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ICARIIN EXERTS AN ANTIDEPRESSANT EFFECT IN AN UNPREDICTABLE CHRONIC MILD STRESS MODEL OF DEPRESSION IN RATS AND IS ASSOCIATED WITH THE REGULATION OF HIPPOCAMPAL NEUROINFLAMMATION

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B. LIU, a  C. XU, a,b  X. WU, a F. LIU, a Y. DU, a J. SUN, a J. TAO c* AND J. DONG a*

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a Department of Integrative Medicine, Huashan Hospital, Fudan University, 12 Middle Urumqi Road, Shanghai 200040, China

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b Department of Respiration, Affiliated Hospital, School of Medicine, Hangzhou Normal University, 16 Wen Zhou Road, Hangzhou 311121, China

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c

Department of General Dentistry, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, 639 Zhi Zao Ju Road, Shanghai 200011, China

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Abstract—Icariin (ICA), a flavonoid extracted from the traditional Chinese herb Herba Epimedii that can freely cross the blood–brain barrier, inhibits neuroinflammation and attenuates oxidative stress damage. Our previous studies demonstrated that icariin exerts an antidepressant-like activity in a social defeat mouse model. However, it is unknown whether icariin is beneficial for the treatment of depression via its modulation of oxidative stress and neuroinflammation. The objective of this study was to investigate the effects of icariin on the depression-like behaviors in an unpredictable chronic mild stress (CMS) model of depression in rats. Rats exposed to CMS showed behavioral deficits in physical state, the sucrose preference test (SPT) and the forced swimming test (FST) and exhibited a significant increase in oxidative–nitrosative stress markers, inflammatory mediators, including tumor necrosis factor-alpha (TNF-a) and interleukin-1b (IL-1b), activation of the nuclear factor kappa B (NF-jB) signaling pathway and increased inducible nitric oxide synthase (iNOS) mRNA expression in the hippocampus, which was reversed by chronic treatment with icariin (20 or 40 mg/kg). Interestingly, icariin negatively regulated the activation of the nod-like receptor protein 3

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(NLRP3) inflammasome/caspase-1/IL-1b axis in the hippocampus of CMS rats. These results confirm that icariin exerts antidepressant-like effects, which may be mediated, at least in part, by enhanced antioxidant status and anti-inflammatory effects on the brain tissue via the inhibition of NF-jB signaling activation and the NLRP3 -inflammasome/caspase-1/IL-1b axis. Our findings provide new information to understand the antidepressant action of icariin, which is targeted to the NLRP3-inflammasom in brain. Ó 2015 Published by Elsevier Ltd. on behalf of IBRO.

Key words: icariin, unpredictable chronic mild stress, depression, NLRP3 inflammasome, hippocampus. 18

*Corresponding authors. Tel: +86-21-52888301 (J. Dong), +86-2123271699 (J. Tao). E-mail addresses: [email protected] (J. Tao), [email protected] (J. Dong).   These two authors contributed equally to this work. Abbreviations: ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; BW, body weight; CAT, catalase; CMS, unpredictable chronic mild stress; ELISA, enzyme-linked immunosorbent assay; FST, forced swim test; HPA, hypothalamic–pituitary–adrenal; ICA, icariin; IL-1b, interleukin-1b; IL-6, interleukin-6; iONS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NF-jB, nuclear factor kappa B; NLRP3, nod-like receptor protein 3; PSS, physical state score of rats coats; ROS, reactive oxygen species; SOD, superoxide dismutase; SPT, sucrose preference test; TBARS, thiobarbituric acid reacting substances; TNF-a, tumor necrosis factor-alpha. http://dx.doi.org/10.1016/j.neuroscience.2015.02.053 0306-4522/Ó 2015 Published by Elsevier Ltd. on behalf of IBRO. 1

INTRODUCTION

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Depression is a multi-causal and life-threatening psychiatric disorder with a very high prevalence in the general population. It is projected that this illness will be the second leading cause of disease burden for developed countries by 2020, according to the World Health Organization (WHO) (Menken et al., 2000). Depression is characterized by altered mood and cognitive functions and recurrent thoughts of death or suicide with a lifetime incidence of 15–25% (Paykel, 2006). It affects not only the directly afflicted but also surrounding people, including their families and friends (Stern, 2012). Although there is extensive research on existing antidepressants that target the serotonin and/or norepinephrine systems, it takes weeks to months to produce a therapeutic response, and only one-third of drug-responsive patients achieve full remission of their depressive symptoms and gain functional recovery (Skolnick, 2002). The in-depth understanding of the mechanisms underlying this disease is required to develop novel treatments for depression. Oxidative stress, defined as a disturbance in the balance between the production of reactive oxygen species (ROS) and antioxidant defense systems, is a critical factor implicated in the pathogenesis of major depression (Maes et al., 2011a; Pandya et al., 2013; Zhang and Yao, 2013). In particular, a number of oxidative disturbances in depressed patients have been reported, including enhanced oxidative damage and decreased antioxidant enzyme levels (Ng et al., 2008;

