The effect of epigallocatechin-3-gallate on morphine-induced memory impairments in rat: EGCG effects on morphine neurotoxicity

Abstract

Aim of study:

This investigation evaluated the capacity of epigallocatechin-3-gallate (EGCG) as the main polyphenolic compound in the green tea extract against memory impairment and neurotoxicity in morphine-treated rats.

Methods:

To measure the EGCG effect (5 and 50 mg/kg, i.p., co-treated with morphine) on spatial learning and memory of morphine-administrated male Wistar rats (45 mg/kg, s.c., 4 weeks), the Morris water maze test was used. Some apoptotic protein levels (Bax, Bcl-2, and cleaved caspase 3) were evaluated in the hippocampus tissue by the Western blot test. Also, oxidative stress status (malondialdehyde level, glutathione peroxidase, and superoxide dismutase activity) was measured in hippocampus tissue.

Results:

The data presented that EGCG treatment (50 mg/kg) inhibited the morphine-induced memory deficits in rats. Also, EGCG administration reduced the apoptosis and oxidative stress in the hippocampus of morphine-treated rats.

Conclusions:

Our data indicate that EGCG can improve memory in morphine-treated rats. Molecular mechanisms underlying the detected effects could be related to the prevention of apoptosis and oxidative stress in the hippocampus of morphine-treated rats.

Keywords

EGCG memory morphine apoptosis oxidative stress

Introduction

Several findings indicate that morphine as a mu-opioid receptor agonist can modulate cognitive functions such as various forms of memory assessment tasks.^1
–3 The hippocampus is a brain region that has an essential task in the progression of learning and memory.⁴ Several findings revealed that chronic morphine or other opiates administration can decrease the hippocampus performance and damage its tissue.^5
–7 Also, numerous investigations have confirmed that chronic exposure to morphine leads to excitation of reactive oxygen species (ROS) generation, inflammation, and apoptosis.^8

–11 Previous findings indicated that oxidative stress mechanisms can participate in opiate-induced apoptosis that results in neurodegeneration in various brain areas like the hippocampus.^11

–15 It has been shown morphine administration in increasing doses may lead to hippocampus CA1, CA2, and CA3 neuronal damage.¹⁶ Also, animal studies have indicated that increases in expression of some proapoptotic proteins such as caspases 3 and Bax as well as decreases in antiapoptotic Bcl-2 protein are involved in morphine-induced apoptosis in the hippocampus.¹⁷

Usage of herbal medicinal compounds, their fractions, and flavonoids medicine for management of oxidative stress, inflammation decrease and apoptosis inhibition have been suggested recently and numerous studies have been performed for assessing such beneficial effects of these remedies.^18
–20 In this context, among the several polyphenols that have been separated from green tea, epigallocatechin-3-gallate (EGCG) has been shown to be the main biologically active compound in green tea.²¹ Several studies revealed that EGCG has numerous pharmacological effects such as antioxidant, vasodilatory, anti-inflammatory, neuroprotective, and anticancer effects.^22
–24

Considering the essential role of the hippocampus in learning and memory which would be affected by chronic morphine administration, the aim of the investigation is to explore the beneficial properties of EGCG on morphine-induced neurotoxicity and memory deficits. Additionally, the effects of EGCG against apoptosis and oxidative stress in the hippocampus of (chronic) morphine-treated rats (chronically) were investigated in this study.

Material and methods

Main chemicals and reagents

EGCG was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary anti-Bax, anti-caspase 3, anti-β-actin, anti-Bcl-2 antibodies were obtained from Abcam (Cambridge, MA, USA). The malondialdehyde (MDA), glutathione peroxidase (GPx) activity, and superoxide dismutase (SOD) activity assay kits were purchased from ZellBio Company (Germany). Morphine sulfate was obtained from Temad Company (Iran).

