L‐Citrulline Ameliorates Iron Metabolism and Mitochondrial Quality Control via Activating AMPK Pathway in Intestine and Improves Microbiota in Mice with Iron Overload

Scope Oxidative stress caused by iron overload tends to result in intestinal mucosal barrier dysfunction and intestinal microbiota imbalance. As a neutral and nonprotein amino acid, L‐Citrulline (L‐cit) has been implicated in antioxidant and mitochondrial amelioration properties. This study investigates whether L‐cit can alleviate iron overload‐induced intestinal injury and explores the underlying mechanisms. Methods and results C57BL/6J mice are intraperitoneally injected with iron dextran, then gavaged with different dose of L‐cit for 2 weeks. L‐cit treatment significantly alleviates intestine pathological injury, oxidative stress, ATP level, and mitochondrial respiratory chain complex activities, accompanied by ameliorating mitochondrial quality control. L‐cit‐mediated protection is associated with the upregulation of Glutathione Peroxidase 4 (GPX4) expression, inhibition Nuclear Receptor Coactivator 4 (NCOA4)‐mediated ferritinophagy and ferroptosis, and improvement of gut microbiota. To investigate the underlying molecular mechanisms, Intestinal Porcine Epithelial Cell line‐J2 (IPEC‐J2) cells are treated with L‐cit or AMP‐activated Protein Kinase (AMPK) inhibitor. AMPK signaling has been activated by L‐cit. Notably, Compound C abolishes L‐cit's protection on intestinal barrier, mitochondrial function, and antioxidative capacity in IPEC‐J2 cells. Conclusion L‐cit may restrain ferritinophagy and ferroptosis to regulate iron metabolism, and induce AMPK pathway activation, which contributes to exert antioxidation, ameliorate iron metabolism and mitochondrial quality control, and improve intestinal microbiota. L‐cit is a promising therapeutic strategy for iron overload‐induced intestinal injury.