Fifteen male Sprague–Dawley rats (4–6 weeks old) were obtained from the Army Military Medical University (SCXK [YU] 2022-0011). Five rats were separated into a cage randomly and further received different treatments. There were three groups in this study, including control, IR, and Gln+IR groups. Rats in the Gln+IR group received daily intraperitoneal injections of Gln (Pricella, China) at a dose of 0.3 g/kg body weight for three consecutive days, while rats in the IR group received equivalent volumes of saline. On the third day, after the final injection, all rats were anesthetized by intraperitoneal injection with nembutal (40 mg/kg bodyweight), and positioned to expose the lower abdomen. The rest of the body was shielded with lead blocks. Rats in the IR and Gln+IR groups were exposed to a single localized abdominal irradiation of 15 Gy using a Co60 source (16). No treatment was performed in the control group. All rats were fed under standard conditions with ambient temperature at 22 ± 2°C, relative air humidity 40–70%, a 12/12 h light-dark cycle and free access to water and diet.

Relevant studies indicate that the peak of acute radiation-induced intestinal injury in rats occurs in 3–7 days (16, 17). In the current study,7 days after irradiation, rats were sacrificed by cervical dislocation after anesthesia with nembutal (40 mg/kg bodyweight), and colon samples were collected. The total length of colon was measured, and samples were immediately snap-frozen in liquid nitrogen for further analyses. During the post-irradiation period, fecal output, body weight, and survival were recorded. The disease activity index (DAI) was calculated according to the scoring criteria showed in Table 1.

Colon specimens from each group were fixed in 4% paraformaldehyde (Servicebio, China), embedded in paraffin, and sectioned transversely. Deparaffinization was performed with xylene, followed by rehydration in graded alcohols. Staining was conducted using an HE Staining Kit (Solarbio, China) and an AB-PAS Staining Kit (Solarbio, China). Microscopic observations were made using an Olympus BX63 microscope (Japan). The histopathological scoring of the colon tissues was performed using the method described by Yoshio Araki et al. (18) (Table 2). GCs were counted in AB-PAS staining sections, and the number of GCs per crypt was statistically analyzed.

Paraffin sections were cut transversely, deparaffinized with xylene, washed with PBS, and underwent antigen retrieval using citrate buffer. The sections were then incubated with 3% hydrogen peroxide and blocked with goat serum. Sections were incubated overnight at 4°C with Lgr5 antibody and Klf4. Goat anti-rabbit secondary antibody was then applied, followed by incubation with diaminobenzidine (DAB) staining kit (ZSGB-BIO, China). Observations were made under an Olympus BX63 microscope (Japan), and positive staining was analyzed using Image J software. The antibodies used are listed in Supplementary Table S1.

The total RNA of colon was extracted by TRIzol (TaKaRa, Japan). Subsequently, the cDNA was synthesized by reverse transcription kit (TaKaRa, Japan) according to the manufacturer’s instruction. To assess the mRNA expression levels of inflammation markers, qPCR was performed by Bio-Rad CFX Maestro system. GAPDH was served as a reference gene. Relative mRNA expression was calculated according to the 2^-ΔΔCt method. Primer sequences are showed in Table 3.

Colon tissues were homogenized, and the total protein was extracted using RIPA lysis buffer. Proteins were denatured with loading buffer at 95°C for 5 min. Then, the protein samples were separated via SDS-PAGE, and transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% skimmed milk at room temperature for 2 hours and incubated with primary antibodies overnight at 4°C. Subsequently, membranes were incubated with secondary antibodies at room temperature for 2 hours. The immunoblotting bands were measured through chemiluminescence detection system, and related expressions of proteins were quantified by ImageJ software. Antibodies used in this study are provided in Supplementary Table S1.

