Oxyberberine, a novel gut microbiota-mediated metabolite of berberine, possesses superior anti-colitis effect: Impact on intestinal epithelial barrier, gut microbiota profile and TLR4-MyD88-NF-κB pathway

Journal Pre-proof Oxyberberine, a novel gut microbiota-mediated metabolite of berberine, possesses superior anti-colitis effect: impact on intestinal epithelial barrier, gut microbiota profile and TLR4-MyD88-NF-␬B pathway Cailan Li, Gaoxiang Ai, Yongfu Wang, Qiang Lu, Chaodan Luo, Lihua Tan, Guosheng Lin, Yuhong Liu, Yucui Li, Huifang Zeng, Jiannan Chen, Zhixiu Lin, Yanfang Xian, Xiaoqi Huang, Jianhui Xie, Ziren Su

PII:

S1043-6618(19)32087-0

DOI:

https://doi.org/10.1016/j.phrs.2019.104603

Reference:

YPHRS 104603

To appear in:

Pharmacological Research

Received Date:

25 September 2019

Revised Date:

11 December 2019

Accepted Date:

13 December 2019

Please cite this article as: Li C, Ai G, Wang Y, Lu Q, Luo C, Tan L, Lin G, Liu Y, Li Y, Zeng H, Chen J, Lin Z, Xian Y, Huang X, Xie J, Su Z, Oxyberberine, a novel gut microbiota-mediated metabolite of berberine, possesses superior anti-colitis effect: impact on intestinal epithelial barrier, gut microbiota profile and TLR4-MyD88-NF-␬B pathway, Pharmacological Research (2019), doi: https://doi.org/10.1016/j.phrs.2019.104603

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Oxyberberine, a novel gut microbiota-mediated metabolite of berberine, possesses superior anti-colitis effect: impact on intestinal epithelial barrier, gut microbiota profile and TLR4-MyD88-NF-κB pathway

a, b

, Gaoxiang Ai a, Yongfu Wang c, Qiang Lu d, Chaodan Luo a, Lihua

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Cailan Li

Tan a, Guosheng Lin a, Yuhong Liu a, Yucui Li a, Huifang Zeng c, Jiannan Chen a,

Guangdong Provincial Key Laboratory of New Drug Development and Research of

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a

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Zhixiu Lin e, Yanfang Xian e, Xiaoqi Huang a, Jianhui Xie f,*, Ziren Su a,*

Chinese Medicine, Mathematical Engineering Academy of Chinese Medicine,

b

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Guangzhou University of Chinese Medicine, Guangzhou, 510006, PR China Department of Pharmacology, Zunyi Medical University, Zhuhai Campus, Zhuhai

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519041, PR China c

The First Affiliated Hospital of Chinese Medicine, Guangzhou University of Chinese

d

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Medicine, Guangzhou, Guangdong, 510405, PR China Department of Pharmaceutical Sciences, Zunyi Medical University, Zhuhai Campus,

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Zhuhai 519041, PR China e

School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong

Kong, Hong Kong, PR China f

Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese

Medicine Syndrome, The Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510120, P.R. China 1

*

Corresponding author

E-mail address: [email protected] (J.H. Xie); [email protected] (Z.R. Su).

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Graphical Abstract

ABSTRACT

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Berberine (BBR), a naturally-occurring isoquinoline alkaloid isolated from several Chinese herbal medicines, has been widely used for the treatment of dysentery and colitis. However, its blood concentration was less than 1%, and intestinal microflora-mediated metabolites of BBR were considered to be the important material basis for the bioactivities of BBR. Here, we investigated the anti-colitis activity and potential mechanism of oxyberberine (OBB), a novel gut microbiota metabolite of BBR, in DSS-induced colitis mice. Balb/C mice treated with 3% DSS in drinking 2

water to induce acute colitis were orally administrated with OBB once daily for 8 days. Clinical symptoms were analyzed, and biological samples were collected for microscopic, immune-inflammation, intestinal barrier function, and gut microbiota analysis. Results showed that OBB significantly attenuated DSS-induced clinical manifestations, colon shortening and histological injury in the mice with colitis, which achieved similar therapeutic effect to azathioprine (AZA) and was superior to BBR. Furthermore, OBB remarkably ameliorated colonic inflammatory response and intestinal

epithelial

barrier

dysfunction.

OBB

appreciably

inhibited

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TLR4-MyD88-NF-κB signaling pathway through down-regulating the protein expressions of TLR4 and MyD88, inhibiting the phosphorylation of IκBα, and the

translocation of NF-κB p65 from cytoplasm to nucleus. Moreover, OBB markedly modulated the gut dysbiosis induced by DSS and restored the dysbacteria to normal

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level. Taken together, the result for the first time revealed that OBB effectively improved DSS-induced experimental colitis, at least partly through maintaining the

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colonic integrity, inhibiting inflammation response, and modulating gut microflora

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profile.

Keywords: Oxyberberine; Gut microflora; Metabolite; Intestinal epithelial barrier;

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TLR4-MyD88-NF-κB; Ulcerative colitis.

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Abbreviations:

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AZA, azathioprine; BBR, berberine; DAI, disease activity index; DSS, dextran sulfate sodium; H&E, hematoxylin and eosin; IBD, inflammatory bowel disease; IFN-γ, interferon-γ; IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; IL-1β, interleukin-1β, IL-6, interleukin-6, IL-10, interleukin-10 and IL-17, JAM-A: junctional adhesion molecule A; interleukin-17; MPO, myeloperoxidase; OBB, oxyberberine; OTUs, operational taxonomic units; PCA, principle component analysis;

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PCoA, principal coordinate analyses; TJs, tight junctions; TNF-α, tumor necrosis factor-α; UC, ulcerative colitis; ZO-1: zonula occludens-1; ZO-2: zonula occludens-2

1. Introduction Ulcerative colitis (UC), a subtype of inflammatory bowel disease (IBD), is a chronic lifelong disorder characterized by relapse-remitting course, idiopathic

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intestinal inflammation, bloody diarrhea, and weight loss that mainly affects the distal colon and rectum [1]. UC is recognized as "changpi", "dysentery" and "diarrhea" in traditional Chinese medicine theory. Its incidence and prevalence has been

continuously rising worldwide, which predominantly afflicts young adults aging

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30-40 years [2]. Patients with chronic inflammation caused by UC tend to exhibit a high risk of developing colorectal cancer [3]. Although the precise etiology remains

barrier

defects,

environmental

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poorly understood, it has been demonstrated to be multifactorial, involving epithelial factors,

antigen

recognition,

deregulated

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immunological responses, and genetic predisposition [4]. Currently, there are no specifically licensed drugs for the treatment of UC. Clinical therapies primarily involved aminosalicylates, immunomodulators, corticosteroids, anti-TNF-α drugs,

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surgery and diet therapy. However, these agents achieve limited success due to the limitations of clinical efficacy and potential toxicity [5]. Hence, it is very significant

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to further search more effective and less toxic agents for the treatment of UC. Berberine (BBR), a quaternary isoquinoline alkaloid derived from several

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traditional Chinese medicinal herbs like Coptidis chinensis Franch., is commonly used as a nonprescription drug to treat gastroenteritis, colitis, diarrhea and dysentery. Obviously, BBR is a promising active component due to its broad array of bioactivities (e.g. anti-inflammation, antidiarrheal and anti-colitis), low toxicity, and low cost [6]. However, the extremely low blood concentration (<1%) of BBR after oral administration in experimental or clinical settings is insufficient to achieve the effects as observed under experiments in vitro, which imposes challenges to explain 4

its excellent and diverse pharmacological actions in clinical trials [7, 8]. Recent studies have reported that almost 45% of BBR metabolites have been identified in plasma, urine, feces, bile and tissues of rats or humans [9], and theses metabolites exhibit similar pharmacological effects to BBR [10], which indicates that the metabolites of BBR might contribute significantly to the pharmacological effects of BBR in vivo. It has been reported that about 43.5% of BBR is metabolized in the intestine, and only 0.14% in the liver, which indicates that gut microflora play a critical role in the

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metabolism of BBR [11]. Intestinal bacteria can convert BBR into metabolites like dihydroberberine, berberubine, demethyleneberberine, jatrorrhizine by reduction, methylation, demethylation, dehydroxylation and other reactions [12, 13], which

exhibit similar pharmacological properties (e.g. anti-inflammatory, antioxidant,

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anti-colitis effects) to BBR [10, 14]. However, the pharmacological activities of these

metabolites are inferior to those of BBR, which is difficult to explain the broad and

may produce more active metabolites.

