(–)-Epigallocatechin Gallate InhibitsLipopolysaccharide-Induced MicroglialActivation and Protects AgainstInflammation-Mediated DopaminergicNeuronal Injury

Rui Li,1,2,3 Yuan-Gui Huang,3 Du Fang,1,2,3 and Wei-Dong Le1,2*1Health Science Center, Shanghai Institute for Biological Science, Chinese Academy of Science, andShanghai Second Medical University, Shanghai, Peoples Republic of China2Institutes of Bio-Medical Sciences, Ruijin Hospital, Shanghai Second Medical University, Shanghai,Peoples Republic of China3Department of Neurology, Xijing Hospital, The Fourth Military Medical University, Xi’an,Peoples Republic of China

Microglial activation is believed to play a pivotal role inthe selective neuronal injury associated with several neu-rodegenerative disorders, including Parkinson’s disease(PD) and Alzheimer’s disease. We provide evidence that(–)-epigallocatechin gallate (EGCG), a major monomer ofgreen tea polyphenols, potently inhibits lipopolysaccha-ride (LPS)-activated microglial secretion of nitric oxide(NO) and tumor necrosis factor-� (TNF-�) through thedown-regulation of inducible NO synthase and TNF-�expression. In addition, EGCG exerted significant protec-tion against microglial activation-induced neuronal injuryboth in the human dopaminergic cell line SH-SY5Y and inprimary rat mesencephalic cultures. Our study demon-strates that EGCG is a potent inhibitor of microglial ac-tivation and thus is a useful candidate for a therapeuticapproach to alleviating microglia-mediated dopaminer-gic neuronal injury in PD. © 2004 Wiley-Liss, Inc.

Key words: green tea polyphenol; nitric oxide; tumornecrosis factor; Parkinson disease; tyrosine hydroxylase

Increasing evidence demonstrates that the ongoinginflammatory process in the brain is critically involved inthe pathogenesis of a variety of neurodegenerative disor-ders, including Parkinson’s disease (PD) and Alzheimer’sdisease (AD) (McGeer et al., 1988, 2001; Vila et al., 2001).Microglial cells, the major resident immunoreactive pop-ulation in the brain, are actively involved in the patho-logical inflammatory events. In response to a variety ofinsults, such as toxicant agents, trauma, ischemia, andmodified cell debris, microglia are promptly activated andproduce an excess of proinflammatory factors, includingtumor necrosis factor-� (TNF-�), interleukin 1� (IL-1�),nitric oxide (NO), and reactive oxygen species, whichmay trigger or exacerbate neuronal death (Hunot et al.,1996; Le et al., 2001).

Selective dopaminergic cell degeneration in the sub-stantia nigra (SN) of the midbrain is the hallmark of PD,the cause of which is not clearly known. Dopaminergiccells are vulnerable to microglial activation-induced injury(Kim et al., 2000). In 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP)-treated mice, a classic animalmodel of PD, microglial activation in the SN precedesdegeneration of dopaminergic neurons, but not vice versa(Czlonkowska et al., 1996). Moreover, administration ofimmunoglobin from patients with PD into the SN of ratscaused selective dopaminergic neuronal loss in vivo (Chenet al., 1998), in which microglial activation may play animportant role (Le et al., 2001). Notably, administration oflipopolysaccharide (LPS), a potent inflammation pro-moter, caused highly selective dopaminergic cell loss in themurine SN that mimicked some pathologic features foundin humans with PD (Gao et al., 2002; Liu et al., 2003).Chronic administration of rotenone reproduced some fea-tures of PD, including selective dopaminergic neuronalloss in the SN as a result of microglial activation in thisregion (Sherer et al., 2003). cDNA microarray analysis inthe MPTP model of PD has documented that the inflam-matory response in the SN is an early event associated withthe neuronal injury (Mandel et al., 2003). Several agents,such as monocycline, naloxone, vasoactive peptide (VIP),

Contract grant sponsor: National Science Foundation of China; Contractgrant number: 30370491; Contract grant sponsor: National Academy ofScience, China; 100-Talent Project; Contract grant number: 2002298.

