Natural ligand 3,3'-diindolylmethane functions as an AHR agonist to modulate angiogenesis and organ development in...

Aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that is crucial for regulating various toxicological responses and physiological functions. Recent studies have highlighted that interactions between AHR and certain natural ligands can modulate inflammation and immune responses, offering therapeutic potential for a wide range of diseases. This research aims to investigate the effects of interactions between AHR and natural ligands on chicken development and to elucidate the importance of avian AHR activation and the application of natural ligands. In vitro reporter gene assays using COS-7 and LMH cell lines were conducted with seven natural ligands to evaluate the transactivation potency of chicken AHR, demonstrating that these natural ligands act as AHR agonists in chickens. Notably, 3,3'-diindolylmethane (DIM), previously reported to have anti-cancer and anti-inflammatory effects in rodents, was identified as the most responsive natural ligand to chicken AHR, as it induced the highest fold change in luciferase activity in both COS-7 and LMH cells, suggesting strong agonistic activity in vitro. Further investigations into the effects of DIM were performed through in ovo experiments, transcriptome analysis, and a shell-less incubation system, assessing its impact on chicken embryo development. In in ovo experiments, although no differences were observed in body indices between the control and DIM-treated groups, significant increases in the weights of the liver, kidney, and intestine were noted in the DIM-treated groups. Transcriptome analysis of 24,667 genes identified 936 differentially expressed genes (DEGs) in the DIM-treated groups, with 76.4 % containing Xenobiotic Response Elements (XREs) in their promoters, suggesting a crucial role for AHR in regulating these genes. Pathway and network analysis revealed that DIM treatment significantly altered the MAPK signaling pathway, Focal adhesion, Nod-like receptor (NLR) signaling pathway, PPAR signaling pathway, and mTOR signaling pathway, which are associated with cell proliferation, angiogenesis, metabolism, and immune regulation. These results highlight the diverse physiological effects of AHR activation by DIM in developing embryos. The shell-less incubation system showed that the DIM-treated group exhibited a significant increase in total blood vessel length and the number of branches during the early developmental stages of chicken embryos. These results suggest that DIM activates the AHR signaling pathway, promoting hepatic cell proliferation and angiogenesis while modulating inflammatory responses during chicken development. These findings highlight the role of AHR in regulating developmental processes beyond its traditional function in response to toxic substances, providing new insights into the utilization of AHR natural ligands in poultry development and disease control.