PBB-72169; No of Pages 9 Pharmacology, Biochemistry and Behavior xxx (2015) xxx–xxx
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Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
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Research article
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Peng Jin a,c,d, Hai-Ling Yu b,⁎, Tian-Lan b, Feng Zhang b, Zhe-Shan Quan a,⁎ a
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Article history: Received 7 January 2015 Received in revised form 30 March 2015 Accepted 3 April 2015 Available online xxxx
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Keywords: Oleoylethanolamide Antidepressant Brain-derived neurotrophic factor Oxidative stress HPA Mice
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Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry of Education, Yanbian University College of Pharmacy, Yanji 133000, PR China College of Medicine, Yanbian University, Park Street 977, Yanji, 133002 Jilin, PR China Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Cancer Research Institute, College of Medicine, Seoul National University, Yongon-dong 28, Chongno-gu, Seoul 110-799, Republic of Korea d Department of Biomedical Science, Ischemic/Hypoxic Disease Institute, Cancer Research Institute, College of Medicine, Seoul National University, Yongon-dong 28, Chongno-gu, Seoul 110-799, Republic of Korea b
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Oleoylethanolamide (OEA) is an endocannabinoid analog that belongs to a family of endogenous acylethanolamides. Increasing evidence suggests that OEA may act as an endogenous neuroprotective factor and participate in the control of mental disorder-related behaviors. In this study, we examined whether OEA is effective against depression and investigated the role of circulating endogenous acylethanolamides during stress. Mice were subjected to 28 days of chronic unpredictable mild stress (CUMS), and during the last 21 days, treated with oral OEA (1.5–6 mg/kg) or 6 mg/kg fluoxetine. Sucrose preference and open field test activity were used to evaluate depression-like behaviors during CUMS and after OEA treatment. Weights of the prefrontal cortex and hippocampus were determined, and the adrenal index was measured. Furthermore, changes in serum adrenocorticotropic hormone (ACTH), corticosterone (CORT) and total antioxidant capacity (T-AOC), brain-derived neurotrophic factor (BDNF), and lipid peroxidation product malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activities in the hippocampus and prefrontal cortex were detected. Our findings indicate that OEA normalized sucrose preferences, locomotion distances, rearing frequencies, prefrontal cortex and hippocampal atrophy, and adrenal indices. In addition, OEA reversed the abnormalities of BDNF and MDA levels and SOD activities in the hippocampus and prefrontal cortex, as well as changes in serum levels of ACTH, CORT, and T-AOC. The antidepressant effects of OEA may be related to the regulation of BDNF levels in the hippocampus and prefrontal cortex, antioxidant defenses, and normalizing hyperactivity in the hypothalamic–pituitary–adrenal axis (HPA). © 2015 Published by Elsevier Inc.
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brain are ideally situated to exert regulatory control over emotional behavior, mood, and stress response (Yu et al., 2011; Hill and Gorzalka, 2009). Malfunctions in the endocannabinoid system may promote the development and maintenance of psychiatric disorders such as depression and panic disorder (Yu et al., 2011; Bambico et al., 2009). A growing body of evidence demonstrates that deficits in endocannabinoid signaling may result in depression-like and anxiogenic behavioral responses. Endocannabinoids are released following the development of depression, causing both neuroprotective and degenerative effects (Viscomi et al., 2010). Pharmacological augmentation of endocannabinoid signaling can produce both antidepressant and anxiolytic behavioral responses (Yu et al., 2011; Hill et al., 2009; Parolaro et al., 2010). Oleoylethanolamide (OEA; Fig. 1) is an endocannabinoid analog that belongs to a family of endogenous acylethanolamides. Acylethanolamides act as agonists of the nuclear peroxisome proliferator-activated receptor alpha (PPARα) (Yu et al., 2011). Increasing evidence suggests that OEA may act as an endogenous neuroprotective factor and participate in the control of reward-related behaviors (Zhou et al., 2012). Systemic
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1. Introduction
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Depression is a major mental disorder associated with clinical symptoms such as persistent negative mood, decreased physical activity, feelings of helplessness, sluggish thought, and cognitive dysfunction. Many psychological, social, environmental, and genetic factors can result in depression (Bilbao et al., 2013), and the incidence of depression has increased, with approximately 5% of the population experiencing a major depressive episode (Yu et al., 2011). Current antidepressant drugs can alleviate only some symptoms of depression, and side effects are common. Therefore, the development of more effective and less toxic antidepressants has attracted significant attention in recent years. The central endocannabinoid system is a neuroactive lipid signaling system in the brain that acts to control neurotransmitter release. The expression patterns of this system throughout limbic regions of the
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⁎ Corresponding authors. E-mail addresses: [email protected] (H.-L. Yu), [email protected] (Z.-S. Quan).
http://dx.doi.org/10.1016/j.pbb.2015.04.001 0091-3057/© 2015 Published by Elsevier Inc.
Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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administration of OEA to rats results in elevated OEA plasma levels, as has also been reported in patients with post-traumatic stress disorder, and improved memory of aversive training experiences (Hauer et al., 2013). These findings indicate that changes in peripheral endocannabinoid concentrations may reflect alterations in central endocannabinoid signaling that alters behavior. Reduced endocannabinoid functionality may be a predisposing factor for major depression, and boosting endocannabinoid tone may be a useful alternative therapeutic approach for depressive disorders (Yu et al., 2011; Parolaro et al., 2010; Su et al., 2011). However, it is not clear whether orally administered OEA is effective against depression, and the regulation of circulating endogenous acylethanolamides during stress needs to be investigated. In the present study, the antidepressant-like effects of OEA were examined in comparison with the antidepressant fluoxetine (Fxt, a serotonin reuptake inhibitor) using a mouse model of chronic unpredictable mild stress. Additionally, in neurochemical studies, changes in the prefrontal cortex and hippocampus of brain-derived neurotrophic factor (BDNF) and lipid peroxidation product malondialdehyde (MDA) levels and superoxide dismutase (SOD) activities, as well as serum levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), and total antioxidant capacity (T-AOC) were determined.
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2. Materials and methods
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2.1. Drugs
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2.4. Open-field test in mice
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Fig. 1. Structure of oleoylethanolamide.
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at one mouse per cage (32 × 18 × 16 cm), and the stressors included the following: (1) horizontal shock for 5 min (1 shock/s); (2) swimming in 4 °C for 1 min; (3) swimming in 25 °C water for 5 min; (4) food deprivation for 14 h; (5) water deprivation for 16 h; (6) wet bedding for 17 h; (7) housed together (5 mice per cage) for 17 h; (8) empty cage for 17 h; (9) cage tilt at 45° for 2 h; (10) room temperature at 45 °C for 5 min; (11) tail suspension for 5 min; (12) foreign object placement for 6 h; (13) flash for 10 min; and (14) reversed light/dark cycle (overnight illumination). The 14 stressors were scheduled semirandomly, with two different stressors daily. Apart from the NC group, the mice were treated with the stressors for 28 days, and the same stressor was not continued, so that the mice could not anticipate the occurrence of specific stressors. Untreated mice served as the NC group, which were housed in another room at 5 mice per cage (32 × 18 × 16 cm). In addition to withdrawing food and water for 24 h before the sugar preference experiment and withdrawing food for 6 h before weighing, the mice were not subjected to additional stimulation. All behavioral tests were performed at 9:00–11:00, in a quiet warm environment, and were carried out in parallel in each experimental group. Biochemical parameters were measured on the last day of the experiment. After CUMS for 7 days, the CUMS-treated mice were randomly divided into five groups: the model control (MC) group; mice treated daily with Fxt or OEA at 1.5, 3, or 6 mg/kg group in 0.3% sodium carboxymethyl cellulose via gavage for 21 days.
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2.2. Animals
2.5. Sucrose preference test
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2.3. Experimental procedures (Fig. 2)
The sucrose preference test procedure was performed as described below (Martin et al., 2013; Lv et al., 2013). Learning and training were performed in each mouse before the experiment. Initially, the mice were acclimatized to sucrose and water for 3 days, and then exposed to 1% sucrose solution (w/v) for 24 h. A bottle of 1% sucrose solution and a bottle of tap water were presented at random positions to each mouse for the next 24 h, followed by food and water deprivation for 24 h. For testing, on day 4, two bottles, one containing 1% sucrose solution and the other containing tap water were weighed and presented to each mouse for 4 h. The position of the two bottles was set randomly. The sucrose preference test was performed at days 7 and 28 of the experiment. The sucrose preference was calculated using the following formula: Sucrose preference = sucrose consumption (g) / water consumption (g) + sucrose consumption (g) × 100%.
