The procedures for all animal experiments were approved by the Ethics Committee of PLA Rocket Force Characteristic Medical Center and were performed in accordance with the Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice aged 6–8 weeks were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). Lgr5-EGFP-IRES-cre-ERT2 mice (The Jackson Laboratory, United States) were crossed with ROSA26-Tdtomato mice (The Jackson Laboratory, United States) to generate Lgr5-EGFP-IRES-cre-ERT2; ROSA26-Tdtomato heterozygous mice. Likewise, Bmi1-CreER mice (The Jackson Laboratory, United States) were crossed with R26R-EYFP mice (The Jackson Laboratory, United States) to generate Bmi1-CreER; R26R-EYFP mice heterozygous mice. PUMA knockout (PUMA KO) mice were kindly provided by Professor Cong (Academy of Military Medical Sciences, China). For the in vivo experiments, DSF (Tetraethylthiuram disulfide, 86720-50G, Sigma-Aldrich, United States), dissolved in vehicle (5% dimethyl sulfoxide, 5% Tween-80 and 90% saline), was treated to mice 1 h before irradiation at a dose of 100 mg/kg. For the in vitro experiments, DSF was dissolved in dimethyl sulfoxide (DMSO) to create concentrated stock solutions at a concentration of 50 mM and stored at −20°C.The stock solution was diluted in culture medium at the time of use. DTC (Ditiocarb sodium, HY-B1637, MedChemExpress, United States), dissolved in vehicle was injected intraperitoneally into mice 1 h before irradiation at a dose of 200 mg/kg. The animals were randomly assigned to different treatment groups. For survival experiments, at least 10 mice/groups were used and at least 3 mice/groups were used for other experiments.

Human intestinal epithelial cell (HIEC) line was purchased from Eallbio Life Sciences (Beijing, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-Invitrogen, United States) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C under 5% CO₂ incubator.

X-ray generated by the Precise linear accelerator (VWAT Elekta, Sweden) in the Radiation Centre (PLA Rocket Force Characteristic Medical Center, Beijing) was used as the source of radiation and radiation was delivered with a 6 MeV linear accelerator. For total body irradiation (TBI), mice were irradiated at a dose rate of 0.6 Gy/min. For abdominal irradiation, a 2 cm area of the anesthetized mouse containing the gastrointestinal tract was irradiated, thus shielding the upper thorax, head, neck, lower extremities and upper extremities as well as protecting a portion of the bone marrow by using lead plate.

The proximal small intestines were gathered and fixed after the mice were sacrificed 3.5 days after irradiation. Paraffin-embedded sections (4 µm) were stained with hematoxylin-eosin (H&E) for observation and histological analysis. Normal mitosis contains condensed chromosomes that show even and symmetrical separation and alignment. Aberrant mitosis contains condensed chromosomes with multipolar spindles, lagging or misaligned chromosomes, anaphase bridges, or micronuclei (Leibowitz et al., 2011). Eight intestinal circumferences were randomly selected from each mouse to count regenerated crypts.

Briefly, 4-μm paraffin-embedded sections were subjected to deparaffinization, antigen retrieval by sodium citrate solution, blocking with goat serum and antibody incubation. The following antibodies were used: anti-BrdU (ab6326, Abcam, United Kingdom); anti-phospho-histone H3 (9701S, CST, United States); anti-cleaved caspase-3 (9664S, CST, United States) and anti-γH2AX (05636, Merck Millipore, Germany). For 5-bromo-2′-deoxyuridine (BrdU) IHC, BrdU (B5002-5G, Sigma–Aldrich, United States) was administered to mice by intraperitoneal injection at a dose of 120 mg/kg 2 h prior to sacrifice. The viability of a surviving crypt was confirmed by incorporation of BrdU into five or more epithelial cells within each regenerative crypt. Eight intestinal circumferences were randomly selected from each mouse to count and positive cells were counted in 5 crypts in each intestinal circumference.