*Correspondence to: Wei-Dong Le, MD, PhD, 1201 Science at Technol-ogy Building, Ruijin Hospital, Shanghai Second Medical University,Shanghai, P.R. China. E-mail: wdle@SIBS.ac.cn

Received 1 June 2004; Revised 4 August 2004; Accepted 16 August 2004

Published online 11 October 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jnr.20315

Journal of Neuroscience Research 78:723–731 (2004)

© 2004 Wiley-Liss, Inc.

dextromethorphan, and silymarin, have been elucidated asdopaminergic neuroprotectives, which is correlated withtheir inhibitory effects on microglial activation (Lu et al.,2000; Wu et al., 2002; Wang et al., 2002; Delgado andGanea, 2003; Liu et al., 2003). These findings suggest thatinhibition of microglial activation is a prospective targetfor dampening neurodegenerative disorders, especiallyPD.

Green tea polyphenols, the major components ofgreen tea, are regarded as the main sources of green tea’sbioactivity. Apart from putative multiple biological prop-erties in antioxidation and antiapoptosis, green tea poly-phenols show their potential neuroprotective effects inanimal models of PD, AD, and ischemic stroke (Pan et al.,2003; Mandel et al., 2004). Previous studies showed thatgreen tea and its polyphenols can protect dopaminergic neu-rons against 1-methyl-4-phenylpyridinium (MPP�)-inducedinjury both in vitro and in vivo (Levites et al., 2001; Pan etal., 2003). Other groups have reported that green teapolyphenols can modulate the proinflammatory cytokines,such as iNOS and TNF-�, and the associated intracellularsignaling pathways in peripheral macrophages (Yang et al.,1998; Calixto et al., 2004). Here we provide evidencethat a monomer of the green tea polyphenols,(–)-epigallocatechin gallate (EGCG), can inhibit themicroglia-induced neuronal death via direct modulationof microglial activation.

MATERIALS AND METHODS

Materials

LPS from Escherichia coli (serotype 0111:B4), DNase I,laminin, poly-L-lysine, EGCG (purity �95%), all-trans-retinoicacid, rabbit monoclonal antibody against iNOS, mouse mono-clonal antibody against tyrosine hydroxylase (TH), and micro-tubule-associated protein-2 (MAP-2) were purchased fromSigma (St. Louis, MO). An enzyme-linked immunosorbentassay (ELISA) kit specific for rat TNF-� was purchased from BDBiosciences Clontech (Palo Alto, CA). Griess reagents werefrom Beyondtime Company (Hang Zhou, China).1,1�-Dioctadecyl-3,3,3�,3�-tetramethyl-indocarbocyanine-LDL(DiI-Ac-LDL) was from Biomedical Technologies (Stoughton,MA). Vectastain ABC Kit and biotinylated secondary antibodieswere obtained from Vector Laboratories (Burlingame, CA).Flasks and plates were purchased from Nunc (Glostrup,Denmark). DMEM, heat-inactivated endotoxin-free fetal bo-vine serum (FBS) and Trizol reagents were obtained from In-vitrogen (Carlsbad, CA). An AMV reverse transcription systemwas purchased from Promega (Madison, WI). The SuperSignaldetection kit was purchased from Chemicon (Temecula, CA).

Microglial Cultures and Cell Purification

Microglia were isolated and purified from Spraque-Dawley rat brains at postnatal days 1–2 (Experimental AnimalCenter of Shanghai). Briefly, after the rat brains were dissectedand the meninges were carefully removed, cerebral corticaltissue was minced mechanically and digested with trypsin(0.25%) and DNase I (0.01%). Dissociated cells were resus-pended in DMEM supplemented with 10% FBS and seeded in

75-cm2 flasks at a density of 5 � 106 cells per flask. The cellswere cultured in 5% CO2 at 37°C incubator, and the mediumwas changed every 3–4 days. Two weeks after seeding, whenthe mixed culture cells were grown at 80% confluence, the flaskswere shaken at 180 rpm in an orbit shaker for 5 hr at 37°C, andthe floating cells were collected and transferred to a new flask toallow adherence for 1 hr before being gently shaken. Theattached cells were collected and plated in 24-well plates previ-ously coated with poly-L-lysine (10 �g/ml) and maintained inDMEM supplemented with 2% FBS for 24 hr before furtherexperimental treatment. Microglial purity was evaluated by DiI-Ac-LDL labeling, a cell marker for microglia, showing �98%purity. To activate the microglia, 500 ng/ml LPS was added tothe cultures. In some wells, EGCG at �1–100 �M was added30 min before LPS treatment to study its effects on microglialinhibition. After 24 hr of incubation with LPS, with or withoutEGCG addition, culture media were centrifuged to remove thedetached cells from the media, and the supernatant was collectedand used as conditioned medium (CM).

