Agomelatine Softens Depressive-Like Behavior Through the Regulation of Autophagy and Apoptosis

Hindawi BioMed Research International Volume 2021, Article ID 6664591, 10 pages https://doi.org/10.1155/2021/6664591

Research Article Agomelatine Softens Depressive-Like Behavior through the Regulation of Autophagy and Apoptosis

Fengpei Chen,1 Shijia Chen ,2 Jie Liu,1 Nashwa Amin,2,3 Weidong Jin ,1,4 and Marong Fang 2

1The Second Clinical Medical College of Zhejiang Chinese Medical University, Hangzhou 310053, China 2Institute of Neuroscience, Zhejiang University School of Medicine, Hangzhou 310058, China 3Department of Zoology, Faculty of Science, Aswan University, Egypt 4Zhejiang Mental Health Center, Tongde Hospital of Zhejiang province, Hangzhou 310012, China

Correspondence should be addressed to Weidong Jin; [email protected] and Marong Fang; [email protected]

Received 12 December 2020; Revised 11 January 2021; Accepted 26 February 2021; Published 18 March 2021

Academic Editor: Andrea Scribante

Copyright © 2021 Fengpei Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Depression is a common and disabling mental disorder with high recurrence rate. Searching for more effective treatments for depression is a long-standing primary objective in neuroscience. Agomelatine (AGO) was reported as an antidepressant with unique pharmacological effects. However, its effects and the underlying mechanism are still unclear. In this study, we sought to evaluate the antidepressant effects of AGO on the chronic restraint stress (CRS) mouse model and preliminarily investigate its effects on the gut microbial metabolites. The CRS model mice were established in 28 days with AGO (60 mg/kg/day, by oral) or fluoxetine (15 mg/kg/day, by oral) administration. The number of behavioral tests was conducted to evaluate the effect of AGO on depression-like behavior alleviation. Meanwhile, the expression of the BDNF/TrkB/pERK signaling pathway, apoptosis, autophagy, and inflammatory protein markers were assessed using western blot and immunofluorescence. Our findings show that AGO can attenuate the depressive-like behavior that significantly appeared in both sucrose preference and forced swimming tests. Additionally, a noticeable upregulation of autophagy including Beclin1 and LC3II, microglial activity marker Iba-1, and BDNF/TrkB/pERK signaling pathways are indicated. An obvious decreased expression of NF-κB, iNOS, and nNOS as well as apoptosis including Bax is observed in AGO administration mice. On the other hand, we found that AGO impacted the rebalancing of short-chain fatty acids (SCFAs) in mouse feces. Altogether, these findings suggest that AGO can exert antidepressant effects in a different molecular mechanism.

1. Introduction tricyclic antidepressants (TCAs), norepinephrine and dopa- mine reuptake blockers, and monoamine oxidase inhibitors Depression is characterized by sadness, anhedonia, low self- which exhibit little or no improvement on depression and esteem, loss of motivation, sleep disruption, loss of appetite, have side effects such as fatigue, sleep disorders, cognitive and other cognitive symptoms [1]. Symptoms of depression disorders, and sexual dysfunctions [3–5]. are highly prevalent, affecting up to 27% of the general popu- AGO is an antidepressant with unique pharmacological lation according to recent meta-analytic data [2]. However, actions. Previous studies reported AGO to act as both a mel- the exact biological, psychological, and social mechanisms atonin agonist and a selective serotonin antagonist [6]. In the underlying the pathogenesis of depression remain largely previous decade, AGO was reported to have at least compa- unknown. Currently, most patients with depression are rable efficacy to other antidepressants in terms of response treated with synthetic drugs including selective serotonin and remission rates and to confer better tolerability and be reuptake inhibitors (SSRI) that act on the monoaminergic sys- associated with fewer side effects [7–9]. In vertebrates, the tem, serotonin-norepinephrine reuptake inhibitors (SNRIs), pineal gland is the main source of melatonin production. 2 BioMed Research International

