Larazotide Acetate Protects the Intestinal Mucosal Barrier from Anoxia/Reoxygenation Injury via Various Cellular Mechanisms

<b>Background/Objective</b>: Larazotide acetate (LA) is a synthetic octapeptide under development as a therapeutic candidate for celiac disease, acting to reduce intestinal permeability and regulate tight junctions (TJs). Although several studies have shown barrier-protective effects, the cellular mechanisms underlying LA's actions in the intestinal epithelium remain unclear. This study aimed to elucidate the mechanistic roles of LA in maintaining intestinal epithelial integrity during cellular injury. <b>Methods:</b> C2BBe1 and leaky IPEC-J2 cell monolayers were pretreated with 10 mM LA and subjected to anoxia/reoxygenation (A/R) injury. Transepithelial electrical resistance (TEER), TJ protein localization, and phosphorylation of myosin light chain-2 (MLC-2) were analyzed. In addition, RNA sequencing was conducted to identify differentially expressed genes and signaling pathways affected by LA treatment. <b>Results:</b> LA pretreatment significantly increased TEER and preserved TJ protein organization during A/R injury. Transcriptomic analysis revealed enrichment of genes related to barrier regulation, small GTPase signaling, protein phosphorylation, proliferation, and migration. LA pretreatment markedly reduced MLC-2 phosphorylation, likely through modulation of the ROCK pathway, consistent with RNA-seq findings. Moreover, LA enhanced cellular proliferation, validating transcriptomic predictions. <b>Conclusions:</b> LA exerts a protective effect on intestinal epithelial integrity by stabilizing tight junctions, reducing MLC-2 phosphorylation, and promoting epithelial proliferation. These findings highlight a novel mechanism for LA and support its therapeutic potential in treating gastrointestinal disorders associated with "leaky gut" and mucosal injury.