N-acetylcysteine attenuates neuroinflammation associated depressive behavior induced by chronic unpredictable mild stress in rat

Accepted Manuscript Title: N-acetylcysteine attenuates neuroinflammation associated depressive behavior induced by chronic unpredictable mild stress in rat Authors: Joneth Fernandes, Girdhari Lal Gupta PII: DOI: Reference:

S0166-4328(18)31740-6 https://doi.org/10.1016/j.bbr.2019.02.025 BBR 11809

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

15 December 2018 29 January 2019 13 February 2019

Please cite this article as: Fernandes J, Lal Gupta G, N-acetylcysteine attenuates neuroinflammation associated depressive behavior induced by chronic unpredictable mild stress in rat, Behavioural Brain Research (2019), https://doi.org/10.1016/j.bbr.2019.02.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

N-acetylcysteine attenuates neuroinflammation associated depressive behavior induced by chronic unpredictable mild stress in rat

Joneth Fernandesa,#, Girdhari Lal Guptaa,#,* Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM's

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NMIMS, V.L. Mehta Road, Vile Parle (W), Mumbai – 400 056, India

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*

Corresponding Author:

Dr. Girdhari Lal Gupta,

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Department of Pharmacology, School of Pharmacy & Technology Management,

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SVKM’s NMIMS, V. L. Mehta Road,

Ph. +91–2242332027

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Email: [email protected]

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Vile Parle (W), Mumbai – 400 056, India

Highlights

Chronic unpredictable mild stress exposure (CUMS) induces neuroinflammation

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authors contributed equally to this work

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#Both

associated depression-like behaviors and neurochemical alterations in rats.

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CUMS provokes deficits in locomotion abilities. N-acetylcysteine ameliorates the sucrose intake in the sucrose preference test, immobility time in the forced swimming test, number of line crossings, rearing in the open field test and locomotor index in the actophotometer and demonstrated the antidepressant-like behaviors in the CUMS rats.

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N-acetylcysteine exhibits antidepressant-like activity by its effects on serotonin levels and pro-inflammatory markers IL-1β, IL-6, and TNF-α in the chronic unpredictable mild stress-exposed rats.

Abstract

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Depression is a heterogeneous disorder and associated with inflammatory responses. The influences of N-acetylcysteine (NAC) on neuroinflammation associated depression-like

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behavior have not been investigated yet, and associated biochemical changes are currently unclear. Therefore, we assessed the effects of NAC on neuroinflammation associated depression-like behavior induced through chronic unpredictable mild stress (CUMS) in rats.

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The antidepressant-like effect of NAC was depicted using the sucrose preference test and the

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forced swimming test (FST) while CUMS-induced alteration in the locomotor index was

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measured using the open field test (OFT) and actophotometer. Our results revealed that CUMS

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exposure markedly aggravated depression-like behavior, the levels of pro-inflammatory

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cytokines IL-1β, IL-6, TNF-α, and reduced the serotonin levels. One-week consecutive NAC (50 and 100 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o., a selective serotonin reuptake inhibitor)

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treatment significantly increased sucrose preference index, reduced immobility time in the FST, and the increased the number of squares crossed, number of rearing in the OFT and

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locomotion in the actophotometer in the CUMS-exposed rats. Moreover, the levels of proinflammatory cytokines in the hippocampus as well as pre-frontal cortex were suppressed, and

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remarkably restored the serotonin levels by NAC (50 and 100 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.) administration. However, NAC (25 mg/kg, p.o.) exerted insignificant protection against CUMS-induced depressive-like behavior and associated neuro-inflammation. This study demonstrates that NAC exhibited the antidepressant-like effect in the CUMS-exposed

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rats, which might be mediated by anti-inflammatory potential and restoring serotonergic responses in the stressed rats.

Abbreviations: CUMS, chronic unpredictable mild stress

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ELISA, enzyme-linked immunosorbent assay SPT, sucrose preference test

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OFT, open field test FST, forced swimming test IL-1β, interleukin-1beta

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IL-6, interleukin-6

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TNF-α, tumor necrosis factor-α

Keywords: Chronic unpredictable mild stress; N-acetylcysteine; neuroinflammation

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associated depressive behavior; cytokines; interleukin-1β; interleukin-6; tumor necrosis factor-

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α. 1. Introduction

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As stated by the World Health Organization, depression affects an estimate of 300

million people of all ages globally and is a highly debilitating, life-threatening psychiatric

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illness [1,2]. Depression is regarded to become the second leading cause of disease burden globally by 2030 [3,4]. Depression leads to pathological stages like low morale, anhedonia, reduced physical activity, helplessness feelings, negative moods, weight loss, and cognitive dysfunctions, etc. [5,6]. Despite significant progress have been made to understand the pathogenesis of depression, still, most of the current antidepressants used in the clinical practice

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do not only demonstrate a limited efficacy but also exert side effects [7,8]. Therefore, the development of safer and effective newer medications is always desired world-wide. Predominantly, the antidepressants based on the monoaminergic deficiency hypothesis have a key role in the clinical practice, however, their influence on the distinct neurochemical systems are also currently in focus to have a better treatment strategy [9]. Increasing evidence has

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claimed that depression is an oxidative and inflammatory disorder [10,11]. Clinical evidence

also supports that inflammation is closely associated with the complicated pathogenesis of

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neuropsychiatric disorders, which include depression [4,12]. Increasing reports have revealed that there is a direct association of expression of pro-inflammatory mediators with the depression-like behavior [11–13]. Moreover, pro-inflammatory cytokines including

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interleukin-1β (IL-1 β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) have been

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elucidated to elevate during the depression in animals [4,13]. Depressive patients have also

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been depicted to show monoaminergic deficiency and elevated levels of pro-inflammatory cytokines in the peripheral circulation and some brain regions [12,14]. Although the

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pathogenesis of depression progression has been elucidated extensively, further studies are still needed to assimilate the relevant molecular mechanism. Additionally, most of the reports on

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depression primarily focus on changes in the brain areas including the hippocampus and

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prefrontal cortex [15–17].

N-acetylcysteine (NAC), a glutamate modulator and antioxidant medication, has been

recognized as a multi-target acting drug. NAC shows beneficial effects in extensive range of

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pharmacological activities, including in psychiatric disorders [18], depressive symptoms [19], bipolar depression [20,21], cannabis use associated anxiety, depression, and sleep quality [22], adolescent

cannabis

compulsive disorder

use depressive in

patients

disorder [24],

[23],

treatment-resistant

cocaine-induced

reinstatement

obsessive[25,26],

depressive behavior in Huntington's disease [27,28], diabetes-induced depression-like Page 4 of 40

behavior and oxidative stress [29], cognitive dysfunction in depression [30], anxiety and oxidative damage induced by unpredictable chronic stress in Zebrafish [31]. Additionally, NAC is also depicted to reduce the alcohol-linked neuro-inflammation in rats [32] and alcohol abstinence-anxiety [33]. Intriguingly, the beneficial roles of NAC in depression pathogenesis have also been reported clinically [34], nonsuicidal self-injury adolescents and young adults

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[35]. Previously, beneficial interactive use of NAC has also been shown to reduce doses of

antidepressant drugs required to produce antidepressant-like effects in rats [36]. Recently, we

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reported that NAC administration improved alcohol abstinence-induced depressive-like behavior in rats [37]. The promising effects of NAC are connected owing to its influences on

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the monoaminergic neurotransmitters and its anti-inflammatory effects [32,37]. The

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antioxidant effects of NAC and its potential link with anti-inflammatory action have also been

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suggested in earlier studies [38,39].

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Chronic unpredictable mild stress (CUMS) consists of mild stressors which imitate the common stressors in routine human life. It results in alterations in neural, endocrine variables

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and behavioral changes [40]. Moreover, the influence of NAC on neuroinflammation associated depressive behavior induced by a chronic animal model of depression have not been

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elucidated yet, and associated biochemical changes are currently unclear. Therefore, the

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purpose of this study was to investigate the underlying mechanisms involved in the antidepressant-like effects of NAC in the CUMS animal model via alterations in proinflammatory cytokines and monoamines in the hippocampus and prefrontal cortex regions of

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the brain.

2. Materials and methods 2.1 Animals and housing

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Male Wistar rats (7–8 weeks, weighing 120–150 g) were obtained from Bharat Serum and Vaccines Ltd, Thane, India, and housed in an environment at 25 ± 2 °C in controlled light (12:12 hr light: dark cycle, light on at 0700 h), and with a relative humidity of 70 ± 10%. Standard laboratory rat pellet diet (Nutrimix Laboratory Animal Feed, Maharashtra, India) and water were provided ad libitum. The rats were acclimatized to the laboratory conditions for 7

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days before the experiments. Prior approval for animal experimentation was obtained from

Institutional Animal Ethics Committee, constituted by the Committee for the Purpose of

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Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India guidelines (CPCSEA/IAEC/P-93/2017).