Of the many substances identified, recent studies suggest that oxetine (FLX), and agomelatine (AGO). All groups were sub- melatonin and its metabolites are highly effective physiolog- jected to CRS except the control group. Fluoxetine (Patheon, ical antioxidants and can also exert direct, non-receptor- France) and agomelatine (Lianyungang, China) were dis- mediated actions, such as the scavenging of a number of free solved in physiological saline (NaCl, 0.9%). Dose and admin- radicals including both reactive oxygen and nitrogen species istration schedules of AGO and FLX were based on previous (ROS and RNS, respectively) [10–13]. results [24]. Specifically, mice in the CRS group were sub- BDNF is associated with neuropsychiatric disorders, such jected to CRS and intragastric administration of 8.0 mL/kg as depression [14] and schizophrenia [15]. BDNF and its saline daily, and AGO and FLX were administered to mice mechanisms are also therapeutic targets of pharmacological at doses of 60 mg/kg body weight/day and 15 mg/kg body agents that are currently used to treat mice with depression weight/day, respectively. Drugs were administered half an [16]. Previous studies have shown that fluoxetine can hour before the CRS regimen. The specific experimental pro- improve depression-like behaviors by regulating the BDNF cedure is shown in Scheme 1. signaling pathway [17]. However, the role of BDNF in the antidepressant process of AGO still requires in-depth 2.3. The Chronic Restraint Stress Model. The CRS model was research. Ample preclinical evidence suggested that exposure based on the Chiba method [25], with slight modification. to acute or chronic stress agents triggers the production of Briefly, mice were individually restricted in a modified, cytokines, which may be related to the physiological pro- well-ventilated tube for 6 h without water and food, and mice cesses mediated by autophagy [18]. The induction of autoph- were exposed to CRS for 28 consecutive days. agy protects neurons and glial cells under stress through the deregulation of various DAMPs and suppression of stress 2.4. Behavioral Test reaction, which will contribute to the alleviation of depres- 2.4.1. Open Field Test (OFT). The apparatus consisted of a sion [18, 19]. The observation that the increase in autophagic white Plexiglas box (45 cm × 45 cm × 45 cm) divided into markers goes along with the reversal of the behavioral effects fi two zones: outer square (periphery) and inner square (center: argues in favor of autophagy being a bene cial component of 25 cm × 25 cm). Each mouse was placed in the center of the the stress response in general [20]. Existing research shows fl fl box and allowed to freely explore the environment in a room that uoxetine could promote the autophagic ux unblocked with dim light for 5 min. The movement of the mouse was in the way of enhancing fusion of autophagosomes with lyso- fi recorded by a video camera (VideoTrack, Viewpoint Inc., somes in primary astrocytes and nally ameliorates depres- France). The total distance covered in the box and the time sion [21]. Similarly, apoptosis, another molecular spent in the central zone were calculated. mechanism that maintains cellular homeostasis, has recently been shown to be closely related to the development of 2.4.2. Elevated Plus Maze (EPM). The elevated plus maze depression [22]. Moreover, autophagy is important in cell apparatus consisted of two opposite-facing closed arms death decisions and can protect cells by preventing them (30 cm × 5 cm × 15 cm), two opposite-facing open arms from undergoing apoptosis [23]. All of these indicate that (30 cm × 5 cm), and a central area (5cm×5cm), and the autophagy and apoptosis may act as key mediators in antide- maze was 50 cm above the ground. For the elevated plus pressant and stress-induced behavioral action. maze, the mouse was placed in the center of the maze facing However, whether AGO can alleviate depression by reg- one of the two closed arms. A 5-minute test was recorded ulating autophagy or apoptosis is currently not clearly under- using an automatic analyzing system (ANY-maze, Stoelting, stood. In the current study, we aimed to investigate (i) the USA). The number of entries and the time spent in the open underlying mechanisms of AGO administration in CRS- arms were recorded using an automatic analyzing system. induced depression, (ii) the changes in the hippocampus and subsequent anxiety behavior, and (iii) the impact of the 2.4.3. Forced Swimming Test (FST). Mice were placed into a AGO regimen on brain inflammation factors and microbial cylinder (12 cm diameter, 25 cm height) filled with 24 ± 1°C metabolites such as SCFAs. water. Water depth was adjusted so that the mice can neither touch the bottom nor jump out. Two mice at a time were 2. Materials and Methods videotaped from the side. A cardboard divider separated the cylinders so that the mice could not see each other during 2.1. Animals. Male C57BL/6N mice (weighing 20 ± 2 g,8 the trials. The mouse activity was video recorded for 6 min, weeks old) were used in this study. The mice were main- and the duration of immobility was recorded during the last tained in a room at 24 ± 1°C with 50%–60% relative humidity 4 min of the 6 min test. After the test session, mice were under a 12 h light/dark cycle (lights on at 09:00) with free placed in a clean cage containing paper towels under a heat access to food and water. All experimental procedures were lamp until dry. Immobility was defined according to criteria approved and performed according to the Animal Care and described [26]. Use Committee of the Institute of Zhejiang University, China. The animal ethics number is ZJU20160267. 2.4.4. Sucrose Preference Test (SPT). The SPT was performed as previously reported [17] with slight modification. Briefly, 2.2. Study Design and Drug Administration. After 2 weeks of mice were single housed and were accustomed to two bottles adaptation, the mice were randomly assigned to 4 groups of 8 of water after two bottles of 2% sucrose for a day. After being mice each: control (CON), chronic restraint stress (CRS), flu- deprived of water and food for 24 h, the mice were exposed to BioMed Research International 3

