doi:10.1016/j.freeradbiomed.2005.04.022 
Copyright © 2005 Elsevier Inc. All rights reserved.
Original Contribution
Protective effect of green tea polyphenols on the SH-SY5Y cells against 6-OHDA induced apoptosis through ROS–NO pathway
Shuhong Guoa, Erwan Bezardb and Baolu Zhaoa, 
, 
aLaboratory of Visual Information processing, Center of Brain & Cognitive Science, Institute of Biophysics, Academia Sinica, Beijing 100101, People's Republic of China
bBasal Gang, CNRS UMR5543, 146 Rue Leo Saignat, 33076 Bordeaux Cedex, France
Received 11 November 2004;
revised 18 April 2005;
accepted 25 April 2005.
Available online 13 May 2005.
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Abstract
Green tea polyphenols (GTP) are thought to help prevent oxidative stress-related diseases, such as cancer, cardiovascular disease, neurodegenerative disease, and aging. We here investigate the protective mechanisms of GTP on SH-SY5Y cells against apoptosis induced by the pro-parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). GTP rescued the changes in condensed nuclear and apoptotic bodies, attenuated 6-OHDA-induced early apoptosis, prevented the decrease in mitochondrial membrane potential, and suppressed accumulation of reactive oxygen species (ROS) and of intracellular free Ca2+. GTP also counteracted the 6-OHDA-induced nitric oxide increase and overexpression of nNOS and iNOS, and decreased the level of protein-bound 3-nitrotyrosine (3-NT). In addition, GTP inhibited the autooxidation of 6-OHDA and scavenged oxygen free radicals in a dose- and time-dependent manner. Our results show that the protective effects of GTP on SH-SY5Y cells are mediated, at least in part, by controlling the ROS–NO pathway.
Keywords: Parkinson's disease; Green tea polyphenols; Neuroprotection; Apoptosis; Reactive oxygen species; Nitric oxide; Peroxynitrite
Abbreviations: 6-OHDA, 6-hydroxydopamine; DAF-2DA, 4,5-diaminofluorescein diacetate; DCF-DA, 2′,7′-dichlorofluoresecein diacetate; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; EGCG, (−)-epigallocatechin gallate; ECG, (−)-epicatechin gallate; EGC, (−)-epigallocatechin; EC, (−)-epicatechin; EDTA, ethylenediaminotetraacetic acid; eNOS, endothelial cell nitric oxide synthase; Fluo-3 AM, Fluo-3 acetoxymethyl ester; GSH, glutathione reduced; GTP, green tea polyphenols; iNOS, inducible nitric oxide synthase; L-NMMA, NG-methy-l-arginine acetate salt; 3-NT, 3-nitro-l-tyrosine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; ONOO−, peroxynitrite; PBS, phosphate-buffered saline; PD, Parkinson's disease; PI, propidium iodide; ROS, reactive oxygen species
Fig. 1. Effect of 6-OHDA and GTP on SH-SY5Y cell viability. Cells were incubated in drug-free medium or medium containing different concentrations of 6-OHDA (A) for 24 h (F(11,35) = 243.2; P = 0). (B) Cells were preincubated with different concentrations of GTP for 1 h, 6-OHDA (100 μM) was added for an additional 24 h (●) or the cells were preincubated with different concentrations of GTP for 1 h and washed with medium for three times, and then 6-OHDA (100 μM) was added for an additional 24 h (■) (F(11,35) = 114.8; P = 0). Data are expressed as percentage of the untreated control ± SE, n = 3. *P < 0.01 significantly different from control cells; #P < 0.01 compared with 6-OHDA group cells by ANOVA.
Fig. 2. Effects of tea catechins on SY5Y cell viability. (A) Cells were incubated in drug-free medium or medium containing different concentrations of tea catechins for 24 h (F(5,89) = 8.67; P < 0.0001). After 1 h cells were pretreated with various concentrations of tea catechins (B) or with various concentrations of tea catechins and then washed with medium for three times (C), with GSH (D) or L-NMMA (E), and then the cells were incubated with 100 μM 6-OHDA for 24 h (B, F(5,89) = 2.93, P < 0.05; C, F(5,89) = 3.48957, P < 0.05; D, F(10,87) = 140.01, P = 0; E, F(7,63) = 63.99, P = 0). Data are expressed as percentage of the untreated control ± SE, *P < 0.05 and **P < 0.01 compared with untreated control cells (A, D, E) or with 6-OHDA group (B, C), ##P < 0.01 compared with 6-OHDA group cells (D, E) by ANOVA.
Fig. 3. Fluorescence micrographs of SH-SY5Y cell nuclei from untreated cells (A); cells exposed to 100 μM 6-OHDA (B) or GTP (C) for 24 h; or cells preincubated with 50 μM (D), 100 μM (E), and 200 μM (F) GTP. The cells were stained with the DNA-binding fluorochrome Hoechst 33258. Scale bar = 50 μm.
