Introduction

Ischemic stroke is a debilitating clinical disorder that affects millions of people, yet lacks effective neuroprotective treatments. There is now considerable evidence indicating that oxidative stress plays an important role in the secondary injury after acute cerebral ischemia, which was characterized by a progressive increase in reactive oxygen species (ROS) [1]. Therefore, the intervention of oxidative damage using compounds with antioxidant properties may relieve or prevent diseases in which oxidative stress is the primary cause [2]. The redox sensitive transcription factor nuclear factor erythroid-2 related factor 2 (Nrf2) plays a key role in the cellular defence against oxidative stress. Activation of Nrf2 has been implicated to protect neurons from ischemic insult [3]. Under basal conditions, Nrf2 is retained in the cytosol by binding to Kelch-like ECH-associated protein 1 (Keap1). In response to oxidative stress, Nrf2 dissociates from Keap1 and translocates to the nucleus [4–7], where it forms a heterodimer with its obligatory partner Maf and binds to the antioxidant response element (ARE) sequence to activate transcription of a large number of antioxidative and electrophile detoxification genes including heme oxygenase-1 (HO-1), NAD(P)H:quinine oxidoreductase 1 (NQO1) and glutamate-cysteine ligase (GCL) [8].

Green tea, a popular beverage derived from tea plant, contains large quantities of the biologically active polyphenols and catechins. Four types of catechins, namely (−)-epicatechin (EC) (−)-epigallocatechin (EGC) (−)-epicatechin-3-gallate (ECG), and EGCG, comprise 30 % of the dry weight of green tea. EGCG is the most abundant catechin and is mainly responsible for the biological properties of green tea such as its potent antioxidant effects [9, 10]. Several studies have indicated that EGCG have protective effects against cerebral ischemia in rats [11]. In a recent study Romeo et al [12] reported that EGCG could protect against oxidative stress-induced H19-7 cell death through its effects on Nrf2 activation. However, it was not clear whether EGCG regulate Nrf2 signaling in the brain. The present study tested the hypothesis that EGCG protect against ischemic brain injury and ameliorate cerebral ischemia-induced oxidative damage by activating the Nrf2/ARE signaling pathway.

Materials and Methods

Drugs and Reagents

(−)-Epigallocatechin gallate (EGCG) and DCFH-DA were purchased from Sigma (St. Louis, MO, USA). Antibodies to Nrf2, HO-1, GCLC, GCLM, anti-mouse-horseradish peroxide (HRP) IgG and anti-rabbit-HRP-IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). In Situ Cell Death Detection Kit was purchased from Roche (Indianapolis, IN, USA). The glutathione assay kit was provided by Beyotime Institute of Biotechnology (Haimen, China).

Animal Preparation and Drug Treatment

All animal experiments were approved by Institutional Animal care and Use Committee of Shandong University. Adult male Sprague–Dawley rats weighing 270–320 g were used for the experiment. Middle cerebral artery occlusion (MCAO) was induced as described previously [13]. Briefly, rats were anesthetized with 10 % chloralhydrate (350 mg/kg, i.p). The left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were isolated. An 18 mm length of nylon suture (φ 0.2 mm) was introduced into the ECA lumen and advanced into the ICA to block the origin of the MCA. Restoration of MCA blood flow in animals subjected to 2 h of MCA occlusion was achieved by withdrawing the suture to the ECA. The sham control rats received the same surgery procedures but did not have the suture inserted. EGCG (40 mg/kg) was given by intraperitoneally injection once daily for three consecutive days prior to surgery. Sham rats received vehicle (0.9 % NaCl, i.p) only. During and after the surgery, rectal temperature was controlled with a homeothermic blanket and kept at 37 °C until the complete recovery of the animal from the anesthesia. 24 h after reperfusion, animals were anesthetized and decapitated for future experiment.