To further investigate the mechanism by which Gln regulates GCs differentiation in RIII, an in vitro ISC radiation model was established using HT-29 cells. The HT-29 cell line, purchased from Wuhan Procell Life Science & Technology Co., Ltd., was cultured in McCoy’s 5A (Pricella, China) medium supplemented with 10% fetal bovine serum (Pricella, China) and 100 kU/L penicillin-streptomycin (Pricella, China), and incubated in a 5% CO2, 37°C incubator. HT-29 cells are capable of expressing various intestinal lineage-specific markers and differentiating along multiple lineages, thus are considered pluripotent, similar to ISC (19). Previous research indicates that, compared to the basal concentration of Gln at 2 mM (the standard concentration in the culture medium), a concentration of 4 mM Gln significantly demonstrates protective effects on HT-29 cells. Therefore, we selected 2 mM Gln as the baseline concentration group and 4 mM Gln as the experimental group concentration to study the effects of Gln on mTOR and Notch1 under radiation conditions.

In this study, to specifically inhibit mTOR signaling, the HT-29 cells were treated with 20 ng/mL RAPA (Solarbio, China). Cells were divided into four groups: 2 mM Gln, 2 mM Gln + RAPA, 4 mM Gln, and 4 mM Gln + RAPA. 4 hours after treatments, all cells were exposed to radiation at dose of 8 Gy using a Co (60) source (20). After an additional 24 hours of culture, cells were collected for further analysis.

To investigate the role of mTOR/notch1 pathway on Gln radio-resistance effects in colon, HT-29 cells were treated with the 10 μmol/L Jagged-1 (MCE, USA) (21), which activates the Notch1 receptor. Four groups were set up: 2 mM Gln, 2 mM Gln + Jagged-1, 4 mM Gln, and 4 mM Gln + Jagged-1. 4 hours after treatments, all cells were exposed to radiation at dose of 8 Gy using a Co (60) source (20). After an additional 24 hours of culture, cells were collected for further analysis.

HT-29 cells were plated in confocal culture dishes and grown to approximately 60% confluency. After 4 hours treatment, cells were fixed with 4% paraformaldehyde, permeabilized using 0.5% Triton X-100 (Beyotime, China). Then, cells were blocked with 10% goat serum (Beyotime, China) and incubated with anti-MUC2 antibody at 4°Covernight. Subsequently, cells were treated with a fluorescent secondary antibody at room temperature for 1 hour. After washing, cells were stained with anti-fade DAPI medium (Beyotime, China) and incubated in dark for 15 minutes. MUC2 expression was observed by confocal microscope.

Statistical analyses were performed using SPSS version 20, and graphs were generated with GraphPad Prism 8. Data are presented as mean ± SD. Comparisons between two groups were carried out by t-test, while one-way ANOVA was applied in multiple groups- comparison. When these assumptions were not met, non-parametric tests were applied. P < 0.05 was considered statistically significant.

To explore the effects of Gln on RIII, classic intestinal injury markers were assessed after irradiation. Rats in the IR group exhibit loose stools and positive fecal occult blood, which were further progressed to diarrhea and rectal bleeding. However, no evident diarrhea or bleeding was found in rats in Gln+IR group, in which only loose stools and positive fecal occult blood were observed (Figure 1A). Additionally, the disease activity index (DAI) was used to evaluate the severity of the disease based on clinical symptoms such as weight loss, stool consistency, and bleeding. Compared with IR group, the DAI was significantly lower in Gln+IR group (Figure 1B). Importantly, colon length was significantly reduced in the IR group, whereas Gln significantly mitigated radiation-induced colon shortening (Figure 1C).

To observe the structural damage of the intestines in RIII rats, HE staining of colon tissues was performed, which revealed that rats in the IR group exhibited extensive erosion, ulceration, inflammatory cell infiltration, and loss of normal crypt architecture. Pre-treatment with Gln significantly mitigated the radiation-induced damage in rat colons, as evidenced by the significantly lower histological scores in the Gln+IR group compared to the IR group (Figure 2A). Inflammatory response is one of the key features of colonic injury. The results showed that transcription levels of inflammatory cytokines IL-1β and IL-6 were significantly higher in the IR group compared to the control group, whereas these levels were significantly reduced in the Gln+IR group compared to the IR group (Figure 2B).

