(+)-Sesamin attenuates chronic unpredictable mild stress-induced depressive-like behaviors and memory deficits via suppression of neuroinflammation

Accepted Manuscript (+)-Sesamin attenuates chronic unpredictable mild stress-induced depressive-like behaviors and memory deficits via suppression of neuroinflammation

Yihang Zhao, Qianxu Wang, Mengzhen Jia, Shangchen Fu, Junru Pan, Chuanqi Chu, Xiaoning Liu, Xuebo Liu, Zhigang Liu PII: DOI: Reference:

S0955-2863(18)30665-X https://doi.org/10.1016/j.jnutbio.2018.10.006 JNB 8084

To appear in:

The Journal of Nutritional Biochemistry

Received date: Revised date: Accepted date:

5 July 2018 10 October 2018 17 October 2018

Please cite this article as: Yihang Zhao, Qianxu Wang, Mengzhen Jia, Shangchen Fu, Junru Pan, Chuanqi Chu, Xiaoning Liu, Xuebo Liu, Zhigang Liu , (+)-Sesamin attenuates chronic unpredictable mild stress-induced depressive-like behaviors and memory deficits via suppression of neuroinflammation. Jnb (2018), https://doi.org/10.1016/ j.jnutbio.2018.10.006

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ACCEPTED MANUSCRIPT (+)-Sesamin attenuates chronic unpredictable mild stress-induced depressive-like behaviors and memory deficits via suppression of neuroinflammation

Yihang Zhao, Qianxu Wang, Mengzhen Jia, Shangchen Fu, Junru Pan, Chuanqi Chu, Xiaoning

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Liu, Xuebo Liu, Zhigang Liu*

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Laboratory of Functional Chemistry and Nutrition of Food, College of Food Science and

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Engineering, Northwest A&F University, Yangling, China

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* Corresponding author: Dr. Zhigang Liu, College of Food Science and Engineering, Northwest

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A&F University, Xinong Road 22, Yangling 712100, China. Tel: +86 29 87092817; Fax: +86 29 87092817; E-mail: [email protected]

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ABBREVIATION

CUMS, chronic unpredictable mild stress; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; NE, noradrenaline; 5-HT, 5-hydroxytryptamine; CNS, central nervous system; MDD, major depressive disorder; MCI, mild cognitive impairment; SE, sesamin; EPM, elevated plus maze test; H&E, hematoxylin-eosin; IHC, immunohistochemical; TEM, transmission electron microscope; PSD-95, postsynaptic density protein 95; BDNF, brain derived neurotrophic factor; NGF, nerve growth factor; NT3, neurotrophin 3; NT4, neurotrophin 4; IBA-1, ionized calcium binding adaptor molecule 1; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; BBB, blood-brain barrier; ZO-1, zonula occludens-1; HPA, hypothalamic–pituitary–adrenocortical.

ACCEPTED MANUSCRIPT ABSTRACT Depression is a mood disorder that is related to neuroinflammation and cognition loss. This study is aimed to determine the potential antidepressant effects of (+)-sesamin, a lignan component of sesame, in a mild stress-induced depression mouse model. CD-1 mice were treated with chronic unpredictable mild stress (CUMS)

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process and orally administrated with sesamin (50 mg/kg/d) for 6 weeks. Behavioral tests including forced swimming test, tail suspension test, open field test, and elevated

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plus maze test demonstrated that sesamin treatment inhibited CUMS-induced mice

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depressant-like behaviors and anxiety, without changing immobility. It was found that sesamin prevented stress-induced decease levels of 5-HT and NE in striatum and

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serum. Cognitive deficits were assessed using Y-maze and Morris water maze test. Sesamin treatment also prevented stressed-induced memory impairments and

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neuronal damages. Consistently, sesamin also enhanced synapse ultrastructure and improved expressions of PSD-95 in stressed mice hippocampus with improving

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neurotrophic factors expression including BDNF and NT3. Moreover, sesamin

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treatment significantly prevented CUMS-induced neuroinflammation by inhibiting over-activation of microglia and expressions of inflammatory mediators including iNOS, COX-2, TNF-α and IL-1β in stressed mice hippocampus and cortex. These

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results illustrated that sesamin markedly improved CUMS-induced depression and memory loss via inhibiting neuroinflammation, which indicate that as food

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component, sesamin might be also a novel potential therapeutic for depression. KEYWORDS: Sesamin; Chronic unpredictable mild stress; Cognition; Depression; Neuroinflammation

ACCEPTED MANUSCRIPT 1 INTRODUCTION Depression is one of the most common mental disorders [1], and the major depressive disorder (MDD) affects over 350 million people worldwide [2]. Numerous research has indicated that depression and anxiety secondary to chronic stress are

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associated with decreased levels of dopamine, norepinephrine (NE), and serotonin (or

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5-hydroxytryptamine, 5-HT) in the brain [3]. Besides, depression could also lead to

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cognitive deficits via aggravating the hippocampus, prefrontal cortex and other

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emotion regulation key brain region nerve atrophy, and decrease synapse function. It is believed that depression is mainly caused by the decrease of NE and 5-HT in the

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central nervous system, and the decline of synaptic content [4, 5]. The clinical treatments or drugs for depression, such as fluoxetine, paroxetine, and citalopram, are

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inhibitors of the reuptake of 5-HT and NE (SNRIs). However, the side effects of these

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drugs [6] including headaches, weight gain or loss, agitation and cognitive

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impairments are need to be solved by new and safe therapeutics. Neuroinflammatory responses are highly associated with the pathology of

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depression [7, 8] and depression-related cognitive impairment. It has been reported that 5-HT and NE levels were observed declined in inflammation-induced mice [9]. Moreover, over-expressions of proinflammatory cytokines including TNF-α, IL-6 and IL-1β have been found to relate to disorders of neuroendocrine function, neurotransmitter metabolism and synaptic plasticity and behaviors in depression [10]. Over-activation of microglia, the major immune cell in central nervous system (CNS), has also been reported to be participated in the pathogenesis of MDD [11, 12].