Assessment of Functional Outcome and Cerebral Infarction

Functional outcome was evaluated using modified neurological severity score (mNSS) [14]. The rats were evaluated 24 h after MCAO. 2,3,5-triphenyltetrazolium chloride (TTC) staining was used to check the brain infarction size 24 h after reperfusion [15]. The colorless TTC was reduced to a red formazan product by dehydrogenases, which were abundant in mitochondria [16]. After anesthesia, the brains were removed and sectioned into 2 mm slices, which were then incubated in a 0.2 % solution of TTC at 37 °C for 30 min. Infarct volume was measured by a blinded observer using digital imaging (digital camera, Olympus MDF-382E) and image analysis software (C imaging 1280 system). The infarct area was calculated by Swanson’s method [17] to correct for edema. The total volumes of both contralateral (VC) and ipsilateral hemisphere (VL) were measured, and the infarct percentage (I) was calculated as % I = 100 × \( \frac{{({\text{V}}_{\text{c}} \text{ - }{\text{V}}_{\text{L}} )}}{{{\text{V}}_{\text{c}} }} \) to avoid mismeasurement secondary to edema.

Hematoxylin and Eosin (HE) Staining

Rats were sacrificed and fixed with formaldehyde perfusion 24 h after reperfusion. Paraffin blocked brains were cut into 4 μM sections. The sections were stained with HE and observed under a light microscope. The representative photographs were captured to determine the neuronal morphology in the cerebral cortex as described [18].

TUNEL Staining

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to visualize DNA fragmentation using an In Situ Cell Death Detection Kit according to the manufacturer’s protocols (Roche Diagnostic, Indianapolis, IN). Briefly, the sections were post-fixed in ethanol-acetic acid (2:1) and then rinsed. Next, sections were incubated with proteinase K (100 µg/ml), rinsed, incubated in 3 % H2O2, permeabilized with 0.5 % Triton X-100, rinsed again, and then incubated in the TUNEL reaction mixture. Sections were then rinsed and visualized using a Converter- horse-radish peroxidase (POD) with 0.02 % 3, 3′-diaminobenzidine (DAB). Sections were then counterstained with Mayer’s hematoxylin and then mounted onto gelatin-coated slides. Slides were air dried overnight at room temperature [19].

Western Blot Analysis for Nrf2, HO-1, GCLC and GCLM

Protein extracts were prepared from ischemic cortex or control brains by homogenization in a RIPA buffer. Protein concentration was determined by bicinchonininc acid (BCA) method. Samples were mixed with loading buffer and boiled for 5 min. Equal amounts of protein were separated by Tris–glycine-SDS-PAGE. The membranes were blocked in 5 % non-fat dried milk in TBS-T, and then were incubated with primary antibody (anti-Nrf2 antibody 1:200; anti-HO-1 antibody 1:100; anti-GCLC antibody 1:100; anti-GCLM antibody 1:100; anti-actin antibody 1:1,000) in blocking buffer overnight at 4 °C. Signals from peroxidase-conjugated secondary antibody were visualized by chemiluminescence, and data, within a linear range, were quantified with Image J software [20].

Measurement of Glutathione Levels

Glutathione (GSH) levels were determined by using Glutathione Quantification Kit. 5, 5′-Dithiobis (2-nitrobenzoic acid) (DTNB) and GSH reacted to generate 2-nitro-5-thiobenzoic acid and glutathione disulfide (GSSG). Since 2-nitro-5-thiobenzoic acid is a yellow colored product, GSH concentration in the sample was determined by reading 412 nm absorbance with a multi-well plate reader.

ROS Determination

The ROS level was determined using a ROS-sensitive fluorescent probe, 2, 7-dichlorodihydro fluorescent diacetate (DCFH-DA). The fluorescence intensity of brain tissues was measured with a fluorescence spectrophotometer (Thermo Scientific Varioskan Flash), with excitation and emission wavelengths of 488 and 525 nm respectively.

Statistical Analysis

All values are presented as the mean ± SD unless otherwise specified. An unpaired t test or analysis of variance (ANOVA) was used for the comparison of means between groups. P < 0.05 was considered statistically significant.

Results

EGCG Improved Cerebral Function after Cerebral Ischemia/Reperfusion Injury

The neurological severity scores were evaluated 24 h after reperfusion. A significant increase in mNSS was found in animals 24 h after focal cerebral ischemia. Compared with cerebral ischemia/reperfusion (I/R) group, rats treated with EGCG had better cerebral functional outcome and lower scores in mNSS (*P < 0.05 vs. I/R group) (Fig. 1).

