Antidepressant effects of a polysaccharide from okra (Abelmoschus esculentus (L) Moench) by anti-inflammation and rebalancing the gut microbiota

Journal Pre-proof Antidepressant effects of a polysaccharide from okra (Abelmoschus esculentus (L) Moench) by anti-inflammation and rebalancing the gut microbiota

Tingxu Yan, Tingting Nian, Zhengzheng Liao, Feng Xiao, Bo Wu, Kaishun Bi, Bosai He, Ying Jia PII:

S0141-8130(19)39679-5

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.138

Reference:

BIOMAC 14165

To appear in:

International Journal of Biological Macromolecules

Received date:

26 November 2019

Revised date:

6 December 2019

Accepted date:

15 December 2019

Please cite this article as: T. Yan, T. Nian, Z. Liao, et al., Antidepressant effects of a polysaccharide from okra (Abelmoschus esculentus (L) Moench) by anti-inflammation and rebalancing the gut microbiota, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.12.138

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© 2019 Published by Elsevier.

Journal Pre-proof Antidepressant effects of a polysaccharide from okra (Abelmoschus esculentus (L) Moench) by anti-inflammation and rebalancing the gut microbiota Tingxu Yana, Tingting Nianb, Zhengzheng Liaob, Feng Xiaoa, Bo Wua, Kaishun Bic, Bosai Hea, and Ying Jiaa* a

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School of Functional Food and Wine, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China b School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China c School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, China

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Correspondence to: Jia, Ying, School of Functional Food and Wine, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang, 110016, PR China. E-mail address: [email protected]

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Abstract The present study aimed to evaluate the antidepressant-like effect of a polysaccharide (OP), which is isolated from okra (Abelmoschus esculentus (L) Moench), in CUMS-induced mice and its possible mechanisms. OPT, FST and TST were employed to examine the anxiety and depressive behavior in CUMS-induced mice and fecal microbiota transplantation (FMT) CUMS-induced mice, while proinflammatory cytokines, TLR4/ NF-κB pathway and MAPKs signaling were detected in both CUMS-induced mice and LPS-induced BV2 cells. The results showed that anxiety- and depressive-like behaviors, gut microbiota dysbiosis and changes of SCFAs, and activation of inflammatory reactions in the colon, serum, and hippocampus of CUMS-induced mice, as well as activation of inflammatory reactions in BV2 cells, could be alleviated by the treatment of OP. The mice that were colonized by OP microbiota showed improved anxiety and depressive behaviors and lower inflammatory response. Furthermore, OP inhibited the expression of TLR4, the nuclear translocation of NF-κB and high levels of proinflammatory cytokines, and enhanced the MAPKs signaling, these effects of OP also observed in LPS-induced BV2 cells. Above all, suggested that the potential mechanism of the antidepressant-like effects of OP was closely correlated with the bidirectional communication of microbiota-gut-brain axis via regulation of inflammation response. Keywords: OP, microbiota, inflammation, depression, TLR4/ NF-κB

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1.Introduction Depression is a major human blight and the primary cause of disability and early mortality from suicide worldwide. This is large because 350 million people suffer from it according to the World Health Organization report. Major depressive disorder (MDD) is a heterogeneous and etiologically complex psychiatric disorder, including genomic and environmental factors, neuroendocrine and neurotransmission abnormalities, inflammation, metabolic dysfunctions and dysbiosis of gut microbiome [1, 2]. It is currently believed that the inflammatory hypothesis is one of the most prevalent topics concerning MDD, and activation of the inflammatory response system acts as a central part of the pathophysiology of depression. One of the most consistent findings on the association between inflammation and depression are increased levels of proinflammatory cytokines in both plasma and CNS among patients with depression, and cytokines such as TNF-α, IL-1β and IL-6 were further proved that could influence the progression and severity of depressive disorders[3]. This increase in cytokines may be further related to abnormalities of Toll-like receptors (TLRs), and TLR4 would be activated by MyD88 pathway and caused the production of proinflammatory cytokines, along with the upregulation of proteins including COX-2, activation of NLRP3 inflammasome, NF-κB and MAPKs signaling, and resulting in inflammatory response[4, 5]. In addition, the gut microbiota is involved in the regulation of host inflammatory responses and can fuel inflammation, through short-chain fatty acids[6]. Moreover, stress can impact the gut microbiota and reshape its composition, affecting the regulation of the proinflammatory cytokines[7]. Peripheral cytokines have been shown to cross the blood-brain barrier by the circumventricular organs[8]. Very recent evidence indicated that gut microbiota communicates with the CNS through neural and immune pathways, and these bidirectional communication pathways constitute the microbiota-gut-brain axis which is vital for depression[9]. A polysaccharide (OP), which is isolated from okra (Abelmoschus esculentus (L) Moench), is one of the major polysaccharides components and the largest proportion of the compounds in okra which has been used as a food for the treatment of intestinal and urethral disorders[10]. Our previous studies had found OP could reverse high-fat-diet-induced metabolic disorder, degeneration of neurons and activation of AMPK signaling pathway, and decrease the high expression of inflammatory cytokines[11]. It is of significance to probe the rebalancing metabolic effects of OP in view of gut microbial and behavioral changes. The present study was designed to investigate the antidepressant-like effects of OP and the possible bidirectional communication mechanisms of the microbiota-gut-brain axis in a chronic, unpredictable, mild stress (CUMS)-induced mouse model. 2.Materials and methods 2.1 Materials OP (Mw 626 kDa) was isolated by using the previous method[12], dissolved in distilled water. The GC-MS results showed that OP contained rhamnose (6.11%), arabinose (5.51%), galactose (7.12%), glucuronic acid (39.25%) and galactosyl acid

