Fisetin alleviates zinc overload-induced intestinal injury by inhibiting ferroptosis and remodeling gut microbiota in weaned piglets.

BACKGROUND: High doses of zinc oxide (ZnO) effectively prevent post-weaning diarrhea and promote growth in weaned piglets, but raise concerns over intestinal injury, metabolic disorders, and risks of fostering bacterial resistance. This study aimed to identify non-chelating polyphenols capable of mitigating zinc toxicity and to elucidate their protective mechanisms, with emphasis on ferroptosis inhibition. RESULTS: Network toxicology predicted ferroptosis as a central mechanism in zinc-induced intestinal injury, which was confirmed in intestinal epithelial cells where zinc overload specifically induced ferroptosis without activating apoptosis, necroptosis, or autophagy. From multiple polyphenols, fisetin (FIS) was identified as a non-chelating candidate that alleviated zinc-induced cytotoxicity, preserved tight junction proteins, and activated the Nrf2-GPX4 axis to inhibit ferroptosis in vitro. In zinc-overloaded weaned piglets, FIS supplementation maintained the growth-promoting effects of high-dose zinc while ameliorating intestinal damage. FIS also attenuated oxidative stress, alleviated inflammation, and inhibited ferroptosis in the jejunum. Furthermore, FIS remodeled the gut microbiota, enriching beneficial taxa Romboutsia (positively correlated with growth performance) and Clostridium_sensu_stricto_1 (positively correlated with the p-Nrf2/Nrf2 ratio), while suppressing the zinc-enriched Anaerovibrio (negatively correlated with GPX4 protein expression). FIS also shifted microbial metabolic pathways toward amino acid and terpenoid metabolism, potentially contributing to the observed ferroptosis defense. CONCLUSION: FIS alleviates high-dose ZnO-induced intestinal injury in weaned piglets through dual modulation, activating the Nrf2-GPX4 axis to inhibit ferroptosis and remodeling the gut microbiota to reinforce this defense. By preserving the growth benefits of zinc while mitigating its toxicity, FIS represents a promising nutritional strategy for sustainable swine production.