AB-PAS staining was used to assess mucus production and evaluate the integrity of the intestinal barrier, particularly by highlighting goblet cells (GCs) involved in mucus secretion. The results showed GCs were significantly decreased in the IR group compared to the control, which was approaching depletion. In contrast, the Gln significantly increased colonic GCs (Figure 3). These results indicated that radiation induced severe loss of intestinal GCs in rats, whereas intraperitoneal Gln significantly attenuates radiation-induced GC depletion.

To investigate the effects of Gln on intestinal stem cells (ISCs) and goblet cell (GC) maturation, immunohistochemical analysis of colon tissues was performed. The results confirm that the expression of Lgr5, a marker of ISCs, is significantly higher in the Gln+IR group compared to the IR group, indicating that pre-administration of Gln protects ISCs. Additionally, the expression of Klf4, a marker of GC maturation, is significantly increased in the Gln+IR group, suggesting enhanced goblet cell differentiation and maturation (Figure 4).

Notch1 and mTOR are key signaling pathways involved in cellular growth, differentiation, and response to stress, making them critical targets in radiation-induced injury and recovery. To investigate the effects of Gln on the mTOR signaling pathway and Notch1 expression in rat intestines, western blot analysis was performed. The results showed that Notch1 expression in the colon was significantly elevated in the IR group compared to the control, but it was significantly reduced in the Gln+IR group. These findings suggest that intraperitoneal Gln administration inhibits radiation-induced Notch1 overexpression in the rat colon. Additionally, Gln significantly increased the phosphorylation of mTOR and its downstream targets, S6K1 and 4E-BP1, compared with the IR group, indicating that Gln activates the mTOR signaling pathway. Phosphorylation of S6K1 and 4E-BP1 plays crucial roles in promoting protein synthesis, cell growth, and survival. Specifically, phosphorylated S6K1 enhances ribosomal biogenesis and translation initiation, while 4E-BP1 phosphorylation relieves its inhibition on eukaryotic translation initiation factor 4E (eIF4E), thereby further promoting protein synthesis. These findings suggest that Gln not only activates the mTOR pathway but also supports cellular processes essential for repair and regeneration following radiation-induced damage (Figure 5).

To examine the effects of mTOR inhibition on Notch1 and MUC2 expression, mTOR was pharmacologically inhibited by RAPA in HT-29 cells. The results show that in the 4 mM group, the expression and phosphorylation levels of mTOR are significantly higher than in the 2 mM group, accompanied by a downward trend in Notch1 expression and upregulated MUC2 expression. After adding RAPA, both the 2 mM+R and 4 mM+R groups exhibit lower mTOR expression and phosphorylation levels compared to the 2 mM and 4 mM groups, respectively. Notch1 expression in the 2 mM+R and 4 mM+R groups is higher than in the 2 mM and 4 mM groups, respectively, while MUC2 expression is inhibited in HT-29 cells. These findings suggest that while Gln activates the mTOR signaling pathway and suppresses Notch1 expression, inhibiting mTOR with RAPA can restore Notch1 expression and inhibit MUC2 expression (Figure 6).

To explore the role of the Notch1/mTOR signaling pathway in regulating the intestinal barrier, HT-29 cells were treated with Jagged-1, a Notch1 agonist. The expression of Notch1 was significantly enhanced in both 2 mM+J and 4 mM+J groups compared with the 2 mM and 4 mM groups, respectively. In addition, MUC2 expressions in both 2 mM+J and 4 mM+J were significantly downregulated. However, no significant changes were observed in mTOR expression or phosphorylation levels. Additionally, Notch1 expression in 4 mM+J group was lower than that in 2 mM+J group. These results suggest that activation of Notch1 does not markedly affect mTOR expression or phosphorylation, while Gln can inhibit Jagged-1–induced Notch1 activation (Figure 7).