EFFECTS AND UNDERLYING MECHANISM OF 5-LIPOXYGENASE INHIBITOR (ZILEUTON) ON MICE DEPRESSIVE-LIKE BEHAVIOR

Objective: Treatment experiment was conducted to investigate the effectiveness and mechanism of the action of zileuton in corticosteroid-induced depressive mice model through neuroinflammation. Methods: The mice were randomly separated into four groups: (Veh+Veh), (Corticosteroid+Veh), (Corticosteroid+ZIL50), and (Corticosteroid+ZIL100). Intraperitoneal injection of corticosterone (CORT) (20 mg/kg for 6 weeks) was used in the mice to induce depression and neuroinflammation diverse from the Veh+Veh group, which was injected only physiological saline. The drug-treated groups (Corticosteroid+ZIL50 and Corticosteroid+ZIL100) were orally administered with the mentioned doses of zileuton. After confirming the effectiveness of zileuton through the behavioral tests, the mechanism of the action of the drug was explored through a set of biochemical assays. Results: Zileuton (50/100 mg/kg) administration improved the performance of the mice in the behavioral experiments (p<0.05 or 0.01). Immunohistochemistry detection of Iba1+ revealed over activation of microglial cells in the corticosteroid-treated mice which was suppressed by the zileuton (50 or 100 mg/kg [p<0.05 or 0.01]). Through Western blotting tests, it had been found that CORT (i.p.) administration led to the increment of the protein 5-Lipoxygenase in the mouse hippocampus associated with neuroinflammation, which was decreased significantly by zileuton (p<0.05 or 0.01). Level of tumor necrosis factor-alpha, interleukin-1 beta, nuclear factor kappa B p65 protein (for neuroinflammation), Bax, and cleaved caspase-3 and TUNEL assay increased, and Bcl-2 expression decreased in the CORT-induced depressive mice. These were significantly reversed by zileuton (50 or 100 mg/kg [p<0.05 or 0.01]). Conclusion: It can be concluded that selective 5-lipoxygenase inhibitor zileuton can efficiently inhibit the depressive-like behavior/activity in CORT-induced depressive mouse model. Moreover, the underlying mechanism may be the inhibition of hippocampal neuroinflammation and apoptosis. **p<0.01 versus CORT+Veh group. These data suggest that 5-Lipoxygenase inhibitor zileuton blocks CORT-induced microglial activation


INTRODUCTION
Major depression and mania are two extremes of emotional disorders, which refer to the pathophysiological changes in mood state [1]. Depression disorder is a severe psychiatric abnormality characterized by a diffusive and chronic low mood that is accompanied by low self-esteem, psychomotor retardation or agitation, change in appetite, and an absence of interest or usually enjoyable pleasant attack [2]. Still now, the pathological mechanism of depression has not been well defined. Decreases of monoamine deficiency, the hypothalamic-pituitary-adrenal (HPA) axis hyperactivity, and neurotrophin are involved in the pathophysiology of depression [3].
Chronic stress leads to the regulation of HPA axis within the neuroendocrine system, and it was observed that the level of corticosterone (CORT) in the cycle disrupted the circadian regulation of CORT secretion and the glucocorticoid (GC) receptor negative recompose circuit [4]. Proved that, chronic stress could cause an imbalance in the HPA axis in neuroendocrine system. In patients with depression, hippocampal feedback was impaired, leading to excessive activity of HPA axis and elevated levels of humeral corticosteroids [5,6]. As a result, Cushing's disease with patients or those receiving long-term pharmacotherapy with GC shows an exceptionally high rate of depression [7]. Stimulation and continued actions of the HPA axis are attenuated through the adverse feedback circulating GC following exogenous CORT administration, and this is generally related to the improvement of psychosomatic disorders, which lead to severe changes in indicative or consistent emotional behavior accompanied by depressive-like symptoms [8,9]. HPA axis is activating by high dose of CORT administration, will increment depressive behavior in rodents, as held by a significantly diminish in sucrose consumption [8].
Arachidonic acid (AA) metabolized into inflammatory molecules called as leukotrienes (LTs) that have been metabolized by 5-lipoxygenase (5-LOX) inhibitor, which are capable mediators of several inflammatory and vascular diseases.
Zileuton attenuates brain inflammation in cerebral ischemia by inhibiting the nuclear factor-kappa B (NF-κB) p65 and downstream inflammatory mediators. However, it is important to further discover the potential mechanism of zileuton's neuroprotection against central nervous system (CNS) disorders.
Given this background, in this research work, we focused to give a glimpse the effects of 5-LOX drug zileuton (50 mg/kg and 100 mg/kg) treatment on the tail suspension test (TST), forced swimming test (FST), novelty suppression feeding test (NSFT), and open-field test (OFT) in mice that show repetition of exposure to CORT. Nowadays, many evidence suggested that neuroinflammation is responsible for the pathophysiology of depression [9,10]. We can show remarkable influence in both patients suffering depression and animal model of depression in the presence of high levels of pro-inflammatory cytokines, for example, interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) [10,11]. These pro-inflammatory cytokines show

