Sage Journals: Discover world-class research

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

References

Sala

Braida

Leone

, et al. Chronic morphine affects working memory during treatment and withdrawal in rats: possible residual long-term impairment. Behav Pharmacol 1994; 5: 570–580.

Farahmandfar

Kadivar

Naghdi

. Possible interaction of hippocampal nitric oxide and calcium/calmodulin-dependent protein kinase II on reversal of spatial memory impairment induced by morphine. Eur J Pharmacol 2015; 751: 99–111.

Spain

Newsom

. Chronic opioids impair acquisition of both radial maze and Y-maze choice escape. Psychopharmacology (Berl) 1991; 105: 101–106.

Girardeau

Benchenane

Wiener

, et al. Selective suppression of hippocampal ripples impairs spatial memory. Nat Neurosci 2009; 12: 1222–1223.

Zhou

Kang

, et al. Chronic morphine treatment impaired hippocampal long-term potentiation and spatial memory via accumulation of extracellular adenosine acting on adenosine A1 receptors. J Neurosci 2010; 30: 5058–5070.

Miladi-Gorji

Rashidy-Pour

Fathollahi

, et al. Voluntary exercise ameliorates cognitive deficits in morphine dependent rats: the role of hippocampal brain-derived neurotrophic factor. Neurobiol Learn Mem 2011; 96: 479–491.

Yang

Wen

Dong

, et al. Effects of cholecystokinin-8 on morphine-induced spatial reference memory impairment in mice. Behav Brain Res 2013; 256: 346–353.

Guzman

Vazquez

Brizuela

, et al. Assessment of oxidative damage induced by acute doses of morphine sulfate in postnatal and adult rat brain. Neurochem Res 2006; 31: 549–554.

Ozmen

Naziroglu

Alici

, et al. Spinal morphine administration reduces the fatty acid contents in spinal cord and brain by increasing oxidative stress. Neurochem Res 2007; 32: 19–25.

10.

Merighi

Gessi

Varani

, et al. Morphine mediates a proinflammatory phenotype via μ-opioid receptor–PKCε–Akt–ERK1/2 signaling pathway in activated microglial cells. Biochem Pharmacol 2013; 86: 487–496.

11.

Motaghinejad

Karimian

Motaghinejad

, et al. Protective effects of various dosage of Curcumin against morphine induced apoptosis and oxidative stress in rat isolated hippocampus. Pharmacol Rep 2015; 67: 230–235.

12.

Hassanzadeh

Habibi-asl

Farajnia

, et al. Minocycline prevents morphine-induced apoptosis in rat cerebral cortex and lumbar spinal cord: a possible mechanism for attenuating morphine tolerance. Neurotox Res 2011; 19: 649–659.

13.

Katebi

Razavi

Alamdary

, et al. Morphine could increase apoptotic factors in the nucleus accumbens and prefrontal cortex of rat brain’s reward circuitry. Brain Res 2013; 1540: 1–8.

14.

Liu

Wang

, et al. Neuronal apoptosis in morphine addiction and its molecular mechanism. Int J Clin Exp Med 2013; 6: 540–545.

15.

Shibani

Sahamsizadeh

Fatemi

, et al. Effect of oleuropein on morphine-induced hippocampus neurotoxicity and memory impairments in rats. Naunyn Schmiedeb Arch Pharmacol 2019; 392: 1383–1391.

16.

Ghafari

Golalipour

. Prenatal morphine exposure reduces pyramidal neurons in CA1, CA2 and CA3 subfields of mice hippocampus. Iran J Basic Med Sci 2014; 17: 155–161.

17.

Boronat

García-Fuster

Garcia-Sevilla

. Chronic morphine induces up-regulation of the pro-apoptotic Fas receptor and down-regulation of the anti-apoptotic Bcl-2 oncoprotein in rat brain. Br J Pharmacol 2001; 134: 1263–1270.

18.

Mercer

Kelly

Horne

, et al. Dietary polyphenols protect dopamine neurons from oxidative insults and apoptosis: investigations in primary rat mesencephalic cultures. Biochem Pharmacol 2005; 69: 339–345.

19.

Hajializadeh

Nasri

Kaeidi

, et al. Inhibitory effect of Thymus caramanicus Jalas on hyperglycemia-induced apoptosis in in vitro and in vivo models of diabetic neuropathic pain. J Ethnopharmacol 2014; 153: 596–603.

20.

Rasoulian

Hajializadeh

Esmaeili-Mahani

, et al. Neuroprotective and antinociceptive effects of rosemary (Rosmarinus officinalis L.) extract in rats with painful diabetic neuropathy. J Physiol Sci 2019; 69: 57–64.

21.

Chu

Deng

Man

, et al. Green tea extracts epigallocatechin-3-gallate for different treatments. BioMed Res Int 2017; 2017: 5615647.

22.

Bondy

. The relation of oxidative stress and hyperexcitation to neurological disease. Proc Soc Exp Biol Med 1995; 208: 337–345.

23.

Dufourny

Leroy

Warembourg

. Differential effects of colchicine on the induction of nitric oxide synthase in neurons containing progesterone receptors of the guinea pig hypothalamus. Brain Res Bull 2000; 52: 435–443.

24.

Pourkhodadad

Alirezaei

Moghaddasi

, et al. Neuroprotective effects of oleuropein against cognitive dysfunction induced by colchicine in hippocampal CA1 area in rats. J Physiol Sci 2016; 66: 397–405.

25.

