Materials

General laboratory chemicals and reagent solutions were purchased from Sigma-Aldrich (St. Louis, MO). ELISA kits for IL-6 and TNF-α were purchased from Bio-legend. ELISA kit for CXCL1 was purchased from R&D systems. All antibodies were purchased from Santacruz unless otherwise specified. LPS was purchased from Sigma Aldrich. Colitis grade DSS (36,000–50,000 M.W) was purchased from MP Bio. UroA was custom synthesized as previously described23.

Mice

C57BL/6 mice were either bred in our animal facility or purchased from Jackson Laboratories. Breeding pairs of Nrf2−/− mice (B6.129x1-Nfe2/2tm1Ywk/J, stock # 0170009) were purchased from Jackson Laboratories and bred at U of L animal facility to generate experimental animals. AhR−/− mice (Model# 9166) were purchased from Taconic Laboratories. We utilized the mice at the ages of between 7–9 weeks age old for colitis experiments. Mice were kept in specific pathogen-free (SPF) barrier conditions with temperature-controlled room with alternate 12 h cycles of dark and light. Animals were allowed free excess to feed and water ad libitum. All studies were performed under approved protocols from Institutional Animal Care and Use Committee (IACUC), University of Louisville, Louisville, KY, USA. Source Data for all the bar graphs are provided as a Source Data File.

Synthetic procedure for synthesis of UAS03

Chemically UroA (3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one) structure has a bridge ester, lactone, and two hydroxyl on two phenyl rings. UroA has a lactone (cyclic ester) bond that connects two phenyl rings and leads to the planar structure. Gastric pH or digestive enzymes can hydrolyze the lactone bond leading to opening of the ring. This will result in losing the planar structure, becomes propeller structure, and potentially loses its activities. To generate more stable and potent compounds, we have synthesized non-hydrolyzable cyclic ether derivative, UAS03 by the following procedure (Supplementary Fig. 21).

Sodium borohydride (0.165 g, 4.38 mmol) was added to dry THF (10 ml), and the mixture was cooled 10 °C before borontrifluoride etherate (0.80 g, 5.7 mmol) was added drop wise over a period of 1 h. Then 3,8-dihydroxy-6H-benzo[c]chromen-6-one (Uro-A) (0.5 g, 2.19 mmol) in THF (5 ml) was added over a period of 10 min. The mixture was allowed to stir for 5 h at 50 °C. The completion of reaction was monitored by thin layer chromatography (TLC). The reaction was quenched with methanol. 3 N aqueous HCl solution (10 ml) was added, and the mixture was gently heated to 50 °C for 30 min. The reaction mixture was adjusted to neutral with 10% NaOH solution, and the volatiles were evaporated under reduced pressure. The crude product was purified by column chromatography using 50% ethylacetae in Hexane with 60–120 mesh silica gel to get pure 6H-benzo[c]chromene-3,8-diol product.

MS (M+1) = 215.2. 1H-NMR (DMSO-d 6 ): δ: 9.49 (2H, s), 7.51–7.50 (1H, d, J = 6.6 Hz), 7.48–7.47 (1H, d, J = 6.6 Hz), 6.75–6.73 (1H, m), 6.61 (1H, s), 6.48–6.46 (1H, m), 6.32 (1H, s), 4.96 (2H, s). 13C-NMR (DMSO-d 6 ): δ: 158.10, 156.71, 154.93, 131.88, 123.86, 122.79, 121.66, 115.72, 114.89, 111.84, 110.07,103.95, 68.18.

