Antidepressant-like effect of zileuton is accompanied by hippocampal neuroinflammation reduction and CREB/BDNF upregulation in lipopolysaccharide-challenged mice
Abstract
Background: Recent studies demonstrated beneficial effects of zileuton, a 5-lipoxygenase (5LO) inhibitor, on some brain diseases in animal models, but the roleof zileuton in the depression remains unknown.Methods: We investigated effects of zileuton on depressive behaviors using tail suspension test (TST), forced swimming test (FST) and novelty-suppressed feedingtest (NSFT) in mice injected with lipopolysaccharide (LPS). The 5LO level, activation of microglia, NF-κB p65, TNF-α, IL-1β, brain-derived neurotrophic factor (BDNF), and c-AMP response element-binding protein (CREB) were determined in the mouse hippocampus.Results: We firstly found that the expression of hippocampal 5LO was graduallyincreased over LPS exposure and was reversed by fluoxetine administration. Zileuton significantly suppressed LPS-induced depressive behaviors, evidenced by the decreases in immobility time in TST and FST, as well as the latency to feed in NSFT. This treatment pronouncedly alleviated LPS-induced neuroinflammatory response, characterized by decreased 5LO, suppressed activation of microglia, decreased NF-κB p65, TNF-α and IL-1β, and significantly increased the ratio of p-CREB/CREB or mBDNF/proBDNF in the hippocampus of the LPS-challenged mice. Conclusions: Zileuton abrogates LPS-induced depressive-like behaviors andneuroinflammation, and enhances CREB/BDNF signaling in the hippocampus, suggesting that zileuton could have potential therapeutic value for depression.
1Introduction
Major depression is a devastating psychiatric disorder with increased risk of suicide, high individual suffering, and an enormous economic burden for the society (Greenberg et al., 2003; Tanti and Belzung, 2010). It will be the third most important disease resulting in disability by 2030 (Organization, 2008). Current antidepressants were developed based on the “monoaminergic hypothesis”, which considers a synaptic deficiency in 5-hydroxytryptamine (5-HT, serotonin) or noradrenaline as the main cause of the disease (Kai et al., 2015). Unfortunately, observable therapeutic benefits of available antidepressants usually take 6-8 weeks to emerge(Uher et al., 2011) and only 30-45% of patients ultimately achieve remittance for current anti-depressive treatment (Uribe et al., 1999). Furthermore, antidepressants are also inundated by side effects and drug–drug interactions (Nestler et al., 1989), which makes patients suffer from new disturbances. Therefore, novel alternative therapeutic approaches are urgently needed to be developed for depressionZileuton is a therapeutic drug approved for treating asthma since 1997 in the USA (Pathway et al., 1999). It is a 5-lipoxygenase inhibitor that is mainly used for the prophylaxis and treatment of chronic asthma for those patients who are age 12 and older (Carter et al., 1991). It has also shown to treat patients with chronic obstructive pulmonary disease, upper airway inflammatory conditions, and dermatological conditions such as acne, pruritic in Sjogren-Larsson Syndrome, and atopic dermatitis (Cingi et al., 2015). Intriguingly, the focus on zileuton has currently been intensified as its novel pharmacological effects, especially, in the central nervous system (CNS).Recent studies demonstrated that oral treatment of zileuton significantly reduced cerebral damage, and the expression of 5LO and neuroinflammatory response in animal models of cerebral ischemia (Shi et al., 2013; Silva et al., 2015; Tu et al., 2009; Tu et al., 2016). In mouse experimental spinal cord injury, zileuton treatment significantly reduced the spinal cord inflammation and tissue injury, neutrophil infiltration, and improved the recovery of limb function. Zileuton also observably ameliorated behavioral deficits, reduced brain Aβ deposition, tau phosphorylation and improves synaptic integrity in the animal models of Alzheimer’s disease (AD) (Chu et al., 2013; Di et al., 2014). In this study, we firstly observed whether zileuton improves lipopolysaccharide (LPS)-induced depressive-like behaviors, hippocampal neuroinflammatory responses and CREB/BDNF pathway in mice.
