 14 d时仍维持在较低水平,与正常对照组比较差异有统计学意义(ZO-1蛋白:10.35±0.80比24.63±3.57, claudin-5蛋白:32.35±3.11比46.43±7.20,均P<0.05).NBP干预可明显改善急性CO中毒导致的大鼠BBB超微结构改变;脑组织ZO-1和claudin-5阳性细胞明显增多,蛋白表达亦明显升高,分别于1 d、3 d起明显高于CO中毒组(1 d ZO-1蛋白:7.57±0.69比3.38±0.30,3 d claudin-5蛋白:20.46±1.42比11.43±0.86,均P<0.05),并随时间延长呈升高趋势.免疫荧光双标染色结果提示,ZO-1和claudin-5蛋白不仅可以在同一细胞中共存,也可以在不同细胞中单独表达.线性回归分析显示,急性CO中毒大鼠脑组织ZO-1与claudin-5表达呈线性正相关(R2=0.917,P=0.022).结论 NBP可能通过上调ZO-1和claudin-5蛋白表达维持急性CO中毒大鼠BBB结构和功能的完整性,进而减轻CO中毒引起的脑损伤.

Objective To explore the effects of N-butylphthalide on the expressions of ZO-1 and claudin-5 in blood-brain barrier (BBB) in rats with acute carbon monoxide (CO) poisoning. Methods A total of 144 adult healthy male Sprague-Dawley (SD) rats were randomly divided into normal control group, CO poisoning group, and NBP treatment group, with 48 rats in each group. The acute CO poisoning model was reproduced in hyperbaric oxygen chamber, and all model rats were given hyperbaric oxygen therapy once daily. The rats in the normal control group were free to breathe fresh air. The rats in NBP treatment group were administered orally NBP 60 mg/kg twice a day at 2 hours after poisoning until death. The rats in normal control group and CO poisoning group were treated with equal amount of pure olive oil. Four rats were sacrificed from each group at 1, 3, 7, 14 days after model reproducing, respectively. The changes in ultrastructure of BBB were observed under transmission electron microscope. The expressions of ZO-1 and claudin-5 proteins were determined by immunofluorescence staining and Western Blot. The localization of the two target proteins was observed by immunofluorescence double staining. The correlation between the two proteins was analyzed by linear regression. Results The ultrastructure of BBB was normal in normal control group, some ZO-1 and a large number of claudin-5 positive cells were observed. The ultrastructure of BBB was seriously injured, ZO-1 and claudin-5 positive cells in brain tissue were significantly decreased, and the expressions of ZO-1 and claudin-5 proteins in brain tissue at 1 day after poisoning in CO poisoning group were significantly lower than those of normal control group (ZO-1 protein:3.38±0.30 vs. 24.50±5.62, claudin-5 protein: 11.38±0.93 vs. 46.35±6.88, both P < 0.05), and although gradually restored, they were maintained at relatively lower levels until 14 days as compared with those in normal control group (ZO-1 protein: 10.35±0.80 vs. 24.63±3.57, claudin-5 protein: 32.35±3.11 vs. 46.43±7.20, both P < 0.05). NBP treatment could significantly alleviate the ultrastructure injury of BBB induced by acute CO poisoning, the amount of ZO-1 and claudin-5 positive cells in brain tissue were significantly increased, as well as the expressions of ZO-1 and claudin-5 proteins were significantly increased, which were significantly higher than those of CO poisoning group from 1 day and 3 days on, respectively (1-day ZO-1 protein: 7.57±0.69 vs. 3.38±0.30, 3-day claudin-5 protein:20.46±1.42 vs. 11.43±0.86, both P < 0.05), and which showed an increase tendency with time prolongation. The results of immunofluorescence double staining showed that ZO-1 and claudin-5 proteins could not only coexist in the same cell, but also could be expressed separately in different cells. Linear regression analysis showed the positive correlation between the expressions of ZO-1 and claudin-5 proteins in brain tissue of rats with acute CO poisoning (R2= 0.917, P = 0.022). Conclusion NBP could markedly improve the ultrastructure and functional integrity of BBB through up-regulating the expressions of ZO-1 and claudin-5 proteins, and then reduce brain damage caused by CO poisoning.