Dietary iron supplementation modulates jejunal epithelial lineage differentiation and enteroendocrine cell-related responses in suckling piglets

Iron is an essential trace element involved in intestinal epithelial homeostasis, but its role in regulating epithelial lineage differentiation during early life remains incompletely understood. This study investigated whether dietary iron modulates jejunal epithelial differentiation, with particular attention to enteroendocrine cell-related responses, in suckling piglets. Twenty-four 7-day-old male suckling piglets were assigned to low-, medium-, and high-iron groups. Jejunal tissues were collected for histological analysis, quantitative real-time PCR, CHGA immunofluorescence staining, and transcriptomic analysis. Porcine jejunal organoids were further used to evaluate the direct effect of iron on epithelial lineage marker expression and the involvement of PPARγ-associated signaling. Dietary iron supplementation did not significantly alter jejunal histomorphology or pathological scores, but it increased the expression of several epithelial lineage marker genes, including LGR5 , LYZ , MUC2 , ALPI , and CHGA . Among these markers, CHGA showed the most pronounced and dose-responsive increase with increasing dietary iron level. High iron supplementation also increased CHGA fluorescence intensity and the number of CHGA-positive cells in the jejunum. Transcriptomic analysis showed that differentially expressed genes between the low- and high-iron groups were enriched in the PPAR signaling pathway, which was globally downregulated in the high-iron group. In porcine jejunal organoids, ferric ammonium citrate increased CHGA expression, whereas activation of PPARγ by troglitazone attenuated iron-induced CHGA upregulation. These findings suggest that dietary iron regulates jejunal epithelial lineage-associated differentiation in suckling piglets, with a preferential effect on CHGA-positive enteroendocrine cell-related responses. PPARγ-associated signaling may represent a potential regulatory node in iron-mediated intestinal epithelial development.