JSH-23 prevents depressive-like behaviors in mice subjected to chronic mild stress: Effects on inflammation and antioxidant defense in the hippocampus
Qi Wang, Xiaomei Dong, Nannan Li, Yan Wang, Xiaofeng Guan, Yiwei Lin, Jiguang Kang, Xia Zhang, Yuchen Zhang, Xiaobai Li, Tianchao Xu
PII: S0091-3057(18)30036-4
DOI: doi:10.1016/j.pbb.2018.04.005
Reference: PBB 72587
To appear in: Pharmacology, Biochemistry and Behavior
Received date: 26 January 2018
Revised date: 17 April 2018
Accepted date: 19 April 2018
Please cite this article as: Qi Wang, Xiaomei Dong, Nannan Li, Yan Wang, Xiaofeng Guan, Yiwei Lin, Jiguang Kang, Xia Zhang, Yuchen Zhang, Xiaobai Li, Tianchao Xu
, JSH-23 prevents depressive-like behaviors in mice subjected to chronic mild stress: Effects on inflammation and antioxidant defense in the hippocampus. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Pbb(2017), doi:10.1016/j.pbb.2018.04.005
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JSH-23 prevents depressive-like behaviors in mice subjected to chronic mild stress: effects on inflammation and antioxidant defense in the hippocampus
Qi Wanga, Xiaomei Donga, Nannan Lia, Yan Wanga, Xiaofeng Guana, Yiwei lina,
Jiguang Kanga, Xia Zhanga, Yuchen Zhanga, Xiaobai Lia,*, Tianchao Xub,*
a Department of Psychiatry, The First Hospital of China Medical University, Shenyang,
Liaoning Province, China
b Department of Medical Psychiatry, General Hospital of Shenyang Military Command, Shenyang, Liaoning Province, China
* Corresponding author: Xiaobai Li, MD, PhD, (155 Nanjing Street, Heping District, Shenyang 110001, PR China. E-mail address: [email protected]); Tianchao Xu, MD, (83 Wenhua Street, Shenhe District, Shenyang 110000, PR China. E-mail address: [email protected])
Abstract
Nuclear factor-kappa B (NF-κB), which is reported to play an important role in the pathogenesis of depression, also has a central role in the genesis and progression of inflammation. Here, we have targeted the nuclear translocation of NF-κB using 4-methyl- N1-(3-phenyl-propyl)-benzene-1,2-diamine (JSH-23) to elucidate its role in depression. We investigated the antidepressant-like effects of JSH-23 in the chronic mild stress (CMS) mouse model, which is a valid, reasonably reliable, and useful model of depression. The antidepressant-like effects of JSH-23 were evaluated using the sucrose preference test (SPT) and the forced swimming test (FST). We also assessed inflammatory markers [interleukin (IL)-6 and tumor necrosis factor-α (TNF-α)] and components of antioxidant defense [superoxide dismutase (SOD) and nuclear factor erythroid-2-related factor 2 (Nrf 2)] in the hippocampus. Fluoxetine, a classical antidepressant, was used in this study as a positive control. Administration of JSH-23 significantly prevented the decreased sucrose preference in the SPT and prevented the increased immobility time in the FST caused by CMS, but had no effect on locomotor activity. Expression of NF-κB p65 protein in the hippocampus was decreased, and elevated levels of IL-6 and TNF-α were reduced, after JSH-23 administration. In addition to its anti-inflammatory effect, JSH-23 treatment increased the expression of SOD and Nrf 2 in the hippocampus, suggesting that it strengthens antioxidant defense. The current study demonstrated that inhibiting the NF- κB signaling cascade using JSH-23 prevented depressive-like behaviors by decreasing inflammation and improving antioxidant defense in the hippocampus. We concluded that NF-κB activation plays an important role in the pathophysiology of depression and that targeting NF-κB signaling may provide a novel and effective therapy for depression. Additional preclinical studies and clinical trials are, however, needed to further elucidate the effects of this therapeutic strategy.