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significant pharmacological activities of BBR. Hence, additional metabolic pathways

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In the present study, for the first time, we found that the intestinal microflora could transform BBR into oxyberberine (OBB) by oxidation reaction. OBB is an oxidized

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protoberberine alkaloid, which as a minor component exists in Coptidis chinensis Franch., and Phellodendron chinense Schneid. [15-17]. It has been revealed that OBB has a number of pharmacological actions including anti-inflammatory [17],

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anti-fungal [18], anti-tumor [19], and anti-arrhythmic [20] effects, among which OBB exhibits superior anti-inflammatory, anti-fungal and anti-arrhythmic activities to BBR.

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Besides, our previous study has found that OBB exhibits superior safety profile as compared to BBR [17]. It is therefore possible to hypothesize that OBB, a novel metabolite of BBR, may be a promising bioactive agent worthy to be explored. Since Coptidis chinensis and its principal active constituent BBR are among the most frequently used traditional Chinese medicines in the therapy of UC [21], this study aimed to assess the potential role of BBR intestinal oxidative metabolite OBB on a colitis murine model induced by dextran sulfate sodium (DSS) and delineate the 5

potential underlying mechanisms. Our results demonstrated that OBB prominently ameliorated DSS-induced acute colitis, which achieved similar therapeutic effect to azathioprine (AZA), and was superior to BBR, via protecting colonic integrity, inhibiting immune-inflammation responses, and modulating gut microbiota. To the best of our knowledge, it is the first time to investigate the anti-UC effect and underlying mechanism of OBB, the gut oxidative metabolite of BBR. This work is envisaged to provide an experimental evidence for the traditional application of Coptidis chinensis and BBR in the treatment of diarrhea and dysentery. Furthermore,

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it may provide support for the research and development of potential novel anti-UC lead compound.

2. Materials and Methods

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2.1 Chemicals and reagents

BBR (purity above 98%) was obtained from Shanghai Jun Rui Biotechnology Co.,

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Ltd (Shanghai, China). DSS (MW: 36000-50000) was purchased from MP Biomedicals (USA). AZA was obtained from Aspen Pharmacare Australia Pty Ltd.

2.2 Animals

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all of analytical grade.

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(Leonards, New South Wales, Australia). Other chemicals, solvents and reagents were

Male Balb/C mice (22-24 g), SD rats (180-200 g) and Kunming (KM) mice (16–20

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g) were purchased from Guangdong Medical Laboratory Animal Center. Mice were kept under pathogen-free conditions at ambient temperature (22 ± 2 °C) in a 12h

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light-dark cycle with free access to standard food and water. The experimental procedures were performed according to the guidelines approved by the Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine. 2.3 The biotransformation of BBR by intestinal microflora The biotransformation of BBR by intestinal microflora was performed according to previous report [13]. Firstly, the fresh feces from normal SD rats and KM mice, and 6

six single intestinal strains (clinical isolates)-beneficial bacteria: Lactobacillus acidophilus (ATCC 4356) and Bifidobacterium longum (ATCC 15697), intermediate bacteria: Streptococcus faecalis (ATCC 19433) and Escherichia coli (ATCC44102), and pathogenic bacteria: Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC26003), which were all purchased from Guangdong culture collection Center (China), were incubated with BBR (100 μg/mL) water solution under anaerobic conditions at 37 °C for 24 h, respectively. Secondly, the metabolites of BBR were detected and identified in the feces of normal SD rats and KM mice,

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respectively, within 24 h after oral administration. Finally, pseudo-germfree SD rat or KM mouse model was established by oral administration with antibiotics following

the established regime [13]. The metabolic transformation of BBR in vivo was studied in pseudo-germfree mice to verify the effect of intestinal microflora on its metabolite.

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The samples were added with acetonitrile and centrifuged at 5,000 g for 20 min. The

supernatant was collected and dried under nitrogen gas, then dissolved in 1 mL

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methanol and filtered through 0.22 𝜇m microporous membrane. The metabolites were

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identified and analyzed by LC-ESI-MS and NMR methods. 2.4 HPLC-ESI-MS analysis

HPLC-ESI-MS analysis was conducted on a Shimadzu UFLC system equipped

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with LCMS-IT-TOF MS system (Shimadzu, Japan) integrating an electrospray ion source interface (ESI). Chromatographic separation was performed using a

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Phenomenex Luna C18 (2) 100A column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of A (acetonitrile) and B (0.5% aqueous formic acid) with a gradient

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elution: 0-20 min, 10%-90% A; 20-30 min, 90%-10% A. The injection volume was 10 𝜇L at a flow rate of 1 mL/min and the column temperature was 30 °C. The complete scan was carried out in the ESI-positive ionization mode within the m/z 50-1000 Da. 2.5 Synthesis and identification of OBB OBB was synthesized according to the previous method with some modifications [22]. Briefly, potassium ferricyanide (154 g) was dissolved in 20% sodium hydroxide 7

solution (1050 mL). Then 33.18 g BBR was added slowly to the suspension and refluxed for 10 h. The precipitate was filtered and scrubbed with 95% ethanol followed by dichloromethane. The yellow crystals dried at 30 °C were collected to afford orange sandy solid (5.3 g). Then, the synthesized sample was characterized by a series of technologies involving NMR spectroscopic analysis (400 MHz for 1H NMR and 101 MHz for 13C NMR,CDCl3) that recorded by Bruker AVANCE III HD 400M (Bruker, Rheinstetten, Germany) and HPLC-ESI-MS that performed as described in Section 2.4.

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2.6 Induction of colitis

As depicted in Fig. 3, after one week of acclimation, the Balb/C mice were

randomly divided into the following groups (n = 17/group): Control group, DSS, DSS

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+ AZA (positive group, 13 mg/kg), DSS + BBR (50 mg/kg), DSS + OBB-L (12.5

mg/kg), DSS + OBB-M (25 mg/kg), and DSS + OBB-H (50 mg/kg). Colitis was

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induced by administration with 3% DSS drinking water for 8 consecutive days. Water consumption was monitored daily. At the same time, the mice in each group were

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given corresponding agent: AZA (13 mg/kg), BBR (50 mg/kg), OBB (12.5, 25 and 50 mg/kg), and corresponding solvent (2% Tween-80 aqueous solution). The dosages of AZA, BBR and OBB were adopted based on previous reports and our pilot trial [14,

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23].

