摘要
建立鱼藤酮(rotenone)诱导的大鼠肾上腺髓质嗜铬细胞瘤细胞(Pheochromocytoma,PC12)损伤的类帕金森病细胞模型,探讨益智仁中的原儿茶酸(PCA)对该类帕金森病细胞模型的保护作用及其可能作用机制.采用四唑盐(MTT)方法检测细胞活力,并且采用乳酸脱氢酶(LDH)方法作为细胞活力的进一步验证;对于细胞形态变化采用Hoechst 33258染色法进行观察;采用细胞内常见的抗氧化酶试剂盒(超氧化物歧化酶(SOD),过氧化氢酶(CAT),谷胱苷肽过氧化物酶(GSH-Px))分别检测细胞内各抗氧化酶含量的变化;对于细胞内活性氧(ROS)水平的变化采用2',7'-二氯荧光素二乙酸(DCF-DA)进行检测;用Caspase-3/CPP32 Colorimetric试剂盒检测细胞内Caspase-3的活性.将0.5μM鱼藤酮作用于PC12细胞48 h后,诱导其产生典型间接氧化应激过程并产生凋亡特征,益智仁中的1 m M原儿茶酸对鱼藤酮诱导的PC12细胞损伤有明显的抗凋亡保护作用,其可能机制是增强细胞内源性抗氧化酶的活力,抑制活性氧的产生,阻止Caspase-3的激活途径,从而达到减少或抑制细胞凋亡的目的.
To establish a damage model induced by rotenone in PC12 cells and assess the effect and its mechanism of protocatechuic acid from Alpinia oxyphylla on protecting PC12 cells from damage induced by rotenone.The cell viability was analyzed by M TT method and further verified by lactate dehydrogenase（ LDH） assay.The morphological changes were observed by Hoechst33258 staining assay.The activities of antioxidant enzymes in PC12 cells was quantified by thesuperoxide dismutase（ SOD） Assay Kit,catalase（ CAT） Assay Kit,and glutathione peroxidase（ GSH-Px） Assay Kit.The relative level of reactive oxygen species（ ROS） was measured by intracellularly oxidizing the dye 2＇,7＇-dichlorofluorescin diacetate（ DCF-DA） to the fluorescent 2＇,7＇-dichlorofluorescin（ DCF） in the presence of oxidants.The activity of caspase-3 was determined using the Caspase-3 / CPP32 Colorimetric Assay Kit.Application with 0.5 μM rotenone for 48 h induced apoptosis by indirect oxidative stress,concentration of 1 m M PCA have the obvious antiapoptotic effect.Protocatechuic acid from Alpinia oxyphylla could significantly protect PC12 cells from damage induced by rotenone.The mechanism of it may be increasing the activities of cellular antioxidant enzymes,suppressing the formation of intracellular ROS,counteracting the activation of caspae-3 so as to prevent the cellular apoptosis.
出处
《辽宁大学学报（自然科学版）》
CAS
2016年第1期61-67,共7页
Journal of Liaoning University：Natural Sciences Edition
