Chemical sources and preparation
Perillaldehyde (PAH) was purchased from AbMole (Shanghai, China) and was dissolved in normal saline for in vivo experiments and in PBS (prepared into a 4 mM stock solution) for the subsequent in vitro experiments. The Nrf2 inhibitor ML385 was purchased from Selleck (Shanghai, China), dissolved in DMSO, and prepared into a 10 mM stock solution.
Animal source and irradiation protocol
Male C57BL/6J mice (6–8 weeks old, 20–22 g) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and housed under standard laboratory conditions in the Soochow University Animal Centre. All animal experiments were conducted following the protocols approved by the Animal Ethics Committee of Soochow University.
The mice were randomly divided into two groups (n = 10 / group): IR and IR + PAH, for survival experiments. PAH dissolved in normal saline was administered to mice in the IR + PAH group by oral gavage at a dose of 100 mg/kg daily for seven consecutive days before TAI (Fig. 1A) (4, 48). Mice in the IR group received an equal volume of normal saline by oral gavage and at the same frequency as the IR + PAH-treated mice. All mice were subjected to 13 Gy TAI using the X-RAD 320iX Biological Irradiator (Precision X-ray, North Branford, CT, USA) at a dose rate of 1.1 Gy / min. The mice were randomly divided into 3 groups (n = 6 / group): Control, IR, and IR + PAH, for the subsequent experiments. The processing procedures of mice in the IR and IR + PAH groups were similar to those described earlier. Mice in the control group were sham-irradiated and received an equal volume of normal saline by oral gavage and at the same frequency as the other groups.
Histological analysis, immunohistochemistry and immunofluorescence staining
The mice intestines were harvested and fixed in 4% paraformaldehyde at 6 hours or 3.5 days after TAI. The intestinal tissues were then paraffin-embedded and then cut into 4 µm sections for hematoxylin-eosin (H&E) staining and periodic acid-schiff (PAS) staining according to the manufacturer's instructions. H&E-stained sections were viewed under an optical microscope, followed by an analysis of the villus height using Image J software.
Immunohistochemistry (IHC) and immunofluorescence (IF) were performed as described by Li M et al.[51]. The primary antibodies used for IHC staining included anti-Lgr5 (1:200; 251487; Abbiotec, San Diego, CA, USA), anti-Ki67 (1:400; CST12202), anti-Nrf2 (1:50; CST12721), anti-E-Cadherin (1:200; CST14472) (Cell Signaling Technology, Beverly, MA, USA), anti-GPX4 (1:400; ab125066; Abcam, Cambridge, MA, USA), anti-ZO-1 (1:50; 61-7300), and anti-Claudin3 (1:100; 34-1700) (Invitrogen, Carlsbad, CA, USA) antibodies. The primary antibodies used for IF staining included anti-γ-H2AX (1:400; CST9718) (Cell Signaling Technology), anti-8-OHdG (1:200; NB600-1508), and anti-lysozyme (1:200; NBP2-6118) (Novus Biological, Littleton, CO, USA) antibodies.
Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay
The intestine sections at 6 h post-TAI were stained using an in situ Cell Death Detection Kit (Roche Diagnostic, Mannheim, Germany) according to the manufacturer's instructions.
Fluorescein isothiocyanate (FITC)-dextran permeability assay
Mice were randomly divided into 3 groups (n = 4 / group): Control, IR, and IR + PAH, and treated as described earlier. The mice were subjected to fasting for 8 hours at three days post-TAI and then administered with 0.2 ml of FITC-dextran (Sigma-Aldrich, Saint Louis, MO, USA) (50 mg/100 g body weight). Blood samples were collected 4 hours after FITC-dextran administration and centrifuged at 960 x g for 5 minutes to obtain the serum. The fluorescence level of FITC was measured using a microplate reader at a wavelength of 492 nm.
Crypt isolation and organoid culture
The small intestines of C57BL/6J mice were cut longitudinally and flushed with cold PBS. The tissues were cut into 2–3 mm pieces, washed 15–20 times with cold PBS, and incubated with Gentle Cell Dissociation Reagent (Stem Cell Technologies, Vancouver, BC, Canada) at room temperature on a rocking platform for 15 minutes. The supernatant was gently aspirated, and the pieces were subsequently resuspended in cold PBS supplemented with 0.1% BSA and passed through a 70 µm cell filter (BD Biosciences, San Diego, CA, USA) to remove the tissue fragments. Crypts for organoid culture were obtained by centrifuging the resuspended sections at 290 x g for 5 minutes at 4°C, followed by resuspending the sections in complete IntestiCult™ Organoid Growth Medium (Stem Cell Technologies) at a density of 500 crypts per 50 µl. The resuspended crypts were subsequently mixed with an equal volume of Matrigel® (BD Biosciences) and then seeded on a prewarmed 24-well plate at a density of 500 crypts per well. Complete IntestiCult™ Organoid Growth Medium (750 µl) was then added to each well. The resulting organoids were treated with complete medium with or without 100 µM PAH 4 hours pre-IR and then exposed to 6 Gy X-rays at a dose rate of 1.1 Gy / min or sham-irradiated. The organoids were finally viewed under an optical microscope, followed by an analysis of the organoids using Image J software. At least 50 organoids were counted.