Adaption CRS and drug administration OFT EPM FST SPT

Sampling

Days Weigh every other day 28 29 30 31 32

Scheme 1: Experimental schedule. Mice were treated for a total of 4 weeks with agomelatine, fluoxetine, or saline. After 4 weeks of treatment, animals underwent a battery of tests relevant to depression. All groups were sacrificed on the same day. Tissues were harvested for further analysis. CRS: chronic restraint stress; OFT: open field test; FST: forced swimming test; SPT: sucrose preference test. one bottle of 2% sucrose and one bottle of water for 2 h dur- occurred overnight at 4°C: Iba1 (Abcam, 1 : 100). The sections ing the dark phase. Bottle positions were switched after 1 h were washed with 0.01 M PBST for 5 min three times on the (for a 2 h test). After 2 hours of testing, water and sucrose following day and then incubated with secondary antibodies consumption was measured. containing FITC goat anti-rabbit IgG (1 : 100, Boster, BA1105) and Alexa Fluor® anti-rabbit 488 for 3 h in the dark 2.5. Western Blot. Mice were sacrificed by cervical disloca- at room temperature. After washing, probes were mounted in tion. The hippocampus was extracted for the evaluation of medium containing DAPI (VECTASHIELD, USA). A fluores- protein level and stored at -80°C until use. Total protein of cence microscope (Olympus BX61, NIKON, Japan) was used the hippocampus was prepared from each group using for taking images. IBa1 staining was obtained with the NIH 1 mL ice-cold RIPA buffer, supplemented with 1% PMSF ImageJ software (https://imagej.net/welcome). (phenylmethanesulfonyl fluoride, Beyotime ST505), protease inhibitors, and phosphatase inhibitors. Probes were ground 2.7. Fecal SCFA Detection: Gas Chromatographic Analysis. and then centrifuged at 12,000 rpm for 30 min at 4°C before The fecal samples were frozen immediately after collection protein supernatants were obtained. The protein concentra- and stored -80°C until analysis. Specifically, 80 mg of each tion was determined by using the BCA assay. 20 μg protein fresh stool sample was weighed, suspended in 500 μL anhy- was electrophoresed on a 10% SDS-PAGE gel and transferred drous alcohol, and homogenized for approximately 5 min. onto a polyvinylidene difluoride (PVDF) membrane. After Then, the sample was incubated at room temperature for blocking with 5% nonfat milk in TBS-Tween 20, membranes 10 min with occasional shaking and centrifuged for 10 min were incubated with primary antibodies overnight at 4°Cas at 12,000 rpm. The supernatant was transferred into 2 mL follows: rabbit anti-NF-κB/p65 (Cell Signaling Technology, screw top vials (Agilent, United States). The concentration 1 : 1,000), rabbit anti-nNOS (Omnimabs, 1 : 500), rabbit of SCFAs (including acetate, propionate, butyrate, isobuty- anti-iNOS (Boster, 1 : 500), rabbit anti-LC3B (Novus, rate, and valeric acid) in mouse fecal samples was determined 1 : 1000), rabbit anti-p62 (Cell Signaling Technology, using an Agilent 7890A gas chromatography unit, fitted with 1 : 1000), rabbit anti-Beclin (Cell Signaling Technology, a high-resolution gas chromatography DB-624 column 1 : 1000), rabbit anti-Bax (Boster, 1 : 500), rabbit anti-Bcl2 (30 m × 0:32 mm × 1:8 μm, Agilent, United States). Nitrogen (ABclonal, 1 : 500), rabbit anti-BDNF (Boster, 1 : 500), rabbit was supplied as a carrier gas at a flow rate of 1.2 mL/min. The anti-TrkB (Abcam, 1 : 1000), rabbit anti-pERK (Cell Signal- initial oven temperature was 100°C, maintained for 5.0 min, ing Technology, 1 : 1000), and mouse anti-GAPDH (ABclo- raised to 200°Cat20°C/min, and finally held at 200°C for nal, 1 : 10000). Then, the membranes were subjected to 5 min. three washing steps with TBS-Tween 20 (each 5 minutes) before incubation with the secondary antibody goat anti- 2.8. Statistical Analysis. Normalization and equal variances rabbit IgG antibody (Bioker, 1 : 5000) and goat anti-mouse between group samples were assessed using the normal Q-Q IgG antibody (Bioker, 1 : 5000) at room temperature for 2 h. plot and homogeneity test for variance, respectively. When After the final washing steps, membranes were subjected to normalization and equal variance between sample groups were enhanced chemiluminescence reaction and bands were achieved, one-way ANOVA with LSD was used. Where nor- quantified by densitometry (Quantity One 4.2, Bio-Rad, Her- malization or equal variance of samples failed, Mann–Whitney cules, CA). Densitometric values of markers were normalized U tests were performed. Each value was presented as a mean to GAPDH levels. Each experiment was performed at least ±SEMand analyzed with one-way ANOVA using SPSS 22.0 three times. The detection and quantification of each band software. A value of P <0:05 was considered significant. were performed using Image Lab software. 3. Results 2.6. Immunofluorescent Staining. Mice were perfused with 4% paraformaldehyde (PFA) after being perfused through the 3.1. AGO Ameliorates CRS-Induced Depressive-Like heart with 50 mL (±) of saline. Brain tissue sections were sub- Behavior. In order to investigate the effect of AGO in the jected to immunohistochemistry analysis. Frozen sections, depressive-like behavior, we sought to first analyze the body about 16 μm thick, were rinsed in 0.01 M PBS for 5 min and weight during CRS and drug treatment. No significant differ- then moved to room temperature for 1 h and blocked with ences were found in the baseline body weight among the four 5% normal goat serum. Incubation with primary antibodies groups. Then, statistical analysis revealed that CRS resulted 4 BioMed Research International