ACCEPTED MANUSCRIPT Therefore, to suppress the acute or chronic neuroinflammation might be one clue to alleviate depression process. The animal models preformed in current study, i.e. the chronic unpredictable mild stress (CUMS) model has already contributed to the elucidation of the pathophysiological mechanisms of depression [13]. CUMS

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represents an acute inflammatory stimulus, and produces depressive-like behaviors in

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mice that is associated with microglial activation in various regions of the mouse

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brain [13].

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(+)-Sesamin is one of the most abundant lignans in sesame (Sesamum Indicum L.) seed and oil [14]. It has been reported that sesamin possesses beneficial effects on

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improving lipid metabolism and liver damages in animal and human study [15-17]. Importantly, sesamin also has anti-inflammatory and potential neuroprotective effects.

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A previous study demonstrated that sesamin inhibited LPS-induced cytokines

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production in murine microglia and BV-2 cells [14]. Sesamin could also prevent blood-brain barrier disruption in a traumatic brain injury mice model [18]. Moreover,

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a latest clinical study indicated that treatment of sesamin with astaxanthin could

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improve mild cognitive impairment (MCI) [19]. The same Japanese research group also found that dietary supplementation of sesamin also promoted healthy volunteers recovery from mental fatigue [20]. Our previous research found that sesame crude extract and sesamol, other lignan in sesame oil, could prevent CUMS-induced mice depressive-like behaviors and synapse functions [21]. Interestingly, a series of research also demonstrated that (-)-sesamin, which is an epimeric isomer lignans with (+)-sesamin, extracted from Asiasari Radix, could reduce anxiety-like behaviors and

ACCEPTED MANUSCRIPT memory deficits induced by chronic electric foot shock in mice [3, 22]. To summarize, sesamin might be a potential candidate to prevent stress-induced mental disorders and cognitive impairments. However, there is little research to demonstrate this hypothesis.

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The present study was aimed at uncovering the effects of dietary sesamin

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supplementation on CUMS-induced depression in a mice model by (a) characterizing

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the effects of sesamin on mice anxiety and depression behavioral tests including

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forced swimming test, tail suspension test, open field test and elevated plus maze test; (b) examining the effects of sesamin on cognitive function of CUMS treated mice

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including Y-maze test and Morris water maze test; (c) uncovering the effects of sesamin on expressions of monoamine neurotransmitter neurotrophic factors and

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hippocampal synapse morphology alterations in the brain; (d) determining the effects

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of sesamin on expressions of postsynaptic dense protein and protein related to synapse function in the brain; (e) measuring the effects of sesamin on expressions of

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inflammatory cytokines in the brain. Above all, it provides novel insights into the

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effects and mechanisms of sesamin on the regulation CUMS-induced depression and cognitive impairments.

ACCEPTED MANUSCRIPT 2 MATERIALS AND METHODS 2.1 Animals Male CD-1 mice (weight: 28-32 g) were obtained from medical laboratory animal center of Xi’an Jiaotong University (Xi’an, Shanxi, China). Mice were

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single-housed per cage for bedding at 22±2 °C under a relative humidity of 50±10 %

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and a twelve-hour light/dark cycle. All mice were fed with a standard diet (AIN-93M)

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and respectively randomized into four groups (n = 10/group): Control, SE, CUMS, CUMS + SE. Different groups of animals were provided with vehicle (0.01 mL/g), SE

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(50mg/kg/day, dissolved in Olive oil) for 6 weeks. Sesamin (98%, S25758) was

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purchased from Yuanye biotechnology (Shanghai, China). All the animals received environmental adaptation for 2 weeks. During the study, food and water were

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provided ad libitum. All procedures were performed in accordance with the guidelines

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of China Council on Animal Care. All of the experimental procedures followed by Guide for the Care and Use of Laboratory Animals: Eighth Edition, ISBN-10:

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0-309-15396-4, and the animal protocol was approved by the animal ethics committee

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of Northwest A&F University. 2.2 Chronic unpredictable mild stress (CUMS) procedures This CUMS regimen was as described previously [21]. In brief, the CUMS protocol consisted of the sequential application of a variety of mild stressors: S1: 5-min cold swimming (at 4 °C), S2: 1-min tail pinch (1 cm from the tip of the tail), S3: 24-h food and water deprivation, S4: overnight illumination, S5: 15-min force swimming (at 23 °C), S6: 24-h cage tilting (30°), and S7:200 mL of water for sawdust

ACCEPTED MANUSCRIPT dampness per cage (sufficient to reach the moisture of the sawdust bedding). These stressors were randomly scheduled over a 1-week period and repeated throughout the 6-week experiment. Non-stressed animals were left undisturbed in their home cages except during housekeeping procedures such as cage cleaning.

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2.3 Animal experiments

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2.3.1 Forced swimming test

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The forced swimming test was conducted based on the original method [23]. Briefly, mice were individually placed in a cylindrical container with warm water at

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25℃, to a depth of 12 cm, and left there for 6 min. A mouse was judged to be

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immobile when it floated in an upright position, and made only small movements to keep its head above water. Immobility time of the last 4 min during a total 6-min

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system mentioned ahead.

