鞣花酸通过降低自噬作用减轻缺氧缺血性脑损伤  

Ellagic acid attenuates hypoxic-ischemic brain injury by alleviating autophagy

作　　者：蔡晨晨 叶丽霞 朱将虎[1] 白俊杰 曾珊珊 陈尚勤[1] 林振浪[1] CAI Chen-chen;YE Li-xia;ZHU Jiang-hu;BAI Jun-jie;ZENG Shan-shan;CHEN Shang-qin;LIN Zhen-lang(Department of Neonatology,The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University,Wenzhou 325027,China)

机构地区：[1]温州医科大学附属第二医院育英儿童医院新生儿科,浙江温州325027

出　　处：《中国病理生理杂志》2019年第2期311-319,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81701489;No.81771624);北京医卫健康公益基金资助项目(No.B17137);温州市科技局基金资助项目(No.Y20160020)

摘　　要：目的:探究鞣花酸(EA)能否通过降低自噬减轻缺氧缺血性脑损伤。方法:将17只Sprague-Daw-ley(SD)大鼠的7日龄乳鼠随机分组为假手术组(n=5)、缺氧缺血性脑病(HIE)组(n=6)和HIE+鞣花酸预给药组(n=6)。将鞣花酸(10 mg/kg)混悬于玉米油(10 m L/kg)中,对预给药组的乳鼠进行灌胃。各组动物手术24 h后取大脑组织切片进行TTC染色和HE染色,以判断鞣花酸在体内的脑保护作用。利用Western blot技术检测损伤侧脑部皮质区的蛋白及PC12细胞中自噬相关指标(beclin-1、P62、LC3-II/-I和Atg5)的表达水平。利用氯化钴(800μmol/L)制作PC12细胞缺氧处理的模型,观察预给药组中鞣花酸(8μmol/L)对氯化钴处理后的细胞保护作用,利用CCK-8法检测细胞活力,流式细胞术检测氧化应激水平,单丹磺酰戊二胺(MDC)免疫荧光染色观察胞内的自噬情况,四甲基罗丹明乙酯(TMRE)荧光染色检测线粒体膜电位水平,以明确鞣花酸是否通过调节自噬机制达到减轻体外神经元缺氧损伤的目的。结果:7日龄乳鼠造模后HIE组脑部梗死面积较大,HE染色显示脑组织水肿及结构完整性破坏,神经元少见;鞣花酸预给药组脑部梗死面积减少,HE染色显示脑组织仅部分损伤,神经元分布较HIE组数量多且结构完整。提取HIE组乳鼠的脑组织皮质蛋白,发现Atg5、beclin-1和LC3-II/-I的蛋白表达水平显著高于正常组(P <0.01),而P62蛋白的表达水平降低(P <0.01);鞣花酸预处理后Atg5、beclin-1和LC3-II/-I蛋白表达水平均低于HIE组(P <0.01),而P62蛋白的表达呈现较高趋势(P <0.01)。氯化钴处理组的PC12细胞具有高氧化应激水平(P <0.01),鞣花酸有降低氧化应激的趋势(P <0.05)。另外,鞣花酸预处理组线粒体膜电位水平升高(P <0.05)。MDC荧光染色发现,相较于氯化钴处理组的MDC高荧光定量与自噬小体数量增多的趋势(P <0.01),鞣花酸确能减少PC12细胞内自噬小体的数量,减轻自噬强度,从而发挥细AIM:To investigate whether ellagic acid(EA)attenuates hypoxic-ischemic encephalopathy(HIE)by down-regulating autophagy.METHODS:In vivo,Sprague-Dawley rats(n=17)were randomly divided into 3 groups:5 rats for sham group,6 rats for HIE group and 6 rats for HIE+EA pretreatment group.The rats in HIE+EA pretreatment group were treated with EA(10 mg/kg,10 mL/kg,suspended in corn oil,ig).After 24 h of operation,the rats from each group were sacrificed and their brains were collected.TTC staining and HE staining were used to define the infarct areas and brain structure.The autophagy-related proteins beclin-1,P62,LC3-II/-I and Atg5 in the cortex in each group were compared by Western blot.In vitro,PC12 cells were divided into 3 groups:control group,CoCl2 group and CoCl2+EA pretreatment group.CoCl2 at 800 μmol/L was added to the PC12 cells to induce an anoxic environment.The PC12 cells were pretreated with EA at 8 μmol/L and the cell viability was measured by CCK-8 assay.The production of reactive oxidative species(ROS)in the cells was detected by flow cytometry with DCFH-DA staining.MDC staining and TMRE staining were applied to reflect the extent of autophagy and the state of apoptosis,respectively.The autophagy-related proteins in PC12 cells were also investigated.RESULTS:In HIE group,7-day-old rats were given the operations and the their large infarct areas in the hemisphere were observed by TTC staining.HE staining displayed the injured hemispheres which contained few neurons,and exhibited edema status and serious structural damage.EA pretreatment decreased the infarct area and alleviated the damage to hemisphere with more visible neurons,compared with HIE group.Compared with sham group,the levels of autophagy-related proteins Atg5,beclin-1 and LC3-II/-I in the cortex were increased(P<0.01),and P62 protein expression was decreased(P<0.01)in HIE group.Compared with HIE group,the protein expression of Atg5,beclin-1 and LC3-II/-I was decreased(P<0.01)and P62 protein expression was increased in HIE+EA pretreatment group(

关 键 词：缺氧缺血性脑病 鞣花酸 自噬 氧化应激 

分 类 号：R743.3[医药卫生—神经病学与精神病学] R363.2[医药卫生—临床医学]