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Male adult Kunming mice (18–22 g; Laboratory Animal Center of Yanbian University, Jilin Province, China) were used. The mice were housed individually in cages for 3 days to adapt to the environment under the following controlled conditions: 12 h light–12 h dark cycles; free access to tap water and food pellets; and ambient temperature and relative humidity at 22 ± 2 °C and 55 ± 5%, respectively. Some of the mice were subjected to the stressors as the depression model. The experimental procedures were approved by the Animal Care and Use Committee of the Institute of Psychology of the Chinese Academy of Sciences and in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.
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The animals were randomly divided into two groups: a chronic unpredictable mild stress (CUMS)-treated group and a normal control (NC) group. The CUMS-treated mice were subjected to a series of variable stressors as described by Su et al. (2011), Farley et al. (2011) and Hu et al. (2010), with slight modifications. The animals were housed
2.6. Serum sample collection
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The mouse BDNF ELISA Kit, mouse ACTH ELISA Kit, and mouse CORT ELISA Kit were purchased from Beijing pines Biotechnology Co., Ltd., China; the MDA, SOD, and T-AOC Kits were purchased from Nanjing Jiancheng Bioengineering Institute, China. The tested compound OEA (synthesized at the College of Pharmacy, Yanbian University, Jilin Province, China) or fluoxetine (Fxt, Enhua Jiangsu Pharmaceutical Co., Ltd., China) was suspended in 0.3% methyl cellulose (Loba-Chemie, Shanghai, China) and administered daily via gavage. All doses were expressed as mg/kg body weight.
The open-field test (OFT) procedure was performed once per week as described below (Elliot et al., 1986; Martin et al., 2013). Each mouse was placed individually in the center of the open-field apparatus, and locomotor activity was assessed. The open-field apparatus was a non-transparent plastic container (80 × 60 × 30 cm), with the underside divided into 10 × 10 cm squares of 48 units without walls. The animals were gently placed in the center of the platform and were allowed to explore the surroundings. Hand-operated counters were used for 3 min to score locomotion (number of line crossings with all four paws) and rearing frequencies (number of times an animal stood on its hind legs). The researchers, blind to the treatment groups, scored the behaviors in the open field. Experiments were performed in a dark room, and the apparatus was illuminated by a 60 W bulb, providing a yellowish light and positioned 1 m above the center of the apparatus. The walls and floor surfaces were thoroughly cleaned with 10% ethanol between the tests.
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After the stressor session, mice were euthanized by decapitation, 186 and blood was collected into the serum separator. Samples were 187
Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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Fig. 2. Experimental procedures. A. Animal groups and treatments. B. Experimental procedures in chronic unpredictable mild stress (CUMS). CUMS procedures were performed for 28 days, followed by drug treatment for 21 days. Vehicle, oleoylethanolamide (1.5, 3, 6 mg/kg), or Fxt (6 mg/kg) were administered for 21 days starting at day 8 after initiating CUMS procedures.
using a microplate reader within 15 min of stop solution addition. The BDNF concentration is expressed as pg/mg brain tissue, and the ACTH and CORT concentrations were expressed as pg/mL and ng/mL serum, respectively.
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2.9. Determination of MDA, SOD, and T-AOC
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The levels of MDA and SOD activity in the supernatants of tissues homogenates were determined with the commercial analysis kits, according to the manufacturer's instructions. The MDA level was expressed as nmol/mg protein of brain tissue, the SOD activity was expressed as units/mg protein of brain tissue, and T-AOC was expressed as units/mL of serum.
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Mice were euthanized by decapitation, and the whole brain was removed quickly on an ice table. The hippocampus and prefrontal cortex were separated under a dissecting microscope, gently washed, and homogenized in a cold phosphate buffer (0.05 mol/L, pH 7.8), and centrifuged at 3500 ×g for 10 min. The supernatant was stored at −80 °C for subsequent biochemical analysis.
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2.8. Determination of BDNF, ACTH, and CORT
2.10. The hippocampus and prefrontal cortex weights and adrenal index
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BDNF, ACTH, and CORT concentration determination was performed using the ELISA method, according to the mouse BDNF, ACTH, or CORT ELISA Kit manufacturer's instructions, respectively. The specific steps used standard wells (50 μL of standard), testing sample wells (10 μL of sample and 40 μL of sample diluent), and blank wells. A total of 100 μL of horseradish peroxidase (HRP)-conjugate reagent was added to each well, and the wells were covered with an adhesive strip and incubated for 60 min at 37 °C. Each well was washed 5 times by filling each well with wash solution (400 μL) using a squirt bottle manifold dispenser. After the last wash, remaining wash solution was removed by aspirating or decanting, inversion of the plate, and blotting against clean paper towels. Chromogen solution A (50 μL) and chromogen B (50 μL) were added to each well. The wells were gently mixed, then incubated for 15 min at 37 °C away from light. After incubation, 50 μL of stop solution was added to each well. Optical density (OD) was obtained at 450 nm
After each animal's blood and brain were removed, the hippocampus and prefrontal cortex were separated and weighed. The bilateral adrenals were immediately removed and weighed. The adrenal index was calculated using the following formula:
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allowed to clot for 30 min at 37 °C before centrifugation for 10 min at 3000 × g at 4 °C. The serum was isolated and stored at − 80 °C for subsequent ACTH, CORT, and T-AOC determination.
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Adrenal indexðgÞ ¼ bilateral adrenals weightðgÞ=mouse body weightðgÞ: 231
2.11. Statistical analysis One-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test was used for other statistical evaluations. Data are presented as the means ± SEM. p b 0.05 was considered statistically significant.
Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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Fig. 3. Changes in physical symptoms and behavioral indicators during CUMS procedure and after OEA treatment. A (a–d). Effect of oleoylethanolamide (OEA) on open-field test performance in chronic unpredictable mild stress (CUMS) mice. A (a and c). The change of crossing and rearing with time in the CUMS procedure; A (b and d). The counts of crossing and rearing in mice after 28 days of CUMS and 21 days of drug treatment. (B). The sucrose preference test after 28 days of CUMS and 21 days of drug-treatment. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA 1.5, 3, 6 (OEA at 1.5, 3, and 6 mg/kg group). Baseline is the mouse ethological indicators before CUMS. ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001 vs. MC, n = 10.
Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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CORT levels (p b 0.05, p b 0.01, respectively) as compared with those 271 of the MC group (Fig. 5). 272
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3.2. Determination of BDNF concentrations in hippocampus and prefrontal cortex
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Fig. 4 shows that, after 28 days of CUMS, BDNF levels in the hippocampus and prefrontal cortex of CUMS model mice were decreased significantly compared with those of the NC group (p b 0.001, p b 0.01, respectively). With drug treatment during the last 21 days, the BDNF levels in the Fxt 6 mg/kg and OEA 1.5, 3, and 6 mg/kg treatment groups were significantly increased (p b 0.01, p b 0.05, p b 0.01, p b 0.01, respectively, in hippocampus; p b 0.05, p b 0.05, p b 0.05, p b 0.01, respectively, in prefrontal cortex) compared with the MC group.
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3.3. Determination of serum ACTH and CORT concentrations
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After 28 days of CUMS and 21 days of drug treatment, serum ACTH and CORT concentrations in the mice in CUMS model group were significantly increased compared with the NC group (p b 0.01, p b 0.01, respectively). The Fxt (6 mg/kg) and OEA (6 mg/kg) groups showed significantly decreased ACTH (p b 0.05, p b 0.01, respectively) and
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Fig. 4. Effect of oleoylethanolamide on brain-derived neurotrophic factor (BDNF) levels in the hippocampus and prefrontal cortex. The BDNF concentration is expressed as pg/mg brain tissue. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA 1.5, 3, 6 (OEA at 1.5, 3, and 6 mg/kg group). ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001 vs. MC, n = 10.