Differentiated Dopaminergic SH-SY5Y Cells andPrimary Mesencephalic Cultures

Neuroblastoma SH-SY5Y cells were seeded at a density of1 � 105/ml and differentiated with 10 �M all-trans-retinoicacid (RA) in DMEM supplemented with 2% FBS for 48 hr in a96-well plate previously coated with poly-L-lysine (10 �g/ml).Culturing of primary neurons from embryonic rat mesenceph-alon was performed according to a method described previously(Crawford et al., 1992), with some modifications. Briefly, themesencephalic region was dissected from embryonic day 14 ratbrain and then minced and digested with trypsin (0.025%) andDNase I (0.01%). After mechanical dissociation by pipetting, thecells were washed twice with DMEM containing 10% FBS, thenseeded at a density of 1.5 � 105 per well in 96-well platespreviously coated with poly-L-lysine (10 �g/ml) and laminin(2 �g/ml). Cells were grown in defined DMEM media for5 days. The cultured cells were then transferred into the indi-cated CM for 48 hr for TH and MAP-2 immunostaining. Thecomposition of the cultures at the time of treatment was ap-proximately 82% neurons in which 25% were TH-immuno-reactive and about 18% were glia.

DiI-Ac-LDL Labeling

DiI-Ac-LDL is taken up specifically by microglia, and theDiI (fluorescent probe) is accumulated in the intracellular mem-branes that can be viewed under fluorescence microscope (Giu-lian and Baker, 1986). Purified microglia were incubated in thepresence of 5 �g/ml DiI-Ac-LDL for 4 hr at 37°C. After carefulremoval of the media, microglia were rinsed with DMEM threetimes and then visualized via fluorescence microscope.

NO Determination in the CM

The production of NO was determined by the measure-ment of nitrite, a stable product of NO, which reflects accumu-lated NO in the media, by using the Griess reagents. Briefly,100 �l of media were mixed with 100 �l of Griess reagent in a96-well plate. After a 15-min reaction, the plate was mounted inan ELISA reader at 540 nm with the reference filter set at

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630 nm. The nitrite concentration was determined from a so-dium nitrite standard curve (Si et al., 1997).

Cell Injury Assay by MTT

We used the 3,[4,5-dimethylthiazol-2-yl]-diphenyl-tetrazolium bromide (MTT) assay to determine the neuronalSH-SY5Y cell viability after the cells were incubated with CMcollected from microglia cultures. To exclude the possibility ofcell injury induced by LPS in the CM, a high concentration ofLPS (1,000 ng/ml) was added to some wells. Briefly, at the endof the indicated experiment, 20 �l of the dye MTT (5 mg/ml)was added to each well and the plates were incubated for 3 hr at37°C. After this incubation, 100 �l of lysis buffer [20% sodiumdodecyl sulfate (SDS) in 50% N,N-dimethylformamide, con-taining 0.5% (v:v) 80% acetic acid and 0.4% (v:v) 1 N HCl] wasadded to each well, and the color intensity (proportional to thenumber of live cells) was assessed with a microplate reader at the570 nm wavelength. Each experiment was performed in tripli-cate.

iNOS Detection by Western Blot

Purified microglia grown at a density of 1 � 106 insix-well plates cultured and treated for the indicated time at37°C were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH7.4, 150 mM NaCl, 0.1% SDS, 1 mM EDTA, 1% TritonX-100, 1% sodium deoxycholate, 1 mM phenylmethylsulfonylfluoride, 5 �g/ml aprotinin, 5 �g/ml leupeptin). After mea-surement of the protein concentration by the Bradford methodwith a kit, 20 �g of protein homogenate was loaded intoSDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 7% gelfor electrophoresis. The separated protein was then transferredto the nitrocellulose (NC) membrane and incubated overnightwith anti-iNOS rabbit antibody (1:2,000). Subsequent steps forWestern blotting detection were performed with the SuperSig-nal detection kit as described in the manufacturer’s guidebook.A duplicated sample was run on a 12% SDS-PAGE and thendetected with �-actin antibody as an internal control.

TNF-� Assay by ELISA

Levels of TNF-� secreted by microglia in the media weredetermined by using an ELISA assay kit provided by BD Bio-sciences Clontech. All the procedures were performed followingthe manufacturer’s instruction manual.