Key words: Depression; Inflammation; Antioxidant defense; Nuclear factor-kappa B
1. Introduction
Major depressive disorder (MDD) is a common, complex, and potentially life- threatening mental disorder, which imposes a severe social and economic burden worldwide. MDD will be the second largest global burden of disease by 2030 (Lopez and Mathers, 2006). Despite many years of extensive investigations and increasing numbers of studies, the pathogenesis of MDD remains unclear. Emerging evidence suggests an important role for inflammation and oxidative stress as main contributors to the neuroprogression that is observed in MDD, where patients show increased systemic inflammatory and oxidative stress biomarkers (Bakunina et al., 2016; Wolkowitz et al., 2011). Immunity and inflammation have been implicated in the pathogenesis of MDD and in mechanisms of antidepressant response (Mocking et al., 2017). Previous studies have demonstrated increased levels of systemic inflammation and activation of the immune system among individuals with MDD (Miller and Raison, 2016). Inflammatory and metabolic dysregulation has been found to be associated with poor response to antidepressants, which could result in worse MDD outcomes (Vogelzangs et al., 2014). Oxidative stress also plays important roles in the pathophysiology of MDD via the actions of free radicals and reactive oxygen and nitrogen species (Bakunina et al., 2016). The level of oxidative stress is a useful parameter for measuring and predicting MDD, as well as for determining the effectiveness of antidepressant therapy (Cicek et al., 2014).
Nuclear factor-kappa B (NF-κB) is a transcription factor that resides in the cytoplasm of every cell and translocates to the nucleus when activated. NF-κB comprises homo- and heterodimeric complexes of the Rel family proteins (p65, c-Rel, RelB, p50, and p52). NF-κB binds to DNA in the promoter region of target genes as a dimer that is usually composed of p50 and p65 (Serasanambati and Chilakapati, 2016). It acts, together with inhibitory kappa B-α (IκB-α) and is expressed in neurons and glial cells in the mature central nervous system (CNS); where p65/p50 is the most abundant dimer and IκB-α is the predominant inhibitor (Gutierrez and Davies, 2011).
NF-κB family members are key regulators of cell proliferation, apoptosis, differentiation, and oncogenesis (Serasanambati and Chilakapati, 2016). In recent years, many studies have shown that NF-κB plays physiological roles in the development and function of the nervous system (Crampton and O’Keeffe, 2013). The effects of NF-κB activity in the CNS are usually associated with control of synaptic plasticity, neurite outgrowth, and neuronal apoptosis (Meffert and Baltimore, 2005). Recent studies have shown, however, that activators of the NF-κB pathway can have opposing effects and can induce either “positive effects” (cell survival, apoptotic cell death, neurite outgrowth, neuronal differentiation and plasticity, and cell proliferation) or “negative effects” (cell death and toxicity) (Mincheva-Tasheva and Soler, 2013). NF-κB had been widely studied in a variety of human diseases, including asthma, atherosclerosis, AIDS, diabetes, Alzheimer ’s disease, and cancer (Zhang et al., 2017).
The NF-κB inflammatory cascade is an important target for diseases associated with acute and chronic inflammatory damage (Wullaert et al., 2011). The NF-κB family of transcription factors plays an important role in the immune system by regulating a variety of processes, ranging from the development and survival of lymphocytes and lymphoid organs to the control of immune responses (Vallabhapurapu and Karin, 2009). Nuclear translocation of p65/p50 and IκB phosphorylation are important steps in the NF-κB pathway that result in inflammation (Karin et al., 2004). Activation of NF-κB plays a central role and is associated with the release of proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, interferon-γ, and tumor necrosis factor-α (TNF-α), which can cause secondary neurotoxicity (Shih et al., 2015). Moreover, NF-κB is a redox-sensitive factor that is closely associated with oxidative stress, and suppression of NF-κB has been shown to improve antioxidant defense (Kumar et al., 2012). The pro-oxidant mechanism of NF-κB involves modulation of nuclear factor erythroid-2-related factor 2 (Nrf 2), which is a key regulator of redox signaling (Wakabayashi et al., 2010). NF-κB
family members are also key regulators of cell proliferation, apoptosis, differentiation, and
oncogenesis (Serasanambati and Chilakapati, 2016). In recent years, many studies have
shown that NF-κB plays physiological roles in the development and function of the
nervous system (Crampton and O’Keeffe, 2013). In the adult CNS, NF-κB is expressed in
both neurons and glial cells. The effects of NF-κB activity in the CNS are usually
associated with control of synaptic plasticity, neurite outgrowth, and neuronal apoptosis
(Meffert and Baltimore, 2005). Recent studies have shown, however, that activators of
the NF-κB pathway can have opposing effects and can induce either “positive effects”
(cell survival, apoptotic cell death, neurite outgrowth, neuronal differentiation and
plasticity, and cell proliferation) or “negative effects” (cell death and toxicity) (Mincheva-
Tasheva and Soler, 2013).