3.4. Determination of T-AOC and MDA levels and SOD activities
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After 28 days of CUMS, serum T-AOC levels in MC mice were significantly decreased (p b 0.001, Fig. 6), MDA concentrations were significantly increased (p b 0.01, p b 0.01 in the hippocampus and prefrontal cortex, respectively; Fig. 7A), and SOD activities were significantly decreased (p b 0.01, p b 0.001 in hippocampus and prefrontal cortex, respectively; Fig. 7B) compared to those in the NC group. After 21 days of drug treatment, the Fxt (6 mg/kg) and OEA (6 mg/kg) groups exhibited significantly increased serum T-AOC levels (p b 0.01, p b 0.01, respectively) compared to the MC group. The Fxt 6 mg/kg and OEA 1.5, 3, and 6 mg/kg treatment groups exhibited significantly decreased MDA concentrations (p b 0.01, p b 0.05, p b 0.01, p b 0.01, respectively, in hippocampus; p b 0.05, p b 0.05, p b 0.05, p b 0.01, respectively, in prefrontal cortex; Fig. 7A) compared to the MC group. Finally, the Fxt 6 mg/kg and OEA 1.5, 3, and 6 mg/kg treatment groups showed significantly increased SOD activities (p b 0.05, p b 0.05, p b 0.001, p b 0.001, respectively) in the hippocampus compared to the MC group, while in the prefrontal cortex, SOD activities were significantly increased only in the Fxt 6 mg/kg and OEA 3 and 6 mg/kg treatment groups (p b 0.05, p b 0.05, p b 0.01, respectively; Fig. 7B).
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3.5. Hippocampus and prefrontal cortex weights and adrenal index
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Depression-like behaviors after 28 days of CUMS and 21 days of OEA (1.5, 3, or 6 mg/kg) treatment were examined (Fig. 3). The NC and CUMS groups showed similar behavioral indicators before CUMS (data not shown). Fig. 3A (b and d) shows that, after 21 days of drug treatment, the exploratory activities of mice (crossing and rearing) in the MC group were significantly lower than those in the NC group (p b 0.001, p b 0.001, respectively). The Fxt 6 mg/kg, and OEA 1.5, 3, and 6 mg/kg treatment groups were increased significantly as compared with the MC group (p b 0.01, p b 0.05, p b 0.01, p b 0.001 in crossing, respectively; p b 0.05, p b 0.05, p b 0.01, p b 0.001 in rearing, respectively). After 28 days of CUMS and 21 days of drug treatment, sucrose preferences were significantly decreased (p b 0.001, Fig. 3B) in the MC mice compared with the NC group. Sucrose preferences in the Fxt 6 mg/kg and OEA 1.5, 3, and 6 mg/kg treatment groups were significantly increased (p b 0.01, p b 0.05, p b 0.01, p b 0.001, respectively; Fig. 3B) as compared with the MC group.
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Fig. 8 shows that the hippocampus and prefrontal cortex weights of mice in the CUMS group were significantly decreased when compared with those in the NC group (p b 0.01, p b 0.05, respectively) after 28 days of CUMS and 21 days of drug treatment. The Fxt 6 mg/kg and OEA 3 and 6 mg/kg treatment groups were significantly increased as compared with the MC group (p b 0.01, p b 0.05, p b 0.01, respectively, in hippocampus; p b 0.05, p b 0.05, p b 0.05, respectively, in prefrontal cortex; Fig. 8). In addition, the adrenal indices of mice in the CUMS model group were significantly higher than those in the NC group (p b 0.01), and that in the Fxt (6 mg/kg), and OEA (6 mg/kg) treatment groups were significantly decreased when compared to that of the MC group (p b 0.05, p b 0.05, respectively; Fig. 9).
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4. Discussion
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Depression is a major psychiatric disorder. Based on the clinical association of depressive episodes and stressful life events, many of the animal models used for the evaluation of antidepressant drug activity include a stress component. CUMS can induce behavioral and physiological abnormalities that are important predictors of depression symptoms that may be associated with cerebral monoamine metabolic abnormalities, hypothalamic–pituitary–adrenal (HPA) axis hyperactivity, and changes in endogenous opioid system function and reward thresholds. These abnormalities are similar to depressive symptoms in humans and are often used as an animal model of the pathogenesis of depression (Su et al., 2011; Campos et al., 2013; Xu et al., 2011). In the present study, the effects of CUMS in mice were evaluated by changes in exploratory activity in the OFT, sucrose preference test. The OFT is commonly used to investigate locomotor activity, exploratory behaviors, and depression-like behaviors in experimental animals. The number of crossings and rearings measured during the OFT reflects an animal's exploratory behavior (Elliot et al., 1986; Martin et al., 2013). The sucrose preference test is used to determine an animals' reward sensitivity and depressive state, which is a core symptom of depression (Farley et al., 2011; Lv et al., 2013). After 28 days of chronic stress exposure, exploratory activities in OFT, sucrose preferences of CUMS-treated mice were significantly changed as compared with the NC group (Fig. 3). This indicates that CUMS treatment led to a series of impairments in mice that are similar
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Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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Some studies have shown that chronic stress and prolonged depression are associated with shrinkage of the hippocampus (Sheline, 2003; Jin et al., 2003) and prefrontal cortex (Drevets et al., 1997; Tanaka et al., 2008). Furthermore, a few studies indicated that pharmacological treatment might reverse the decrease in prefrontal cortex and hippocampal volume in patients with depression (Tanaka et al., 2008; McEwen, 2012). These studies are consistent with our results, in which the weight reduction of the hippocampus and prefrontal cortex in CUMS mice was observed, and chronic treatment with Fxt or OEA partially normalized these changes (Fig. 8). Weight reductions may be due to stress-elevated apoptotic markers in the CNS, which are related to neurochemical changes that may be involved in the initiation of stress disorders (Gao et al., 2014). Fxt and OEA may prevent apoptosis and promote regeneration of hippocampal and prefrontal cortex neurons by neurochemical mechanisms, thereby reversing the atrophy of the hippocampus and prefrontal cortex due to stress. Exposure to stress has been shown to affect cortical and hippocampal function adversely by deregulating the expression of neurotrophic factors (Lemtiri-Chlieh and Levine, 2010; Binder and Scharfman, 2004). BDNF is a neurotrophin that exhibits well-characterized effects on neuronal survival and phenotypic differentiation during development (Heyman et al., 2012; McAllister et al., 1999; Lessmann and Mittmann, 2008). BDNF has also emerged as a potent synaptic modulator involved in many forms of activity-dependent synaptic plasticity and may play key roles in learning and memory (Lu, 2003; Tyler et al., 2002). BDNF and its receptor, the tropomyosin receptor kinase B (trkB) receptor, are expressed throughout the nervous system, with the highest levels in the neocortex and hippocampus (Masana et al., 1993; Conner et al., 1997). Stress has been shown to decrease the expression of BDNF, while antidepressant treatment increases the expression of BDNF (Zhao and Levine, 2014). The restoration of BDNF levels is crucial for the rescue of synaptic plasticity in animals (Masana et al., 1993; Conner et al., 1997). The genetic deletion of CB1 receptors leads to decreased BDNF expression, which suggests that the endocannabinoid system exerts a tonic control over BDNF expression (Bilkei-Gorzo, 2012). This hypothesis is supported by our results (Fig. 4). In the present study, BDNF levels were decreased in the hippocampus and prefrontal cortex of CUMS mice. The BDNF levels were normalized after chronic application of Fxt at 6 mg/kg or OEA at 1.5–6 mg/kg. Oxidative stress is known to contribute to neuronal degeneration in the CNS, and evidence suggests that oxidative stress in the rat brain may play an important role in the pathogenesis of anxiety and depression (Réus et al., 2010; Cekić et al., 2010). Reactive oxygen species (ROS)
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to the symptoms observed in depressed patients, such as loss of acute activity and curiosity, anhedonia. After 21 days of OEA treatment, the numbers of crossing and rearing frequencies increased in the OFT, indicating improvements in acute activity. In addition, increased sucrose preferences were observed, indicating normalization of reward sensitivities in mice. The mechanisms underlying depression are very complicated, and its pathogenesis remains unclear. Stress induces several alterations in the central nervous system (CNS), and the classical “monoamine hypothesis” cannot explain the entire pathogenesis of depression. The hippocampus is one of several limbic structures that are implicated in mood disorders. The hippocampus has intrinsic connections with the prefrontal cortex, which is more directly related to emotion and cognition, and contributes to other major symptoms of mood disorders (Réus et al., 2010; Duman and Monteggia, 2006). Furthermore, the hippocampus and prefrontal cortex receive information from multisensory systems and the higher cortex, and play an important role in emotion and behavior, as well as learning and memory. Hence, the hippocampus and prefrontal cortex may be targets of psychological stress and display structural plasticity after both acute and chronic stress (Gao et al., 2014; Dwivedi, 2009).
