一氧化氮合酶抑制剂N-硝基-L-精氨酸甲酯对局灶性脑缺血再灌注大鼠缺血半暗带区细胞自噬的影响

Effects of nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester on the expressions of autophagy in the penumbra of rats following focal cerebral ischemia/reperfusion

目的探究一氧化氮合酶(nitric oxide synthase,NOS)抑制剂N-硝基-L-精氨酸甲酯(Nω-nitro-L-arginine methyl ester,LNAME)对大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)模型大鼠再灌注后24 h脑内一氧化氮(nitric oxide,NO)和自噬相关蛋白Beclin1、LC3B表达的影响,探讨L-NAME保护脑缺血再灌注损伤的机制。方法 18只健康雄性SD大鼠采用随机数字表法分为假手术(Sham)组、MCAO组,和MCAO+L-NAME组,每组6只。使用改良线栓法制作大鼠右侧大脑中动脉缺血90min再灌注模型。术中监测大鼠肛温,使其维持在正常范围。于再灌注后24 h处死大鼠,迅速取脑,用免疫荧光染色检测小鼠脑组织冰冻切片缺血半暗带区Beclin1和LC3B的表达水平,用免疫荧光双标分别将Beclin1和LC3B与NO介导的蛋白质损伤标志物3-硝基酪氨酸(3-nitrotyrosine,3-NT)进行共定位。结果 Sham组大鼠没有3-NT染色阳性细胞,偶见Beclin1和LC3B染色阳性细胞,MCAO组小鼠脑组织缺血半暗带区3-NT、Beclin1、LC3B均大量表达(P<0.05)。与MCAO组相比,给予L-NAME治疗后,缺血再灌注大鼠脑组织半暗带区3-NT、Beclin1、LC3B表达均明显减少(P<0.05)。缺血再灌注小鼠脑组织半暗带区,Beclin1和LC3B分别与3-NT免疫荧光染色共定位。结论 L-NAME通过抑制NOS活性,减少NO的产生,直接避免神经元的氧化应激损伤;同时,氧化应激损伤的减弱也避免了自噬的进一步激活,起到了间接的神经保护作用。

Objective To explore the effects of nitric oxide synthase（ NOS） inhibitor Nω-nitro-L-arginine methyl ester（ L-NAME） on the production of nitric oxide（ NO） and the expression of Autophagy-related proteins Beclin1 and LC3 B in the brain tissues of rats with middle cerebral artery occlusion（ MCAO）/reperfusion injury and to investigate the mechanism of neuroprotective effect induced by LNAME. Methods Eighteen male Sprague Dawley rats were divided into 3 groups randomly： Sham group,MCAO group,and L-NAME group（ n = 6）. MCAO was induced by suture method. The rats were underwent 90 min of right MCAO,and then reperfusion by withdrawing filament. Rectal temperature was monitored and kept in normal range during the operation. The rats were sacrificed and the brains were harvested at 24 h after reperfusion. The expressions of Beclin1 and LC3 B were detected by immunofluorescent staining. And the spatial location of 3-nitrotyrosine（ 3-NT）, the biomarker of NO-mediated protein damage, and Beclin1/LC3 B were detected respectively by double immunofluorescence labeling. Results In Sham group,no 3-NT positive cells were observed. Little Beclin1 positive cells and LC3 B positive cells distributed in the brain tissues. Compared with Sham group,the number of Beclin1 positive cells and LC3 B positive cells increased significantly in the penumbra of MCAO group（ P〈0. 05）. Compared with MCAO group,the number ofBeclin1 positive cells and LC3 B positive cells decreased significantly in the penumbra of rats after received L-NAME on 24 h after reperfusion（ P〈0. 05）. Beclin1 and LC3 B-positive immunoreactive cells were colocalized with 3-NT separately in the penumbra of ischemia/reperfusion rats. Conclusion L-NAME reduced the production of NO by inhibiting the activity of NOS,which could prevent neurons from oxidative stress injury directly. At the same time,L-NAME also inhibited the expression of autophagy,which might play an indirect role in neuroprotective effect.