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O’Donnell et al., 2014). There is increasing evidence that repeated and unpredictable stress results in lipid and protein damage, as well as decreased antioxidant enzyme activities and glutathione levels in the brain tissue of rats (Madrigal et al., 2002; Sahin and Gumuslu, 2004), whereas chronic treatment with antidepressants reduces the levels of oxidative stress markers and increases several endogenous antioxidants (Maes et al., 2011a,b). Moreover, other studies in depressed populations have shown that antioxidants (radical scavengers), such as N-acetyl-cysteine (NAC), may have antidepressant properties because they normalize the antioxidant concentrations (Maes et al., 2011a; Pandya et al., 2013). In addition, a large body of evidence suggests that many inflammatory mediators, such as tumor necrosis factor-alpha (TNF-a) and interleukin-1 beta (IL-1b), are involved in depression both in animal models (Lopresti et al., 2012) and in patients with depression (Rawdin et al., 2013) and that administration of interleukin-6 (IL-6), IL-1b, TNF-a or lipopolysaccharide (LPS) can result in depression-like and anxiety-like behaviors in animal models (Bluthe et al., 2000; Dantzer et al., 2008; Sukoff et al., 2012). A similar manifestation of depressive symptoms is also observed in patients undergoing immunotherapy for hepatitis C or cancer with IL-2 and INF-c, which provide strong evidence of a causal relationship between inflammation and depression (Dutcher et al., 2000; Capuron et al., 2001). Moreover, antidepressant drugs, such as fluoxetine and paroxetine, reduce the inflammatory cytokines in the brain and blood (Hwang et al., 2008). Therefore, disturbances in the balance between oxidative stress and the antioxidant defense system and inflammation is proposed as a crucial biological and cellular pathogenesis mechanism of depression (Xu et al., 2014). Anti-oxidative and anti-inflammatory compounds are therefore potential agents for the development of novel antidepressants. There is increasing interest in the antidepressant effect of herbs because treatment of depression with conventional antidepressants results in complete remission in only 50% of individuals (Nestler et al., 2002). Icariin (ICA), a flavonoid extracted from the Chinese herb Berberidaceae epimedium L. has a wide range of pharmacological actions, including anti-inflammatory and antioxidant activity in the central nervous system (Luo et al., 2007; Guo et al., 2010). Moreover, previous studies by Pan et al. have demonstrated the antidepressant-like effects of icariin (Pan et al., 2006, 2007, 2010, 2013). Pharmacokinetically, icariin is widely distributed in the body, including the brain, indicating that icariin pass through the blood–brain barrier (Li and Wang, 2008). Our previous studies demonstrated that icariin exerts an antidepressant-like activity in a social defeat mouse model (Wu et al., 2011) and also has a protective effect against corticosterone-induced apoptosis in primary cultured rat hippocampal neurons (Liu et al., 2011). We have also shown that icariin significantly inhibits peripheral and lung inflammation in LPS-challenged mice and in the supernatant of LPS-treated RAW 264.7 macrophages (Xu et al., 2010). Recently, we demonstrated that icariin

alleviates airway inflammation and anxiety behaviors in a murine model of combined social disruption stress and ovalbumin challenge by modifying the function of the hypothalamic–pituitary–adrenal (HPA)-axis (Li et al., 2014). However, the therapeutic effect of icariin on the unpredictable chronic mild stress (CMS) model of depression, and whether the antidepressant-like action of icariin is involved in the inhibition of oxidative stress and inflammation, remain unknown and require evaluated to determine its utility for the treatment or prevention of depression. Therefore, because inflammation and oxidative stress are involved in the pathophysiology of mood disorders, the present study was performed to identify the molecular mechanism of icariin as an antidepressant, which may be related to disturbances between oxidative stress and the antioxidant defense system and inflammation in the central nervous system. An unpredictable CMS model of depression in rats was used, which will help determine whether icariin is a useful novel therapeutic for depression.

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EXPERIMENTAL PROCEDURES

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Animals and housing

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Male Sprague–Dawley (SD) rats with initial weights of 120– 140 g (6 weeks old) were purchased from Shanghai SLAC Co. (Shanghai, China). All rats were housed singly at an average room temperature of 22 ± 1 °C and humidity of 50–60% with a 12-h light/dark cycle (lights on from 6:00 am to 6:00 pm) and given access to food and water ad libitum. The animals were acclimatized for at least 7 days before use in the experiments. All experiments were conducted in accordance with guidelines of Animal Care and Use Committee at Huashan Hospital of Fudan University. Every effort was made to minimize the number of animals used and their suffering.

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Drugs and reagents

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The icariin was purchased from the Shanghai Ronghe Medical Science Co., Ltd. (Shanghai, China). The purity of icariin was 98.93%, as verified by high-performance liquid chromatography (HPLC). The icariin was dissolved in DMSO and diluted in saline. The final concentration of DMSO used was less than 1% (v/v). Fluoxetine was provided by Eli Lilly and Company (Suzhou, China) and diluted with saline solution to the final concentration of 10 mg/ml. Rat IL-1b and TNF-a enzyme-linked immunosorbent assay (ELISA) kits were obtained from eBioscience (San Diego, CA, USA).