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Fig. 5. Effect of oleoylethanolamide on serum adrenocorticotropic hormone and corticosterone levels. (A). Effect of oleoylethanolamide on serum adrenocorticotropic hormone (ACTH) levels; (B). Effect of oleoylethanolamide on serum corticosterone (CORT) levels. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA (OEA at 6 mg/kg group). ⁎ p b 0.05, ⁎⁎ p b 0.01 vs. MC, n = 10.
Fig. 6. Effect of oleoylethanolamide on serum total antioxidant capacity (T-AOC) levels. T-AOC is expressed as units/mL for serum. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA (OEA at 6 mg/kg group). ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001 vs. MC, n = 10.
Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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Fig. 8. Effect of oleoylethanolamide on the weight of the hippocampus and prefrontal cortex of mice. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA 1.5, 3, 6 (OEA at 1.5, 3, and 6 mg/kg group). ⁎ p b 0.05, ⁎⁎ p b 0.01 vs. MC, n = 10.
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elevating activities of SOD, reducing MDA levels in the hippocampus and prefrontal cortex, and elevating serum T-AOC levels (OEA at 6 mg/kg). In fact, OEA treatment reversed lipid peroxidation in the hippocampus and the prefrontal cortex of Kun-Ming mice induced by CUMS, and enhanced the body's antioxidant capacity. The HPA axis includes both central and peripheral components, namely the hypothalamus, pituitary, and adrenal glands, which control the stress responses of an individual. It is a major integrated system that maintains body homeostasis (Boyle et al., 2005). Stress is processed through the cerebral cortex, and the transfer of signals to the hypothalamus through the HPA axis induces an increase in blood glucocorticoid (GCS) levels. In this way, behavioral and psychological stress can induce an increase in blood GCS, suggesting that HPA axis dysfunction may be important in the pathogenesis of depression (Wingenfeld and Wolf, 2011). Therefore, when the body is under long-term stress, the HPA axis is continuously hyperactive, and by acting on the adrenal cortex,
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are free radicals or reactive anions/molecules containing oxygen atoms, and an overload of free radicals may cause cell damage by enzyme inactivation, lipid peroxidation, and DNA modification (Liu et al., 1996). Recent studies have showed that ROS may play a role in the physiopathology of depression. Rats subjected to chronic mild stress showed increases in superoxide production in the hippocampus, prefrontal cortex, and cortical brain (Eren et al., 2007), and in humans, elevated ROS in the plasma of patients with major depression has been demonstrated (Fontella et al., 2005). SOD is a major antioxidant enzyme, and can degrade superoxides into hydrogen peroxide (H2O2). MDA is a product of the breakdown of unsaturated fatty acids into their essential chains through oxidation and is accepted as a reliable marker of oxidative stress. T-AOC includes nonenzymatic and enzymatic antioxidant systems (Mokoena et al., 2010; Júnior et al., 2009) and reflects the level of antioxidant capacity. In this study, we demonstrated an elevation in the level of lipid oxidation production of MDA, a decrease in SOD activities in the hippocampus and prefrontal cortex, and a reduction of serum T-AOC in CUMS mice. We confirmed that chronic treatment with the antidepressant Fxt or OEA at 1.5–6 mg/kg improved oxidative stress parameters, such as
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Fig. 7. Oleoylethanolamide effects on MDA levels and SOD activities in the hippocampus and prefrontal cortex. (A). Malondialdehyde (MDA) levels in the hippocampus and prefrontal cortex. (B). Superoxide dismutase (SOD) activities in the hippocampus and prefrontal cortex. The MDA levels are expressed as nmol/mg protein of brain tissues. The SOD activity is expressed as units/mg protein of brain tissues. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA 1.5, 3, 6 (OEA at 1.5, 3, and 6 mg/kg group). ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001 vs. MC, n = 10.
Fig. 9. The adrenal index of the groups. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA 1.5, 3, 6 (OEA at 1.5, 3, and 6 mg/kg group). ⁎ p b 0.05, ⁎⁎ p b 0.01 vs. MC, n = 10.
Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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This work was supported by the National Natural Science Foundation of China (No. 81460217) and the Yanbian University Natural Science Foundation of China (201322).
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In conclusion, OEA can improve animal depression-like behaviors, but its novel and complex mechanism may be related to regulation of the homeostasis of BDNF levels in the hippocampus and prefrontal cortex, in which the HPA axis and antioxidant defenses play a crucial role. OEA is a small molecule and is lipophilic. It easily penetrates the brain after peripheral administration, and may be clinically beneficial as an endocannabinoid analog antidepressant. Although OEA shows potential as an antidepressant that reverses cerebral functional abnormalities and ameliorates depression, further research should be performed on the specific pathways by which OEA affects BDNF levels, the HPA axis, and antioxidant defenses.
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induces excessive secretion of glucocorticoids. Persistent hyperthyroidism of the HPA axis can seriously affect the function and structure of the body's adrenal glands. After exposure to 20 days of chronic stress, rats show adrenal cortex hyperplasia, medullary atrophy, and an increase in the adrenal index (Liu et al., 2011). In recent years, numerous studies have demonstrated that the HPA axis may be important for the clinical efficacy of antidepressant treatment, and the HPA axis is an emerging target in the treatment of depression (Locatelli et al., 2010). In the present study, ACTH and CORT levels in CUMS-treated mice were increased significantly when compared with the NC group, and OEA (6 mg/kg) and Fxt (6 mg/kg) reduced ACTH and CORT levels significantly in CUMS-treated mice compared with the NC group. Indeed, in this respect, OEA at 6 mg/kg was more effective than Fxt at 6 mg/kg (Fig. 5). Moreover, the adrenal index was increased significantly compared with the NC group, and OEA (6 mg/kg) and Fxt (6 mg/kg) significantly reduced the adrenal index in CUMS-treated mice, as compared with the NC group (Fig. 9). These results are consistent with the above mentioned neurochemical studies on serum ACTH and CORT after CUMS and drug treatments in mice. Long-term chronic stress induces ACTH and CORT increases, adrenal hyperplasia, and increased adrenal indices. OEA normalized these changes, indicating that OEA can reduce hyperactivity of the HPA axis to ameliorate the effects of stress. In summary, in the present study, we determined the antidepressantlike effects of OEA in a mouse CUMS depression model, and compared these effects in CUMS mice treated with the antidepressant Fxt, a serotonin reuptake inhibitor. Our results confirm that OEA at 1.5–6 mg/kg which increased locomotor activity and sucrose preferences, increased BDNF levels and SOD activities, and decreased MDA levels in the hippocampus and prefrontal cortex, while OEA at 3–6 mg/kg which reversed atrophy of the hippocampus and prefrontal cortex were observed. OEA at 6 mg/kg reduced serum ACTH and CORT levels, increased T-AOC levels, and reversed adrenal hyperplasia in CUMS-treated mice, and OEA at 6 mg/kg was more effective in improving some indicators than Fxt at 6 mg/kg. These results suggest that OEA may normalize a hyperactive HPA axis more effectively than Fxt, and increased BDNF levels improve exploratory activity, reward sensitivity, and anhedonia. As stated above, the reversal of abnormal secretion of BDNF and stress-induced HPA axis hyperactivity, as well as improving the body's antioxidant capacity appears to mediate the effects of OEA on antidepressant-like behavior in mice, and may be the mechanisms for the antidepressant activity of OEA.