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2.7 Daily observation and sample collection Mice were monitored daily for general appearance, body weight, stool consistency,

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and rectal bleeding. Disease activity index (DAI) was evaluated following previous method (Table 1) [24]. Twenty-four hours after the last drug administration, the blood samples were collected from the retro-orbital venous plexus with a glass capillary under anesthesia. Mice were then sacrificed and the colon was immediately obtained to measure the colon length (between the proximal rectum and the ileocecal junction). Besides, 1 cm of the distal colon tissues was collected for histologic examination. The remaining colon tissues were stored at -80 °C for further analysis. 8

2.8 Histopathological evaluation Proximal and distal colons were harvested and fixed in 4% paraformaldehyde, and embedded in paraffin. Five-μm-thick tissue sections were stained with hematoxylin and eosin (H&E) for light microscopic examination using a DP73 light microscope (Olympus, Tokyo, Japan). Colonic mucosa damage score was assessed as previously described (Table 2) [6, 24]. 2.9 Measurement of colonic MPO activity and cytokine concentrations

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Colonic tissues were homogenized in phosphate buffered saline (pH 7.4) and centrifuged at 14,000 g for 15 min at 4 °C. Then the supernatants were collected for the measurement of myeloperoxidase (MPO) activity and cytokine concentrations. MPO activity was measured using a myeloperoxidase assay kit (Jiancheng

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Bioengineering Institute, Nanjing, China). The levels of cytokines, including TNF-α,

IL-6, IFN-γ, IL-1β, IL-10 and IL-17 were quantified using corresponding commercial

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ELISA kit (Beijing Chenglin Biology Co. Ltd., Beijing, China). All procedures were

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performed according to the manufacturer's instructions.

2.10 Measurement of serum IgA, IgG and IgM levels The serum was obtained from the collected blood samples through centrifugation at

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3,500 g for 15 min at 4 °C. Then, the levels of immunoglobulin IgA, IgG and IgM were measured by QuantiCyto® ELISA kit (NeoBioscience, Shenzhen, China)

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according to the protocols accompanying the kit.

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2.11 Quantitative real-time PCR analysis Total RNAs of colon tissues were extracted using TRIzol (Invitrogen Life

Technologies, MA, USA) following the manufacturer's protocol. cDNA was obtained from RNA samples using Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Quantitative real-time PCR was then performed by FastStart Universal SYBR Green Master (Roche, Germany or Switzerland). The relative gene expression was measured and normalized 9

to GADPH expression using 2−ΔΔCt method. The PCR primer sequences were shown in Table 3. 2.12 Western blot analysis Colon total protein, cytoplasmic protein or nuclear protein was extracted in accordance with the manufacturer’s instructions. The target antibodies used for Western blot were as follows: rabbit polyclonal antibodies against TLR4, MyD88, p65, IκΒα, phosphorylated IκΒα (p-IκΒα), zonula occludens-1 (ZO-1), zonula occludens-2

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(ZO-2), occludin, claudin 1, junctional adhesion molecule A (JAM-A), β-actin, and Histone H3 (Affinity Biosciences, OH, USA). Relative protein expression was

measured and normalized to the expression of β-actin (total protein and cytoplasmic protein) or histone (nuclear protein) using Image J software.

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2.13 Molecular docking simulation

The molecular docking simulation was conducted to investigate the potential

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interaction between candidate compounds (BBR and OBB) and the target proteins (TLR4, MyD88 and NF-κB) by using AutoDock version 4.2.6 programs (The Scripps

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Research Institute, USA). The three-dimensional crystal structures of target proteins were obtained from the RCSB Protein Data Bank (https://www.rcsb.org/), and the

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three-dimensional structures of BBR and OBB were explored from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The size of the grid box was set at 30 Å (x, y, z) with 0.375 Å grid spacing. Binding free energy (kcal/mol) was calculated

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using the Lamarckian Genetic Algorithm (LGA). Ligand-protein complexes with the lowest free energy of conformation were regarded as the most favorable structure. The

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results were visualized by a PyMOL program (Schrödinger LLC, New York, NY, USA).

2.14 Analysis of cecal microbiota composition by 16S RNA gene sequencing Eight different cecal contents of each group were randomly collected and stored at -80 °C. The metagenomic DNA was extracted using the OMGA-soil DNA kit according to the manufacturer’s instruction. Hypervariant V4 region of bacterial 16S 10

rRNA

gene

was

amplified

using

the

5’-GTGCCAGCMGCCGCGGTAA-3’)

primers and

515F

(: 806R

(5’-GGACTACHVGGGTWTCTAAT-3’). Then the PCR products were purified with Ampure XP beads. The quality of sequencing libraries was detected by Agilent 2100. Purified amplicons were sequenced by MiSeq System (Illumina). Raw fastq data were filtered low-quality by QIIME61 (v1.17). Then the paired-end reads were overlapped using flash software. Operational taxonomic units (OTUs) were clustered by USEARCH (v7. 0.1090) with 97% homology. The taxonomy profiling was analyzed

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by RDP classifier (v2.2) comparing the Greengene Database with a 0.5 threshold. The alpha diversity analysis was performed using Mothur (v1.31.2). Two-dimensional

principle component analysis (PCA) and principal coordinate analysis (PCoA) plots

with a weighted uniFrac matrix were used to assess the variation (β-diversity distance)

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between experimental groups. Different taxa microbes were identified based on

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taxon-based analysis and LEfSe analysis. 2.15 Statistical analysis

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Data were presented as mean ± standard error of the mean (S.E.M.) of three independent experiments. The statistical significance of differences between two groups was analyzed by Student's t-test. The differences between multiple groups

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were analyzed by one-way ANOVA followed by Dunnett’t test using SPSS software (v. 19.0, SPSS, Chicago, IL, USA). A value of P < 0.05 or P < 0.01 was defined as

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statistically significant.

3. Results

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3.1 The metabolism of BBR by intestinal microflora As shown in Fig. 1, LC-MS analysis showed that BBR was converted to a new

metabolite (OBB, LC-ESI-MS m/z: 352.1 [M+H], 337.1, 322.1, 308.1) by gut microflora in normal and pseudo-germfree mice or rat. The conversion rate of OBB by intestinal microflora in normal SD rats and KM mice was 12.42% and 17.03%, respectively. After oral administration with BBR, the content of OBB was 1.49% and 1.75% in fresh feces of normal SD rats and normal KM mice within 24 h, respectively. 11

And the content of OBB was significantly decreased in pseudo-germfree mice, which was 0.05% and 0.21% in pseudo-germfree SD rats and KM mice within 24 h, respectively (Fig. 1B). Moreover, 6 kinds of single intestinal bacterium (beneficial bacteria: Bifidobacterium longum, Lactobacillus acidophilus; intermediate bacteria: Streptococcus Faecalis, Escherichia coli; harmful bacteria: Pseudomonas aeruginosa, Staphylococcus aureus) all could convert BBR to OBB, among which the intermediate bacteria Streptococcus Faecalis provided the most productivity for the conversion of BBR into OBB (Fig. 1C).

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3.2 Synthesis and identification of OBB

The molecular formula of synthetic orange sandy solid was deduced to be

C20H17NO5 by LC-ESI-MS at m/z: 352.1 [M+H]+, 337.1, 322.1, 308.1. Its chemical 13

C NMR were recorded as follows: 1H NMR (400 MHz,

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shifts of 1H NMR and

CDCl3): δ 7.32 (1H, d, J = 8.6 Hz, H-11), 7.28 (1H, s, H-12), 7.21 (1H, s, H-13), 6.71

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(1H, s, H-l), 6.70 (1H, s, H-4), 6.00 (2H, s, OCH2O), 4.29 (2H, m, J = 6.2 Hz, H-6), 4.01 (3H, s, OCH3), 3.95 (3H, s, OCH3), 2.89 (2H, m, J = 6.2 Hz, H-5).

