Objective  To investigate the protective effect and molecular mechanism of trehalose in ionizing radiation (IR)-induced small intestinal damage.

Methods  Eight-week-old male C57 BL/6 J mice were randomly divided into four groups, with each group containing six mice: control (no treatment given), trehalose-treated (consumption of 2 g/100 ml seaweed sugar water), IR (whole abdominal irradiation group treated with 13 Gy IR), and trehalose+IR groups (trehalose+total abdominal irradiation treatment with 13 Gy IR). Daily monitoring of body weight was performed after 13 Gy irradiation and 2 g/100 ml trehalose drinking water treatment. After 14 days, the mice were euthanized, and samples from the blood, small intestine, and colon were collected. Intestinal injury was assessed via hematoxylin-eosin (HE) and periodic acid-schiff (PAS) staining. The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in the serum and small intestine tissues were quantified through enzyme-linked immunosorbent assay (ELISA). Experiments on mouse small-intestine epithelial cell proliferation, cell clone formation, cell apoptosis rate, reactive oxygen species (ROS) determination, and DNA double-strand breaks (DSBs) detection were conducted to detect cell viability, clone formation ability, apoptosis, ROS levels, and the number of DSBs, respectively. The expression levels of autophagy-related genes and proteins were determined via quantitative real-time polymerase chain reaction (qRT-PCR) method and Western blot method. Inter group comparison was achieved through two independent sample t-tests.

Results  The animal study results demonstrated that compared with the irradiation group, trehalose significantly alleviated the weight loss, facilitated the recovery of irradiated mice, and promoted its recovery (t=4.064, P<0.05), the trehalose+IR group exhibited milder colon shortening degree of mice, and statistically significant difference (t=4.044, P<0.05). In addition, the HE and PAS staining evaluation results indicated that trehalose can alleviate intestinal mucosal damage in mice after IR. Compared with the irradiation group, the trehalose+irradiation group presented longer villi, deeper crypts, and more goblet cells. The two groups exhibited a statistically significant difference (t=4.373, 8.414, 6.515, all P<0.05). ELISA demonstrated that trehalose substantially decreased the TNF-α and IL-6 levels in mouse serum and small intestinal tissues after IR, and the differences were statistically significant (t=4.475, 8.686, 3.993, 6.007, all P<0.05). In vitro, the results of cell proliferation and cloning experiments revealed that trehalose promoted the activity and clone formation ability of mouse small-intestine MODE-K cells after IR (0.885±0.127) vs. (0.644±0.151), (97.330±5.937) vs. (64.000±7.324), with the differences showing statistical significance (t=2.566, 4.411, all P<0.05). The findings of the apoptosis detection experiment indicated that trehalose inhibited the apoptosis rate of mouse small intestine MODE-K cells after IR treatment (15.270±0.647) % vs. (9.334±0.854) %, and the difference was statistically significant (t=8.315, P<0.05). According to ROS assay and DSB detection experiments, trehalose reduced intracellular ROS levels and DSBs after IR (t=8.884, 4.802, both P<0.05). The findings of qRT-PCR and Western blot analysis showed that compared with the IR group, the trehalose+IR group showed significantly increased expressions of autophagy-related genes and proteins transcription factor EB (TFEB) and microtubule-associated proteins 1 light chain 3 beta in the small-intestine tissue and MODE-K mouse cells (208.210±26.143) vs. (8.986±5.362), (13.970±4.587) vs. (5.815±1.873), and the differences were statistically significant (t=8.875, 8.461, all P<0.05).

Conclusion  Trehalose alleviates IR-induced intestinal damage in mice through the regulation of TFEB-mediated autophagy activation.