AhR agonists based on calixarene as carriers for the treatment of radiation enteritis

Objective To investigate the therapeutic effects of intestinal azoreductase-activated azo-calix4arene (referred to as azoC4A) combined with aryl hydrocarbon receptor (AhR) agonist β-naphthoflavone (BNF) on radiation enteritis (RE) in mice.

Methods Fluorescence titration was utilized to determine the host–guest recognition capability between azoC4A and AhR agonist BNF. Using luciferase reporter gene to detect the activation ability of host guest recognition complexes on AhR in human liver cancer cell line AhR Luc-HepG2 (referred to as AhR Luc-HepG2 cells). In the animal experiment, 24 female C57BL/6 mice were randomly divided into four groups using a randomized block design: radiation-only, azoC4A, BNF, and azoC4A-BNF groups, with six mice per group. After 1 week of purified diet, the BNF, azoC4A, and azoC4A-BNF groups were orally administered daily doses by gavage with BNF (15 mg/kg), azoC4A (84 mg/kg), and azoC4A-BNF (BNF equivalent dose of 15 mg/kg), respectively. Seven days after drug administration, mice received abdominal 15 Gy ¹³⁷Cs γ-ray irradiation. Mice were euthanized on the third day post-irradiation, and colon, small intestine, and blood samples were collected. Real-time quantitative polymerase chain reaction (PCR) was used to detect AhR activation levels in intestinal tissues, while inflammatory markers of RE (diamine oxidase and tumor necrosis factor-α levels) and intestinal pathological changes were evaluated. Student′s t-test was used for pairwise comparisons of metric data that conforms to normal distribution between groups.

Results Fluorescence titration results revealed that the non-covalent binding coefficient between AhR agonist BNF and azoC4A was 6×10⁷ (mol/L)⁻¹. When the concentration ratio of azoC4A to BNF was 3∶2, the binding rate reached 80%. In the luciferase reporter gene assay using AhR-Luc HepG2 cells, the luminescence intensity of the BNF group increased to 3.6-fold compared with the control group (288±17 vs. 79±30), with the difference being statistically significant (t=10.480, P<0.001). Meanwhile, the difference in luminescence intensity between the azoC4A-BNF and the control groups (125±13 vs. 79±30, t=2.451, P=0.063) was not statistically significant. The colon length in the azoC4A-BNF group was significantly longer than that in the radiation-only group ((49.6±2.9) mm vs. (44.8±3.4) mm, t=2.400, P<0.05). The plasma diamine oxidase contents in the azoC4A-BNF and BNF groups were significantly reduced ((132.5±40.0) U/L vs. (32.8±22.4) U/L vs. (27.6±7.8) U/L, t=4.399, 3.731; both P<0.05) compared with the radiation-only group. Meanwhile, the tumor necrosis factor-α (TNF-α) contents in the azoC4A-BNF group were significantly lower than that in the control group ((9.0±2.2) pg/ml vs. (13.5±2.3) pg/ml, t=2.721, P<0.05). The reelative expression of cytochrome P450 family 1 subfamily a member 1(Cyp1a1) in the intestinal tissues of the azoC4A-BNF group, BNF group and radiation-only group were 8.49±1.08, 5.49±0.83, −0.30±1.08. Moreover, the differences in the azoC4A-BNF and BNF groups were statistically significant (t=3.430, 3.018; both P<0.05) compared with the radiation-only group.

Conclusions The non-covalent combination of azo-calixarene with AhR agonist BNF demonstrated therapeutic efficacy against RE injury in mice. Furthermore, azo-calixarene, serving as a water-soluble carrier, addressed the poor water solubility and targeting issues of AhR agonists, providing a novel approach for the application of AhR ligands.