2Materials and methods
LPS was from Sigma-Aldrich (05:B5, St. Louis, MO). Antibodies were purchased from several companies: anti-5LO was obtained from Abcam (Cambridge, USA); anti-IL-1β, anti-TNF-α and anti-BDNF were obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany); anti-NF-κB p65, anti-CREB and anti-pCREB were from Cell Signaling Technology, Inc. (Massachusetts ,USA); anti-β-actin, anti-Histone H3 and anti-Iba1 were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Secondary antibodies were from Bioworld Technology Co. Ltd. (Minnesota, USA). The nucleoprotein extraction kit was from Sangon Biotech Co., Ltd. (Shanghai, China) and streptavidin-biotin complex (SABC) immunohistochemistry kit was fromBoster Biotechnology Co., Ltd. (Wuhan, China). Fluoxetine (FLX) was obtained from Jiangsu Hengrui medicine Co., Ltd. Zileuton (ZIL) was purchased from Meilun Biotechnology Co., Ltd. (Dalian, China). All other chemicals were of analytical grade and commercially available.Male Institute of Cancer Research (ICR) mice (Yangzhou University Medical Center, China), weighing about 18-22 g (6-8 weeks), were used in our experiments because it could reduce the confounding variables associated with the estrous cycle of females (Uz et al., 2008a). Mice were housed in an air-conditioned room with controlledtemperature (22±2℃), humidity (55±5%), 12 h light/dark cycles and ad libitum foodand water. All experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The procedures were approved by the Animal Care and Use Committee of China Pharmaceutical University.In the first experiment, mice were treated with FLX (10 mg/kg, i.g.) once a day for 7 consecutive days, then they were injected with LPS (0.5 mg/kg, i.p.) 30 minutes after last FLX administration, the expression of 5LO in mouse hippocampus was detected 24 h after LPS exposure.
In the second experiment, the mice were divided into five groups as follows: Veh+Veh (0.9% saline and solvent); LPS+Veh (LPS 0.5 mg/kg and solvent); LPS+ZIL25 (LPS 0.5 mg/kg and zileuton 25 mg/kg); LPS+ZIL50 (LPS 0.5 mg/kg and zileuton 50 mg/kg); LPS+ZIL100 (LPS 0.5 mg/kg and zileuton 100 mg/kg). Mice except Veh+Veh group were injected with LPS (0.5 mg/kg, i.p.), 1 hourlater they were treated with ZIL once daily for 2 days. ZIL for intragastric administration (i.g.) was dissolved in absolute alcohol and diluted with normal saline before intragastric injection. 0.5% alcohol (V/V) had no significant effect on animals by itself. LPS for intraperitoneal injection (i.p.) was dissolved in PBS.The OFT is used to confirm that antidepressant effect is not a result from the stimulation of general motor activity. The apparatus of OFT was made up of an acrylic plate (50×50×40 cm) that surrounded by a 40-cm-high opaque plastic wall, which was divided into 144 equal squares. Each mouse was placed onto a corner square of the arena with facing the corner, and was allowed to freely explore the open field for 6 min per trial, during which the number of squares crossed with paws (crossing) was counted as locomotor activities. After each mouse was tested, the odors on acrylic plate were cleaned with 70 % ethanol.The apparatus consisted of a plastic box (50×50×20 cm) and a fluorescent light (450 lx) was placed at the center of the arena. The floor was covered with approximately 2 cm of wooden bedding and the animals were deprived of food for 24 h before the test. A single pellet of food (regular chow) on a white paper platform was placed at the center of the box. Mice were placed respectively in a corner of the maze while a stopwatch was immediately started. The assessment of interest (chewing) was scored when the mouse was sitting on its haunches and biting food with the use of forepaws.The mice were moved to their home cage immediately after the test. The amount of food consumed within 6 min (home cage food consumption) was measured.The TST was carried out as described previously (Cryan et al., 2005).