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(42.01%). LPS from E. coli 055:B5 was obtained from Sigma-Aldrich (St. Louis, Missouri) and dissolved in saline in a tube. ELISA kits of TNF-α, IL-6, and IL-1β were purchased from Mlbio Biology (Shanghai, China). Standards: Acetic acid (CAS No.64-19-7, ≥99.7%), propionic acid (CAS No.79-09-4, ≥99.5%), isobutyric acid (CAS No.79-31-2, ≥99.5%), butyric acid (CAS No.107-92-6, ≥99.0%), isovaleric acid (CAS No.503-74-2, ≥99.0%), valeric acid (CAS No.109-52-4, ≥99.0%), hexanoic acid (CAS No.142-62-1, ≥99.0%) and heptanoic acid (CAS No.111-14-8, ≥99.0%) were all purchased from Sigma-Aldrich (St. Louis, Missouri). All other chemicals and reagents were of analytical grade. 2.2 Animals and experimental protocol 90 male C57BL/6 mice (18-22 g) were purchased from the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China). The animals were housed (4 mice/cage, 12 h light/dark cycle) under pathogen-free conditions in temperature (22-24°C) and humidity (55 ± 10%), and allowed free access to food and water. The study was carried out in compliance with the National Institutes of Health and institutional guidelines for the humane care of animals and was approved by the Animal Care Committee of Shenyang Pharmaceutical University (Protocol No.: SYPU-IACUC- C2018-11-2-101). After acclimatizing the animals for 7 days, male C57BL/6 mice were randomly divided into 9 groups (n=10/group) for two sets of experiments. 2.2.1 Experiment 1: 6 groups of mice in the first set of experiments: (1) CON (control group not subjected to any stress), (2) CUMS (CUMS group subjected to the CUMS procedure), (3) OP (OP treatment+CUMS group), (4) CON-D (control-donor group not subjected to any stress), (5) CUMS-D (CUMS-donor group subjected to the CUMS procedure), (6) OP-D (OP treatment+CUMS-donor group); Mice were treated with vehicle or OP (400 mg/kg, i.g.) for 14 days. The dose and the method of administration of OP were chosen based on our previous study[11]. After 4 weeks of CUMS, the CON group, the CUMS group and the OP group were subjected to a series of behavioral tests OFT, FST and TST in sequence, between two of which test were allowed to rest for 2 h. After the finish of the behavioral tests, mice were used to collect serum and cecum content. Blood samples from eye socket were collected and then centrifuged at 4500 rpm for 15 min. The serum and cecum contents were harvested and stored at -80℃. Finally, mice were euthanized by decapitation, and whole-brain (stored at 4% paraformaldehyde), hippocampus and colon were rapidly removed and stored at -80°C. The CON-D, CUMS-D and OP-D groups, did not perform the behavioral tests, and the cecum content from donor mice was collected and mixed from the same group, then diluted 40-fold in PBS immediately. After centrifugation for 5 min at 4°C, the supernatant was collected under sterile conditions and stored at -80℃ under FMT. 2.2.2 Chronic unpredictable mild stress (CUMS) protocol CUMS was performed as previously described[13]. CUMS consisted of exposure to a variety of unpredictable stressors (randomly), including (1) 24 h food deprivation, (2) 24 h water deprivation, (3) 1 h exposure to an empty bottle, (4) exposure to an