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Parolaro D, Realini N, Vigano D, Guidali C, Rubino T. The endocannabinoid system and psychiatric disorders. Exp Neurol 2010;224:3–14. Réus Gislaine Z, Stringari Roberto B, de Souza Bruna, Petronilho Fabrícia, Dal-Pizzol Felipe, Hallak Jaime E, et al. Harmine and imipramine promote antioxidant activities in prefrontal cortex and hippocampus. Oxid Med Cell Longev 2010;3:325–31. Sheline YI. Neuroimaging studies of mood disorder effects on the brain. Biol Psychiatry 2003;54:338–52. Su ZH, Li SQ, Zou GA, Yu CY, Sun YG. Urinary metabonomics study of anti-depressive effect of Chaihu-Shu-Gan-San on an experimental model of depression induced by chronic variable stress in rats. J Pharm Biomed Anal 2011;55:533–9. Tanaka J, Horiike Y, Matsuzaki M, Miyazaki T, Ellis-Davies GC, et al. Protein synthesis and neurotrophin-dependent structural plasticity of single dendritic spines. Science 2008; 319:1683–7. Tyler WJ, Alonso M, Bramham CR, Pozzo-Miller LD. From acquisition to consolidation: on the role of brain-derived neurotrophic factor signaling in hippocampal-dependent learning. Learn Mem 2002;9:224–37.
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Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
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Research article
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Peng Jin a,c,d, Hai-Ling Yu b,⁎, Tian-Lan b, Feng Zhang b, Zhe-Shan Quan a,⁎ a
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Article history: Received 7 January 2015 Received in revised form 30 March 2015 Accepted 3 April 2015 Available online xxxx
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Keywords: Oleoylethanolamide Antidepressant Brain-derived neurotrophic factor Oxidative stress HPA Mice
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Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry of Education, Yanbian University College of Pharmacy, Yanji 133000, PR China College of Medicine, Yanbian University, Park Street 977, Yanji, 133002 Jilin, PR China Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Cancer Research Institute, College of Medicine, Seoul National University, Yongon-dong 28, Chongno-gu, Seoul 110-799, Republic of Korea d Department of Biomedical Science, Ischemic/Hypoxic Disease Institute, Cancer Research Institute, College of Medicine, Seoul National University, Yongon-dong 28, Chongno-gu, Seoul 110-799, Republic of Korea b
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Oleoylethanolamide (OEA) is an endocannabinoid analog that belongs to a family of endogenous acylethanolamides. Increasing evidence suggests that OEA may act as an endogenous neuroprotective factor and participate in the control of mental disorder-related behaviors. In this study, we examined whether OEA is effective against depression and investigated the role of circulating endogenous acylethanolamides during stress. Mice were subjected to 28 days of chronic unpredictable mild stress (CUMS), and during the last 21 days, treated with oral OEA (1.5–6 mg/kg) or 6 mg/kg fluoxetine. Sucrose preference and open field test activity were used to evaluate depression-like behaviors during CUMS and after OEA treatment. Weights of the prefrontal cortex and hippocampus were determined, and the adrenal index was measured. Furthermore, changes in serum adrenocorticotropic hormone (ACTH), corticosterone (CORT) and total antioxidant capacity (T-AOC), brain-derived neurotrophic factor (BDNF), and lipid peroxidation product malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activities in the hippocampus and prefrontal cortex were detected. Our findings indicate that OEA normalized sucrose preferences, locomotion distances, rearing frequencies, prefrontal cortex and hippocampal atrophy, and adrenal indices. In addition, OEA reversed the abnormalities of BDNF and MDA levels and SOD activities in the hippocampus and prefrontal cortex, as well as changes in serum levels of ACTH, CORT, and T-AOC. The antidepressant effects of OEA may be related to the regulation of BDNF levels in the hippocampus and prefrontal cortex, antioxidant defenses, and normalizing hyperactivity in the hypothalamic–pituitary–adrenal axis (HPA). © 2015 Published by Elsevier Inc.
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brain are ideally situated to exert regulatory control over emotional behavior, mood, and stress response (Yu et al., 2011; Hill and Gorzalka, 2009). Malfunctions in the endocannabinoid system may promote the development and maintenance of psychiatric disorders such as depression and panic disorder (Yu et al., 2011; Bambico et al., 2009). A growing body of evidence demonstrates that deficits in endocannabinoid signaling may result in depression-like and anxiogenic behavioral responses. Endocannabinoids are released following the development of depression, causing both neuroprotective and degenerative effects (Viscomi et al., 2010). Pharmacological augmentation of endocannabinoid signaling can produce both antidepressant and anxiolytic behavioral responses (Yu et al., 2011; Hill et al., 2009; Parolaro et al., 2010). Oleoylethanolamide (OEA; Fig. 1) is an endocannabinoid analog that belongs to a family of endogenous acylethanolamides. Acylethanolamides act as agonists of the nuclear peroxisome proliferator-activated receptor alpha (PPARα) (Yu et al., 2011). Increasing evidence suggests that OEA may act as an endogenous neuroprotective factor and participate in the control of reward-related behaviors (Zhou et al., 2012). Systemic
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Depression is a major mental disorder associated with clinical symptoms such as persistent negative mood, decreased physical activity, feelings of helplessness, sluggish thought, and cognitive dysfunction. Many psychological, social, environmental, and genetic factors can result in depression (Bilbao et al., 2013), and the incidence of depression has increased, with approximately 5% of the population experiencing a major depressive episode (Yu et al., 2011). Current antidepressant drugs can alleviate only some symptoms of depression, and side effects are common. Therefore, the development of more effective and less toxic antidepressants has attracted significant attention in recent years. The central endocannabinoid system is a neuroactive lipid signaling system in the brain that acts to control neurotransmitter release. The expression patterns of this system throughout limbic regions of the
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⁎ Corresponding authors. E-mail addresses: [email protected] (H.-L. Yu), [email protected] (Z.-S. Quan).
http://dx.doi.org/10.1016/j.pbb.2015.04.001 0091-3057/© 2015 Published by Elsevier Inc.
Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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administration of OEA to rats results in elevated OEA plasma levels, as has also been reported in patients with post-traumatic stress disorder, and improved memory of aversive training experiences (Hauer et al., 2013). These findings indicate that changes in peripheral endocannabinoid concentrations may reflect alterations in central endocannabinoid signaling that alters behavior. Reduced endocannabinoid functionality may be a predisposing factor for major depression, and boosting endocannabinoid tone may be a useful alternative therapeutic approach for depressive disorders (Yu et al., 2011; Parolaro et al., 2010; Su et al., 2011). However, it is not clear whether orally administered OEA is effective against depression, and the regulation of circulating endogenous acylethanolamides during stress needs to be investigated. In the present study, the antidepressant-like effects of OEA were examined in comparison with the antidepressant fluoxetine (Fxt, a serotonin reuptake inhibitor) using a mouse model of chronic unpredictable mild stress. Additionally, in neurochemical studies, changes in the prefrontal cortex and hippocampus of brain-derived neurotrophic factor (BDNF) and lipid peroxidation product malondialdehyde (MDA) levels and superoxide dismutase (SOD) activities, as well as serum levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), and total antioxidant capacity (T-AOC) were determined.
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at one mouse per cage (32 × 18 × 16 cm), and the stressors included the following: (1) horizontal shock for 5 min (1 shock/s); (2) swimming in 4 °C for 1 min; (3) swimming in 25 °C water for 5 min; (4) food deprivation for 14 h; (5) water deprivation for 16 h; (6) wet bedding for 17 h; (7) housed together (5 mice per cage) for 17 h; (8) empty cage for 17 h; (9) cage tilt at 45° for 2 h; (10) room temperature at 45 °C for 5 min; (11) tail suspension for 5 min; (12) foreign object placement for 6 h; (13) flash for 10 min; and (14) reversed light/dark cycle (overnight illumination). The 14 stressors were scheduled semirandomly, with two different stressors daily. Apart from the NC group, the mice were treated with the stressors for 28 days, and the same stressor was not continued, so that the mice could not anticipate the occurrence of specific stressors. Untreated mice served as the NC group, which were housed in another room at 5 mice per cage (32 × 18 × 16 cm). In addition to withdrawing food and water for 24 h before the sugar preference experiment and withdrawing food for 6 h before weighing, the mice were not subjected to additional stimulation. All behavioral tests were performed at 9:00–11:00, in a quiet warm environment, and were carried out in parallel in each experimental group. Biochemical parameters were measured on the last day of the experiment. After CUMS for 7 days, the CUMS-treated mice were randomly divided into five groups: the model control (MC) group; mice treated daily with Fxt or OEA at 1.5, 3, or 6 mg/kg group in 0.3% sodium carboxymethyl cellulose via gavage for 21 days.