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an impact on the plasticity and neurotransmission, therefore, suppress the neurogenesis in the brain, and appliance of antidepressants suppresses these cytokines activation. The conversion of AA to 5-hydroxyperoxyeicosatetraenoic acid is carried out in the presence of a pro-inflammatory enzyme named 5-LOX and afterward the catalysis, it also produces hydroxyeicosatetraenoic acid (5-HETE). Therefore, it metabolized in several LTs [12]. 5-LOX and the LTs in the CNS perform both neuromodulatory and neuroendocrine effects [13] and lead to inflammatory response characterized by the increments of IL-1β, TNF-α, and NF-κB. Nowadays, a mass of researchers focused on 5-LOX. 5-LOX is responsible for the various type of CNS damage such as cerebral ischemia, cognitive deficit, traumatic brain injury, and anxiety by implicating its novel pathophysiological role [14][15][16]. Therefore, the relationship and the mechanism between 5-LOX and depressive-like behaviors need to be clarified. During this study, we first observed whether 5-LOX inhibitor zileuton prevents depressive behaviors and neuroinflammatory responses in CORT (corticosteroid)-induced mice model [17].
Furthermore, we tend to additionally evaluate whether the behavioral antidepressant-like effects of zileuton associated with alterations in hippocampal cell proliferation and neuronal commitment, as well as astrocytic hyperactivation.

Materials
The pure drug zileuton was generously obtained from Dalian Meilun Biological Co., Ltd., China, CORT (corticosteroid) was purchased from Dalian Meilun Biological Co., Ltd., China, streptavidin-biotin complex (SABC) immunohistochemistry (IHC) kit was purchased from Wuhan Boster Biological Technology, Ltd., China. Trizol reagents and bovine serum albumin were purchased from Nanjing SunShine Biotechnology Co., Ltd., China; Western blot markers were obtained from Thermo Scientific, USA. Chemiluminescence detection reagents were purchased from Tanon Science and Technology Co., Ltd., China. Caspase-3 rabbit monoclonal antibody, Bcl-2 rabbit monoclonal antibody, and Bax rabbit monoclonal antibody were purchased from Cell Signaling Technology Ltd., USA.

Animals
Institute of Cancer Research mice were used for this experiment, took 12 mice in each group. All experiments were conveyed according to NIH guide for the care, and animal care committee in China Pharmaceutical University approved the use of laboratory animal (NIH publications No. 80-23, received, 1996) and the procedures. All animals were placed on a 12-h dark/light cycle with free access to water and standard chow and adapted for 1 week before the start of the experiment.

FST
The FST was performed [18]. In short, separately placed the mice in a glass cylinder (diameter 19 cm, height 25 cm) containing water (25±1°C) to a depth of 13 cm and allowed to swim for 6 min. Record the behavior of the mice and explored by TSE behavioral software. After a 6 min test, removed the mouse from the tank and thoroughly dried with dry cloth then returned to the colony room in their home cage. Change the tank's water after each swim session. Judged the immobility time as the absence of active, unwanted behavior, such as jumping, swimming, rearing or diving. Measured the total immobility time during the final 4 min of a 6 min test season by an observer blind to the treatment conditions.
TST TST was performed [19]. After FST, mice were conducted 24 h. Shortly, moved the mice from housing room to the testing room in their home cages, at least 1 h before testing to allow for adapt to the new environment. During the dark period of the circadian cycle, mice were tested. Each mouse was hanged by his or her tail with adhesive tape to the hook in the soundproof box. The total immobility period during 6 min test was explored by ANY-MAZE software. The immobility period during initial 2 min of the 6 min task was discounted while the last 4 min task was explored statistically.

OFT
The OFT was performed [20]. The mice were conducted 24 h after TST. The open field contained (40×40×38 cm) square arena with clean Plexiglas wall and floor. At first, mouse was placed in one corner of the open space and during a 5 min test, session allowed to move arena freely. Measured peripheral and central activities using a computerassisted activity system with software to easily the data collection and analysis.