NF-κB had been widely studied in a variety of human diseases, including asthma,
atherosclerosis, AIDS, diabetes, Alzheimer ’s disease, and cancer (Zhang et al., 2017).
To the best of our knowledge, however, very few studies have focused on mood disorders. The hippocampus plays an important role in stress and emotion (Rebecca et al., 2012). In recent years, studies on the role of NF-κB in the hippocampus have shown that it regulates neurogenesis and neuritogenesis and neuritogenesis and was involved in learning and memory (Crampton and O’Keeffe, 2013). NF-κB has also been associated with MDD. In patients with MMD, psychosocial stressor challenge was shown to increase plasma NF-κB DNA binding in peripheral blood mononuclear cells (Pace et al., 2006). Activation of NF-κB represents a downstream effector of the neuroendocrine response to stressful psychosocial events and links changes in the activity of the neuroendocrine axis to the cellular response (Bierhaus et al., 2003). IL-1β/NF-κB signaling was found to be activated by acute or chronic stress (Koo et al., 2010). In the
hippocampus, NF-κB regulated neurogenesis and neuritogenesis and was involved in
learning and memory. As a ubiquitous transcriptional factor, NF-κB can regulate the
expression of many genes involved in numerous cell functions (Crampton and O’Keeffe, 2013; Meffert and Baltimore, 2005). A few of the targets of NF-κB, such as protein kinase
A (PKA), metabotropic glutamate receptor 2 (mGlu2), and brain-derived neurotrophic
factor (BDNF) (Barco et al., 2005; Cuccurazzu et al., 2013; Kaltschmidt et al., 2006), are
associated with synapses and their plasticity has been reported in the brain. We
speculate, therefore, that NF-κB plays an important role in the pathogenesis of MDD and that modulators of NF-κB signaling could be used to treat MDD.
The aromatic diamine 4-methyl-N1-(3-phenyl-propyl)-benzene-1,2-diamine (JSH-23) inhibits nuclear translocation of the NF-κB p65/p50 heterodimer, without affecting IκBα degradation (Shin et al., 2004). JSH-23 inhibits nuclear translocation and NF-κB transcriptional activity in lipopolysaccharide (LPS)-stimulated macrophage-like RAW
264.7 cells with an IC50 value of 7.1 μM (Ariassalvatierra et al., 2011; Shin et al., 2004).
In the present study, we investigated the antidepressant-like effects of JSH-23 in the chronic mild stress (CMS) mouse model, which is a valid, reasonably reliable, and useful model of depression (Willner, 1997). The antidepressant-like effects of JSH-23 were evaluated using the sucrose preference test (SPT) and the forced swimming test (FST). We also measured levels of inflammatory markers IL-6 and TNF-α) and antioxidant defense markers [superoxide dismutase (SOD) and Nrf 2 in the hippocampus (Liu et al., 2016; Wakabayashi et al., 2010). Fluoxetine, a classical antidepressant drug, was used as a positive control.
2. Materials and methods
2.1. Animals
Twenty-seven male C57BL/6J mice (6–8 weeks old) were obtained from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). The mice were housed individually under standard conditions (21 ± 1°C and 55 ± 2% humidity) on a 12 h light/dark cycle, with lights on at 7:00 AM. The mice had free access to food and water and were allowed to acclimatize to laboratory conditions for 1 week prior to the experiments. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals: (1) Use of appropriate species, quality, and number of
animals; (2) avoidance or minimization of discomfort, distress, and pain to animals; (3) provision of appropriate animal husbandry directed and performed by qualified persons; and (4) experimentation on living animals only by or under the close supervision of qualified and experienced persons.