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2.2 Drugs doses and routes of administration

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N-acetylcysteine (Loba Chemie), fluoxetine (Divis Pharmaceuticals Pvt. Ltd, India), were

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procured from the respective sources. NAC and fluoxetine were dissolved in 0.5% w/v of

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sodium–carboxymethylcellulose (Na-CMC) in distilled water. Oral administration of NAC and

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psychiatric patients.

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fluoxetine were selected as it is considered as the most preferred route of administration in the

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2.3 Experimental Design

All behavioral studies were executed between 09:00 and 17:00 h in the naive experimental rats.

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After acclimatization to the laboratory conditions, 36 rats were randomly divided into six groups: (1) Unstressed + vehicle (control); (2) CUMS + vehicle; (3-5) three NAC-treated groups (25, 50, and 100 mg/kg of NAC + CUMS); and (6) CUMS + 10 mg/kg, p.o. of standard drug fluoxetine. The doses of NAC and fluoxetine were selected on the basis of available scientific reports [37,41–46]. NAC and fluoxetine were administrated once a day by oral route during the last seven days of the CUMS exposure (from day 21 to day 27). The rats in the Page 6 of 40

normal control group and CUMS group received an equivalent volume of 0.5% Na-CMC (10 ml/kg, p.o., once a day, from day 21 to day 27). One-hour after the drug administration, behavioral tests were executed, and thereafter animals were sacrificed for further biochemical determinations. The detailed schedule of the experimental illustration is presented in Figure 1.

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2.4 Chronic unpredictable mild stress procedure Rats were exposed to a variety of mild environmental or psychosocial stressors as described

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previously [47–49] with minor modifications as per the schedule in table 1. Briefly, the control group rats were housed with 3 rats per cage with free access to food and water and were left unstressed in their home cages with the exception of general cleaning and handling. However,

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CUMS and drug-treated groups of rats were isolated in the individual cages and exposed to the

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following nine stressors for 27 days: (1) Warm swimming at 40°C for 5 min; (2) 24 h of water

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deprivation; (3) 24 h of the cage tilting (30°); (4) tail pinching for 1 min (1 cm from the tip of

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the tail); (5) 24 h of food deprivation; (6) overnight illumination; (7) overhang (10 min); (8)

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cold swimming at 8 °C for 5 min; (9) physical restraint for 2 h. These stressors were executed once daily between 09:00 h and 17:00 h (except 24 h of stressors) with a variable unpredictable

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sequence for consecutive 27 days and each stressor was repeated three times in 27 days. Prophesy and adjustment to any particular stressor were arrested by not repeating the same

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stressor for the two consecutive days (Table 1).

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2.5 Behavioral tests

After the termination of the CUMS procedure, trails were executed in a sound-attenuated room using the behavioral paradigm in the order listed in Figure 1, i.e., open field test (OFT) on day 28, actophotometer test on day 29, sucrose preference test (SPT) on day 30 and 31, and forced swim test (FST) on day 33 and 34. Only one test was performed each day keeping a 24 h lapse between the sucrose preference test and the forced swim test. The behavioral trials were Page 7 of 40

performed by a trained observer who was unaware of the experimental groups. Antidepressant fluoxetine (a selective serotonin reuptake inhibitor) was used as the standard drug in the treatment of CUMS-induced depression-like behavior [50].

2.5.1 Open-field test

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The OFT was performed under bright ambient room light to assess the locomotor and

exploratory behavior of the rats as previously reported [48]. The open field apparatus consisted

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of a four-sided black box (96 x 96 x 50 cm). The floor was divided into 16 equal squares by white lines of 6 mm thicknesses, and the four central squares were defined as the center area.

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The rats were placed individually into the center area and allowed to explore the unfamiliar

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arena for 5 min. The number of total squares crossed, number of rearing (number of times the

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rat raised on its hind legs), and grooming episodes (washing of the coat) were registered. Post-

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exposure of each rat, the OFT apparatus was wiped with a 50% ethanol to abolish any sign of

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olfactory cues.

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2.5.2 Spontaneous locomotor activity

Locomotion of each rat was investigated in an actophotometer (Dolphin) for the period of 5

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min. The actophotometer is equipped with light-emitting probes on the two adjacent walls and photo-detector on the other two walls. A count is registered by the instrument on the

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interruption of the light beams by the rat movement, and total counts were recorded digitally. The photocells were tested before initiation of the trail. The apparatus was wiped with 50% alcohol between tests to remove any sign of olfactory cues.

2.5.3 Sucrose preference test

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The SPT was used to observe anhedonic-like behavior and executed as reported earlier with minor modifications [40,51]. Initially, rats were trained to consume 2% w/v sucrose solution for 24-hours by placing 2 bottles of sucrose solution in each cage to prevent neophobia-induced changes in the preference. After a 12-hour period of food and water deprivation, all rats were given free access to two standard drinking bottles, one containing 2.5% sucrose and the other

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with tap water for a period of three hours. The placement of the tap water and sucrose solution

bottle was switched at the midway point of the three-hour test to prevent side preferences. The

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sucrose and water intake were measured, and sucrose preference was presented as the percentage of total volume of fluid consumed (percentage of sucrose intake/sucrose intake plus

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water intake).

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2.5.4 Forced swim test

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To evaluate the depression-like behavior, frequently used FST model was standardized in our laboratory [37,52,53]. Rats were subjected singly in a transparent cylinder (60 cm in height and

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25 cm in diameter) with water at 25 ± 2 °C and a depth of 30 cm. The FST paradigm comprised 2 sections: an initial 15-min pre-test (a day before the test) and a 5-min test on the following

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day. The immobility time was recorded if they remained static in an upright position or floated with only enough motion necessary to keep their head above the water level. After each session,

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the rats were wiped with a towel and kept into heated cages for 10 min, and thereafter returned

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to their home cages.

2.6 Animal sacrifice, tissue collection Post-behavioral analysis on day 35, rats were sacrificed by decapitation to remove the brain and isolated brain tissues i.e. the hippocampus and prefrontal cortex. The brain tissues were frozen (−80 °C) for further analysis. One half of each prefrontal cortex and the hippocampus

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from both the hemispheres was utilized for the estimation of serotonin levels, and other half was separately employed for the determination of pro-inflammatory cytokines.

2.7 HPLC analysis of brain tissue homogenate to measure serotonin levels This method was standardized for the estimation of serotonin in the brain tissue in our

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laboratory [49]. The prefrontal cortex and hippocampus were homogenized in 2 ml of 0.1N

perchloric acid and centrifuged at 8000×g for 20 min at 4 °C separately. The supernatant of the

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tissue homogenate was collected and stored at –80 °C until use. This supernatant was further filtered using a 0.45 µm membrane filter (Durapore, Millipore). Chromatography was

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performed with HPLC (SHIMADZU: LC-2010 CHT, Shimadzu, Kyoto, Japan), equipped with

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25 cm length, 4.6 mm internal diameter Kromasil C18 Column (particle size 5 mm) (Kromasil

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CA). Chromatographic detection was achieved at 280 nm wavelength by injecting 20 mL of

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the clear tissue homogenate in triplicates and using 0.05% formic acid and acetonitrile (90:10 v/v) as the mobile phase with 1 ml/min flow rate. Standard serotonin weighing 10 mg was

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diluted to 10 ml with the mobile phase to make a 1000 µg/mL stock solution. The stock solution was further diluted with the mobile phase to get 100, 200, 300 and 400 µg/mL solutions. These

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four dilutions were scanned in triplicates at 280 nm, and the chromatographs were recorded to obtain the retention time and peak area under the curve. The calibration curve was obtained by

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plotting the peak area under the curve versus concentration. Additionally, NAC and fluoxetinetreated rats brain tissue homogenate samples were scanned in triplicates at 280 nm. Finally, the

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concentration of serotonin was calculated using the calibration curve.

2.8 Determination of inflammatory cytokine levels by ELISA Another half of the brain tissues i.e. the hippocampus and prefrontal cortex were separately chilled in ice-cold sterile saline (0.9%). Subsequently, each tissue was homogenized in the 10-

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time volume of ice-cold 0.1 M phosphate buffer at pH 7.4 and centrifuged at 2500×g for 15 min at 4 °C. The obtained supernatant was separated and stored at −80 °C until proinflammatory cytokines estimations. The assay kits for Rat IL-1β ELISA, Rat IL-6 ELISA, and Rat TNFα ELISA were purchased from Krishgen Biosystems, India. The cytokine IL-1β, IL6, and TNFα in the hippocampal as well as in the prefrontal cortex were detected in duplicate

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with commercial ELISA kits according to the manufacturer's instructions. The mean absorbance was determined for each set of duplicate standards and samples. The levels of

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cytokines IL-1β, IL-6, and TNFα were expressed as pg/mg of the total protein.