Animals

Adult, albino male Wistar rats (230 ± 10 g) were separately maintained in polycarbonate cages in Rafsanjan University of Medical Sciences colony room with a 12-h light and 12-h dark cycle at 22 ± 2°C. Food and water were provided ad libitum except during the behavioral examinations. The animal experimental protocols were permitted by Rafsanjan University of Medical Sciences Research Committee. Our animal protocols were done in agreement with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publications No. 80–23).

Experimental design

The rats were randomly inserted to one of the four experimental groups (n = 8):

Saline group (non-morphine): normal saline-treated rats (1.2 ml/kg, i.p., 4 weeks).

Morphine + saline group: morphine (45 mg/kg, s.c., 4 weeks) and normal saline (1.2 ml/kg, i.p., 4 weeks) treated rats.¹⁵

Morphine + EGCG groups: The rats co-treated with morphine (45 mg/kg, s.c., 4 weeks) and 5 or 50 mg/kg EGCG (1.2 ml/kg, i.p., 4 weeks).

Morris water maze

To examine the hippocampus-dependent spatial learning and memory, the Morris water maze (MWM) apparatus was used. The MWM apparatus was a tank with black color (60 cm in height and, 140 cm in diameter). The tank was filled with water (22 ± 1°C) and geographically separated into four conceptual quadrants. A black platform with black color (10 cm in diameter) was located 2 cm under the water surface in one of the quadrant center (target quadrant). The device was positioned in the center of a room with four visual clues placed on the walls nearby the tank.

For four consecutive days, the rats performed four trials per each day. Each animal was randomly located in the pool of water facing the tank wall from four quadrant points. For each animal, the travel distance and escape latency of each animal was recorded in the 60s. The spatial probe examination was done 24 h after the navigation trial. The hidden platform was removed in the probe examination; we recorded the swim time in the target quadrant (place of the removed hidden platform). Also, the swim speed was recorded in the probe examination. We used the automatic video tracking system to evaluate the rats locomotion (Ethovision software; version 7.1, Noldus Information Technology, the Netherlands).

Hippocampus tissue dissection

The rats were euthanized after the MWM test under deep anesthesia by exposure to CO₂ atmosphere. The animals’ brain was rapidly removed and the hippocampus was dissected carefully. The hippocampus was homogenized in ice-cold lysis buffer (including 1-mM EDTA, 10-mM Tris-HCl, 0.1% sodium dodecyl sulfate (SDS), 0.1% Na deoxycholate, 1% NP-40, and protease inhibitor cocktail).²⁵ The homogenized tissue was centrifuged at 14,000 r/min at 4°C for 20 min. The concentration of protein was evaluated by Bradford method in each collected supernatant.

Hippocampus oxidative stress status assessment

To examine the hippocampus oxidative stress levels, the GPx enzyme activity and the SOD enzyme activity were measured. Also, the MDA amount was assessed in hippocampus tissue. GPx, SOD, and MDA levels were evaluated by commercial assay kits (ZellBio). The homogenates supernatant was examined in strict accord with the instructions in the assay kits.

Western blot assessment

To evaluate some apoptosis biochemical biomarkers (Bax, Bcl-2, and caspase-3 protein), Western blotting test was used in morphine-treated rats. Briefly, the protein samples were electrically separated by 12.5% polyacrylamide gel and transferred to the polyvinylidene fluoride (PVDF) membrane. The membranes were blocked (overnight at 4°C) in Tris-buffered saline with Tween20 (TBST) (150-mM NaCl, 20-mM Tris-HCl, and 0.1% Tween20; pH 7.4) with 5% nonfat milk. Then, the PVDF membranes were incubated with monoclonal rabbit anti-Bax, anti-Bcl-2, and anti-caspase 3 (1:1000) for 3 h at room temperature. A blocking buffer was used as antibody diluent. The blots were washed three times with TBST, then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Abcam, 1:5000) for 1 h at room temperature. The blots were detected by an enhanced chemiluminescence method. The band densitometry was evaluated by ImageJ analyzing software (Freeware version V 1.51A plug-ins, downloaded from the NIH). Beta-actin (β-Actin) immunoblotting (1:5000, Abcam) was used as a loading control.