2.2. Experimental design and drug treatment
The CMS procedure was performed as previously described (Willner et al., 1987; Zhang et al., 2015). Briefly, the mice were subjected to various stressors for six weeks, using a semi-random schedule (Fig. 1A). The stress regime for each week consisted of food or water deprivation (16 h); cage tilted at 45°(12 h); soiled cage (200 mL of water spilled onto bedding, 8 h); and stroboscopic lighting (400 flash/min in the dark, 8 h). The mice were subjected to one of these stressors on each day of the week, with the same stressor not applied for two consecutive days. The control mice were housed in a separate room and had no contact with the stressed animals.
After the third week of the CMS paradigm, drug or vehicle (0.9% saline) was administered intraperitoneally in a total volume of 10 mL/kg between 8:00 AM and 10:00 AM. The mice were randomly divided into four groups: 1) control + vehicle (CON, n = 7, mice received 0.9% NaCl); 2) CMS + vehicle (CMS, n = 7, mice received 0.9% NaCl); 3) CMS + fluoxetine [n = 7, mice received fluoxetine (10 mg/kg, Eli Lilly and Company, Indianapolis, IN, USA)] (Isingrini et al., 2012); 4) CMS + JSH-23 [n = 6, mice treated with JSH (3 mg/kg, Tokyo Chemical Industry Co., Ltd., Japan)] (Kumar et al., 2011).
2.3. Body weight
The body weight of the mice in each of the four groups was recorded every week to evaluate their nutritional status.
2.4. Behavioral procedures
2.4.1. SPT
The SPT was performed as previously described (Zhang et al., 2016), and started 24 h after the last drug administration. Briefly, 72 h before the test, the mice were habituated
to two drinking bottles containing 1% sucrose solution. Following deprivation of water and food for 12 h, the mice were then allowed free access to either of two bottles containing 1% sucrose solution or water for 12 h. The position of the bottles was switched every 4 h to prevent the effects of position preference on drinking behavior. Sucrose preference was calculated as: sucrose intake (g)/total liquid intake (g).
2.4.2. Locomotor activity test
Locomotor activity tests were conducted to exclude false positive results that could arise if fluoxetine or JSH-23 reduced the immobility time, which is used as an indicator of depressive-like behavior in the FST, by acting as psychostimulants. Locomotor activity was measured as described previously (Wang et al., 2017), using an open field test, which began 24 h after the SPT. The open field was 60 x 60 x 40 cm, constructed of plywood, painted white, and placed in a dimly lit room. The open field was divided into nine equal squares drawn on the floor of the arena. A camera located on the ceiling above the apparatus recorded the animals’ movements. The mice were gently placed in the center square and left to explore for 10 min. Before and after each test, the open field arena was cleaned with a 10% alcohol solution and allowed to dry. The distance the animals moved, and the number of squares crossed (with all four paws), were analyzed using a video-tracking program (EthoVision, version 7.0; Noldus Information Technology, Wageningen, The Netherlands).
2.4.3 FST
The FST was carried out as previously described (Zomkowski et al., 2006) and began 24 h after the locomotor activity test. The mice were individually placed in a glass cylindrical container (21 cm in height and 12 cm in diameter, total volume ~1,000 mL) that was filled with water (22 ± 1°C) to a depth of 12 cm. Each mouse was exposed to a test session for 6 min and judged to be immobile when it remained floating passively in the water without struggling. The duration of immobility was recorded during the last 4
min of the test. Latency to the first immobility was also recorded, starting immediately after placing the mouse in the water.
2.5. Brain sample collection
Twenty-four hours after the behavior test, the mice were sacrificed by neck dislocation and the brains were rapidly dissected on ice. Dissections were carried out according to The Rat Brain In Stereotaxic Coordinates (Paxinos and Watson, 2004). The hippocampus was harvested, washed, weighed, and then stored at −70°C.
2.6. Biochemical assays
2.6.1. Enzyme-linked immunosorbent assay (ELISA)
Hippocampal tissues were homogenized and centrifuged at 1,000 × g for 30 min. Protein levels of IL-6 and TNF-α in the supernatants were quantified using ELISA kits (Boster Biological Technology Co., Ltd., Wuhan, China), according to the manufacturer’s protocol. Absorbance was measured at 450 nm.