Apoptosis was assessed in small intestinal sections from mice post radiation by using a TUNEL apoptosis assay kit (C1098, Beyotime, China). The detection method was performed according to the manufacturer’s instructions. The number of TUNEL positive cells per crypt was calculated in more than 50 crypts per mouse.

To examine the contribution of intestinal stem cells to tissue regeneration and label the Lgr5⁺ ISC and the Bmi1⁺ ISC lineages, Lgr5-EGFP-IRES-cre-ERT2; ROSA26-Tdtomato mice and Bmi1-CreER; R26R-EYFP mice were injected intraperitoneally with tamoxifen in corn oil, and intestinal tissue was harvested on day 7 post-IR. The intestine was cut open and fixed in 4% formaldehyde. After cleaning the intestine, it was embedded in 4% agarose, and a 150 μm thick section was prepared using a vibrating sectioning mechanism (5100 plus, Campden, Germany). The experimental method is described in detail (Snippert et al., 2011). Lineage generation efficiency was quantified by calculating the GFP⁺ or YFP⁺ villi arising from Lgr5⁺ or Bmi1⁺ ISCs.

The method of isolating intestinal crypts has been previously described (Mahe et al., 2013). Briefly, a segment of proximal intestine representing a 10-cm region from 2 to 12 cm distal to the pyloric sphincter was collected for crypt isolation. After flushing with ice-cold PBS, the small intestines were chopped into small pieces and then placed into cold DPBS containing 2 mM EDTA for 30 min. The epithelial layer was liberated from the stroma using a mixer, and the stroma was then discarded. The solution was filtered through a 70 μm strainer to remove the villus fraction and collect the crypt fraction.

Isolated small intestinal crypt cells were collected at 0.5, 4 and 24 h following TBI. In brief, protein lysis, quantification, electrophoresis, membrane transfer, blocking, antibody incubation and visualization using a BIO-RAD ChemiDoc MP imaging system (California, United States) were performed. The following antibodies were used: anti-γH2AX (ab2893, Abcam, United Kingdom) and anti-Vinculin (A01207, BOSTER, China).

HIECs were seeded onto glass bottom cell culture dishes (801002, NEST, China), and pretreated with or without 0.05 μM DSF for 12 h before 6 Gy radiation. Cells were collected at indicated time points (0, 0.5, 1, 2, 4, 12 and 24 h after radiation), fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with goat serum, and incubated with anti-γH2AX antibody (05636 Merck Millipore, Germany) overnight at 4°C, followed by incubation with Alexa Fluor 488 (A0428, Beyotime, China), Hoechst (C1026, Beyotime, China) counterstaining and observed by confocal microscopy (LSM-700, ZEISS, Germany). The γH2AX-focis were analyzed from the images of IF staining by using ImageJ software 1.8.0 (National Institutes of Health, United States).

GraphPad Prism 8.0 (GraphPad Software, San Diego, California) was used for statistical analyses. All data were obtained from at least two repetitions and were presented as the mean ± SEM unless otherwise specified. Comparisons between groups were performed using an unpaired two-tailed Student’s t-test. Log-rank analysis was performed for survival analysis. Asterisks represent the p values as follows: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. p < 0.05 was considered statistically significant.

We first evaluated the radioprotective effect of DSF using a total-body irradiated mouse model. Mice were injected intraperitoneally with DSF at a dose of 100 mg/kg and then subjected to 12 Gy irradiation 1 h later. All vehicle-treated mice died within 4.5 days after irradiation with an average weight loss of 25% by 4 days, which is characteristic of lethal intestinal toxicity. In contrast, DSF-treated mice died within 5–11 days after irradiation with the average survival time prolonged from 4 to 7 days and less weight loss (Figures 1A,B). To exclude the interference of the effects of IR-induced hematopoietic failure on death, we used total-abdominal irradiation (TAI) model. After abdominal exposure to 12 Gy radiation, 81.25% (13/16) of the vehicle-treated mice died within 6 days, while 56.3% (9/16) of the DSF-treated mice survived more 30 days (Figure 1C). We further assessed the histological changes in the small intestine following radiation by H&E staining. The 12 Gy radiation exposure resulted in a decrease of approximately 90% surviving crypts at 3.5 days, whereas DSF treatment increased the percentage of surviving crypts by approximately 3-fold compared to the vehicle-treated group (Figures 1D,E). Vehicle alone has no radioprotection to the mouse small intestine (Supplementary Figure S1). Oral DSF pretreatment also increased regenerated crypts after irradiation (Supplementary Figure S2). In addition, DTC, which is a thiol metabolite of DSF, protected intestinal crypt cells after irradiation (Supplementary Figure S3). The average villus height in intestinal sections shortened to 48% of the unirradiated villi at 5 days after TBI, while DSF pretreatment increased villus height to nearly 81% (Figures 1F,G). These findings suggested that DSF protects against radiation-induced intestinal injury.

