Methods
Cell culture
Caco-2bbe2 (ATCC, Rockville, MD) and m-ICCl2 (a kind gift from Dr. Alain Vandewalle, INSERM U773, Paris, France; (Bens et al., 1996)) cells were grown under standard cell culture conditions as previously described (Sheth, Samak, Shull, Seth & Rao, 2009a). Experiments were conducted using cells grown in Transwell inserts of varying diameters (6.5 or 12 mm) for 3–4 or 15–17 days.
Epithelial barrier function
Transepithelial macromolecular permeability was evaluated by measuring the unidirectional flux of FITC-inulin as previously described (Sheth, Samak, Shull, Seth & Rao, 2009a).
Animals
All animal studies were performed under the protocols approved by the University of Tennessee Health Science Center (UTHSC) Institutional Animal Care and Use Committee (IACUC). In the first study, 12-14 week-old adult wild type (WT) and Lpar2-/- mice were subjected to total body irradiation (TBI; 9.5 Gy). WT andLpar2-/- mice were randomized into Sham (sham-treated), and IR (irradiated) groups. The integrity of colonic epithelial tight junction and adherens junction were examined at 2 hours after TBI. In the first study, mice were subjected to TBI with or without RP-1 administration (0.1 mg/kg, i.p., single dose 24 hours before irradiation, pretreatment group). Control mice were subjected to similar conditions without radiation (sham-treated). In the second study, mice were randomized into three groups: Sham (control), IR (irradiated with the administration of vehicle), and IR+ RP-1 (irradiated with the administration of RP1 at 24 hours post-irradiation). The integrity of colonic epithelial tight junction, adherens junction, and endotoxemia was examined at 2 and 4 hours post-irradiation. In the third study, mice were subjected to partial body irradiation, in which 5% of the bone marrow was shielded (PBI-BM5). Experimental groups consisted of Sham (control), IR (irradiated with the administration of vehicle), and IR+RP-1 (irradiated with intraperitoneal administration of RP1; 3 mg/kg, s.c.; at 24 hours post-irradiation). At varying times (28, 52, 76, and100 hours) after irradiation, the integrity of colonic epithelial tight junction, adherens junction, and actin cytoskeleton was examined. At 28 hours post-irradiation, colons were collected and analyzed for oxidative stress and epithelial junctional integrity.
Irradiation
Cells were irradiated with 2, 5, or 10 Gy γ-irradiation from a137Cs source (using a J.L. Shepherd & Assoc. Mark I, Model 25, San Fernando, CA, USA) at a dose rate of 440 cGy/min. After irradiation, the culture medium was replaced with fresh complete culture medium. For TBI, mice were subjected to radiation (9.5 Gy at a dose rate of ~76 cGy/min). For the PBI model, mice were anesthetized with 87 mg/kg ketamine and 13 mg/kg xylazine IP and placed in a plexiglass restrainer so that the legs below the knee were shielded. The shielding protected tibiae, fibulae, and the paws from radiation that contained ∼5% of the bone marrow. Mice placed into the isodose field of the irradiator were irradiated in groups of eight. In this model, 15.7 Gy delivered at a dose rate of 147 cGy/min yielded empirical ∼LD85/10 mortality. Radiation field mapping and calibration by ion chamber dosimetry were done by the manufacturer. In addition, routine validation and quality control measurements of exposure rates and exposure rate mapping in the chamber at positions of interest were conducted by a certified health physicist using a calibrated RadCal 0.6 cc therapy grade ion chamber/electrometer system. High-dose thermoluminescent dosimeters were used in most irradiations to validate the actual dose delivered to the mice (calibrated at MD Anderson Cancer Center Radiation Dosimetry Services). The isodose field was validated using Gafchromic film for high-dose dosimetry (10-50 Gy, Ashland Inc., Covington, KY). At the end of the experiment, gut permeability was measured as described below. Colon segments were stored frozen for further analyses.
Gut permeability in vivo
Mucosal barrier dysfunction in vivo was evaluated by measuring gut permeability to FITC-inulin (6 kDa). On the last day of the experiment, mice were injected with FITC-inulin (50 mg/ml solution; 2 µl/g body weight) via the tail vein. One hour after injection, blood samples were collected by cardiac puncture under isoflurane anesthesia, and plasma was isolated using heparin sulfate anticoagulant. Luminal contents from the colon and ileum were flushed with 0.9% saline. Fluorescence in plasma and the luminal flushing was measured using a fluorescence plate reader. Fluorescence values in the luminal flushing were normalized to fluorescence values in corresponding plasma samples and calculated as the percent of the amount injected.
Immunofluorescence microscopy
Caco-2 and m-ICC12 cell monolayers were permeabilized with 0.2% Triton X-100, blocked, and stained for different junctional proteins as described in a prior study (Shukla et al., 2018). Fluorescence was examined by using a Zeiss LSM 510/710 laser scanning confocal microscope. Cryo-sections of the colon (10 µm thickness) were fixed in acetone and methanol mixture (1:1) at 20oC for two minutes and rehydrated in 14 mM phosphate-buffered saline (PBS). Sections were permeabilized with 0.5% Triton X-100 in PBS for 15 minutes and blocked in 4% non-fat milk in TBST (20 mM Tris, pH 7.2 and 150 mM NaCl). Sections were incubated for one hour with primary antibodies (mouse monoclonal anti-occludin and rabbit polyclonal anti-ZO-1 antibodies or mouse monoclonal E-cadherin and rabbit polyclonal anti-β-catenin antibodies), followed by incubation for one hour with the secondary antibodies (AlexaFluor-488-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG antibodies from Molecular Probes, Eugene, OR) containing Hoechst 33342 dye. In all cases, images from x-y (1 μm) sections were collected using LSM Pascal or Zen software (White Plains, NY, USA). Images from optical sections were stacked using ImageJ software (NIH, Bethesda, MD, USA) and processed with Adobe Photoshop (Adobe Systems, San Jose, CA, USA).