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CMS procedure and drug treatments

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The CMS-exposed and control groups were housed in separate rooms under similar conditions. For our CMS procedure, we used various stressors, of which the sequence was intentionally designed to maximize unpredictability. All CMS rats were exposed to one-two stressor each day for 35 days. Briefly, CMS consisted of exposure to a variety of the following unpredictable

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stressors: food deprivation (24 h), overnight water deprivation (18 h) followed by 1 h of empty water bottle replacement exposure, cage tilt (45°) for 18 h, overnight illumination (60-W lamp) for 13 h, soiled cage (200 ml water in 100-g sawdust bedding) for 21 h, forced swimming at 12 °C for 5 min, physical restraint (placing the animal in a plastic tube and adjusting it with plaster tape on the outside to restrain the animal) for 2 h and pair-housing for 24 h. These stressors were randomly scheduled over a 1-week period and repeated throughout the 5-week experiment. The details of the CMS procedure, including variety, time, and length of activity are shown in Table 1. Different groups of animals were administered with vehicle (saline 1 ml/100 g), icariin (20 and 40 mg/kg), and fluoxetine (10 mg/kg), respectively. Icariin and fluoxetine were administered by oral gavage (p.o.) once daily for the 35 days of the CMS procedure. The behavioral testing was performed at least 16–18 h after the last dose to avoid the acute effects of drug treatment. The experimental design is displayed in Fig. 1.

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Sucrose preference test (SPT)

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The CMS protocol decreases the rodent preference for sweet solutions (Willner, 2005), which is hypothesized to represent anhedonia, a core symptom of major depression (Willner, 1997). This anhedonic behavior is commonly assessed in rats via the SPT and can be reversed by chronic antidepressant treatment (Bhatt et al., 2014). At the beginning of the SPT, all rats were habituated to consume 1% sucrose solution by providing the sucrose solution as the only drinking fluid for 48 h. The rats were deprived of food and water for 23 h prior to the test. Two graduated burets were placed on the cage filled with either 1% sucrose solution or tap water. The rats were allowed to drink freely for 1 h, and the volume of sucrose and tap water consumed was recorded. The preference for sucrose was calculated as (ml sucrose/total ml consumed) * 100. SPT was administered once per week to all groups.

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Physical state score (PSS)

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This procedure was performed as described previously (Alonso et al., 2004). The physical state of coat was evaluated weekly until the end of the CMS procedure. The coat state is recorded using a scale from 1 to 3 as follows: the score is 3 if in a good state (the fur is smooth and shiny, with no tousled), 1 if in a bad state (piloerection and/or dirty fur on most of the body), 2 if intermediate between the 0 and 3. Each measure was scored by an experimenter blinded to the treatment group.

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Forced swim test (FST)

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The procedure used was based on that initially described by Slattery and Cryan (2012). Two swim sessions were conducted: an initial 15-min pretest, followed 24 h later by a 5-min test. Briefly, on the first day (pretest), rats were forced to swim individually in a clear Plexiglas cylinders (50-cm height, 18-cm diameter) that were filled with water (22–24 °C, 30 cm depth) for 15 min. On the second day

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(test), the rats were placed in the same condition for 5 min. Following each swim session, the rats were towel dried and returned to their home cages. The test sessions were recorded videotaped and later scored by an observer who was blinded to the groups of animals. The cumulative time spent in immobile posturing (minimal effort to keep head above water) during the 5-min test was recorded.

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Brain homogenate preparation

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All rats were decapitated immediately after the end of the FST procedure between 11:00 am and 1:00 pm to avoid fluctuations in the hormone levels. The animals were sacrificed by an overdose of sodium pentobarbital (50 mg/kg body weight, i.p.). The entire hippocampus of each brain was quickly collected on dry ice and stored at 70 °C until assayed. The tissue samples were then homogenized with ice-cold 0.1 M phosphate buffer (pH 7.4) 10 times (w/v). The homogenate was centrifuged at 2500g (4 °C) for 15 min to remove the cellular debris, and aliquots of the supernatant were separated and used for biochemical estimations. Biochemical estimations of the brain homogenate were performed after completion of all behavioral assessments.

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Estimation of MDA assay level

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The MDA content in the rat hippocampus was determined using the thiobarbituric acid method with an assay kit from Nanjing Jiancheng Bioengineering Institute according to the manufacturer’s instructions. The MDA content was assayed in the form of thiobarbituric acid reacting substances (TBARS). A total of 100-ll supernatant was mixed with 1 ml 20% trichloroacetic acid and 1.0 ml 0.1% TBARS reagent and incubated at 95 °C for 80 min. After cooling on ice, the mixture was centrifuged at 1000g for 20 min. The absorbance of the supernatant was measured at 532 nm using a microplate reader (Biotek, USA). The amount of TBARS was determined using tetraethoxypropane as a standard. The content of TBARS, as an index of MDA, is expressed as the nanomole per milligram protein.

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Estimation of nitrite level

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The levels of the nitric oxide (NO) derivative nitrite were determined in the rat hippocampus with the Griess reagent. A nitrite detection kit (Beyotime Biotech Inc., Jiangsu, China) was used according to the instructions provided by the manufacturer. The samples were assayed in triplicate, and a standard curve using NaNO2 was generated for each experiment for quantification. Briefly, 50-ll supernatant or standard NaNO2 was mixed with 100-ll Griess reagent in a 96-well plate. Then, the optical density (OD) was read with a microplate reader (Biotek, USA) at 540 nm. The concentration of nitrite in the brain tissue is calculated using a NaNO2 standard curve and expressed as micromole per milligram of protein.