2.6.2. Western blotting
Hippocampal tissues were suspended in lysis buffer, supplemented with a complete protease and phosphatase inhibitor cocktail set (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, China), and centrifuged at 12,000 rpm for 30 min at 4°C. Following tissue homogenization, protein concentration was measured using a bicinchoninic acid test kit (Nanjing KeyGen Biotech. Co., Ltd.). Samples were resolved on 7.5%–10% Criterion SDS-PAGE (Nanjing KeyGen Biotech. Co., Ltd.), followed by transfer to 45 μm PVDF membranes. Transfer was performed at 4°C in buffer containing 25 mM TRIS, 192 mM glycine, and 20% methanol. The following primary antibodies were used: mouse anti-NF- κB p65 antibody (1:500, ImmunoWay Biotechnology Company, Plano, TX, USA, YM3111); rabbit anti-SOD antibody (1:500, ImmunoWay Biotechnology Company, YT5337); mouse anti-Nrf 2 antibody (1:500; ImmunoWay Biotechnology Company, YM0590); mouse anti-GAPDH antibody (1:25,000, Abcam, Cambridge, MA, USA, Ab181602). Secondary anti-goat antibody (Abbkine Scientific Co., Ltd., USA, A23920)
was used at a dilution of 1:8,000. Densitometric analysis was performed using VisionWorks LS software (UVP, USA). The ratio of individual proteins to GAPDH was then determined and these values were compared for statistical significance.
2.7. Statistical analysis
Statistical analysis was performed using SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA). Potential differences between the mean values were analyzed using one-way analysis of variance (ANOVA) followed by the LSD post hoc test. Values are presented as mean ± S.E.M and values of p < 0.05 were considered to be statistically significant. 3. Results 3.1. Body weight As shown in Fig. 1B, there was no significant difference in body weight between the CON group and the CMS group at any time point (p < 0.05). The body weight gain in the CMS group at 4 weeks was lower than in the CMS + JSH-23 group (p = 0.028). There were no differences between the CMS group and the CMS + JSH-23 group or the CMS group and the CMS + fluoxetine group at weeks 5 and 0.6 (p < 05). These results indicate that CMS does not affect weight gain in mice. JSH-23 increased the weight gain of mice subjected to CMS in the short term. Fig. 1. Illustration of experimental procedures (A) Changes in body weight gain of mice over 6 weeks. (B) Data are presented as mean (n = 6–7 mice per group). *p < 0.05, CMS + JSH-23 vs. CMS group. 3.2. Locomotor activity In the locomotor activity test, there was no significant difference between groups in total distance moved [F(3,23) = 1.727, p = 0.189] or number of crossings [F(3,23) = 1.517, p = 0.237] (Fig. 2), indicating that neither fluoxetine nor JSH-23 affected locomotor activity. Fig. 2. Effect of JSH-23 and fluoxetine on total distance moved (A) and number of crossings (B) in locomotor activity test. Data are presented as mean ± S.E.M. (n = 6–7 mice per group). 3.3. Depressive-like behavior in SPT and FST Sucrose preference was significantly decreased after 6 weeks of CMS [F(3,23) = 7.252, p = 0.001] (Fig. 3) and the effect of stress was prevented by fluoxetine (p = 0.002) or JSH-23 (p = 0.001). There was, however, no significant difference between groups in total liquid intake [F(3,23) = 0.895, p = 0.459]. In the FST test, stress significantly increased immobility time [F(3,23) = 3.552, p = 0.017] and decreased latency to immobility [F(3,23) = 2.821, p = 0.041], but these effects were prevented by fluoxetine (p = 0.009, p = 0.042, respectively) and JSH-23 (p = 0.020, p = 0.015, respectively). These findings demonstrated that fluoxetine and JSH-23 were able to reduce depressive-like behavior induced by CMS. Fig. 3. Effect of JSH-23 and fluoxetine on SPT (A and B) and FST (C and D). Data are presented as mean ± S.E.M. (n = 6–7 mice per group). *p < 0.05, **p < 0.01 vs. CON group. $p < 0.05, $$p < 0.01 vs. CMS group. 3.4. Expression of NF-κB p65 in the hippocampus Expression of NF-κB p65 was increased by CMS [F(3,20) = 3.537, p = 0.023] (Fig. 4). Expression of NF-κB p65 was decreased in both the CMS + fluoxetine group and the CMS + JSH-23 group compared with the CMS group (p = 0.048 and p = 0.008, respectively). Fig. 4. Effect of JSH-23 and fluoxetine on level of NF-κB p65 in hippocampus. Expression of GAPDH served as an internal reference control in western blot experiments. Data are expressed as % of control and presented as mean ± S.E.M. (n = 6 mice per group). *p < 0.05, vs. CON group. $p < 0.05, $$p < 0.01 vs. CMS group. 