NSFT
After habituation in the testing room, mice were subjected to the NSFT test [21]. The testing instrument consists of a plastic box (50×50×20 cm), covered by wooden bed 2 cm. Twenty-four hours before this behavioral test, all food was removed from their cage. At testing time, a single pellet of food was placed on a white paper platform in the center of the testing box. The mouse has placed in the center of the box. The mouse was placed in the corner of the box and instantly started the stopwatch. The latency to eat (it means the mouse sitting on its haunches and biting the pellet with the use of forepaws) was recorded.

Tissue preparation and immunohistochemical analyses
Tissue preparation, at first anesthetized the mice and perfused transcardially with 0.1 M phosphate-buffered saline (PBS) (pH-7.4), contains 4% paraformaldehyde and 5 U/ml heparin. Then, the brain was collected and fixed in 4% paraformaldehyde for 18-20 h followed by 30% sucrose solution for 24 h. After then, the brain was dissected and embedded into optimal cutting temperature compound on dry ice and cryosectioned at 30 μm. Following the manufacturer's instructions, immunohistochemical staining was performed using SABC IHC kit. After washing, the section with 0.1 M PBS solution heated the sections on water bath for 4 h in 0.3% Triton X-100 at 60°C. Then again washed the section and treated with 3% H 2 O 2 at normal room temperature for 10 min and washed with PBS for 3×5 min followed by blocking 5% BSA for 30 min. After then, the sections were incubated overnight at 4°C with primary antibody for Iba 1 (rabbit immunoglobulin G [IgG], 1:1000) diluted in 5% BSA. Next day, washed the sections in PBS for 3×5 min at room temperature and incubated with biotinylated mouse anti-rabbit IgG for 20 min. After then, again rewashed in PBS (3×5 min, 37°C) and incubated with SABC at 37°C for 20 min. Again, sections were washed in 0.1 M PBS for 4×5 min and mounted on the glass slides. Then added the diaminobenzidine followed by gradient dehydration ([1] 70% ethanol for 5 min, [2] 95% ethanol for 5 min, [3] 100% ethanol for 2×5 min, and [4] xylene for 2×5 min). Next, sections were covered with DPX mounting solution and cover glass. At last, photomicrographs were obtained using a Nikon DS-Fi2 camera connected to a Nikon Eclipse Ti microscope and analyzed by the Image-Pro Plus software.

Total protein and nuclear protein extraction
Homogenized the mouse hippocampus in an ice-cold radioimmunoprecipitation assay buffer, it contains 0.1% phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 12,000 g for 15 min and collected the supernatant. Total protein concentration was determined. The supernatant was used to determine using the bicinchoninic acid protein kit and stored at −20°C.
Nucleoprotein extraction kit was used for nuclear extraction. Shortly, chopped the mouse hippocampus into small pieces, then homogenized in the ice-cold hypotonic buffer, contains 1% PMSF, 0.5% phosphate inhibitor, 0.1 % DL-Dithiothreitol (DDT) and then centrifuged at 4°C 3000 g for 5 min. Then washed the precipitate with hypotonic buffer solution and centrifuged at 4°C, 5000 g for 5 min. Finally, 0.2% lysis buffer containing 1% PMSF, 0.1% DDT, and 0.5% phosphatase buffer was added into the precipitate, cooled for 20 min, and centrifuged at 4°C, 15,000 g for 10 min. Subjected the supernatant nuclear extract was detected by WB assay for NF-κB p65 and histone H3 using as the control.

Statistical analysis
Data shown are expressed as mean±standard error of the mean using analysis of variance (ANOVA) software to analyze the behavioral tests (TST, OFT, FST, and NSFT). All other data were analyzed by one-way ANOVA for various comparisons followed by Dennett's post hoc analyzer. All analyses were carried out using the SPSS 22.0. Considered that, p<0.05 was statistically significantly difference between the groups.

Zileuton blocks CORT-induced microglial activation
It informed that CORT activates the mediated inflammatory response through microglia, whose changes are characterized by improved cell numbers and conformational changes, as well as size enlargement and thicker processes [22]. To study the effects of hippocampal 5-LOX enzyme on microglia, we detected the activation levels of microglia by immunohistochemical method. As shown in figure, the number of IBA1 positive cells in the hippocampus of CORT-induced mice was significantly prolonged (Fig. 3a, F(3,12)=13.115, p<0.01) compared to the Veh+Veh group, while zileuton (50 mg/kg or 100 mg/kg) significantly decreased IBA1 staining cells (Fig. 3b CORT+Zil50: p<0.05 and CORT+Zil100: p<0.01).
Activated microglia and NF-κB pathway may regulate various proinflammatory cytokines involved in the onset of depression [24] to

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investigate the effect of zileuton on the neuroinflammatory process induced by CORT.