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Table 1. Time and length of stressors used in the CMS procedure Stressors

Sunday

Monday

Food deprivation Water deprivation Empty water bottle Cage tilt (45°) Overnight illumination Soiled cage Forced swimming Physical restraint Pair-housing

09:00

09:00

18:00

16:00 07:00

Tuesday

Wednesday

Thursday

09:00 10:00 10:00–11:00

09:00

16:00 09:00

12:00

Assay of superoxide dismutase (SOD) activity

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The SOD activity was measured using a kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s protocol. In a phosphate-buffered reaction mixture, xanthine and xanthine oxidase were incubated at 37 °C for 20 min in the presence of samples, blanks, SOD standards, and WST-1. During the reaction, SAR is generated from xanthine and oxygen. SOD catalyzes the dismutation of SAR, thereby reducing the amount available to oxidize WST-1 to WST-1 formazan. The sample results were compared against an SOD standard reference curve after the absorbance was read at 450 nm with a microplate reader. A unit of SOD was defined as the amount of enzyme causing 50% inhibition of the NBT reduction rate. The SOD activity is expressed as the units of nitrite per milligram protein.

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Estimation of catalase (CAT) activity

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The activity of CAT was measured using a CAT detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. CAT activity was measured using the ammonium molybdate spectrophotometric method, which is based on the observation that ammonium molybdate rapidly terminates the H2O2 degradation reaction catalyzed by CAT and reacts with the residual H2O2 to generate a yellow complex, which is monitored as the absorbance at 405 nm. The CAT activity is expressed as units of nitrite per milligram protein.

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16:00

10:00

16:00 07:00

09:00

10:00–12:00

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Saturday

5 min/rat

Fig. 1. Schematic representation of the CMS experimental procedure and behavioral test. BW, body weight; SP, sucrose preference test; PSS, physical state score of rat coats; FST, forced swim test.

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18:00 09:00

Friday

10:00–12:00 10:00

10:00

Measurement of cytokine release by ELISA

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To detect the levels of pro-inflammatory factors in the hippocampus, brain homogenates were obtained from the hippocampi, and separated by centrifugation at 14,000g for 5 min at 4 °C to remove cellular debris. The supernatant was stored at 80 °C until use. The concentrations of IL-1b and TNF-a in the stored supernatant were measured using a sandwich ELISA kit (Duo-Set; R&D Systems, Minneapolis, MN, USA).

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Real time transcriptase-polymerase chain reaction

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Total RNA was extracted from the rat hippocampus using Trizol reagent (Invitrogen Life Technologies, USA) followed by treatment with RNase-free DNaseI (Invitrogen Life Technologies, USA). Reverse transc ription was performed with the One-Step RNA-PCR Kit (Takara), according to the manufacturer’s protocol. Quantitative real-time PCR was performed on a 7300 Real-Time PCR System using the SYBR Green PCR Master Mix. After the addition of primers (Table 2 and template DNA to the master, the PCR thermal cycle parameters were as follows: 95 °C for 10 min, 40 cycles of 60 °C for 60 s, and 95 °C for 15 s, and a melting curve from 60 to 95 °C to ensure amplification of a single product. The relative expression of genes of interest was calculated and expressed as 2DDCT, in which 2DDCT = [(CT target gene  CT endogenous control) test sample  (CT target gene  CT endogenous control) control sample].

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Western blot analysis

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Protein extracts were obtained from the right lobes of the hippocampus, and the protein concentrations were quantified by BCA (Beyotime Biotech Inc., Jiangsu, China). A total of 40–80-lg protein samples were dissolved with an equal volume of loading buffer (0.1 M Tris–HCl buffer (pH 6.8) containing 0.2 M DTT, 4% SDS, 20% glycerol and 0.1% bromophenol blue), separated on 10% SDS–PAGE and then electrotran sferred at 90 V to a PVDF membrane. The membranes were blocked with TBST containing 5% non-fat dried milk for 1 h at room temperature followed by incubation with primary antibodies at 4 °C overnight. The primary antibodies used were rabbit anti-NF-jB p65 (1:1000, Beyotime Biotech Inc., Jiangsu, China), mouse anti-iNOS, rabbit anti-NLRP3, rabbit anti-ASC, rabbit

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Table 2. Primer sequences used in real-time-PCR analysis

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Gene

GenBank accession no.

Sequence (50 –30 )

TNF-a

NM_012675.3

IL-1b

NM_031512.2

CD11b

NM_012711.1

iNOS

NM_012611.3

b-Actin

NM_031144.2

CCCAATCTGTGTCCTTCTA (forward) CACTACTTCAGCGTCTCGT (reverse) AAGATGGAAAAGCGGTTTG (forward) GGGAAGGCATTAGGAATAG (reverse) AGGAGTGTGTTTGCGTGTC (forward) CTTGGTGTTCTTGCGGACT (reverse) CTCAGGCTTGGGTCTTGTT (forward) TGTTGTTGGGCTGGGAATA (reverse) CCTCTATGCCAACACAGT (forward) AGCCACCAATCCACACAG (reverse)

anti-IL-1b, rabbit anti-caspase-1 p10 (1:400, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-b-actin, rabbit anti-lamin B1 (1:1000, Bioworld Technology Co., Ltd, Nanjing, China). The blots were washed extensively with TBST and incubated with secondary antibodies in TBST/5% non-fat dried milk for 1 h at room temperature. After washing, the signal was detected using an enhanced chemiluminescence method (ECL kit, Millipore, USA). The membranes were imaged and analyzed using the Quantity One Image Analysis Software (Syngene, U.K.).