3.5. Levels of inflammation and antioxidant defense in hippocampus Expression levels of IL-6 [F(3,20) = 4.351, p = 0.034] and TNF-α [F(3,20) = 6.033, p = 0.001] were increased by CMS (Fig. 5 A,B) and these effects of stress was prevented by JSH-23 (p = 0.002 and p = 0.001, respectively) and fluoxetine (p = 0.014 and p = 0.012, respectively). Expression levels of SOD [F(3,20) = 11.181, p ＜ 0.001] and Nrf 2 [F(3,20) = 3.918, p = 0.007] were decreased by CMS (Fig. 5D, C) and these effects of stress were also prevented by JSH-23 (p ＜ 0.001 and p = 0.015, respectively) and fluoxetine (p = 0.001 and p = 0.027, respectively). Fig. 5. Effect of JSH-23 and fluoxetine on inflammation (A and B) and antioxidant defense (C and D) in the hippocampus. Expression of GAPDH served as an internal reference control in western blot experiments. Data are expressed as % of control and presented as mean ± S.E.M. (n = 6 mice per group). *p < 0.05, **p < 0.01 vs. CON group. $p < 0.05, $$p < 0.01 vs. CMS group. 4. Discussion The pathogenesis of MDD comprises complex interrelated metabolic and neurochemical processes (Mcintyre et al., 2009; Réus et al., 2016). The importance of inflammatory processes in different diseases, including MDD, has been the subject of increasing investigation. NF-κB is one of the primary transcription factors that amplifies the inflammatory response to damage (Vallabhapurapu and Karin, 2009) and, as a redox-sensitive factor, NF-κB is also closely associated with oxidative stress (Kumar et al., 2012). In this study, consistent with previous findings (Koo et al., 2010) we found that the depressive-like behaviors caused by exposure to CMS were mediated by NF-κB signaling. We also found that exposure to CMS increased levels of inflammation and oxidative damage in the hippocampus, which is an important structure involved in stress and emotion (Rebecca et al., 2012). Importantly, these effects could be reversed by treatment with the NF-κB inhibitor JSH-23, providing evidence that targeting NF-κB signaling may provide a novel and effective therapy for depression. In the present study, mice were subjected to a 6 week CMS program and were treated with drugs for the last 3 weeks. compared with the CON group, sucrose preference was significantly decreased in the CMS group, suggesting that CMS induced responses related to anhedonia, which is a core symptom of MDD. This result was in agreement with our previous report (Zhang et al., 2015). Immobility time was increased and latency to immobility was significantly decreased by CMS in the FST, which models despair, another core symptom of MDD (Petit-Demouliere et al., 2005). Consistent with previous studies (Gosselin et al., 2017; Grønli et al., 2005), we found no significant difference between the four groups in the locomotor activity test. The results supported the idea that the behavioral alterations observed in mice exposed to CMS were not caused by impairment of locomotor activity or by deficient performance in behavioral procedures caused by locomotor impairments. Previous studies have reported that CMS in mice resulted in reduced body weight or no change in body weight (Marco et al., 2017; Willner et al., 1996) and, in the present study, we found that CMS did not affect weight gain in mice. These discrepancies may result from different stress schedules, such as intensity and duration of stimuli, or different genetic backgrounds of the mouse strains used. The hippocampus is an important encephalic structure involved in chronic stress- induced depressive-like behaviors (Yue et al., 2017). Within the hippocampus, the p65/p50 heterodimer is the most abundantly expressed form of NF-κB and p65- dependent transcriptional activity known to regulate dendritic cells (Crampton and O'Keeffe, 2013). In the present study, we found that expression of NF-κB p65 in the hippocampus was increased after 6 weeks CMS. A limitation of this work is that the specific mechanism of stress-induced NF-κB activation was not