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C-NMR

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(101 MHz, CDCl3): δ 160.22 (C-8), 151.50 (C-9), 149.56 (C-10), 148.53 (C-3), 147.44 (C-2), 135.71 (C-14), 132.42 (C-8a), 130.12 (C-12a), 123.83 (C-14a), 122.41 (C-12), 119.46 (C-4a), 119.04 (C-11), 108.02 (C-4), 104.79 (C-1), 101.53 (OCH2O),

NMR,

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101.42 (C-13), 61.72 (OCH3), 56.94 (OCH3), 39.46 (C-6), 28.79 (C-5). The data of 1H C-NMR and LC-ESI-MS spectrometry revealed that the synthetic orange

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sandy solid was OBB, which were in agreement with previous report [16]. The purity of OBB was determined to be above 98% by HPLC.

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3.3 Anti-colitis activity of OBB 3.3.1 General observations As shown in Fig.2, DSS-treated colitis mice exhibited marked colitis symptoms characterized by shaggy hair, low vitality, body weight loss, diarrhea, and occult fecal blood, which were analogous to the symptoms of human UC (Fig. 2B-D). The DAI score remarkably increased after DSS intake as compared to that of the control group 12

(Fig. 2E, P < 0.01). By contrast, pretreatment with OBB (12.5, 25, and 50 mg/kg) significantly alleviated these colitis symptoms and decreased DAI score in a dose-dependent manner (P < 0.05 vs. DSS). Noticeably, the therapeutic effect of OBB-M (25 mg/kg) was similar to that of AZA (13 mg/kg) and superior to that of BBR (50 mg/kg, P < 0.05). Besides, colon length was deemed as an important indicator of the severity of colorectal inflammation. As shown in Fig. 2F-G, the colon length was significantly shortened in colitis group as compared to that of the control group (P < 0.01). By

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contrast, DSS-induced colon shortening was observed to be markedly improved by pretreatment with OBB (12.5, 25, and 50 mg/kg, all P < 0.01) and two positive drugs

(BBR and AZA, both P < 0.05). These results indicated that pretreatment with OBB

3.3.2 OBB suppressed colon tissue injury

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exerted pronounced protective effect against DSS-induced experimental colitis.

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As shown in Fig. 3A, in the control group, the colon tissues showed intact mucosa, submucosa, muscular layer and outer membrane. By contrast, the DSS-induced colitis

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group was characterized by inflammatory cell infiltration, epithelial cell destruction, and mucosal thickening, which resulted in a lower microscopic score compared to the control group (5.00 ± 0.58 vs. 0.27 ± 0.15，P < 0.01, Fig. 3B). However, consistent

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with the effects on general observations, OBB (12.5, 25 and 50 mg/kg) pretreatment groups were observed to effectively restore the crypt architecture epithelium and

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reduce severe histologic inflammation as compared to the colitis model group. Notably, OBB-H (50 mg/kg) exhibited similar effect to AZA, and exerted superior

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effect in ameliorating these deteriorating pathological changes to BBR (P < 0.05). MPO activity is an important marker of neutrophile granulocyte infiltration. As

shown in Fig. 3C, MPO activity was remarkably elevated in DSS-treated colitis group as compared to the control group (0.79 ± 0.05 vs. 0.44 ± 0.03, P < 0.01). However, pretreatment with OBB significantly inhibited the elevated MPO activity in a dose-dependent manner (P < 0.01 vs. DSS). And OBB-H (50 mg/kg) exhibited similar inhibitory effect to AZA (13 mg/kg) and superior effect to BBR (50 mg/kg, P < 0.01). 13

The results manifested a beneficial effect of OBB on DSS-induced experimental colitis via preventing inflammatory infiltration. 3.3.3 OBB improved intestinal barrier function As shown in Fig. 4A-B, as compared with the control group, the mRNA expression of mucin-1 and mucin-2 was significantly down-regulated in DSS-treated colitis mice (P < 0.01). However, the expression of these two genes was prominently up-regulated by pretreatment with OBB (12.5, 25 and 50 mg/kg) and positive drugs (BBR and

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AZA) in a dose-dependent manner (P < 0.05 vs. DSS). Additionally, as shown in Fig. 4C-H, all tight junctions (TJs) proteins (including ZO-1, ZO-2, occludin, JAM-A, claudin-1) were observably suppressed in DSS-treated colitis mice compared with those of the control mice (P < 0.05), indicating that the TJs structure was disrupted.

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By contrast, OBB and positive drugs promoted the expression of the above five TJs proteins in a dose-dependent manner. Notably, OBB exhibited more evident

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protective effect than BBR at the same dose (P < 0.05). These results indicated that OBB might prevent DSS-induced disruption of intestinal integrity by enhancing the

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expressions of mucin mRNA and TJs proteins.

3.3.4 Effects of OBB on the productions of immune-inflammatory cytokines

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As illustrated in Fig. 5A-F, the levels of pro-inflammatory cytokines, including IL-6, IL-1β, IL-17, TNF-α, and IFN-γ were significantly increased in DSS-treated

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colitis mice as compared to those of the control group (P < 0.05). However, these elevated pro-inflammatory cytokines were dramatically decreased by OBB (12.5, 25

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and 50 mg/kg), BBR (50 mg/kg) and AZA (13 mg/kg) in a dose-dependent manner (P < 0.05 vs. DSS). It was noteworthy that OBB-H (50 mg/kg) exhibited pronounced effect in suppressing these inflammatory cytokines, even superior to the positive agents AZA and BBR. 3.3.5 Effects of OBB on serum IgA, IgG and IgM levels As shown in Fig. 5G–I, the levels of IgA, IgG, and IgM in the serum of DSS-induced colitis mice were significantly higher than those of the control group (P 14

< 0.05). However, the elevated levels of IgA, IgG and IgM were significantly decreased by OBB (12.5, 25 and 50 mg/kg), BBR (50 mg/kg) and AZA (13 mg/kg) in a dose-dependent manner (all P < 0.05 vs. DSS). Furthermore, OBB-H (50 mg/kg) exerted a more significant inhibitory effect on the IgA production than BBR (50 mg/kg) (P < 0.05). The above results revealed that OBB might exhibit significant anti-UC effect, at least in part, by compensating DSS-induced immune dysregulation in UC mice.

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3.3.6 Effects of OBB on TLR4-MyD88-NF-κB signaling pathway As depicted in Fig. 6, the expression of TLR4, MyD88, p-IκBα and p65 (nucleus)

was significantly augmented, whereas the expression of IκBα and p65 (cytoplasmic) was markedly decreased in DSS-treated colitis group as compared with those of the

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control group (all P < 0.01). However, pretreatment with OBB (5, 10 and 20 mg/kg),

BBR and AZA all significantly increased the expression of p65 (cytosolic), decreased

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the expression of p65 (nuclear), and reduced the ratio of p-IκBα/IκBα in a dose-dependent manner (all P < 0.05 vs. DSS). These results manifested that

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pretreatment with OBB might down-regulate the protein expression of TLR4 and MyD88, inhibit IκBα phosphorylation and the translocation of NF-κB p65 from cytoplasm to nucleus, thereby suppressing the TLR4-MyD88-NF-κB signaling

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pathway. This mechanism might contribute to regulating inflammation and immune function to reduce intestinal mucosal inflammation and colonic mucosal injury.

