A total of 90 6-week-old female (weight, 18–21 g) BALB/c mice were obtained from Dooyeol Biotech and housed at 23±2°C and relative humidity of 50±5%, with artificial illumination from 08:00-20:00 and 13–18 air changes per h. The mice were classified into three main categories: control, LDR IR 1 h before, and LDR IR 24 h before. The mice were housed in groups (n=3 mice per group) and received standard laboratory feed and water at all times. The experiment involved continuous monitoring of animal health and behavior, conducted twice daily. All experimental procedures were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, 8th edition, revised 2011) (16) and a protocol approved by the Institutional Animal Care and Use Committee of the Dongnam Institute of Radiological and Medical Sciences (DIRAMS; approval nos. DI-2015-002 and DI-2021-002).

Efforts were made to reduce suffering and distress among the animals by providing analgesics (xylazine; Rompun^®; 10 mg/kg; Bayer Koreaa; via intraperitoneal injection) or anesthetics (alfaxalone; 85 mg/kg (17); Jurox Pty Ltd.; via intraperitoneal injection) as needed as well as establishing certain housing conditions. If experimental animals exhibited visible alterations as well as a weight loss >20%, they were sacrificed in accordance with criteria for humane endpoints. Following the administration of respiratory anesthetic induction using 5% isoflurane for 60 sec, the mouse appeared to be in an unconscious state. The dose rate of isoflurane was reduced by 2% for maintenance anesthesia and euthanasia was carried out by applying a cervical dislocation. Death was confirmed through a physical examination, which revealed the absence of the cardiac and respiratory activity. A total of 90 mice were used in the experiments. No mice were euthanized due to humane endpoints and no mice were found dead throughout the experiment.

The LDR IR exposure was similar to that previously described (10,11). The DIRAMS LDR IR facility used radiation exposure with a ¹³⁷Cs source (370 GBq). The mouse cages were positioned on shelves with dose rates of 6.0 mGy/h; the total accumulative radiation dose of the set-up places was 10, 100 and 500 mGy. The group exposed to LDR IR at 10 mGy undergo three exposures, each occurring at intervals of 3 days. In addition, mice were high-dose rate (HDR) irradiated with a single 500 mGy dose using 6 MV high energy photon rays (Elekta Instrument AB) at 3.8 Gy/min. Sham-irradiated mice were treated similarly but without radiation. After 1 and 24 h of irradiation, the mice were sacrificed and intestinal tissue samples were taken.

The small intestine was fixed in 10% neutral buffered formaldehyde for 24 h at room temperature and four distinct jejunal segments were sliced into 4 µm thick pieces and placed on glass slides. To examine morphology, jejunal slices were stained with hematoxylin and eosin for 2 and 1 min at room temperature. All samples were sectioned and reoriented in successive slices to examine the morphological alterations and ascertain which ones had the longest villi. This method was chosen because it produced more reliable results than standard techniques, which only measured the 10 longest villi in a single slice per sample (4,18). A total of 10 jejunal sections, including the height of the basal lamina and the length of the 10 longest villi, were measured from each animal. The stained sections were analyzed using a Motic Easyscan Digital Slide Scanner (Motic Incorporation, Ltd.).

The induction of colitis in murine models by DSS (cat. no. 160110; 36–50 kDa; MP Biomedicals) was similar to that previously described (19); 5% DSS dissolved in drinking water was used as the treatment method for mice for 7 days. In the control groups, mice were given normal drinking water.

Mice (n=1 mouse per group) were sacrificed and their jejunum tissue were removed. TRIzol^® reagent (cat. no. 15596026; Thermo Fisher Scientific, Inc.) was used for total RNA extraction, according to the manufacturer's instructions. RNase-Free DNase I (cat. no. EN0521; Thermo Fisher Scientific, Inc.) was used to degrade genomic DNA. Total RNA levels in the samples were measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). BioAnalyzer2100 (Agilent Technologies, Inc.) analyzed sample size, quantification and integrity, eliminating those with a RNA integrity number <7. Following the manufacturer's instructions, NEXTflex Poly (A) Beads (Bioo Scientific Corp.) was used to isolate mRNA from total RNA samples.