Fig. 1
figure 1

EGCG improves the test scores in MCAO rats. Data represent mean ± SD from 8 rats in each group. ## P < 0.01 versus sham; **P < 0.01 versus I/R group

Full size image

EGCG Decreased Cerebral Infarct Size after Cerebral I/R Injury

Infarct volume determined by postmortem TTC staining at 24 h were 23.4 ± 2.9 % in the I/R group (## P < 0.01 vs. sham group), while 8.95 ± 1.65 % in the EGCG group (**P < 0.01 vs. I/R group). No cerebral infarction was observed in the sham group (Fig. 2a, b).

Fig. 2
figure 2

EGCG decreases the extent of cerebral infarction in MCAO rats. a TTC staining was used to evaluate the infarction volume. The red area was healthy tissue and the white area was infarct tissue. b Infarction volume of the brain from EGCG group were less than that of the I/R group. n = 8 in each group, ## P < 0.05 versus sham; **P < 0.05 versus I/R group (Color figure online)

Full size image

EGCG Attenuated Neuronal Injury in Ischemic Cortex after Cerebral I/R Injury

Hematoxylin and eosin (HE) staining was an effective and easy way to evaluate cell viability and was used in this study to evaluate neuronal cell death after MCAO. Compared with I/R group, the number of survived cells in cerebral cortex was significantly increased in rats treated with EGCG (Fig. 3a, b). The number of survived cells were 1,287.4 ± 233.6 per mm2 in the control group, while 119.4 ± 32.6 per mm2 in I/R group (## P < 0.01 vs. sham group). For the EGCG treatment group, the number was 817.7 ± 26.3 per mm2 (**P < 0.01 vs. I/R group).

Fig. 3
figure 3

EGCG reduces the neuronal death in the ischemic cortex. a Representative image of HE staining performed on sections from control and ischemic cortex. Compared with I/R group, EGCG treatment significantly increased the number of survived neurons in the ischemic cortex. The semi-quantative data were shown in (b). ## P < 0.05 versus sham; **P < 0.05 versus I/R group

Full size image

The protective effect of EGCG against cerebral ischemic damage was further confirmed by TUNEL staining. Fig. 4a, b showed TUNEL staining in samples collected 24 h after reperfusion. In the MCAO group, the apoptotic cells were characterized by a round and shrunken shape with strong staining in the nucleus. Some occasional TUNEL-positive cells (4.7 ± 5.4 per mm2) were common in the sham groups. Compared with I/R group (1,002.2 ± 117.8 per mm2), number of TUNEL-positive cells (490.8 ± 73.2 per mm2) was significantly reduced with EGCG treatment (**P < 0.01 vs. I/R group).

Fig. 4
figure 4

EGCG attenuates cerebral I/R-induced neuronal apoptosis. a Representative image of TUNEL staining performed on sections from sham and ischemic cortex. b The apoptotic cells in ischemic cortex from EGCG treatment group were much less than those in I/R group. ## P < 0.05 versus sham; **P < 0.05 versus I/R group

Full size image

EGCG Induced the Expression of Nrf2 and ARE-regulated Genes in Ischemic Cortex Following MCAO

To determine whether the observed protective effect of EGCG was accompanied by activation of the Nrf2/ARE pathway in MCAO rats, we analyzed the protein levels of Nrf2 and its down-stream targets by western blot analysis. Compared with sham group, the Nrf2 expression in I/R group relatively increased about 1.5-fold 24 h after cerebral I/R, while in the EGCG treatment group it significantly increased (about 4.5-fold of the sham group) compared with I/R group (*P < 0.05 vs. I/R group) (Fig. 5a). We next determined whether EGCG also induced the expression of endogenous ARE-regulated genes. As shown in Fig. 5b, rats treated with EGCG exhibited an obvious increase in the protein levels of HO-1, GCLC and GCLM in the ischemic cortex compared to I/R group (*P < 0.05 vs. I/R group).