Statistical analysis

We evaluated the normality distribution of the data using the Kolmogorov–Smirnov test. The data were shown as mean ± standard error of the mean. The result from the MWM test was done by one- or two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. The obtained data in Western blot analysis and oxidative stress status evaluations were examined by one-way ANOVA followed by Tukey’s post hoc test. A p value of less than 0.05 (p < 0.05) was considered as statistical significance.

Results

The effects of EGCG on hippocampus-dependent spatial learning and memory

Figure 1 shows the latency time and reduction of traveled distance in experimental groups to find the hidden escape platform during the training trial in the MWM test. Collectively, statistic comparisons during 4 days training trial between the animals revealed that morphine-treated animals have more latency time (Figure 1(a)) and traveled a higher distance (Figure 1(b)) than saline-treated rats (p < 0.001), presenting a weak spatial learning performance because of morphine treatment. However, 50 mg/kg of EGCG could decrease the travel distance and escape latency in morphine-administrated rats (p < 0.05) (Figure 1). It is notable that administration of 5 mg/kg EGCG did not decrease the morphine side effects on travel distance and escape latency in rats (Figure 1).

Figure 1.

The effect of EGCG administration on spatial learning in the morphine-treated rats. Each line represents the average of escape latency (a) and traveled distance (b) to find the hidden platform in the MWM test for four consecutive trial days. Each value is the mean ± SEM. The difference in escape latency and travel distance between various experimental groups over the time course of the study was analyzed by repeated-measures two-way ANOVA followed by Tukey’s post hoc test. n = 8 per group. ^&&& p < 0.001 versus saline (non-morphine treated) group; ^α p < 0.05 compared with morphine group (for overall row comparison between the experimental groups during the 4 days). **p < 0.01 and ***p < 0.001 versus saline group; ^# p < 0.05 and ^## p < 0.01 compared with morphine group (for comparison between the experimental groups in each day). EGCG: epigallocatechin-3-gallate; MWM: Morris water maze; SEM: standard error of the mean; ANOVA: analysis of variance.

The probe trial test was shown in Figure 2 helping the MWM test. As presented in Figure 2(a), morphine-treated animals didn’t remember the location of the escape platform and consequently spent less time in the target quadrant than the saline-treated rats (p < 0.001). This finding indicates that chronic morphine administration significantly impaired the spatial memory. Also, morphine-treated rats with 50 mg/kg of EGCG significantly spent more time in the target quadrant than the morphine group (p < 0.01) (Figure 2(a)). There were any significant differences between morphine and morphine + 5 mg/kg EGCG-treated rats in time spent in the target quadrant (Figure 2(a)). However, there are no significant differences seen among the swimming speed in all rats (Figure 2(b)), representing that animals do not show any locomotor impairments in all experimental groups. It is notable that we have not seen any significant changes in the body weight between the experimental groups during and at the end of the study.

Figure 2.

The effect of EGCG treatment on spatial memory in morphine-treated rats. The percentage of time spent in the target quadrant (a) and the swimming speed (b) in the probe task in the MWM test. Each value is the mean ± SEM. The result from the probe part of MWM test was done by one-way ANOVA followed by Tukey’s post hoc test. n = 8 per group. **p < 0.01 and ***p < 0.001 versus saline (non-morphine treated) group; ^## p < 0.01 compared with morphine group. EGCG: epigallocatechin-3-gallate; MWM: Morris water maze; SEM: standard error of the mean; ANOVA: analysis of variance.

The effect of EGCG on hippocampus lipid peroxidation concentration

Oxidative stress damage was measured following chronic morphine treatment via lipid peroxidation concentration assessment, which was evaluated as MDA levels. As illustrated in Figure 3, chronic morphine administration induced an elevation in hippocampus MDA concentration and this increase was significant versus saline-treated group (p < 0.001). Treatment with 50 mg/kg of EGCG reduced the MDA concentration in the morphine-treated rats (p < 0.01). No significant difference was seen among MDA levels in morphine and morphine + 5 mg/kg EGCG-treated rats (Figure 3).