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2.9 Statistical analysis

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The data were statistically analyzed using GraphPad Prism Software version 7.00, San Diego, California, USA, and expressed as mean ± standard error of the mean (S.E.M.) (n = 6).

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Data were analyzed by one-way analysis of variance (ANOVA), followed by post-hoc Tukey’s

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Results:

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multiple comparison tests. Differences with p < 0.05 were considered statistically significant.

3.1 Effects of CUMS and NAC in the Open field test

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As illustrated in Fig. 2, reduction in the number of squares crossed and the number of rearing were noticed significantly in the CUMS-exposed rats, and those were reversed by one-week

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treatment with NAC (50 and 100 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.) in the CUMSexposed rats. One-way ANOVA followed by post-hoc Tukey’s multiple comparison tests depicted significant differences in the number of square crossing, and the number of rearing [F (5, 30) = 47.85, p < 0.0001] in the OFT. Rats in the CUMS group demonstrated a significant reduction in the number of squares crossed (p < 0.0001; Fig. 2) and the number of rearing (p

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< 0.01; Fig. 2) in the OFT as compared to the normal control rats. Treatment with NAC (50 and 100 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.) for seven consecutive days significantly increased the number of squares crossed (p < 0.001, p < 0.0001, p < 0.0001; Fig. 2), and the number of rearing (p < 0.05, p < 0.01, p < 0.01; Fig. 2) respectively in the OFT as compared to the CUMS-exposed rats. However, no significant effects on the number of squares crossed,

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and the number of rearing were observed on the treatment with a lower dose of NAC (25 mg/kg,

p.o.) when compared to the vehicle-treated CUMS-exposed rats (p > 0.05; Fig. 2).

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Furthermore, there were no significant differences in the numbers of grooming among all NAC and fluoxetine-treated groups (p > 0.05; Fig. 2) in the CUMS as well as normal control rats.

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Additionally, the separate experiment was executed to confirm the drugs escalating locomotor

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index which may endanger false positive behavioral results. In that study, rats were not exposed

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to the CUMS procedure (normal rats), and on the one-week treatment with NAC (25, 50, and

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100 mg/kg, p.o.) did not alter the total number of squares crossed (p > 0.05), the number of rearing (p > 0.05) and number of grooming (p > 0.05) in the OFT and locomotor score in the

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actophotometer (p > 0.05) when compared with vehicle-treated normal control group (Data presented as supporting information, Fig. 1S & 2S), indicating that elevated locomotion in the

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OFT and reduced immobility in the FST on the NAC treatment in the CUMS-exposed rats

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cannot be regarded as a psychostimulant action.

3.2 Effect of NAC treatment in the CUMS rats on locomotion activity

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As shown in Fig. 3, the influences of CUMS and NAC on the locomotion were also

investigated using actophotometer. Application of one-way ANOVA depicted a significant difference in the locomotion count [F (5, 30) = 39.34, p < 0.0001]. Post-hoc Tukey’s multiple comparison tests depicted that CUMS group showed a significant reduction in the locomotion count as compared to the control rats in the actophotometer (p < 0.0001; Fig. 3). NAC (50 and

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100 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.) treatment for seven consecutive days significantly increased the locomotion count (p < 0.001, p < 0.0001, p < 0.0001; Fig. 3) in the actophotometer as compared to the CUMS-exposed rats. However, no significant reversal in the locomotor count was exerted after treatment with NAC (25 mg/kg, p.o.) as compared with

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CUMS-exposed rats (p > 0.05; Fig. 3).

3.3 Influences of NAC on the percent of sucrose consumption

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As presented in Fig. 4, significant differences were depicted among the treatments groups on the percent of sucrose consumption [F (5, 30) = 12.57, p < 0.0001]. One-way ANOVA

followed by post-hoc Tukey’s multiple comparison tests demonstrated the significant decrease

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in the percent of sucrose consumption in the CUMS-exposed rats in comparison with normal

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control group (p < 0.0001; Fig. 4). In this study, one-week administration of NAC (50 and 100

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mg/kg, p.o.) and fluoxetine (10 mg/kg, p.o.) revealed an antidepressant-like effect, as sucrose

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intake in CUMS-exposed rats significantly raised (p < 0.01, p < 0.0001, p < 0.001; Fig. 4)

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respectively as compared with the vehicle-treated CUMS-exposed rats. However, no significant alteration was observed in the percentage sucrose consumption on the treatment

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with NAC (25 mg/kg, p.o.) when compared to CUMS group (p > 0.05; Fig. 4).

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3.4 Influences of NAC on depressive-like behavior in rats In the FST (Fig. 5), one-way ANOVA revealed significant differences among various

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groups in the immobility time [F (5, 30) = 44.81, p < 0.0001]. Post-hoc Tukey’s multiple comparison tests demonstrated that CUMS-exposed rats depicted the significant increase in the immobility time as compared to the normal control rats (p < 0.0001; Fig. 5). Chronic treatment with the NAC (50 and 100 mg/kg, p.o.) and fluoxetine (10 mg/kg, p.o.) significantly decreased the immobility duration in the CUMS-exposed rats when compared with the vehicle-treated

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CUMS-exposed rats (p < 0.001, p < 0.0001, p < 0.0001; Fig. 5) respectively in the FST rat model. Moreover, NAC (25 mg/kg, p.o.) did not exert any significant differences in the duration of immobility in the CUMS-exposed rats (p > 0.05; Fig. 5).

3.5 Influences of NAC on the serotonin levels in the hippocampus and prefrontal cortex

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As shown in Fig. 6, one-way ANOVA depicted significant differences among the treatments groups on the serotonin levels in the hippocampus [F (5, 30) = 55.95, p < 0.0001],

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and prefrontal cortex [F (5, 30) = 42.16, p < 0.0001]. Post-hoc Tukey’s multiple comparison

test showed that CUMS-exposed rats exhibited significant decrement in the serotonin levels in the hippocampus (p < 0.0001; Fig. 6A), and prefrontal cortex (p < 0.0001; Fig. 6B) as

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compared to the normal control group. One-week NAC (50 and 100 mg/kg, p.o.) or fluoxetine

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(10 mg/kg, p.o.) administration improved the level of serotonin in the hippocampus (p < 0.05,

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p < 0.001, p < 0.0001; Fig. 6A) as well as in the prefrontal cortex (p < 0.05, p < 0.001, p <

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0.0001; Fig. 6B) respectively in the CUMS-exposed rats as compared to the vehicle-treated

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CUMS-exposed rats, thus induce the antidepressant-like activity. 3.6 Influences of NAC on the pro-inflammatory cytokines

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Next, the effect of NAC on the pro-inflammatory cytokine levels in the hippocampus

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and prefrontal cortex were examined. One-way ANOVA exerted significant differences in the pro-inflammatory cytokine IL-1β [F (5, 30) = 20.4, p < 0.0001], IL-6 [F (5, 30) = 59.57, p < 0.0001], and TNFα [F (5, 30) = 42.64, p < 0.0001] in the hippocampal of rats as compared to

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the normal control group. Additionally, one-way ANOVA demonstrated differences among the treatment groups on the pro-inflammatory cytokine IL-1β [F (5, 30) = 20.4, p < 0.0001], IL-6 [F (5, 30) = 36.56, p < 0.0001], and TNFα [F (5, 30) = 21.33, p < 0.0001] in the prefrontal cortex. The post-hoc Tukey’s multiple comparison test showed that exposure of the rats to the chronic CUMS depicted significant elevation in the pro-inflammatory cytokine IL-1β (p <

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0.0001; Fig. 7A), IL-6 (p < 0.0001; Fig. 7B), and TNFα (p < 0.0001; Fig. 7C) levels in the hippocampus and IL-1β (p < 0.0001; Fig. 8A), IL-6 (p < 0.0001; Fig. 8B), and TNFα (p < 0.0001; Fig. 8C) levels in the prefrontal cortex. One-week treatment with NAC (50 and 100 mg/kg, p.o.) and fluoxetine (10 mg/kg, p.o.) markedly attenuated IL-1β (p < 0.001, p < 0.0001, p < 0.0001; Fig. 7A), IL-6 (p < 0.0001, p < 0.0001, p < 0.0001; Fig. 7B), and TNFα (p <

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0.001, p < 0.0001, p < 0.0001; Fig. 7C) levels in the hippocampus respectively. Additionally, NAC (50 and 100 mg/kg, p.o.) and fluoxetine (10 mg/kg, p.o.) also significantly reversed the

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pro-inflammatory cytokine IL-1β (p < 0.001, p < 0.0001, p < 0.0001; Fig. 8A), IL-6 (p < 0.01, p < 0.0001, p < 0.0001; Fig. 8B) and TNFα (p < 0.05, p < 0.0001, p < 0.01; Fig. 8C) levels in

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the prefrontal cortex respectively as compared to the vehicle-treated CUMS-exposed rats.