Cell cultures

Human colon epithelil carcinoma cell lines, HT29 (ATCC # HTB-38TM) and Caco2 cells (ATCC # HTB-37TM) were maintained in DMEM-high glucose and EMEM-high glucose (Cornings; 10-009CV) respectively, supplemented with 10% fetal bovine serum, 1X penicillin-streptomycin solution (100 U/ml penicillin, and 100 µg/ml streptomycin; Sigma Aldrich) in a humidified atmosphere (5% CO 2 , 95% air, 37 °C). Mouse bone marrow derived macrophages (BMDMs) were isolated and cultured using the following procedure59. Briefly, mice were killed by CO 2 anesthesia, rinsed in 70% ethanol and bone marrow was isolated from tibias and femurs. Bone marrow cells were plated (2 × 106 cell/ml) in DMEM-high glucose (HyClone) supplemented with 10% FBS, 1% glutamine, 1X penicillin-streptomycin solution and 50 ng/mL mouse M-CSF (R&D Systems Inc., Minneapolis, MN) for 7 days for differentiation.

Measurements of IL-6 and TNF-α levels in BMDM

BMDM were plated in 96 (10,000 cells/well) and 12 wells (0.1 × 106 cells/well) plate for ELISA and RNA isolation. To evaluate the anti-inflammatory properties, BMDMs were stimulated with E. coli-derived lipopolysaccharides (LPS; O55:B5; Sigma) at 50 ng/mL concentration for six hours alone or in combination with UroA or UAS03 at indicated concentrations (0.01, 0.1, 1, 10, 25, and 50 μM) in quadruplicates. For cytokine production via ELISA, the supernatant was collected and centrifuged at 10,000 × g for 10 min at 4 °C to pellet down any cell and cytokines were quantified using IL-6 and TNF-α specific ELISA kit (Biolegend) following manufacturer’s instruction.

LPS-induced peritonitis

Male mice (C57BL/6J; 6–8 weeks old) were randomly divided in 3 groups viz. vehicle (0.25% sodium carboxymethylcellulose (CMC)), UroA and UAS03. UroA and UAS03 groups received oral gavage of respective compounds (20 mg/kg in 100 μl of volume) at 0, 6, 12, 18, and 24 h. Vehicle group received same volume of CMC at same time. After 24 h, mice were injected intra-peritoneally with LPS (2 mg/kg; Sigma-Aldrich). Post 4 h LPS challenge, mice were killed and blood was collected. The serum was prepared using BD Microtainer separator tubes. The serum samples were analyzed for IL-6 and TNF-α using respective ELISA assay kit (Biolegend).

Real-time PCR

Total RNA was isolated from cells/tissue using Maxwell® 16 LEV simplyRNA tissue kit (Promega) and reverse transcribed with TaqMan™ Reverse transcription Kit (Applied Biosystems, CA, USA). The transcribed cDNA (after dilution) was mixed with 100 nM gene specific primers (Real time primers LLC) and 1X SYBR green reaction mix (Power SYBR® Green PCR Master Mix; Applied Biosystems, CA, USA). Changes in gene expression was analyzed using CFX96TM Real-Time System (Bio Rad) and fold change in expression was calculated using 2-ΔΔCT method using GAPDH/β-actin as house keeping gene and normalized with untreated control.

In vitro permeability study

For in vitro cellular permeability studies, Caco2 cells or HT29 cells (2 × 104 cells/cm2) were seeded in 24-well Transwell® plates (Cornings; USA), on polyester membrane filters (pore size 0.4 µm, surface area 1.12 cm2)60. Culture medium was added to both apical and basal chamber and the medium was changed every other day up to 21 days for Caco2 cells or 5–7 days for HT29 cells. For Caco2 cells, transepethelial electrical resistance (TEER) was calculated using EMD Millipore Millicell-ERS2 Volt-Ohm Meter (Millipore). Filters (with cell monolayer) showing more than 600 Ω.cm2 were used for permeability study. After cells reach desired confluence (monolayered cells), cells were pre-treated with vehicle (0.01% DMSO) UroA (50 μM) and UAS03 (50 μM) for 24 h. After treatment, monolayer was washed with PBS to remove any residual drug and 200 μL of LPS (50 ng/ml in HBSS) was added to each well and incubated for 2 h. After LPS treatment, the monolayer was washed with PBS twice and 200 μL of FITC-Dextran (FD-4; Sigma Aldrich, USA) solution (1 mg/mL in HBSS) was added. After 2 h, a sample from the basal chamber was withdrawn and FD4 concentration was determined using fluorescence 96-wells plate reader at excitation and emission wavelengths were 480 and 525 nm, respectively.