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empty cage (without sawdust bedding), (5) grouped housing, (6) 24 h soiled cage (200 mL water in 100 g sawdust bedding), (7) level shaking for 30 min, (8) 5 min cold swimming (5°C), (9) nip tail for 1 min. All stresses were applied individually and continuously, day and night. The control animals were housed in a separate room and had no contact with the stressed groups. To prevent habituation and to ensure the unpredictability of the stressors, all stresses were randomly scheduled, repeated throughout the 4-week experiment. The control group mice were left undisturbed except for necessary procedures such as routine cage cleaning. 2.2.3 Experiment 2 3 groups of mice in the second set of experiments: (1) FMT+CON, (2) FMT+CUMS, (3) FMT+OP. All the mice were given an antibiotic cocktail of vancomycin (0.125 mg/day) and neomycin, gentamicin metronidazole, ampicillin (0.25 mg/day each) once daily for 2 weeks by gavage. 200 μL of donor supernatant from the donor mice of Experiment 1 was administrated to all mice every other day for the next 2 weeks by gavage as well after a three-day washout period. The behavioral tests were performed after the FMT procedure. The hippocampus was collected and immediately stored at -80°C for further detection. 2.3 Behavioral studies 2.3.1 Open field test (OFT) In order to eliminate potential effects of drug administration on spontaneous locomotor activity, the animals were carried on the open-field test as the previous study described[14]. In a word, mice were individually placed into a white plastic box (40 cm × 40 cm × 60 cm) with the floor divided into 12 equal squares. Each mouse was placed individually into the center of the arena and allowed to freely explore the open field for 6 min per trial. The total distance was counted as locomotor activities. The boxes were cleaned with 50% ethanol after each trial. 2.3.2 Elevated plus maze test (EPM) The EPM test was performed according to previously reported with little modifications[15]. The EPM consisted of two open (8 × 17 cm) and two closed (8 × 17 × 30 cm) arms, which were connected by a central platform (8 × 8 cm) elevated 30 cm above floor level. The mice were placed on the central platform using cupped open hands, facing an open arm. The number of entries made into the open arms and closed arms during a 5 min period was counted, and the time spent in the open arms was determined by an observer blinded to treatment. The percentage of entries made into the open arms (%) was calculated. Entry into an arm was considered valid only when all four paws of the mouse were inside that arm. The apparatus was thoroughly cleaned with 70% ethanol after each trial to avoid the residual odors. 2.3.3 Tail suspension test (TST) The TST was carried out on the basis of previously reported methods[16]. In brief, mice were suspended 60 cm above the floor by their tails for 6 min and the immobility time was set down during the last 4 min. 2.3.4 Forced swimming test (FST)

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FST was carried out as previously described[17]. In brief, each mouse was individually placed into a transparent glass container (height: 25 cm, diameter: 15 cm) filled with water (depth: 15 cm). Each mouse was forced to swim for 6 min and the immobility time was recorded during the final 4 min. 2.4 Determination of cytokines in colon, serum, and hippocampus The concentrations of TNF-α, IL-6, and IL-1β in hippocampus, serum and colon were measured by ELISA kits in accordance with the manufacturer's instructions. The results were exhibited as pg/mL. 2.5 Histopathological examination The colons were soaked in 4% paraformaldehyde in 0.1 mol/L PBS (pH 7.4) for 48 h, dehydrated, embedded in paraffin, cut into 5μm slices transversely with a microtome and stained with hematoxylin and eosin (H.E.) and analyzed by a light microscope. 2.6 16S rRNA analysis of fecal microbiota The experiments including extracting the total DNA from cecum content samples and analyzing microbial composition via 16S rRNA sequencing analysis were undertaken by Sangon Biotech (Shanghai, China). To analyze the taxonomic composition of the bacterial community, the V3-V4 region of the 16S rRNA gene was selected for the subsequent pyrosequencing. The V3–V4 region of the bacterial 16S rRNA gene was amplified by PCR using the following primer pair: 341F, CCCTACACGACGCTCT TCCGATCTG, and 805R, GACTGGAGTTCCTTGGCACCCGAGAATTCCA. The collected outcomes were analyzed on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) according to standard instructions. 2.7 SCFAs concentration analysis The concentrations of SCFAs (including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, hexanoic acid, and heptanoic acid) in mice cecum content samples were determined by GC-MSTQ8040 (Agilent, USA), fitted with a DB-FFAP column (30 m × 0.25 mm × 0.25 µm, Agilent, United States). Standard solutions of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, hexanoic acid, and heptanoic acid were prepared at 1, 0.4, 0.2, 0.1, 0.05, 0.01, 0.01 and 0.005 mg/mL respectively. And the internal standard substance of 2-methylbutyric acid was prepared at 1μg/mL. Each fecal sample was soaked in 10 mL double distilled water by vortexing and centrifuging at 5,000 rpm for 20 min at 4°C. Then the 450 µL liquid of supernatant was added by 100 µL concentrated HCl and 500 µL internal standard solution, vortexing for 1 min and centrifuged at 15,000 rpm for 10 min, then the mixture was reserved in -20°C. 2.8 Fluorescence quantitative real-time-PCR The transcription levels of TLR4, IKKα and NF-κB p65 in the brain and was determined with quantitative real-time-PCR. Total RNA was isolated using TRIpure Lysis Buffer (BioTeke, Bejing, China). Reverse transcription was performed with 2 μg RNA using Super M-MLV and RNase inhibitor (BioTeke, Bejing, China). qRT-PCR was carried on Fluorescence quantitative PCR ExicyclerTM 96 (BIONEER, Korea). Reaction procedures ere as follow: an initial step at 94°C for 5 min, 40 cycles of 94°C for 10 s, 60°C for 20 s and 72°C for 30 s. The primers used were as the following: TLR4, fwd 5′-AGCAGGTGGAATTGTATCGC- 3′, rev 5′-TCAGGTCC AAGTTGCCGTTT-3′; IKKα, fwd