Briefly, mice were tested during the dark period of the circadian cycle. They were allowed to adapt to the new environment for at least 1 h before testing. Each mouse was suspended by taping the tail with a hook 50cm above the bottom in a soundproof box. The total immobility time during the 6-min test was computed by ANY-MAZE software. The immobility time during the first 2 min was discounted while the last 4-min task was statistically analyzed.The FST was conducted to assess the despair behavior of the mice. The apparatus was made up of cylinder (diameter 15cm, height 25cm) containing 15cm of watermaintained at 25±1℃. The animals were not able to touch the bottom. Each mousewas subjected individually to an inescapable cylinder for 6 min during the test session. Immobility time was defined as each mouse remained floating with all limbs motionless, except that which was necessary to keep the mice head above water. Water was replaced between each trial. The total immobility time was calculated by ANY-MAZE software. The immobility time during the initial 2 min of the 6- min task was discounted while the last 4-min task was statistically analyzed.Mice were transcardially perfused with normal saline and then killed by cervicaldislocation. The hippocampus were immediately collected and homogenized in 0.5 ml of RIPA buffer (50 mM Tris–HCl (pH 7.4), 150 nM NaCl, 1 mM PMSF, 1 mM EDTA, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS) which contains 0.1% PMSF. After the homogenization was centrifuged at 12,000 rpm for 15 min at 4°C, the dissolved proteins were collected from the supernatant. The protein concentrations were determined using Coomassie blue-based assay reagent. Protein extracts were separated by a SDS polyacrylamide gel electrophoresis, followed by transferring onto a PVDF membrane. 5 % skim milk in Tris buffer saline was used to block the membranes, which then were incubated at 4 °C overnight with respective primary antibodies diluted in 5 % skim milk for anti-5LO antibody (1:1,000), anti-TNF-α (1:500), anti-IL-1β (1:1,000), anti-BDNF (1:1000), or β-actin (inner control, 1:3,000).
After washed by Tris buffered saline-tween 20 (TBST), the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000) for 2 h at room temperature. Enhanced chemiluminescence detection reagents and a gel imaging system (Tanon Science & Technology Co, Ltd., China) were used to visualize the immunoreactive bands. Nuclear extracts were carried out by nucleoprotein extraction kit (Sangon Biotech, China). Mouse hippocampus were collected and homogenized in ice-cold hypotonic buffer, which contains 0.5% phosphatase inhibitor, 1 % phenylmethylsulphonyl fluoride, and 0.1 % DL-dithiothreitol, and then the homogenization was centrifuged at 4 °C, 5,000 rpm for 5 min. Finally, 0.2 ml lysis buffer containing 0.5 % phosphatase inhibitor, 1 % phenylmethylsulphonyl fluoride and 0.1 % DL-dithiothreitol were addedinto the precipitate, chilled for 20 min, and centrifuged at 4 °C, 15,000×g for 10 min. The supernatant nuclear protein extract was subjected to Western blot for assays of NF-κB p65 (1:1,000), p-CREB （Ser133）(1:1,000), CREB (1:1,000) and histone H3(1:1,000) was used as a loading control. For analyzing hippocampal microglia activation, mice anesthetized and transcardially perfused with PBS, followed by 4 % formaldehyde fixation. The brains were dissected, post-fixed in 4 % paraformaldehyde for 24 h at 4 °C and then cryoprotected in a 30 % sucrose solution for an additional 24 h.
