艾芬地尔预先给药对脑缺血再灌注损伤大鼠缺血半暗带区iNOS和细胞凋亡的影响

Influence of ifenprodil pretreatment on focal cerebral ischemia-reperfusion-induced iNOS expression and activity and apoptosis in the ischemic penumbra in rats

目的 观察艾芬地尔预先给药对脑缺血再灌注损伤大鼠缺血半暗带区诱导型一氧化氮合酶（iNOS）和细胞凋亡的影响,探讨艾芬地尔脑保护的可能机制.方法 健康雄性SD大鼠54只,体重280～320 g,周龄9～10周,随机分为3组（n=18）：假手术组（S组）;缺血再灌注组（IR组）缺血前即刻腹腔注射与艾芬地尔等容量的生理盐水;艾芬地尔预先给药组（IF组）缺血前即刻腹腔注射艾芬地尔10 mg/kg（5 mg/ml）.IR组和IF组制备大鼠大脑中动脉闭塞再灌注损伤模型,阻闭2 h后恢复再灌注48 h.于再灌注结束时进行神经功能评估,随后断头取脑,测定梗死核心区和缺血半暗带区iNOS表达、iNOS酶活性及NO含量,观察细胞凋亡情况.结果 与S组比较,IR组和IF组神经功能缺陷评分升高,缺血半暗带区和梗死核心区iNOS表达上调,iNOS活性升高,NO含量升高,凋亡细胞计数增多（P〈0.01）;与IR组比较,IF组梗死核心区域减小,神经功能缺陷评分降低,缺血半暗带区和梗死核心区iNOS表达下调,iNOS活性降低,NO含量降低,缺血半暗带区凋亡细胞计数减少（P〈0.05）.与梗死核心区比较,缺血半暗带区IR组和IF组iNOS活性和NO含量降低,凋亡细胞计数增高（P〈0.05）.结论 艾芬地尔预先给药通过下调缺血半暗带区iNOS蛋白表达,降低iNOS活性和NO含量,减少细胞凋亡,从而发挥脑保护作用.

Objective To explore the possible mechanism for the neuroprotective effect of ifenprodil by investigating its effects on inducible nitric oxide synthase （iNOS） expression and activity and apoptosis in the ischemic penumbra following focal cerebral ischemia-reperfusion （I/R） in rats.Methods Fifty-four adult male SD rats weighing 280-320 g were randomly divided into 3 groups （ n = 18 each） ： I sham operation group （group S） ; II focal cerebral I/R group （group I/R） and Ⅲ ifenpradil preconditioning group （group IF） received intraperitoneal ifenprodil 10 mg/kg before focal cerebral I/R. Focal cerebral I/R was induced by middle cerebral artery occlusion （MCAO） . A 3-0 nylon thread with rounded tip was inserted into right internal jugular vein and threaded cranially until resistance was met. MCAO was maintained for 2 h. At 48 h after reperfusion, the animals were assessed for neurological function which was scored （0 = no functional deficit, 4 = unable to crawl, unconscious） and then decapitated. The brains were immediately removed for microscopic examination and determination of iNOS protein expression and activity, NO content and apoptosis in the ischemic core （IC） and penumbra （IP）. Results Ifenprodil pretreatment significantly decreased the cerebral infarct size and neurological scores in group IF as compared with group I/R. In group I/R the iNOS activity was increased compared with group S.The iNOS activity and NO content were significantly lower in IP than in IC in group IR and IF. The TUNEL-positive cells were also mainly confined to IP. Compared with group I/R, in group IF the iNOS protein expression was significantly down-regulated in IC and IP and the iNOS activity and NO content in IC and IP were suppressed and TUNEL-positive cells were significantly reduced in IP. Conclusion Ifenprodil pretreatment has protective effect against cerebral I/R injury by inhibiting iNOS protein expression in IP, suppressing iNOS activity and NO content and reducing apoptosis.