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5′-ACCGTGAACATCCTCTG- 3′, rev 5′-CTGCT CTGGTCCTCATT-3′; NF-κB p65, fwd 5′-CGGCCTCATCCACATGAACT- 3′, rev 5′- GAACGTGAAAGGGGTTATTG-3′; β-actin, fwd 5′-CTGTGCCCATCTACGAG GGCTAT-3′, rev 5′-TTTGATGTCACGCACGATTTCC-3′. Primers were designed with Primer Express Software. Data were analyzed via the 2 △△ CT method. 2.9 BV-2 microglia cell culture and treatment The BV2 microglia were cultured in DMEM medium supplemented with 10% FBS, at 37 °C in an incubator with 5% CO2. The cells were passaged 2 to 3 times each week, and cells passaged over 3 times could be used for the experiments. Potential cytotoxicity of OP was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Beyotime Institute of Biotechnology) assay. BV2 cells were seeded into 96-well plate and incubated overnight. BV2 cells were pretreated for 2 h with OP (0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, 200 μg/mL) and then stimulated with LPS (1 mg/mL) for 24 h. Cells were then incubated in 0.5 mg/mL MTT solution and the absorbance at 570 nm was measured using a microplate reader (M1000, TECAN, Austria GmbH, Austria). Three independent experiments were performed. 2.10 Measurement of cytokines of BV2 microglia cell BV2 cells were seeded into 96-well plate and incubated overnight. BV2 cells were pretreated for 2 h with OP (12.5, 25, 50, 100 μg/mL) and then stimulated with LPS (1 μg/mL) for 24 h. The supernatant was collected and used to test the NO. And TNF-α, IL-6, and IL-1β were determined by ELISA and RT-PCR. 2.11 Immunofluorescence (IF) Staining analyses BV2 cells were seeded into 96-well plate and incubated overnight. BV2 cells were pretreated for 2 h with OP (25, 50 μg/mL) and then stimulated with LPS (1μg/mL) for 24 h. The cells were washed with PBS, fixed with 4% polyformaldehyde PBS for 30 minutes, then infiltrated with 0.1% Triton X-100 at room temperature for 10 minutes. The cells were then blocked by 3% BSA and incubated overnight with primary antibodies anti-p65 overnight. The second antibody CY3 anti-rabbit (1:2000; Abcam, USA) immunoglobulin were incubated at 24 ℃ for 1 hour. Cells were washed with PBS three times. The images were collected by fluorescence microscopy (Leica Microsystems Trading Ltd). IOD was analyzed by image J 64 and image-pro plus 6.0 2.12 Western Blot (WB) Analysis Total protein was extracted from BV2 cells and hippocampal tissues using RIPA Lysis and Extraction Buffer. Protein concentrations were determined by using a BCA kit. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for approximately 90 min before being transferred to the nitrocellulose filter membrane. The membranes were blocked with 5% skim milk dissolved in Tris-buffered saline with Tween 20 (TBST) at room temperature for 2 h and probed with antibodies against COX2 (1:500), NLRP3 (1:1000), IκBα (1:1000), iNOS (1:1000), ERK1/2 (1:1000), JNK (1:1000), P38 (1:1000), p-ERK1/2 (1:1000), p-JNK (1:1000), p-P38 (1:1000), TLR4 (1:1000), PI3K (1:1000), AKT (1:1000), GSK3β (1:1000), P-PI3K (1:1000), p-AKT (1:1000), p-GSK3β (1:2000), and β-actin, overnight at 4℃. The next day, membranes were washed three times in TBST and then

Journal Pre-proof incubated with the corresponding HRP-labeled secondary antibodies (1: 3000). After washing the membranes three times with TBST, ECL reagent was used to identify immunoreactive bands. The signals were detected by analyzed using Image pro plus 2.13 Statistical analysis

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All experimental data were represented as mean ± SEM and analyzed by one-way analysis of variance (ANOVA) followed by Tukey multiple comparison tests using GraphPad Prism 8 (GraphPad Software, USA). The analysis results were only reported when a significant difference was noticed. A value of p˂0.05 was considered statistically significant. 3.Results 3.1 Effects of OP on depressive-like behavior of CUMS-induced mice Data of OFT (Fig.1A) showed that there are no significant differences in the total locomotor distance among all groups, which indicated that either CUMS procedure nor OP treatment cause any influence on the spontaneous locomotor activity of mice. Time in the center of OFT was remarkably less in the CUMS group than in the CON group (p<0.001), and this result was ameliorated by administration with OP (p<0.001), illustrating that OP could against the anxiety symptom induced by CUMS (Fig.1B). This speculation was confirmed by the decrease of entries in open arms (Fig.1C, p<0.001) and time spent in open arms (Fig.1D, p<0.05) of the OP mice in EPM. The immobility time was extremely increased in CUMS-induced mice in TST (Fig.1E, p<0.01) and FST (Fig.1F, p<0.001) compared with the control mice. And OP treatment reversed the above changes (p<0.05, p<0.001) and suggested a promising antidepressant-like effect.