The brains were embedded into optimal cutting temperature compound (Tissue-Tek, Torrance, CA) and cryosectioned (30 μm). Sections were washed with PBS (3 × 5 min), and then blocked with 0.3 % Triton X-100 for 4 h at 60 °C and then treated with 3 % H2O2 at room temperature for 30 min. After washed with PBS (3 × 5 min), sections were blocked with 5 % BSA for 30 min and incubated in anti-Iba1 (1:1000) primary antibody diluted in 5 % BSA overnight at 4 °C. Sections were washed with PBS (3 × 5 min) and further incubated with biotinylated mouse anti-rabbit IgG (40 min, 37 °C) and then washed again (3 × 5 min). Sections were incubated with strept avidin-biotin complex (20 min, 37 °C) and washed with PBS (4 × 5 min). Diaminobenzidine (DAB) was applied as a final chromogen for protein detection. After gradient dehydration (70 % ethanol, 5 min; 95 % ethanol, 5 min; 100 % ethanol 5 min; xylene 5 min), imaging (200× and 400×) was performed using a Nikon DS-Fi2 camera connected to a Nikon Eclipse Timicroscope. Microglia activation was quantified by Image-Pro Plus software. The number ofmicroglia in the hippocampus was measured, followed by the microglial Iba1-positive area to generate the ratio of microglial staining to hippocampus area (% area occupied). The mean values from 5 sections analyzed in each animal were used for statistical analysis.All data shown are expressed as mean± standard error of the mean (SEM). The data normality was assessed by Kolmogorov-Smirnov test by SPSS. All the data comparisons among multiple groups were made with one-way ANOVA followed by Scheffe’s post hoc test. p<0.05 was considered statistically significant. 3Results Since 5LO is a pro-inflammatory enzyme, we investigated whether the exposure to LPS (0.5 mg/kg, i.p.), an inflammation inducer, can change the expression of 5LO in the mouse hippocampus. As shown in Fig.1, the expression of 5LO was significantly increased in LPS-induced mouse hippocampus (F(3,12)=15.959, P<0.01). Fascinatingly, FLX pre-treatment significantly prevented upregulation of the hippocampal 5LO expression induced by LPS (P<0.01) in the LPS-challenged mic, and administration of FLX alone did not affect the expression of 5LO in mouse hippocampus (P>0.05). These results suggest that 5LO might participate in LPS-induced depressive-like behaviors.To evaluate whether 5LO inhibitor zileuton alleviates LPS-induced depression-like behaviors, we performed different behavior tests. As expected, mice exposed to LPS displayed an increment of immobility time in the TST (Fig 2A, F(4,35)=5.782, P<0.05). Treatment with zileuton (50 mg/kg or 100 mg/kg) significantly reduced the immobility time in the TST (Fig 2A, P<0.05) compared to the LPS-treated control group. Similar findings were acquired in the FST. In comparison with the Veh+Veh group, mice treated with LPS spent dramatically longer immobility time (Fig 2B, F(4,35)=6.294, P<0.01), which was diminished by zileuton treatment (25 mg/kg, 50 mg/kg or 100 mg/kg) in the FST. (Fig 2B, LPS+ZIL25: P<0.05, LPS+ZIL50 or LPS+ZIL100: P<0.01). In addition, the anxiety-related behaviors were evaluated using the NSFT. The results showed that LPS caused a significant increment in the latency to feed in the novel environment (Fig 2C, F(4,35)=6.108, P<0.01) and zileuton treatment (50 mg/kg or 100 mg/kg) pronouncedly decreased the latency to feed in the mice injected with LPS (Fig 2C, P<0.01). No significant differences were observed in home cage consumption index (Fig 2D, F(4,35)=0.349, P>0.05) and the locomotor activities in the OFT (Fig 2E, F(4,35)=1.065, P>0.05) among groups.
