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Home > The selenium-containing compound 3-((4-chlorophenyl)selanyl) -1-methyl-1H-indole reverses depressive-like behavior induced by acute restraint stress in mice: modulation of oxido-nitrosative stress and inflammatory pathway
The selenium-containing compound 3-((4-chlorophenyl)selanyl) -1-methyl-1H-indole reverses depressive-like behavior induced by acute restraint stress in mice: modulation of oxido-nitrosative stress and inflammatory pathway
Angela Maria Casaril1,2 & Micaela Domingues1 & Suely Ribeiro Bampi1 & Darling de Andrade Lourenço1 &
Nathalia Batista Padilha3 & Eder João Lenardão3 & Mariana Sonego4 & Fabiana Kommling Seixas4 & Tiago Collares4 &
Cristina Wayne Nogueira5 & Robert Dantzer 2 & Lucielli Savegnago1
Introduction
Exposure to stress induces behavioral, emotional, and cognitive modifications that can increase the risk of progression of neuropsychiatric disorders such as major depressive disorder (Joëls et al. 2006). Although glucocorticoids (GCs) released by the adrenal cortex in response to stress are claimed to have immunosuppressive and anti-inflammatory properties, recent data indicate they can also have proinflammatory influence on the immune system (Elenkov 2008).
Stress can activate the innate immune system through the activation of Toll-like receptor 4 (TLR4) by damage-associated molecular patterns (DAMPs) that are generated from insults to tissues, or by pathogen-associated molecular patterns (PAMPs), which can be a result of bacterial translocation. Activated TLR4 promotes the activation of glycogen synthase kinase 3 beta (GSK-3β) (Jope et al. 2017), which can also be activated by increases in cortisol (Dobarro et al. 2013). Among its many actions, activation of GSK-3β by phosphorylation at tyrosine 216 residue prevents the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) from the cytosol to the nucleus (Kensler et al. 2007). Consequently, Nrf2 does not bind to the antioxidant response element (ARE), thereby reducing the transcription of antioxidant enzymes, such as heme oxygenase-1 (HO-1), super- oxide dismutase (SOD), and catalase (CAT) (Kensler et al. 2007). Additionally, GSK-3β promotes the activation of tran- scription factors in the inflammatory response, such as nuclear factor kappa B (NF-κB), thereby increasing the production of pro-inflammatory cytokines in the central nervous system, espe- cially by microglia and astrocytes (Jope et al. 2017). Pro- inflammatory cytokines are also responsible for the activation of the enzymes indoleamine-2,3-dioxygenase (IDO) (Schwarcz and Pellicciari 2002) and inducible nitric oxide synthase (iNOS), further contributing to impaired neurotransmission and oxidative imbalance by increased reactive species (RS) generation. Excessive formation of free radicals and/or defects in the antiox- idant defense can damage lipids, proteins, and nucleic acids, which in turn are implicated in major depressive disorder (Maes et al. 2011), highlighting the importance of the antioxidant balance in the central nervous system.
Selenium is an essential trace element for human health and exerts most of its action as integral constituent of selenoproteins that are widely implicated in redox signaling (Brigelius-Flohé and Flohé 2017). In turn, selenium- containing organic compounds have emerged as promising biologically active molecules (Casaril et al. 2015; Domingues et al., 2018 ; Singh et al. 2016; Masaki et al. 2016; Kil et al. 2017; Gandin et al. 2018; Sudati et al. 2018). Recently, Vieira et al. (2015) reported that the synthetic organoselenium com- pound 3-((4-chlorophenyl)selanyl)-1-methyl-1H-indole (CMI) has in vitro antioxidant activity, while Casaril et al. (2017b) showed that it potently inhibits inflammation-associated oxi- dative stress (i.e., hypochlorous acid, peroxynitrite, and hydro- gen peroxide). In addition, Birmann et al. (2018) have demon- strated that the antinociceptive effect of CMI is mediated by the monoaminergic, opioidergic, and adenosinergic systems, pointing out the promising role of CMI in modulating different pathways for the treatment of pain and inflammation. Further studies revealed that CMI prevents the depressive-like behav- ior induced by lipopolysaccharide (LPS) in mice (Casaril et al. 2017a).
In light of the potential antioxidant and anti-inflammatory profile of CMI, we hypothesized that this organoselenium compound should be able to counteract stress-induced depres- sion-like behavior in mice. We used for this purpose a model of acute restraint stress in mice that had already been validated (Pesarico et al. 2015; Thakare et al. 2016; Surkin et al. 2018). We show here that CMI has antidepressant properties in this model that are associated with downregulation of stress- induced inflammation and oxidative stress.