Semiquantitative RT-PCR for the Detection ofMicroglial TNF-� mRNA

To determine whether the inhibition of microglial secre-tion of TNF-� was the result of regulation of TNF-� transcrip-tion by EGCG, purified microglia (at a density of 1 � 106) werestimulated with LPS (500 ng/ml), with or without EGCGpretreatment. After 24 hr incubation, total microglial RNA wasextracted with Trizol reagents. cDNA was synthesized from1 �g of the total RNA with a reverse transcription kit by AMVreverse transcriptase. PCR of the cDNA was performed at thedenatured conditions for 45 sec at 94°C, annealing for 45 sec at48°C for TNF-� and 55°C for GADPH, with an extension for45 sec at 72°C, and at the end of 35 cycles a further extensionfor 5 min at 72°C. Amplification of the housekeeping enzymeGAPDH gene was always involved to serve as a control

of reaction efficacy. Primers for TNF-� were 5�-CCA-ACAAGGAGGAGAAGT-3� for forward and 5�-GTATGA-AGTGG- CAAATCG-3� for reverse (product fragment323 bp), whereas those for GAPDH were 5�-GCTGGG-GCTCACCTGAAGGG-3� for forward and 5�-GGATGA-CCTTGCCACGCC-3� for reverse (product fragment 343 bp).The synthesized PCR products were separated by electrophore-sis on a 2% agarose gel and analyzed by Gel-Pro analyzer version3.1 software (Media Cybernetics). The ratio of arbitrary unit oftarget genes over GAPDH was used for expressing the relativelevels of mRNA expression.

MAP-2 and TH Immunocytochemistry in PrimaryMesencephalic Cultures

To investigate neuronal injury in primary neuronal cul-tures from embryonic rat mesencephalon induced by activatedmicroglia, CM, MAP-2, and TH immunohistochemistry wereused to stain neurons and dopaminergic neurons specifically inthe cultures. After the treatment, cultured cells were washedwith phosphate-buffered saline (PBS) three times and fixed with4% paraformaldehyde for 30 min at room temperature. Primaryantibody against MAP-2 (rabbit anti-rat, 1:2,000 in dilution)was incubated with cultured primary mesencephalic cells andthen detected with Alex Fluor 488-labeled goat anti-rabbitsecond antibody and viewed under a fluorescence microscope.Primary antibody against TH (1:6,000 in dilution) was detectedsubsequently with an ABC kit and developed with diaminoben-zidine (DAB) staining. For quantitation of MAP-2-and TH-immunoreactive cells, 10 fields per well (113 mm2 surface area)were counted with a premarked frame lens. The size of field was1 mm2, and the 10 fields consisted of 10% of the whole surfaceof the well.

Statistical Analysis

All values were shown as mean SEM. Statistical signif-icance between groups was assessed by using one-way ANOVA,followed by post hoc Duncan multiple comparisons with theSPSS 10.0 program (SPSS Inc., Chicago, IL). P 0.05 wasconsidered significant.

RESULTS

EGCG Inhibits LPS-Induced MorphologicalChange of Microglia

Highly purified (�98%, as assessed by DiI-ac-LDLlabeling) microglia, when grown in DMEM supplementedwith 2% FBS for 2 days, appeared typically ramified andeither bipolar or unipolar, and these were considered“resting microglia” (Fig. 1A). After incubation with500 ng/ml LPS for 24 hr, most microglia underwentdramatic morphological changes characteristic of activatedmicroglia which appeared round with enlarged and amoe-boid cell bodies (Fig. 1B). Pretreatment with �1–100 �MEGCG partially abolished LPS-induced morphologicalchanges in the cultured microglia (Fig. 1C). EGCG alonedid not induce any visible morphological changes in mi-croglia.

EGCG Inhibits Microglia and Protects DA Neurons 725

EGCG Inhibits NO and TNF-� Release FromLPS-Activated Microglia

The cultured resting microglia produced min-imal NO and TNF-�. In response to LPS exposure(500 ng/ml), microglia released significantly high levels ofNO and TNF-�. The media levels of NO released fromLPS-activated microglia were elevated in a time-dependent manner with an increase of 1.5-fold at 3 hr,2.5-fold at 6 hr, 4.2-fold at 12 hr, and 5.1-fold at 24 hr.Pretreatment with EGCG (10 �M) significantly inhibitedmicroglial secretion of NO by 74–86% at the indicatedtime points (Fig. 2). TNF-� levels were also increased inthe media of cultured microglia following the 24 hr in-cubation with 500 ng/ml LPS, showing 28-fold higherthan that of resting microglia (Fig. 3). EGCG (1, 10, and100 �M) pretreatment significantly inhibited TNF-� lev-els in the media of LPS-treated microglia cultures by 67%(for 1 �M) to 91% (for 100 �M), as indicated by ELISAassay for TNF-� (Fig. 3).