Preparation of the detergent-insoluble fraction
Actin-rich detergent-insoluble fraction was prepared as described previously (Rao, Basuroy, Rao, Karnaky & Gupta, 2002). Mucosal scrapping from the colon and ileum were incubated on ice for 15 minutes with lysis buffer-CS (Tris buffer containing 1% Triton-X100, 2 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml bestatin, 10 µg/ml pepstatin-A, 10 µl/ml of protease inhibitor cocktail, 1 mM sodium vanadate and 1 mM PMSF). Briefly, mucosal lysates were centrifuged at 15,600 x g for 4 min at 4°C to sediment the high-density actin-rich detergent-insoluble fraction. The pellet was suspended in 100 µl of preheated lysis buffer-D (20 mM Tris buffer, pH 7.2, containing 10 µl/ml of protease inhibitor cocktail, 10 mM sodium fluoride, 1 mM sodium vanadate and 1 mM PMSF), sonicated to homogenize the actin cytoskeleton, and heated at 100°C for 10 min. Protein content was measured by the BCA method (Pierce Biotechnology, Rockford, IL). Triton-insoluble and soluble fractions were mixed with an equal volume of 2X concentrated Laemmli’s sample buffer, heated at 100°C for 5 min, and 25-40 µg protein samples were used for SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis.
Immunoblot analysis
Triton-soluble and insoluble fractions were separated by SDS-polyacrylamide gel electrophoresis (7%) and transferred to PVDF membranes as described in a previous study (Rao, Basuroy, Rao, Karnaky & Gupta, 2002). Membranes were immunoblotted for different proteins using specific antibodies for different tight junction and adherens junction proteins with β-actin as the housekeeping protein in combination with HRP-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibodies. Blots were developed using the ECL chemiluminescence reagent (Pierce, Rockford, IL) and quantitated by densitometry using ImageJ software. The density for each band was normalized to the density of the corresponding actin band.
Protein thiol assay
Protein thiols in colonic sections were monitored as described previously (Shukla et al., 2018). Briefly, reduced protein thiols were evaluated by staining cryosections of the colon with BODIPY FL-N -(2-aminoethyl) maleimide (FLM) and confocal microscopy at excitation and emission wavelengths (490 nm and 534 nm, respectively). For oxidized protein thiols, the reduced protein thiol was first alkylated with N-ethylmaleimide followed by reduction of oxidized protein thiols with tris (2-carboxyethyl) phosphine prior to staining with FLM. Control staining was done after N-ethylmaleimide treatment. Fluorescence images were collected and quantitated using ImageJ software.
RNA extraction and RT-qPCR
RNA was isolated from the colon by using the TRIzol kit (Invitrogen, Carlsbad, CA, USA) and quantified using a NanoDrop photometer. Total RNA (1.5 μg) was used for the generation of cDNA using the ThermoScript RT-PCR kit for first-strand synthesis (Invitrogen). Quantitative PCR (qPCR) reactions were performed using cDNA mix (cDNA corresponding to 35 ng RNA) with 300 nmole primers in a final volume of 25 μl of 2× concentrated RT2 Real-Time SYBR Green/ROX master mix (Qiagen, Germantown, MD, USA) in an Applied Biosystems QuantStudio 6 Flex Real-Time PCR instrument (Norwalk, CT, USA). The cycle parameters were: 50 °C for 2 min, one denaturation step at 95 °C for 10 min, and 40 cycles of denaturation at 95 °C for 10 s, followed by annealing and elongation at 60 °C. The relative gene expression of each transcript was normalized to the GAPDH gene transcripts using the ΔΔCt method. Sequences of primers used for qPCR are provided in the Supplemental Information section (Table-1).
Plasma endotoxin assay
Plasma endotoxin concentrations were measured using Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Scientific, Cat# 88282) according to the manufacturer’s instructions.
Statistical Analysis
All data are expressed as Mean ± SEM. The differences among multiple groups were first analyzed by ANOVA (Prism 6.0, GraphPad, Inc. San Diego, CA, USA). When statistical significance was detected, Tukey’s t-test was used to determine the statistical significance between multiple testing groups and the corresponding control. Statistical significance was established at 95% confidence.
Materials
Hoechst 33342 dye and BODIPY FL-N -(2-aminoethyl) maleimide were purchased from Life Technologies (Grand Island, NY, USA). N-ethylmaleimide and tris (2-carboxyethyl) phosphine were from Sigma-Aldrich (St Louis, MO, USA). All other chemicals were purchased from either Sigma-Aldrich or Thermo Fisher Scientific (Tustin, CA, USA). Anti-ZO-1, anti-occludin, anti-claudin-2 (Cldn-2) and anti-claudin-3 (Cldn-3) antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Anti-E‐Cadherin and anti-β‐catenin antibodies were purchased from BD Biosciences (Billerica, MA, USA). Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG and anti-β-actin antibodies were obtained from Sigma-Aldrich (St Louis, MO, USA). AlexaFlour‐488‐conjugated anti‐mouse IgG and Cy3‐conjugated anti‐rabbit IgG were purchased from Molecular Probes (Eugene, OR, USA). The anti-nrf2 antibody was purchased from Abcam (Cambridge, MA, USA).The anti-cofilinps3 antibody was purchased from Cell Signaling Technology (Danvers, MA, USA).