25 ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎ ⁎⁎⁎⁎⁎⁎ 24 ⁎⁎⁎ ⁎⁎ Forced swimming test ⁎⁎⁎ ⁎⁎⁎ ⁎⁎ 250 ⁎⁎ ⁎⁎ 23 ⁎⁎⁎ ⁎ 200 22 150

Body (g) weight 100 21 50

20 time (s) Immobility 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 CON CRS AGO FLX Day

CON AGO CRS FLX (a) (b) Sucrose preference test Open feld test ⁎⁎ ns Open feld test ns 100 ⁎ ⁎⁎ ns ns ns ns 2500 15 ns ns ns 80 2000 60 1500 10 40 1000 5 Total distance Total 20 (cm) travelled 500

0 0 (s) area in center Time 0 Sucrose consumption (%) consumption Sucrose CON CRS AGO FLX CON CRS AGO FLX CON CRS AGO FLX (c) (d) (e) Elevated plus maze Elevated plus maze ns ns ns ns 20 ns ns 60 ns ns 15 40 10 20 5 Open arms entry Open arms

0 (s) arms in open Time 0 CON CRS AGO FLX CON CRS AGO FLX (f) (g)

Figure 1: CRS-induced depression-like behaviors in mice. (a) Effects of CRS and drug treatment on body weight are presented. (b) The immobility time in FST after CRS, AGO, and FLX treatment is shown. (c) SPT after CRS and drug treatment is depicted. (d) Total distance traveled in the OFT after CRS and drug treatment is presented. (e) Time spent in the center area in OFT after CRS and drug treatment is presented. (f) Open arms entry in the EPM is shown. (g) Time in open arms in the EPM after CRS and drug treatment is shown. ∗P <0:05; ∗∗P <0:01; ∗∗∗P <0:001; ns = not significant. The values represent mean ± SEM. n =8. in retardation of the body weight gain starting with day 9 into and time spent in the open arms (Figures 1(f) and compared to the control group, while AGO and FLX treat- 1(g)). These experiments suggest that AGO restores the ment did not counteract the effect (Figure 1(a)). At the depressive-like behavior after CRS. end of the chronic stress period, a battery of well- established behavioral tests aimed at measuring anxiety- 3.2. Effect of AGO Treatment on BDNF/TrkB and ERK1/2 like behavior (OFT, EPM) and depression-like behaviors Activity. Recent studies demonstrate that BDNF-TrkB signal- (SPT, FST) were conducted. Mice in the CRS group ing is required for promoting proliferation [27] of newborn showed significant longer freezing time in FST neurons in the adult hippocampus in response to antidepres- (Figure 1(b)) and lower sucrose preference (Figure 1(c)) sant administration. Previous studies were conducted in compared with the control mice. AGO and FLX signifi- order to determine if AGO administration alters the level cantly restored the above changes. Unfortunately, no sig- of BDNF in the hippocampus [14], a region implicated nificant effect was observed in the locomotor activity and in the actions of antidepressants. Indeed, expression of the time spent in the central area (Figures 1(d) and BDNF and TrkB declined in the hippocampus of mice 1(e)). In addition, the CRS procedure did not alter entries subjected to CRS as depicted in Figures 2(a)–2(c). This BioMed Research International 5

BDNF BDNF⁎ TrkB 2.0 ⁎ 2.5 ⁎⁎ TrkB ⁎ ns ns ⁎ ⁎ 1.5 2.0 pERK1/2 1.5 GAPDH 1.0 1.0 0.5 FLX CRS 0.5 AGO CON Relative density /control density Relative 0 /control density Relative 0 CON CRS AGO FLX CON CRS AGO FLX (a) (b) (c) pERK 1/2 Beclin1 1.5 ⁎⁎ BeclinI ⁎⁎ LC3I 2.0 ns ns ⁎ LC3II ⁎⁎ ns 1.0 p62 1.5 ⁎

0.5 GAPDH 1.0

FLX 0.5 CRS AGO CON

Relative density /control density Relative 0 0 CON CRS AGO FLX /control density Relative CON CRS AGO FLX (d) (e) (f)

LC3II p62 Bc12 ⁎⁎⁎ ⁎ ⁎⁎ 2.5 ⁎⁎ 2.0 ⁎⁎ ns ns Bax 2.0 1.5 GAPDH 1.5 ns 1.0 FLX 1.0 CRS AGO 0.5 CON 0.5 0 0 /control density Relative Relative density /control density Relative CON CRS AGO FLX CON CRS AGO FLX (g) (h) (i) Bcl2/Bax NF-�B/p65 NF-�B/p65 ⁎⁎ 1.5 ⁎ 2.0 ⁎⁎ ⁎ ⁎⁎ ns iNOS ns ⁎ 1.5 1.0 nNOS 1.0 GAPDH 0.5 0.5 FLX CRS AGO CON 0 Relative density /control density Relative

Relative density /control density Relative 0 CON CRS AGO FLX CON CRS AGO FLX (j) (k) (l) iNOS nNOS ⁎ ⁎ ⁎ ⁎ 2.0 2.5 ⁎⁎ ⁎ ns ns 1.5 2.0 1.5 1.0 1.0 0.5 0.5 0 0 /control density Relative Relative density /control density Relative CON CRS AGO FLX CON CRS AGO FLX (m) (n)