The NEXTflex Rapid Directional RNA-Seq Library Prep Kit 2.0 (Bioo Scientific Corp.) was used to create an RNA sequencing library in accordance with the manufacturer's protocols. In brief, mRNA was chemically fragmented after isolation from 1 µg of total RNA using NEXTflext Poly (A) Beads. Following the creation of the double-stranded cDNA from the fragmented mRNA, the library was purified using magnetic beads made by Agencourt AMPure XP (Beckman Coulter, Inc.), 3′-end adenylation and sequencing adapter ligation. The experiment used the Agilent 2100 Bioanalyzer and reverse transcription-quantitative (RT-q) PCR to verify each library's successful preparation before moving on to massively parallel deep sequencing. HiSeq 2500 sequencing platform (Illumina, Inc.) was used for paired-end sequencing with a typical read size of 100 nucleotides. The Illumina base-calling pipeline processed the raw sequencing data and two technical duplicates were created and sequenced for every sample.

Adaptor and low-quality bases were trimmed with Cutadapt ver. 1.3 (20) and Trim Galore ver. 0.44 (https://www.bioinformatics.babraham.ac.uk) and the clean reads were aligned to the reference rat genome (rn5) downloaded from Ensemble (www.ensembl.org) with STAR ver. 2.5 (21). Mapped data from the bam file was imported to StrandNGS ver. 2.9 (StrandNGS.com) for further expression analysis. Gene quantitation was normalized using DESEQ (22) and baseline correction to the median of gene expression and log2 transform were applied. Differentially expression genes (DEGs) were determined by log2-transformed fold changes (≥1 or ≤-1) with one-way analysis of variance (ANOVA; false discovery rate <0.1). To investigate Gene ontology (GO) enrichment of DEGs, we utilized Homer ver. 4.5 (23) and Metascape webserver (http://metascape.org) (24), with Mus musculus used as the input species and analyzed GO biological process (GO:BP) dataset to identify upregulated genes. Only terms meeting the criteria of P<0.05, a minimum count of 3 and an enrichment factor >2 was considered significant. The most statistically significant term in a cluster was selected to represent that cluster.

Using an ISOGEN kit (311-02501; Nippon Gene, Tokyo, Japan), total RNA was isolated from jejunal samples. RT-qPCR analyses were performed as described in a previous study (4). RT-qPCR was performed duplicate using CFX Opus 96 instrument (Bio-Rad Laboratories) and TOPreal^™ SYBR Green qPCR 2× PreMIX (Cat. No. RT500M; Enzynomics) and the according the manufacturer's guidelines. The RT-qPCR reaction was carried out in a 25 µl reaction, including 5 µl of cDNA (40 ng), 1 µl of each primer (6 µM), 12.5 µl of 2× SYBR green PreMix, and 5.5 µl of double distilled water. The thermal cycling profile comprised a preincubation step at 94°C for 10 min, followed by 45 cycles of denaturation (94°C, 15 sec), annealing (55–60°C, 30 sec), and elongation (72°C, 20 sec). The software generated amplification curves and determined threshold cycle values. Primer candidates were acquired utilizing an application available on the PRIMER3 ver. 0.4.0 website (https://bioinfo.ut.ee/primer3-0.4.0/). The primers were validated using the NCBI BLAST (basic local alignment search tool) ver. +2.15.0 database (https://blast.ncbi.nlm.nih.gov/). The expression levels of mRNAs for ATPase NA⁺/K⁺ transporting subunit alpha 4 (ATP1A4), glucose-6-phosphatase catalytic subunit 2 (G6PC2), mucin 6 (MUC6), mucin 20 (MUC20) and transient receptor potential cation channel subfamily V member 6 (TRPV6) were measured, normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression and expressed relative to the corresponding mean value for the jejunum tissue of control mice that was unexposed to radiation using the 2^−ΔΔCq method (25). Mouse primers used in analysis were as follows: ATP1A4-(NM_013734) forward 5′-CTGGTGAGCTGAATCAGAAACC-3′ and reverse 5′-AAGACCCTTGGTCAAGTCCAC-3′; G6PC2-(NM_001289857) forward 5′-CAGGAGGACTACCGGACTTAC-3′ and reverse 5′-TCAACTGAAACCAAAGTGGGAA-3′; MUC6-(NM_001368953) forward 5′-AGCCCACATTCCCTATCAGC-3′ and reverse 5′-CACAGTGGAAGATTGCGAGAG-3′; TRPV6-(NM_022413) forward 5′-AGGGGTTAATACTCTGCCTATGG-3′ and reverse, 5′-GCACCTCACATCCTTCAAACTT-3′; GAPDH-(NM_008084.2) forward 5′-TCCATGACAACTTTGGCATT-3′ and reverse 5′-GTTGCTGTTGAAGTCGCAGG-3′.

