Distinct Effects of Tea Catechins on 6-Hydroxydopamine-Induced Apoptosis in PC12 Cells

Archives of Biochemistry and Biophysics Vol. 397, No. 1, January 1, pp. 84 –90, 2002 doi:10.1006/abbi.2001.2636, available online at http://www.idealibrary.com on

Distinct Effects of Tea Catechins on 6-HydroxydopamineInduced Apoptosis in PC12 Cells Guangjun Nie, 1 Chaofang Jin,* ,1 Yuanlin Cao, Shengrong Shen,* and Baolu Zhao 2 Laboratory of Visual Information Processing, Department of Molecular and Cell Biophysics, Institute of Biophysics, Academia Sinica, Beijing 100101, China; and *Department of Tea, Zhejiang University, Hangzhou 310029, China

Received August 16, 2001, and in revised form October 10, 2001; published online December 4, 2001

Green tea polyphenols have aroused considerable attention in recent years for preventing oxidative stress related diseases including cancer, cardiovascular disease, and degenerative disease. Neurodegenerative diseases are cellular redox status dysfunction related diseases. The present study investigated the different effects of the five main components of green tea polyphenols on 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells, the in vitro model of Parkinson’s disease (PD). When the cells were treated with five catechins respectively for 30 min before exposure to 6-OHDA, (ⴚ)-epigallocatechins gallate (EGCG) and (ⴚ)-epicatechin gallate (ECG) in 50 –200 ␮M had obvious concentration-dependent protective effects on cell viability, while (ⴚ)-epicatechin (EC), (ⴙ)-catechin ((ⴙ)-C), and (ⴚ)-epigallocatechin (EGC) had almost no protective effects. The five catechins also showed the same pattern described above of the different effects against 6-OHDA-induced cell apoptotic characteristics as analyzed by cell viability, fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis methods. The present results indicated that 200 ␮M EGCG or ECG led to significant inhibition against typical apoptotic characteristics of PC12 cells, while other catechins had little protective effect against 6-OHDA-induced cell death. Therefore, the classified protective effects of the five catechins were in the order ECG>EGCGⰇEC>(ⴙ)-CⰇEGC. The antiapoptotic activities appear to be structurally related to the 3-gallate group of green tea polyphenols. The present data indicate that EGCG and ECG might be potent neuroprotective agents for PD. © 2001 Elsevier Science

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G. Nie and C. Jin contributed equally to this work. To whom correspondence should be addressed at Institute of Biophysics, Academia Sinica, 15 Datun Road, Chaoyang District, Beijing 100101, China. Fax: 8610-64871293. E-mail: [email protected]. 2

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Key Words: Parkinson’s disease; apoptosis; green tea polyphenols; 6-OHDA; antioxidant; PC12 cells.

Parkinson’s disease (PD) 3 is a neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra zona compacta and a decrease in the neurotransmitter dopamine content in the striatum (1, 2). Arvid Carlsson and his colleagues discovered that PD resulted from a marked lack of dopamine in certain parts of the brain and the supplement with L-dopa had efficiently therapeutic benefits for treatment of the disease (3). Very recently, neuroprotective strategies have been introduced as well (4 – 6). Since the causes of PD are mainly oxidative stress and mitochondrial dysfunction (7, 8), antioxidants, free radical scavengers, monoamine oxidase inhibitors, and other such drugs have the potential for therapeutic development used to cure PD. Green tea polyphenols, as natural antioxidants and efficient free radical scavengers, have diverse pharmacological activities, such as antimutagenic and anticarcinogenic effects (9 –11). In the central nervous system, oral administration of green tea polyphenols or flavonoid-related compounds has preventive effects on iron-induced lipid peroxide accumulation and age-related accumulation of neurotoxic lipid peroxides in rat brain (12, 13). 3 Abbreviations used: EGCG, (⫺)-epigallocatechins gallate; ECG, (⫺)-epicatechin gallate; EGC, (⫺)-epigallocatechin; EC, (⫺)-epicatechin; (⫹)-C, (⫹)-catechin; 6-OHDA, 6-hydroxydopamine; ROS, reactive oxygen species; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide); PI, propidium iodide; EB, ethidium bromiole; PD, Parkinson’s disease; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; Abs, absorbance; NAC, N-acetylcysteine; ARE, antioxidant response element; TAE, Tris-Acetate-EDTA buffers; DPPH, 1,1-diphenyl-2-picrylhydrazyl; AMVN, 2,2⬘-azobis(2,4dimethylvaleronitrile); MPAK, mitogen-activated protein kinase.