Figure 2: Effect of AGO treatment on BDNF/TrkB and pERK1/2 in autophagy, apoptosis, and inflammation in CRS mice. (a) Representative western blot results of BDNF, TrkB, and pERK1/2 are shown; densitometry analysis of BDNF (b), TrkB (c), and pERK1/2 (d) in the hippocampus is presented. (e) Representative western blot results of LC3II, Beclin1, and p62 are shown; densitometry analysis of Beclin1 (f), LC3II (g), and p62 (h) in the hippocampus. (i) Representative western blot results of Bcl2 and Bax are shown; (j) densitometry analysis of the Bcl2/Bax ratio in the hippocampus. (k) Representative western blot results of NF-κB/p65, iNOS, and nNOS; densitometry analysis of NF-κB/p65 (l), iNOS (m), and nNOS (n) in the hippocampus. ∗P <0:05; ∗∗P <0:01; ∗∗∗P <0:001; ns = not significant. The values represent mean ± SEM. n =3-5. was corroborated with decreased levels of active ERK1/2 Moreover, there was no significant change between the (Figures 2(a) and 2(d)). Interestingly, AGO restored the AGO and FLX groups, indicating that these two drugs CRS-induced decrease to levels achieved by FLX regimens. protect similarly against CRS-induced depressive 6 BioMed Research International symptoms by interfering with BDNF and TrkB expression istration resulted in a decrease in IBa1 expression in the hip- and ERK activity in the hippocampus. pocampal region (Figures 3(a) and 3(b)). Importantly, AGO treatment ameliorated IBa1 expression compared with that 3.3. AGO Promotes CRS-Induced Autophagy in the of CRS mice (Figures 3(a) and 3(b)) in line with data pre- fi ff Hippocampus. To con rm the protective e ect of AGO on sented in Figure 2. hippocampal autophagy in the CRS model, we sought to investigate the putative protective mechanisms of AGO. 3.7. Changes in SCFA Concentrations. Based on the Therefore, the expression level of autophagy markers “microbiota-gut-brain” axis, we determined the effects of LC3II, p62, and Beclin1 was analyzed by western blotting. AGO administration on short-chain fatty acids. The SCFA Our experiments showed that Beclin1 was downregulated (acetate, propionate, isobutyrate, butyrate, and valeric acid) in the CRS group (Figures 2(e) and 2(f)), which was concentration in mouse fecal samples was analyzed by GC restored in AGO-treated mice comparable to the levels and is shown in Figure 4. Of all investigated fatty acids, only found in animals subjected to FLX regimens. Similarly, isobutyrate showed a difference between CRS and control. LC3 II expression in the hippocampus increased in mice Administration of AGO mainly decreased the concentration subjected to AGO and FLX treatment after CRS of acetate, valeric acid, and total SCFAs and not isobutyrate. (Figures 2(e) and 2(g)). Given that LC3II and Becline1 On the other hand, high levels of isobutyric acid induced by can only reflect the initialization of autophagy, we also CRS were relieved by drug treatment indicating that estab- detected the protein level of p62, which is the best- lished AGO or FLX regimens can attenuate depressive-like characterized molecule and degradation product of behaviors via the modulation of microbiota-driven SCFA autophagy. As shown in Figures 2(e) and 2(h), hippocam- levels. pal p62 levels were increased in CRS mice but significantly decreased after treatment with AGO and FLX. 4. Discussion The results suggest that stress led to lower levels of autophagy after the chronic restraint stress; this was ame- In our present study, we found that AGO treatment for 4 liorated by AGO treatment having higher levels than weeks alleviated depression-like