Cell culture
The human intestinal epithelial cell line HIEC-6 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in DMEM (HyClone, Hudson, NH, USA) supplemented with 10% FBS (Biological Industries, Cromwell, CT, USA) and 1% (v/v) penicillin-streptomycin (Beyotime, Shanghai, China) at 37°C in a humidified 5% CO2 atmosphere. The cells were then treated with complete medium with or without 100 µM PAH 4 hours pre-IR.
Cell viability assay
The viability of the HIEC-6 cells was evaluated using the cell counting kit-8 (CCK-8; Beyotime) following the manufacturer's instructions. The HIEC-6 cells (2000 cells/well) were first seeded into 96-well plates and incubated for 24 hours. The cells were then subjected to varying treatments. CCK-8 solution (10 µL) was then added to the cells, followed by incubation for 2 hours at 37°C. The optical density (OD) of the cells was finally measured at 450 nm wavelength using a microplate reader.
Colony formation assay
HIEC-6 cells were seeded in 6-well plates in triplicate at densities of 200–2000 cells/well depending on the radiation dose. The cells were cultured overnight, treated with or without 100 µM PAH for 4 hours, and then subjected to 0, 2, 4, 6, and 8 Gy X-ray radiation. The medium containing drugs was then immediately replaced with fresh medium, and the cells were subsequently cultured at 37°C for 1–2 weeks to form colonies. The cell colonies were stained with crystal violet and viewed under a microscope. Viable colonies consisted of at least 50 cells were counted.
Cell death and lipid peroxidation assays
HIEC-6 cells were seeded in 6-well plates in triplicate. The cells were cultured overnight, treated with or without 100 µM PAH for 4 hours, and then subjected to 6 Gy X-ray radiation. The cells were then collected 48 h post-radiation and subjected to various assays. The cell death assay was done by staining the cells with PI (Beyotime) according to the manufacturer’s instructions. The lipid peroxidation assay was done by incubating the cells with 5 µM BODIPY™ 581/591 C11 fluorescence probe (D3861, Invitrogen, Carlsbad, CA, USA) for 30 minutes at 37°C. The level of cell death and lipid peroxidation was then analyzed by FACSVerse flow cytometry (BD Biosciences).
Intracellular ROS assay
HIEC-6 cells were seeded in 6-well plates in triplicate. The cells were cultured overnight, treated with or without 100 µM PAH for 4 hours, and then preincubated with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (BD Biosciences, San Jose, CA, USA) for 30 min at 37°C according to the manufacturer’s instructions. The cells were then subjected to 6 Gy X-ray radiation and collected after 15 minutes, followed by an analysis of the intracellular ROS level using FACSVerse flow cytometry.
Oxidant and antioxidant index assay
Small intestine tissues of mice were harvested at 3.5 days post-radiation. The MDA content and SOD activity were then measured using commercially available kits (ZCIBIO Technology, Shanghai, China). The level of GSH in HIEC-6 cells was determined using a GSH and GSSG Assay kit (Beyotime) following the manufacturer’s instructions.
Quantitative real-time PCR (qRT-PCR)
Total RNA from HIEC-6 cells was isolated using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol, followed by cDNA synthesis using the PrimeScript™ RT Reagent Kit (Perfect Real Time) (TAKARA, Otsu, Japan). Real-time PCR was performed using SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) on a 7500 Real-Time PCR cycler (Applied Biosystems, Foster City, CA, USA). The expression levels of PTGS2 and GPX4 were normalized to that of β-actin. All primers used (Supplementary Table S1) were synthesized by Sangon Biotech (Shanghai, China).
Western blot analysis
Protein levels of HIEC-6 cells were analyzed using the Western blot assay according to the published methods [52]. The primary antibodies used were anti-Nrf2 (1:1000; 16396-1-AP; Proteintech Group, Chicago, IL, USA), anti-Slc7A11 (1:1000; CST12691), anti-HO-1 (1:1000; CST43966) (Cell Signaling Technology), and anti-GPX4 (1:2000; ab125066; Abcam). The anti-tubulin antibody (1:1000; 66031-1-Ig; Proteintech Group) was used as the loading control.
Statistical analysis
All statistical analyses were done using GraphPad Prism 8.0 (San Diego, CA, USA), and the data were presented as means ± SD. Differences between groups were calculated using the unpaired two-tailed Student's t-test. Survival analysis was done using the Kaplan-Meier method and the log-rank test. The significance threshold was set at P < 0.05.