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Data analysis

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The data are presented as mean ± SEM and analyzed with the SPSS 20.0 software. The significance of the difference between the controls and samples treated with various drugs was determined by a one-way ANOVA followed by the least significant difference (LSD) for post hoc comparisons. A p < 0.05 was considered statistically significant.

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RESULTS

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Effects of icariin on depression-related behaviors in CMS model rats

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To determine whether icariin has a potential antidepressant effect, we used CMS model rats, which have depression-like behaviors. As shown in Fig. 2, at the beginning of the experiment, there was no significant difference among the groups for body weight (BW) [F(4,45) = 0.888, p > 0.05)], SPT [F(4,45) = 0.470, p > 0.05], and PSS [F(4,45) = 0.173, p > 0.05]. CMS animals exhibited a slowdown in the rate of BW increase at the 3rd week of the CMS procedure compared to non-stressed animal [F(4,45) = 31.785, p < 0.01)], which was ameliorated by treatment with icariin (p < 0.05) or fluoxetine (p < 0.01) at the 4th week, and this effect was continued to the end of the experimental procedure (Fig. 2A). Moreover, as shown in Fig. 2B, we found that rats exposed weeks to CMS for 3 weeks display a significantly reduced preference for sucrose solution [F(4,45) = 2.339, p < 0.05)] compared to non-stressed rats. Icariin (20 mg/kg) or fluoxetine treatment reversed the anhedonia induced by CMS (p < 0.05), and the effect was sustained for the following weeks. After

5 weeks of treatment, icariin (20 and 40 mg/kg) and fluoxetine significantly restored the sucrose consumption compared to the CMS animals (p < 0.01). As shown in Fig. 2C, after CMS for 1 week, the stressed rats showed a significant decline of their physical state as indicated by their coats, due to stress [F(4,45) = 3.839, p < 0.05)] compared to the nonstressed control group, and the effect of stress lasted until the end of the 5-week CMS [F(4,45) = 8.680, p < 0.001)]. By contrast, the decline of physical state of the animal was significantly improved by icariin (20 and 40 mg/kg) and fluoxetine after 5 weeks of treatment [icariin (20 mg/kg) and fluoxetine, p < 0.05; icariin (40 mg/kg), p < 0.01]. In the forced swimming test, CMS rats spent more time immobile over the 5-min test period compared to the non-stressed control group [F(4,25) = 7.547, p < 0.001, Fig. 2D]. Icariin (40 mg/kg) and fluoxetine decreased the CMS-induced increase of immobile time in the FST (p < 0.05, p < 0.01, respectively). These results indicate that chronic mild stress successfully induced depression-like behavior and that icariin has an anti-depressive effect in a CMS rat model of depression.

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Effects of icariin on oxidative–nitrosative stress markers in hippocampus

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As shown in Fig. 3, the amount of SOD in the CMS rats was significantly decreased compared to the control group in hippocampal tissues [F(4,25) = 3.024, p < 0.05]. Treatment with 40 mg/kg icariin significantly increased the SOD contents in the hippocampus compared to the CMS rats (p < 0.05). In the CMS group, there was a significant increase in the CAT levels in the hippocampal tissue [F(4,25) = 4.272, p < 0.01] compared to the control group. Icariin (40 mg/kg) increased the CMS-induced decrease of the CAT level. CMS also caused significant oxidative–nitrosative damage as evidenced by an increased in lipid peroxidation (MDA) (p < 0.01) and nitrite concentration (p < 0.01) in the hippocampus compared to the nonstressed group. Icariin (40 mg/kg) treatment significantly attenuated the oxidative–nitrosative stress markers (reduced MDA, nitrite levels) in the hippocampus (p < 0.05) compared to the CMS group. However, a lower dose of icariin (20 mg/kg) caused a significant

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Fig. 2. Icariin treatment ameliorates depression-like behaviors in CMS model rats. (A) The body weight was measured during the CMS procedure. (B) The sucrose preference was tested during the CMS procedure. (C) The coat score was decreased in CMS model rats. (D) The immobility time was measured in the FST (n = 6). The results are the mean ± SEM (n = 10 per group in BW, SPT and PSS). ⁄p < 0.05, ⁄⁄p < 0.01 vs. Control; # p < 0.05, ##p < 0.01 vs. CMS. CMS, chronic mild stress; Flx, fluoxetine.

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change only in the CAT activity in the hippocampus (p < 0.05). These results indicate that icariin possesses potential antioxidant effects in a CMS model of depression.

expression [F(4,10) = 4.164, p < 0.05, Fig. 4E] in the hippocampus, and the treatment of the rats with icariin (40 mg/kg) inhibited the mRNA expression of CD11b (p < 0.05). These results indicate that icariin modulates the microglial activation and central expression of inflammatory mediators in the hippocampus of CMS rats.