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4. Discussion

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In the current study, we assessed the antidepressant-like effect of NAC and also scrutinized the possible mechanism of action in a rat model of CUMS exposure-induced depression. Our

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major findings comprise that CUMS resulted in the elevation of inflammation and decreased serotonin level in the hippocampus as well as in the prefrontal cortex of the rats. NAC-treatment

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rats.

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efficiently ameliorated neuroinflammation associated depressive behavior in CUMS-exposed

The CUMS model is a well-established and an authentic animal model of depression

because of its ability to induce stress in the most unpredictable manner [46,49,54]. It can be

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related to humans suffering from depression due to the routine unpredictable stressors of human life with good predictive, face, and construct validity [40]. Hence, CUMS model was employed to assess the antidepressant-like effects of NAC in this study. However, the influence of NAC on depression-like behavior has been investigated earlier in several other animal models [27,28,37]. Page 15 of 40

Here, our results revealed that rats exposed to CUMS for 27 days, demonstrated a remarkable decrease in the sucrose intake in the SPT, immobility time in the FST, the number of line crossings, rearing in the OFT, locomotor index in the actophotometer, and these behavioral alterations in the CUMS rats were regarded as a depression-like behavior. In the earlier reports, the SPT was used to demonstrate the anhedonia-like behavior in animals (loss

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of responsiveness to rewards) and was considered as a core symptom of depression [40,46,55]. In the sucrose preference test, rats were given access to a highly preferred sucrose solution or

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were given a choice between a sucrose solution and drinking water. Sucrose preference decreases over weeks of CUMS exposure but can be restored to normal levels by chronic

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treatment with antidepressant drugs [10]. Furthermore, we found that one-week consecutive

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NAC (50 and 100 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.) treatment significantly increased

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sucrose preference index and effectively attenuated the depression-like behavior of CUMS-

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exposed rats.

The FST animal model was used to evaluate the antidepressant-like effect of the drugs due

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to its ease of use, high predictive validity, and good reliability [37,52,53]. In the present study, we detected an increase in the immobility time in the FST in the CUMS-exposed rats. Despair

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behavior in our study is consistent with the result of the previous studies [10,56]. One-week

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NAC (50 and 100 mg/kg) or fluoxetine (10 mg/kg) treatment significantly reduced immobility time in the FST, indicating antidepressant-like effects of NAC in the CUMS animal model, whereas 25 mg/kg of NAC did not alter the CUMS-induced increase in the immobility time in

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the FST. Our results are in concordance with earlier studies which reveal that NAC exerts antidepressant and antianhedonic effect in several animal models of depression [57,58]. Therefore, the results confirmed the antidepressant-like activity of NAC in the CUMS-induced depression.

Page 16 of 40

We, further, confirmed that whether the decreased immobility in the FST on the administration of NAC was presented by the antidepressant-like effect or the locomotorstimulant activity. So we used the OFT and actophotometer animal models to evaluate the locomotor, exploratory behavior [47,59]. In this study, CUMS-exposed rats showed significant impairment in the number of crossing, rearing in the OFT and locomotor count in the

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actophotometer. Treating CUMS-exposed rats with NAC (50 and 100 mg/kg) or fluoxetine (10

mg/kg) reversed locomotion, as evident from the significantly higher number of line crossings,

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rearing in the OFT and remarkable restoration of the locomotion count in the actophotometer. However, in normal control rats, one-week treatment with NAC did not alter the locomotor

U

activity when compared to vehicle-treated normal control group, suggesting that NAC

N

administration have no effects on the locomotor activity in normal control animals. These

A

results are in accordance with the previous studies implying the antidepressant-like effect of

M

NAC may not be involved by the locomotor-stimulant activity [37,59]. Interestingly, previous studies have indicated that chronic stress plays a crucial role in the

ED

progression of depression associated inflammation and disturbance of brain neurotransmitters [12,54,60,61]. Numerous investigations have confirmed that increased inflammatory cytokines

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in the brain played a crucial role in the pathogenesis of depression [12,54,62–65]. Therefore,

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pro-inflammatory cytokines may represent the apparent target for the treatment of depression. NAC is also reported to attenuate plasma markers of oxidative stress, ischemia, and reperfusion-induced burst production of reactive oxygen species and the consequent oxidative

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stress and inflammation in diabetic myocardial ischemia and diabetic retinopathy [38,39]. Previous studies have reported that the cytokine-induced behavioral alterations are associated with changes in the metabolism of serotonin levels in the hippocampus and prefrontal cortex [4,12,17,64,66,67]. The pro-inflammatory cytokines also are known to increase the expression and function of the transporters (reuptake pumps) for serotonin, noradrenaline reuptake by Page 17 of 40

activating the MAPK (mitogen-activated protein kinases) pathways such as p38 protein expression and leading to decreased synaptic availability of serotonin and depressive-like behavior [68]. Therefore, we decided to measure the levels of cytokines and serotonin in the hippocampus as well as in the prefrontal cortex of the CUMS-exposed rats, and following treatment with NAC and fluoxetine. Our results revealed that CUMS markedly aggravated pro-

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inflammatory markers IL-1β, IL-6, TNF-α, and depression-like behavior. These findings are

consistent with those analogous results obtained after similar paradigms of CUMS [69,70].

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Clinical studies revealed that pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α were significantly elevated during the depression [69,71,72]. The administration of TNF-α was

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also reported to produce a depressive-like behavior in the forced swimming test [73]. The

N

results of our study clearly demonstrated that NAC (50 and 100 mg/kg) or fluoxetine (10

A

mg/kg) exerted an anti-inflammatory and neuroprotective effect in the brain. Interestingly,

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NAC treatment at 100 mg/kg completely reversed the CUMS-induced increase in the IL-1β, IL-6, and TNF-α levels as compared with CUMS-exposed rats in the hippocampus as well as

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in the prefrontal cortex. NAC has also been demonstrated to ameliorate the pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α in several other inflammatory diseases [74,75].

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However, the effects of NAC on the CUMS-induced depression-like behavior and altered cytokines have not been screened yet. We speculated that NAC also inhibited neuro-

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inflammation of the hippocampus as well as the prefrontal cortex in the CUMS rats via modulating pro-inflammatory cytokines and serotonin levels. IL-1β, IL-6, and TNF-α are the

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most widely investigated cytokines as a potent mediator of inflammation and associated depression. The involvement of IL-1β, IL-6, and TNF-α cytokines to ingress the brain via leaky regions in the blood-brain barrier has been demonstrated earlier in the pathogenesis of the depression [12,63,64]. These cytokines also dysregulated the metabolism of relevant neurotransmitters like as serotonin and disruption of synaptic plasticity through alterations of

Page 18 of 40

brain-derived neurotrophic factor (BDNF) and its receptor, tropomycin receptor kinase B (TrkB) which plays a key role in the pathophysiology of depression in the prefrontal cortex and hippocampus. Pro-inflammatory cytokines are reported to decrease BDNF proteins availability and also interfere with TrkB receptor phosphorylation, BDNF signaling and further attenuating the downstream activation of phospholipase Cγ1 (PLCγ1) and extracellular regulated signal

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kinase (ERK), thus diminished neurogenesis and neuroplasticity. The putative role of NAC

could be due to the reduction in the levels of pro-inflammatory cytokines and promoting the

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availability of BDNF for cell survival and neurogenesis [68,76]. However, activation of the kynurenine pathway of tryptophan metabolism may be involved in the pathogenesis of the

U

depression [77]. In addition, nuclear factor-κB (NF-kB) has recently emerged as an important player in the pathogenesis of depression, with its influences in neurogenesis, synaptic

N

transmission, plasticity, and behavioral actions. The pro-inflammatory cytokine TNF-α is

A

reported to binds to its receptor and results in activation of IκB kinase β (IKKβ), which

M

phosphorylates IκB (inhibitory protein), allowing NF-κB (p65 and p50 subunits) to translocate

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to the nucleus. Activated NF-kB, through protein-protein interaction, associated with glucocorticoid receptor, thus interfering binding to DNA and promote antineurogenic and

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stress responses [76,78].

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Increasing evidence suggested that monoaminergic deficiency especially serotonergic activity has been regarded to trigger depressive disorders and most of the existing antidepressants work by increasing the serotonin and/or noradrenaline levels [79,80]. In the

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present study, we also observed a significant reduction in serotonin levels in the hippocampus as well as in the prefrontal cortex of the CUMS-exposed rats. Further, one week NAC (50 and 100 mg/kg) or fluoxetine (10 mg/kg) treatment remarkably restored the serotonin levels in the hippocampus as well as in the prefrontal cortex of the CUMS-exposed rats. These observations are consistent with the previous reports wherein NAC has not only modulated glutamate Page 19 of 40

neurotransmission but also has an interactive role in modulating serotonin levels for antidepressant-like effects [37,81].