EGCG Down-Regulates iNOS in ActivatedMicroglia

To determine 1) whether increased NO release inthe activated microglia culture media was the result ofelevated microglial-derived iNOS expression and 2)whether the inhibition of NO production in activatedmicroglia by EGCG was associated with down-regulationof microglial cytoplasmic iNOS, purified microglia werecollected after 24 hr of incubation with LPS (500 ng/ml),

Fig. 1. Morphological changes in purified microglia labeled with DiI-Ac-LDL. Rat microglia were incubated for 1 day with vehicle (A), LPS(500 ng/ml; B), or EGCG (10 �M; C) 30 min before LPS challenge.Note that, after being treated with LPS, microglia became larger andround, whereas cells pretreated with EGCG maintained a “resting”appearance. Scale bar � 100 �m.

Fig. 2. NO release from microglia cultures and the inhibitory effect ofEGCG. Purified microglia were challenged with 500 ng/ml LPS inDMEM media for 24 hr, and EGCG (10 �M) was added 30 min beforeLPS administration. Nitrite levels in the microglia culture media col-lected at different time points were determined by Griess reaction.Values were obtained from three independent experiments. *P 0.05compared with LPS treatment at the same time point.

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with or without pretreatment with EGCG. Cell extractswere run on SDS-PAGE and processed for immunoblotanalysis to detect iNOS protein. The protein levels ofiNOS were undetectable in the resting microglia (data notshown) and in the resting microglia treated with EGCGalone (Fig. 4). After exposure to LPS (500 ng/ml) for24 hr, iNOS levels were remarkably elevated (Fig. 4) butwere significantly inhibited by EGCG pretreatment in adose-dependent manner, with a remarkable inhibitory ef-fect at 1 �M (41% inhibition) and 10 �M (63% inhibition)EGCG and a more dramatic inhibitory effect at 100 �MEGCG (82%), as controlled by �-actin (Fig. 4).

Down-Regulation of TNF-� mRNA Transcriptionin Microglia by EGCG

Resting microglia expressed a noticeable amount ofTNF-� transcription, which was used as a basal level tocompare with the level of activated microglia after ex-posed to LPS (500 ng/ml) for 24 hr (Fig. 5, lane 1). LPSaddition stimulated a prompt increase in TNF-� mRNAby 5.8-fold. Pretreatment with 1, 10, and 100 �M EGCGsignificantly suppressed LPS-induced elevation of TNF-�mRNA. EGCG alone did not affect TNF-� mRNAtranscription (Fig. 5).

Reduced SH-SY5Y Neuronal Injury by CM FromEGCG Pretreated Microglial Cultures

Human neuroblastoma SH-SY5Y cells typically ex-hibit rhombus-like morphology with or without short

processes when grown in the DMEM supplemented 10%FBS (Fig. 6A). In response to the addition of 10 �M RAfor 48 hr to the DMEM media with low concentration ofFBS (2%), SH-SY5Y cells became thin and fusiform-like,with prolonged neurite-like processes and increased cell–cell contacts (Fig. 6B–D). After differentiation, neuronalSH-SY5Y cultures were transferred into resting or acti-vated microglia CM for 24 hr. As indicated by the MTTassay, LPS-activated microglia CM caused a remarkablereduction in cell viability by 51%. Media from cells pre-treated with 1, 10, and 100 �M EGCG before LPSstimulation ameliorated the reduction of cell viability by24%, 36%, and 67% respectively (Fig. 6E). High-concentration LPS (1,000 ng/ml) alone did not causedetectable changes in SH-SY5Y cell viability.