Zileuton administration alone has no effects on normal mice (Supplement Figure). Taken together, 5LO inhibitor zileuton can significantly alleviate LPS-induced depressive-like behaviors. To determine that antidepressant-like effect of zileuton is involved in brain 5LO, weexamined 5LO level in the hippocampus, a key brain region for depression, in the LPS-challenged mice, using Western blot. The results showed that zileuton treatment (50 mg/kg or 100 mg/kg) obviously reduced 5LO expression (Fig 3A and 3B, F(4,15)=13.082, P<0.01) in this brain region, which indicates that antidepressant-like effect of zileuton is accompanied by hippocampal 5LO downregulation in the LPS-induced animal model.It has been reported that LPS mediates neuroinflammatory response via the microglial activation, which undergo an alteration characterized by cell numbers increment and conformational changes, including size enlargement and thicker processes (Graeber and Streit, 2010).To investigate the effect of zileuton on microglia, we assessed microglial activation by immunohistochemical detection of microglia marker, Iba-1. Compared with the Veh+ Veh group, LPS exposure significantly increased the number of Iba1-positive cells in mouse hippocampus (Fig 3C and 3D, F(4,25)=5.190, P<0.01),which was significantly decreased by zileuton treatment (P<0.01). These data indicated that zileuton suppressed LPS-induced microglial activation in the mouse hippocampus.Evidence has shown that 5LO mediates inflammatory response by regulating NF-κB pathway (Kazmi et al., 1995), which could be activated by LPS-triggered the nuclear translocation of p65 subunit (Dallot et al., 2005). Here, we are curious about whether zileuton inhibits LPS-activated NF-κB pathway. Consistent with previous studies,LPS activated NF-κB pathway, characterized by a p65 subunit increment in the nuclear (Fig.4A and 4B,F(4,15)=16.834, P<0.01), whereas zileuton treatment significantly decreased the levels of nuclear NF-κB p65 in the hippocampus of mice injected with LPS (P<0.01).Activated 5LO, microglia or NF-κB pathway could regulate various neurotoxin mediators and pro-inflammatory cytokines involved in the pathogenesis of depression (Drevets et al., 2008). To explore the effect of zileuton on the neuroinflammatory process induced by LPS, pro-inflammatory cytokines such as TNF-α and IL-1β were assayed by Western blot analysis. As shown in Fig 4C-E, LPS exposure significantly increased the generations of TNF-α and IL-1β in the hippocampus (Fig.4D, F(4,15)=19.674, P<0.01; Fig.4E, F(4,15)=17.579, P<0.01), whereas zileuton treatmen obviously decreased the levels of TNF-α and IL-1β (P<0.01). These results indicated that zileuton attenuated LPS-induced neuroinflammatory response.Zileuton increases the CREB activation and the ratio of mBDNF/proBDNF CREB, a nuclear transcription factor, is a key player in multiple intracellular signaling pathways. On neuronal stimulation, CREB is normally phosphorylated at Ser133 and activated, which is involved in the pathology of depression(Guo et al., 2014; Liu et al., 2016). As shown in Fig 5, the levels of p-CREB/CREB in the hippocampus were obviously decreased in the group of LPS+Veh, and significantly increased in both groups of LPS+ZIL50 and LPS+ZIL100 (Fig. 5 A and 5B, F(4,15)=12.496, LPS+ZIL50 or LPS+ZIL100: P<0.01).The best acknowledged transcriptional target of CREB is BDNF, which is known toemerge as an important synaptic modulator of synaptogenesis and synaptic plasticity (Tong et al., 2012; Yu et al., 2013). BDNF affects neurons bilaterally by the transition of processor BDNF (proBDNF), the neurotoxic form of BDNF, to mature BDNF (mBDNF), which enhances the synaptic plasticity and neuronal survival (Harte-Hargrove et al., 2013). The present results showed that the ratio of mBDNF/proBDNF in the hippocampus was significantly decreased in the group of LPS+Veh (Fig. 5C and 5D, F(4,15)=8.210, P<0.01), whereas markedly increased in both groups of LPS+ZIL50 and LPS+ZIL100 (LPS+ZIL50: P<0.05, LPS+ZIL100:P<0.01). These data suggested that anti-depression of zileuton was associated with the CREB/BDNF signaling pathway. 