behaviors via activating stressed animal. autophagy and the BDNF-TrkB pathway in the hippocampus of CRS mice. Moreover, an obvious decrease in inflammation 3.4. AGO Decreases CRS-Induced Apoptosis in the including NF-κB, iNOS, and nNOS as well as apoptosis Hippocampus. To confirm the role of AGO treatment in the including Bax is confirmed. It implies the possibility that reg- apoptosis mechanism, we investigated the hippocampal Bax ulating autophagy and the BDNF-TrkB pathway may be an and Bcl-2 protein levels to evaluate apoptosis. As shown in alternative mode of action for AGO besides its role in modu- Figures 2(i) and 2(j), CRS resulted in a decrease in the lating melatonin. Bcl2/Bax ratio in the hippocampus. Administration of AGO CRS is a noninvasive protocol, which is very similar to was associated with a significantly increased Bcl2/Bax ratio human psychological stress [29] and therefore renders as a in the hippocampus. Altogether, these findings suggest that reliable animal model of studying the depression with AGO might improve depressive symptoms in the CRS mouse good replicability. Our findings revealing lower body model by maintaining essential autophagy and inhibiting weight gain and depressive-like behaviors (FST and SPT) excessive apoptosis. after CRS are consistent with previous studies [30]. Con- 3.5. AGO Treatment Alleviates CRS-Induced Inflammation in tradicting literature reported either the failure of CRS to the Hippocampus. Ample preclinical and clinical evidence generate an anxiety-like behavior or an important role of fl – showed that external stress is accompanied by an increased CRS in in icting such behavioral changes [31 33]. Rea- production of proinflammatory cytokines and their sons for this discrepancy might be attributed to the upstream regulators. NF-κB is a transcription factor reported parameters of the applied restraint stress (e.g., frequency to regulate a plethora of proinflammatory cytokines, such as and duration) [34]. In short, the successful establishment iNOS [28]. Our experiments show that NF-κB subunit of depression model mice allowed us to study the antide- p65/RelA is elevated after CRS in the hippocampus pressant mechanism of AGO. (Figures 2(k) and 2(l)). Furthermore, our data show that BDNF-related signaling pathways have been reported iNOS and nNOS are also elevated after CRS (Figures 2(k), to play an essential role in the pathogenesis of stress- 2(m), and 2(n)). Most importantly, administration of AGO induced depression-like behaviors [35]. In the present partially restored both p65 and iNOS/nNOS levels, which study, we found that expression levels of BDNF/TrkB in fi were comparable with FLX treatment (Figures 2(k), 2(m), the hippocampus of the CRS group were signi cantly and 2(n)). Altogether, these findings implicate NF-κBin downregulated. This is consistent with the current clinical CRS-induced inflammation. research results [36]. Such BDNF/TrkB [37] integration leads to the activation of some signaling pathways includ- 3.6. Effect of AGO Treatment on Alterations of Microglia in ing extracellular signal-regulated kinase 1/2 (ERK1/2) the Mouse Hippocampus. To investigate the effect of AGO which is consistent with our findings that CRS-induced and FLX on microglia in the hippocampus, immunofluores- depressive-like behaviors and the consequent downregula- cence staining for hippocampal sections was performed. tion of BDNF/TrkB and ERK1/2 activity could be reversed IBa1 was used as a specific marker for microglia. CRS admin- by AGO administration. BioMed Research International 7