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As shown in Table 4 and Fig. 7, molecular docking simulation further indicated that OBB interacted with the active site of TLR4, MyD88 and NF-κB, with the binding

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energy of -5.66, -4.21 and -4.27 kcal/mol, respectively. Meanwhile, BBR could also insert into the active pocket of TLR4, MyD88 and NF-κB, with the binding energy of -5.52, -4.09 and -4.09 kcal/mol, respectively. BBR formed several hydrogen bonds with the amino acid ARG 295 and ILE 298 of TLR4, GLU 183 and TRP 205 of MyD88, and SER 317, ASN 339 and ASP 294 of NF-κB. OBB also made hydrogen contact with ARG 295, GLU 299 and ILE 298 of TLR4, GLU 183 and ARG 180 of MyD88, and ASP 294, ASN 339 and SER 317 of NF-κB. These results indicated that 15

BBR and OBB revealed distinct affinities to the binding sites of TLR4, MyD88 and NF-κB, therefore disturbing the TLR4-MyD88-NF-κB signaling pathway activation, which was congruent with the in vivo assay. 3.3.7 OBB modulated the structure of gut microbiota A bar-coded pyrosequencing run was conducted to investigate the changes in the gut microbiota from UC model mice after pretreatment with BBR, AZA and OBB-L. The result indicated that 1425958 usable reads and 640 OTUs were obtained from 40

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samples. The number of OTUs in DSS group was significantly decreased in comparison with that of the control group (P < 0.01), which was unimproved by pretreatment with OBB, BBR and AZA (Fig. 8A). However, the number of OTUs in

OBB group was similar to that of AZA, which was obviously higher than that of the

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BBR group. In addition, Venn diagram revealed that 312 OTUs coexisted in the five groups (Fig. 8B).

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Based on different alpha diversity indices, as shown in Table 5, in contrast to the control group, significant reduction of both microbial species richness (Observed

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species, Ace and Chao1) and diversity (Shannon and Simpson) were observed in DSS group (P < 0.01). The decreased alpha diversity in DSS-induced colitis group was unimproved by OBB, BBR and AZA, which was in agreement with the above OTUs

na

results. However, both PCA and PCoA demonstrated that a marked overall structural shift of gut microbiota in samples of DSS-induced colitis mice compared with that of

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the normal mice. Whereas the overall gut microbiota structure in the treatment groups (AZA, BBR, and OBB-L) was similar to that of the control group. Notably, the

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OBB-L group was more similar to the control group than BBR group (Fig. 9A and B). The system clustering tree revealed significant differences among the five groups, and OBB-L-treated group was clustered separately from the DSS group, indicating that pretreatment with OBB inhibited the DSS-induced gut microbiota structural changes in colitis mice (Fig. 9C). As shown in Fig. 8C, the phylogenetic classification of OTUs revealed a total of 9 phyla were shared by all samples, with Bacteroidetes, Firmicutes and Proteobacteria 16

being the most predominant phyla. Lower abundance of Bacteroidetes, and higher abundance of Firmicutes and Proteobacteria were observed in DSS-treated colitis group as compared with those of the control group, which was consistent with previous reports [25, 26]. However, following pretreatment with OBB-L, AZA, or BBR, the abundance of Bacteroidetes and Firmicutes almost returned to the normal level (Table 6). Moreover, a total of 31 genera were identified in all samples (Fig. 8E). Besides, the relative abundance of Clostridium, Coprobacillus, Dehalobacterium, Dorea, Parabacteroides, Paraprevotella, Ruminococcus, Staphylococcus

and

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Helicobacter was significantly increased, whereas Prevotella was significantly decreased in the DSS colitis group as compared to those of the control group (P < 0.05). However, the dysbacterium microflora structure was favorably harmonized by pretreatment with OBB-L (Table 6).

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In addition, LefSe (LDA effect size) analysis was used to identify statistically

significant biomarkers and dominant microbiota in each group. As shown in Fig. 8D,

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Firmicutes was found to be the major phylum of gut microbiota in the DSS colitis group, while Bacteroidetes in the OBB-L and control groups, Proteobacteria,

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Streptococcaceae, Enterococcaceae, and Erysipelotrichale in BBR group, and Firmicutes and Deferribacteres in AZA group, were identified to be the predominant

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intestinal flora, respectively.

4. Discussion

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BBR represents one of the most studied naturally occurring protoberberine alkaloids, and is attracting increasing attention from investigators due to its excellent

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and diverse pharmacological actions. BBR has low oral bioavailability (< 1%) due to poor aqueous solubility, and therefore many approaches have been adopted to solve the problems of poor bioavailability. Nanotechnology has been proposed as an exceptionally valuable tool widely used in biology, medicine, chemistry, agriculture and food industry due to its several advantages such as targeting drug delivery, reduced toxic side effects, and improved drug stability and bioavailability [27-30]. Hence, in recent years, remarkable growth researches have been focused on the 17

nanoformulation of BBR to improve its bioavailability and therapeutic effect [31-33]. However, the underlying mechanisms remain unclear in regard to its extremely low plasma concentration-effect relationship. Of note, low oral bioavailability but significant bioactivity is a conundrum still not solved for numerous clinically effective natural products and synthetic drugs, such as scutellarin [34], paclitaxel [35], ginsenosides [36], curcumin [37], doxorubicin [38] and so on. Therefore, clarifying the pharmacodynamic material basis and molecular mechanism of BBR in vivo is one of the key scientific issues in further medical research.

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BBR is presumably poorly absorbed by intestinal epithelial cells [39], and the liver is known to be the major organ contributing to drug metabolism and bioconversion. However, the AUC0–t

value of BBR after intraduodenal administration was

h

significantly less than that after intraportal administration, indicating that the

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intestinal first pass metabolism of BBR was tremendous in rats [11]. Nevertheless, the role of gut microbiota in drug action has still been underestimated. In fact, gut

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microbiota-mediated biotransformation is critical for some drugs, especially those with poor oral bioavailability, to exert their toxicities or therapeutic effects in vivo [40,

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41]. Gut microbiota harbor many types of metabolic enzymes (oxidase, reductase, esterase, etc.), allow triggering a series of metabolic reactions of drugs, including

na

oxidation, reduction, intramolecular cyclization, isomerization and rearrangement, which contribute significantly to both bacteria and the host [42]. In humans, chronic administration of BBR results in the following primary gut

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microbiota metabolites: berberrubine, dihydroberberine, thalifendine, columbamine, demethyleneberberine, and jatrorrhizine [12]. These metabolites exhibit similar

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pharmacological effects to BBR, such as anti-inflammatory, antimicrobial, antitumor, anti-colitis and hepatoprotective activities [10, 14]. Dihydroberberine, the reduced metabolite

of

BBR

by

gut

microbiota-derived

nitroreductases,

was

its

intestine-absorbable form, which has been only found in the feces of rats and subsequently reverted back to BBR through oxidization in intestine [13]. Consistently, the present study showed that the intestinal microbiota converted BBR into OBB in the intestine via oxidation reaction with a high inter-individual variability. An 18

antibiotic-pretreated animal possess a relatively reduced amount of microbiota, which has been widely used to investigate the relationship between host and microbial xenobiotic metabolism [43]. As expected, after oral administration of antibiotics, the biotransformation of BBR into OBB was markedly suppressed, which was in accord with previous reports [13, 44]. In our previous study, we have found that OBB can be detected as a minor component in Coptis chinensis [17], which has been listed in the Chinese Pharmacopoeia and widely used for the treatment of gastroenteritis, including

