谷氨酰胺联合氟比洛芬酯对大鼠局灶性脑缺血再灌注损伤的影响

Protective effects of glutamine combined with flurbiprofen axetil on focal cerebral ischemia-reperfusion injury in rats

目的探讨谷氨酰胺联合氟比洛芬酯对大鼠局灶性脑缺血再灌注损伤的保护作用及机制。方法改良线栓法制作大鼠大脑中动脉闭塞(MCAO)2 h/再灌注24 h模型。将60只雄性Sprague-Dawley大鼠随机分为5组,每组12只。假手术组(Sham组)不插入线栓,其余操作与缺血再灌注组相同。缺血再灌注组(IR组)分别于插入线栓前1 h和15 min静脉注射0.9%氯化钠溶液1 mL作为对照。谷氨酰胺组(Gln组)于插入线栓前1 h静脉注射0.75 g/kg谷氨酰胺1 mL,插入线栓前15 min静脉注射0.9%氯化钠溶液1 mL。氟比洛芬酯组(FPA组)于插入线栓前1 h静脉注射0.9%氯化钠溶液1 mL,插入线栓前15 min静脉注射10 mg/kg氟比洛芬酯1 mL。谷氨酰胺联合氟比洛芬酯组(Gln+FPA组)于插入线栓前1 h静脉注射0.75 g/kg谷氨酰胺1 mL,插入线栓前15 min静脉注射10 mg/kg氟比洛芬酯1 mL。MCAO 2 h/再灌注24 h后2,3,5-氯化三苯基四氮唑(TTC)染色计算梗死灶率,苏木精-伊红染色观察脑组织学形态学变化,免疫组织化学法检测脑组织热休克蛋白(HSP)70、核转录因子(NF)-κB蛋白。结果 Gln、FPA和Gln+FPA组的梗死灶率显著低于IR组(P值分别<0.05、0.01),Gln+FPA组又显著低于Gln和FPA组(P值均<0.05)。与IR组相比,3个治疗组的脑组织损伤均减轻,Gln+FPA组的减轻程度最明显。与Sham组比较,IR、FPA、Gln和Gln+FPA组的海马组织和半暗带的HSP70阳性细胞表达显著增加(P值均<0.01);与IR和FPA组比较,Gln和Gln+FPA组的海马组织和半暗带的HSP70阳性细胞表达显著增加(P值均<0.01);但Gln组与Gln+FPA组间以及FPA组与IR组间海马组织和半暗带的HSP70阳性细胞表达的差异无统计学意义(P值均>0.05)。与Sham组相比,IR、Gln、FPA和Gln+FPA组大鼠的缺血半暗带NFκ-B蛋白表达显著增加(P值分别<0.01、0.05);与IR组相比,Gln、FPA和Gln+FPA组大鼠的缺血半暗带中NF-κB蛋白表达均显著减少(P值分别<0.05、0.01);与Gln和FPA组相比,Gln+FPA组大鼠的缺血半暗带中NFκ-B蛋白表达进一步减少(P值分别<0.01、0.05)。结论谷氨酰胺和氟比洛芬酯预处理均能不同程度地减轻大鼠局灶性脑缺血再灌注损伤,两者联合应用具有增强作用。

Objective To investigate the protective effects of glutamine combined with flurbiprofen axetil（FPA） on focal cerebral ischemia-reperfusion injury（CIRI） in rats and the related mechanisms.Methods A modified middle cerebral artery occlusion（MCAO） was used to establish a 2 h/reperfusion 24 h CIRI model in rats.Totally 60 Sprague-Dawley rats were evenly randomized into 5 groups,namely,Sham group（sham operation）,IR group（normal saline＋local cerebral ischemia reperfusion）,Gln group（glutamine 0.75 g/kg ＋ local cerebral ischemia and reperfusion）,FPA group（FPA 10 mg/kg ＋local cerebral ischemia and reperfusion）,and Gln＋FPA group（glutamine 0.75 g/kg＋FPA 10 mg/kg＋ local cerebral ischemia and reperfusion）.After 24 h reperfusion,the histological changes were observed through H-E staining;the infarction ratios of the brain were measured by TTC staining;and the expressions of HSP70 and NF-κB protein were examined by immunohistochemical technique.Results The brain infarction ratios in three treated groups were significantly decreased compared with that in IR group（P0.01 or 0.05）,and that in Gln＋FPA group was significantly decreased than those in Gln and FPA groups（P0.05）.Compared with IR group,the histological damages were significantly alleviated in the 3 treated groups,with the slightest one found in Gln＋FPA group.Compared with Sham group,the expression of HSP70 proteins in the hippocampus or IP area in the other 4 groups were significantly increased（P0.01）.Compared with IR and FPA groups,Gln and Gln＋FPA groups had significantly higher expression of HSP70 proteins（P0.01）,and there were no significant differences between Gln and Gln＋FPA groups（P0.05） or IR group and FPA group（P0.05）.Compared with Sham group,NF-κB protein expression in IP area of the other four groups were significantly increased（P0.01 or 0.05）.Compared with IR group,NF-κB expression in the three treated groups were significantly decreased（P0.01 or 0.05）.Compared with Gln and FPA groups,the expression of NF-κB in Gln＋FPA group was significantly decreased（P0.01 or 0.05）.Conclusion Pretreatment with glutamine or flurbiprofen axetil can protect the neurons from focal CIRI in rats to different degrees,and the combination of them can achieve a better effect.