乙酰胆碱介导的内皮超极化因子对海马神经元缺氧/再给氧损伤的保护作用  

Neuroprotective effects of acetylcholine-mediated EDHF in primarily cultured hippocamal neurons insulted by hypoxia-reoxygenation

作　　者：吴剑[1] 陈志武[1] 

机构地区：[1]安徽医科大学药理学教研室,安徽合肥230032

出　　处：《中国药理学通报》2007年第12期1624-1629,共6页Chinese Pharmacological Bulletin

基　　金：安徽省人才专项基金资助项目(No2005Z.031)

摘　　要：目的研究乙酰胆碱（acetylcholine,ACh）介导的大鼠大脑中动脉内皮释放的内皮超极化因子（endothelium-de-pendent hyperpolarizing factor,EDHF）对海马神经元缺氧/再给氧损伤的保护作用。方法用原代培养的大鼠海马神经元细胞建立缺氧/再给氧损伤模型,取大鼠大脑中动脉（mid-dle cerebral artery,MCA）血管段,PGI2和NO的阻断剂NG-ni-tro-L-argininemethyl ester（L-NAME）和indomethacin（Indo）预处理后,用ACh刺激血管内皮释放EDHF,用四甲基偶氮唑盐（MTT）染色吸光度和乳酸脱氢酶（LDH）活性作为细胞损伤指标,用激光共聚焦显微镜检测细胞内游离Ca^2＋浓度。结果与正常对照组相比,缺氧/再给氧损伤组细胞MTT染色吸光度明显降低,上清液中LDH活性和细胞内Ca^2＋浓度则明显升高。1μmol·L^-1ACh合用含血管内皮的MCA血管段（MCA/Endo）或ACh＋MCA/Endo＋L-NAME＋Indo均可抑制缺氧/再给氧致海马细胞MTT染色吸光度降低、培养上清液中LDH活性及细胞内Ca^2＋浓度的升高,但单用1μmol·L^-1ACh或MCA/Endo却无上述抑制作用,合用1μmol·L^-1ACh和去内皮MCA血管段（MCA/-Endo）也无明显抑制作用。K^＋在25-35μmol·L^-1范围内,可明显减弱ACh＋MCA/Endo＋L-NAME＋Indo对缺氧再给氧致海马神经细胞MTT染色吸光度降低、上清液LDH活性及细胞内Ca^2＋浓度升高的抑制作用,但Ba^2＋没有明显影响。结论假定的EDHF对原代培养的海马神经元缺氧/再给氧损伤具有保护作用,其机制可能与抑制缺氧/再给氧引起的钙超载有关。Aim To study the protective effects of Presumptive endotheliumartmendependent hyperpolarizing factor（EDHF） released from the rat middle cerebral arteries （MCA）, which was mediated by acetylcholine （ACh）, on primarily cultured hippocampal neurons subjected to hypoxia-reoxygenation injury. Methods Primarily cultured hippocampal neurons was insulted by hypoxia-reoxygenation; EDHF was produced in rat MCA ring by 1 μmol·L^-1 ACh in the presence of NG-nitro-L-argininemethyl ester （L-NAME, a NOS inhibitor） and indomethcacin （ Indo, a COX inhibitor） ; MTT absorbance and the LDH activity were served as the cell injury index, The level of free calcium fluorescence intensity in the cultured hippocampal neurons was monitored by laser scanning confocal microsope. Results Compared with nomal group, MTT absorbance were decreased significantly, LDH activity in the supemate culture fluid and the Ca^2＋ in hippocampal neurons increased significantly in model group. The conjoined use of 1 μmol·L^-1 ACh and the endothelium-intacted MCA （MCA/Endo） or ACh ＋ MCA/Endo ＋ L-NAME ＋ Indo can repress both the decrease of MTT absorbance and the increases of LDH activity in the supemate culture fluid and the Ca^2＋ in neurons which resulted from hypoxia-reoxygenation injury;Neither 1 μmol·L^-1 ACh alone nor MCA/Endo alone has the similar effects mentioned above, the conjoined use of 1 μmol·L^-1 ACh and the endothelium-denuded MCA （ ACh ＋ MCA/-Endo ） also has little effect. K^＋ which concentration is between 25 - 35 μmol·L^-1 can significantly attenuate the effects afforded by the conjoined use of 1 μmol·L^-1 ACh, MCA/Endo, L- NAME and Indo, but it is not the same situation when it comes to Ba^2＋. Conclusions Presumptive EDHF can protect primarily cultured rat hippocamal neurons insulted by hypoxia-reoxygenation and the mechanism is partially concerned with the inhibition of calcium overload.

关 键 词：内皮依赖性超极化因子 海马神经元 缺氧/再给氧 大脑中动脉 钙离子 

分 类 号：R-332[医药卫生] R322.81