Figure 3.

The effect of EGCG treatment on lipid peroxidation in the hippocampus of morphine-treated rats. Each value is the mean ± SEM. n = 8 per group. ***p < 0.001 versus saline (non-morphine treated) group; ^## p < 0.01 compared with morphine group. EGCG: epigallocatechin-3-gallate; SEM: standard error of the mean; MDA: malondialdehyde.

The effect of EGCG on GPx and SOD activity levels in the hippocampus

As shown in Figure 4, the GPx and SOD activity levels were significantly reduced in the hippocampus tissue of the morphine-administrated animals versus saline-treated group (p < 0.001 and p < 0.01, respectively). Treatment with 50 mg/kg of EGCG significantly enhanced the GPx (p < 0.05, Figure 4(a)) and SOD activity (p < 0.05, Figure 4(b)) levels in the hippocampus of morphine-treated rats. There was any significant difference among hippocampus GPx and SOD activity levels in morphine and morphine + 5 mg/kg EGCG-administrated groups (Figure 4(a) and (b)).

Figure 4.

The effect of EGCG treatment on GPx (a) and SOD (b) activity in the hippocampus of morphine-treated rats. Each value is the mean ± SEM. n = 8 per group. **p < 0.01 and ***p < 0.001 versus saline (non-morphine treated) group; ^# p < 0.05 compared with morphine group. EGCG: epigallocatechin-3-gallate; GPx: glutathione peroxidase; SOD: superoxide dismutase; SEM: standard error of the mean.

The effect of EGCG on apoptosis in the hippocampus of experimental groups

Using Western blot analysis, we assessed the effect of EGCG on morphine-induced hippocampus tissue injury by evaluation of caspase 3 and Bax (as two proapoptotic proteins) and Bcl-2 (as an antiapoptotic protein) expression in hippocampus tissue. Molecular examination showed that chronic morphine administration leads to significant elevation in Bax (p < 0.001), cleaved caspase 3 (p < 0.01), and reduced Bcl-2 protein expression (p < 0.01) versus to saline-treated group. While 50 mg/kg EGCG administration prohibited morphine-induced Bax and cleaved caspase 3 (p < 0.001 and p < 0.05, respectively) protein expression (Figure 5(a) and (b)). In addition, treatment with 50 mg/kg of EGCG leads to an increase in the Bcl-2 protein expression level in morphine-treated groups (p < 0.05) (Figure 5(c)). According to Figure 5(d), EGCG at the dose of 50 mg/kg significantly reduced the Bax: Bcl-2 ratio in morphine-treated rats (p < 0.05).

Figure 5.

Western blot analysis of the Bax (a), cleaved caspase-3 (b), Bcl-2 (c), and Bax/Bcl-2 ratio (d) proteins in the hippocampus of morphine-treated animals. Each value in the graph represents the mean ± SEM band density ratio for each group. Beta-actin was used as an internal control. n = 8 per group. *p < 0.05, **p < 0.01, and ***p < 0.001 versus saline (non-morphine treated) group. ^# p < 0.05 and ^## p < 0.01 versus morphine group. SEM: standard error of the mean.

Discussion

This study exanimated the effects of EGCG against memory defect, oxidative stress, and apoptosis in hippocampus tissue in rats treated with chronic morphine. Our study showed that the neuroprotective properties of EGCG against morphine-induced hippocampus injury is associated with the decrease of oxidative stress (GPx and SOD activities elevation as well as decreasing of MDA content) and apoptosis (by the prevention of cleaved caspase-3 protein expression and Bax/Bcl-2 ratio).