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D E F Fig.1 Effects of OP on depressive and anxiety behavior of CUMS-induced mice. (A) Total distance in OFT, (B) Center distance in OFT, (C) Entries in open arm in EPM, (D) Time spent in open arm in EPM, (E) Immobility time in TST, (F) Immobility time in FST. The data represented the values of mean±SEM from 10 mice/group. **p<0.01, *** p<0.001 vs. CON group, #p<0.05, ###p<0.001 vs. CUMS group. 3.2 Effects of OP on cytokines in colon, serum, and hippocampus of CUMS-induced mice

Journal Pre-proof As shown in Fig.2, the levels of TNF-α (2A) and IL-6 (2B) were increased by CUMS in the colon (p<0.01, p<0.05, p<0.05), plasma (p<0.01, p<0.05, p<0.05) and hippocampus (p<0.01, p<0.05, p<0.05), and increased level of IL-1β (2C) in colon (p<0.05). OP treatment significantly lowered the above-elevated levels of cytokines, which indicated that OP could directly regulate inflammation in the CNS and peripheral regions. CUMS * ##

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Fig.2 Effects of OP on cytokines in colon, serum, and hippocampus of CUMS-induced mice. (A) TNF-α, (B) IL-6 and (C) IL-1β. The data represented the values of mean±SEM from 10 mice/group. *p<0.05, **p<0.01 vs. CON group, #p<0.05, ##p<0.01 vs. CUMS group. 3.3 Effects of OP on histopathological changes in the colon of CUMS-induced mice Due to the rising proinflammatory factors of the colon, we investigated the histopathological changes of colon. The morphological changes of glandular cells and intestinal mucosa were mainly examined by H.E. As shown in Fig.3, CUMS procedure induced the mice colon glandular cells row messy and damaged the integrity of intestinal mucosa. Interestingly, OP treatment partly inhibited histopathological damages.

CON CUMS OP Fig.3 Effects of OP treatment on CUMS-induced histopathological changes in the colon (H.E. staining, 100×). 3.4 Effects of OP on intestinal microflora profiles and composition of CUMS-induced mice Due to the interactions between proinflammatory cytokines in colon and gut microflora, we applied 16S rRNA sequencing analysis to investigate the changes of gut microbiota in the CUMS-induced mice. Shannon index (Fig.4A) and Chao1 index (Fig.4B) and were decreased and Simpson index (Fig.4C) was increased in the CUMS group (p<0.05, p<0.05, p<0.05), and OP treatment reversed the above variations (p<0.05, p<0.05, p<0.05). Further, the composition of gut microbiota of each fecal sample based on different levels was exhibited (Fig.4D-4M). From the quantitative pictures, at the phylum level, the relative proportions of Bacteroidetes (p<0.05) and

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Actinobacteria (p<0.05) were increased, whereas Firmicutes (p<0.05) were decreased in the CUMS-induced depressive mice, and OP administration alleviated the phylum alterations (p<0.05, p<0.05, p<0.05). At the class level, the relative abundance of Bacteroidia (p<0.05) and Actinobacteria (p<0.05) were elevated, when the Clostridia were declined in the CUMS group (p<0.05), and the OP also reversed the changes of class (p<0.05, p<0.05, p<0.05). At the order level, we found the relative ratios of Bacteroidales have risen (p<0.05), when Clostridiales were decreased in the CUMS mice (p<0.05) and the levels were changed by OP (p<0.05, p<0.01). At the family level, we found that the levels of Lachnospiraceae (p<0.001) and Lactobacillaceae (p<0.05) were decreased, Bacteroidaceae (p<0.05) was elevated in the CUMS group, and OP treatment improved the situation (p<0.001, p<0.05, p<0.05). At the genus level, the relative abundance of Barnesiella (P<0.01) and Bacteroides (p<0.05) were increased, Lactobacillus (p<0.05) was reduced in CUMS mice, and these changes were also reversed by OP supplementation (p<0.05, p<0.05, p<0.05).

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Fig.4 Effects of OP treatment on gut microbiota changes of CUMS-induced mice. (A). Shannon index, (B) Chao1 index, (C) Simpson index, (E) Samples community distribution barplots of phylum (D, E), class (F, G), order (H, I), family (J, K) and genus (L, M). As was shown in the graph, different colors correspond to the names of different classified levels. The width of each color expressed the ration of relative abundance. *p<0.05, **p<0.01, ***p<0.001 vs. CON group. #p<0.05, #p<0.01, ### p<0.001 vs. CUMS group.

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3.5 Effects of OP on levels of SCFAs of CUMS-induced mice GC-MS was employed to detect the concentrations of SCFAs (including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, hexanoic acid, and heptanoic acid) in mice cecum content samples. As shown in Fig.5, CUMS procedure increased the level of isovaleric acid (p<0.001) when decreased the levels of acetic acid (p<0.001), propionic acid (p<0.001) and butyric acid (p<0.001), and then OP treatment reversed these changes (p<0.001, p<0.001, p<0.001, p<0.001).

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Fig.5 Effects of OP on CUMS-induced SCFAs changes in cecum content samples. The data represented the values of mean±SEM from 6 mice/group. *p<0.05, **p<0.01 vs. CON group. ###p<0.001 vs. CUMS group. 3.6 Effects of OP on the TLR4/NF-κB signaling pathway in the hippocampus of CUMS-induced mice RT-PCR results (Fig.6A) showed that the mRNA expressions of TLR4, NF-κB p65 and IKKα were significantly upregulated in the CUMS group, and OP restored these variations. Based on the results of western blot (Fig.6B), IκBα was activated by OP inhibiting the phosphorylation of IKKα, which blocked the nucleus translocation of NF-κB to induce further inflammation response. Moreover, we also found OP could reduce the neuroinflammation might by regulating the downstream cascades, like NLRP3, MymD88, and PI3K/AKT/GSK3β signaling.