DISCUSSION
Through the present study provides evidence that the neuroprotective effect of zileuton has shown in CORT-induced depressive mice model through modulating loss of behavioral activities, activation of microglia, neuroinflammation, and cell apoptosis. Specifically, CORT led to the significant increase of hippocampal 5-LOX protein, produced several behavioral activities including increased immobility time in the FST and TST test, and increased the latency to feed in the NSFT in mice. It was accompanied by the hippocampal inflammatory response, activated microglia, increased the expression of TNF-α, IL-1β, NF-κB p65, increased cellular apoptosis caspase-3, Bax, TUNEL, and decreased Bcl-2. 5-LOX inhibitor drug zileuton markedly attenuated these effects by inducing CORT. These results suggesting that antidepressant effect of 5-LOX inhibitor reduced neuroinflammation. In addition, increasing evidence suggested that a strong association in-between neuroinflammation and depression, which was marked by, increased levels of pro-inflammatory cytokines in the CNSs. The 5-LOX is broadly expressed in the CNS. It localizes mainly in neuronal cells [25,26]. In recent study had suggested in various regions of the brain, including the hippocampus. Where its levels was increased. We first identified the expression of hippocampal 5-LOX in depressive mice model by induced CORT, which was used to model inflammation with depressive disorders.
Therefore, we found that after the administration of zileuton produced an antidepressive effect in the behavioral tests, exhibited by inhibiting the CORT stress-induced increase in immobility time in the FST and TST and to the difference in locomotor activities by observed in OFT. In addition, depression often with anxiety in patients, so we estimated the anxiety-like behavior by the selection of NSFT. Zileuton treatment significantly opposite the anxiety-like behaviors induced by CORT, characterized by reducing the increment of latency to feed by CORT. Moreover, zileuton exhibited an antidepressant effect.
Treatment with zileuton is one of the 5-LOX inhibitors, successfully attenuated histopathological and biochemical changes by induced CORT microglial associated neuroinflammation in intraperitoneal route [24,27]. However, histological changes in microglia have considered being a pathophysiological role of depression [28]. 5-LOX effective inflammatory mediator, which plays a crucial role in the asthma patient and other inflammatory diseases [29,30].
Evidence focuses that apoptosis as an essential faction contributing depression pathogenesis [41] not only inhibits the inflammatory reactions but also blocks the apoptotic processes as well [42].
Here, this experiment increase of CORT caused increment of pro-apoptotic molecules such as caspase-3, Bax, TUNEL immunofluorescence assay, and approximately Bcl-2 reduction in the mouse hippocampus. However, the treatment of zileuton (50 mg/kg and 100 mg/kg) significantly gives such response in comparison to model group. Therefore, it concluded that 5-LOX could be a promising target for the treatment of depression.

CONCLUSION
The effect of 5-lipoxygenase was founded by clinical studies targeted against neuroinflammatory cascade with the synthesis pathway in the pathogenesis of depression.
However, the study was limited to usage of the research of depressive mice model, and must be needed to discover them for depression. It was research to explore by Utilization of CORT (corticosteroid) induced mouse model of depression. My research show neuroinflammation was strongly related to behavioral activities. In this research, activation of microglia was frequent. Increases the level of proinflammatory cytokines (such as TNF-α, IL-1β) and pro-apoptotic molecules (cleaved caspase-3 and TUNEL) observed while reduces in the antiapoptotic molecule (Bcl-2) reserved in the CORT (corticosterone in an intraperitoneal injection) induced mice, possibly through the NF-κB signaling pathway.
5-LOX inhibitor zileuton treatment, at high doses (100 mg/kg) is more than zileuton (50 mg/kg), reversed/reduced such changes and showed some outcome in the behavioral tests.
This experiment illustrates that 5-LOX enzyme as an emerging therapeutic target in depressive mouse model and motivates 5-LOX inhibitor such as Zileuton, basically used in anti-asthmatic drug, to be promoted for the treatment of depression, which can minimize the mental, clinical social and economic liability of depression.

AUTHORS' CONTRIBUTIONS
Both authors have contributed to reviewing the preparation and editing of the manuscript. c d e f