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目的:研究褐藻多糖硫酸酯(fucoidan)对过氧化氢(H2O2)诱导的大鼠嗜铬细胞瘤细胞株(pheochromocytomacells,PC12)凋亡的影响,探讨fucoidan对阿尔茨海默病(Alzheimer′s disease,AD)的作用机制。方法:50μg.L-1神经生长因子(nerve growth factor,NGF)诱导分化PC12细胞(differentiated PC12,dPC12)7 d,0.5 mmol.L-1H2O2刺激30 min造成dPC12细胞凋亡。磺酰罗丹明B(sulforhodamine B sodium salt,SRB)法检测不同质量浓度fucoidan(0.1,1,10,50,100 mg.L-1)对细胞存活的影响,筛选适宜药物浓度;荧光底物法检测凋亡细胞内caspase-3的活性及细胞浆中溶酶体组织蛋白酶D的活性;酶-底物法检测不同质量浓度fucoidan(0.01,0.1,1,10,100 mg.L-1)对人肝脏溶酶体组织蛋白酶D活性的影响。结果:fucoidan能够拮抗H2O2所致细胞损伤,以10 mg.L-1作用最为明显;并能降低异常升高的凋亡执行酶caspase-3的活性。fucoidan对H2O2损伤后dPC12细胞胞浆内和人肝脏提取的溶酶体组织蛋白酶D活性均具有抑制作用。结论:fucoidan可以抑制H2O2诱导的dPC12细胞凋亡,其作用机制可能与抑制溶酶体组织蛋白酶D的活性有关。
Objective: To investigate the effect of fucoidan on the apoptosis of rat hepatocellular carcinoma cell line pheochromocytomacells (PC12) induced by hydrogen peroxide (H2O2), and to investigate the effect of fucoidan on the apoptosis of Alzheimer’s disease , AD) mechanism of action. Methods: DPC12 cells were induced apoptosis by differentiating PC12 cells (differentiated PC12, dPC12) by nerve growth factor (NGF) for 7 d and stimulation with 0.5 mmol.L-1H2O2 for 30 min. The effect of fucoidan (0.1,1,10,50,100 mg.L-1) on cell survival was detected by sulforhodamine B sodium salt (SRB) method, and the suitable drug concentration was screened. The fluorescence substrate method was used to detect the apoptosis The activity of intracellular caspase-3 and the activity of lysosomal cathepsin D in cytoplasm. The enzyme-substrate method was used to detect the effect of fucoidan (0.01,0.1,1,10,100 mg.L-1) Effect of body cathepsin D activity. Results: Fucoidan could antagonize the H2O2-induced cell injury, and the most obvious effect was at 10 mg.L-1. The fucoidan could reduce the activity of caspase-3, an abnormally elevated apoptosis-inducing enzyme. Fucoidan has an inhibitory effect on the activity of lysosomal cathepsin D extracted from cytoplasm and human liver of dPC12 cells after H2O2 injury. Conclusion: fucoidan can inhibit the apoptosis of dPC12 cells induced by H2O2, which may be related to the inhibition of lysosomal cathepsin D activity.