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Fig.7 Western blot analysis of OP on the MAPK signaling pathway in the hippocampus of CUMS-induced mice. The data represented the values of mean±SEM. *p<0.05, ** p<0.01 vs. CON group. #p<0.05, ##p<0.01 vs. CUMS group. 3.8 Transplantation of CUMS or OP microbiota on depressive-like behavior in the recipient mice Compared with the mice colonized with the CON microbiota, the mice colonized with the CUMS microbiota exhibited increased anxiety and depressive symptoms in OFT, EPM, TST, and FST (Fig.8). However, the OP-recipient mice spent longer time in the center zone (p<0.01) in OFT, more entries (p<0.05) and time in the open arms (p<0.05) in EPM, higher immobility during the TST (p<0.05) and FST (p<0.01) compared to the CUMS recipient mice.

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Fig.8 Transplantation of CUMS or OP microbiota on depressive and anxiety behavior of CUMS-induced mice. (A) Total distance in OFT, (B) Center distance in OFT, (C) Entries in open arm in EPM, (D) Time spent in open arm in EPM, (E) Immobility time in TST, (F) Immobility time in FST. The data represented the values of mean±SEM from 10 mice/group. *p<0.05, **p<0.01 vs. FMT+CON group, #p<0.05, ##p<0.01 vs. FMT+CUMS group. 3.9 Transplantation of CUMS or OP microbiota on hippocampal cytokines levels in the recipient mice

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As shown in Fig.9, the levels of TNF-α, IL-1βand IL-6 were increased in CUMS-recipient mice (p<0.01, p<0.05, p<0.05). However, the above-elevated levels of cytokines were significantly lowered in OP-recipient mice, which indicated that OP involved in the mechanism of neuroinflammation and gut microbiota.

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Fig.9 Transplantation of CUMS or OP microbiota on hippocampal cytokines levels in the recipient mice. (A) TNF-α, (B) IL-6 and (C) IL-1β. The data represented the values of mean±SEM from 10 mice/group. *p<0.05, **p<0.01 vs. FMT+CON group, #p<0.05, ## p<0.01 vs. FMT+CUMS group. 3.10 Effects of OP on histopathological changes in the colon of CUMS-induced mice The histopathological changes of the colon after FMT were investigated as shown in Fig.10. The mucosal epithelial structure of FMT+CON mice was intact and clear, without necrosis or ulcer. In the FMT+CUMS group, mucosa defects were observed in colon, and a large number of inflammatory cells infiltrated in the mucosa, submucosa and muscle layer. The mucosal ulcer surface was healed and the infiltration of inflammatory cells in mucosa and submucosa was reduced in FMT+OP mice.

FMT+CON FMT+CUMS FMT+OP Fig.10 Transplantation of CUMS or OP microbiota histopathological changes in the colon (H.E. staining, 100×). 3.11 Effects of OP on NO production in LPS stimulated BV2 microglial cells The safety of OP for BV2 microglia was determined by MTT assay. As shown in Fig. 11A, cell viability was not significantly different between control and OP-treated groups with different doses (0, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 μg/mL), indicating OP has acceptable safety. All data are expressed as the percentage normalized to the control group. In order to analyze the suppressive effect of OP on NO production, BV2 microglial cells were pretreated with different concentrations of OP for 2 h, followed by

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treatment with LPS for 24 h, before determination of the levels of NO in the culture media by NO assay. LPS markedly increased NO production in BV2 microglial cells. On the other hand, OP significantly decreased NO production in LPS-stimulated BV2 microglial cells. In particular, 25, 50 and 100 μg/mL OP significantly decreased the LPS induced NO production; however, this downregulation did not reach the level observed for untreated control (Fig. 12B).

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A B Fig.11. Effect of OP on NO release in BV2 cells. (A) MTT assay. (B) NO release in BV2 cells. The data represented the values of mean±SEM (n=3). **p<0.01, ***p<0.001 vs control group, ##p<0.01, ###p<0.001 vs LPS group. 3.12 Effects of OP on inflammatory response in LPS stimulated BV2 microglial cells To evaluate the effects of OP on inflammatory response in BV2 cells, pro-inflammatory cytokines were investigated. Secreted IL-1β, IL-6 and TNF-α in culture medium were assessed by ELISA. Statistical results (Fig.12A, 12C, 12E) showed that the three cytokines were induced greatly at 24 h of stimulation, while the release of IL-6 and TNF-α were significantly decreased after OP protection Relative mRNA expressions of the IL-1β, IL-6 and TNF-α gens in BV2 were assayed by quantitative real-time PCR kit with β-actin as internal control. Consistent with the result of ELISA, the mRNA of IL-6 and TNF-α were decreased after OP treatment (Fig.12B, 12F).