Materials and methods
Drugs and reagents
CMI (Fig. 1A) was prepared and characterized at the Laboratory of Clean Organic Synthesis at the Federal University of Pelotas, according to Vieira et al. (2015). The RNA extraction reagent was purchased from Ambion (Life Technology, USA). The oligonucleotides were synthesized by Exxtend Biotecnologia Ltda (Campinas, Brazil). All other chemicals were of analytical grade and were obtained from Servylab and WF Científica (Brazil). CMI was dissolved in canola oil (a non-polar and inert substance) and administered intragastrically (i.g.) at a constant volume of 10 ml/kg body weight.
Animals
Behavioral experiments were performed in Swiss male mice (25–30 g; 10–13-week-old), maintained at 22–25 °C and 40– 60% relative humidity with free access to water and food, under a 12:12-h light/dark cycle (lights on at 7:00 a.m.). The studies were performed in accordance with protocols ap- proved by the Committee on the Care and Use of Experimental Animal Resources at the Federal University of Pelotas, Brazil (4034-2017).
Acute restraint stress
The physical restraint was performed in mice as previously reported by Pesarico et al. (2015), with minor modifications. Briefly, mice were subjected to immobilization for 240 min using an individual rodent restraint device made of Plexiglas fenestrate, restraining all physical movement and causing minimum pain. Mice were deprived of food and water during the physical stress. After the restraint stress, mice were put back in their home cage and received canola oil or CMI 10 min later. They were submitted to behavioral testing 30 min later and euthanized immediately after.
Experimental design
Animals were randomly divided into six experimental groups (n = 6/group) and the physical stress was performed for 240 min (Fig. 1B). The behavioral tasks—open field test (OFT), tail suspension test (TST), new object exploration test (NOET), and splash test—were carried out 280 min after the beginning of the acute restraint stress (ARS) protocol, and the vehicle or CMI (1 or 10 mg/kg, orally) was given 10 min after the physical restraint (Pesarico et al. 2015). Previous data also support the 30-min pre-treatment to evaluate the antidepressant-like activity of CMI (Casaril et al. 2017a) and other selenium-containing compounds (Martinez et al. 2014; Pinto Brod et al. 2016). All the observations were done by an observer blinded to the treatment and the behavioral tests were scored manually. Following the behavioral assessment, mice were anesthetized (inhalation of isoflurane) before blood col- lection by cardiac puncture (Parasuraman et al. 2010). After that, mice were killed by cervical dislocation, followed by brain removal (residual blood and blood cells can be present in the samples) and isolation of the prefrontal cortex (PFC) and total hippocampus (HC) for analysis (Sunkin et al. 2013).
Behavioral tests
Open field test
The open field test (OFT) was carried out before the other behavioral tests (Walsh and Cummins 1976) to assess the possible effect of the treatments on the locomotor activity. Mice were placed individually in the center of a box (30 × 30 × 15 cm) divided into nine quadrants of equal areas, and observed for 5 min to report their locomotor (scored by the number of segments crossed with the four paws) and explor- atory activities (expressed by the number of time the mice stood on rear limbs).
Tail suspension test
The total duration of immobility in the tail suspension test (TST) was measured according to the method described by Steru et al. (1985). Mice that were both acoustically and visu- ally isolated from each other were suspended 50 cm above the floor by adhesive tape placed approximately 1 cm from the tip of their tail. During the first 2 min of habituation, the latency time to immobility was recorded (i.e., time for the first immo- bility episode), and during the last 4 min, the immobility du- ration (defined as the absence of the escape attempt behavior) was observed.
New object exploration test
For this test, mice were allowed to explore a novel object for 15 min in an arena 18 × 25 cm2 surrounded by plastic walls (Strekalova et al. 2004). The object had a complex texture surface (artificial flower, 2.5 × 2.5 × 4 cm3) and it was fixed to the center of the arena. The total duration of time spent exploring the object was scored, and at the end of each test session, the arena was cleaned with a 70% ethanol solution.
Splash test
The grooming behavior of mice was observed as a mea- surement of motivational and self-care activities, which are considered to be deficient in depressive patients. A 10% sucrose solution was squirted on the dorsal coat of each mice and the grooming activity (including nose/face grooming, head washing, and body grooming) was re- corded for 5 min (Freitas et al. 2013).