CM From EGCG-Pretreated Microglial CulturesReduces Neuronal Injury of PrimaryMesencephalic Cells

In the primary mesencephalic cultures, the transfer ofCM from LPS-treated microglia caused marked depletionof MAP-2-immunoreactive cells (30% depletion) andTH-immunoreactive cells (76% depletion; Fig. 7B,F),

Fig. 3. EGCG-mediated inhibition of microglial TNF-� release in themedia at 24 hr. Unprovoked microglia (resting microglia) producedminimal levels of TNF-� in the media. Incubation with 500 ng/ml LPSfor 24 hr induced a marked elevation (about 17-fold over control) ofTNF-� in the media. Preincubation with EGCG (�1–100 �M) in-hibited LPS-induced elevation of TNF-� in media by approximately67%, 84%, and 91% for 1, 10, and 100 �M, respectively. EGCG alonehad no effect on microglial TNF-� release. Values were obtained fromthree independent experiments. *P 0.01, **P 0.001 comparedwith LPS treatment.

Fig. 4. EGCG dose dependently inhibits LPS-induced iNOS increasein the microglia. After the appropriate treatment, 1 � 106 purifiedmicroglial cells were lysed with RIPA buffer, and 20 �g cytoplasmicproteins was separated by SDS-PAGE (7%), transferred to a NC mem-brane, and detected with a monoclonal antibody against iNOS. Thesame volume of homogenate was run on SDS-PAGE, with �-actindetection serving as an internal control (A). Densitometer analysis ofthe Western blot in A is shown in B. *P 0.01, **P 0.001compared with LPS treatment.

EGCG Inhibits Microglia and Protects DA Neurons 727

compared with untreated cell cultures (Fig. 7A,E), indi-cating that the predominant injured cell type in the cul-tures was TH-immunoreactive neurons. CM fromEGCG-pretreated microglial cultures 30 min prior to LPSadministration significantly ameliorated this depletion(Fig. 7C,G), whereas CM from EGCG-alone-treated mi-croglia had no effect on MAP-2 and TH immunoreactiv-ity (Fig. 7D,H). Quantitative MAP-2- and TH-immunoreactive cells analysis showed that CM fromEGCG-pretreated microglia in the primary mesencephaliccultures significantly reduced the loss of MAP-2-positivecells with 10 and 100 �M of EGCG pretreatment(Fig. 7I). Such CM administration ameliorated the loss ofTH-immunoreactive cells by 26% (1 �M EGCG), 53%(10 �M EGCG), and 73% (100 �M EGCG), respectively.

DISCUSSIONThe current understanding that chronic inflammation

in the brain plays an important role in the pathogenesis ofneurodegenerative disorders is based largely on mechanisticstudies of microglial activation-induced neuronal injury. Mi-croglia constitute approximately 10–15% of the cellular pop-ulation in the brain (Barron, 1995). Activation of microglia

Fig. 6. Activated microglia CM-induced cell injury of SH-SY5Y cellsand the protection of EGCG. A: SH-SY5Y in DMEM supplementedwith 10% FBS; note the rhombus-like morphology with or withoutshort processes. B: Addition of all-trans-retinoic acid (10 �M) inDMEM with 2% FBS for 48 hr induced an apparent cell differentiation.C: Incubation of CM from LPS-activated microglial culture for 24 hrsignificantly compromised cell viability and neurites. D: CM frommicroglial cultures pretreated with EGCG (10 �M) before LPS admin-istration largely protected against cell injury. E: Viable cells werecounted with the MTT assay, and values shown are percentage ofcontrol. Lanes 1–5 represent the CM from untreated microglia (restingmicroglia), CM from LPS (500 ng/ml) stimulated microglial culturesand CM from 1 �M, 10 �M, and 100 �M of EGCG-pretreatedmicroglial cultures, respectively. Lanes 6 and 7 represent the treatmentof EGCG (10 �M) alone and 1,000 ng/ml LPS, respectively. Viabilityof untreated differentiated SH-SY5Y cells were used as controls(lane 8). *P 0.05, **P 0.001 compared with the CM from LPS(500 ng/ml)-treated microglia (lane 2). #P � 0.05 compared with theresting microglia derived CM (lane 1). Scale bar � 50 �m.

Fig. 5. Down-regulation of reactive microglial TNF-� mRNA byEGCG. Purified microglia (1 � 106) were treated with or withoutEGCG for 30 min and then stimulated with 500 ng/ml LPS for 24 hr.Total RNA isolation, cDNA synthesis, and the PCR procedure aredescribed in the text. GAPDH served as an internal control (A). Figuresare representative of three independent experiments. Densitometeranalysis of the Western blot in A is shown in B. *P 0.05, **P 0.01compared with LPS treatment.