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Effects of icariin on NF-jB activation and inducible nitric oxide synthase (iNOS) mRNA and protein levels in the hippocampus

468

NF-jB activation and its nuclear translocation following CMS were determined by monitoring NF-jB-p65 localization in the cytosolic and nuclear fractions of the hippocampus in controls and drug-treated groups. In the nuclear fraction, CMS caused a significant increase in the NF-jB-p65 levels in the hippocampus compared to the non-stressed group [F(4,10) = 16.191, p < 0.001, Fig. 5A]. Moreover, icariin (40 mg/kg) treatment inhibited the nuclear translocation of p65 (p < 0.05), which is involved in the inflammatory response, in the hippocampus induced by CMS. To determine whether NF-jB activation resulted in the activation of downstream targets, iNOS mRNA and protein level were measured respectively by Real-Time PCR and Western blot. CMS induced a significant increase in the hippocampal iNOS mRNA and protein levels [F(4,10) = 2.900, p < 0.05,

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Effects of icariin on neuroinflammation in the hippocampus As shown in Fig. 4, CMS caused a significant increase in the IL-1b and TNF-a concentrations [F(4,25) = 2.505, p < 0.01, F(4,25) = 3.972, p < 0.01, respectively, Fig. 4A, B] in the hippocampus of rats compared to the non-stressed group. Similarly, CMS treatment also markedly increased the mRNA levels of TNF-a and IL-1b [F(4,10) = 3.440, p < 0.01, F(4,10) = 2.603, p < 0.05, respectively, Fig. 4C, D] in the hippocampus. Treatment of the rats with icariin (40 mg/kg) markedly attenuated the concentrations and mRNA expression of TNF-a and IL-1b (p < 0.05) in the hippocampus of rats compared to the CMS-treated control group. However, a lower dose of icariin (20 mg/kg) did not cause any significant differences except for TNF-a mRNA expression (p < 0.05) compared to the CMS-treated control group. The effect of icariin on microglial activation was also evaluated by examining the gene expression of the microglial cell surface marker, CD11b. The results revealed that CMS induced CD11b mRNA

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Fig. 3. Icariin treatment attenuates the oxidative–nitrosative stress damage in the hippocampus of CMS model rats. Oxidant stress was assessed by measuring the activities of SOD (A) and CAT (B), and the levels of MDA (C) and NO (D) in the hippocampus. The results are the mean ± SEM (n = 6 per group). ⁄p < 0.05, ⁄⁄p < 0.01 vs. Control; #p < 0.05, ##p < 0.01 vs. CMS. CMS, chronic mild stress; Flx, fluoxetine.

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F(4,10) = 4.008, p < 0.01, respectively, Fig. 5B, C] relative to the control group. Treatment of the animals with fluoxetine and icariin (40 mg/kg) significantly inhibited of the mRNA expression of iNOS compared to the CMS-treated control group (p < 0.05). However, icariin (20 and 40 mg/kg) fail to inhibit CMS-induced iNOS protein upregulation. These results indicate that icariin has a potential anti-inflammatory effect in a CMS model of depression. Effects of icariin on nod-like receptor protein 3 (NLRP3) inflammasome in the hippocampus of CMS rats IL-1b is a critical mediator of depression-like behavior caused by acute and chronic stress. The NLRP3 inflammasome is responsible for the maturation of IL-1b. As discussed above, the hippocampal IL-1b protein concentration and mRNA expression level in CMS rats were significantly increased compared to control rats, which indicates that the NLRP3 inflammasome may be activated in the brain. Furthermore, CMS significantly

increased the protein expression of IL-1b by Western blot analysis (Fig. 6A). As predicted, all three components of the NLRP3 inflammasome showed significantly higher protein expression levels in the CMStreated control group compared to the control group, including NLRP3 (Fig. 6B), apoptosis-associated specklike protein containing a caspase recruitment domain (ASC) (Fig. 6C), and caspase-1 (Fig. 6D). Furthermore, icariin (40 mg/kg) and fluoxetine significantly inhibited the upregulation of NLRP3, IL-1b and ASC and caspase-1 activation in the hippocampus of CMS rats. These data suggest that the NLPR3 inflammasome has key roles in CMS-induced depression-like behaviors and that icariin modulates NLPR3 inflammasome activity.

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In the present study, we demonstrated that icariin treatment ameliorated depression-like behaviors and attenuated oxidative stress damage and neuroinflammation of the hippocampus in CMS-induced depression rat model. Moreover, icariin inhibited the NF-jB signaling pathway

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Fig. 4. Icariin treatment partially inhibits CMS-induced neuroinflammation in the hippocampus of rats. Icariin inhibits the production of TNF-a (A) and IL-1b (B) in the hippocampus of CMS model rats, determined by ELISA. Icariin also decreases the relative mRNA level of IL-1b (C) and TNF-a (D) in the hippocampus of stressed rats, evaluated by real-time PCR. (E) Icariin alleviates the activation of microglia (CD11b) in the hippocampus induced by CMS. The results are the mean ± SEM (n = 6 per group). ⁄p < 0.05, ⁄⁄p < 0.01 vs. Control; #p < 0.05, ##p < 0.01 vs. CMS. CMS, chronic mild stress; Flx, fluoxetine.