Biochemical evaluation
Tissue processing
The PFC and HC were separated in two hemispheres in order to submit each sample to all biochemical determinations
(Casaril et al. 2017a). The right hemispheres were immersed in TRIzol, maintained at − 80 °C, and were submitted to the quantitative real-time polymerase chain reaction (qRT-PCR).
The left hemispheres were homogenized in 50 mM Tris-HCl, pH 7.4 (1:10, w/v). The homogenate was centrifuged at 2500g for 10 min at 4 °C, and the supernatant fraction was used for the determination of reactive oxygen species (ROS) forma- tion, thiobarbituric acid reactive species (TBARS) levels, ni- tric oxide metabolites (NOx), catalase (CAT), and superoxide dismutase (SOD) activities.
Plasma corticosterone assay
Determination of plasma corticosterone levels was performed according to Zenker and Bernstein (1958). Briefly, aliquots of plasma were incubated with chloroform and centrifuged for 5 min at 2500 rpm, followed by addition of 0.1 M NaOH and another round of centrifugation. After the addition of the fluo- rescence reagent (H2SO4 and ethanol 50%), samples were centrifuged (5 min at 10,000g) and incubated at room temper- ature for 2 h. Fluorescence intensity emission, corresponding to plasma corticosterone levels, was recorded at 540 nm (with 247-nm excitation) and corticosterone levels were expressed as nanogram per milliliter.
Determination of the reactive oxygen species formation
Quantification of reactive oxygen species (ROS) levels in the PFC and HC of mice was performed according to Loetchutinat et al. (2005). Briefly, aliquots of the homogenate supernatant were incubated with 1 mM dichloro-dihydro- fluorescein diacetate (DCHF-DA) and 10 mM Tris-HCl pH 7.4. The oxidation of DCFH-DA to fluorescent dichlorofluorescein (DCF) is measured for the detection of intracellular ROS. The DCF fluorescence intensity emission was recorded at 520 nm (with 480-nm excitation) and ROS levels were expressed as arbitrary units (AU) of fluorescence.
Thiobarbituric acid reactive species assay
Lipid peroxidation in the PFC and HC was measured by the formation of thiobarbituric acid reactive species (TBARS) during an acid-heating reaction, as described by Ohkawa et al. (1979). An aliquot of the homogenate supernatant was incubated with 8.1% SDS, 0.8% TBA, and acetic acid/HCl (pH 3.4) at 95 °C during 2 h. Malondialdehyde (MDA) was used as a biomarker of lipid peroxidation. Absorbance was measured at 532 nm, and the results were expressed as nanomoles of MDA per gram of tissue.
Nitric oxide in neural tissue
The total nitric oxide (NO) metabolites, NOx, in the PFC and HC of mice were determined by the Griess reaction as an indicator of nitrate/nitrite production (Lima-Junior et al. 2013). Briefly, aliquots of the homogenate were incubated with equal volume of the Griess reagent for 5 min at 25 °C, and the nitrite concentration was determined by measuring the optical density at 550 nm in reference to a standard curve of NaNO2 solution. Results were expressed as nanomoles of NOx per gram of tissue.
Evaluation of superoxide dismutase activity
The measurement of superoxide dismutase (SOD) activity is based on the capacity of SOD to inhibit autoxidation of adren- aline to adrenochrome, as described by Misra and Fridovich (1972). The color reaction was detected spectrophotometrical- ly at 480 nm and the enzymatic activity was expressed as units per milligram of protein.
Evaluation of catalase activity
Catalase (CAT) activity was assessed spectrophotometrically by the method described by Aebi (1984), which involves monitoring the disappearance of H2O2 in the presence of S1 at 240 nm. Enzymatic activity was expressed in units per milligram of protein (1 U decomposes 1 μmol of H2O2 per minute at pH 7 at 25 °C).
Quantitative real-time polymerase chain reaction
Total mRNA was extracted in the HC and PFC right hemi- spheres using TRIzol (Invitrogen™, Carlsbad, USA) followed by DNase treatment with DNA-free® kit (Ambion™, USA) and mRNA quantification. The cDNA synthesis was per- formed using High Capacity cDNA Reverse Transcription kit (Applied Biosystems™, UK) according to the manufac- turer’s protocol. The amplification was made with UltraSYBR Mix (COWIN Bioscience Co., Beijing, China) using the Stratagene Mx3005P and the oligonucleotides were obtained from Exxtend Biotecnologia Ltda, Campinas, Brazil. Gene expressions were normalized using GAPDH as a reference gene and the conditions for the reaction involved 95 °C for 15 s, 60 °C for 60 s, and 72 °C for 30 s. The 2ΔΔCT (delta-delta comparative threshold) method was used to normalize the fold change in gene expressions.