5. Conclusions

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Overall, this study demonstrates that NAC attenuates the antidepressant-like effect in the CUMS-exposed rats, which might be mediated by its anti-inflammatory potential and ability

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to restore serotonergic responses in the hippocampus as well as prefrontal cortex of stressed rats. However, further studies are required to identify the gene and protein expression alteration

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for the influence of NAC to attenuate CUMS-induced depression.

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Disclosure statement

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Funding information

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We declare that we have no conflict of interest.

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This study was supported by Shobhaben Pratapbhai Patel School of Pharmacy & Technology

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Management, SVKM's NMIMS, V.L. Mehta Road, Vile Parle (W), Mumbai – 400 056, India

Appendix A. Supporting information Supporting information related to this article is provided.

Page 20 of 40

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X.L. Zhu, J.J. Chen, F. Han, C. Pan, T.T. Zhuang, Y.F. Cai, Y.P. Lu, Novel

ED

[1]

M

References

antidepressant effects of Paeonol alleviate neuronal injury with concomitant alterations in BDNF, Rac1 and RhoA levels in chronic unpredictable mild stress rats,

[2]

PT

Psychopharmacology (Berl). (2018) 1–15. doi:10.1007/s00213-018-4915-7. World Health Organization, Depression and other common mental disorders: global

CC E

health estimates, World Heal. Organ. (2017) 1–24. doi:CC BY-NC-SA 3.0 IGO.

[3]

N. Eshel, J.P. Roiser, Reward and punishment processing in depression, Biol.

A

Psychiatry. 68 (2010) 118–124. doi:10.1016/j.biopsych.2010.01.027.

[4]

Z.Q. Li, Z.Y. Yan, F.J. Lan, Y.Q. Dong, Y. Xiong, Suppression of NLRP3 inflammasome attenuates stress-induced depression-like behavior in NLGN3-deficient mice, Biochem. Biophys. Res. Commun. 501 (2018) 933–940. doi:10.1016/j.bbrc.2018.05.085.

[5]

H.M. Jia, Q. Li, C. Zhou, M. Yu, Y. Yang, H.W. Zhang, G. Ding, H. Shang, Z.M. Zou,

Page 21 of 40

Chronic unpredictive mild stress leads to altered hepatic metabolic profile and gene expression, Sci. Rep. 6 (2016) 1–10. doi:10.1038/srep23441. [6]

D.M. Kokare, P.S. Singru, M.P. Dandekar, C.T. Chopde, N.K. Subhedar, Involvement of alpha-melanocyte stimulating hormone (α-MSH) in differential ethanol exposure and withdrawal related depression in rat: Neuroanatomical-behavioral correlates, Brain Res. 1216 (2008) 53–67. doi:10.1016/j.brainres.2008.03.064. K. Yan, Y.-B. Chen, J.-R. Wu, K.-D. Li, Y.-L. Cui, Current Rapid-Onset

IP T

[7]

Antidepressants and Related Animal Models, Curr. Pharm. Des. 24 (2018).

[8]

SC R

doi:10.2174/1381612824666180727115222.

D.M. Gerhard, E.S. Wohleb, R.S. Duman, Emerging treatment mechanisms for depression: focus on glutamate and synaptic plasticity, Drug Discov. Today. 21 (2016)

[9]

U

454–464. doi:10.1016/j.drudis.2016.01.016.

J.W. Murrough, C.G. Abdallah, S.J. Mathew, Targeting glutamate signalling in

N

depression: Progress and prospects, Nat. Rev. Drug Discov. 16 (2017) 472–486.

A

doi:10.1038/nrd.2017.16.

M

[10] T. Qin, F. Fang, M. Song, R. Li, Z. Ma, S. Ma, Umbelliferone reverses depression-like behavior in chronic unpredictable mild stress-induced rats by attenuating neuronal

ED

apoptosis via regulating ROCK/Akt pathway, Behav. Brain Res. 317 (2017) 147–156. doi:10.1016/j.bbr.2016.09.039.

PT

[11] M. Berk, L.J. Williams, F.N. Jacka, A.O. Neil, J.A. Pasco, S. Moylan, N.B. Allen, A.L. Stuart, A.C. Hayley, M.L. Byrne, M. Maes, So depression is an inflammatory

CC E

disease, but where does the inflammation come from?, BMC Med. 11 (2013) 200. doi:10.1186/1741-7015-11-200.

[12] C.L. Raison, L. Capuron, A.H. Miller, Cytokines sing the blues: inflammation and the

A

pathogenesis of depression., Trends Immunol. 27 (2006) 24–31. doi:10.1016/j.it.2005.11.006.

[13] Y.M. Liu, J.D. Shen, L.P. Xu, H.B. Li, Y.C. Li, L.T. Yi, Ferulic acid inhibits neuroinflammation in mice exposed to chronic unpredictable mild stress, Int. Immunopharmacol. 45 (2017) 128–134. doi:10.1016/j.intimp.2017.02.007. [14] J.C. Felger, Z. Li, E. Haroon, B.J. Woolwine, M.Y. Jung, X. Hu, A.H. Miller, Page 22 of 40

Inflammation is associated with decreased functional connectivity within corticostriatal reward circuitry in depression, Mol. Psychiatry. 21 (2016) 1358–1365. doi:10.1038/mp.2015.168. [15] F.P. MacMaster, N. Carrey, L.M. Langevin, N. Jaworska, S. Crawford, Disorderspecific volumetric brain difference in adolescent major depressive disorder and bipolar depression, Brain Imaging Behav. 8 (2014) 119–127. doi:10.1007/s11682-013-

IP T

9264-x. [16] W.Y. Zhang, K.Y. Wang, Y.J. Li, Y.R. Li, R.Z. Lu, Chronic stress causes protein

kinase C epsilon-aldehyde dehydrogenase 2 signaling pathway perturbation in the rat

(2018) 1225–1230. doi:10.4103/1673-5374.235060.

SC R

hippocampus and prefrontal cortex, but not in the myocardium, Neural Regen. Res. 13

[17] Y. Zhang, K. Fan, Y. Liu, G. Liu, X. Yang, J. Ma, Cathepsin C aggravates

U

neuroinflammation involved in disturbances of behaviour and neurochemistry in acute

A

1702. doi:10.1007/s11064-018-2564-1.

N

and chronic stress-induced murine model of depression, Neurochem. Res. 43 (2018)

M

[18] A. Minarini, S. Ferrari, M. Galletti, N. Giambalvo, D. Perrone, G. Rioli, G.M. Galeazzi, N -acetylcysteine in the treatment of psychiatric disorders: current status and

ED

future prospects, Expert Opin. Drug Metab. Toxicol. 13 (2017) 279–292. doi:10.1080/17425255.2017.1251580.

PT

[19] M. Porcu, M.R. Urbano, W.A. Verri, D.S. Barbosa, M. Baracat, H.O. Vargas, R.C.B.R. Machado, R.R. Pescim, S.O.V. Nunes, Effects of adjunctive N acetylcysteine on depressive symptoms: Modulation by baseline high-sensitivity C-

CC E

reactive protein, Psychiatry Res. 263 (2018) 268–274. doi:10.1016/j.psychres.2018.02.056.

A

[20] B. Panizzutti, C. Bortolasci, K. Hasebe, S. Kidnapillai, L. Gray, K. Walder, M. Berk, M. Mohebbi, S. Dodd, C. Gama, P. V. Magalhães, S.M. Cotton, F. Kapczinski, A.I. Bush, G.S. Malhi, O.M. Dean, Mediator effects of parameters of inflammation and neurogenesis from a N -acetyl cysteine clinical-trial for bipolar depression, Acta Neuropsychiatr. (2018) 1–8. doi:10.1017/neu.2018.13. [21] P.K. Ellegaard, R.W. Licht, H.E. Poulsen, R.E. Nielsen, M. Berk, O.M. Dean, M. Mohebbi, C.T. Nielsen, Add-on treatment with N-acetylcysteine for bipolar Page 23 of 40

depression: a 24-week randomized double-blind parallel group placebo-controlled multicentre trial (NACOS-study protocol), Int. J. Bipolar Disord. 6 (2018) 11. doi:10.1186/s40345-018-0117-9. [22] Y.-I. Hser, L.J. Mooney, D. Huang, Y. Zhu, R.L. Tomko, E. McClure, C.-P. Chou, K.M. Gray, Reductions in cannabis use are associated with improvements in anxiety, depression, and sleep quality, but not quality of life, J. Subst. Abuse Treat. 81 (2017)

IP T

53–58. doi:10.1016/j.jsat.2017.07.012. [23] R.L. Tomko, A.K. Gilmore, K.M. Gray, The role of depressive symptoms in treatment of adolescent cannabis use disorder with N-Acetylcysteine, Addict. Behav. 85 (2018)

SC R

26–30. doi:10.1016/j.addbeh.2018.05.014.