RNA sequencing

Total RNA was isolated from HT29 cells treated with vehicle and UroA (50 μM) (n = 3) for 24 h and RNA was isolated using Trizol based lysis followed by Qiagen RNeasy kits. The isolated RNA was checked for integrity (RIN>9.5) using the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA) and quantified using a Qubit fluorometric assay (Thermo Fisher Scientific, Waltham, MA). Poly-A enriched mRNASeq libraries were prepared following Illumina’s TruSeq Stranded mRNA LT library preparation protocol (Illumina Inc., San Diego, CA) using 1 µg of total RNA. All 15 samples were individually barcoded and quantitated with the KAPA Library Quantitation Kit for Illumina Platforms (Kapa Biosystems, Wilmington, MA) in conjunction with an Agilent Bioanalyzer DNA 1000 analysis (Agilent Technologies, Santa Clara, CA) for fragment size determination. The average fragment size was approximately 300 bp. 1.8 pM of the pooled libraries with 1% PhiX spike-in was loaded on one NextSeq 500/550 75 cycle High Output Kit v2 sequencing flow cell and sequenced on the Illumina NextSeq 500 sequencer. The quality of the 1 × 75 bp sequences was checked using FASTQC (version 0.10.1)61. Trimming was not necessary with the median quality score above 30 (error probability = 0.001 or 1 base call in 1000 is predicted to be incorrect) across the entire length of the read and the lower quantile above a score of 20 (error probability = 0.01) at the end of the read where there is an expected decrease in quality. The raw reads for each sample were directly aligned to the Homo sapiens (hg38) reference genome assembly (hg38.fa) using tophat2 (version 2.0.13)62, generating alignment files in bam format. Optional parameters include –no-coverage-search and –library-type fr-firststrand. The human ENSEMBL63 transcriptome gtf v82 was used for transcript identification, resulting in 60,903 total genes. Supplementary Table 1 indicates the number of raw reads successfully aligned for each of the samples. On average, 26 million reads were aligned per sample with a mean alignment rate of 97%. Following sequence mapping, differentially expressed genes were determined using tuxedo suite of programs including cuffdiff2 (version 2.2.1)64,65 with the optional parameter –library-type fr-firststrand. The RNA-seq data was deposited in gene data base (GEO # GSE113581).

Immunoblots (western blots)

The total protein lysates were collected either from colon tissue/cells using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, USA) and quantified using BCA protein quantification kit (Thermo Scientific) as per instructional manual. Total protein (20–50 μg) of was resolved on NuPAGETM 4–12% Bis-Tris gel (Novex Life technologies) and transferred to polyvinylidene difluoride membrane (0.22 μm pore; Millipore, USA). After blocking with 5% (w/v) skim milk powder (containing 1X TBS) for 1 h, the membrane was then incubated with respective antibodies at 4 °C overnight (dilutions of respective antibodies is given in Table 1). Next day, respective secondary antibody conjugated with Horseradish peroxidase were probed and the chemiluminescent substrate was used to detect the protein bands (ImageQuant LAS 4000). Densitometry analysis of bands were done using ImageJ software. Anti-bodies for Cldn4, Ocln, Cldn1, Cyp1A1, AhR, HO1, NQO1, Keap1, β-actin, and Lamin B were purchased from Santa Cruz Biotechnologies (USA) and Nrf2 from Novus Biologicals (USA). Source and list of antibodies are provided in Table 1. The uncropped images of important immunoblots are shown in Supplementary Fig. 22.