Alzoubi

Gerges

Aleisa

, et al. Levothyroxin restores hypothyroidism-induced impairment of hippocampus-dependent learning and memory: behavioral, electrophysiological, and molecular studies. Hippocampus 2009; 19: 66–78.

26.

Ahmadi-Soleimani

Azizi

Gompf

, et al. Role of orexin type-1 receptors in paragiganto-coerulear modulation of opioid withdrawal and tolerance: a site specific focus. Neuropharmacology 2017; 126: 25–37.

27.

Kaeidi

Azizi

Javan

, et al. Direct facilitatory role of paragigantocellularis neurons in opiate withdrawal-induced hyperactivity of rat locus coeruleus neurons: an in vitro study. PLoS One 2015; 10: e0134873.

28.

Kim

Ham

Hong

, et al. Brain reward circuits in morphine addiction. Mol Cells 2016; 39: 645–653.

29.

Naghibi

Hosseini

Khani

, et al. Effect of aqueous extract of Crocus sativus L. on morphine-induced memory impairment. Adv Pharmacol Sci 2012; 2012: 494367.

30.

Mozafari

Shamsizadeh

Fatemi

, et al. CX691, as an AMPA receptor positive modulator, improves the learning and memory in a rat model of Alzheimer’s disease. Iran J Basic Med Sci 2018; 21: 724–730.

31.

Barnes

CA.

Spatial learning and memory processes: the search for their neurobiological mechanisms in the rat. Trends Neurosci 1988; 11: 163–169.

32.

Lieberwirth

Pan

Liu

, et al. Hippocampal adult neurogenesis: its regulation and potential role in spatial learning and memory. Brain Res 2016; 1644: 127–140.

33.

Vorhees

Williams

. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 2006; 1: 848–858.

34.

Atici

Cinel

, et al. Opioid neurotoxicity: comparison of morphine and tramadol in an experimental rat model. Int J Neurosci 2004; 114: 1001–1011.

35.

Eisch

Barrot

Schad

, et al. Opiates inhibit neurogenesis in the adult rat hippocampus. Proc Natl Acad Sci U S A 2000; 97: 7579–7584.

36.

Zhou

Yan

, et al. Protective role of taurine against morphine-induced neurotoxicity in C6 cells via inhibition of oxidative stress. Neurotox Res 2011; 20: 334–342.

37.

Rozisky

Laste

de Macedo

, et al. Neonatal morphine administration leads to changes in hippocampal BDNF levels and antioxidant enzyme activity in the adult life of rats. Neurochem Res 2013; 38: 494–503.

38.

Qiusheng

Yuntao

Rongliang

, et al. Effects of verbascoside and luteolin on oxidative damage in brain of heroin treated mice. Pharmazie 2005; 60: 539–543.

39.

Das

Ratty

. Studies on the effects of the narcotic alkaloids, cocaine, morphine, and codeine on nonenzymatic lipid peroxidation in rat brain mitochondria. Biochem Med Metab Biol 1987; 37: 258–264.

40.

Janero

. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990; 9: 515–540.

41.

Limon-Pacheco

Gonsebatt

. The role of antioxidants and antioxidant-related enzymes in protective responses to environmentally induced oxidative stress. Mutat Res 2009; 674: 137–147.

42.

Circu

. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med 2010; 48: 7497–7462.

43.

Kaeidi

Rasoulian

Hajializadeh

, et al. Cisplatin toxicity reduced in human cultured renal tubular cells by oxygen pretreatment. Ren Fail 2013; 35: 1382–1386.

44.

Kaeidi

Taati

Hajializadeh

, et al. Aqueous extract of Zizyphus jujuba fruit attenuates glucose induced neurotoxicity in an in vitro model of diabetic neuropathy. Iran J Basic Med Sci 2015; 18: 301–306.

45.

Kannan

Jain

. Oxidative stress and apoptosis. Pathophysiology 2000; 7: 153–163.

46.

Tan

Bai

, et al. Epigallocatechin-3-gallate attenuates acrylamide-induced apoptosis and astrogliosis in rat cerebral cortex. Toxicol Mech Methods 2017; 27: 298–306.

47.

Pei

Huang

, et al. (-)-Epigallocatechin-3-gallate inhibits arsenic-induced inflammation and apoptosis through suppression of oxidative stress in mice. Cell Physiol Biochem 2017; 41: 1788–1800.

48.

Ding

Zhao

, et al. EGCG ameliorates the suppression of long-term potentiation induced by ischemia at the Schaffer collateral-CA1 synapse in the rat. Cell Mol Neurobiol 2012; 32: 267–277.

49.

Ding

Man

, et al. Protective effects of a green tea polyphenol, epigallocatechin-3-gallate, against sevoflurane-induced neuronal apoptosis involve regulation of CREB/BDNF/TrkB and PI3K/Akt/mTOR signalling pathways in neonatal mice. Can J Physiol Pharmacol 2017; 95: 1396–1405.

50.

Assunção

Santos-Marques

Carvalho

, et al. Green tea averts age-dependent decline of hippocampal signaling systems related to antioxidant defenses and survival. Free Radic Biol Med 2010; 48: 831–838.

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

Abstract

Aim of study:

Methods:

Results:

Conclusions:

Keywords

Introduction

Material and methods

Main chemicals and reagents

Animals

Experimental design

Morris water maze

Hippocampus tissue dissection

Hippocampus oxidative stress status assessment

Western blot assessment

Statistical analysis

Results

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

The effect of EGCG on hippocampus lipid peroxidation concentration

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

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

Discussion

Conclusions

Footnotes

Author contributions

Declaration of conflicting interests

Funding

ORCID iDs

References