cGMP通路激活对小鼠脑缺血后神经发生的作用  

The effect of cGMP pathway activation on neurogenesis after cerebral ischemia in mice

作　　者：校欢 承欧梅[2] 韩萍[3] 谭瑞[4] XIAO Huan;CHENG Oumei;HAN Ping;TAN Rui(Department of Pharmacy,the Second Affiliated Hospital of Chengdu Medical College,China National Nuclear Corporation 416 Hospital,Chengdu,Sichuan 610066,China;Department of Neurology,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China;Department of Medical Service,Chongqing General Hospital,Chongqing University,Chongqing 401147,China;Department of Pharmacy,Banan Affiliated Hospital of Chongqing Medical University,Chongqing 401320,China)

机构地区：[1]成都医学院第二附属医院·核工业四一六医院药学部,四川成都610066 [2]重庆医科大学附属第一医院神经内科,重庆400016 [3]重庆大学附属人民医院医务处,重庆401147 [4]重庆医科大学附属巴南医院药剂科,重庆401320

出　　处：《检验医学与临床》2024年第17期2470-2475,共6页Laboratory Medicine and Clinic

基　　金：国家自然科学基金面上项目(81871002)。

摘　　要：目的观察小鼠脑缺血后海马体神经发生的变化及其与环磷酸鸟苷(cGMP)相关调节机制。方法76只C57BL/6雄性小鼠按照随机数字表法分为假手术组和模型组,每组38只,采用双侧颈总动脉夹闭法建立脑缺血模型。采用Morris水迷宫检测其学习记忆功能,采用苏木精-伊红(HE)染色法检测海马体CA1区病理变化,采用免疫荧光法检测海马体齿状回神经发生标志物5′-溴脱氧尿嘧啶核苷(BrdU)、双肾上腺皮质激素(DCX)、BrdU/神经元核抗原(NeuN)阳性细胞数的表达,采用比色法、硝酸还原法分别检测海马体一氧化氮合酶(NOS)活性和一氧化氮(NO)水平,采用酶联免疫吸附试验(ELISA)检测cGMP水平和磷酸二酯酶(PDE)9活性,采用Western bolt法检测cGMP依赖性蛋白激酶G(PKG)、脑源性神经营养因子(BDNF)蛋白表达。结果多变量方差分析结果显示,在训练2~5 d,在相同训练天数模型组小鼠逃避潜伏期的时间同假手术组相比明显延长,差异均有统计学意义(F=13.683、8.625、73.266、90.327,P<0.05)。在第6天的空间探索试验中,假手术组、模型组小鼠穿越平台次数分别为(6.67±1.37)、(1.67±0.51)次,模型组小鼠穿越平台次数明显少于假手术组,差异有统计学意义(t=8.300,P<0.05)。模型组海马体CA1区锥体神经元数目明显减少,差异有统计学意义(P<0.05),提示脑缺血模型建立成功;与假手术组比较,模型组小鼠海马体马齿状回神经发生标志物BrdU、DCX、BrdU/NeuN阳性细胞数明显增加,同时NOS活性下降,NO生成减少,PDE9活性降低,而cGMP水平增加,PKG和BDNF蛋白表达上调,差异均有统计学意义(P<0.05)。结论PDE9活性降低,cGMP-PKG信号通路激活可能参与促进小鼠脑缺血后海马体神经发生过程。Objective To observe the changes of hippocampal neurogenesis after cerebral ischemia and its mechanism related to cyclic guanosine phosphate(cGMP)in mice.Methods A total of 76 C57BL/6 male mice were randomly divided into sham group and model group,with 38 mice in each group.The model of cerebral ischemia was induced via bilateral common carotid artery occlusion.Morris water maze was applied to assess spatial learning and memory of the mice,and Hematoxylin-Eosin(HE)staining was executed to appraise the pathological changes in the hippocampal CA1 region.Immunofluorescence was implemented to detect the count of positive cells of 5'-Bromo-2-deoxy Uridine(BrdU),doublecortin(DCX)and BrdU/Neuro-specific nuclear-binding protein(NeuN),the markers of neurogenesis in hippocampal dentate gyrus.The Nitric oxide synthase(NOS)activity and nitric oxide(NO)content were inspected by colorimetric and nitric acid reduction methods respectively.The cGMP level and phosphodieterase(PDE)9 activity were examined by ELISA kits,while cGMP dependent protein kinase G(PKG)and brain-derived neurotrophic factor(BDNF)protein expression were determined by Western bolt.Results During the 2-5 days training period,the escape latency of the model group mice was significantly prolonged compared to the sham group,the differences were statistically significant(F=13.683,8.625,73.266,90.327,P<0.05).In the space exploration experiment on the 6th day,the number of times the sham group and model group mice crossed the platform were(6.67±1.37)and(1.67±0.51)times respectively.The number of times the model group mice crossed the platform was significantly less than that of the sham group,the difference was statistically significant(t=8.300,P<0.05).The number of pyramidal neurons in the hippocampal CA1 region of the model group reduced significantly(P<0.05),indicating the successful establishment of the cerebral ischemia model.Compared with the sham group,the model group showed a significant increase in the number of BrdU,DCX,BrdU/NeuN positive cells in the hi

关 键 词：脑缺血 海马体 神经发生 一氧化氮 磷酸二酯酶 环磷酸鸟苷 

分 类 号：R446.9[医药卫生—诊断学] R743[医药卫生—临床医学]