Table 1 List of antibodies used for western blots Full size table

Confocal imaging

HT29 or CaCo2 or BMDM cells (50,000 cells/well) were plated on to 8-well chambered slides (154534PK; ThermoFisher Scientific) allowed them to grow overnight. The cells were induced with vehicle (0.01% DMSO) or UroA (50 µM) or UAS03 (50 µM) for desired time points and fixed with cold methonol. The AhR or Nrf2 or Cldn4 stained with respective anti-bodies (1:200 dilution) followed by fluorecently labeled (Alexa flour 594 for AhR and Alexa flour 488 for Nrf2 and Cldn4) secondary ab (1:500 dilution; ThermoFisher Scientific). The nucleus was stained with DAPI (Sigma Aldrich). The confocal images were captured using Nikon A1R confocal microscope using ×60 magnification lense with appropriate laser channels.

AhR-reporter assay

AhR-reported assay was performed using AhR Reporter Assay system (Indigo Biosciences). The AhR Reporter cells (expressing luciferase under AhR promoter) as well as positive control MeBio (AhR ligand) compound were provided in the kit. The cells were treated with Vehicle or UroA or UAS03 or ellagic acid or MeBio for 6 h and luminoscence was measured according to manufacture’s instructions.

Nrf2-reporter assay

ARE-luciferase plasmid vector was obtained from Cayman Chemicals. HT29 cells were transfected at 50% confluency using lipofectamine 3000 reagent (ThermoFisher Scientific). Briefly, cells were seeded in 6-well plates (0.5 × 106 cells) and grown for 24 h. The transfection complex containing 1 µg of plasmid DNA and transfection reagent was added to each well in absence of FBS. After 6 h medium containing 10% FBS was added and cells were incubated for another 16–18 h. These cells were treated with vehicle (0.01% DMSO) or UroA (50 µM) or UAS03 (50 µM) or sulforaphane (10 µM) for 24 h. After incubation with inducers, cells were lysed and firefly luciferase activities (luminiscence) were measured with Luciferase Assay System (Promega) using mutiwell plate luminometer (BMG, LABTECH).

Measurements of Cyp1A1 enzyme activty (ex vivo)

Mice were treated with Vehicle or UroA or UAS03, BNF, or FICZ daily for 1 week at indicated concentration either through oral or i.p. route. After 1 week, mice were euthanized and the colon and liver tissues were dissected. Microsomes from these tissues were prepared using the following procedure66. For hepatic microsomes, liver was first perfused with 0.9% sodium chloride solution and excised out. Adhering blood and saline was removed by blotting on tissue paper and tissue was homogenized in tissue homogenization buffer (50 mM Tris-HCl, pH 7.4 with 250 mM sucrose). Homogenate was centrifuged at 10,000 × g for 30 min at 4 °C. supernatant obtained was further centrifuged at 105,000 × g for 60 min at 4 °C. The pellet was washed with homogenization buffer and centrifuged again at 105,000 × g for 60 min at 4 °C. The pellet was suspended in homogenization buffer and used for protein and CYP assay. For intestinal microsome preparation, intestine was removed and washed with 0.9% sodium chloride. The intestine was longitudinally cut open to expose mucosal layer and mucosa was scrapped with help of glass slide. The scraped tissue was collected in homogenization buffer (50 mM Tris-HCl buffer containing glycerol (20% v/v), protease inhibitor (1%) and heparin (3 U/ml)). This suspended mucosa was homogenized and centrifuged at 10,000×g for 20 min at 4 °C. Supernatant obtained was further centrifuged at 105,000 × g for 60 min at 4 °C. The pellet was washed with buffer and centrifuged again at 105,000 × g for 60 min at 4 °C. The pellet was suspended homogenization buffer and used for protein and CYP enzymes assays.

