Objective To investigate the effects and the mechanisms of κ-carrageenan on radiation-induced intestinal injury in mice.
Methods A localized abdominal irradiation model was established using 40 C57BL/6J mice, which were assigned to four groups (n=10 per group) using a simple random sampling method: control, irradiation (IR) only, IR+low-dose κ-carrageenan (32.5 mg/kg), and IR+high-dose κ-carrageenan (65.0 mg/kg). Mice were exposed to a single 12 Gy dose of 137Cs γ-rays to the abdomen, with body weight recorded every 3 days. The mice were euthanized 12 days after irradiation, and the samples of their small intestine and colon tissues were collected. For the fecal microbiota transplantation (FMT) model, 30 C57BL/6J mice were divided into donor (n=10) and recipient (n=20) groups using a simple random sampling method. The donor group mice were gavaged daily with 200 μl of high-dose (65.0 mg/kg) κ-carrageenan, while the recipient group mice were further randomized into the IR+FMT-control group (n=10, gavaged daily with 200 μl of fecal microbiota from normal mice) and the IR+FMT-κ-carrageenan group (n=10, gavaged daily with 200 μl of fecal microbiota from the donor group) using a simple random sampling method. After 12 days of gavage, the mice were euthanized, and the samples of their small intestine and colon tissues were collected. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to quantify the expression of intestinal barrier function-related genes (Krüppel-like factor 4 (KLF4), zonula occludens-1 (ZO-1), Occludin, and mucin 2 (MUC2)) and pro-inflammatory-related genes (interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)). Hematoxylin-eosin staining was used to assess the structure of the mouse small intestine, and 16S ribosomal ribonucleic acid (rRNA) sequencing was performed to analyze the effects of radiation and κ-carrageenan on the gut microbiota of mice. Comparison of quantitative data between two groups were performed using the independent samples t-test.
Results Compared with the IR only group, the IR+low-dose κ-carrageenan group exhibited lower body weight and shorter colon length, but these differences were not statistically significant (t=1.78 and 1.39; both P>0.05). However, IR+high-dose κ-carrageenan significantly exacerbated colon shortening compared with the IR only group (t=2.70, P<0.05). Hematoxylin-eosin staining showed that the small intestinal villi in the IR+low-dose κ-carrageenan group were shortened, inter-villus spaces were enlarged, and villous swelling appeared compared with those in the IR only group. In the IR+high-dose κ-carrageenan group, the severity of radiation-induced intestinal injury was further aggravated. RT-qPCR results indicated that, compared with the IR only group, the relative mRNA expression levels of intestinal barrier function-related genes (KLF4, ZO-1, Occludin, and MUC2) were significantly downregulated in the IR+low-dose κ-carrageenan group (t=2.68–4.96, all P<0.05). Although pro-inflammatory-related genes (IL-6 and TNF-α) were upregulated, the differences in the IR+low-dose κ-carrageenan group were not significant (t=1.71, 1.42; both P>0.05). In the IR+high-dose κ-carrageenan group, the downregulation of the relative mRNA expression levels intestinal barrier function-related genes (KLF4, ZO-1, Occludin, and MUC2) and the upregulation of pro-inflammatory-related genes (IL-6 and TNF-α) were further intensified and the differences were statistically significant (t=2.24–4.85, all P<0.05). FMT results demonstrated that mice in the IR+FMT (κ-carrageenan) group exhibited a significantly shorter colon length (t=2.73, P<0.05) than mice in the IR+FMT (control) group. In the small intestine, the relative mRNA expression levels of intestinal barrier function-related genes (KLF4, ZO-1, and Occludin) were significantly downregulated (t=5.14, 2.73, 2.31; all P<0.05). Conversely, the relative mRNA expression levels of pro-inflammatory-related genes (IL-6 and TNF-α) were significantly elevated (t=2.87, 9.63; both P<0.05). 16S rRNA sequencing revealed that radiation significantly reduced the relative abundance of probiotics, including Lachnoclostridium, Lachnospiraceae, and Blautia (t=3.06, 3.51, 2.46; all P<0.05), compared with the control group. Compared with the IR only group, the relative abundance of Lachnoclostridium significantly decreased in the IR+high-dose κ-carrageenan group (t=2.08, P<0.05).
Conclusion κ-Carrageenan exacerbates radiation-induced intestinal injury in mice by influencing the gut microbiota.
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