邻氨基苯甲酸抑制TRPM2通道对脑缺血再灌注损伤小鼠神经功能及小胶质细胞活化的影响  

Effects of Anthranilic Acid Inhibiting TRPM2 Channel on Neurological Function and Activation of Microglia in Mice with Cerebral Ischemia-reperfusion Injury

作　　者：仇莽莽 焦岩 王芳姣 张燕[2] 郑乐怡 马康 梁雪云[3] 文玉军[2] 余建强[4] 何仲义[1] 牛建国[1,2] QIU Mangmang;JIAO Yan;WANG Fangjiao;ZHANG Yan;ZHENG Leyi;MA Kang;LIANG Xueyun;WEN Yujun;YU Jianqiang;HE Zhongyi;NIU Jianguo(Department of Human Anatomy and Histology Embryology,School of Basic Medicine,Ningxia Medical University,Yinchuan 750004,China;Ningxia Key Laboratory of Cerebrocranial Diseases,Ningxia Medical University,Yinchuan 750004,China;Ningxia Human Stem Cell Research Institution of General Hospital,Ningxia Medical University,Yinchuan 750004,China;Ningxia Collaborative Innovation Center of Regional Characteristic Traditional Chinese Medicine,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学基础医学院人体解剖与组织胚胎学系,银川750004 [2]宁夏医科大学宁夏颅脑疾病重点实验室,银川750004 [3]宁夏医科大学总医院干细胞研究所,银川750004 [4]宁夏医科大学宁夏特色中医药协同创新中心,银川750004

出　　处：《宁夏医科大学学报》2021年第6期577-584,共8页Journal of Ningxia Medical University

基　　金：宁夏重点研发计划项目(2019BFH02003)。

摘　　要：目的探究N-(对戊基肉桂酰基)邻氨基苯甲酸(ACA)抑制瞬时受体电位通道M2(TRPM2)对小鼠脑缺血再灌注损伤小鼠神经功能及小胶质细胞活化的影响。方法将48只C57BL/6雄性小鼠随机分为假手术组(Sham)、大脑中动栓塞再灌注组(MCAO/R)和TRPM2通道抑制组(ACA)。采用线栓法建立小鼠脑缺血再灌注损伤模型,采用Bederson评分法筛选损伤程度一致的模型进行后续实验。ACA组小鼠缺血2 h后腹腔注射25 mg·kg^(-1)ACA(TRPM2通道抑制剂)。再灌注24 h后,采用Bederson评分评价小鼠神经功能,2,3,5-氯化三苯基四氮唑(TTC)染色评价小鼠脑梗死率,激光散斑血流成像监测小鼠大脑皮质脑血流量,苏木精—伊红染色法(HE)染色观察各组小鼠缺血侧颞侧皮质区、海马CA1、CA3及缺血半暗带区形态学变化;Nissl染色观察各组小鼠缺血侧颞侧皮质区、海马CA1、CA3及缺血半暗带区神经元数量的变化;免疫荧光染色观察半暗带区离子钙结合衔接分子1(Iba-1)及诱导型一氧化氮合酶(iNOS)的表达,酶联免疫吸附法(ELISA)检测小鼠血清中肿瘤坏死因子-α(TNF-α)的表达。结果与Sham组相比,MCAO/R组小鼠Bederson评分、脑梗死率均上升(P均<0.05);与MCAO/R组相比,ACA组小鼠Bederson评分、脑梗死率降低,大脑皮质缺血侧脑血量增加(P均<0.05)。HE及Nissl染色结果发现:与Sham组相比,MCAO/R组在缺血侧颞侧皮质区、海马CA1、CA3及缺血半暗带区均可见组织结构紊乱,细胞核固缩,嗜酸性细胞及淋巴细胞多见,神经细胞数量明显减少;与MCAO/R组相比,ACA组缺血侧颞侧皮质区、海马CA1、CA3及缺血半暗带区组织结构明显改善,炎性细胞明显减少,神经元数目明显增加,Iba-1、iNOS荧光强度减少,TNF-α表达量减少(P均<0.05)。结论ACA抑制TRPM2通道对脑缺血再灌注损伤小鼠具有保护作用,可能与ACA抑制小胶质细胞活化并减少炎性因子的分泌有关。Objective To explore the effect of N-(p-pentylcinnamoyl)anthranilic acid inhibiting transient receptor potential channel M2(TRPM2)on neurological function and activation of microglia in mice with cerebral ischemia-reperfusion injury.Methods Forty-eight C57BL/6 male mice were randomly divided into sham operation group(Sham),cerebral arterial embolization reperfusion group(MCAO/R)and TRPM2 channel inhibition group(ACA).The mouse model of cerebral ischemia-reperfusion injury was established by the suture method,and the models with the same degree of injury were screened by the Bederson score method for follow-up experiments.Mice in the ACA group were intraperitoneally injected with 25 mg·kg^(-1) ACA[N-(p-pentylcinnamoyl)anthranilic acid,TRPM2 channel inhibitor]after 2 hours of ischemia.After 24 hours of reperfusion,the Bederson score was used to evaluate the neurological function of the mice,the 2,3,5-triphenyltetrazolium chloride(TTC)staining was used to evaluate the cerebral infarction rate of the mice,the laser speckle blood flow imaging was used to monitor the mouse brain cortical cerebral blood flow,the Hematoxylin-eosin(HE)staining was used to observe the morphological changes of the ischemic temporal cortex,hippocampus CA1,CA3 and ischemic penumbra in each group of mice,the Nissl staining was used to observe the changes in the number of neurons in the ischemic temporal cortex,hippocampus CA1,CA3 and ischemic penumbra of each group of mice,the immunofluorescence staining was used to observe the expression of ionized calcium binding adapter molecule 1(Iba-1)and inducible nitric oxide synthase(iNOS)in the penumbra area,and enzyme-linked immunosorbent assay(ELISA)was used to detect mouse serum tumor necrosis factor-α(TNF-α)expression.Results Compared with the Sham group,the Bederson score of the MCAO/R group increased(P<0.05),and the cerebral infarction rate increased significantly(P<0.05);compared with the MCAO/R group,the Bederson score of the ACA group decreased(P<0.05),the cerebral blood volume on the isch

关 键 词：TRPM2 脑缺血再灌注损伤 小胶质细胞 炎症因子 

分 类 号：R743.31[医药卫生—神经病学与精神病学]