没食子酸对H2O2诱导的SH-SY5Y神经细胞损伤保护作用的研究

神经药理学报 ›› 2012, Vol. 2 ›› Issue (2): 7-14.

没食子酸对H₂O₂诱导的SH-SY5Y神经细胞损伤保护作用的研究

郭春燕¹，张丹参²

1 河北北方学院药学系，张家口，075000，中国
2 河北科技大学，石家庄，050018，中国

出版日期:2012-04-26 发布日期:2013-11-01
通讯作者: 张丹参，女，教授，博士生导师；研究方向：神经药理学；Tel：+86-0311-81668016，E-mail:zhangds2011@ 126. com
作者简介:郭春燕，女，教授，博士；研究方向：中药有效成分药理学；Tel：+86-0313-4029423，E-mail: guochy0311@163.com
基金资助:
国家自然基金课题(No. 81274005)，国家自然基金课题(No.81073120)，河北省自然科学基金项目(No.C2011405014)，河北省自然科学基金项目(No. C2009001026)

Protective Effects of Gallic Acid Against Hydrogen Peroxide-induced Oxidative Damage in SH-SY5Y Cells

GUO Chun-yan¹, ZHANG Dan-shen²

1 Department of Pharmacy, Hebei North University, Zhangjiakou, 075000,China
2 Hebei University of Science and Technology, Shijiazhuang, 050018, China

Online:2012-04-26 Published:2013-11-01
Contact: 张丹参，女，教授，博士生导师；研究方向：神经药理学；Tel：+86-0311-81668016，E-mail:zhangds2011@ 126. com
About author:郭春燕，女，教授，博士；研究方向：中药有效成分药理学；Tel：+86-0313-4029423，E-mail: guochy0311@163.com
Supported by:
国家自然基金课题(No. 81274005)，国家自然基金课题(No.81073120)，河北省自然科学基金项目(No.C2011405014)，河北省自然科学基金项目(No. C2009001026)

摘要/Abstract

摘要： 目的：以H₂O₂诱导SH-SY5Y细胞氧化损伤为模型，探讨没食子酸对H₂O₂诱导SH-SY5Y细胞氧化损伤的影响及其机制。方法：筛选没食子酸(gallic acid，GA)对SH-SY5Y细胞无毒剂量范围，以此剂量范围研究没食子酸对H₂O₂诱导SH-SY5Y细胞氧化损伤的保护作用。将细胞分为5组:正常对照组(Control)，H₂O₂模型组(Model)，GA 5µmol•L^-1组(GA1)、GA 10µmol•L^-1组(GA2) 和GA25µmol•L^-1组(GA3)。GA组先加入GA预处理1 h后再加入终浓度为200 µmol•L^-1H₂O₂。Hoechst 33258 染色观察凋亡细胞的形态学特征；碘化丙啶染色流式细胞仪检测细胞周期构成比；diaphorase催化的INT显色反应检测乳酸脱氢酶(lactate dehydrogenase, LDH)释放量；DCFH-DA检测细胞内活性氧(reactive oxygen species, ROS)；利用天冬氨酸蛋白水解酶-3(caspase-3)可以催化底物Ac-DEVD-pNA的反应检测caspase-3活性。结果：过氧化氢组与正常对照组相比，终浓度为200 μmol•L^-1 H₂O₂作用于细胞24 h后，细胞活力下降为65%，差异具有统计学意义(P<0.01)，细胞凋亡率上升(P<0.01)；明显出现了细胞凋亡的形态学特征；增殖指数(proliferation index, PI)降低(P<0.05)； LDH释放量和细胞内ROS增加(P<0.01)，caspase-3活性增强(P<0.01)。与H₂O₂损伤组相比，终浓度为5～25 µmol•L^-1GA能显著改善上述指标的变化(P<0.05)。结论：GA在一定剂量范围内对H₂O₂诱导的 SH-SYSY神经细胞损伤具有明显的保护作用，其保护效应可能与清除ROS，减轻DNA氧化损伤，抑制线粒体通路介导的细胞凋亡有关。

关键词: 没食子酸, 过氧化氢, SH-SY5Y, 细胞凋亡, ELISA(enzyme linked immunosorbent assay)

Abstract: Objective：This study examined the neuroprotective effects of GA on SH-SY5Y cells injured by H₂O₂ and the molecular mechanisms underlying these neuroprotective effects. Method:CCK-8 assay was performed to determine the non-toxic dose range of gallic acid in SH SY5Y cells. Cultured human neuroblastoma SH-SY5Y cells were subjected to oxidative damage with H₂O₂ in the presence and absence of non-toxic doses of GA. The growth of the cells was analyzed by plotting the cell growth curves and by CCK-8 assay. The cell morphological changes were observed by inverted optical microscope. Typical morphological features of apoptotic cells were detected using Hoechst 33258 staining. FCM was used to analyze cell cycle alteration using propidium iodide staining; the release rate of LDH was determined by diaphorase-INT reaction. ROS production was determined by DCFH-DAfluorescence. 8-OHdG production was determined by ELISA. Caspase-3 activity was determined by its capacity to catalyze the substrate Ac-DEVD-pNA. Results: H₂O₂ treatment produced dose-dependent and time-dependent cytotoxicity compared with that of the normal control. 200 μmol•L^-1 H₂O₂ exposure for 24 h decreased the cell viability to 65% of control (P<0.01), the typical changes of cell apoptosis were seen, proliferation index was significantly decreased (P<0.05), and the apoptosis index was significantly increased(P<0.01). In addition, H₂O₂ significantly increased the LDH release and the level of intracellular ROS. H₂O₂-induced oxidative stress also increased the caspase-3 activity (P<0.01). GA (5～25 µmol•L^-1) significantly and dose-dependently ameliorated the abovementioned cellular and biochemical changes (P<0.05).Conclusion: Our study revealed that GA has neuroprotective effects against H₂O₂-induced oxidative damage，and the potential mechanisms could involve eliminating ROS, attenuating DNA oxidative damage and inhibiting mitochondria mediated apoptosis.

Key words: Gallic acid, Hydrogen Peroxide, SH-SY5Y, Apoptosis, ELISA

中图分类号:

R965

郭春燕，张丹参. 没食子酸对H₂O₂诱导的SH-SY5Y神经细胞损伤保护作用的研究[J]. 神经药理学报, 2012, 2(2): 7-14.

GUO Chun-yan1, ZHANG Dan-shen2. Protective Effects of Gallic Acid Against Hydrogen Peroxide-induced Oxidative Damage in SH-SY5Y Cells[J]. Acta Neuropharmacologica, 2012, 2(2): 7-14.

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没食子酸对H₂O₂诱导的SH-SY5Y神经细胞损伤保护作用的研究

Protective Effects of Gallic Acid Against Hydrogen Peroxide-induced Oxidative Damage in SH-SY5Y Cells

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