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period was the photosensors and analyzed using a computerized video tracking

2.3.2 Tail suspension test

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The tail suspension test was performed as previously described [24]. Mice were

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suspended by the tail for 6 min by attaching them to a horizontal bar (distance from the floor is 50 cm) with adhesive tape placed 2 cm from the tip of the tail in a visually isolated area. A mouse was considered immobile when it floated in an upright position and made only very slight movements that were necessary to keep its head above water. The time of immobility of tail-suspended mice during the last 4 min was measured with timers. This procedure was conducted by the same observer, who was blind to the specific experimental groups and strictly followed the test standard

ACCEPTED MANUSCRIPT procedures. 2.3.3 Elevated plus maze test The elevated plus maze test was to evaluate the degree of animal anxiety [25], using the natural reluctance of rodents to explore open spaces [26]. The elevated

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plus-maze apparatus consisted of four arms: two open arms (30×8 cm) and two closed

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arms of the same size, with 15-cm-high black walls elevated 70 cm above the floor.

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The open and closed arms were connected with a central square (8×8 cm) to form a plus sign. Animals were placed into an open field box (30×30×40cm) for 5min, and

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then placed on exploratory platform so that their percentage of open arm entries and

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time spent exploring open arms were allowed to detect EPM for 5min. The maze was thoroughly cleaned with ethanol and allowed to dry between subjects in order to

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eliminate any odor cues. The percentage of open arm entries and the time spent on the

every animal tested.

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2.3.4 Open field test

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open arms of mice were recorded. Apparatus was cleaned with 70% alcohol between

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The locomotor activity was assessed via open field test as previously described [27]. The apparatus consisted of an open box (31×31×30 cm) with black surface over the wall and the bottom is divided into 25 equal squares. Before the test, mice was adapted to the surrounding environment in the center for 2 min. After adjustment, mice were tested for 5 min to record the total distance and the number of crossing squares by a computerized video-tracking system mentioned ahead. 2.3.5 Y-maze test

ACCEPTED MANUSCRIPT The Y-maze was mainly used in animal discrimination learning, working memory and reference memory test, performed as previous described [28]. The Y-maze consists of three arms (start arm, novel arm, other arm) of (35×16×16 cm), with 120 ° to each other. During the learning period, mice moved freely in the start

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arm and the other arm for 8 min. An arm entry was counted when the hind paws of the

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mouse were completely within the arm. The series of total arm entries and

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spontaneous alternation were calculated. Successful alternation was defined as consecutive entries into a new arm before returning to the two previously visited arms.

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Percent alternation was calculated as Alternation %= [Number of successful

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alternations/ (Total arm entries − 2)] × 100 2.3.6 Morris water maze test

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The Morris water maze was used to study the functional evaluation of brain

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region related to spatial learning and memory, performed as previously described with minor modifications [29]. The task was conducted in a circular tank (diameter: 150

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cm and height: 35 cm) filled with opaque water (23-25 °C). A 4.5-cm-diameter and

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14.5-cm-height platform was placed at the center of 1st quadrant of the pool. This test included 2 periods: initial spatial training and probe test. The pool was divided into four quadrants of equal area. There were four prominent visual cues on each side of four quadrants of the pool. On the first day, all mice were trained to stand on the platform for 60 s to realize the existence of the hidden platform and memorize the environment. During the following 3 d, the hidden platform trials were used to evaluate spatial learning ability, with black-dyed water and the platform submerged

ACCEPTED MANUSCRIPT 0.5-1.0 cm below the water surface. On each day, the mouse was placed into the water maze at one of the four quadrants and allowed to swim freely until they found and climbed onto the platform. If the mice failed to escape within 60 s, they were gently conducted to the platform and allowed to stay there for 30 s. Each mice was subjected

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to four trials per day, and the starting position was different for each trial. Escape

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latency (s) was recorded by video-tracking system. In order to assess the spatial

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retention of the location of the hidden platform, a probe trial was conducted 24 h after the last acquisition session. On the 4th day, the platform was removed from the pool

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and each mice was allowed one 60 s swim probe trial. Finally, the data of the time

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spent in the target quadrant and the number of platform crossings were recorded by the photosensors and analyzed using a computerized video-tracking system (Super

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2.4 Real Time-qPCR

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Maze software, Shanghai Xinruan Information Technology Co. Ltd, China).

To quantify the mRNA expression levels of the regulation genes of neurotrophic

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factors and inflammatory cytokines and protein, total RNA from brain tissues were

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isolated by Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacture’s instruction. Up to 5 μg of total RNAs isolated from tissues were reverse transcribed by using PrimeScriptTM RT Master Mix reverse transcription kit (TaKaRa PrimeScript RT Master Mix, Dalian, China) and the CFX96TM real-time system (Bio-Rad, Hercules, CA). Gene-specific mouse primers were used as mentioned in Table 1. Ct values were normalized to GAPDH, and the relative gene expression was calculated with the 2-Ct method.

ACCEPTED MANUSCRIPT 2.5 ELISA assays The serum, hippocampal, cortical and striatum of the brain 5-HT and NE levels were quantified by 5-HT or NE ELISA kits (Mouse 5-HT Elisa kit, Mouse NE Elisa kit, Xinle Biology Technology, Shanghai, China).