Protein determination
Protein concentration was measured according to the method of Lowry et al. (1951) using serum bovine albumin as a standard.
Statistical analysis
All experimental data are presented as mean ± standard error of the mean (SEM). Comparisons between stress exposure and treatment were performed by two-way anal- ysis of variance (ANOVA). When ANOVA revealed a significant main effect, Tukey’s post hoc test was used for between-group comparisons. Pearson’s correlation analysis was performed to investigate any possible rela- tionship between the immobility time in the TST and neu- rochemical data. Probability values less than 0.05 (p < 0.05) were considered statistically significant. The statistical analysis was accomplished using GraphPad Prism version 7.0 for Windows, GraphPad Software (San Diego, CA, USA).
Protein determination
Protein concentration was measured according to the method of Lowry et al. (1951) using serum bovine albumin as a standard.
Statistical analysis
All experimental data are presented as mean ± standard error of the mean (SEM). Comparisons between stress exposure and treatment were performed by two-way anal- ysis of variance (ANOVA). When ANOVA revealed a significant main effect, Tukey’s post hoc test was used for between-group comparisons. Pearson’s correlation analysis was performed to investigate any possible rela- tionship between the immobility time in the TST and neu- rochemical data. Probability values less than 0.05 (p < 0.05) were considered statistically significant. The statistical analysis was accomplished using GraphPad Prism version 7.0 for Windows, GraphPad Software (San Diego, CA, USA).
The TST is widely used to measure depression-like behavior in rodents, while assessing the antidepressant- like response from several pharmacological classes of drugs (Browne and Lucki 2013). As depicted in Fig. 2C, mice submitted to physical stress showed increased dura- tion of immobility when compared to the vehicle-treated group. Noteworthy, this depressive-like behavior was re- versed by the administration of CMI independently of the dose (ARS × CMI interaction; F(2,30) = 16.70, p < 0.001). Administration of CMI in non-stressed mice did not influ- ence immobility time.
Corroborating with this result, stressed mice showed re- duced latency for the first immobility episode, when com- pared to the vehicle-treated group (Fig. 2D). On the contrary, treatment with CMI at both doses was able to reverse the decreased latencies (ARS × CMI interaction; F(2,30) = 15.4, p < 0.001). Administration of CMI to non-stressed mice did not influence latency time.
Regarding the new object exploration test (NOET) (Fig. 2D), a two-way ANOVA of the exploratory time revealed a significant ARS × CMI interaction (F(2,30) = 13.20, p < 0.001). Concerning self-care and grooming (Fig. 2E), a two-way ANOVA revealed a significant ARS × CMI interaction (F(2,30) = 7.06, p = 0.003). Administration of CMI to non-stressed mice did not influ- ence these behaviors.
Increased circulating glucocorticoid levels induced by ARS were restored by treatment with CMI
To document the involvement of the HPA axis in our experi- ments, plasma levels of corticosterone were measured in mice submitted to ARS. The results presented in Fig. 2G show that the restraint-induced increase in corticosterone levels was sig- nificantly reversed by CMI (ARS × CMI interaction; F(2,30) = 23.90, p < 0.001) while the administration of CMI to non- stressed mice had no effect.
Discussion
The present study shows for the first time that a single admin- istration of CMI is able to reverse the behavioral response to restraint in the TST, splash test, and NOET by a mechanism dependent, at least in part, on the modulation of oxidative stress and neuroinflammation. Indeed, we observed that CMI regulated the SOD and CAT activities in the PFC and HC of stressed mice, alongside with a decrease in plasma levels of corticosterone. Additionally, we reported here that CMI reversed the increases in TBARS, ROS, and NOx forma- tion in the PFC and HC of mice submitted to ARS. Noteworthy, CMI was also able to restore the mRNA expres- sion of GR, NF-κB, iNOS, TNF-α, IDO, GSK-3β, and BDNF in the PFC and HC of mice. A summary of these results is shown in Table 2. Thus, the beneficial effects of CMI on behavior are associated with its capacity to modulate oxidative stress and neuroinflammation caused by immobili- zation stress. The hypothesis of a causal role of oxidative stress and neuroinflammation in the behavioral effects of re- straint is supported by the significant correlations between the biochemical endpoints and immobility time in the TST.