Ethoxyresorufin-O-deethylase (EROD) assay

The microsomal proteins (0.5 mg) were mixed with 200 μL Tris buffer (0.1 M, pH 7.4) containing ethoxyresorufin (0.01 mM). To start reaction, NADPH (0.1 mM) was added and incubated at 37 °C for 10 min. After 10 min, reaction was terminated by adding equal volume of acetonitrile and reaction mixture was centrifuged at 13,000 × g for 10 min at 4 °C. Supernatant was used to determine resorufin by measuring fluorescence (Ex. 530 nm, Em. 580 nm). Pure resorufin (Sigma Aldrich) was used to generate standard curve.

P450-Glo Cyp1A1 luminiscence assay

The above microsomes (20 μg) were used for P450-Glo Cyp1A1 luminiscence assays as per manufacturer’s instructions.

Measurement of Cyp1A1 enzyme activities in vitro

EROD assay: HT-29 cells (15,000 cells/well) treated with vehicle, UroA and UAS03 (24 h), were rinsed with HBSS buffer, and then fresh HBSS buffer was added along with 5 μM of 7-ethoxyresorufin. Cells were further incubated at 37 °C for 1 h. After the incubation time, fluorescence (Exc. 530 nm, Em. 580 nm) was measured and product (resorufin) formed was calculated from calibration standard and normalized with protein concentration.

P450-Glo Cyp1A1 luminiscence assay: HT29 cells (25,000 cells/well) were plated in 48 well plate. Cell were then treated with UroA (0.1, 1, 10, 25, and 50 µM) or UAS03 (0.1, 1, 10, 25, and 50 µM) or FICZ (0.1, 1, 10, 25, and 50 nM) for 24 h. After treatment, cells were washed to remove any residual drugs, and fresh medium containing Cyp1A1 substrate (as per protocol provided with kit Cat.# V8751; Promega) for 3 h. After incubation, 25 µl of culture medium was removed from each well and transferred to a 96-well white opaque plate and 25 µl of luciferin detection reagent was added to initiate the luminescence reaction and plate was incubated at room temperature for 20 min. After incubation, luminescence was recorded in luminometer. The data reported as fold change over vehicle treatment.

Small interfering RNA (siRNA) mediated knockdown experiment

The AhR siRNA (SR300136) and Cyp1A1 siRNA (SR301093) was purchased from Origene. For knockdown experiments, HT29 cells (0.5 × 106 cells/well) were plated in 6 well plate and grown for 24 h. The AhR, Cyp1A1 and control-siRNA was transfected into HT29 cells using Lipofectamine ® RNAiMAX reagent (ThermoFisher Scientific) as per intruction given. After 24 h of transfections, cell were induced with vehicle (0.01% DMSO), UroA (50 µM), and UAS03 (50 µM) for 24 h. After treatment with inducers, cells were lysed using RIPA buffer and total protein was used to analyse the expression of AhR, Cyp1A1 and Cldn4 by western blot.

Cyp1A1 deletion by CRISPR/Cas9 method

HT29 cells (1.5 × 105) were plated in 6-well in antibiotic free standard growth medium 24 h prior to transfection. At 60% confluency cells, cells were co-transfected with 2 µg each of CRISPR/Cas9 KO Plasmid (sc-400511-KO-2; Santa cruz) and HDR Plasmid (sc-400511-HDR-2; Santa cruz) using UltraCruz® Transfection Reagent (sc-395739; Santa Cruz). Medium was replaced with selective medium (containing 4 µg/mL puromycin) 96 h post transfection. Transfection was confirmed with fluorescence microscopy and western blot (CYP1A1). The double postive cells for GFP and RFP were sorted using MoFlo XDP sorting instrument (Beckman Coulter). The deletion of Cyp1A1 in these sorted was confirmed by western blots. These cells were then plated in 6-well plate for in standard medium for evaluating the effect of UroA/UAS03 on Cldn4 expression. After 24 h of UroA/UAS03 treatment cells were harvested for protein and Cldn4 expression was investigated along with normal HT29 cells.