0003-9861/01 $35.00 © 2001 Elsevier Science All rights reserved.

EFFECTS OF TEA CATECHINS ON APOPTOSIS

FIG. 1.

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Chemical structures of green tea polyphenols.

Salah et al. investigated the antioxidant activities of tea catechins against aqueous phase radicals and as chain-breaking antioxidants (14). Our previous data indicated that green tea polyphenols showed antioxidant activities higher than those of vitamin C and vitamin E (15). The studies of the relationship between chemical structures and their antioxidant activities of (⫺)-epigallocatechin gallate (EGCG), (⫺)-epicatechin gallate (ECG), (⫺)-epigallocatechin (EGC), and (⫺)-epicatechin (EC), the four components of green tea polyphenols (Fig. 1), demonstrated that the galloyl group in the C ring was very important for their antioxidant properties against lipid peroxidation in synaptosomes (16, 17). Tea catechins show excellent antioxidative activities against both hydrophilic and hydrophobic free radicals. However, their antioxidative activities are dependent on not only their chemical structures but also the different microenvironments, which is consistent with the observation of vitamin E, GSH, NAD(P)H, and several plant phenols’ antioxidants (18). These typical antioxidants also show prooxidant properties under some special conditions (18). Therefore, it is important to determine the potential neuroprotective capabilities of green tea polyphenols in the different reactive systems and to elucidate the relationship between their structures and functions.

6-Hydroxydopamine (6-OHDA)-induced apoptosis in rat pheochromocytoma PC12 cells was well established as an in vitro model for PD. 6-OHDA is a selective catecholaminergic neurotoxin (19) and the cytotoxicity of this neurotoxin is based on the damage of dopaminergic neurons in two ways: it easily forms free radicals and it is a potent inhibitor of the mitochondrial respiratory chain complexes I and IV (20). Under physiologic conditions, 6-OHDA is rapidly and nonenzymatically oxidized by molecular oxygen to form hydrogen peroxide and the corresponding p-quinone (21). The former can react with iron(II) to form the reactive and damaging hydroxyl free radical. In view of this autoxidation process and the formation of reactive oxygen species (ROS) by 6-OHDA, EGCG and other catechins are expected to have beneficial effects on the oxidative stress-mediated pathogenesis process. In the present study, we studied the effects of five catechins of green tea polyphenols on 6-OHDA-induced apoptosis and the relationship between their structures and functions. MATERIALS AND METHODS Materials. PC12 cells were supplied by Dr. Xiaomin Wang at the Medical School of Beijing University in China. RPMI 1640 cell cul-

