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

Abstract

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

CLC Number:

R965

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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