Despite several reports having examined the ability of promising drugs to protect behavioral and biochemical mod- ifications induced by ARS (Moretti et al. 2013; Freitas et al. 2014; Ai et al. 2017), few positive results have been obtained so far (Pesarico et al. 2015). Therefore, our study was de- signed to help to fulfill this gap and if possible increase the range of potential treatments for major depressive disorder. We show here that CMI has clearly the ability to treat stress- induced depression. This effect is independent of the dose at least in the range of doses tested in the present experiments. This lack of a dose-response effect has already been docu- mented for other selenium-containing compounds (Pinto Brod et al. 2016; Domingues et al. 2018) and neuroprotective agents (Rosa et al. 2018).
The ARS is a widely used animal model to induce depressive-like behavior (Thakare et al. 2016; Surkin et al. 2018) akin to major depressive disorder symptoms, partially by targeting the brain antioxidant and inflammatory systems (Buynitsky and Mostofsky 2009; Spiers et al. 2016; Jope et al. 2017; Surkin et al. 2018). Indeed, the establishment of brain oxidative stress as a result of increased activity of nicotin- amide adenosine dinucleotide phosphate (NADPH) oxidase (NOX) (Schiavone et al. 2009) and/or mitochondrial impair- ment (Jevtić et al. 2016) has been linked to neuropathogenetic alterations induced by different types of stress in rodents. In addition, stress procedure has been shown to cause alterations in amyloid beta (Aβ) release, contributing to the induction of depressive-like behavior in rodents (Morgese et al. 2017). The depressive-like behaviors induced by Aβ occur via impact on serotonergic and neurotrophin levels, HPA axis activation, and microglial TLR4 signaling (Justice et al. 2015; Morgese et al. 2014; Ledo et al. 2016). In the present study, we focused on the ability of CMI to counteract depressive-like behaviors via modulation of oxidative alterations and neuroinflamma- tion, opening a window for future investigations related to mitochondrial function and Aβ signaling.
Hyperactivity of the HPA axis is the most common alter- ation found in patients with major depressive disorder (Stetler and Miller 2011). Increased levels of circulating corticoste- rone with concentrations in the range of those reported in other studies (Spiers et al. 2016) confirm the effectiveness of the ARS protocol employed in the present study. Additionally, the downregulation of GR mRNA expression in the PFC and HC of mice submitted to ARS could contribute to the reduced negative feedback loop that controls GC secretion. In accordance with this interpretation, we observed that the acute administration of CMI reversed the increasing plasmatic levels of corticosterone and upregulated the GR mRNA ex- pression in the PFC and HC of mice submitted to ARS. These findings, together with the significant negative correlation with immobility time in the TST, show the ability of CMI to modulate hyperactivity of the HPA axis, an effect that could play a role in its antidepressant properties.
Stress is known to activate intracellular pathways involved in increasing free radical production. GC can induce neuronal oxidative stress by enhancing mitochondrial respiration and oxidative phosphorylation (You et al. 2009). NO is a neuromodulator able to control the release of corticosterone (Rettori et al. 2009), thus further influencing the stress re- sponse (Gądek-Michalska et al. 2016), including in the model of ARS (Chen et al. 2016; Surkin et al. 2018). Overall, RS produced during the stress promote lipid peroxidation, as ev- idenced by increased MDA formation (Maes et al. 2011; Niki 2012), that in fact is the major consequence of the oxidative stress in the brain (Niki 2012). In line with this, the present study showed a higher production of ROS, TBARS, and NOx and decreased mRNA expression of iNOS in the PFC and HC of mice submitted to the ARS, when compared to the non- stressed control. Interestingly, these oxidative alterations in mice subjected to ARS were reversed by a single dose of CMI. Considering that CMI was already capable of preventing oxidative alterations in LPS-challenged mice (Casaril et al. 2017a), this antioxidant effect of CMI is perhaps not surprising, but still very promising in terms of recovery from acute stressors. Since behavioral disturbances may be a reflection of increased oxidative alterations in the brains of stressed mice, we propose that the blockade of depression- like behavior by CMI treatment is mediated by its ability to decrease ROS, NOx, and TBARS generation, a hypothesis that is supported by the significant correlation among these variables.
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