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FIG. 2. Effects of 6-OHDA on PC12 cell viability. PC12 cells were incubated in drug-free medium or medium containing different concentrations of 6-OHDA (0, 100, 250, 300, and 500 ␮M). Cell viability was estimated by MTT assay after the cells were treated with 6-OHDA for 12, 24, and 36 h. Data are means ⫾ SD; n ⫽ 7. *P ⬍ 0.01 compared with normal cells (6-OHDA ⫽ 0). ture medium, newborn calf serum, donor horse serum, Hepes, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from GIBCO Life Technologies. 6-OHDA, trypsin, sodium pyruvate, dimethyl sulfoxide (DMSO), propidium iodide (PI), ethidium bromiole (EB), agarose, Proteinase K, DNA mark, Hoechst 33258, and poly-D-lysine (30,000 –70,000) were obtained from Sigma. EGCG, ECG, EGC, EC, and (⫹)-C were kindly provided by Zhejiang University. All other chemicals made in China were analytical grade. Cell culture. The rat pheochromocytoma PC12 cells were incubated in RPMI 1640 medium supplemented with 10% heat-inactivated donor horse serum, 5% heat-inactivated newborn calf serum, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 10 mM Hepes, 1.0 mM sodium pyruvate, and 1.5 g/L NaHCO 3. All experiments were performed in cells plated at a density of 2 ⫻ 10 5 cells/ml on 96-well plates or at a density of 2 ⫻ 10 6 cells/ml in poly-D-lysine-coated 35-mm dishes or 25-cm 2 flasks. The cells were incubated at 37°C in 5% CO 2/95% air with 100% relative humidity. After 36 – 48 h, the cells were exposed to 6-OHDA alone or pretreated with different concentrations of the five catechins for 30 min. Measurement of cell viability by MTT assay. MTT assay was performed as described previously with modification (22). After a period of incubation, the viable cells could convert the soluble dye MTT to insoluble blue formazan crystals. Briefly, at the indicated time after the treatment, 1 mg MTT (200 ␮l of a 5 mg/ml stock solution in phosphate-buffered saline (PBS)) was added to 1 ml medium and incubation continued at 37°C for 3 h. MTT was removed and the colored formazan was dissolved in DMSO. The absorbance (Abs) at 590 nm of each aliquot was determined using a Bio-Rad 3350 microplate reader. The viability of PC12 cells in each well was presented as the percentage of control cells. Morphological changes. The changes in nuclear morphology of apoptotic cells were investigated by labeling the cells with the nuclear stain Hoechst 33258 (HO33258) and examining them under fluorescent microscopy. Briefly, the PC12 cells preplated in 35-mm dishes (5 ⫻ 10 5 cells/dish) were treated with 6-OHDA at the different concentrations. Catechins were added 30 min before 6-OHDA addition if necessary. The treatments were continued for 24 h, and then the cells were fixed, stained with Hoechst 33258 (3 ␮mg/ml), and observed under fluorescence microscopy. The apoptotic cells were distinguished from control cells by the presence of a fragmented or highly condensed nucleus. Flow cytometry analysis. Flow cytometry analysis was performed as described previously with minor modification (23). Cells were

trypsinized as a single cell suspension, harvested by centrifugation, and washed with PBS. After being fixed in ice-cold 70% ethanol at ⫺20°C overnight, the cells were collected by 300g centrifugation for 5 min, washed twice with PBS, resuspended in PBS supplemented with RNase A (100 ␮g/ml), and incubated at 37°C for 30 min. Then the cells were stained with PI (3 ␮g/ml) at 4°C for 30 min and analyzed by flow cytometry (Coulter EPICS XL, USA). Analysis of DNA fragmentation. After the treatment, the cells were precipitated by centrifugation at 300g, washed twice with PBS, then incubated with lysis buffer (100 mM NaCl; 10 mM Tris–HCl, pH 8.0; 25 mM EDTA, pH 8.0; 0.5% SDS), and treated with Proteinase K (0.1 mg/ml) at 50°C for 10 h. The nuclear lysates were extracted twice with phenol– chloroform–isoamyl ethanol (25:24:1). Then DNA was precipitated with 0.1-volume of 10 mM ammonium acetate and 2 vol ice-cold ethanol at ⫺20°C overnight. The DNA was pelleted by centrifugation at 12,000g for 20 min at 4°C, washed twice with 70% ethanol, and dried in air. After the DNA was resuspended in 30 ␮l TE (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) and 4 ␮l RNase A for another 1.5 h at 37°C, DNA was electrophoresed in a 1.2% agarose gel in TAE buffer (40 mM Tris–HCl, 20 mM acetic acid, and 1 mM Na 2EDTA, pH 8.0). The gel was stained with 0.5 ␮g/ml EB, visualized by UV transillumination, and photographed. Statistical analysis. Data were analyzed by the Student t test and presented as means ⫾ standard deviations. P ⬍ 0.05 was considered significant.

RESULTS

Different Effects of Five Catechins on PC12 Cell Viability PC12 cell viability was greatly reduced when exposed to 6-OHDA and the cytotoxicity of 6-OHDA was concentration and time dependent (Fig. 2). The survival rate of PC12 was about 63 and 45% when the cells were treated with 250 ␮M of 6-OHDA for 24 and 36 h, respectively. Treatment with catechins for 30 min before PC12 cells were exposed to 250 ␮M 6-OHDA resulted in the cell viability being rescued (Fig. 3). At 50 –100 ␮M, all catechins had little effect on the cell viability. While at 200 ␮M, their effects were signifi-

FIG. 3. Effects of five catechins on 6-OHDA-induced decrease of PC12 cell viability. Cell viability was estimated by MTT assay after treatment with 6-OHDA for 24 h. Different concentrations of five catechins were added 30 min before 250 ␮M 6-OHDA treatments. Data are means ⫾ SD; n ⫽ 7. *P ⬍ 0.05 and *P ⬍ 0.01 compared with control cell group (without tea catechins).