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diarrhea, abdominal pain, and colitis for a long history. BBR, an eminent component of Coptis chinensis, has been reported to exert pronounced therapeutic effect against

colitis through inhibiting inflammatory responses, modulating the gut microbiota and

attenuating mucosal lesions [6]. Interestingly, it has been reported that OBB exerts

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superior anti-inflammatory, anti-fungal, and antiarrhythmic activities to BBR, with

more favorable safety profile [17, 18, 20]. When BBR was metabolized to its oxidized

re

derivative OBB, the structure of C-8 quaternary ammonium turned to more active lactam ring, and the lipophilicity would be enhanced and easier to be absorbed

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through biofilm, which was beneficial to enhance its biological activity. Therefore, in this study, OBB was expected to exhibit superior anti-colitis effect to BBR.

na

We employed a widely used mouse colitis model induced by DSS, a classical chemical inducer for UC, which closely resembled clinical symptoms of human UC [45]. In the present study, pretreatment with OBB remarkably alleviated the clinical

ur

symptoms of colitis such as weight loss, diarrhea, stool character, and fecal blood. Of note, BBR failed to suppress the weight loss, which might be associated with its lipid-

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and glucose-lowering properties [46]. Besides, the shortened colon length, often regarded as an indicator of inflammation [47], was observed in DSS-induced colitis group. Furthermore, neutrophils play a critical role in the pathogenesis of colitis [48]. MPO, the peroxidase abundantly expressed in neutrophil granulocytes, has been observed to have high level in the colon of UC patients [49]. In this study, histopathological evaluation demonstrated that DSS induced inflammatory infiltration and destroyed the epithelial barrier of colon. In contrast, pretreatment with OBB 19

significantly alleviated mucosal inflammation and tissue destruction as evident by extended colon length, improved histological score and decreased MPO activity, which was in line with previous studies on BBR [6, 14]. Noteworthily, OBB exerted superior effect to BBR. These data suggested that OBB effectively alleviated DSS-induced UC in mice. Intestinal epithelial barrier dysfunction is implicated to play a crucial role in the pathogenesis of UC [50]. The intestinal epithelial barrier, evolving to maintain a balance between absorbing nutrients and preventing the entry of toxins and luminal

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bacteria, consists of mucus layer, epithelial cells and intercellular junctions [51]. Epithelial mucus layer, composed of mucins (primarily including mucin-1 and mucin-2) released by epithelial goblet cells, is the first line of defense that prevents

invading pathogens, which is dramatically decreased in UC patients [52]. Mucin-2

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knock-out mice exhibit an abnormal morphology, epithelial ulcer, an active

inflammatory response and an increase in proliferation, which spontaneously develop

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colitis [53]. TJs are intercellular complexes located at the most apical part of the junctional complex of intestinal epithelial cells that play crucial roles in epithelial

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permeability, cell-cell adhesion and paracellular motion [54]. TJs are consisted of transmembrane protein (e.g., occludin, claudins and junctional adhesion molecule A

na

JAM-A) and intracellular membrane or scaffolding protein (e.g., zonula occludens, and intracellular regulatory molecules [kinases and actin]) [55]. Destruction of TJs could result in increased colonic permeability to harmful bacteria and toxins, leading

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to intestinal inflammation response, diarrhea and thus evoking the occurrence and development of UC [52]. Hence, therapeutic restoration of the mucosal barrier

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function may provide an invaluable contribution to the treatment of UC. Previous studies have indicated that BBR and its metabolites, including berberubine and palmatine, attentuated barrier function disruption and apoptosis in the colon epithelium of DSS-induced colitis mice [14, 47]. Similarly, in the present study, the down-regulated expressions of mucin mRNA and TJ proteins (including ZO-1, ZO-2, occluding, claudin-1 and JAM-A) in DSS-induced colitis mice were significantly suppressed by OBB, and its inhibitory effect was similar to that of AZA, while 20

superior to the counterpart of BBR. These results demonstrated that the protective effect of OBB against DSS-induced colitis might be tightly associated with the enhancement of synthesis of mucus and improvement of the epithelial TJs. Inflammatory response, characteristic of intestinal injury in IBD, plays a critical role in the pathogenesis of UC [52]. Pro-inflammatory cytokines (e.g. TNF-α, IL-1β and IFN-γ) have been reported to suppress the TJs proteins (e.g. ZO-1, ZO-2 and occluding) with vicious cycles, resulting in further deteriorated gut epithelial barrier dysfunction and aggravated intestinal inflammation [56]. Elevated levels of

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pro-inflammatory cytokines, such as TNF-α and IL-1β could be detected in the colonic tissue of UC patients. IFN-γ and IL-17 secreted by Th17 cells could induce

the expressions of pro-inflammatory cytokines and chemokines, resulting in intestinal/colonic tissue damage [57]. Therefore, regulation of these inflammatory

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mediators may afford an important strategy for the therapy of UC.

Emerging evidence suggests that the inflammatory response is primarily regulated

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by the TLR4-MyD88-NF-κB pathway [58]. An increased intestinal permeability in UC patients allows toxic substances and pathogenic microorganisms to cross the

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intestinal wall, resulting in the activation of TLR4 [59]. TLR4, a member of the Toll-like receptor family of proteins, has been observed to be up-regulated in animals

na

and humans with IBD [60, 61]. Activation of TLR4 initiates the activation of MyD88, an important adaptor molecule essential for TLR signaling. This in turn triggers the activation

of

downstream

NF-κB

signaling

pathway

and

synthesis

of

ur

pro-inflammatory cytokines, including IL-6, IL-1β and TNF-α that contribute to the development of IBD [62]. Moreover, NF-κB also plays a vital role in the disruption of

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epithelial barrier function and interaction between mucosal immune system and gut microbiota [63]. Our data suggested that pretreatment with OBB dramatically inactivated TLR4,

MyD88, phosphorylated IκBα, and the migration of NF-κB p65 from cytoplasm to nucleus. OBB efficaciously suppressed the expression of NF-κB downstream molecules, including TNF-α, IL-6, IL-1β, IFN-γ, IL-17, and IL-10 in the colons of DSS-induced colitis mice. Notably, OBB exerted superior anti-inflammatory effect to 21

BBR. These results combined with molecular docking simulation suggested that the alleviative effect of OBB against DSS-induced colitis might be closely associated with the improvement of inflammatory status via inhibiting TLR4-MyD88-NF-κB signaling pathway. Gut microbiota, an important component of the intestinal barrier, is vital for host physiological processes related to maturation of intestinal mucosal barrier function, development of the immune system, nutrient absorption, and energy metabolism [64, 65]. Inflammatory diseases of the gastrointestinal tract are closely associated with gut

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microbiota dysbiosis, characterized by expansion of facultative anaerobic bacteria of the Enterobacteriaceae family (phylum Proteobacteria). Gut microbiota dysfunction could cause the increased permeability of the intestinal epithelial cell, trigger mucosal

inflammatory response, and promote the development of colitis [66, 67]. Therefore,

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regulation of the intestinal microbiota has been deemed as one of therapeutic strategies for UC patients. It has been reported that there is an interactive relationship

re

between drug and gut microflora: intestinal microflora-mediated drug metabolism and gut microflora-targeted alteration by drug [68, 69]. BBR has been reported to regulate

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gut microbiota structure and reduce its diversity in UC mice and therefore relieve the symptoms of UC [6]. In the present study, DSS-induced colitis mice exhibited a lower

na

relative abundance of Bacteroides, but higher of harmful bacteria such as Firmicutes and Proteobacteria, in comparison with the control group. By contrast, OBB, the gut microbiota metabolite of BBR, substantially recovered the relative abundance of

ur

bacteria, including Anaeroplasma, and Odoribacter, and decreased pathogenic bacteria,

comprising

Parabacteroides,

Paraprevotella,

Ruminococcus,

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Staphylococcus Butyricicoccus, Clostridium, Coprobacillus, Enterococcus, and Dehalobacterium. This suggested that pretreatment with OBB effectively maintained the homeostasis of intestinal microbiota through shifting the gut microbiota structure of UC mice.