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and NLRP3-inflammasome activation, which may contribute to the suppression of hippocampal neuroinfl ammation in the CMS-induced depression model. These findings further confirmed the antidepressant-like effect of icariin, which provides insight into the use of icariin for depression treatment. In addition, the 5-week CMS procedure induced hippocampal NLRP3 inflammasome activation in rats, which provides additional evidence for

the regulatory mechanism for IL-1b-related neuroinflam mation in this animal model of depression. The CMS procedure used in this study is a validated animal model of human depression (Hill et al., 2012), which is hypothesized to reproduce many of the complex symptoms of human depression, such as increased physiological changes (Guilloux et al., 2011), decreased sucrose preference and loss of motivation (Strekalova

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Fig. 5. Icariin treatment inhibits the CMS-induced increase of NF-jB p65 nuclear translocation and iNOS gene expression in the hippocampus of rats. (A) Representative immunoblots and relative level for NF-jB p65 and lamin B1 in the hippocampus of rats. (B) Representative immunoblots and relative level for iNOS and b-actin in the hippocampus of rats. (C) Relative mRNA level of iNOS in the hippocampus of rats, evaluated by realtime PCR. The results are the mean ± SEM (n = 3 per group). ⁄p < 0.05, ⁄⁄p < 0.01 vs. Control; #p < 0.05, ##p < 0.01 vs. CMS. CMS, chronic mild stress; Flx, fluoxetine.

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et al., 2004). Therefore, this model is often used to analyze the cellular and molecular mechanisms underlying the pathophysiology of depression and for identifying the mechanism of antidepressants. Here, we confirm that rats exposed to CMS for 5 weeks exhibited many depressive behavioral phenotypes, such as slower BW gain, reduced preference for sucrose, marked degradation of the physical state of the animal’s coat and increased immobility time in the FST, which are consistent with a previous report (Liu et al., 2009). The PSS of the coat is easy to score, rapidly observed and reproducible and is a good index of the response of the animal to CMS (Alonso et al., 2004). Icariin, a biologically active component

purified from the Chinese herbal plant Epimedium, has become one of the most widely used medicinal plant products in China. Icariin has a wide range of pharmacological actions, including anti-inflammatory (Li et al., 2014) and antioxidant actions (Luo et al., 2007) and antidepressant-like activity (Pan et al., 2007, 2010). Our previous experiments also showed that icariin exerts an antidepressant-like activity in a social defeat mouse model (Wu et al., 2011). In this study, we provide further evidence for the efficacy of icariin in a CMS-induced depression model in rats, as evidenced by increased BW gain, improved physical state of the animal’s coat, increased preference for sucrose and mobility time in the FST.

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Fig. 6. Icariin treatment inhibits the CMS-induced NLRP3 inflammasome in the hippocampus of rats. (A) Icariin and Flx inhibit the protein level of IL-1b in the hippocampus of stressed rats. (B) Icariin and Flx treatment decrease the relative NLRP3 protein level in the hippocampus compared to the CMS group. (C) Icariin and Flx inhibit the protein level of ASC in the hippocampus of CMS model rats. (D)The increase of caspase-1 induced by CMS in the hippocampus is inhibited by icariin and Flx treatment. b-Actin is used as a loading control, and the protein expression is the relative density units normalized to b-actin. The results are the mean ± SEM (n = 3 per group). ⁄p < 0.05, ⁄⁄p < 0.01 vs. Control; #p < 0.05, ##p < 0.01 vs. CMS. CMS, chronic mild stress; Flx, fluoxetine.

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Oxidative stress results in the production of ROS and reduces the antioxidants level in a particular cell (Rinwa and Kumar, 2013), and plays an important role in major depression and may constitute a common pathogenic mechanism for several psychiatric disorders (Maes

et al., 2011a). The major antioxidant enzymes, including SOD and CAT, are the first line of the antioxidant defense system against ROS in vivo during oxidative damage. MDA is another well-known indicator of oxidative damage under conditions of oxidative stress. The