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2.6 H&E staining and immunohistochemical staining

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Histological brain sections, which were fixed in 4% (v/v) paraformaldehyde,

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embedded in paraffin. The brain sections rehydrated by xylene and descending levels of ethanol (100, 90, 80, and 70% ethanol) for 5 min in each grade, and after 5 washes

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in PBS (pH 7.4) for 4 min each. For histopathology stain, the tissues were stained

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with haematoxylin and eosin (H&E) dyestuff. For immunohistochemical (IHC) staining, Tris-EDTA Buffer Epitope Retrieval Method was

used to

permeabilize

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the tissues, and carried out to perform antigen retrieval. The paraffin sections were

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quenched by 3% peroxide-methanol, for 10 min to eliminate the confounding peroxidase. After being blocked to prevent nonspecific staining, the sections were

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incubated with the primary antibody overnight at 4 °C. Then tissues were washed 5

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times in PBS, the paraffin sections were incubated with the corresponding secondary antibody diluted according to the manufacturer’s recommendations for 25 min at 37 °C, which was coupled to the horseradish peroxidase–streptavidin (Streptavidin Peroxidase Link Detection Kits; Zhongshan Golden Bridge Biotechnology, Beijing, China) for 15 min at room temperature, and colored with DAB kit (Zhongshan Golden Bridge Biotechnology) reaction for 15 min at room temperature without light. The mounted sections were observed under a light microscope (Olympus, Tokyo,

ACCEPTED MANUSCRIPT Japan). The representative images were captured from the CA1 region of hippocampus and PrL region of cortex. 2.7 Immunofluorescence Brain sections were incubated with primary antibodies : PSD-95 (3450) (1:200,

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Cell Signaling Technology, Inc. Beverly, MA) at 4 °C overnight, then washed thrice

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with PBS, and incubated with Alexa Fluor (555)-conjugated anti-rabbit (4413)

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secondary antibody at 37 °C for 20 min. Immunofluorescence images were evaluated on an inverted fluorescent microscope (Olympus, Tokyo, Japan) (×200). The

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representative images were captured from the CA1 region of hippocampus.

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2.8 Electron microscopy for structural analysis of the hippocampus Structural analysis of the hippocampus was observed via electron microscopy as

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previously described [21]. Transmission electron microscope (TEM) analysis was

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guided after the collection of CA1 region of hippocampus. The hippocampus was split and treated in a cold fixative solution made up of 2.5% glutaraldehyde (pH 7.2) at

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4 °C for 4 h. After washing with PBS (0.1 mol/L, pH 7.2) thrice. Then the specimens

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were post-fixed in 1% OsO4 (in 0.2 mol/L PBS, pH 7.2) at 4 °C for 1 h and washed again with PBS (0.1 mol/L, pH 7.2) thrice. The specimens were dehydrated for 15-20 min each in a graded series of ethanol solutions (30, 50, 70, 80, 90, and 100%) and then transferred to acetone for 20 min incubation. Materials were then permeated in an acetone−resin mixture (1:1) for 1 h at 25 °C and then transferred to an acetone−resin mixture (1:3) overnight. Ultrathin sections were placed in the region which were closed to the embedded blocks and kept away from the dorsal rim area,

ACCEPTED MANUSCRIPT stained with uranyl acetate and alkaline lead citrate for 15 min, and then observed using TEM (JEOL, Tokyo, Japan). 2.9 Statistical Analysis Data were reported as mean ± SEM of at least three independent experiments.

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Significant differences between mean values were determined by Two-way ANOVA

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with sesamin treatment (0 and 50 mg/kg) and stress (control/CUMS) as factors.

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Post-hoc test was performed using Tukey test for multiple comparison test by Graphpad Prism 6.0 software. Means were considered to be statistically significant if

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p<0.05.

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3 RESULTS

3.1 Effects of sesamin on depressive like behaviors and anxiety in CUMS-treated mice

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In order to examine potential antidepressant effects of sesamin, the

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CUMS-induced depression-like mice model was employed in present study (Fig. 1A). During the experiment, mice showed no changes in bodyweight (Fig. 1B). Results

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showed that 42-days CUMS elicited depressive like behavior. The assessments of

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depression related behaviors were performed by forced swimming test and tail suspension test (Fig. 1C&D). Sesamin significantly decreased the CUMS-induced rise of immobility time (p<0.05) during forced swimming test and tail suspension test, which indicated that sesamin could improve the depressive state caused by chronic stress. The stress and the interaction of sesamin treatment and stress significantly affected results (p<0.01). Furthermore, the activity of freedom movement in mice was observed by open

ACCEPTED MANUSCRIPT field test (Fig. 1E&F). All treatments had no effects on crossings number and the total distance of animals moved in the test, which indicated that both CUMS and sesamin did not affect the independent activity of mice. To assess anxiety behavior in mice, the elevated plus maze test was measured in

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terms of percentage of open arm entries of mice and time spent exploring open arms

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(Fig. 1G&H). Results demonstrated that mice anxiety behavior was stimulated by

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42-days CUMS significantly (p<0.05). Sesamin treatment had an increase influence on percentage of open arm entries of mice (p<0.05) and the interaction of sesamin and

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stress also had significant impacts on this parameter (p<0.01), indicating that sesamin

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improved the anxiety behavior of stressed mice.