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underlies a large spectrum of pathological events in the cen-tral nervous system, especially in PD and AD, in which theactivation of microglia is often concomitant with selectiveneuronal death. Under pathological conditions, microglia donot merely engulf the destructed cell debris and fragments butare actively involved in the cell injury process. For instance,in the MPTP mouse model of PD, microglial activation

occurred as early as 1 day after the MPTP injection, whereasthe depletion of dopaminergic neurons was not pronounceduntil 7 days after the MPTP treatment (Czlonkowska et al.,1996). More interestingly, the same pattern can be observedin LPS-induced dopaminergic neuronal injury. Administra-tion of LPS in the rat brain triggered a rapid activation ofmicroglia, followed by a delayed and gradual loss of nigraldopaminergic neurons that began after 4–6 weeks andreached 70% at the end of the tenth week (Gao et al., 2002).Although activated microglia release some beneficial factors,such as glia-derived neurotrophic factor, it is generally ac-cepted that microglial activation in the brain can trigger orexacerbate neuronal damage.

Our findings indicate that phagocytosis of activatedmicroglia might not be necessary in the process of neuro-nal injury. The fact that CM from LPS-activated microgliaculture caused a significant reduction of SH-SY5Y cellviability and a loss of TH-positive cells in primary mes-encephalic cultures implies that molecules produced andreleased by activated microglia can directly cause neuronalinjury and death. Activated microglia release a number ofmolecules, including free oxygen radicals, NO, and proin-flammatory cytokines, such as TNF-� and interleukin-1�,which can synergistically impair neurons (Jeohn et al.,1998). It has been reported that CM from the LPS- oradvanced glycation end products (AGEs)-activated micro-glia caused neuronal cell death and retraction of neuriteextension in the differentiated neuroblastoma cell lineNeuro2a (Munch et al., 2003). In our study, SH-SY5Ycell injury induced by CM from activated microglia maynot be mediated by the LPS in the media, insofar as highconcentrations of LPS did not cause detectable cell death(Fig. 6). In primary mesencephalic cultures, CM fromLPS-activated microglial injured MAP-2-immunoreactivecells, which represented mature neurons in the primarymesencephalic culture system. However, TH-immuno-reactive cells were the major victims of the injury (Fig. 7I),indicating that dopaminergic neurons were more vulner-able to inflammation-induced cell injury.

We have previously reported that modified dopami-nergic cell membrane fragments, as well as IgG from PDpatients, can activate cultured microglia (Le et al., 2001).In addition, zymosan A (McMillian et al., 1997), tissueplasminogen activator (TPA; Siao and Tsirka, 2002),

Š

Fig. 7. CM-induced MAP-2- and TH-immunoreactive cell injury inprimary mesencephalic cultures. A–D show MAP-2-immunoreactivecells, and E–H show TH-immunoreactive cells. A,E: MAP-2- andTH-immunoreactive cells in untreated primary mesencephalic cultures,respectively. Incubation with the CM from LPS-treated (500 ng/ml,48 hr) microglia cultures significantly resulted in injury of MAP-2 (B)-and TH (F)-immunoreactive cells. Addition of CM from the microgliacultures pretreated with 10 �M EGCG 30 min before LPS remarkablyattenuated the cell injury (C,G); EGCG alone did not compromiseneurons in the cultures (D,H). Quantitative analysis of MAP-2- andTH-immunoreactive cells is shown in I. Data were obtained from threeindependent experiments. *P 0.05 compared with LPS-treated cul-tures; **P 0.05 compared with the control. Scale bars � 200 �m.

EGCG Inhibits Microglia and Protects DA Neurons 729

AGEs (Munch et al., 2003), rotenone (Sherer et al., 2003),and many others are potent microglial activators. Verysimilarly to LPS, these agents induce microglia from aresting to an activated state, resulting in increased gener-ation of free radicals, NO and elevated proinflammatoryfactors, leading to the neuronal injury and death. Thesefindings imply that microglial activation may follow thesame signal pathways, at least in the later stages, which mayprovide an opportunity to develop different approaches toinhibit microglial activation as a strategy to block theneuronal injury.