investigated. Interestingly, “apparently contradictory” effects can be caused by NF-κB. For example, BDNF- mediated activation of NF-κB has been reported to promote neuronal survival, neurite outgrowth, and myelin formation (Caviedes et al., 2017; Gutierrez et al., 2005) but NF-κB activation has also been reported to induce “negative” effects, such as cell toxicity and death (Bagnati et al., 2016; Ju et al., 2014; Koo et al., 2010). An earlier study found that both acute and chronic stress can activate IL-1β/NF-κB signaling, resulting in impairment of hippocampal neurogenesis and thereby contributing to depressive-like behaviors (Koo et al., 2010). Further work needs to be carried out to investigate the specific mechanism of NF-κB activation caused by stress. NF-κB is a transcription factor that serves as a master switch for turning on certain immune and inflammatory responses (Serasanambati and Chilakapati, 2016). NF-κB activation is implicated in neuroinflammation. In the CNS, activation of NF-κB plays a central role and is associated with the release of proinflammatory cytokines, such as IL- 1β, IL-6, interferon-γ, and TNF-α, which can cause secondary neurotoxicity (Shih et al., 2015). Proinflammatory cytokine receptors are highly aggregated in brain regions associated with emotion, such as the hippocampus and prefrontal lobe (Parnet et al., 2002). Proinflammatory cytokines exert harmful effects on adult neurogenesis in the hippocampus (Zaben et al., 2017), with IL-6 and TNF-α being the most widely investigated cytokines. Consistent with the results of an earlier study (Manikowska, 2014), we found that levels of IL-6 and TNF-α were significantly increased by CMS. NF-κB- mediated production of IL-6 has been shown to cause depression-like behaviors and to decrease cell proliferation in the hippocampus (Monje et al., 2011). Inhibition of the NF- κB/IL-6/STAT3 pathway has also been implicated in depressive-like behaviors caused by LPS (Liao et al., 2017). Activated NF-κB induces expression of TNF-α, which activates signal transduction pathways leading to activation of NF-κB, forming a link between inflammation and depressive-like behaviors (Elnahas et al., 2016). The bidirectional relationship between NF-κB and proinflammatory cytokines provides a positive regulatory loop to amplify the inflammatory responses (Bremner and Heinrich, 2005). As well as its inflammatory effect, NF-κB has been reported to hamper defense against oxidative stress. The pro-oxidant mechanism of NF-κB involves modulation of transcription factor Nrf 2, which is a key regulator of redox signaling (Wakabayashi et al., 2010). NF-κB inhibits Nrf 2 by competing for the transcription co-activator CREB binding protein-p300 complex (Liu et al., 2008). In the present study, CMS increased NF-κB activation, which led to decreased expression of Nrf 2 in the hippocampus. SOD is the first detoxification enzyme and the most powerful antioxidant in the cell. SOD is a key endogenous antioxidant enzyme and is a component of first line defense system against reactive oxygen species (Alscher et al., 2002). The expression of SOD was decreased in the CMS group, indicating that antioxidant defense ability in the hippocampus was reduced by stress. CMS has been shown to increase oxidative damage and decrease the activity of antioxidant enzymes (Che et al., 2015). CMS has also been shown to increase production of oxidants and lead to an imbalance between SOD and catalase activities, which contributes to depressive-like behaviors (Lucca et al., 2009). Furthermore, in the hippocampus, activation of NF-κB is associated with neurogenesis and neuronal growth (Crampton and O'Keeffe, 2013). Fluoxetine, a classical antidepressant, was used in this study as a positive control. Administration of fluoxetine has been shown to significantly suppress LPS-induced phosphorylation of NF-κB p65 in BV-2 cells (Yang et al., 2014). Consistent with our results, previous studies found that fluoxetine could reverse the depressive-like behavior caused by CMS and could reduce levels of inflammation and oxidative stress in the hippocampus (Moretti et al., 2012; Sakr et al., 2015).The present study was designed to investigate the antidepressant effects of JSH-23. Members of