3.2 Effects of sesamin on levels of serotonin and norepinephrine in brain and serum

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To determine whether the antidepressant effects of sesamin were related with

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levels of these hormones, both NE and 5-HT expressions were detected in brain. 5-HT is commonly thought to be a contributor to mood and cognition, and NE is mainly

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responsible for cognitive alertness and reward system [30]. As shown in Fig. 2, 5-HT

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and NE concentrations in the striatum and serum tended to decrease in the CUMS group, compared with the control group. However, sesamin treatment significantly prevented the decrease in 5-HT and NE in striatum (26.3%, 218.9% respectively, p<0.05) in CUMS-treated mice, but had no effect on hippocampus and cortex. The interaction of sesamin and stress has significant effects on NE and 5-HT levels in striatum (p<0.01). 3.3 Effects of sesamin on CUMS-induced cognition deficits

ACCEPTED MANUSCRIPT To investigate the effects of sesamin on CUMS-induced spatial and related forms of learning and memory, the behavioral changes were further observed via Morris water maze and Y-maze tests. The changes in the escape latency and escape distance to reach the hidden platform were examined in this trial (Fig. 3A&B), during the test

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period, there was no trend in escape latency in training session for 3 days. However,

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the mean latency was significantly prolonged in the CUMS group as compared with

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the control group (p<0.05). Meanwhile, treatment with sesamin markedly ameliorated the effects of CUMS on escape latency (p<0.05), indicating a better memory. In terms

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of escape distance, there was a downward trend in water-maze training. Sesamin

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treatment took a shorter distance to find the platform, compared to the CUMS group. After 3 days, a probe trial was performed with the platform removed (Fig. 3C) to

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evaluate the retention of memory, and number of platform crossings and time spent in

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the target quadrant were measured. As shown in Fig. 3D&E, mice in CUMS group showed a decrease number of platform crossings and spent less time in the target

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quadrant remarkably (p<0.05), compared with the control group. The number of

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platform crossings and average spent time in the target quadrant in sesamin treatment group significantly generated a substantial increase as compared with those in CUMS group (p<0.05). All the sesamin treatment (p<0.05), stress (p<0.05), and their interaction (p<0.05) had significant effects on the time spent in the target quadrant. These data suggested that sesamin could recover the damage of chronic stress-induced learning and memory impairments. Y maze test is a behavioral study on working memory alteration in animals.

ACCEPTED MANUSCRIPT Analyses of the spontaneous alternation percentage and total arm entries in Y maze task (Fig. 3F&G) showed CUMS group significantly decreased spontaneous alternation and total arm entries compared with control group (p<0.05), indicating an impairment of working memory. Supplement of sesamin dramatically improved

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memory formation in CUMS-treated mice, decreasing significantly spontaneous

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alternation and total arm entries (p<0.05). The sesamin treatment (p<0.05) and stress

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factor (p<0.05), but no their interaction (p>0.05), significantly influenced spontaneous alternation and total arm entries.

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3.4 Effects of sesamin on the expression levels of neurotrophic factors and

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hippocampal synapse morphology alterations

To detect the neuronal integrity and orderliness, H&E staining and

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immunohistochemistry of BDNF protein were used to perform. As shown in Fig. 4A,

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histology of the hippocampal CA1 region revealed that shrinkage of nuclei and shrinking neurons were found in CUMS mice. Administration of sesamin could

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inhibit the mentioned above histopathological damages and recover normal

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arrangement of neurons in hippocampus. BDNF is an essential neurotrophin in CNS that regulates neural development and neuronal function, and BDNF levels were observed to decline in depression and neurodegenerative disorders [31]. As shown in Fig. 4A, sesamin treatment also improved BDNF expression in CUMS-treated mice hippocampus. The mRNA levels of other neurotrophic factors in hippocampus including NT3, NT4, and NGF were also investigated. NT3, NT4, and NGF are a group of neurotrophic factors of the NGF

ACCEPTED MANUSCRIPT family which has activity on certain neurons of the peripheral and central nervous system. [32, 33]. Compared with control group, CUMS caused a marked decrease in BDNF mRNA expression in hippocampus (65.9%, p<0.05), while treatment with sesamin increased BDNF mRNA expression (288.6%, p<0.05) (Fig. 4B). Consistently,

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significantly decreased mRNA expression of NT3 in hippocampus were observed in

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CUMS group (49.2%, p<0.05) relative to control group. Supplementation of sesamin

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increased mRNA expression of NT3 (53.9%, p<0.05) compared with CUMS-treated mice (Fig. 4C). The interaction of sesamin treatment and stress factors had significant

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effects on mRNA expressions of BDNF and NT3 (p<0.05).However, all treatments

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had no influence on mRNA expression of NT4 and NGF (Fig. 4D&E). 3.5 Effects of sesamin on ultrastructure of synapse and expressions of postsynaptic

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dense protein in hippocampus

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The ultrastructure of hippocampus synapse was examined using transmission electron microscopy and immunofluorescence staining of PSD-95 (Fig. 5A&B). As

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shown in Fig. 5C&D, compared to control group, CUMS markedly decreased (18.8%,

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p<0.05) the length of postsynaptic density. Sesamin treatment significantly enhanced the length of postsynaptic density in hippocampus compared to CUMS group (26.5%, p<0.05). The interaction of sesamin treatment and stress factors had significant effects on PSD length (p<0.01). In addition, the mRNA levels of PSD-95 in hippocampus and cortex were investigated (Fig. 5E&F). Significantly declined levels of PSD-95 in hippocampus were observed in CUMS group (34.8%, p<0.05) relative to control group. Supplement of sesamin improved levels of PSD-95 (65.6%, p<0.05) compared

ACCEPTED MANUSCRIPT with CUMS group. The interaction of sesamin treatment and stress factors had significant effects on hippocampal PSD-95 mRNA expression (p<0.01). 3.6 Effects of sesamin on the mRNA expression of inflammatory proteins and cytokines in brain induced by CUMS

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Recent research suggests depression is accompanied by neuroinflammation, and

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characterized by microglial activation [12]. To examine microglial activation in CNS,

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immunohistochemistry of IBA-1 protein and mRNA level were detected in hippocampus and cortex. IBA-1 is a microglia/macrophage-specific calcium-binding

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protein, which is a marker of activated microglia [34]. Immunohistochemistry of

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IBA-1 staining results showed that CUMS significantly increased expression of IBA-1 in hippocampal CA1 region and cortex in comparison with control. However,

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expression of IBA-1 was decreased in sesamin treated mice in the region of brain (Fig.