It has been demonstrated that different brain areas participate in mediating the opioid properties.^26
–28 Several previous findings revealed that animals with morphine administration have memory defects.^6,7,29 However, the exact mechanisms of morphine-induced memory defects remain indefinite. In this case, it is recommended that the chronic morphine administration may disturb the brain elements function such as hippocampus.^1,5,17 It is well recognized that the hippocampus plays an essential role in memory process and spatial learning.^30
–32

Several experimental behaviors that are related to the hippocampus-dependent have been examined primarily with lesion or inactivation experiments. The MWM test is one of the main robust measures of hippocampal function for assessing spatial learning and memory.³³

Our findings demonstrated that morphine treatment (chronically) leads to significant learning and memory defects in rats. This is in agreement with previous studies.^1,5,29 This phenomenon was confirmed by increasing the time of escape latency and reducing the time spent in the quadrant of the target point for morphine-administrated animals compared to the saline rats in the MWM test.

It is well established that the morphine administration leads to considerable cellular toxicity in hippocampus tissue.^11,17,34,35 The exact underlying mechanisms of this toxicity is not yet completely known. It is believed that morphine-induced cytotoxicity (and/or other opioids) can result from oxidative stress and ROS generation.^11,36

In some areas of brain tissues, the oxidative stress induced by morphine is revealed by changes in cellular antioxidant status. Moreover, it has been revealed that morphine administration can lead to the diminishing of the hippocampus antioxidant enzymatic activities of GPx and SOD.^11,37 Increase in MDA concentration in the various brain regions including the hippocampus is also associated with chronic opioid consumption.^11,37
–39 Lipid peroxides are unstable oxidative stress indicators in neurons that decompose to form more reactive and complex compounds such as MDA, natural lipid peroxidation bi-products.⁴⁰

The molecular apoptosis mechanisms in various cell types, such as neurons, can be initiated by ROS generation, cellular antioxidant system disruption, or cellular degradation processes.^41

–45 It has been indicated that the opiates can lead to apoptosis in neurons of brain. Thus, it has been demonstrated that the administration of morphine chronically can cause a significant elevation in the apoptosis pathway protein expression (Bax and caspase 3) while decreases the Bcl-2 protein expression, thereby stimulating the hippocampus tissue apoptosis.^11,14,17

In this study, EGCG exerted antioxidant, antiapoptotic, and spatial learning and memory increment effects in rats treated with morphine chronically. Several previous studies have shown antioxidant and antiapoptotic properties of oleuropein. In agreement with our findings, He et al. showed that EGCG reduces the MDA concentration and also elevates the antioxidant enzymes activities level (SOD, GPx, and catalase) and apoptosis levels (reduction in Bax: Bcl-2 ratio, a cleaved caspase-3 protein expression) in rat cerebral cortex apoptosis induced by acrylamide.⁴⁶ Also, it has been demonstrated that the administration of EGCG in arsenic-treated mice could reduce the T-cell apoptosis biochemical markers (such as caspase 3) as well as serum oxidative stress status levels (by decreasing the MDA, and SOD, CAT enzymatic activity).⁴⁷ Furthermore, Ding et al. showed that EGCG administration can reduce the ischemia-induced hippocampus CA1 area damage in rats and modulated the synaptic transmission as well as improvement of the induction of long-term potentiation (LTP).⁴⁸ Also, in another study, it has been shown that EGCG by decreasing Bax: Bcl-2 ration and elevating the brain-derived neurotrophic factor (BDNF) protein expression can reduce the sevoflurane-induced rat hippocampus neuronal apoptosis.⁴⁹ In a study, Assunção and colleagues demonstrated that green tea powder (the main source of EGCG) supplementation elevated the Bcl-2, BDNF protein expression, and antioxidant defense system in the hippocampus tissue of aged rats.⁵⁰

Conclusions

Collectively, the present study showed that EGCG can reduce spatial learning and memory impairments in rats treated with chronic morphine. The reason may be related to the protective effect of the EGCG against morphine-induced hippocampus oxidative stress and apoptosis.

Footnotes

Author contributions

AK conceived and designed the experiments; SS, AK, and JH performed the experiments; AK, ASH, MR, MA, and JH analyzed the data; ASH, VSH and JH contributed reagents/materials/analysis tools; and AK and SS wrote the article.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Kerman Neuroscience Research Center and Rafsanjan University of Medical Sciences [Grant Number: 96171].

ORCID iDs

I Fatemi

A Kaeidi

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