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DNA was degraded to fragments of low molecular weight and subsequently leaked out from the cells, and DNA content was decreased. When a number of apoptotic cells were stained with a DNA-specific fluorochrome, propidium iodide (PI), a special DNA peak (usually called sub-G 1 peak or apoptotic peak) appeared. This peak is thought to be one of the characteristics of apoptosis. Figures 5 and 6 show the ratios of apoptotic cells. 6-OHDA caused 32.5% cells to undergo apoptosis. One hundred and two hundred micromolar EGCG or ECG inhibited the cytotoxicity of 6-OHDA significantly. The survival ratios were about 88.9 and 95.4% for 100 and 200 ␮M EGCG, respectively; In the case of 100 and 200 ␮M ECG, the survival ratios were about 79.6 and 96.6%, respectively. The survival ratios of the cells in other catechin cases were lower than 65% in contrast. Therefore, the protective effect of the five catechins was classified in the order ECG⬎EGCGⰇEC(⫹)C⬎EGC. The results are consistent with the results of the cell viability analysis described previously. The Results of Agarose Gel Electrophoresis of DNA

FIG. 4. Fluorescence micrographs of PC12 cell nuclei from untreated cells (A); cells exposed to 250 ␮M 6-OHDA for 24 h (B); or cells preincubated with 200 ␮M EGCG (C), ECG (D), EC (E), (⫹)-C (F), or EGC (G). The cells were stained with the DNA-binding fluorochrome Hoechst 33258. Scale bar ⫽ 30 ␮m.

cantly enhanced: the cell viability rate was more than 90% when the cells were treated with EGCG or ECG before being damaged by 6-OHDA; while in the cases of EC or (⫹)-C, the survival rate was about 70 – 80%. However, EGC decreased the cell viability to about 50% (Fig. 3). Therefore, the decreased order of the protective effects of the five catechins was classified as ECGⱖEGCGⰇECⱖ(⫹)-C⬎EGC. Protective Effects of Green Tea Polyphenols on the Changes in Nuclear Morphology Apoptotic nuclei indicated by condensed nuclei and nuclear fragmentation was apparent after exposure to 250 ␮M 6-OHDA for 24 h (Fig. 4B). These changes in nuclear characteristics of apoptosis were rescued significantly in the cells treated with 200 ␮M EGCG or ECG (Figs. 4C and 4D). Other catechins in 200 ␮M had little effect (Figs. 4E– 4G). Protective Effect of Green Tea Polyphenols on Apoptosis of PC12 Cells Flow cytometry was further performed to determine the apoptotic cells. In the cells undergoing apoptosis,

One of the important hallmarks of apoptosis is DNA fragmentation into multiples of 180 –200 bp, which appears as a typical “DNA laddering” pattern on DNA electrophoresis gel. The obvious DNA ladder was observed in the cells treated with 250 ␮M of 6-OHDA for 24 h (Fig. 7). Figure 7 shows the results of DNA electrophoresis in the cells pretreated with the five catechins at a concentration of 200 ␮M before exposure to 6-OHDA. Catechins inhibited 6-OHDA-induced DNA fragmentation. 6-OHDA-induced DNA fragmentation was rescued by 200 ␮M EGCG or ECG, but not by other catechins at the experimental concentrations. DISCUSSION

The selective loss of dopaminergic neurons in the substantia nigra is the direct cause of neurodegeneration in PD. Recently, apoptosis has also been suggested to be involved in this process (24, 25). In vitro, 6-OHDA-induced apoptosis in PC12 cells, which secrete dopamine and possess a dopamine transporter, is frequently used as a cell model for PD. 6-OHDA is a mitochondrial complex I inhibitor, and it can reproduce PD-like cell damage in vivo (26). The mechanism of 6-OHDA-induced toxicity has been summarized in two ways (20, 21, 27–29). (1) One is via extracellular toxicity: 6-OHDA generates H 2O 2 and other ROS through autooxidation to damage cells (28 –32). (2) The other way is that 6-OHDA is taken up by PC12 cells via the dopamine transporter (26) and its effect on PC12 cells is to generate intracellular ROS (33) either by direct mitochondrial respiratory chain complexes I and IV inhibition or by enzymatic deamination through mono-