Fig. 4. Cell apoptosis and necrosis detected by flow cytometry. SH-SY5Y cells were incubated in drug-free medium (A) or medium containing 100 μM 6-OHDA (B) or GTP (C) for 24 h; or cells were preincubated with 50 μM (D), 100 μM (E), and 200 μM (F) GTP and then exposed to 6-OHDA for 24 h. The results shown in (G) are the means and SE for three independent experiments.
Fig. 5. GTP attenuate 6-OHDA-induced accumulation of ROS. Cells were exposed to 6-OHDA without or with different concentrations of GTP for 24 h. Data are the means ± SE of a ratio between fluorescence intensity and protein content. n = 3. F(5,17) = 704.8, P < 0.0001. **P < 0.01 compared with control cells, ## P < 0.01 compared with 6-OHDA-treated cells.
Fig. 6. GTP attenuate 6-OHDA-induced decrease of mitochondrial membrane potential. Cells exposed to 6-OHDA with or without GTP were incubated with rhodamine 123, and then the fluorescence intensity was measured. MMP of control is defined as 100% and the data are expressed as percentage of the untreated control ± SE, n = 5, F(5,29) = 76.9, P < 0.0001. *P < 0.05, **P < 0.01 compared with control cells, ##P < 0.01 compared with 6-OHDA-treated cells.
Fig. 7. GTP attenuate 6-OHDA-induced elevation of intracellular [Ca2+]i in SY5Y. Cells exposed to 6-OHDA and (or) GTP were incubated with Fluo-3 AM, and [Ca2+]i was measured. Data are the means ± SE of a ratio between fluorescence intensity and protein content. n = 5. F(5,29) = 32.4, P < 0.0001. *P < 0.01 compared with control cells, # P < 0.01 compared with 6-OHDA-treated cells.
Fig. 8. GTP attenuate 6-OHDA-induced increase of intracellular NO in SY5Y. Cells were incubated with DAF-2DA, and then the fluorescence intensity was recorded. Data are expressed as percentage of the untreated control ± SE, n = 8, F(5,47) = 38.5, P < 0.0001. **P < 0.01 compared with control cells, #P < 0.05, ##P < 0.01 compared with 6-OHDA-treated cells.
Fig. 9. Effect of GTP and 6-OHDA on the expression of nNOS and iNOS. SY5Y cells were exposed to 6-OHDA (100 μM) with or without various concentrations of GTP for 24 h, and the nNOS and iNOS were detected in the cell lysates by Western blot. (A) The protein levels and date analysis for nNOS (B) and iNOS (C). The data are analyzed by the ratio of the band intensity of nNOS and iNOS over that of actin, expressed as ratio ± SE, n = 5, F(5,29) = 32.4, P < 0.0001. *P < 0.05 and **P < 0.01 compared with control cells, ##P < 0.01 compared with 6-OHDA-treated cells.
Fig. 10. GTP attenuate 6-OHDA-induced elevation of protein-bound 3-NT in SY5Y. Cells were exposed to 6-OHDA and (or) GTP, and then 3-NT was measured (F(5,17) = 704.8, P < 0.0001) (A). (B) The standard curve of 3-NT. *P < 0.05, **P < 0.01 compared with control cells, ##P < 0.01 compared with 6-OHDA-treated cells.
Fig.11. Effect of GTP on the generation of ROS during the autooxidation process of 6-OHDA. ROS generation was detected using luminol in vitro. The integral of the signal peak reflects the formation of total ROS. Inserted figure shows the signal intensity of autooxidation of 25 μM 6-OHDA with or without different concentrations of GTP. Data are expressed as the ratio of integral area of the peaks ± SE, n = 5, F(7,39) = 76.9, P = 0. *P < 0.05 and **P < 0.01 compared with 6-OHDA group by ANOVA.
Fig. 12. Effect of GTP on the generation of p-quinone free radicals during the autooxidation process of 6-OHDA. (A) 10 mM 6-OHDA and different concentrations of GTP were incubated for 10 min at room temperature in vitro (F(4,24) = 236.8, P < 0.0001). (B) 10 mM 6-OHDA and 10 mM GTP were incubated for the indicated time periods (F(4,24) = 254.8, P < 0.0001). Data are expressed as the scavenge rate of GTP ± SE, n = 5, *P < 0.01 compared with 6-OHDA group by ANOVA.
Fig. 13. A hypothetical model diagram of the potential targets of GTP or EGCG is suggested in cell signaling pathways affected by 6-OHDA-induced oxidative stress: (1) direct inhibition of oxidized 6-OHDA and/or scavenging of ROS; (2) inhibiting the negative effect of 6-OHDA on both PKC and ERK1/2, and EGCG can direct phosphorylative activation of PKC; (3) attenuating NF-κB translocation to the nucleus, and inhibiting it activation; (4) modulating the expression of cell death and cell cycle genes; and (5) modulating the intracellular NO level and inhibiting the generation of peroxynitrite.
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