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6A).

To further investigate the effects of sesamin on inflammation induced by chronic

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stress in brain, the hippocampal and cortical inflammatory related gene expressions

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were measured including COX-2, iNOS, TNF- and IL-1β. As shown in the Fig. 6B-I, CUMS significantly increased the mRNA levels of COX-2 and iNOS production in cortex (44.6%, 100.1% respectively, p<0.05), compared to control group. Notably, sesamin treatment dramatically improved COX-2 and iNOS mRNA expression in CUMS cortex (39.1%, 61.5% respectively, p<0.05). The interaction of sesamin treatment and stress factors also had significant effects on COX-2 and iNOS expressions in cortex (p<0.05). Besides, in hippocampus, the mRNA levels of iNOS

ACCEPTED MANUSCRIPT treated with CUMS were dramatically increased (65.4%, p<0.05), compared to control group. Sesamin supplementation substantially suppressed CUMS-elevated proportions of iNOS in hippocampus (52.0%, p<0.05), indicating that sesamin inhibited the expression of inflammatory protein. Consistently, sesamin also alleviated

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the enhanced expression of proinflammatory cytokines TNF- and IL-1β. All the

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sesamin treatment, stress factor and their interaction had significant effect on TNF-

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and IL-1β expressions in cortex (p<0.01). The sesamin treatment (p<0.05) and stress (p<0.05), but not their interaction, (p>0.05) significantly affected TNF- and IL-1β

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expressions in hippocampus.

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4 DISCUSSION

In the current study, it demonstrated that sesamin treatment improved

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CUMS-induced anxiety and depressive-like behaviors by forced swimming test, tail

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suspension test and elevated plus maze test. Morris water maze test and Y-maze test also revealed that sesamin rescued spatial learning abilities, reference memory and memory

in

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working

stress-induced

mice.

Consistently,

the

monoamine

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neurotransmitter NE was improved in striatum. The levels neurotrophic factor BDNF in hippocampus was also increased by sesamin, which was consistent to the enhancement of PSD-95 expressions in depressed mice. Moreover, sesamin treatment suppressed overexpression of IBA-1 and decreased COX-2, iNOS, TNF-α, and IL-1β levels in brain. These results indicated that sesamin partly reversed stress-induced neuroinflammation in brain. The neuroprotective effects of sesamin has been well-documented [35-38].

ACCEPTED MANUSCRIPT Sesamin treatment (15 mg/kg and 30 mg/kg) prevented kainic acid-induced status epilepticus in rats [35]. Sesamin (20 mg/kg) attenuated neuronal apoptosis and damages in a unilateral striatal 6-hydroxydopamine model of PD [39]. Sesamin treatment (50 or 100 mg/kg) was reported to possess improvement effects in an aging

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mice model [40]. Interestingly, (-)-sesamin (25 or 50 mg/kg), the epimeric isomer

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lignan of (+)-sesamin has also been reported to prevent stress-induced anxiety and

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memory impairment in a rodent model. The current study revealed that 50 mg/kg/d sesamin treatment could also ameliorate chronic mild stress-induced depression-like and

improve

learning and

memory

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behaviors

via

attenuating

microglial

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activation-mediated inflammation. Interestingly, as shown in Fig. 1 and Fig. 3, sesamin treatment had no effects on control group animals. Consistently, previous

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research also demonstrated that sesamin treatment has no significant effects on

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control or sham group animals in 6-hydroxydopamine-induced neurotoxicity, cerebral artery occlusion and nociception animal models [39, 41, 42].

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The distribution and metabolism of sesamin have been well-examined in recent

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decades [43, 44]. After administration, sesamin incorporated into the liver and then transported to the brain and other tissues, which indicated that sesamin possesses blood-brain barrier (BBB) permeability [43]. In addition, it has been reported that sesamin (30 mg/kg/d) could also alleviate controlled cortical impact injury-induced BBB disruption via enhancing tight junction proteins ZO-1 and occluding expression in a mice model [18]. Oxidative stress and neuroinflammation in MDD are mechanistically linked to the presence of BBB hypermeability [45]. In the present

ACCEPTED MANUSCRIPT study, sesamin alleviated CUMS-induced depressive-like behaviors, and this effect might also relate to its protective role on BBB in brain, which should be further investigated in the future study. Chronic stressful life events are high risk for MDD, and exposure to CUMS has

[21,

46].

CUMS

also

lead

to

impairment

of

5-HT

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neuroinflammation

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been demonstrated to generate free radicals resulting in oxidative stress and

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neurotransmission and dysfunction of hypothalamic–pituitary–adrenocortical (HPA)

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axis [47]. 5-HT plays a pivotal role in regulating mood, sleep, and cognition [48]. In the present study, CUMS decreased the 5-HT level in serum, which might also be

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related to the cognitive impairments. Sesamin improved 5-HT and NE levels in striatum but not in serum, which might partly explained its beneficial effects on

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depressive like behaviors. However, the levels of 5-HT and NE did not changed in

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cortex or hippocampus in stressed mice which indicated that the cognitive improvement effects of sesamin might be explained by its other bioactivities.

