Green Tea Extract Ameliorates Learning and Memory Deficits in Ischemic Rats via Its Active Component Polyphenol Epigallocatechin-3-gallate by Modulation of Oxidative Stress and Neuroinflammation

Abstract

Ischemic stroke results in brain damage and behavioral deficits including memory impairment. Protective effects of green tea extract (GTex) and its major functional polyphenol (-)-epigallocatechin gallate (EGCG) on memory were examined in cerebral ischemic rats. GTex and EGCG were administered 1 hr before middle cerebral artery ligation in rats. GTex, EGCG, and pentoxifylline (PTX) significantly improved ishemic-induced memory impairment in a Morris water maze test. Malondialdehyde (MDA) levels, glutathione (GSH), and superoxide dismutase (SOD) activity in the cerebral cortex and hippocampus were increased by long-term treatment with GTex and EGCG. Both compounds were also associated with reduced cerebral infraction breakdown of MDA and GSH in the hippocampus. In in vitro experiments, EGCG had anti-inflammatory effects in BV-2 microglia cells. EGCG inhibited lipopolysaccharide- (LPS-) induced nitric oxide production and reduced cyclooxygenase-2 and inducible nitric oxide synthase expression in BV-2 cells. GTex and its active polyphenol EGCG improved learning and memory deficits in a cerebral ischemia animal model and such protection may be due to the reduction of oxidative stress and neuroinflammation.

Figure 1

Schedule of drug treatment and experiment orders. Green tea extract (GTex), EGCG, and PTX were administrated orally 1 h before the surgery. The oral administration to rat continued once daily for 7 days and 1 h prior to training or testing. Four to seven days after surgery, the spatial memory test (SMT) of the Morris water maze (MWM) was performed 4 trials a day for 3 consecutive days, followed 24 h later (day 7) by the reference memory test (RMT). Rats were sacrificed immediately after the behavioral test.

Figure 2

HPLC chromatograms of the GTex at 280 nm. Trace: (a) standard, (b) GTex. BHQ: tert-butylhydroquinone as an internal standard. (EGCG: (−)-epigallocatechin gallate, ECG: (−)-epigallocatechin, EGC: (−)-epicatechin gallate).

Figure 3

Effects of GTex and EGCG on cerebral infarction. (a) Effect of GTex (30~300 mg/kg, p.o.) groups and EGCG (10 mg/kg, p.o.) on cerebral infarct area at 24 h after reperfusion. The pale area represents infarct tissue and the red area normal tissue. (b) Infarction area by TTC staining (n = 6 in each group). I/R: ischemia/reperfusion control group. Each vertical bar represented mean ± S.E. *P < 0.05, ***P < 0.001 compared to I/R group. Scale bar = 1 cm.

Figure 4

Effect of GTex (30~300 mg/kg, p.o.), EGCG (10 mg/kg, p.o.), and pentoxifylline (PTX, 100 mg/kg, p.o.), on the swimming time took to reach the hidden platform of the Morris water maze in the ischemia/reperfusion (I/R) rats. **P < 0.01, ***P < 0.001 compared to the sham group. ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 compared to I/R group (n = 6 in each group).

Figure 5

Effect of GTex (30~300 mg/kg, p.o.), EGCG (10 mg/kg, p.o.), and pentoxifylline (PTX, 100 mg/kg, p.o.), on the time spent in the target quadrant in ischemia/reperfusion (I/R) rats. The performance of each rat was tested 24 hours after the final training day in a probe trial (60 sec) during which the platform was removed. *P < 0.05 compared to the sham group. ^# P < 0.05, ^## P < 0.01 compared to I/R group (n = 6 in each group).

Figure 6

Inhibitory effect of EGCG on LPS-induced NO production in BV-2 cells incubated with LPS (0.5 μg/mL) in the presence or absence of indicated concentration of EGCG. Accumulated nitrite in the culture medium was determined by the Griess reaction. Each vertical bars represented mean ± S.E. ***P < 0.001 compared to LPS only group.

Figure 7

Effects of EGCG (2, 10, and 25 μM) on expression of COX-2 and iNOS in BV-2 cells treated with lipopolysaccharide (LPS, 0.5 μg/mL) for 24 h. Cultures were pretreated with EGCG for 1 h before the addition of LPS treatment. Bars represent the mean ± SE from three independent experiments. Densitometry analyses are presented as the relative ratio of protein/β-actin protein and are represented as percentages of the LPS only group. ***P < 0.001 compared to LPS only.