摘要
背景:葛根素在体外有抗缺血再灌注损伤的作用,改善微循环,抑制血小板聚集的作用,但葛根素保护脑缺血神经元损伤是否与细胞凋亡有关尚不清楚。目的:在细胞水平观察葛根素在体外模拟脑缺血再灌注诱导的神经元损伤中的保护作用。设计:随机对照的实验。单位:华中科技大学同济医学院基础医学院生物化学与分子生物学系。对象:实验于2002-03/11在华中科技大学同济医学院基础医学院生物化学与分子生物学系实验室完成。以培养的鼠成神经瘤细胞株N2a细胞为观察对象。方法:将培养的N2a细胞放入通有体积分数为0.05的CO2和体积分数为0.95的N2的37℃培养箱中培养90min后,加入葛根素(0.5mmol/L)后正常培养24h。采用MTT法观察细胞的生存能力,采用Annexin-V染色法检测早期细胞凋亡程度,收集培养上清分析乳酸脱氢酶酶活性反应细胞膜通透性。免疫印迹分析半胱氨酸天冬氨酸蛋白酶3的表达;同时检测半胱氨酸天冬氨酸蛋白酶3的活性。主要观察指标:各组细胞早期细胞凋亡程度,乳酸脱氢酶酶活性,半胱氨酸天冬氨酸蛋白酶3的表达和活性。结果:葛根素能显著提高N2a细胞模拟缺血再灌注24h后的存活率;显著降低培养液中乳酸脱氢酶活性;显著降低缺血再灌注的N2a细胞的凋亡程度(P<0.01);与此同时,葛根素可显著降低缺血再灌注诱导的半胱氨酸天冬氨酸蛋白酶3的活性及表达(P<0.01)。结论:葛根素具有神经保护作用,可显著抑制脑缺血再灌注诱导的N2a细胞凋亡,其作用机制与显著抑制半胱氨酸天冬氨酸蛋白酶3的表达及活性有关。
BACKGROUND： Puerarin tunetions to relieve the injury caused by ischemia reperfusion, improve the mieroeirculation, and inhibit the agglutination of platelet. But it is not clear yet that whether its protection on neurons relates to the apoptosis of cells. OBJECTIVE： To observe the protection of puerarin on neurons injured by mimic ischemia reperfusion in vitro. DESIGN： A random control study. SETTING： Laboratory of Bioehemistry and Moleculobiology Department of Preclinieal Medicine College of Tongji Medival College of Huazhong University of Science and Technology. PARTICIPANTS： The experiment was done on March 11, 2002. The cultured N2a cells of rat neuromablast were observed. METHODS： Ninety minutes after the euhured N2a cells were put into the 37℃ incubator with 0.05 CO2 and 0.95 N2（v/v）, puerarin 0.5 mmol/L was added for culture for 24 hours. The methyl thiazolyl tetrazolium （MTT） method was used to observe the surviving ability of cells. The Annexin-V staining was adopted to exam the severity of apoptosis in the early stage and the supernate was collected to analyze the activity of lactate dehydrogenase （LDH） to reflect the permeability of cytomembrane. The immunoblotting method was applied to analyze the expression of caspase-3, at the same time, its activity was measured. MAIN OUTCOME MEASURES： The severity of cell apoptosis, the activity of I,DH, the expression and activity of easpase-3. RESULTS： Puerarin couht improve significantly the surviving rate of N2a cells 24 hours after reperfusion, decrease remarkahly the activity of LDH in the culture fluid, lessen obviously the N2a apnptosis （P 〈 0.01）, at the same time, reduce tremendously the activity and expression of caspase-3 induced by ischemia reperfusion （P 〈 0.01）. CONCLUSION： Puerarin can protect nerves, inhibit obviously the N2a apoptosis induced by ischemia reperfusion. The meehanism is that it inhibits greatly the expression and activity of easpase-3.
出处
《中国临床康复》
CSCD
北大核心
2005年第33期190-192,F0003,共4页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助(30100057)~~