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Fig.12 The effect of OP on inflammatory response in LPS stimulated BV2 microglial cells. (A) The protein levels of TNF-α. (B) The mRNA levels of TNF-α. (C) The protein levels of IL-6. (D) The mRNA levels of IL-6. (E) The protein levels of IL-1β. (F)The mRNA levels of IL-1β. The data represented the values of mean±SEM. *p<0.05, ** p<0.01, vs control group, #p<0.05, ##p<0.01 vs LPS group. 3.13 Effects of OP on nuclear translocation of the NF-κB in LPS stimulated BV2 microglial cells NF-κB is a transcription factor known to directly regulate many proinflammatory genes. The results of IF confirmed that LPS treatment significantly increased the nuclear translocation of the NF-κB; however, pretreatment with OP (25, 50 μg/mL) suppressed LPS-induced NF-κB activity (Fig.13).

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Fig.13. Effects of OP on the nuclear translocation of NF-κB detected by immunofluorescence staining with anti-p65 antibody (green) and DAPI (nuclei, blue). Magnification: ×200. Scale bar, 50μm. 3.14 Effects of OP on the NF-κB and MAPK signaling pathway in LPS stimulated BV2 microglial cells Fig.14 showed that LPS stimulation increased the nuclear translocation and OP inhibited this action, which is consistent with the IF results and western blot results in the hippocampus of CUMS-induced mice. Besides that, OP could also reduce the expression of MAPK signaling which also in line with the previous data. Furthermore, OP could significantly downregulate the expressions of COX2 and iNOS.

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Fig.14 The effect of OP on the NF-κB and MAPK signaling pathway in LPS stimulated BV2 microglial cells. (A) Nucleus NF-κB. (B) Cytosolic NF-κB. (C) TLR4. (D) ERK1/2. (E) JNK. (F) P38, (G) COX2, (H) iNOS. The data represented the values of mean±SEM. * p<0.05, **p<0.01, ***p<0.01vs control group, #p<0.05, ##p<0.01, ###p<0.001 vs LPS group. Discussion In the current work, we offer new insights into the molecular mechanisms underlying the development of depression induced by CUMS in mice. The major findings were as follows. (1) OP could alleviate depressive and anxiety behavior and reduce the rising proinflammatory cytokines in the colon, serum, and hippocampus caused by CUMS procedure. (2) The gut microbiota profiles and composition were regulated by OP treatment. OP decreased the level of isovaleric acid when increased the levels of acetic acid, propionic acid, and butyric acid. (3) Along with CUMS-induced the elevated proinflammatory cytokines, TLR4/NF-κB and MAPKs signaling, and its downstream cascades increased in hippocampus, while CUMS-induced those effects almost reversed by OP. (4) FMT could lighten the symptoms of depression and anxiety of CUMS-recipient mice, and restore the histopathological damage in colon as well. (5) The results of BV2 microglial cells further proved that OP could inhibit the NO production, inflammatory response and the nuclear translocation induced by LPS, together with the downregulation of MAPKs signaling. These findings illustrated that the antidepressant-like effects of OP in CUMS-induced mice maybe via inhibiting inflammatory response of microbiota-gut-brain axis. Clinical and preclinical studies have confirmed the close relationship between MDD and chronic low-grade inflammatory response. Elevated peripheral blood inflammatory cytokines were found in patients with depression, which have been shown to access the brain and interact with virtually every pathophysiologic domain

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known to be involved in depression[18]. Consistent with the published data, we found that the levels of proinflammatory cytokines were increased in serum and hippocampus of CUMS-induced mice. Besides we also observed elevated cytokines and histopathological damages in colon. This intestinal inflammation refers to the impairment of the intestinal mucosal barrier, leading to the entry of endotoxin and proinflammatory cytokines in the blood system[19], which proved by the increased cytokines in serum and hippocampus in our study. Previous studies reported that a reduction in microbial diversity or the complete absence of the gut microbiota alters normal immune system function[20]. According 16S rRNA sequencing analysis of gut microbiota in the CUMS-induced mice, we found that OP treatment significantly increased Shannon index and Chao1 index, decreased Simpson index, which indicating OP could restore the gut microbial diversity. This hypothesis was also proved by our FMT results. Further analysis of the composition of gut microbiota showed that OP downregulated the relative proportions of Bacteroidetes and Actinobacteria, upregulated Firmicutes at the phylum level. Fecal samples from patients with depression results showed that Bacteroidetes and Actinobacteria were significantly more abundant in MDD subjects[21], which in line with our data. In addition, Firmicutes can ferment carbohydrates to a variety of SCFAs, and the lack of these SCFAs can lead to decreased intestinal barrier function, which significant correlates with stress-induced behavioral changes[22]. At the class level, some metabolic end products of Clostridia, such as butyrate, confer benefit to the host by providing nutrition for colonocytes[23]. Numerous studies have implicated Bacteroidales is one of the predominant bacterial phyla at the colonic mucosal surface, could promote the production of IL-6 in a MyD88-dependent fashion[24]. Significant downregulation of Clostridiales has impact on the acute inflammation, and the decrease of Clostridiales and Lachnospiraceae, the expansion of Bacteroidaceae during inflammation correlated with a reduction of transcripts related to butyrate formation[25-27]. Previous studies showed that increased gut Lactobacillaceae attenuated circulating proinflammatory cytokines in mice[28], which directly demonstrated OP could alleviate the inflammatory response in colon as well. At the genus level, there was a significant positive correlation between the relative abundance of Barnesiella[29] and inflammation, and a negative correlation with Lactobacillus[30]. Additionally, SCFAs, mainly butyrate, propionate and acetate, are regarded as mediators in the communication between the intestinal microbiome and the immune system[31]. SCFAs could inhibit the nuclear transcription factor NF-κB, downregulate the proinflammatory factors, and inhibit the intestinal inflammation[32]. SCFAs are key molecules in modulating microglia maturation and function, as well as depression[33]. In our study, SCFAs were significantly increased in OP treated mice, associated with the enhanced abundance of Clostridiales and Lachnospiraceae. Above all indicated that OP-triggered SCFAs generation and anti-inflammation response by modulation of gut microbiota positively contributed to the antidepressant-like effect in CUMS-induced mice. Among the various signal transduction pathways implicated in depressive inflammation, the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) pathway