DAPI 1ba 1 Merge CON CRS AGO FLX

(a) Ibal expression 2.0 ⁎ ⁎ ⁎⁎ ns 1.5

1.0

0.5 Cell numbers/ control Cell numbers/

0.0 CON CRS AGO FLX (b)

Figure 3: Immunofluorescence results indicated variation of microglial activation after CRS and AGO treatment. (a) Representative images of Iba1 (green) and DAPI (blue). Merged images are shown in the right panel. Scale bar = 100 μm. (b) Quantification analysis of Iba1-positive cells in the DG of the hippocampus. ∗P <0:05; ∗∗P <0:01; ns = not significant. Values are expressed as mean ± SEM. n =3.

In fact, autophagy and apoptosis constitute a strategy to the cell. Preclinical experiments have found that signs of adapt to and cope with the stress. The two affect each other decreased autophagy appeared in both chronic unpredict- and form a complex molecular regulatory network [38]. able stress and LPS-related depression model [41, 42]. Stress is known to predispose rodents to depressive-like Our results confirmed that CRS does cause differences in behaviors via inducing apoptosis in the hippocampus of the expression of autophagy and apoptotic factors in the mice [39]. Studies have shown that knocking out the proa- hippocampus; i.e., CRS results in increased p62 and poptotic factor Bax can reduce anxiety and depression-like decreased Beclin1. Unfortunately, we did not find any dif- behaviors in a mouse model of stress [40]. Analogously, ferences upon LC3II expression. However, the features of enhanced apoptosis was detected in the hippocampus of depression could be prevented through increased autoph- rats tested in a repeated unpredictable stress model of agy and antiapoptosis activity as a result of AGO or FLX depression [22]. The results of the present study indicate administration. This is consistent with our previous that AGO acts as an antidepressant partly through its role research on the antidepressant mechanism of FLX [43]. in improving the ratio of Bcl2/Bax. In many cases, autoph- Therefore, we think that this might represent a potential agy usually manifests well before apoptosis “dismantles” antidepressant mechanism orchestrated by AGO. 8 BioMed Research International

