山莨菪碱对家兔全脑缺血再灌注后脑线粒体损伤的保护作用

背景山莨菪碱是从茄科唐古特莨菪中提取的一种生物碱,是一种良好的细胞保护剂.而线粒体功能变化是反映细胞损伤的最敏感指标之一.目的观察山莨菪碱对家兔全脑缺血再灌流后脑线粒体损伤的影响,探讨山莨菪碱的脑保护作用.设计完全随机对照实验.单位华中科技大学同济医学院附属同济医院.材料实验于2002-09/12在华中科技大学同济医学院附属同济医院急诊科实验室完成.选择30只健康家兔,随机分为假手术组、缺血再灌流组、山莨菪碱组,每组10只.方法采用"结扎双侧椎动脉和夹闭双侧颈总动脉+体循环低血压"建立全脑缺血再灌流模型,缺血20 min,再灌流2 h.山莨菪碱组于再灌流前1 min由股静脉给予山莨菪碱,剂量为10 mg/kg,并以5 mg/h速度维持治疗2 h.缺血再灌流组再灌流期间给予等量生理盐水治疗;假手术组只接受手术,但不结扎和夹闭血管.①采用氧化电极法测定脑线粒体呼吸功能,包括呼吸控制率,磷氧比,氧化磷酸化效率.②采用氧电极法测定脑线粒体内还原型烟酰胺腺嘌呤二核苷酸氧化酶,琥珀酸氧化酶,细胞色素C氧化酶活性.③采用原子吸收光谱法测定脑线粒体钙含量.④采用改良八木国夫法测定脑线粒体丙二醛含量.主要观察指标①各组家兔大脑皮质线粒体呼吸功能、呼吸链氧化酶活性变化.②各组家兔大脑皮质线粒体内钙和丙二醛含量.结果30只家兔均进入结果分析.①各组家兔大脑皮质线粒体呼吸控制率、磷氧比、氧化磷酸化效率缺血再灌流组和山莨菪碱组低于假手术组[烟酰胺腺嘌呤二核苷酸链呼吸控制率2.34±0.18,3.58±0.29,4.07±0.38,P＜0.05～0.01;磷氧比1.77±0.10,2.23±0.14,2.41±0.17,P＜0.05～0.01;氧化磷酸化效率(527±0.78),(8.03±1.30),(9.63±1.50)μkat/g,P＜0.05～0.01;黄素腺嘌呤二核苷酸链呼吸控制率1.47±0.23,2.53±0.28,2.84±0.36,P＜0.05～0.01;磷氧比0.88±0.09,1.58±0.11,1.73±0.17,P＜0.05～0.01;氧化磷酸化效率(6.05±1.13),(7.47±1.40),(8.62±1.60)μkat/g,P＜0.05～0.01],山莨菪碱组明显高于缺血再灌注组(P＜0.01).②各组家兔大脑皮质还原型烟酰胺腺嘌呤二核苷酸氧化酶、琥珀酸氧化酶、细胞色素C氧化酶活性缺血再灌流组和山莨菪碱组低于假手术组[还原型烟酰胺腺嘌呤二核苷酸氧化酶(2.62±0.35),(4.55±0.48),(5.07±0.60)μkat/g;琥珀酸氧化酶(1.48±0.17),(1.83±0.22),(2.10±0.28)μkat/g;细胞色素C氧化酶(5.03±1.12),(7.62±1.23),(9.00±1.53)μkat/g,(P＜0.05～0.01)],山莨菪碱组明显高于缺血再灌注组(P＜0.01).③各组大鼠脑线粒体钙和丙二醛含量缺血再灌流组和山莨菪碱组高于假手术组[钙(2.36±0.23),(1.39±0.17),(1.22±0.12)mg/g;丙二醛[(36.38±10.42),(22.69±9.56),(19.74±7.26)μmol/g,(P＜0.05～0.01)].山莨菪碱组低于缺血再灌流组(P＜0.01).结论山莨菪碱可通过钙拮抗、抑制脂质过氧化、稳定线粒体膜,减轻线粒体结构损伤,并促进缺血再灌流后线粒体呼吸功能、呼吸链酶活性的恢复,从线粒体水平发挥脑保护作用.