The intestinal crypt compartment is the basic unit for intestinal epithelial reconstruction (Barker et al., 2012). To clarify the effects of DSF on crypt regeneration, we monitored proliferation kinetics using the BrdU pulse assay, which is a thymidine analog specifically incorporated into the DNA of S-phase proliferating cells. In the vehicle-treated group, regenerated crypts significantly decreased when mice were exposed to progressively higher doses of radiation. In the DSF-treated group, regenerated crypts were increased by 134% (10 Gy), 328% (12 Gy) and 315% (14 Gy), respectively. (Figures 2A,B). These results suggested that DSF protects against RIII in mice exposed to a wide range of irradiation doses. A dose of 12 Gy was used in the subsequent experiments. We continued to use a BrdU incorporation assay to explore the regeneration of crypts from the start of irradiation to 84 h later (Figures 2C–E). Crypt proliferation in vehicle-treated mice was decreased by nearly 89% (from 1322 to 150) at 48 h after TBI followed by the appearance of regenerated crypts at 84 h. Interestingly, compared to the vehicle group, crypt proliferation in DSF-treated mice was inhibited within 24 h, but then it gradually recovered until exceeded at 48 h, reaching nearly 3 times of the crypt proliferation in the control group at 84 h. We further analyzed the effect of DSF on cellular proliferation by phospho-histone H3 staining, which reaches a maximum during mitosis (Ribalta et al., 2004). Forty-8 h after irradiation, there were more phospho-histone H3⁺ cells in the DSF group than in the vehicle group (Figures 2F,G). These data demonstrated that DSF alters intestinal proliferation kinetics in response to radiation, which suppresses crypt regeneration in the early phase and promotes crypt regeneration in the later phase.

The leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) is a marker of ISCs, and the Lgr5⁺ ISCs are indispensable for IR-induced intestinal epithelium regeneration (Metcalfe et al., 2014). To assess the effect of DSF on Lgr5⁺ ISCs after irradiation, we used Lgr5-EGFP-IRES-creERT2 mice in which the allele was knocked in by integrating an enhanced green fluorescent protein (EGFP)-IRES-creERT2 cassette after Lgr5 (Barker et al., 2007). Radiation reduced the loss of more than 70% Lgr5-GFP cells by 48 h, which was significantly suppressed in the DSF group (Figures 3A,B). At the same time, the fraction of Lgr5⁺ crypts increased by over 30% in the DSF-treated group (from 52% to 84% of unirradiated level, Figure 3C). To further clarify whether DSF exerts radioprotection via Lgr5⁺ ISC-mediated epithelial regeneration, we generated Lgr5-eGFP-IRES-CreERT2; Rosa26-TdTomato mice by crossing Lgr5-eGFP-IRES-CreERT2 mice and Rosa26-TdTomato mice (carrying a conditional Tdtomato allele at the Rosa 26 locus that is activated by Cre recombinase) reporter mice. We injected these mice with tamoxifen to induce recombination in Lgr5-expressing cells 24 h before and after 12 Gy TBI. The mice pretreated with DSF produced 1.6 times more progeny than the vehicle group by 7 days of lineage tracing (Figures 3D,E). Similarly, we analyzed Bmi1 cells, which are considered another type of stem cell (Sangiorgi and Capecchi, 2008), by lineage tracing in Bmi1-CreER; Rosa26-YFP mice (Figure 3F). We observed mild protection by DSF against the loss of progeny generation by Bmi1⁺ cells (Figure 3G). Taken together, these results suggested that DSF protects intestinal stem cells.