The jejunum was homogenized in buffer H (50 mM β-glycerophosphate, 1.5 mM ethylene glycol tetra acetic acid, 0.1 mM Na₃VO₄, 1 mM dithiothreitol, 10 µg/ml aprotinin, 2 µg/ml pepstatin, 10 µg/ml leupeptin, and 1 mM phenylmethanesulfonyl fluoride, pH 7.4) for western blotting examination. The protein content of each sample was determined using a Bio-Rad protein assay reagent (cat. no. 500-0006; Bio-Rad Laboratories, Hercules, CA, USA). Western blotting was performed as described previously (10). Briefly, each sample's total protein equivalent was separated on 10% SDS-polyacrylamide gels (40 µg of protein per lane) and transferred to an Immobilon-PSQ transfer membrane (cat. no. ISEQ00010; MilliporeSigma). The membrane was soaked for 1 h at room temperature in a blocking solution containing 5% non-fat milk. Primary antibodies against ATP1A4 (diluted 1:1,000; cat. no. STJ117451; St John's Laboratory Ltd.), G6PC2 (diluted 1:1,000; cat. no. bs-13386R; BIOSS), MUC6 (diluted 1:1,000; cat. no. 224329; United States Biological) and TRPV6 (diluted 1:1,000; cat. no. ACC-036; Alomone Labs) were incubated at 4°C with consistent rocking overnight. After four 10-min Tris-buffered saline (TBS) and 0.1% Tween-20 (TBS-T) washes, the membrane was incubated with secondary polyclonal anti-rabbit antibodies (diluted 1:10,000; cat. no. 12-348; MilliporeSigma) in TBS-T buffer at ambient temperature for 1 h. Using a chemiluminescence reagent (cat. no. 34580; SuperSignal West Pico PLUS; Thermo Fisher Scientific) and the ChemiDoc MP Imaging system (Bio-Rad Laboratories, Inc.), the labeling of the secondary antibody conjugated with horseradish was detected. The membrane was probed with β-actin (diluted 1:5,000; cat. no. A5441; MilliporeSigma), a housekeeping gene, to measure protein expression levels. Using Image Lab ver. 6.0.1 (Bio-Rad Laboratories, Inc.) software, protein bands were quantified.

Results are presented as mean ± standard error of mean (SEM). Radiation-induced response screening was analyzed using one-way analysis of variance (ANOVA) with additional Tukey's post hoc testing. Dose-dependent response validation, frequency-dependent response validation and evaluation of correlation between LDR IR and inflammatory bowel disease (IBD) were analyzed using two-way ANOVA followed by Tukey's post hoc testing. P<0.05 was considered to indicate a statistically significant difference.

To screen for genes changed by LDR IR exposure, RNA-seq was performed 1 and 24 h after irradiation at 10 mGy (Fig. 1A). At 1 h following radiation exposure, 12 enriched GO:BP terms were obtained when 0 Gy was compared with 1 h after exposure to 10 mGy (Fig. 1B and Table I). Furthermore, there was an increase in the BP associated with the digestion system. After 24 h of radiation exposure, 14 enriched GO:BP terms were obtained when 0 Gy was compared with 24 h after 10 mGy (Fig. 1C and Table II). Moreover, there was an observed increase in the BP associated with the immune system. However, no meaningful biological process demonstrated an expected increase across a period of 1 and 24 h following exposure. RNA-seq was used to find genes responsive to LDR IR exposure to identify DEGs affected in the LDR IR jejunum (364 DEGs upregulated in the 1 h after LDR irradiated jejunum; 697 DEGs upregulated in the 24 h after LDR of the jejunum, with 23 genes in common with the comparison of 1 and 24-h IR groups; Fig. 1D and Table III).