5. Conclusion

22

The results showed that the interaction between intestinal microorganisms and drugs could change the structure and efficacy of drugs. OBB, a metabolite of intestinal microflora of BBR, prominently alleviated DSS-induced clinical symptoms, inflammation, and colonic injury, and the protective effect may be partially associated with protection of intestinal barrier integrity, modulation of gut microbiota, inhibition of the

TLR4-MyD88-NF-κB signal transduction pathway

and subsequent

down-regulation of inflammatory mediators. Notably, the results exhibited that OBB achieved similar therapeutic effect as compared to AZA, and superior to BBR, which

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suggested that OBB may have the potential to be further developed into a promising therapeutic agent for the treatment of UC. However, in-depth and definitive investigations, e.g. long-term safety and pharmacokinetics evaluations, should be

Conflict of interest

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The authors declare no conflicts interest.

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investigated prior to possible future clinical trials.

Acknowledgments

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This work was supported by grants from Science and Technology Development Special Project of Guangdong Province (2017A050506044), Science and Technology Plan Project of Guangzhou (201704030028), and Characteristic Cultivation Program

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ur

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for Subject Research of Guangzhou University of Chinese Medicine (XKP2019007).

23

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experimental colitis in mice by inhibiting Th1/Th17 response. Int Immunopharmacol. 2014;20(2):337-45. [58] Zhou JT, Tan LH, Xie JH, Lai ZQ, Huang YF, Qu C, et al. Characterization of brusatol self-microemulsifying drug delivery system and its therapeutic effect against dextran sodium sulfate-induced ulcerative colitis in mice. Drug Deliv. 2017;24(1):1667-79. [59] Bhattacharyya S, Gill R, Chen ML, Zhang F, Linhardt RJ, Dudeja PK, et al. Toll-like receptor 4 mediates induction of the Bcl10-NF kappa B-interleukin-8 inflammatory pathway by carrageenan in human intestinal epithelial cells. J Biol Chem. 2008;283(16):10550-8.

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of Campylobacter jejuni Gastroenteritis Reveals Key Pro-inflammatory and Tissue Protective Roles for Toll-like Receptor Signaling during Infection. Plos Pathog. 2014;10(7): e1004264.

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barrier in the pathogenes of chronic intestinal inflammation: lessons learned from animal models and human genetics. Front Immuno. 2013;4. [64] de Medina FS, Romero-Calvo I, Mascaraque C, Martinez-Augustin O. Intestinal

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Inflammation and Mucosal Barrier Function. Inflamm Bowel Dis. 2014;20(12):2394-404. [65] Jandhyala SM, Talukdar R, Subramanyam C, Vuyyuru H, Sasikala M, Reddy DN. Role of the

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normal gut microbiota. World J Gastroenterol. 2015;21(29):8787-803.

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[69] Zimmermann M, Zimmermann-Kogadeeva M, Wegmann R, Goodman AL. Mapping human

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microbiome drug metabolism by gut bacteria and their genes. Nature 2019;570(7762):462-7.

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Table 1 Disease activity index (DAI) Weight loss (%)

Stool consistency

Occult blood

Score

None

Normal

Negative

0

1-5

-

-

1

Hemoccult +

2

5-10

Loose stools -

-

3

> 20

Diarrhoea

Gross bleeding

4

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10-20

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Table 2 Histological colitis score Inflammation

Lymphocyte

Colon wall

infiltration

aberrant

0%

None

0

Crypt aberrant extent None

Score

Normal Basal 1/3 damage

10%

Mucosa

1

Moderate

Basal 2/3 damage

10-25%

Submucosa

2

Crypt lost; surface

25-50%

Severe epithelium present Crypt and surface

> 50%

Transmural

3

-

4

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Mild

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epithelium lost

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Table 3 Primers for real-time PCR Primer sequences (5’-3’)

Gene Mucin-1

Product size (b.p.)

F：AGGCTCCGTGGTGGTAGAATCG

362

R：AGCGTCCGTGAGTGTGGTAGG Mucin-2

F：GTGGAGGTACAGGTGAACAAGCG

400

R：TGATGAGGTGGCAGACAGGAGAC GAPDH

F：AATGGTGAAGGTCGGTGTGAACG

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R：TCGCTCCTGGAAGATGGTGATGG

235

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Table 4 Hydrophobic interactions and docked amino acid residues of target proteins with BBR and OBB No.

Ligand

Target protein

Binding

(PDB ID)

energy

H-bond

Ligand

Amino acid

H-bond

atoms

residue

length (Å)

2

BBR

OBB

TLR4 (3FXI)

TLR4 (3FXI)

-5.52

2

-5.66

3

C-9O

ARG 295

2.1

C-2O

ILE 298

3.6

C-8O C-2O

BBR

MyD88 (4DOM)

-4.09

2

C-9O

ARG 295

2.2

GLU 299/ILE 298

3.2/3.6

GLU 183

2.6

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1

OBB

-4.21

2

NF-κB (1NFI)

-4.09

3

NF-κB (1NFI)

-4.27

4

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6

BBR

MyD88 (4DOM)

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5

OBB

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4

TRP 205

2.0

C-2O

GLU 183

2.7

C-8O

ARG 180

2.1

C-3O

SER 317/ASN 339

1.9/3.1

C-9O

ASP 294

2.9

C-9O

ASP 294

2.8

C-8O

ASP 294

3.0

C-3O

ASN 339/SER 417

3.1/2.1

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C-3O

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Table 5

Group

Sobs

Ace

Chao

Shannon

Simpson

Control

319.38±14.77

357.22±12.65

358.50±13.73

4.07±0.14

0.04±0.01

DSS

258.50±9.48

294.95±10.14

303.31±13.14

3.40±0.12

0.08±0.01

##

##

#

##

##

262.50±13.27

308.53±11.88

314.27±16.23

3.32±0.15

0.10±0.02

BBR

216.88±15.40

248.36±15.24

251.00±15.22

(50 mg/kg)

*

*

*

OBB-L

253.25±14.76

291.46±14.68

293.33±15.44

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Alpha diversity index of different groups

AZA (13 mg/kg)

Data are expressed with x ± SEM (n = 8).

##

0.12±0.01

3.29±0.11

0.10±0.01

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(12.5 mg/kg)

3.09±0.10

P < 0.01 vs. Control group，* P < 0.05 vs. DSS

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group.