Because of the pivotal role of microglial activation inseveral neurodegenerative disorders, intervention againstmicroglial activation constitutes an effective way to pro-tect against neuronal injury, ultimately to impede theinitiation and progression of these disorders. Our studydemonstrates for the first time that a monomer originatedfrom green tea polyphenols, EGCG, has potent inhibitoryeffects on microglial activation as indicated by the mor-phologic study and assays of NO and TNF-�, the two keyproinflammatory and cytotoxic factors. Furthermore, ourresults show that EGCG-exerted microglial deactivation isneuroprotective in differentiated neuronal SH-SY5Y cellsand in primary mesencephalic cultures.

Green tea, rich in polyphenols, is one of the mostwidely consumed beverages in the world. EGCG is themost abundant catechin in tea and has been regarded as themajor source of the polyphenols’ bioactivities. In additionto its complex effects on cardiovascular diseases, stroke,cancer, hip fractures in women, and rheumatoid arthritis(Hertog et al., 1997; Sesso et al., 1999; Nakachi et al.,2000; Yang et al., 2000; Pan et al., 2003), recent studieshave focused on the neuroprotective properties of tea andtea extracts. Epidemiological findings show that consump-tion of green tea is positively associated with lower risk ofPD (Chan et al., 1998; Ascherio et al., 2001). Severalstudies reported that green tea polyphenols, more specif-ically EGCG, exerted neuroprotective actions against neu-rotoxicants [MPP�, 6-hydroxydopamine (6-OHDA), and�-amyloid] in neuronal cultures (Choi et al., 2001; Leviteset al., 2002; Nie et al., 2002; Pan et al., 2003). Studies invivo revealed that EGCG attenuated MPTP-induced do-paminergic neuron injury and global ischemia-inducedneuronal loss (Lee et al., 2000; Levites et al., 2001). Theunderlying neuroprotective mechanism is believed to beclosely associated with its antioxidant property, free radicalscavenging, iron chelating, catechol-O-methyltransferase ac-tivity reduction, protein kinase C (PKC) or extracellularsignal-regulated kinases activation, and cell survival/cell cyclegene modulation (Lee et al., 2000; Levites et al., 2001, Pan etal., 2003). Detailed data from genomic and proteomicanalysis also showed that antiinflammatory actions and therelated cell signal pathways, such as PKC activation andnuclear factor-�B (NF-�B) regulation, of the EGCG un-derlie its molecular neuroprotective mechanisms (Leviteset al., 2002; Mandel et al., 2003). However, becauseactivated microglia are the cardinal donor of free radicalsand inflammatory factors in the brain, our findings that

EGCG possesses inhibitory effects on microglial genera-tion of NO and TNF-� in vitro can be a reasonableexplanation for its neuroprotective effects in vivo.

We demonstrated neuroprotective effects of EGCGagainst microglia-induced dopaminergic cell injury in dif-ferentiated neuronal SH-SY5Y cells and in primary mes-encephalic cell cultures, and such neuroprotection wasclosely related to the EGCG’s direct inhibition on micro-glial activation. Although the detailed mechanisms under-lying EGCG’s inhibitory effects of microglia are not yetknown, we propose three possibilities. First, in the mo-lecular structure of EGCG contains a phenolic gallop thatis considered to be closely linked with its antiinflammatoryactivity (Ma and Kinneer, 2002). Second, the upstream ofTNF-� and iNOS mRNA transcription, NF-�B, is be-lieved to be the early response of inflammation. Green teapolyphenols intervenes with NF-�B activation and nu-clear translocation induced by 6-OHDA or LPS in severalcell lines (Lin and Lin, 1997; Levites et al., 2002). Finally,based on our finding that EGCG can potently inhibitmicroglial NO and TNF-� generation, and given thatreactive oxidative stress and inflammation are formed to bea vicious cycle, suppression of the two key factors, maylimit the deleterious feedback. However, we cannot ruleout the possibility of EGCG-induced changes in mole-cules other than the inhibition of NO and TNF-� releasedfrom microglia contributing to the EGCG-mediated neu-roprotection.

Because EGCG can penetrate the brain (Suganumaet al., 1998; Manal et al., 2002) and in light of the potentaction against microglial activation, EGCG may be a pro-spective candidate to alleviate microglial activation-involved neurodegenerative process. Our study also opensthe way for a broader spectrum of putative therapeuticapplications of EGCG.

ACKNOWLEDGMENTWe thank Dr. Y.Y Zhang (Shanghai Institute of

Immunology) for his valuable technical support in thisexperiment.

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