[24] D.L.C. Costa, J.B. Diniz, G. Requena, M.A. Joaquim, C. Pittenger, M.H. Bloch, E.C. Miguel, R.G. Shavitt, Randomized, double-blind, placebo-controlled trial of N-

U

acetylcysteine augmentation for treatment-resistant obsessive-compulsive disorder, J.

N

Clin. Psychiatry. 78 (2017) e766–e773. doi:10.4088/JCP.16m11101.

A

[25] J. Jastrzębska, M. Frankowska, M. Filip, D. Atlas, N-acetylcysteine amide (AD4)

M

reduces cocaine-induced reinstatement, Psychopharmacology (Berl). 233 (2016) 3437– 3448. doi:10.1007/s00213-016-4388-5.

ED

[26] M. Frankowska, J. Jastrzębska, E. Nowak, M. Białko, E. Przegaliński, M. Filip, The effects of N-acetylcysteine on cocaine reward and seeking behaviors in a rat model of

PT

depression, Behav. Brain Res. 266 (2014) 108–118. doi:10.1016/j.bbr.2014.02.044. [27] D.J. Wright, L.J. Gray, D.I. Finkelstein, P.J. Crouch, D. Pow, T.Y. Pang, S. Li, Z.M.

CC E

Smith, P.S. Francis, T. Renoir, A.J. Hannan, N-acetylcysteine modulates glutamatergic dysfunction and depressive behavior in Huntington’s disease, Hum. Mol. Genet. 25 (2016) 2923–2933. doi:10.1093/hmg/ddw144.

A

[28] M. Berk, N-acetylcysteine for huntington’s?, Aust. N. Z. J. Psychiatry. 49 (2015) 1071–1072. doi:10.1177/0004867415606208.

[29] G.Z. Réus, M.A.B. dos Santos, H.M. Abelaira, S.E. Titus, A.S. Carlessi, B.I. Matias, L. Bruchchen, D. Florentino, A. Vieira, F. Petronilho, L.B. Ceretta, A.I. Zugno, J. Quevedo, Antioxidant treatment ameliorates experimental diabetes-induced depressive-like behaviour and reduces oxidative stress in brain and pancreas, Diabetes.

Page 24 of 40

Metab. Res. Rev. 32 (2016) 278–288. doi:10.1002/dmrr.2732. [30] A. Carvalho, K. Miskowiak, T. Hyphantis, C. Kohler, G. Alves, B. Bortolato, P. G. Sales, R. Machado-Vieira, M. Berk, R. McIntyre, Cognitive dysfunction in depression – pathophysiology and novel targets, CNS Neurol. Disord. - Drug Targets. 13 (2015) 1819–1835. doi:10.2174/1871527313666141130203627. [31] R. Mocelin, M. Marcon, S. D’ambros, J. Mattos, A. Sachett, A.M. Siebel, A.P.

IP T

Herrmann, A. Piato, N-Acetylcysteine reverses anxiety and oxidative damage induced by unpredictable chronic stress in Zebrafish, Mol. Neurobiol. (2018) 1–8.

SC R

doi:10.1007/s12035-018-1165-y.

[32] R. Schneider, S. Bandiera, D.G. Souza, B. Bellaver, G. Caletti, A. Quincozes-Santos, E. Elisabetsky, R. Gomez, N-acetylcysteine prevents alcohol related neuroinflammation in rats, Neurochem. Res. 42 (2017) 2135–2141.

U

doi:10.1007/s11064-017-2218-8.

N

[33] R. Schneider, C.F. Santos, V. Clarimundo, C. Dalmaz, E. Elisabetsky, R. Gomez, N-

A

acetylcysteine prevents behavioral and biochemical changes induced by alcohol

M

cessation in rats, Alcohol. 49 (2015) 259–263. doi:10.1016/j.alcohol.2015.01.009. [34] M. Berk, O.M. Dean, S.M. Cotton, S. Jeavons, M. Tanious, K. Kohlmann, K. Hewitt,

ED

K. Moss, C. Allwang, I. Schapkaitz, J. Robbins, H. Cobb, F. Ng, S. Dodd, A.I. Bush, G.S. Malhi, The efficacy of adjunctive N-acetylcysteine in major depressive disorder:

PT

A double-blind, randomized, placebo-controlled trial, J. Clin. Psychiatry. 75 (2014) 628–636. doi:10.4088/JCP.13m08454.

CC E

[35] K.R. Cullen, B. Klimes-Dougan, M. Westlund Schreiner, P. Carstedt, N. Marka, K. Nelson, M.J. Miller, K. Reigstad, A. Westervelt, M. Gunlicks-Stoessel, L.E. Eberly, NAcetylcysteine for nonsuicidal self-injurious behavior in adolescents: an open-label

A

pilot study, J. Child Adolesc. Psychopharmacol. 28 (2018) 136–144. doi:10.1089/cap.2017.0032.

[36] L. Costa-Campos, A.P. Herrmann, L.K. Pilz, M. Michels, G. Noetzold, E. Elisabetsky, Interactive effects of N-acetylcysteine and antidepressants, Prog. NeuroPsychopharmacology Biol. Psychiatry. 44 (2013) 125–130. doi:10.1016/j.pnpbp.2013.02.008.

Page 25 of 40

[37] R. Yawalkar, H. Changotra, G.L. Gupta, Protective influences of N-acetylcysteine against alcohol abstinence-induced depression by regulating biochemical and GRIN2A, GRIN2B gene expression of NMDA receptor signaling pathway in rats, Neurochem. Int. 118 (2018) 73–81. doi:10.1016/j.neuint.2018.04.011. [38] G.Y. Tsai, J.Z. Cui, H. Syed, Z. Xia, U. Ozerdem, J.H. McNeill, J.A. Matsubara, Effect of N-acetylcysteine on the early expression of inflammatory markers in the

IP T

retina and plasma of diabetic rats, Clin. Exp. Ophthalmol. 37 (2009) 223–231. doi:10.1111/j.1442-9071.2009.02000.x.

[39] S. Wang, C. Wang, F. Yan, T. Wang, Y. He, H. Li, Z. Xia, Z. Zhang, N-Acetylcysteine

SC R

attenuates diabetic myocardial ischemia reperfusion injury through inhibiting excessive autophagy, Mediators Inflamm. 2017 (2017) 1–10. doi:10.1155/2017/9257291.

U

[40] P. Willner, The chronic mild stress (CMS) model of depression: History, evaluation

N

and usage, Neurobiol. Stress. 6 (2017) 78–93. doi:10.1016/j.ynstr.2016.08.002.

A

[41] S. Lebourgeois, M.C. González-Marín, J. Jeanblanc, M. Naassila, C. Vilpoux, Effect

M

of N-acetylcysteine on motivation, seeking and relapse to ethanol self-administration, Addict. Biol. 23 (2018) 643–652. doi:10.1111/adb.12521.

ED

[42] N.A. Soliman, D.H. Zineldeen, M.A. Katary, D.A. Ali, N-acetylcysteine a possible protector against indomethacin-induced peptic ulcer: crosstalk between antioxidant,

PT

anti-inflammatory, and antiapoptotic mechanisms., Can. J. Physiol. Pharmacol. 95 (2017) 396–403. doi:10.1139/cjpp-2016-0442.

CC E

[43] D.G. MacHado, M.P. Cunha, V.B. Neis, G.O. Balen, A. Colla, J. Grando, P.S. Brocardo, L.E.B. Bettio, J.C. Capra, A.L.S. Rodrigues, Fluoxetine reverses depressivelike behaviors and increases hippocampal acetylcholinesterase activity induced by

A

olfactory bulbectomy, Pharmacol. Biochem. Behav. 103 (2012) 220–229. doi:10.1016/j.pbb.2012.08.024.

[44] S. Sharma, G.L. Gupta, Effect of hydroalcoholic extract of Centella asiatica and its synergy with N – acetyl cysteine on marble – burying behavior in mice : Implications for obsessive – compulsive disorder, Austin J Pharmacol Ther. 4 (2016) 2–5. [45] X. Liu, C. Liu, Baicalin ameliorates chronic unpredictable mild stress-induced

Page 26 of 40

depressive behavior: Involving the inhibition of NLRP3 inflammasome activation in rat prefrontal cortex, Int. Immunopharmacol. 48 (2017) 30–34. doi:10.1016/J.INTIMP.2017.04.019. [46] G.L. Wang, Y.P. Wang, J.Y. Zheng, L.X. Zhang, Monoaminergic and aminoacidergic receptors are involved in the antidepressant-like effect of ginsenoside Rb1 in mouse hippocampus (CA3) and prefrontal cortex, Brain Res. 1699 (2018) 44–53.

IP T

doi:10.1016/j.brainres.2018.05.035. [47] L. Cai, R. Li, W.J. Tang, G. Meng, X.Y. Hu, T.N. Wu, Antidepressant-like effect of

geniposide on chronic unpredictable mild stress-induced depressive rats by regulating

1332–1341. doi:10.1016/j.euroneuro.2015.04.009.