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Table 6

The relative abundance of mice gut microbiota in Phylum and Genus of various groups. Taxonomy

Control

DSS

1

Bacteroidetes

Phylum

51.48 ± 6.57

43.37 ± 7.13

2

Firmicutes

Phylum

39.20 ± 4.97

45.30 ± 6.56

3

Akkermansia

Genus

0.01 ± 0.01

0.00 ± 0.00

4

Bacteroides

Genus

7.64 ± 1.96

5

Clostridium

Genus

6

Coprobacillus

7

AZA

BBR

OBB-L

61.86 ± 6.05 *

55.24 ± 4.32

64.23 ± 5.73 *

31.35 ± 4.54 *

22.52 ± 3.36 **

24.46 ± 3.68 **

0.00 ± 0.00

7.34 ± 2.89 *

0.04 ± 0.03 &

16.02 ± 3.43

28.34 ± 5.92 *

29.87 ± 3.18 *

31.07 ± 4.52 *

1.11 ± 0.18

2.35 ± 0.30 ##

1.89 ± 0.75

1.42 ± 0.28

0.99 ± 0.23 *

Genus

0.00 ± 0.00

0.68 ± 0.19 ##

0.26 ± 0.11*

0.30 ± 0.10 *

0.17 ± 0.07 **

Dehalobacterium

Genus

0.23 ± 0.04

0.41 ± 0.07 #

0.31 ± 0.11

0.18 ± 0.06 *

0.17 ± 0.05 *

8

Dorea

Genus

0.12 ± 0.03

2.23 ± 0.65 #

1.30 ± 0.32

0.88 ± 0.42

1.49 ± 0.53

9

Helicobacter

Genus

2.19 ± 0.82

6.23 ± 1.12 #

1.65 ± 0.43 *

3.76 ± 1.16

2.90 ± 0.83

10

Parabacteroides

Genus

0.06 ± 0.02

1.06 ± 0.20 ##

0.32 ± 0.17 **

0.66 ± 0.21

0.31 ± 0.07 **

11

Paraprevotella

Genus

0.00 ± 0.00

9.88 ± 2.29 ##

6.69 ± 1.02

2.50 ± 1.07 **

5.61 ± 1.02*

12

Prevotella

Genus

3.74 ±1.48

0.17 ± 0.16 #

0.42 ± 0.23

0.07 ± 0.06

1.21 ± 0.61

13

Ruminococcus

Genus

1.71 ± 0.46

3.33 ± 0.59 #

1.64 ± 0.49 *

2.00 ± 0.25 *

1.97 ± 0.37 *

14

Staphylococcus

Genus

0.00 ± 0.00

0.15 ± 0.04 ##

0.00 ± 0.00 *

0.01 ± 0.01 *

0.00 ± 0.00 *

pr

Species

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Pr

e-

No.

Data are expressed as x ± SEM (n = 8). ## P < 0.01 vs. Control group, * P < 0.05, ** P < 0.01 vs. DSS group, & P < 0.05 vs. BBR.

36

Figure legends Fig. 1. Generation of OBB by the gut microbiota. (A) BBR was metabolized into OBB by gut microbiota. (B) The OBB content (%) in the rat or mice faeces after BBR administration in vivo and in vitro for 24 h. (C) BBR was converted into OBB in vitro by 6 intestinal bacteria strains [Bifidobacterium longum, Lactobacillus acidophilus, Escherichia coli, Streptococcus Faecalis, Staphylococcus aureus, Pseudomonas aeruginosa]. Oral administration of antibiotics generated pseudo germ-free (PGF) rats

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or mice. Data are presented as mean ± SEM of 6 mice (A) or three times (B&C) in each group.

Fig. 2. OBB ameliorated the symptoms of DSS-induced colitis in mice. (A) Schematic diagram of the experimental design. (B) Daily bodyweight changes from day 1 to 9.

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(C) Stool consistency score. (D) Rectal bleeding score. (E) DAI score. (F)

Macroscopic appearances of colon tissues. (G) The lengths of colon. Data are

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presented as the mean ± SEM (n = 17). # P < 0.05, ## P < 0.01 vs. Control group, * P <

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0.05, ** P < 0.01 vs. DSS group, & P < 0.05, && P < 0.01 vs. BBR. Fig. 3. Ameliorative effect of OBB on colon tissue injury in mice with DSS-induced colitis. (A) Histological changes (H&E staining images of colonic sections at original

na

magnification 200×, IFI: inflammatory infiltration); (B) Histological score. (C) MPO activity. Data are shown as mean ± SEM (n = 5-7). ## P < 0.01 vs. Control group, * P <

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0.05, ** P < 0.01 vs. DSS group, & P < 0.05 vs. BBR group. Fig. 4. OBB protected intestinal epithelial barrier by modulating TJs proteins. Effect

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of OBB on the mRNA levels of mucin-1(A) and mucin-2 (B). (C) Representative Western blotting images of TJs protein, and the relative protein expressions were normalized to β-actin. (D-H) Changes in the relative protein expression levels of ZO-1, ZO-2, occludin, JAM-A, and claudin-1 were measured respectively. Data are shown as mean ± SEM (n = 3). # P < 0.05, ## P < 0.01 vs. Control group, * P < 0.05, ** P < 0.01 vs. DSS group, & P < 0.05, && P < 0.01 vs. BBR group.

37

Fig. 5. Effect of OBB on the immune-inflammation status in DSS-induced colitis mice. Effect of OBB on colonic inflammatory cytokines TNF-α (A), IL-1β (B), IL-6 (C), IL-17 (D), IFN-γ (E) and IL-10 (F), and serum immunoglobulins IgA (G), IgG (H) and IgM (I) in mice with DSS-induced colitis was determined by ELISA. Data are expressed as the mean ± SEM (n = 7-10). # P < 0.05, ## P < 0.01 vs. Control group, * P < 0.05, ** P < 0.01 vs. DSS group, & P < 0.05, && P < 0.01 vs. BBR group. Fig. 6. Effect of OBB on the activation of TLR4-MyD88-NF-κB signaling pathway in DSS-induced colonic tissues. (A) Representative Western blotting images of TLR4,

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MyD88, cytoplasmic p65, nuclear p65, p-IκBα and IκBα. Changes in the relative

protein expression levels of TLR4 (B), MyD88 (C), nuclear p65 (D), cytoplasmic p65

(E), and p-IκBα/IκBα ratio (F) were measured. Data are shown as the mean ± SEM (n

-p

= 3). # P < 0.05, ## P < 0.01 vs. Control group, * P < 0.05, ** P < 0.01 vs. DSS group.

Fig. 7. Molecular docking simulation of BBR into the active site of TLR4 (A), OBB

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in the active site of TLR4 (B), BBR binding with MyD88 (C), OBB binding with MyD88 (D), BBR interacting with NF-κB (E), and OBB interacting with NF-κB (F).

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Fig. 8. Effect of OBB on intestinal microbiome homeostasis. (A) OTU number for each group of samples; (B) OTU venn analysis; (C) Relative contribution of phylum

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in each group; (D) Characteristics of microbial community composition using LEfSe analysis; (E). Relative contribution of genus in each group.

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Fig. 9. β-diversity analysis in fecal samples. (A) PCA analysis. (B) PCoA analysis; (C) Cluster tree.

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Fig. 10. Summary scheme of the mechanisms underlying the inhibitory effect of OBB on DSS-induced colitis. (A) Normal intestinal tract, (B) DSS-induced colitis, (C) OBB-pretreated colitis. OBB, a novel gut microbiota metabolite of BBR, significantly modulated the dysbacteriosis induced by DSS and restored the dysbacterium to normal level. Meanwhile, OBB protected intestinal epithelial barrier disruption induced by DSS, via up-regulating the mucin mRNA expression and TJs protein expressions, including ZO-1, ZO-2, JAM-A, claudin-1, occludin. Furthermore, OBB 38

remarkably

blocked

TLR4-MyD88-NF-κB

signaling

pathway,

through

down-regulating the protein expression of TLR4 and MyD88, inhibiting the phosphorylation of IκBα and the migration of NF-κB p65 from cytoplasm to nucleus,

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and thus decreased colonic inflammatory cytokine levels induced by DSS.

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