知母总皂苷对脑缺血再灌注损伤的保护机制

Protective mechanism of timosaponin in cerebral ischemia-reperfusion injury

目的:评价知母总皂苷对于大鼠脑缺血再灌注损伤的保护作用,观察其对内皮素1及内皮型一氧化氮合酶的影响。方法:实验于2004年在南方医科大学药理学实验室进行,大鼠132只数字表法随机分为5组:①知母总皂苷50,100mg/kg组(n=30):灌胃相应剂量的知母总皂苷,1次/d,连续7d,7d后线栓法制备急性局灶缺血模型。②己酮可可碱组(n=12,仅进行神经功能评分及脑梗死体积测定):灌胃45.88mg/(kg·d)己酮可可碱,连续7d,7d后同前造模。③模型组(n=30):灌胃等量生理盐水7d,7d后同前造模。④假手术组(n=30):灌胃等量生理盐水7d,7d后手术,但不插线栓塞血管。造模24h观察大鼠神经功能损害并评分,取缺血侧脑组织用TTC染色后比较脑梗死体积,用放射免疫法测定缺血脑组织中内皮素1含量,免疫组织化学法比较脑组织中内皮型一氧化氮合酶阳性表达。结果:87只大鼠进入结果分析。①脑组织梗死体积百分比:知母总皂苷50,100mg/kg组和己酮可可碱组均小于模型组[(21.56±5.03)%,(23.80±3.59)%,(18.50±3.74)%,(28.61±2.76)%,P<0.05,0.01]。②神经功能评分:知母总皂苷50,100mg/kg组和己酮可可碱组均低于模型组(P<0.05,0.01)。③脑组织内皮素1含量:模型组显著高于对照组[(0.68±0.19),(0.31±0.11)ng/g,P<0.01];知母总皂苷50,100mg/kg组均低于模型组[(0.43±0.06),(0.36±0.20)ng/g,P<0.05,0.01],但2组间比较差异无显著性意义。④内皮型一氧化氮合酶阳性表达:知母总皂苷50,100mg/kg组阳性细胞数增多、表达强度增强。结论:知母总皂苷对于脑缺血再灌注后引起的脑损伤具有确定的保护作用,其保护机制可能涉及减少内皮素1的释放、增强内皮型一氧化氮合酶表达、改善缺血脑组织中血管内皮功能及血供有关。

AIM： To study the protective effect and mechanism of timosaponin on cerebral ischemia-reperfusion injury in rats, and observe the influence of timosaponin on endotbelin 1 （ET-1） and endothelial nitricoxide synthase （eNOS）. METHODS： The research was carried out in Department of Pharmacology, Southern Medical University in 2004. We divided： 132 rats into 5 groups at random： ①Timosaponin groups （n=30）： The rats were treated with intragastfic infusion of 50, 100 mg/kg timosaponin, once a day continuously for 7 days, and then the middle cerebral artery occlusion （MCAO） was established in rats by filament method.②Pentoxifylline （PTX） group （n=t2）： The rats were injected with 45.88 mg/kg PTX daily intragastrically for successive 7 days, and then the acute local ischemia model was established identically as above. In addition, the neurological deficits and cerebral infarction area were estimated.③Model group （n=30）： The rats were injected with equal dose of saline for 7 days, and then the model establishment was the same as above.④Sham group （n=30）： The rats were injected with equal dose of saline for 7 days, and then the model establishment was the same as above, but without the endovascular filament for occlusion. Twenty-four hours after modeling, the neurological deficits of all the rats were scored, and ischemic brain tissues were fetched to compare the infarction areas after TTC staining. Radio immunoassay （RIA） was used to assay the level of ET-1 in iscbemic tissues, and immunohistocbemical method was applied to detect the expression of eNOS. RESULTS： Totally 87 rats entered into the result analysis.①Percentage of infarction area： It was lower in timosaponin groups and PTX group than in model group [（21.56±5.03）%, （23.80±3.59）%, （18.50±3.74）%, （28.61 ±2.76）%, P 〈 0.05, 0.01].②Score of neurological deficits： It was also lower in timosaponin groups and PTX group than in model group （P 〈 0.05, 0.01）. ③Level of ET-1： The level of model group was higher than that of control group significantly [（0.68±0.19）, （0.31±0.11） ng/g, P 〈 0.01], and higher than those of timosaponin groups [（0.43±0.06）, （0.36±0.20） ng/g, P 〈 0.05, 0.01]. However, there was insignificant difference in ET-1 level between 50 mg/kg and 100 mg/kg timosaponin groups. ④Expression of eNOS： Timosaponin at dosages of 50 and 100 mg/kg could increase the number of positive cells and the expression of eNOS in ischemic tissues. CONCLUSION： Timosaponin has definitive cerebral protective effect against cerebral ischemia-reperfusion injury, and the protective mechanism is probably related with the reduction of ET-1 level, elevation of eNOS expression, and improvement of endothelial function along with blood supply in isehemie tissues.