Limosilactobacillus reuteri orchestrates IAA-mediated mitochondrial homeostasis and stem cell renewal against intestinal oxidative injury in aged hens

Abstract Background Intestinal oxidative stress compromises gut health and production performance in laying hens. Although Limosilactobacillus reuteri ( L. reuteri ) has been shown to mitigate oxidative stress, the underlying mechanisms remain incompletely understood. This study therefore investigated the protective effects and mechanisms of L. reuteri against diquat-induced intestinal oxidative injury in aged hens. Methods A total of 270 70-week-old Jinbai hens were divided into control (CON), diquat (DQ), and L. reuteri + diquat (LRD) groups with 6 replicates each. Birds in CON and DQ groups received a basal diet, while those in LRD group received basal diet supplemented with L. reuteri . After a 10-week pretreatment, DQ and LRD hens were intraperitoneally injected with diquat, while CON hens were injected intraperitoneally with an equivalent amount of saline solution. Results Diquat challenge significantly compromised intestinal barrier integrity, as evidenced the downregulated mRNA and protein expression of MUC2, ZO-1 and Occludin ( P < 0.05). Notably, these adverse impacts were markedly reversed by L. reuteri pretreatment. Moreover, L. reuteri reshaped the gut microbiota by increasing the abundance of Lactobacillus ( P < 0.05). Metabolomic analysis revealed significant enrichment of tryptophan metabolism, with a notable upregulation of indole-3-acetic acid (IAA). At the mitochondrial level, L. reuteri reduced mitochondrial ROS production, restored ATP content ( P < 0.05), and downregulated mitochondrial biogenesis-related genes expression of PGC-1α and NRF1 and balanced mitochondrial dynamics genes expression of MFN1 and DRP1 ( P < 0.05). Furthermore, L. reuteri elevated GSH-Px and T-AOC activities via activation of the Nrf2 pathway, and reduced IL-1β and IL-6 levels by suppression of NF-κB signaling ( P < 0.05). In vitro organoid culture further demonstrated IAA significantly reduced mitochondrial ROS production and restored ATP levels ( P < 0.05) and stimulated stem cell proliferation. These changes inhibited apoptosis and promoted intestinal proliferation, thereby alleviating intestinal oxidative injury. Conclusion L. reuteri alleviates intestinal oxidative damage via modulation of the gut microbiota–IAA axis, which coordinates mitochondrial homeostasis and stem cell renewal, thereby mitigating oxidative stress and enhancing barrier function. These findings highlight its potential as a probiotic therapeutic agent for mitigating oxidative stress and preserving gut health in aging poultry.