4Discussion The present study demonstrated that oral administration of zileuton exerted antidepressant effects in the LPS-induced depression model mice, and the antidepressant effect of zileuton was accompanied by attenuation of hippocampalinflammatory response indicated by reductions of IL-1β，TNF-α, and NF-κB p65, as well as decreased microglia activation and 5LO expression. This treatment also increased levels of p-CREB/CREB and mBDNF/proBDNF in the hippocampus ofLPS-challenged mice. Although previous studies reported the antidepressant effects of 5-lipoxygenase activating protein (FLAP) inhibitor MK-886 and 5-lipoxygenase deficiency on the forced swimming behavior of mice (Uz et al., 2008b), to our knowledge, the present study is the first to suggest that zileuton exert antidepressant effects on LPS-challenged mice, which is involved in reduction of neuroinflammatoryresponse and enhancement of CREB/BDNF pathway in the hippocampus.Growing evidence suggests that neuroinflammation plays a crucial role in the pathophysiology of depression (Dantzer et al., 2008; Hashimoto, 2015; Raison et al., 2010). Various bacterial and viral infections are associated with a range of depressive symptoms (Yirmiya et al., 1999). Many of these infectious pathogens have a special affinity for the brain, where they induce microglial activation (Rock et al., 2004). These pathogens also induce the secretion of proinflammatory cytokines (Vollmerconna et al., 2004), whose plasma levels are correlated with depressive symptomatology (Dowlati et al., 2010; Haapakoski et al., 2015). Consistently, experimental administration in humans of immune challenges that are known to activate microglia [e.g., LPS(endotoxin)] induces depressive symptoms, whose severity is highly correlated with elevated blood levels of inflammatory cytokines (Grigoleit et al., 2011; Harrison et al., 2009; Reichenberg et al., 2001). LPS, an endotoxin, is the major component of the outer membrane of Gram-negative bacteria. In this study, a single LPS challenge, mimicking an acute inflammatory event, activated strong peripheral immune response, and subsequently signaled the brain to evoke central inflammation, resulting in microglial activation together with depression-like behavior. Zileuton, an orally available selective and specific 5LO inhibitor, not only produced antidepressant effects, but also inhibited activation ofmicroglia and elevation of proinflammatory cytokines such as L-1β，TNF-α and NF-κB p65 in the LPS-challenged mice.5LO is a proinflammatory enzyme that catalyzes the conversion of arachidonic acid to5-hydroxyperoxy-eicosatetraenoic acid (5-HPETE) and subsequently to hydroxyl-eicotetraenoic acid 5-HETE), which can be then metabolized in different leukotrienes (Rådmark et al., 2007). Leukotrienes initiate immune cell chemotaxis and are critical molecular players in the inflammatory pathophysiology (Kanaoka and Boyce, 2014). 5LO is found in vasculature, endothelial cells, as well as the CNS, in both neuron and glia (Chu and Praticò, 2009). Some studies showed that 5LO was involved in cerebral ischemia, AD, traumatic brain injury, anxiety, and other the CNS disorders (Di et al., 2014; Giannopoulos et al., 2013; Ikonomovic et al., 2008; Joshi and Praticò, 2011; Shi et al., 2013). Interestingly, 5LO expression was significantly up-regulated in the hippocampus, being more susceptible to depression, in LPS-challenged mice, and zileuton, similar to antidepressant fluoxetine, pronouncedly suppressed this upregulation. This result supported previous report that 5LO inhibition and deficiency was associated with antidepressant activity(Uz et al., 2008a). It is well known that neurotrophins serve as important regulators in the pathophysiology of depression, and the most extensively studied neurotrophin is BDNF, which is upregulated in the hippocampus by antidepressant treatment and is sufficient to produce antidepressant behavioral responses (Son et al., 2012; Taliaz et al., 2010; Wang et al., 2008).BDNF activates several intracellular pathways, the most common of which is the mitogen-activated protein kinases and/or extracellular-regulated kinase cascade(Peng et al., 2008), which leads to an increase of CREB. Moreover, CREB is able to modify BDNF transcription and the increase in CREB/BDNF pathway could be the molecular basis for the improvement ofneurogenesis, synaptic plasticity, memory and mood (Li et al., 2009; Mariga et al., 2017). The recent studies demonstrated that LPS-induced pro-inflammatory cytokine mediated the impairment of BDNF function (Custódio et al., 2017). Our findings revealed the improving effects of zileuton on the downregulation of the CREB/BDNF pathway in depressive-like mice challenged with LPS. Therefore, our data suggests for the first time that the neurotrophic CREB/BDNF pathway may participate in antidepressant-like effects of zileuton on this model of depression.Taken together, our findings confirm the beneficial effects of zileuton on LPS-induced depression-like behaviors. Although further research is necessary to fully clarify the underlying mechanisms, our data implicates for the first time that regulation of hippocampal neuroinflammatory response and CREB/BDNF pathway may contribute to its therapeutic effects. In conclusion, our findings may provide a new insight into the antidepressant-like effect of zileuton, indicating a novel therapeutic value of it for major depression.