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FIG. 5. Flow cytometric analysis of PC12 cell after 24-h treatment: normal cells (1); exposure to 250 ␮M 6-OHDA only (2); and before 250 ␮M 6-OHDA treatment, addition of 200 ␮M EGCG (3), ECG (4), EGC (5), EC (6), or (⫹)-C (7). The x axis is DNA content and the y axis is the number of cells.

amine oxidase. But through our experimental observation and the results from Soto-Otero (21), we see that generation of H 2O 2 and other ROS through 6-OHDA autooxidation plays an essential role in 6-OHDA-induced apoptosis. Spectrophotometric monitoring of the autooxidation of 6-OHDA showed a rapid increase (within several minutes) in absorbance at 480 nm and the corresponding cell culture solution manifested a rapid formation of red chromophores (21). Our data indicate that the catechins, especially EGCG and ECG, are potent neuroprotective agents

FIG. 6. The percentage of apoptotic PC12 cells after 24-h treatment, determined by flow cytometry. The cells were exposed to 250 ␮M 6-OHDA only or preincubated with 50 –200 ␮M EGCG, ECG, EGC, EC, or (⫹)-C for 30 min before 6-OHDA treatment. All the experiments were done three times independently. *P ⬍ 0.05 and **P ⬍ 0.01 in comparison with 6-OHDA damaged cell groups.

and have inhibitory effects on 6-OHDA-induced toxicity. One of the possible mechanisms may be their directly scavenging ROS produced either outside or inside the cell or both. Our previous data have shown that green tea polyphenols can scavenge different kinds of reactive oxygen species and organic free radicals, for example, superoxide anion, hydroxyl radical, singlet oxygen, DPPH, AMVN, and lipid free radicals (15–17). Sulfhydryl compounds, such as N-acetylcysteine (NAC), glutathione (GSH), and cysteine, especially cysteine, can remove the H 2O 2 formed during the autooxidation of 6-OHDA and reduce the reaction rate (21). Green tea polyphenols may react with H 2O 2 directly or prevent the Fenton reaction between Fe 2⫹ and H 2O 2 to form hydroxyl radicals. Therefore, the presence of green tea polyphenols appears to represent a significant protective agent against oxidative stress. It is believed that the scavenging efficiency of the catechins depends on their chemical structures, their oxidation potential, their acid dissociation constant, the diffusion rate of the molecules in the cellular membrane system, and the concentrations available inside the cells. The studies on the structure–antioxidant activity relationship of catechins (EGCG, ECG, EGC, and EC, and their corresponding epimers) suggest that the active sites of green tea polyphenols reacted with oxygen free radicals are an ortho-hydroxyl group in the B-ring and the galloyl moiety in the C-ring (16). The gallate group in the C-ring is the most important factor contributing to the antioxidant efficiency. The study of

EFFECTS OF TEA CATECHINS ON APOPTOSIS

FIG. 7. Agarose gel electrophoresis of DNA extracted from cells exposed to 250 ␮M 6-OHDA for 24 h. DNA mark (1), normal cells (2), cells damaged with 250 ␮M 6-OHDA only (3), or cells pretreated with 200 ␮M EGCG (4, 6), ECG (5), EGC (7), EC (8), or (⫹)-C (9) and then treated with 250 ␮M 6-OHDA. The molecular weights of the DNA markers are shown on the right side of the figure.

