Objective To investigates the radiation protective effect of azo-calix4arene (azoC4A) activated by gut azo-reductase and combined with the AhR agonist β-naphthoflavone (BNF) in mice with radiation-induced enteritis.

Methods The non-covalent binding ability of azo-calix4arene and the AhR agonist BNF was determined using fluorescence titration. The AhR activation capability of the host-guest recognition complex was evaluated in the AhR reporter gene cell line HepG2. For the animal experiments, 24 female C57BL/C mice were randomly divided into 4 groups (single irradiation group, azoC4A group, BNF group, and azoC4A-BNF group, 6 mice per group). After one week of purified diet, the mice were gavaged daily with BNF (15 mg/kg), azoC4A（84 mg/kg）or azoC4A-BNF (equivalent BNF dose, 15 mg/kg). After 7 days of dosing, the mice were irradiated with Cs137 γ-rays at a dose of 15 Gy. On the third day post-irradiation, the mice were euthanized, and samples of the colon, small intestine, and blood were collected. RT-PCR was used to assess AhR activation levels in intestinal tissues, while inflaatory markers and pathological changes in the gut were evaluated. Data were analyzed using one-way ANOVA, and pairwise comparisons were made with the Student′s t-test.

Results Fluorescent titration results indicated that the non-covalent binding constant of the AhR agonist BNF with azoC4A was 6×10⁷ M⁻¹. The binding reached 80% when the concentration ratio of azoC4A to BNF was 3∶2. In the AhR-Luc HepG2 cell-based luciferase reporter gene assay, the spontaneous luminescence intensity of the azoC4A-BNF group was significantly lower than that of the BNF group (125±13 vs. 288±17, t=13.220, P<0.001), and there was no statistically significant difference compared to the Ctrl group (125±13 vs. 79±30, t=2.481, P=0.0704). The length of colon was significantly higher in the azoC4A-BNF complex group than that of the single irradiation group in the mouse (49.6±2.9) mmol/L vs.(44.8±3.4) mmol/L, t=2.400, P<0.05) . Furthermore, the plasma Diamine oxidase(DAO) content was significantly lower in the azoC4A-BNF group and the BNF group compared with the radiation group (32.8±22.4) U/L vs. (27.6±7.8) U/L vs. (132.5±40) U/L; t=4.399, 3.731; P<0.05. Meanwhile, the TNF-α level in the azoC4A-BNF group also decreased significantly (13.49±2.26) pg/ml vs. (9.05±4.94) pg/ml, t=2.721, P=0.035. Furthermore, the gene expression level of Cyp1a1 in the mouse intestine of the azoC4A-BNF group was found to be 8-folds higher than that observed in the BNF group and 431-folds higher than the radiation group (8.49±1.08 vs. 5.49±0.83 vs.-0.30±1.08; t=3.430, 3.018; both P<0.05). Conclusions The non-covalent binding of azo-calixarenes with the AhR agonist BNF was utilized to achieve therapeutic effects on radiation-induced intestinal injury in mice. At the same time, the azo-calixarenes, as a water-soluble carrier, solved the issues of poor solubility and targeting of AhR agonists, providing a new approach for the application of AhR ligands.