To verify changes in genes and proteins caused by LDR IR exposure, RT-qPCR and western blot were performed on mice 1 and 24 h after irradiation at 10 mGy (Fig. 2A). A total of 23 DEGs were selected for qPCR detection to validate the outcome of RNA-seq analysis: APOL2, ATP1A4, CCL2, CCL20, CLEC4E, CRLF1, G6PC2, INS, ITIH4, KLHL3, MUC20, MUC6, NPHS1, PPY, REG1B, SERPINA10, SLC40A1, SLC9B2, SPX, TNFRSF8, TRPV6, UMOD and WNK3. Among these, qPCR results showed that only ATP1A4, G6PC2, MUC6 and TRPV6 levels increased significantly 24 h after LDR IR (Fig. 2B). The protein expression levels of ATP1A4, G6PC2, MUC6, MUC20 and TRPV6 were examined (Fig. 2C and D). G6PC2 expression showed significant upregulation in the 1 and 24 h following 10 mGy radiation exposure to the jejunum. MUC6 was also significantly upregulated in the 24 h following LDR IR exposure. However, MUC20 was not detectable by western blotting and ATP1A4 and TRPV6 did not alter after LDR IR exposure. Therefore, it was hypothesized that G6PC2 and MUC6 are important genes triggered by LDR IR exposure.

To further validate the dose-dependent change in G6PC2 gene expression, qPCR analysis was performed on mice intestinal tissues exposed to LDR IR (10, 100 and 500 mGy) and HDR (500 mGy; Fig. 3A). In the jejunum, G6PC2 gene expression was significantly increased following 10 and 100 mGy exposure compared with the sham group (Fig. 3B). However, 500 mGy of LDR IR and 500 mGy of HDR IR did not alter G6PC2 expression levels. Furthermore, G6PC2 expression of the colon also did not change after LDR IR and HDR IR (Fig. 3C). Therefore, it was concluded that G6PC2 only reacts at LDR IR and this reaction is limited to the jejunum. In an additional experiment, the expression level of G6PC2 was validated by repeated LDR IR treatment (Fig. 3D). A single 10 mGy exposure significantly increased G6PC2 expression after 24 h and three 10 mGy exposures increased G6PC2 expression explosively after 24 h (Fig. 3E), suggesting that frequent exposure to LDR effectively increases G6PC2 expression.

To validate the dose-dependent change in MUC6 gene expression, qPCR analysis was performed on mice intestinal tissue exposed to LDR IR (10, 100 and 500 mGy) and HDR (500 mGy; Fig. 4A). Identifying MUC6 mRNA expression for dose-dependent alterations revealed outcomes comparable to G6PC2. In the jejunum, MUC6 gene expression was significantly higher following 10 mGy exposure compared with the sham group (Fig. 4B). However, 100 and 500 mGy of LDR IR and 500 mGy of HDR IR did not alter MUC6 expression levels. Furthermore, MUC6 expression of the colon also did not change following LDR IR and HDR IR (Fig. 4C). Therefore, it can be concluded that MUC6 only reacts at LDR IR and that this reaction is limited to the jejunum. In an additional experiment, the expression level of MUC6 was validated by repeated LDR IR treatment (Fig. 4D). A single 10 mGy exposure significantly increased MUC6 expression after 24 h and three 10 mGy exposures increased MUC6 expression markedly after 24 h (Fig. 4E), suggesting that frequent exposure to LDR IR effectively increases MUC6 expression.

In order to establish the effect of LDR IR on the intestines with inflammatory conditions, infectious bowel disease was induced by DSS, the jejunal area received radiation and the expression levels of G6PC2 and MUC6 were verified (Fig. 5A). DSS-induced IBD and LDR IR did not cause morphological alterations in the small intestine when processed independently or even after combined treatments; subsequently, neither the length of the villus nor the number of crypts changed, observed 24 h after irradiation (Fig. 5B). Thus, DSS and LDR IR in the jejunum did not act as a powerful stimulus to alter morphological modifications. However, after 24 h, repeated irradiation of LDR IR and DSS significantly boosted G6PC2 expression. Furthermore, when the two stimuli were combined, G6PC2 expression increased with greater efficacy, observed 1 and 24 h after irradiation (Fig. 5C). The expression of MUC6 was significantly increased by exposure to LDR IR and stimulation with DSS. In addition, the combination of these two stimuli led to a substantial increase in MUC6 expression (Fig. 5D).