four catechins scavenging lipid peroxidation of synaptosomes also showed the same trend when scavenging lipid free radicals and the decreased order is ECG⬎EGCG⬎EC⬎EGC (16). The antioxidant activity of phenols is shown to be closely related to their redox potentials (34, 35). EGCG and ECG with the gallate group possess the lowest oxidation peak potentials (0.23 and 0.27 V vs SCE, respectively) and have the most antioxidative activities. While EC without the gallate group possesses the higher oxidation peak potential (0.33 V vs SCE) and is the least active in scavenging lipid free radicals (36). Because the molecular polarity affects the penetrating efficiency of the biological membrane greatly, the difference in hydrophobicity of the catechins may contribute to the different inhibitory effects on 6-OHDA autooxidation. We measured the partition coefficients of the green tea polyphenols in octanol/water and liposome systems (unpublished data). The decreased hydrophobic order of them is ECG⬎GCG⬎(⫹)-C⬎C⬎GC. The results show that EGCG and ECG are more hydrophobic than EC, (⫹)-C, and EGC. From the partition results, we can find that most green tea polyphenols distribute in the octanol or lipsome phase, especially in the liposome phase. That is, the more

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hydrophobic the green tea polyphenols, the more they distribute into the membrane system. Therefore, in the PC12 cell system, we propose that most green tea polyphenols penetrate the cell membrane when the partition reactions reach equilibrium. At the same time, the more hydrophobic the green tea polyphenols, the faster the partition reactions reach equilibrium. In conclusion, the molecular polarity of the five catechins is deemed to affect their ability to suppress 6-OHDAinduced cytotoxicity. Some evidence recently shows that green tea polyphenols can induce and activate some special gene expression and modulate the cellular apoptosis signaling (37, 38). At low concentrations, EGCG and ECG activation of MAPK leads to antioxidant response element (ARE) gene expression including phase II detoxifying enzymes, which can increase the ability of the cellular antioxidant defense system. The induction of ARE reporter gene appears to be structurally related to the 3-gallate group of green tea polyphenols (38), suggesting that EGCG and ECG are more effective neuroprotective agents in activating phase II detoxifying enzymes than EGC, (⫹)-C, and EC, which lack the 3-gallate group in their structures. Unlike most naturally occurring catecholamines, 6-OHDA undergoes rapid autooxidation at or near neutral pH, giving rise to quinoidal compounds. In the presence of reducing agents such as ascorbate, redox cycling takes place, with a large consumption of oxygen and the production of superoxide and other reactive species such as the hydroxyl radicals (39). These free radicals will attenuate the antioxidant’s defense system of the cells and aggravate the cell damage. There are some antioxidants, such as vitamin E, GSH, NAD(P)H, and several plant phenols (18), that can be observed as prooxidants under some special conditions, for example, the accumulation of free status of copper or iron under pathological conditions. It can be postulated that ascorbate– copper or iron–ascorbate complex can recycle the formation of free radicals and stimulate cell damage and even lead to cell apoptosis. The relatively antioxidative capacities, the bioavailability, the ratio of antioxidant and free transition metal, and the microenvironment together determine the fate of the antioxidants. Green tea polyphenols can pass through the brain– blood barrier to exert neuroprotective effects. Sixty minutes after the oral administration of green tea polyphenols, low concentrations of EGCG were present in plasma, liver, and brain (40, 41), although most of the green tea polyphenols were absorbed into the gastrointestinal tract. Yoneda et al. showed that the oral administration of a flavonoid-rich tea extract prevents iron–salt-induced lipid peroxide accumulation in the rat brain (13). Inanami et al. reported that oral administration of rooibos tea containing flavonoid derivatives

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suppresses age-related accumulation of neurotoxic lipid peroxides in rat brain and that oral administration of (⫺)catechin protects against ischemia–reperfusion-induced neuronal death in Mongolian gerbils (42). All these results suggest that green tea polyphenols can pass through the brain– blood barrier and penetrate into the brain to exert neuroprotective effects. Our data indicate that EGCG and ECG possess neuroprotective properties. EGCG is the main component of green tea polyphenols and the contribution of EGCG to the total antioxidant activity of green tea polyphenols almost takes one-third of all the antioxidant activities. EGCG may play the major role in the neuroprotective effects against 6-OHDA-induced cell toxicity. In conclusion, the results of this study show that EGCG and ECG have neuroprotective effects on 6-OHDA-induced apoptosis in PC12 cells and these neuroprotective effects depend on their chemical structures. The mechanism may be related to their antioxidant activities and/or regulation of special gene expression properties. ACKNOWLEDGMENTS We appreciate Dr. Qi Chen from the Salk Institute for Biological studies La Jolla, California, for reviewing this manuscript. This work was supported by a grant from the National Natural Science Foundation of China.

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