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has recently aroused more attention due to its important roles in depression[34]. TLR4-mediating innate immune response was associated with neuroinflammation[35]. Based on the results of western blot and immunofluorescence staining, we found CUMS induced a significant increase of TLR4 expression and nuclear translocation of NK-κB in the hippocampus, and the similar tendency was also observed in the LPS-induced BV2 cells. However, these effects were reversed by OP treatment. Once TLR4 activated by its ligands, it can activate the NF-κB signaling pathway through MyD88 adaptor, and linked to the transcription of many proinflammatory mediators[36, 37].Increased inflammatory cytokine production may further stimulate activation of the NF-κB signaling, leading to further release of cytokines. Following cellular stimulation by immune-modulatory factors, the IκB phosphorylation is mediated by the IKK, which frees the NF-κB dimer to undergo stable translocation into the cellular nucleus[38], our data showed that OP could positively inhibit this procedure which suggesting TLR4/NF-κB might be the target of OP in the brain. Moreover, the NLRP3 inflammasome is a cytosolic receptor protein especially located in macrophage and microglia, which cleaves pro-IL-1β to the mature IL-1β[39]. The NLRP3 inflammasome together with NF-κB pathway regulates IL-1β transcription and function[40]. In our study, OP could significantly reduce the expression of NLRP3 and the decrease of IL-1β was also observed. Akt is reported as a phosphorylation factor on IκB, which functions on the activation of NF-κB. Meanwhile, AKT acts as a central mediator of PI3K signals and the PI3K/AKT signaling pathway plays crucial parts in cell proliferation, growth, survival and immune response[41]. Western blot results showed that PI3K, AKT, GSK3β were all down-regulated under OP treatment. One interpretation of those observations is that OP inactivated PI3K/AKT/GSK3β signaling pathway, then regulated inflammatory cytokines via NF-κB. In addition to the NF-κB, MAPK signaling pathways also play an essential role in the innate immune response[42]. The identified MAPK pathways include ERK1/2, JNK, and p38 MAPK pathways, which activate downstream inflammatory signaling pathways[43]. ERK1/2 is involved in the signaling response that results in the synthesis of IL-8, IL-1β, IL-6, and TNF-α[44]. Combined with the results of CUMS-induced mice and LPS-induced BV2 cells, OP could up-regulate the expressions of phosphorylation of ERK1/2, JNK, and p38 which indicated MAPKs pathways were involved in the anti-inflammation effects of OP. Conclusion Our present study represents the first evidence that OP significantly alleviates depressive-like behaviors in CUMS-induced mice. There was a definitely bidirectional relationship between gut microbiota and depression. The predominant effect was associated with the inactivation of inflammatory reactions in the colon, serum, hippocampus as well as BV2 cells, and the down-regulation of TLR4/ NF-κB pathway, the promotion of MAPKs signaling, and rebalance of intestinal flora. OP has a predominant anti-inflammatory and anti-depressive effects against CUMS. Our findings suggested the potential mechanism of the antidepressant-like effects of OP was closely correlated with the bidirectional communication of microbiota-gut-brain axis via regulation of inflammation response.

Journal Pre-proof Conflict of interest statement The authors have no conflict of interest to declare. Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 81573580). Key Laboratory of polysaccharide bioactivity evaluation of TCM of Liaoning Province. Key techniques study of consistency evaluation of drug quality and therapeutic effect (18-400-4-08) and Liaoning Distinguished Professor Project for Ying Jia (2017). Precise screening technology of Chinese traditional medicine anti-depressant active ingredients (2017LZD01). The Doctoral Scientific Research Foundation of Liaoning Province(2019-BS-233). References [1]

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Author statement Tingxu Yan: Conceptualization, Writing-original draft, Investigation Tingting Nian.: Investigation, Writing- Original draft preparation. Zhengzheng Liao: Resources, Investigation. Feng Xiao: Methodology.: Bo Wu: Software, Validation.: Kaishun Bi, Bosai He and Ying Jia: WritingReviewing and Editing, Supervision