the NF-κB family are normally kept inactive in the cytoplasm by binding to a member of the IκB family of inhibitory proteins (Crampton and O'Keeffe, 2013). JSH-23 is a cell-permeable diamino compound that selectively blocks nuclear translocation of NF-κB p65 and its transcription activity, without affecting IκB-α degradation (Shin et al., 2004). After 3 weeks administration, sucrose preference in the SPT was significantly increased and immobility time in the FST was decreased, indicating that JSH-23 exerted an anti-depressant effect. We also found that JSH-23 had no effect on locomotor activity. Chronic unpredictable stress has been shown to activate NF-κB signaling and decrease proliferation of neural stem-like cells in the subgranular zone of the dentate gyrus of the hippocampus, an effect that can be reversed by infusion of JSH-23 (i.c.v., minipump) (Koo et al., 2010). An earlier study also found that NF-κB activity is enhanced in hippocampal tissue of mice subjected to constant darkness. Blocking the NF-κB pathway using pyrrolidine dithiocarbamate (PDTC), a selective inhibitor of IκBα phosphorylation, led to reduced depressive-like behavior and decreased inflammation in the hippocampus (Monje et al., 2011). Chronic unpredictable mild stress was shown to lead to phosphorylation of the NF-κB subunit p65 which can be blocked by NLRP3 gene knockout (Su et al., 2017). NLRP3 is a multi-protein complex of the innate immune system, and serves as an upstream regulator of the IL-1β signaling pathway (Cassel et al., 2009; Song et al., 2017). Beta-hydroxybutyrate, an endogenic NLRP3 inflammasome inhibitor, suppresses LPS induced inflammation by inhibiting NF-kB activation (Fu et al., 2016), and can improve the depressive and anxiety behaviors in depressed rats (Yamanashi et al., 2017). Inhibition of NF-κB transcriptional activity by blocking nuclear translocation reduced levels of IL-6 and TNF-α in JSH-23-treated mice, as seen in our results. An in vitro study showed that JSH-23 suppressed LPS-induced expression of proinflammatory cytokines (IL-6, IL-1β, TNF-α) and expression of inflammation related enzymes (iNOS and COX-2). JSH-23 has also been shown to inhibit LPS-induced apoptosis (Shin et al., 2004). NF-κB compromises the antioxidant machinery by affecting Nrf 2 activity. Both of these transcription factors interact at several point to control the expression of different target proteins, such as heme oxygenase 1 (HO-1), human NAD(P)H:quinone oxidoreductase (NQO 1) and SOD (Innamorato et al., 2008; Liu et al., 2008). In the present study, we found that expression of both SOD and Nrf 2 was significantly increased after 6 weeks CMS. Interestingly, upregulation of the transcription factor Nrf 2, the guardian of redox homeostasis that enhances HO-1 activity, modulated the inflammatory response in the brain (Innamorato et al., 2008). 5. Conclusions Activation of NF-κB plays an important role in the pathophysiology of depression. The present study demonstrated that inhibition of the NF-κB signaling cascade using JSH-23 prevented depressive-like behaviors by decreasing inflammation and improving antioxidant defense in the hippocampus (Fig. 6). Our findings provide evidence that targeting the NF-κB signaling pathway may offer a novel and effective therapy for depression. Additional preclinical studies and clinical trials are needed to better elucidate the effects of this therapeutic strategy. Fig. 6. Schematic showing the different steps involved in the phosphorylation, activation, and translocation of NF-κB. Nuclear translation is the most important step that leads to direct interaction between active NF-κB subunits and DNA, generating proinflammatory mediators. NF-κB in the nucleus also causes a decline in Nrf 2 activity because NF-κB reduces binding of Nrf 2 to its binding domain on the antioxidant response element. 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Highlights • Chronic mild stress caused depressive-like behaviors in mice. • Chronic mild stress increased inflammation and induced antioxidant defense in the hippocampus. • Inhibiting the NF-κB signaling cascade using JSH-23 prevented depressive-like behaviors by decreasing inflammation and improving antioxidant defense in the hippocampus.