SC R

the hypothalamus-pituitary-adrenal axis, Eur. Neuropsychopharmacol. 25 (2015)

[48] J.F. Ge, W.C. Gao, W.M. Cheng, W.L. Lu, J. Tang, L. Peng, N. Li, F.H. Chen, Orcinol

U

glucoside produces antidepressant effects by blocking the behavioural and neuronal

N

deficits caused by chronic stress, Eur. Neuropsychopharmacol. 24 (2014) 172–180.

A

doi:10.1016/j.euroneuro.2013.05.007.

M

[49] G.L. Gupta, J. Fernandes, Protective effect of Convolvulus pluricaulis against neuroinflammation associated depressive behavior induced by chronic unpredictable

ED

mild stress in rat, Biomed. Pharmacother. 109 (2019) 1698–1708. doi:10.1016/j.biopha.2018.11.046.

PT

[50] Y. Lu, C.S. Ho, R.S. McIntyre, W. Wang, R.C. Ho, Effects of vortioxetine and fluoxetine on the level of Brain Derived Neurotrophic Factors (BDNF) in the hippocampus of chronic unpredictable mild stress-induced depressive rats, Brain Res.

CC E

Bull. 142 (2018) 1–7. doi:10.1016/j.brainresbull.2018.06.007.

[51] M. Erburu, I. Muñoz-Cobo, T. Diaz-Perdigon, P. Mellini, T. Suzuki, E. Puerta, R.M.

A

Tordera, SIRT2 inhibition modulate glutamate and serotonin systems in the prefrontal cortex and induces antidepressant-like action, Neuropharmacology. 117 (2017) 195– 208. doi:10.1016/j.neuropharm.2017.01.033.

[52] D.M. Kokare, M.P. Dandekar, P.S. Singru, G.L. Gupta, N.K. Subhedar, Involvement of α-MSH in the social isolation induced anxiety- and depression-like behaviors in rat, Neuropharmacology. 58 (2010) 1009–1018. doi:10.1016/j.neuropharm.2010.01.006.

Page 27 of 40

[53] R.D. Porsolt, G. Anton, N. Blavet, M. Jalfre, Behavioural despair in rats: A new model sensitive to antidepressant treatments, Eur. J. Pharmacol. 47 (1978) 379–391. doi:10.1016/0014-2999(78)90118-8. [54] C. Zhang, Y.P. Zhang, Y.Y. Li, B.P. Liu, H.Y. Wang, K.W. Li, S. Zhao, C. Song, Minocycline ameliorates depressive behaviors and neuro-immune dysfunction induced by chronic unpredictable mild stress in the rat, Behav. Brain Res. 356 (2019) 348–357.

IP T

doi:10.1016/j.bbr.2018.07.001. [55] J. Xue, H. Li, X. Deng, Z. Ma, Q. Fu, S. Ma, L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3

SC R

inflammasome-mediated inflammatory cytokines and central neurotransmitters,

Pharmacol. Biochem. Behav. 134 (2015) 42–48. doi:10.1016/j.pbb.2015.04.014. [56] Y. Song, R. Sun, Z. Ji, X. Li, Q. Fu, S. Ma, Perilla aldehyde attenuates CUMS-induced

U

depressive-like behaviors via regulating TXNIP/TRX/NLRP3 pathway in rats, Life

N

Sci. 206 (2018) 117–124. doi:10.1016/j.lfs.2018.05.038.

A

[57] I. Smaga, B. Pomierny, W. Krzyzanowska, L. Pomierny-Chamioło, J. Miszkiel, E.

M

Niedzielska, A. Ogórka, M. Filip, N-acetylcysteine possesses antidepressant-like activity through reduction of oxidative stress: Behavioral and biochemical analyses in

ED

rats, Prog. Neuro-Psychopharmacology Biol. Psychiatry. 39 (2012) 280–287. doi:10.1016/j.pnpbp.2012.06.018.

PT

[58] C.O. Arent, G.Z. Réus, H.M. Abelaira, K.F. Ribeiro, A. V Steckert, F. Mina, F. DalPizzol, J. Quevedo, Synergist effects of n-acetylcysteine and deferoxamine treatment on behavioral and oxidative parameters induced by chronic mild stress in rats,

CC E

Neurochem. Int. 61 (2012) 1072–1080. doi:10.1016/j.neuint.2012.07.024.

[59] X. Zu, M. Zhang, W. Li, H. Xie, Z. Lin, N. Yang, X. Liu, W. Zhang, Antidepressant-

A

like effect of Bacopaside I in mice exposed to chronic unpredictable mild stress by modulating the hypothalamic–pituitary–adrenal axis function and activating BDNF signaling pathway, Neurochem. Res. 42 (2017) 3233–3244. doi:10.1007/s11064-0172360-3.

[60] H. Gao, X. Zhu, Y. Xi, Q. Li, Z. Shen, Y. Yang, Anti-depressant-like effect of atractylenolide I in a mouse model of depression induced by chronic unpredictable mild stress, Exp. Ther. Med. (2017) 1574–1579. doi:10.3892/etm.2017.5517. Page 28 of 40

[61] V.S. Velingkar, G.L. Gupta, N.B. Hegde, A current update on phytochemistry, pharmacology and herb–drug interactions of Hypericum perforatum, Phytochem. Rev. 16 (2017) 725–744. doi:10.1007/s11101-017-9503-7. [62] C. Park, E. Brietzke, J.D. Rosenblat, N. Musial, H. Zuckerman, R.M. Ragguett, Z. Pan, C. Rong, D. Fus, R.S. McIntyre, Probiotics for the treatment of depressive symptoms: An anti-inflammatory mechanism?, Brain. Behav. Immun. 73 (2018) 115–124.

IP T

doi:10.1016/j.bbi.2018.07.006. [63] N. Lichtblau, F.M. Schmidt, R. Schumann, K.C. Kirkby, H. Himmerich, Cytokines as biomarkers in depressive disorder: Current standing and prospects, Int. Rev.

SC R

Psychiatry. 25 (2013) 592–603. doi:10.3109/09540261.2013.813442.

[64] J.D. Rosenblat, J.M. Gregory, R.S. McIntyre, Pharmacologic implications of inflammatory comorbidity in bipolar disorder, Curr. Opin. Pharmacol. 29 (2016) 63–

U

69. doi:10.1016/j.coph.2016.06.007.

N

[65] R. Haapakoski, J. Mathieu, K.P. Ebmeier, H. Alenius, M. Kivimäki, Cumulative meta-

A

analysis of interleukins 6 and 1β, tumour necrosis factor α and C-reactive protein in

doi:10.1016/j.bbi.2015.06.001.

M

patients with major depressive disorder, Brain. Behav. Immun. 49 (2015) 206–215.

ED

[66] E.A. Rahim, Z. Mohd-Zain, J. Hussaini, E.H. Wong, L.L. Nyein, N. Parasakthi, A. Adnan, N.K. Palanisamy, Adherence of Streptococcus pneumoniae and expression

PT

analysis of neuraminidase gene (NanA and NanB) after interaction of A549 human lung epithelial cells with pneumococcal strains of various serotypes, Malays. J.

CC E

Microbiol. 13 (2017) 210–216. doi:10.1007/978-0-585-37970-8_8. [67] B. Sperner-Unterweger, C. Kohl, D. Fuchs, Immune changes and neurotransmitters: Possible interactions in depression?, Prog. Neuro-Psychopharmacology Biol.

A

Psychiatry. 48 (2014) 268–276. doi:10.1016/j.pnpbp.2012.10.006.

[68] M. Adzic, Z. Brkic, M. Mitic, E. Francija, M.J. Jovicic, J. Radulovic, N.P. Maric, Therapeutic strategies for treatment of inflammation-related depression, Curr. Neuropharmacol. 15 (2017). doi:10.2174/1570159X15666170828163048. [69] P. Martinez, L. Lien, S. Zemore, J.G. Bramness, S.P. Neupane, Circulating cytokine levels are associated with symptoms of depression and anxiety among people with

Page 29 of 40

alcohol and drug use disorders, J. Neuroimmunol. 318 (2018) 80–86. doi:10.1016/j.jneuroim.2018.02.011. [70] Y.M. Liu, L. Niu, L.L. Wang, L. Bai, X.Y. Fang, Y.C. Li, L.T. Yi, Berberine attenuates depressive-like behaviors by suppressing neuro-inflammation in stressed mice, Brain Res. Bull. 134 (2017) 220–227. doi:10.1016/j.brainresbull.2017.08.008. [71] A. Dey, P. Hankey Giblin, Insights into macrophage heterogeneity and cytokine-

IP T

induced neuroinflammation in major depressive disorder, Pharmaceuticals. 11 (2018) 64. doi:10.3390/ph11030064.