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Interestingly, the level of BDNF, a neurotrophin that regulates neural development

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and neuronal function, enhanced in depressive mice. BDNF is mediator of synapses function and neuroplasticity via [49] BDNF-TrkB signaling. In the present research, the expression of PSD-95 and synaptic ultrastructure revealed that sesamin improved stress-induced cognitive deficits might be through regulating the BDNF expression (Fig. 4). Besides, loss of BDNF or the “neurotrophin hypothesis of depression” is an essential theory in depression and anxiety treatment [50]. A recent post-mortem study found that a significant elevation of BDNF and NT3 levels in MDD patient brain [51].

ACCEPTED MANUSCRIPT In the present study, sesamin improved both BDNF and NT3 expressions in stress-induced mice hippocampus, which might partly be explained its beneficial effects on depressive-like behaviors. Lots of reports have indicated that depression may be a failure to adapt to

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stress-stimulated neuroinflammatory responses [52]. Microglia is the major immune

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cell in CNS, and emerging evidence demonstrated that over-activation and senescence

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of microglia are also associated with depression progress [53]. Sesamin has been

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reported to prevent microglia activation in a diabetic retinopathy mice model [36]. Previous study also found that sesamin could suppress microglia over-activation via

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inhibited MAPK signaling [54] in BV-2 murine microglial cells and primary-cultured rat microglia. Consistently, our data demonstrated that sesamin suppressed microglial

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activation with significantly decreased expressions of neuroinflammation marker

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iNOS and COX-2, and levels of cytokines IL-1β and TNF-α (Fig. 6) in CUMS-treated mice hippocampus and cortex. As neuroinflammation also plays a pivotal role in

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insulting cognitive function [55], these results might explain the protective effects of

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sesamin on both depression and cognitive impairments. Another lignan from sesame seed and sesame oil, sesamol, also exhibits neuroprotective effects. Previous of our research reported that sesamol could prevent systemic inflammation-induced amyloidogenesis and neuronal damages in a rodent model [56]. Sesamol could also prevent high energy density diet-stimulated insulin resistance and disorders of glucose/lipid metabolism [57]. Similarly, previous studies also revealed that sesamin prevented high-fat diet-induced dyslipidemia and obesity

ACCEPTED MANUSCRIPT [58]. Lots of reports also indicated that sesamin could also attenuate insulin resistance and diabetic retinopathy [59, 60] in diabetes animal models. Besides, the lipid-lowering effects of sesamin have also been well-documented [15, 61, 62]. However, the molecular targets of sesamin or sesamol have not yet been fully

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understood. Previous of our research found that sesamol could bind to the C-terminus

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of p50 subunit and DNA binding region of the nuclear transcriptional factor NFκB,

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which is the regulator of cellular inflammatory process [56]. Sesamin has also been demonstrated to down-regulate NFκB activation [55]. Even though the present work

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revealed that sesamin inhibited inflammatory responses, yet the accurate binding

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target of sesamin still need to be further determined.

In conclusion, our present results found that sesamin could prevent chronic

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stress-induced mice anxiety, depressive-like behaviors, synaptic plasticity, and

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memory impairments by improving monoamine neurotransmitter and neurotrophic factors, which could be partly explained by its preventive effects on inflammation

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responses in CNS. This research indicated that sesamin might be a novel

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neuroprotective phytochemical and nutraceutical in future depression and neurodegenerative diseases research. ACKNOWLEDGEMENTS This work was supported by grants from the National Key Research and Development Program of China (No. 2016YFD0400601), National Natural Science Foundation of China (No.81803231), Key Research and Development Plan of Shaanxi, China (2018NY-100), China postdoctoral science foundation (2018T111104),

ACCEPTED MANUSCRIPT and the Fundamental research funds for the central universities (2452017141) supported this research. AUTHOR CONTRIBUTIONS Conception and design of research: ZL, XL; Performed experiments: YZ, QW,

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MJ; Analyzed data: ZL, YZ; Interpretation of results of experiments: ZL, XL; Prepared figures: ZL, JP; Drafted manuscript: ZL, YZ

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CONFLICT OF INTEREST

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The authors declare that there are no conflicts of interest.

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ACCEPTED MANUSCRIPT TABLES Table 1 Primer sequences used for semi-quantitative RT-PCR analysis. Primer, 5’-3’ Gene

Forward

Reverse

CTCCGCCATGCAATTTCCACT

GCCTTCATGCAACCGAAGTA

Nt3

GTTCCAGCCAATGATTGCAA

GGGCGAATTGTAGCGTCTCT

Nt4

CAAGGCTAAGCAGTCCTATGT

CAGTCATAAGGCACGGTAGAG

Nfg

CGACTCCAAACACTGGAACTCA

GCCTGCTTCTCATCTGTTGTCA

Psd-95

TCTGTGCGAGAGGTAGCAGA

AAGCACTCCGTGAACTCCTG

Iba-1

TGATGAGGATCTGCCGTCCAAACT

Cox-2

GAAGTCTTTGGTCTGGTGCCT

GCTCCTGCTTGAGTATGTCG

Nos2

GGAGCGAGTTGTGGATTG

CCAGGAAGTAGGTGAGGG

Tnf-

CCCTCACACTCAGATCATCTTCT

GCTACGACGTGGGCTACAG

Il-1

TGACGGACCCCAAAAGAHTGA

TCTCCXACAGCCACAATGAGT

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SC

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Bdnf

TCTCCAGCATTCGCTTCAAGGACA

ACCEPTED MANUSCRIPT FIGURE LEGENDS Fig. 1 Effects of sesamin on depressive like behaviors and anxiety in CUMS-treated mice Three month-old male CD-1 mice were randomized into four groups (n = 10 per

