自噬抑制剂3-MA对脑缺血再灌注损伤大鼠脑内炎症反应的影响  

Effect of autophagy inhibitor 3-MA on inflammatory response in the brain of rats with cerebral ischemia/reperfusion injury

作　　者：安琪 杨楠[1] 朱玥荃 师文娟[1] 黄敏琪 赵咏梅[1] An Qi;Yang Nan;Zhu Yuequan;Shi Wenjuan;Huang Minqi;Zhao Yongmei(Central Laboratory,Xuanwu Hospital,Capital Medical University,Beijing Geriatric Medical Research Center,Key Laboratory of Neurodegenerative Diseases of Ministry of Education,Beijing 100053,China)

机构地区：[1]首都医科大学宣武医院中心实验室、北京市老年病医疗研究中心神经变性病教育部重点实验室,北京100053

出　　处：《首都医科大学学报》2024年第6期1008-1015,共8页Journal of Capital Medical University

基　　金：国家自然科学基金项目(81971095,81620108011)。

摘　　要：目的研究自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)对局灶性脑缺血再灌注大鼠缺血侧脑组织趋化因子受体2(C-X-C chemokine receptor type 2,CXCR2)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、环氧化酶2(cyclooxygenase-2,COX2)的影响,探究脑缺血再灌注损伤过程中自噬对炎症的作用。方法将雄性SD大鼠采用随机数字表法分为:假手术(Sham)组、大脑中动脉梗死(middle cerebral artery occlusion,MCAO)组和MCAO再灌注24 h+3-MA(3-MA)组,每组5只。按照改良线栓法制作MCAO大鼠模型,模拟缺血90 min。3-MA组大鼠于MCAO前腹腔注射10 mg/kg 3-MA。所有组别大鼠均再灌注24 h。达到再灌注时间后将大鼠处死,迅速断头、取脑,应用Western blotting法检测大鼠缺血侧脑组织CXCR2蛋白水平,免疫荧光染色法检测脑组织冰冻切片缺血半暗带区炎症因子TNF-α、IL-1β及COX2阳性细胞数量,并分别对IL-1β、COX2进行细胞定位。结果(1)MCAO组大鼠再灌注24 h缺血脑组织CXCR2蛋白水平比Sham组明显升高(P<0.05)。3-MA组大鼠缺血侧脑组织CXCR2蛋白水平比MCAO组显著减少(P<0.05)。(2)Sham组大鼠脑内偶见TNF-α、IL-1β和COX2阳性细胞,而MCAO组大鼠脑缺血半暗带区TNF-α、IL-1β和COX2阳性细胞数量均比Sham组显著升高(P<0.05)。相较于MCAO组,给予自噬抑制剂3-MA的缺血大鼠脑组织半暗带区中TNF-α、IL-1β和COX2阳性细胞数量均显著降低(P<0.05)。(3)IL-1β和COX2在脑缺血大鼠缺血半暗带区内分别与神经元标志物NeuN共定位。结论自噬抑制剂3-MA降低脑缺血再灌注大鼠缺血侧脑组织中CXCR2表达以及炎症因子TNF-α、IL-1β和COX2的阳性细胞数量,从而减轻脑缺血后的炎症反应,说明自噬是促进脑缺血后炎症反应的因素之一。Objective To investigate the effects of the autophagy inhibitor 3-methyladenine(3-MA)on C-X-C chemokine receptor type 2(CXCR2),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and cyclooxygenase-2(COX2)in the ischemic brain tissue of rats subjected to transient focal cerebral ischemia/reperfusion,in order to explore the role of autophagy in inflammation during cerebral ischemia/reperfusion injury.Methods The male Sprague Dawley rats were randomized into:sham group,middle cerebral artery occlusion(MCAO)group and MCAO+3-MA(3-MA)group,with n=5 for each group.A model of MCAO was induced by using the intraluminal suture method.The rats underwent 90 minutes of ischemia.Rats in 3-MA group were intraperitoneally injected with 10 mg/kg of 3-MA before MCAO.All groups of rats underwent reperfusion for 24 h.After reperfusion time,the rats were quickly decapitated,and their brains were removed.The levels of CXCR2 protein in the ischemic brain tissue were detected by Western blotting.The number of positive cells of TNF-α,IL-1β,and COX2 were detected by immunofluorescence staining in the ischemic penumbra,and the cellular localization of IL-1βand COX2 was evaluated respectively.Results①Compared with sham group,the level of CXCR2 protein was upregulated obviously in the ischemic brain tissue of MCAO rats after 24 h reperfusion(P<0.05).The level of CXCR2 protein decreased significantly in the ischemic brain tissue of 3-MA group compared with that of MCAO group(P<0.05).②There were few TNF-α,IL-1β,and COX2 positive cells in the sham group.Compared with sham group,the number of TNF-α,IL-1β,and COX2 positive cells in the ischemic penumbra of MCAO group rats obviously increased after 24 h of reperfusion(P<0.05).Compared with MCAO group,the number of positive cells of TNF-α,IL-1βand COX2 decreased significantly in the ischemic penumbra of the MCAO+3-MA group rats(P<0.05).③The IL-1βand COX2 positive cells were colocalized with NeuN,a general neuronal marker,in the ischemic penumbra of cerebral ischemia/reperfusio

关 键 词：脑缺血再灌注损伤 炎症 自噬 

分 类 号：R743.3[医药卫生—神经病学与精神病学]