Intestinal epithelium apoptosis induced by radiation occurs in two distinct phases as follows: radiation exposure rapidly activates p53-PUMA-dependent apoptosis via the intrinsic pathway, which peaks 3–6 h following radiation; and a delayed wave of p53-independent apoptosis emerges at 24 h after irradiation (Kirsch et al., 2010). Therefore, we determined crypt cell apoptosis by TUNEL and cleaved caspase-3 (CC3) staining 4 and 24 h after 12 Gy TBI (Figures 4A–F). Unexpectedly, apoptotic cells (marked by TUNEL or CC3 staining) in DSF-treated mice occurred to a similar degree at 4 h after radiation compared to vehicle-treated mice. However, 24 h after radiation, the total number of apoptotic crypt cell in the DSF-treated group was reduced by half compared to that in the control group. More specifically, apoptosis at cell positions 4, 5 and 6 above the crypt base was suppressed by 36, 10 and 16%, respectively, in DSF-treated mice at 24 h. To further exclude the possibility that DSF exerts radioprotective effects on intestinal recovery and is involved in preventing early intrinsic apoptosis after irradiation, PUMA KO mice were treated with DSF or vehicle and were subjected to 12Gy TBI. The results showed that DSF significantly promoted crypt regeneration, nearly doubling the crypt (Figures 4G,H). These results suggested that DSF promotes intestinal regeneration after irradiation through additional mechanisms that are not directly related to suppression of early intrinsic apoptosis.

Incomplete repair of IR-induced DNA DSBs leads to the accumulation of DNA lesions and genome instability, and aberrant mitosis may occur when the cell cycle reinitiates after irradiation (Vitale et al., 2011). Delayed phase (24 h) γH2AX foci are considered a surrogate for unrepaired DSBs(Hua et al., 2012). To examine unsolved DNA lesions at 24 h post irradiation, we first detected DNA DSBs by γH2AX staining and found that DSF dramatically decreased the proportion of γH2AX-positive crypt cells in irradiated (Figures 5A,B). To further clarify the effect of DSF on IR-induced DNA damage repair kinetics, we isolated intestinal crypts at different time points after irradiation and analyzed γH2AX expression using immunoblotting. At 0.5 h after irradiation, the rapidly elevated expression of γH2AX was similar between the DSF treatment group and the control group. At 4 h after irradiation, however, the γH2AX level of the DSF treatment group was significantly lower than that of the control group (Figures 5C,D). In addition, the DSBs in the HIECs at different time points after irradiation were detected through γH2AX immunofluorescence staining. γH2AX foci formation occurs rapidly in irradiated cells, reaching a maximum at 0.5 h and then declining gradually within 24 h following irradiation (Figures 5E,F). We observed the similar number of γH2AX foci between two groups at 0.5 h after radiation, however, the number of γH2AX foci in cells treated with DSF at a concentration of 50 nM, which had no cytotoxic effect (Supplementary Figure S4), were significantly lower than in vehicle-treated cells at each subsequent time point. These results indicate that DSF protects crypt cells by facilitating DNA repair rather than directly suppressing DNA DSBs induced by irradiation. Moreover, the effect of DSF on the mitotic progression of intestinal crypt cells was examined by H&E staining (Figures 5G,H). Twenty-4 h after 12 Gy TBI, most of the mitotic crypt cells were aberrant because the chromosomes were arranged in an asymmetrical manner (Kirsch et al., 2010; Leibowitz et al., 2011), whereas DSF treatment suppressed aberrant mitosis by 40% compared to the vehicle group. These findings provide evidence that DSF prevents accumulation of DNA damage in intestinal crypt cells, thereby avoiding genome instability.