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group): Control group, SE treatment (50mg/kg/day), CUMS, and CUMS+SE

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treatment (50mg/kg/day). (A) Schematic figure of the treatment protocol; after

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42-days treatment. S1-S7 stands for treatments: S1: 5-min cold swimming (at 4 °C), S2: 1-min tail pinch (1 cm from the tip of the tail), S3: 24-h food and water

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deprivation, S4: overnight illumination, S5: 15-min force swimming (at 23 °C), S6:

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24-h cage tilting (30°), and S7:200 mL of water for sawdust dampness per cage (sufficient to reach the moisture of the sawdust bedding); (B) Bodyweight; (C) Forced

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swimming test; (D) Tail suspension test; (E&F) Open field test (crossings number and

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the total distance); (G&H) Elevated plus maze test (percentage of open arm entries and time spent exploring open arms) were detected as described in Materials and

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methods section. Data presented as mean ± SEM, n≥5. Means with different letters (a,

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b, c) are significantly different from each other (p<0.05). Fig. 2 Effects of sesamin on levels of serotonin and norepinephrine in brain and serum

(A) 5-HT levels in hippocampus; (B) 5-HT levels in cortex; (C) 5-HT levels in striatum; (D) 5-HT levels in serum; (E) NE levels in hippocampus; (F) NE levels in cortex; (G) NE levels in striatum; (H) NE levels in serum were detected. Data presented as mean ± SEM, n≥5. Means with different letters (a, b, c) are significantly

ACCEPTED MANUSCRIPT different from each other (p < 0.05). Fig. 3 Effects of sesamin on CUMS-induced cognition deficits The animals were performed assessments of cognitive functions via the Morris water maze tests and Y-maze as described in the Methods section. For water maze test, (A)

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escape latency; (B) escape distance in place navigation test as well as (C) mouse

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trajectory during the probe trial; (D) the number of platform crossings; (E) the time

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spent in the target quadrant were recorded. For Y-maze test, (F) the spontaneous alternations; (G) the total arm entries were recorded. Data presented as mean ± SEM,

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n≥5. Means with different letters (a, b, c) are significantly different from each other

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(p<0.05).

Fig. 4 Effects of sesamin on the expression levels of neurotrophic factors and

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hippocampal synapse morphology alterations

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(A) Representative images of H&E staining and immunohistochemistry of BDNF protein in CA1 region of hippocampus (images were captured from 12 slices from 3

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mice per group); (B) mRNA levels of BDNF in hippocampus; (C) mRNA levels of

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NT3 in hippocampus; (D) mRNA levels of NT4 in hippocampus; (E) mRNA levels of NGF in hippocampus were detected as described in Materials and methods section. Data presented as mean ± SEM, n≥5. Means with different letters (a, b, c) are significantly different from each other (p < 0.05). Fig. 5 Effects of sesamin on ultrastructure of synapse and expressions of postsynaptic dense protein in hippocampus (A) Representative electron micrograph of synaptosomal fractions from CA1 region

ACCEPTED MANUSCRIPT of hippocampus. Electron micrograph analysis were performed on 12 slices from 3 animals per group; (B) Representative images of immunofluorescence staining of PSD-95 and DAPI in CA1 region of hippocampus. Representative images of immunofluorescence staining were captured from 12 slices from 3 mice per group;

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(C&D) the length and width of PSD; (E&F) mRNA levels of PSD-95 in

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hippocampus and cortex were detected as described in Materials and methods section,

significantly different from each other (p<0.05).

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n≥5. Data presented as mean ± SEM. Means with different letters (a, b, c) are

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cytokines in brain induced by CUMS

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Fig. 6 Effects of sesamin on the mRNA expression of inflammatory proteins and

(A) Representative images of immunohistochemistry of IBA-1 protein in CA1 region

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of hippocampus and PrL region of cortex. Representative images were captured from

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12 slices from 3 mice per group; (B&C) mRNA levels of COX-2 in hippocampus and cortex; (D&E) mRNA levels of iNOS in hippocampus and cortex; (F&G) mRNA

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levels of TNF- in hippocampus and cortex; (H&I) mRNA levels of IL-1 in

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hippocampus and cortex were detected as described in Materials and methods section. Data presented as mean ± SEM, n≥5. Means with different letters (a, b, c) are significantly different from each other (p<0.05).

ACCEPTED MANUSCRIPT (+)-Sesamin attenuates chronic unpredictable mild stress-induced depressive-like behaviors and memory deficits via suppression of neuroinflammation

HIGHLIGHTS: Sesamin attenuated CUMS-induced depression-like behavior and cognitive

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

Sesamin enhanced monoamine neurotransmitters and BDNF levels in

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

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impairments

hippocampus

Sesamin improved the ultrastructure of synapse and PSD expressions in

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

Sesamin suppressed stress-induced microglial over-activation and cytokines

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expression

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

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depressed mice

Figure 1

Figure 2

Figure 3

Figure 4

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Figure 6