Protective effects of anisodamine on brain mitochondrial damage after complete cerebral ischemia and reperfusion in rabbits

BACKGROUND: An isodamine, a kind of alkaloid, is extracted from Anisodus tanguticus (Maxim.) Pascher and is also a good protective agent of cell. However, functional change of mitochondrion is the most sensitive index reflecting cell injury.OBJECTIVE: To study the effects of anisodamine on brain mitochondrial damage following global cerebral ischemia and reperfusion in domestic rabbits and explore its mechanism.DESIGN: Totally randomized controlled trials.SETTING: Emergency Department of Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology.MATERIALS: The experiment was carried out in the laboratory of Emergency Department, Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology, from September to December in 2002. Thirty healthy domestic rabbits of either sex were used and randomized into sham-operation group, ischemia-reperfusion group and anisodamine group with 10 rabbits in each group.METHODS: The models of complete cerebral ischemia and reperfusion injury in rabbits were established by ligation of bilateral common carotids and vertebral arteries with systemic hypotension, ischemia lasting for 20 minutes followed by 2-hour reperfusion. Anisodamine group was injected with anisodamine at a dose of 10 mg/kg body mass via femoral vein one minute before reperfusion, and lasted for 2 hours at a dose of 5 mg/hour by micro-pump. Ischemia-reperfusion group was treated with normal saline of the same volume. Sham-operation group only underwent used to determine mitochondrial respiratory functions, including respiratory control rate (RCR), the ratio of adenosine diphosphate to oxygen nicotinamide adenine dinucleotide hydrogenated (NADH) oxidase, succinate oxidase and cytochrome C oxidase were measured by the oxygenmethod of Yagi.drial calcium (Ca2+) and malondiadhyde (MDA) in cortex.reperfusion group and anisodamine group, RCR, ADP/O, OPR levels were lower than those in sham-operation group [nicotinamide adenine dinucleotide chain: RCR: 2.34±0.18,3.58±0.29,4.07±0.38,P ＜ 0.05-0.01;ADP/O: 1.77±0.10,2.23±0.14,2.41±0.17,P ＜ 0.05-0.01; OPR: (5.27±0.78),(8.03±1.30), (9.63±1.50)μkat/g, P ＜ 0.05-0.01; flavin adenine dinucleotide chain: RCR: 1.47±0.23,2.53±0.28,2.84±0.36,P ＜ 0.05-0.01;ADP/O: 0.88±0.09,1.58±0.11,1.73±0.17 ,P ＜ 0.05-0.01; OPR: (6.05±1.13),(7.47±1.40), (8.62±1.60)μkat/g,P ＜ 0.05-0.01], and those were higher in chemia-reperfusion group and anisodamine group, the activities of respiratory chain oxidase of NADH, succinate and cytochrome C were lower than those in sham-operation group [NADH: (2.62±0.35), (4.55±0.48), (5.07±0.60)μkat/g;succinate: (1.48±0.17), (1.83±0.22), (2.10±0.28)μkat/g; cytochrome C:(5.03±1.12), (7.62±1.23), (9.00±1.53)μkat/g, P ＜ 0.05-0.01], and those were higher in anisodamine group than in ischemia-reperfusion group, the content of mitochondrial Ca2+ [(2.36±0.23), (1.39±0.17),(1.22±0.12) mg/g] and MDA [(36.38±10.42), (22.69±9.56), (19.74±7.26)μmol/g,(P ＜ 0.05-0.01 )] was higher than that in sham-operation group, and it was lower in anisodamine group than in ischemia-reperfusion group (P ＜ 0.01).CONCLUSION: Anisodamine can protect the brain against ischemiareperfusion injury at the level of mitochondria by antagonism of Ca2+, inhibition of lipid peroxidation, stabilization of mitochondrial membrane, alleviation of mitochondrial damage, and improvement of motochondrial respiratory functions and the activities of enzymes of respiratory chain.