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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 has anti-ischemic reperfusion injury in vitro, improves microcirculation and inhibits platelet aggregation. However, it is unclear whether puerarin protects cerebral ischemic neurons against apoptosis. OBJECTIVE: To observe the protective effect of puerarin on the neuronal damage induced by cerebral ischemia-reperfusion in vitro. Design: Randomized controlled experiments. Unit: Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology. PARTICIPANTS: The experiment was performed at the Laboratory of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology from March to November 2002. The cultured mouse neuroblastoma cell line N2a cells were observed. METHODS: The cultured N2a cells were cultured in a 37°C incubator with a volume fraction of 0.05 in CO2 and a volume fraction of 0.95 for 90 min, and then puerarin (0.5 mmol/L) was added for 24 h. MTT assay was used to observe the viability of the cells. Annexin-V staining was used to detect the degree of early cell apoptosis. The culture supernatant was collected to analyze the lactate dehydrogenase enzyme activity and cell membrane permeability. Western blot analysis of caspase 3 expression; simultaneous detection of caspase 3 activity. MAIN OUTCOME MEASURES: Early cell apoptosis, lactate dehydrogenase enzyme activity, and caspase-3 expression and activity in each group. RESULTS: Puerarin could significantly increase the survival rate of N2a cells after simulated ischemia-reperfusion for 24h; significantly decreased the activity of lactate dehydrogenase in culture medium; and significantly decreased the apoptosis of N2a cells after ischemia-reperfusion (P<0.01). At the same time, puerarin significantly reduced the activity and expression of caspase 3 induced by ischemia-reperfusion (P<0.01). CONCLUSION: Puerarin has a neuroprotective effect and can significantly inhibit the apoptosis of N2a cells induced by cerebral ischemia-reperfusion. The mechanism is related to the significant inhibition of the expression and activity of caspase-3.