SC R

[72] C.A. Köhler, T.H. Freitas, B. Stubbs, M. Maes, M. Solmi, N. Veronese, N.Q. de

Andrade, G. Morris, B.S. Fernandes, A.R. Brunoni, N. Herrmann, C.L. Raison, B.J. Miller, K.L. Lanctôt, A.F. Carvalho, Peripheral alterations in cytokine and chemokine levels after antidepressant drug treatment for major depressive disorder: systematic

U

review and meta-analysis, Mol. Neurobiol. 55 (2018) 4195–4206. doi:10.1007/s12035-

N

017-0632-1.

A

[73] M.P. Kaster, V.M. Gadotti, J.B. Calixto, A.R.S. Santos, A.L.S. Rodrigues, Depressive-

M

like behavior induced by tumor necrosis factor-α in mice, Neuropharmacology. 62 (2012) 419–426. doi:10.1016/j.neuropharm.2011.08.018.

ED

[74] D. Alexandropoulos, G. V. Bazigos, I.P. Doulamis, A. Tzani, P. Konstantopoulos, N. Tragotsalou, A. Kondi-Pafiti, T. Kotsis, N. Arkadopoulos, V. Smyrniotis, D.N. Perrea,

PT

Protective effects of N -acetylcystein and atorvastatin against renal and hepatic injury in a rat model of intestinal ischemia-reperfusion, Biomed. Pharmacother. 89 (2017)

CC E

673–680. doi:10.1016/j.biopha.2017.02.086. [75] W. Zhai, R. Feng, L. Huo, J. Li, S. Zhang, Mechanism of the protective effects of Nacetylcysteine on the heart of brain-dead Ba-Ma miniature pigs, J. Hear. Lung Transplant. 28 (2009) 944–949. doi:10.1016/j.healun.2009.05.006.

A

[76] A. Caviedes, C. Lafourcade, C. Soto, U. Wyneken, BDNF/NF-κB Signaling in the Neurobiology of Depression, Curr. Pharm. Des. 23 (2017) 3154–3163. doi:10.2174/1381612823666170111141915. [77] E.Y. Bryleva, L. Brundin, Kynurenine pathway metabolites and suicidality, Neuropharmacology. 112 (2017) 324–330. doi:10.1016/j.neuropharm.2016.01.034.

Page 30 of 40

[78] J.W. Koo, S.J. Russo, D. Ferguson, E.J. Nestler, R.S. Duman, Nuclear factor-kappaB is a critical mediator of stress-impaired neurogenesis and depressive behavior., Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 2669–74. doi:10.1073/pnas.0910658107. [79] D.C. Blanchard, K. Meyza, Risk assessment and serotonin: Animal models and human psychopathologies., Behav. Brain Res. 357–358 (2019) 9–17. doi:10.1016/j.bbr.2017.07.008.

treatment strategies, Cell. Physiol. Biochem. 31 (2013) 761–777.

SC R

doi:10.1159/000350094.

IP T

[80] U.E. Lang, S. Borgwardt, Molecular mechanisms of depression: Perspectives on new

[81] E. Poleszak, P. Wlaź, B. Szewczyk, A. Wlaź, R. Kasperek, A. Wróbel, G. Nowak, A complex interaction between glycine/NMDA receptors and serotonergic/noradrenergic antidepressants in the forced swim test in mice, J. Neural Transm. 118 (2011) 1535–

A

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PT

ED

M

A

N

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1546. doi:10.1007/s00702-011-0630-9.

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Figure Captions Figure 1: Detailed illustration of the experiment. CUMS, chronic unpredictable mild stress; NAC, N-acetylcysteine; OFT, open field test; SPT, sucrose preference test; FST, forced

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swimming test; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α.

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Figure 2: Effects of CUMS and NAC treatment on the (A) number of total squares crossed,

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(B) number of rearing, and (C) number of grooming episodes in the open field test (OFT). Results are shown as mean ± SEM (n = 6) by vertical bars, where **p < 0.01, **** p < 0.0001 ##

p < 0.01,

###

p < 0.001,

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(Compared with normal control group), #p < 0.05,

####

p < 0.0001

(Compared with vehicle-treated CUMS-exposed rats), One-way analysis of variance

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(ANOVA), followed by post-hoc Tukey’s multiple comparison test. CUMS, chronic

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unpredictable mild stress; NAC, N-acetylcysteine; OFT, open field test.

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Figure 3: Effect of CUMS and NAC treatment on the locomotor index in the actophotometer.

###

p < 0.001,

####

p < 0.0001 (Compared to vehicle-treated CUMS-

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normal control group),

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Results are shown as mean ± SEM (n = 6) by vertical bars, where ****p < 0.0001 (Compared to

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exposed rats), One-way analysis of variance (ANOVA), followed by post-hoc Tukey’s multiple

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comparison test. CUMS, chronic unpredictable mild stress; NAC, N-acetylcysteine.

Figure 4: Influences of CUMS and NAC on the percent of sucrose preference. After the administration of NAC (25, 50, and 100 mg/kg, p.o.), fluoxetine (10 mg/kg, p.o.) or vehicle, rats were subjected to the SPT. Results are shown as mean ± SEM (n = 6) by vertical bars, where

****

p < 0.0001 (Compared to normal control group), Page 33 of 40

##

p < 0.01,

###

p < 0.001,

####

p<

0.0001 (Compared to vehicle-treated CUMS-exposed rats), One-way analysis of variance (ANOVA), followed by post-hoc Tukey’s multiple comparison test. CUMS, chronic

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unpredictable mild stress; NAC, N-acetylcysteine; SPT, sucrose preference test.

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Figure 5: Influences of CUMS and NAC on the immobility time in the FST. After the administration of NAC (25, 50, and 100 mg/kg, p.o.), fluoxetine (10 mg/kg, p.o.) or vehicle,

****

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where

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rats were subjected to the FST. Results are shown as mean ± SEM (n = 6) by vertical bars, p < 0.0001 (Compared to normal control group),

###

p < 0.001,

####

p < 0.0001

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(Compared to vehicle-treated CUMS-exposed rats), One-way analysis of variance (ANOVA), followed by post-hoc Tukey’s multiple comparison test. CUMS, chronic unpredictable mild

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stress; NAC, N-acetylcysteine; FST, forced swimming test.

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Figure 6: Effects of CUMS and NAC on the serotonin level in the (6A) hippocampus, and (6B) prefrontal cortex in the CUMS-exposed rats. Results are shown as mean ± SEM (n = 6) by vertical bars, where **** p < 0.0001 (Compared to normal control group), #p < 0.05, ###p < 0.001, ####p < 0.0001 (Compared to vehicle-treated CUMS-exposed rats), One-way analysis of variance (ANOVA), followed by post-hoc Tukey’s multiple comparison test. CUMS, chronic

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unpredictable mild stress; NAC, N-acetylcysteine.

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IP T SC R U N A M ED PT CC E A Figure 7: Influences of CUMS and NAC on pro-inflammatory cytokines in the hippocampus. (7A) IL-1β levels, (7B) IL-6 levels (7C) TNF-α levels. Results are shown as mean ± SEM (n =

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6) by vertical bars, where ####

****

p < 0.0001 (Compared to normal control group),

###

p < 0.001,

p < 0.0001 (Compared to vehicle-treated CUMS-exposed rats), One-way analysis of

variance (ANOVA), followed by post-hoc Tukey’s multiple comparison test. CUMS, chronic unpredictable mild stress; NAC, N-acetylcysteine; IL-1β, interleukin-1β; IL-6, interleukin-6;

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TNF-α, tumor necrosis factor-α.

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Figure 8: Influences of CUMS and NAC on pro-inflammatory cytokines in the prefrontal cortex. (8A) IL-1β levels, (8B) IL-6 levels (8C) TNF-α levels. Results are shown as mean ± SEM (n = 6) by vertical bars, where ****p < 0.0001 (Compared to normal control group), #p < 0.05,

##

p < 0.01,

###

p < 0.001,

####

p < 0.0001 (Compared to vehicle-treated CUMS-exposed Page 38 of 40

rats), One-way analysis of variance (ANOVA), followed by post-hoc Tukey’s multiple comparison test. CUMS, chronic unpredictable mild stress; NAC, N-acetylcysteine; IL-1β,

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interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α.

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Table 1: Chronic unpredictable mild stress stressors protocol with specified days

Stressors

Days 1

16

25

24 h of water deprivation

2

15

21

24 h of cage tilting (30°)

3

12

24

Tail pinching for 1 min (1 cm from the tip of the tail)

4

10

23

24 h of food deprivation

5

11

Overnight illumination

6

14

Overhang (10 min)

7

13

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Warm swimming at 40 °C for 5 min

22 20

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19

Cold swimming at 8 °C for 5 min

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Physical restraint for 2 h

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8

17

26

9

18

27