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2.3. Experimental procedures (Fig. 2)
The sucrose preference test procedure was performed as described below (Martin et al., 2013; Lv et al., 2013). Learning and training were performed in each mouse before the experiment. Initially, the mice were acclimatized to sucrose and water for 3 days, and then exposed to 1% sucrose solution (w/v) for 24 h. A bottle of 1% sucrose solution and a bottle of tap water were presented at random positions to each mouse for the next 24 h, followed by food and water deprivation for 24 h. For testing, on day 4, two bottles, one containing 1% sucrose solution and the other containing tap water were weighed and presented to each mouse for 4 h. The position of the two bottles was set randomly. The sucrose preference test was performed at days 7 and 28 of the experiment. The sucrose preference was calculated using the following formula: Sucrose preference = sucrose consumption (g) / water consumption (g) + sucrose consumption (g) × 100%.
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Male adult Kunming mice (18–22 g; Laboratory Animal Center of Yanbian University, Jilin Province, China) were used. The mice were housed individually in cages for 3 days to adapt to the environment under the following controlled conditions: 12 h light–12 h dark cycles; free access to tap water and food pellets; and ambient temperature and relative humidity at 22 ± 2 °C and 55 ± 5%, respectively. Some of the mice were subjected to the stressors as the depression model. The experimental procedures were approved by the Animal Care and Use Committee of the Institute of Psychology of the Chinese Academy of Sciences and in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.
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The animals were randomly divided into two groups: a chronic unpredictable mild stress (CUMS)-treated group and a normal control (NC) group. The CUMS-treated mice were subjected to a series of variable stressors as described by Su et al. (2011), Farley et al. (2011) and Hu et al. (2010), with slight modifications. The animals were housed
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The mouse BDNF ELISA Kit, mouse ACTH ELISA Kit, and mouse CORT ELISA Kit were purchased from Beijing pines Biotechnology Co., Ltd., China; the MDA, SOD, and T-AOC Kits were purchased from Nanjing Jiancheng Bioengineering Institute, China. The tested compound OEA (synthesized at the College of Pharmacy, Yanbian University, Jilin Province, China) or fluoxetine (Fxt, Enhua Jiangsu Pharmaceutical Co., Ltd., China) was suspended in 0.3% methyl cellulose (Loba-Chemie, Shanghai, China) and administered daily via gavage. All doses were expressed as mg/kg body weight.
The open-field test (OFT) procedure was performed once per week as described below (Elliot et al., 1986; Martin et al., 2013). Each mouse was placed individually in the center of the open-field apparatus, and locomotor activity was assessed. The open-field apparatus was a non-transparent plastic container (80 × 60 × 30 cm), with the underside divided into 10 × 10 cm squares of 48 units without walls. The animals were gently placed in the center of the platform and were allowed to explore the surroundings. Hand-operated counters were used for 3 min to score locomotion (number of line crossings with all four paws) and rearing frequencies (number of times an animal stood on its hind legs). The researchers, blind to the treatment groups, scored the behaviors in the open field. Experiments were performed in a dark room, and the apparatus was illuminated by a 60 W bulb, providing a yellowish light and positioned 1 m above the center of the apparatus. The walls and floor surfaces were thoroughly cleaned with 10% ethanol between the tests.
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Fig. 2. Experimental procedures. A. Animal groups and treatments. B. Experimental procedures in chronic unpredictable mild stress (CUMS). CUMS procedures were performed for 28 days, followed by drug treatment for 21 days. Vehicle, oleoylethanolamide (1.5, 3, 6 mg/kg), or Fxt (6 mg/kg) were administered for 21 days starting at day 8 after initiating CUMS procedures.
using a microplate reader within 15 min of stop solution addition. The BDNF concentration is expressed as pg/mg brain tissue, and the ACTH and CORT concentrations were expressed as pg/mL and ng/mL serum, respectively.
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The levels of MDA and SOD activity in the supernatants of tissues homogenates were determined with the commercial analysis kits, according to the manufacturer's instructions. The MDA level was expressed as nmol/mg protein of brain tissue, the SOD activity was expressed as units/mg protein of brain tissue, and T-AOC was expressed as units/mL of serum.
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Mice were euthanized by decapitation, and the whole brain was removed quickly on an ice table. The hippocampus and prefrontal cortex were separated under a dissecting microscope, gently washed, and homogenized in a cold phosphate buffer (0.05 mol/L, pH 7.8), and centrifuged at 3500 ×g for 10 min. The supernatant was stored at −80 °C for subsequent biochemical analysis.
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BDNF, ACTH, and CORT concentration determination was performed using the ELISA method, according to the mouse BDNF, ACTH, or CORT ELISA Kit manufacturer's instructions, respectively. The specific steps used standard wells (50 μL of standard), testing sample wells (10 μL of sample and 40 μL of sample diluent), and blank wells. A total of 100 μL of horseradish peroxidase (HRP)-conjugate reagent was added to each well, and the wells were covered with an adhesive strip and incubated for 60 min at 37 °C. Each well was washed 5 times by filling each well with wash solution (400 μL) using a squirt bottle manifold dispenser. After the last wash, remaining wash solution was removed by aspirating or decanting, inversion of the plate, and blotting against clean paper towels. Chromogen solution A (50 μL) and chromogen B (50 μL) were added to each well. The wells were gently mixed, then incubated for 15 min at 37 °C away from light. After incubation, 50 μL of stop solution was added to each well. Optical density (OD) was obtained at 450 nm
After each animal's blood and brain were removed, the hippocampus and prefrontal cortex were separated and weighed. The bilateral adrenals were immediately removed and weighed. The adrenal index was calculated using the following formula:
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allowed to clot for 30 min at 37 °C before centrifugation for 10 min at 3000 × g at 4 °C. The serum was isolated and stored at − 80 °C for subsequent ACTH, CORT, and T-AOC determination.
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2.11. Statistical analysis One-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test was used for other statistical evaluations. Data are presented as the means ± SEM. p b 0.05 was considered statistically significant.
Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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Fig. 3. Changes in physical symptoms and behavioral indicators during CUMS procedure and after OEA treatment. A (a–d). Effect of oleoylethanolamide (OEA) on open-field test performance in chronic unpredictable mild stress (CUMS) mice. A (a and c). The change of crossing and rearing with time in the CUMS procedure; A (b and d). The counts of crossing and rearing in mice after 28 days of CUMS and 21 days of drug treatment. (B). The sucrose preference test after 28 days of CUMS and 21 days of drug-treatment. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA 1.5, 3, 6 (OEA at 1.5, 3, and 6 mg/kg group). Baseline is the mouse ethological indicators before CUMS. ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001 vs. MC, n = 10.
Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
P. Jin et al. / Pharmacology, Biochemistry and Behavior xxx (2015) xxx–xxx
CORT levels (p b 0.05, p b 0.01, respectively) as compared with those 271 of the MC group (Fig. 5). 272
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3.2. Determination of BDNF concentrations in hippocampus and prefrontal cortex
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Fig. 4 shows that, after 28 days of CUMS, BDNF levels in the hippocampus and prefrontal cortex of CUMS model mice were decreased significantly compared with those of the NC group (p b 0.001, p b 0.01, respectively). With drug treatment during the last 21 days, the BDNF levels in the Fxt 6 mg/kg and OEA 1.5, 3, and 6 mg/kg treatment groups were significantly increased (p b 0.01, p b 0.05, p b 0.01, p b 0.01, respectively, in hippocampus; p b 0.05, p b 0.05, p b 0.05, p b 0.01, respectively, in prefrontal cortex) compared with the MC group.
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After 28 days of CUMS and 21 days of drug treatment, serum ACTH and CORT concentrations in the mice in CUMS model group were significantly increased compared with the NC group (p b 0.01, p b 0.01, respectively). The Fxt (6 mg/kg) and OEA (6 mg/kg) groups showed significantly decreased ACTH (p b 0.05, p b 0.01, respectively) and
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Fig. 4. Effect of oleoylethanolamide on brain-derived neurotrophic factor (BDNF) levels in the hippocampus and prefrontal cortex. The BDNF concentration is expressed as pg/mg brain tissue. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA 1.5, 3, 6 (OEA at 1.5, 3, and 6 mg/kg group). ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001 vs. MC, n = 10.