NF-κB EMSA assay

RAW 264.7 cells or BMDM were plated in 100 mm dishes (1 × 106) in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin. Cells were allowed to grow for 24 h and after incubation, cells were treated with LPS (50 ng/mL) with and without UroA (50 µM) and UAS03 (50 µM) for 6 h. After treatments, culture medium was removed and washed with PBS. Cells were scraped and pelleted down in PBS. Supernatant was discarded and pellet was used for isolation of nuclear and cytosolic protein using NE-PER Nuclear and Cytoplasmic kit (Thermo Scientific; Cat #78833). Later nuclear protein (2 µg) was used for EMSA using Non-Radioactive EMSA Kits with IR Fluo-Probes for Nuclear factor kappa B p65 (Viagene Biotech Inc Cat # IRTF282 60).

Colon explant culture

Colon tissue pieces (0.5–1 cm length) from wild type (C57BL/6) or Nrf2−/− or AhR−/− mice were cultured in triplicates for 24 h in complete DMEM-high glucose medium (supplemented with 10% fetal bovine serum, 1X penicillin-streptomycin solution) in a humidified atmosphere in the presence of vehicle (0.01% DMSO), UroA (50 µM) or UAS03 (50 µM). The tissues were processed for protein preparation (tissue lysates with RIPA plus buffer) or total RNA isolation. These tissue lysates or RNA were used to determine the expression of Nrf2, Cldn4 and AhR.

Tissue processing for RNA and protein analysis

Mice were treated with as described in results section. Mice were euthanized with CO 2 asphyxiation followed by cervical dislocation. Colon was dissected out and luminal contents were flushed out with cold PBS (containing PMSF and Sodium orthvandate). Small portion of colon was snap frozen in liquid nitrogen and stored at −80 °C for RNA analysis. For preparation of protein samples, colon was opened longitudinally and mucosa was scraped in ice-cold 1X PBS using pre-chilled glass slide and centrifuged at 300 × g for 10 min at 4 °C. Supernatant was discarded and pellet was suspended in RIPA buffer (containing 1X protease inhibitor) and vortexed at high speed. After 30 min incubation on ice, samples were centrifuged at 13,000 × g for 20 min at 4 °C. Supernatant was collected and protein was quantified using BCA protein quantification kit. The lysates were used appropriately for western blots.

28-day repeated dose toxicity study

To evaluate toxicity of UroA and UAS03, we performed 28-days repeated dose toxicity study. Mice were fed (oral gavage) with UroA (20 and 40 mg/kg/day) and UAS03 (20 and 40 mg/kg/day) daily for 28 days. Body weight, food, and water intake were assessed weekly. After 28 days, mice were killed and gross examination of all major organs were performed. Blood was collected to obtain serum. Serum alanine aminotransferase (ALT) and asparate aminotransferase (AST) were analyzed using ALT/ AST kit (BioVision) as per instructional manual.

2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced colitis

Male C57BL/6 or Nrf2−/− mice (6–8 week old age mice) were anesthetized with ketamine/xylazine (100 mg/12.5 mg/kg IP) mixture and administered with single dose of TNBS (2.5 mg/mice; Sigma Aldrich, USA) in 50% ethanol. After administration of TNBS, mice were held upside down for 30–60 s to ensure proper distribution of TNBS in the colon. Control group received 50% ethanol without TNBS. Mice with TNBS were randomly divided into three groups, viz. vehicle (0.25% sodium carboxymethylcellulose (CMC)), UroA and UAS03. UroA or UAS03 was resuspended in 0.25% sodium-CMC at desired concentrations. The mice were given orally Veh or UroA or UAS03 in 100 μl at desired concentrations (4 or 20 mg/kg/body weight). The treatment started after 12 h of TNBS administration and every 12 h thereafter up to 72 h. The experiment was terminated post 60 h TNBS, where AhR−/− mice were involved. In some experiments, we treated only once at post 12 h TNBS administration. TNBS administered and control mice were euthanized for tissue and plasma collection after 80 h of TNBS/ethanol treatment. Mice were examined for colitis phenotype.