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role of oxidative stress in the pathophysiology of major depression leads to use of antioxidants as therapeutics for depression (Scapagnini et al., 2012). Clinical reports of patients with depression showed elevated plasma and/or urine oxidative stress markers and reduced antioxidant enzymes levels, which returned to normal after chronic treatment with antidepressants (Xu et al., 2014). Recent evidence also indicates that a number of antioxidants targeting oxidative/nitrosative stress may have clinical efficacy in the treatment of patients with depression (Zhang and Yao, 2013). Similarly, in the present study, CMS resulted in a decrease in SOD and CAT and an increase in MDA in the hippocampus of rats, which was reversed by icariin and fluoxetine. Repeated treatment with icariin, which inhibited the CUMS-induced depression-like behavior, also restored CMS-induced lipid peroxidation, suggesting a potential relationship between both events. In addition to oxidative stress, neuroinflammation also influences neuronal functions in the pathophysiology of depression (Berk et al., 2013). Neuroinflammation is both a cause and consequence of chronic oxidative stress (Zhou et al., 2014). Generally, appropriate levels of NO play important roles in neuroprotection, but the overproduction of NO contributes to neurotoxicity. Furthermore, iNOS may generate NO that is important in the inflammatory response. In addition, proinflammatory cytokines, including IL-1b, IL-6, and TNF-a, that are induced by injury, infection and psychological stress have been implicated in depressive behavior in rodent models and depressed patients. Of these factors, IL-1 is an important cytokine involved in the modulation of neuroendocrine systems, particularly the HPA. One possible signaling cascade that mediates the effects of IL-1b is NF-jB, which is activated by IL-1b and other cytokines both in peripheral immune cells and the brain. NF-jB also regulates the expression of pro-inflammatory enzymes, including iNOS, COX-2 and pro-inflammatory cytokines. In our study, CMS induced a significant increase in nuclear localization of NF-jB p65 and iNOS mRNA and protein levels in hippocampal brain regions, which suggested that activated NF-jB in the nucleus may interact with kappaB elements in the NOS2 50 flanking region, triggering iNOS gene transcription. These findings are consistent with human studies, which demonstrate that social stress /anxiety increases NF-jB signaling and that this effect is enhanced in depressed patients. Furthermore, treatment with a higher dose of icariin (40 mg/kg) attenuated the IL-1b and TNF-a mRNA and protein levels, blocked NF-jB translocation to the nucleus, and down-regulated the production of NO, which contributes to its potent anti-inflammatory properties and are consistent with our previous report that icariin decreases the production of pro-inflammatory cytokines and nuclear factor kappa B (NK-jB) activation in LPS-induced acute inflammatory responses in vitro and in vivo (Xu et al., 2010). Although icariin treatment inhibited the production of proinflammatory cytokine that may lead to the induction of iNOS gene expression, unaltered iNOS protein level in our study may result from mutual regulation between

cytokines and glucocorticoids. Glucocorticoids may be important negative regulators of NF-jB in the brain (De Bosscher et al., 2003) which is a transcriptional factor for the iNOS gene. Recent evidence suggests that the pathophysiology of depression may be associated with the NLRP3 inflammasome (Iwata et al., 2013; Lu et al., 2014; Pan et al., 2014; Zhang et al., 2014). NLRP3 inflammasome activation links cytokines, psychological stress and depression (Iwata et al., 2013; Maslanik et al., 2013). The activated NLRP3 inflammasome is detected in mononuclear blood cells from patients with MDD (Alcocer-Gomez et al., 2014). The NF-jB pathway is an important downstream regulator of IL-1b signaling. The NLRP3 inflammasome couples with the nuclear factor kappa B (NK-jB) inflammatory pathway to mediate IL-1b transcription and function (Bauernfeind et al., 2009). Other studies has suggested that CMS results in NLRP3 inflammasome activation in the hippocampus of CMS model mice (Lu et al., 2014) and the prefrontal cortex in CUMS-induced depression model rats (Pan et al., 2014). Our current findings indicate that CMS treatment causes NLRP3 up-regulation and caspase-1 activation in the hippocampus of rats, suggesting that the NLRP3-inflammasome/caspase-1/IL-1b axis is associated with the pathogenesis of depression, which is consistent with previous studies (Lu et al., 2014; Pan et al., 2014; Zhang et al., 2014) and further supports this hypothesis. To the best of our knowledge, this is the first report that icariin treatment inhibits activation of the NLRP3 inflammasome, accompanied by a decrease of the IL-1b and caspase-1 protein levels in the hippocampus in CMS-induced depression model rats. These results suggest that icariin elicits its antidepressant effects partially through attenuating or reversing CMS-induced activation of the NF-jB signaling pathway and the NLRP3-inflammasome/caspase-1/IL-1b axis. These data extend previous findings of the role of neuroinflammation in the pathophysiology of depression. These results provide valuable information with regard to the inflammatory hypothesis underlying major depression, which is critical for the development of new and effective pharmacotherapeutic approaches that selectively target these key mechanisms in major depression. However, further studies are required to fully elucidate the association between their anti-inflammatory and antidepressant-like effects and to determine their clinical effectiveness in patients suffering from depression associated with neuroinflammation.

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The present study demonstrates that CMS produces depression-like behavior and causes changes in the oxidative stress and inflammatory response in the hippocampus of rats and that icariin attenuates or reverses these effects, which may be mediated, at least in part, by increased antioxidant status and an anti-inflammatory effect in the brain tissue via inhibition of NF-jB signaling activation and the NLRP3inflammasome/caspase-1/IL-1b axis.

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CONFLICT OF INTEREST

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We declare that we have no duality or conflict of interest

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Acknowledgments—This study was funded by grants from the National Natural Science Foundation of China (No. 81102562).Chinese Ministry of Education Fund for Doctor Discipline Scientific Research (No. 20110071120072). Zhejiang Province Natural Science Fund (No. Y2111111); Zhejiang Province Traditional Chinese medicine health science and technology project (No. 2012ZA105); Hangzhou Medical research and Key Special disease project (No. 20110833B32); Zhejiang Province Medical and health science and technology project (No. 2014KYB196).

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(Accepted 27 February 2015) (Available online xxxx)

Please cite this article in press as: Liu B et al. Icariin exerts an antidepressant effect in an unpredictable chronic mild stress model of depression in rats and is associated with the regulation of hippocampal neuroinflammation. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.02.053

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