3.4. Determination of T-AOC and MDA levels and SOD activities
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After 28 days of CUMS, serum T-AOC levels in MC mice were significantly decreased (p b 0.001, Fig. 6), MDA concentrations were significantly increased (p b 0.01, p b 0.01 in the hippocampus and prefrontal cortex, respectively; Fig. 7A), and SOD activities were significantly decreased (p b 0.01, p b 0.001 in hippocampus and prefrontal cortex, respectively; Fig. 7B) compared to those in the NC group. After 21 days of drug treatment, the Fxt (6 mg/kg) and OEA (6 mg/kg) groups exhibited significantly increased serum T-AOC levels (p b 0.01, p b 0.01, respectively) compared to the MC group. The Fxt 6 mg/kg and OEA 1.5, 3, and 6 mg/kg treatment groups exhibited significantly decreased MDA concentrations (p b 0.01, p b 0.05, p b 0.01, p b 0.01, respectively, in hippocampus; p b 0.05, p b 0.05, p b 0.05, p b 0.01, respectively, in prefrontal cortex; Fig. 7A) compared to the MC group. Finally, the Fxt 6 mg/kg and OEA 1.5, 3, and 6 mg/kg treatment groups showed significantly increased SOD activities (p b 0.05, p b 0.05, p b 0.001, p b 0.001, respectively) in the hippocampus compared to the MC group, while in the prefrontal cortex, SOD activities were significantly increased only in the Fxt 6 mg/kg and OEA 3 and 6 mg/kg treatment groups (p b 0.05, p b 0.05, p b 0.01, respectively; Fig. 7B).
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3.5. Hippocampus and prefrontal cortex weights and adrenal index
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Depression-like behaviors after 28 days of CUMS and 21 days of OEA (1.5, 3, or 6 mg/kg) treatment were examined (Fig. 3). The NC and CUMS groups showed similar behavioral indicators before CUMS (data not shown). Fig. 3A (b and d) shows that, after 21 days of drug treatment, the exploratory activities of mice (crossing and rearing) in the MC group were significantly lower than those in the NC group (p b 0.001, p b 0.001, respectively). The Fxt 6 mg/kg, and OEA 1.5, 3, and 6 mg/kg treatment groups were increased significantly as compared with the MC group (p b 0.01, p b 0.05, p b 0.01, p b 0.001 in crossing, respectively; p b 0.05, p b 0.05, p b 0.01, p b 0.001 in rearing, respectively). After 28 days of CUMS and 21 days of drug treatment, sucrose preferences were significantly decreased (p b 0.001, Fig. 3B) in the MC mice compared with the NC group. Sucrose preferences in the Fxt 6 mg/kg and OEA 1.5, 3, and 6 mg/kg treatment groups were significantly increased (p b 0.01, p b 0.05, p b 0.01, p b 0.001, respectively; Fig. 3B) as compared with the MC group.
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Fig. 8 shows that the hippocampus and prefrontal cortex weights of mice in the CUMS group were significantly decreased when compared with those in the NC group (p b 0.01, p b 0.05, respectively) after 28 days of CUMS and 21 days of drug treatment. The Fxt 6 mg/kg and OEA 3 and 6 mg/kg treatment groups were significantly increased as compared with the MC group (p b 0.01, p b 0.05, p b 0.01, respectively, in hippocampus; p b 0.05, p b 0.05, p b 0.05, respectively, in prefrontal cortex; Fig. 8). In addition, the adrenal indices of mice in the CUMS model group were significantly higher than those in the NC group (p b 0.01), and that in the Fxt (6 mg/kg), and OEA (6 mg/kg) treatment groups were significantly decreased when compared to that of the MC group (p b 0.05, p b 0.05, respectively; Fig. 9).
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Depression is a major psychiatric disorder. Based on the clinical association of depressive episodes and stressful life events, many of the animal models used for the evaluation of antidepressant drug activity include a stress component. CUMS can induce behavioral and physiological abnormalities that are important predictors of depression symptoms that may be associated with cerebral monoamine metabolic abnormalities, hypothalamic–pituitary–adrenal (HPA) axis hyperactivity, and changes in endogenous opioid system function and reward thresholds. These abnormalities are similar to depressive symptoms in humans and are often used as an animal model of the pathogenesis of depression (Su et al., 2011; Campos et al., 2013; Xu et al., 2011). In the present study, the effects of CUMS in mice were evaluated by changes in exploratory activity in the OFT, sucrose preference test. The OFT is commonly used to investigate locomotor activity, exploratory behaviors, and depression-like behaviors in experimental animals. The number of crossings and rearings measured during the OFT reflects an animal's exploratory behavior (Elliot et al., 1986; Martin et al., 2013). The sucrose preference test is used to determine an animals' reward sensitivity and depressive state, which is a core symptom of depression (Farley et al., 2011; Lv et al., 2013). After 28 days of chronic stress exposure, exploratory activities in OFT, sucrose preferences of CUMS-treated mice were significantly changed as compared with the NC group (Fig. 3). This indicates that CUMS treatment led to a series of impairments in mice that are similar
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Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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Some studies have shown that chronic stress and prolonged depression are associated with shrinkage of the hippocampus (Sheline, 2003; Jin et al., 2003) and prefrontal cortex (Drevets et al., 1997; Tanaka et al., 2008). Furthermore, a few studies indicated that pharmacological treatment might reverse the decrease in prefrontal cortex and hippocampal volume in patients with depression (Tanaka et al., 2008; McEwen, 2012). These studies are consistent with our results, in which the weight reduction of the hippocampus and prefrontal cortex in CUMS mice was observed, and chronic treatment with Fxt or OEA partially normalized these changes (Fig. 8). Weight reductions may be due to stress-elevated apoptotic markers in the CNS, which are related to neurochemical changes that may be involved in the initiation of stress disorders (Gao et al., 2014). Fxt and OEA may prevent apoptosis and promote regeneration of hippocampal and prefrontal cortex neurons by neurochemical mechanisms, thereby reversing the atrophy of the hippocampus and prefrontal cortex due to stress. Exposure to stress has been shown to affect cortical and hippocampal function adversely by deregulating the expression of neurotrophic factors (Lemtiri-Chlieh and Levine, 2010; Binder and Scharfman, 2004). BDNF is a neurotrophin that exhibits well-characterized effects on neuronal survival and phenotypic differentiation during development (Heyman et al., 2012; McAllister et al., 1999; Lessmann and Mittmann, 2008). BDNF has also emerged as a potent synaptic modulator involved in many forms of activity-dependent synaptic plasticity and may play key roles in learning and memory (Lu, 2003; Tyler et al., 2002). BDNF and its receptor, the tropomyosin receptor kinase B (trkB) receptor, are expressed throughout the nervous system, with the highest levels in the neocortex and hippocampus (Masana et al., 1993; Conner et al., 1997). Stress has been shown to decrease the expression of BDNF, while antidepressant treatment increases the expression of BDNF (Zhao and Levine, 2014). The restoration of BDNF levels is crucial for the rescue of synaptic plasticity in animals (Masana et al., 1993; Conner et al., 1997). The genetic deletion of CB1 receptors leads to decreased BDNF expression, which suggests that the endocannabinoid system exerts a tonic control over BDNF expression (Bilkei-Gorzo, 2012). This hypothesis is supported by our results (Fig. 4). In the present study, BDNF levels were decreased in the hippocampus and prefrontal cortex of CUMS mice. The BDNF levels were normalized after chronic application of Fxt at 6 mg/kg or OEA at 1.5–6 mg/kg. Oxidative stress is known to contribute to neuronal degeneration in the CNS, and evidence suggests that oxidative stress in the rat brain may play an important role in the pathogenesis of anxiety and depression (Réus et al., 2010; Cekić et al., 2010). Reactive oxygen species (ROS)
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to the symptoms observed in depressed patients, such as loss of acute activity and curiosity, anhedonia. After 21 days of OEA treatment, the numbers of crossing and rearing frequencies increased in the OFT, indicating improvements in acute activity. In addition, increased sucrose preferences were observed, indicating normalization of reward sensitivities in mice. The mechanisms underlying depression are very complicated, and its pathogenesis remains unclear. Stress induces several alterations in the central nervous system (CNS), and the classical “monoamine hypothesis” cannot explain the entire pathogenesis of depression. The hippocampus is one of several limbic structures that are implicated in mood disorders. The hippocampus has intrinsic connections with the prefrontal cortex, which is more directly related to emotion and cognition, and contributes to other major symptoms of mood disorders (Réus et al., 2010; Duman and Monteggia, 2006). Furthermore, the hippocampus and prefrontal cortex receive information from multisensory systems and the higher cortex, and play an important role in emotion and behavior, as well as learning and memory. Hence, the hippocampus and prefrontal cortex may be targets of psychological stress and display structural plasticity after both acute and chronic stress (Gao et al., 2014; Dwivedi, 2009).