DSS-induced colitis

Acute experimental colitis in mice was induced by giving 3% (w/v) colitis grade DSS (MP Biomedicals) in drinking water for 7 days. Control animal received drinking water without DSS. All colitis group mice were randomly divided into three groups viz. vehicle treated (0.25% Na-CMC), UroA (20 mg/kg/day) and UAS03 (20 mg/kg/day) on the 4th and 6th day of DSS treatment. After 7 days, animals were put back on regular water for a period of 7 days. For chronic DSS colitis model, we used three cycles of 2.0% (w/v) DSS and each DSS cycle consisted or 7 days followed by 10 days of regular water and mice were treated with UroA (20 mg/kg/day) on every 4th and 6th day of DSS cycle.

Assessment of colitis severity and tissue collection

Mice were evaluated daily for change in body weight, stool consistency, and rectal bleeding and score was given and combined to obtained disease activity index67. After euthanasia, the colon was removed and flushed with PBS containing (1 mM PMSF and 0.2 mM sodium orthovanadate). Colon length and colon weight were measured and small parts of colon were excised for myeloperoxidase (MPO) activity and RNA isolation. Tissues for MPO and RNA extraction were snap frozen in liquid nitrogen and stored in −80 °C until further analysis. Tissue for histological examination was stored in 10% phosphate buffered saline formalin. Blood was collected and serum was separated by centrifugation at 3500 × g for 15 min. Serum cytokines (IL-6, TNF-α; Biolegend) and chemokines (CXCL1; R&D Systems) levels were measured by ELISA according to manufacturer’s instructions.

In vivo intestinal permeability assay

The gut barrier function was evaluated by in vivo intestinal permeability using FITC-Dextran (MW 4000; FD4, Sigma-Aldrich, USA)68. Briefly, mice were orally administered with FITC-dextran (60 mg/100 gm body weight). Mice were fasted for 4 h prior to euthanization. The FITC-dextran concentration in serum was determined using the standard curve of FITC-dextran in serum (excitation, 485 nm; emission, 525 nm; BMG LABTECH).

Myeloperoxidase (MPO) activity

The MPO activity in the colons was determined using the following procedure69. Briefly, colon tissue was homogenized in 0.5% (w/v) hexadecyltrimethylammonium bromide (H6269; Sigma-aldrich, USA) in 50 mM PBS, pH 6.0. This homogenate underwent 3 freeze-thaw cycles and 10–15 s sonication to obtain homogenous suspension. The supernatant from this suspension was collected after centrifugation at 13000 × g for 20 min at 4 °C. The supernatant (10 µl) was then added to 50 mM potassium phosphate buffer (pH 6.0) containing 0.167 mg/ml o-dianisidine (Sigma-Aldrich, USA) and 0.0005% H 2 O 2 (Sigma-Aldrich USA) and absorbance was taken at 450 nm (BMG, LABTECH) at 2 min interval. Units of MPO in each sample was determined by considering that one unit (U) of MPO = 1 µmol of H 2 O 2 split with molar extinction coefficient of 1.13 × 10−2 nm/min and MPO in each sample calculated by using [ΔA(t 2 − t 1 )]/Δmin × (1.13 × 10−2) formula and MPO units were normalized with per mg tissue.

Histopathology

Collected colon tissue were fixed in 10% buffered formaldehyde solution overnight and fixed tissue underwent standard histopathological processing. Briefly, after fixation tissue underwent dehydration and cleaning with xylene before paraffin embedding. The paraffin section of 5 µm were cut (Leica microtome) and stained for H&E staining. The H&E images were captured using Aperio Scanscope. H&E sections were scored blindly using index scoring described by Erben et al.70.