Fig. 5
figure 5

EGCG increases the expression of Nrf2 and its downstream proteins in ischemic cortex. a Expression of Nrf2 was induced by EGCG in the ischemic cortex of MCAO rats. The results were mean ± SD of three different experiments. *P < 0.05 versus I/R group. b EGCG increased the expression of HO-1, GCLM and GCLC. The results were mean ± SD of three different experiments. # P < 0.05 versus sham; *P < 0.05 versus I/R group

Full size image

EGCG Induced GSH Synthesis And Attenuated Oxidative Damage in Ischemic Cortex Following MCAO

We measured the levels of GSH and ROS at 24 h after MCAO in order to test if the in vivo activation of Nrf2/ARE signaling by EGCG was able to protect against I/R-induced oxidative damage. EGCG pretreatment protected against the I/R-associated oxidative stress by maintaining GSH levels at close to baseline values 24 h after reperfusion (**P < 0.01 vs. I/R group) (Fig. 6a). As an index of ROS formation, we utilized the fluorescent probe DCFH-DA to measure levels of ROS in the cortex of I/R-injured animals. As shown in Fig. 6b, there was a significant increase in ROS formation at 24 h after I/R injury (## P < 0.01 vs. sham group). In turn, EGCG pretreatment significantly decreased the I/R-induced ROS formation (*P < 0.05 vs. I/R group). Altogether, these results strongly suggest that the pretreatment with EGCG protected MCAO rats against I/R-associated oxidative damage in ischemic cortex.

Fig. 6
figure 6

EGCG induces GSH synthesis and inhibits cerebral I/R-induced ROS production in MCAO rats. a EGCG led to an increase in GSH synthesis in ischemic cortex of MCAO rats. b EGCG treatment inhibited I/R-induced ROS production in MCAO rats. # P < 0.05 versus sham; *P < 0.05 versus I/R group

Full size image

Discussion

In the present study, we demonstrated that EGCG pretreatment has protective effects on cerebral I/R injury in a rat MCAO model demonstrated by improved neurologic scores, reduced infarct volume and ameliorated neuronal apoptosis, increased GSH biosynthesis and decreased ROS content. The mechanism was possibly attributed to activating Nrf2 expression.

Although the mechanism of cerebral I/R injury is complicated, the overproduced free radicals is one of the most important start factors, which can cause severe damage of biological macromolecules and lead to apoptosis or necrosis of cells and tissues. Therefore, antioxidants have been considered in prevention and treatment of stroke and certain agents with antioxidant effects did have neuroprotective effects [21].

Green tea is one of the most popular beverages worldwide and its habitual consumption has long been associated with health benefits. Epidemiological studies showed a protective effect of habitual green tea consumption in the cardiovascular and cerebrovascular systems with a significant reduction in the incidence of stroke. Most of the beneficial effects of green tea were attributed to its polyphenolic flavonoids, including EC, EGC, ECG and the major flavonoid EGCG [22]. Purified EGCG has been the focus of research in recent years. A number of studies on different models have demonstrated that EGCG has protective effects on intestinal I/R injury [23], spinal cord injury [24], cisplatin-induced nephrotoxicity [25] and so on. Particularly, recent years have witnessed the protective effect of EGCG on central nervous system, especially on the brain injury [26]. While several previous studies have shown that activation of Nrf2 can protect against cerebral I/R injury, it remains unclear how EGCG effectively protect neurons against oxidative stress-induced toxicity. It was hypothesized that the antioxidant effect of green tea was mainly responsible for its disease-modifying properties. In this study, we demonstrated for the first time that EGCG effectively suppressed the oxidative damage and this protective effect was dependent on stimulation of the Nrf2 mediated anti-oxidative pathways.

The results of the present study have demonstrated that the pretreatment of cerebral I/R rats by EGCG markedly increased Nrf2 expression and improved antioxidant status and histopathologic properties in brain tissue. Many questions related to the antioxidant effects of EGCG still need to be clarified. And the exact molecular mechanism needs to be further explored.

Conclusion

Taken together, the results of this study support the hypothesis that EGCG has robust protective effects and can potently protect against the cerebral I/R injury through activation of Nrf2/ARE pathway. Further studies in models of cerebral I/R injury are thus warranted to further identify other molecules involved in the protective effect and to clarify potential cross talk between upstream and downstream signaling molecules.