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Fig. 5. Effect of oleoylethanolamide on serum adrenocorticotropic hormone and corticosterone levels. (A). Effect of oleoylethanolamide on serum adrenocorticotropic hormone (ACTH) levels; (B). Effect of oleoylethanolamide on serum corticosterone (CORT) levels. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA (OEA at 6 mg/kg group). ⁎ p b 0.05, ⁎⁎ p b 0.01 vs. MC, n = 10.
Fig. 6. Effect of oleoylethanolamide on serum total antioxidant capacity (T-AOC) levels. T-AOC is expressed as units/mL for serum. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA (OEA at 6 mg/kg group). ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001 vs. MC, n = 10.
Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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Fig. 8. Effect of oleoylethanolamide on the weight of the hippocampus and prefrontal cortex of mice. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA 1.5, 3, 6 (OEA at 1.5, 3, and 6 mg/kg group). ⁎ p b 0.05, ⁎⁎ p b 0.01 vs. MC, n = 10.
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elevating activities of SOD, reducing MDA levels in the hippocampus and prefrontal cortex, and elevating serum T-AOC levels (OEA at 6 mg/kg). In fact, OEA treatment reversed lipid peroxidation in the hippocampus and the prefrontal cortex of Kun-Ming mice induced by CUMS, and enhanced the body's antioxidant capacity. The HPA axis includes both central and peripheral components, namely the hypothalamus, pituitary, and adrenal glands, which control the stress responses of an individual. It is a major integrated system that maintains body homeostasis (Boyle et al., 2005). Stress is processed through the cerebral cortex, and the transfer of signals to the hypothalamus through the HPA axis induces an increase in blood glucocorticoid (GCS) levels. In this way, behavioral and psychological stress can induce an increase in blood GCS, suggesting that HPA axis dysfunction may be important in the pathogenesis of depression (Wingenfeld and Wolf, 2011). Therefore, when the body is under long-term stress, the HPA axis is continuously hyperactive, and by acting on the adrenal cortex,
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are free radicals or reactive anions/molecules containing oxygen atoms, and an overload of free radicals may cause cell damage by enzyme inactivation, lipid peroxidation, and DNA modification (Liu et al., 1996). Recent studies have showed that ROS may play a role in the physiopathology of depression. Rats subjected to chronic mild stress showed increases in superoxide production in the hippocampus, prefrontal cortex, and cortical brain (Eren et al., 2007), and in humans, elevated ROS in the plasma of patients with major depression has been demonstrated (Fontella et al., 2005). SOD is a major antioxidant enzyme, and can degrade superoxides into hydrogen peroxide (H2O2). MDA is a product of the breakdown of unsaturated fatty acids into their essential chains through oxidation and is accepted as a reliable marker of oxidative stress. T-AOC includes nonenzymatic and enzymatic antioxidant systems (Mokoena et al., 2010; Júnior et al., 2009) and reflects the level of antioxidant capacity. In this study, we demonstrated an elevation in the level of lipid oxidation production of MDA, a decrease in SOD activities in the hippocampus and prefrontal cortex, and a reduction of serum T-AOC in CUMS mice. We confirmed that chronic treatment with the antidepressant Fxt or OEA at 1.5–6 mg/kg improved oxidative stress parameters, such as
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Fig. 7. Oleoylethanolamide effects on MDA levels and SOD activities in the hippocampus and prefrontal cortex. (A). Malondialdehyde (MDA) levels in the hippocampus and prefrontal cortex. (B). Superoxide dismutase (SOD) activities in the hippocampus and prefrontal cortex. The MDA levels are expressed as nmol/mg protein of brain tissues. The SOD activity is expressed as units/mg protein of brain tissues. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA 1.5, 3, 6 (OEA at 1.5, 3, and 6 mg/kg group). ⁎ p b 0.05, ⁎⁎ p b 0.01, ⁎⁎⁎ p b 0.001 vs. MC, n = 10.
Fig. 9. The adrenal index of the groups. NC (normal control group), MC (model control group, vehicle group), Fxt (fluoxetine at 6 mg/kg group), OEA 1.5, 3, 6 (OEA at 1.5, 3, and 6 mg/kg group). ⁎ p b 0.05, ⁎⁎ p b 0.01 vs. MC, n = 10.
Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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This work was supported by the National Natural Science Foundation of China (No. 81460217) and the Yanbian University Natural Science Foundation of China (201322).
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In conclusion, OEA can improve animal depression-like behaviors, but its novel and complex mechanism may be related to regulation of the homeostasis of BDNF levels in the hippocampus and prefrontal cortex, in which the HPA axis and antioxidant defenses play a crucial role. OEA is a small molecule and is lipophilic. It easily penetrates the brain after peripheral administration, and may be clinically beneficial as an endocannabinoid analog antidepressant. Although OEA shows potential as an antidepressant that reverses cerebral functional abnormalities and ameliorates depression, further research should be performed on the specific pathways by which OEA affects BDNF levels, the HPA axis, and antioxidant defenses.
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induces excessive secretion of glucocorticoids. Persistent hyperthyroidism of the HPA axis can seriously affect the function and structure of the body's adrenal glands. After exposure to 20 days of chronic stress, rats show adrenal cortex hyperplasia, medullary atrophy, and an increase in the adrenal index (Liu et al., 2011). In recent years, numerous studies have demonstrated that the HPA axis may be important for the clinical efficacy of antidepressant treatment, and the HPA axis is an emerging target in the treatment of depression (Locatelli et al., 2010). In the present study, ACTH and CORT levels in CUMS-treated mice were increased significantly when compared with the NC group, and OEA (6 mg/kg) and Fxt (6 mg/kg) reduced ACTH and CORT levels significantly in CUMS-treated mice compared with the NC group. Indeed, in this respect, OEA at 6 mg/kg was more effective than Fxt at 6 mg/kg (Fig. 5). Moreover, the adrenal index was increased significantly compared with the NC group, and OEA (6 mg/kg) and Fxt (6 mg/kg) significantly reduced the adrenal index in CUMS-treated mice, as compared with the NC group (Fig. 9). These results are consistent with the above mentioned neurochemical studies on serum ACTH and CORT after CUMS and drug treatments in mice. Long-term chronic stress induces ACTH and CORT increases, adrenal hyperplasia, and increased adrenal indices. OEA normalized these changes, indicating that OEA can reduce hyperactivity of the HPA axis to ameliorate the effects of stress. In summary, in the present study, we determined the antidepressantlike effects of OEA in a mouse CUMS depression model, and compared these effects in CUMS mice treated with the antidepressant Fxt, a serotonin reuptake inhibitor. Our results confirm that OEA at 1.5–6 mg/kg which increased locomotor activity and sucrose preferences, increased BDNF levels and SOD activities, and decreased MDA levels in the hippocampus and prefrontal cortex, while OEA at 3–6 mg/kg which reversed atrophy of the hippocampus and prefrontal cortex were observed. OEA at 6 mg/kg reduced serum ACTH and CORT levels, increased T-AOC levels, and reversed adrenal hyperplasia in CUMS-treated mice, and OEA at 6 mg/kg was more effective in improving some indicators than Fxt at 6 mg/kg. These results suggest that OEA may normalize a hyperactive HPA axis more effectively than Fxt, and increased BDNF levels improve exploratory activity, reward sensitivity, and anhedonia. As stated above, the reversal of abnormal secretion of BDNF and stress-induced HPA axis hyperactivity, as well as improving the body's antioxidant capacity appears to mediate the effects of OEA on antidepressant-like behavior in mice, and may be the mechanisms for the antidepressant activity of OEA.
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Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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Please cite this article as: Jin P, et al, Antidepressant-like effects of oleoylethanolamide in a mouse model of chronic unpredictable mild stress, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.04.001
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