⁎⁎ ⁎ 60 ⁎⁎ ns ns 0.051 ⁎⁎ 40 ns 20 � mol/g)

4 ns ⁎⁎ ⁎ 2 ns ⁎ ⁎ 0.6 ns ns Te levels of SCFAs ( SCFAs of Te levels 0.3 0 Acetate Propionate Iso-butyrate Butyrate Valeric acid Total SCFAs

Con AGO CRS FLX

Figure 4: Eﬀects of AGO on CRS-induced SCFA changes in fecal samples. ∗P <0:05; ∗∗P <0:01; ∗∗∗P <0:001; ns = not signiﬁcant. The values represent mean ± SEM. n =8.

SCFAs are the key molecules that modulate microglial important role of these cellular events in depression, and maturation and function, as well as depression [44–46]. In therefore might be used as an antidepressive drug in CRS. other studies, the mechanism of melatonin receptor expression may involve the action of SCFAs [47], and this effect does not necessarily affect the signals of brain neu- Data Availability rons. It may be manifested by altering brain inflammation, fi ff The data used to support the ndings of this study are avail- a ecting neuronal BDNF or autophagy pathways. Results able from the corresponding author upon request. from this study indicate that the CRS procedure can significantly increase isobutyrate level in feces and decrease IBa1 expression in the hippocampus indicating that isobu- Conflicts of Interest tyrate plays an important role in the CRS procedure and fl may be involved in regulating the expression of IBa1. As All authors have no con icts of interest to disclose. microglia are the resident immune cells in the brain, they normally respond to various neuropathological stimuli, Acknowledgments including stress, injury, and infection [48]. Once activated, a rapid proliferation in microglial densities and morpho- The National Natural Science Foundation of China (grant logical changes result, followed by the release of proin- numbers 81871063 and 81971769) supported this study. flammatory and anti-inflammatory cytokines [49]. In this We are grateful to the Core Facilities of Zhejiang University study, we found that CRS-induced depression is accompa- and Institute of Neuroscience and Institute of Agrobiology nied by elevated proinflammatory cytokine regulators such and Environmental Sciences, Zhejiang University, for pro- as transcription factor NF-κB or increased iNOS, in line viding technical assistance. with previous studies [50]. As a novel antidepressant, AGO can rebalance the above changes to exert an antidepressant effect. In other words, AGO can exert antidepres- References sant effects through immune pathways. More importantly, [1] E. Falkum, G. Pedersen, and S. Karterud, “Diagnostic and Sta- for the changes in the content of isobutyric acid induced tistical Manual of Mental Disorders, Fourth Edition, paranoid by CRS, we found that AGO could not reverse the disor- personality disorder diagnosis: a unitary or a two-dimensional dered concentration but FLX did. The possible reasons construct?,” Comprehensive Psychiatry, vol. 50, no. 6, pp. 533– might be related to the different molecular mechanisms 541, 2009. of the two drugs. [2] J. Wang, X. Wu, W. Lai et al., “Prevalence of depression and depressive symptoms among outpatients: a systematic review 5. Conclusion and meta-analysis,” BMJ Open, vol. 7, no. 8, article e017173, 2017. Taken together, these data provide further evidence for a [3] Z. Aszalos, “Effects of antidepressants on sleep,” Orvosi Heti- beneficial role of AGO and its effects in the hippocampus lap, vol. 147, no. 17, pp. 773–783, 2006. fi under stressful conditions and con rmed that AGO can [4] S. S. Morimoto, D. Kanellopoulos, K. J. Manning, and G. S. protect the CRS mice from depression. AGO may funnel Alexopoulos, “Diagnosis and treatment of depression and cog- its antidepressant effect through part of the molecular nitive impairment in late life,” Annals of the New York Acad- pathways delineated in the above, further confirming the emy of Sciences, vol. 1345, no. 1, pp. 36–46, 2015. BioMed Research International 9

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