Paeoniflorin improved intestinal barrier function and immune homeostasis of broiler chickens challenged with Eimeria by modulating gut microbiota and suppressing M1 macrophage-associated inflammatory responses.

Coccidiosis caused by Eimeria parasites impairs intestinal function and disrupts immune homeostasis in broilers, leading to substantial economic losses in poultry production. Paeoniflorin (PFN), a monoterpene glycoside with known anti-inflammatory and immunomodulatory properties, has not been extensively studied against avian coccidiosis. This study aimed to investigate whether dietary PFN could mitigate Eimeria-induced intestinal injury by modulating gut microbiota and macrophage polarization. A total of 288 one-day-old male Cobb 500 broilers were randomly assigned to 4 groups in a 2 × 2 factorial design: control (CTR), PFN supplementation (250 mg/kg), coccidial challenge (CC), PFN supplementation and coccidial challenge (PFNCC). On d 8 and d 10, birds in CC and PFNCC groups were orally challenged with live attenuated Eimeria vaccine at 30-fold the recommended dose. Samples were collected on d 14. In vitro, gut microbiota from healthy broilers was cultured with or without PFN under aerobic and anaerobic conditions for 16S rRNA sequencing. The PFN supplementation did not significantly affect growth performance (P > 0.05), but reduced fecal oocyst shedding and serum diamine oxidase activity, increased goblet cell counts, and upregulated tight junctions (occludin and zonula occludens-1) expression in Eimeria-challenged broilers (P < 0.05). The PFN downregulated ileal expression of M1 macrophage markers (tumor necrosis factor (TNF)-α, interferon-γ, inducible nitric oxide synthase and chemokine (CC motif) ligand 17) (P < 0.05). Transcriptomic analysis revealed that PFN partially reversed Eimeria-induced transcriptional reprogramming, with enrichment of immune-related pathways including Toll-like receptor and nuclear factor-κB (NF-κB) signaling. In vitro, PFN attenuated lipopolysaccharide (LPS)-induced M1 macrophage polarization by suppressing NF-κB, IL-12 and TNF-α expression, while reducing lactate dehydrogenase release (P < 0.05). Microbiota analysis showed that PFN increased Akkermansia in challenged birds in vivo (P < 0.05). In vitro, PFN increased Firmicutes and Lactobacillus while reducing Bacteroidetes and Escherichia-Shigella under anaerobic conditions (P < 0.05). In conclusion, dietary PFN alleviated Eimeria-induced intestinal injury and inflammation in broilers by suppressing NF-κB-mediated M1 macrophage-associated inflammatory responses and modulating gut microbiota toward a barrier-supportive configuration.