Modeling a leaky gut In vitro: Sodium butyrate-induced barrier disruption of Caco-2 monolayers

Butyrate exhibits a dual effect on gut permeability, promoting epithelial health at low concentrations but disrupting barrier integrity at higher levels. This disruption at higher concentrations can be exploited, in vitro, to create gut barrier models with increased permeability. This study aimed to unravel the mechanism of action of sodium butyrate (NaB, 50–125 mM) on human intestinal enterocyte monolayers using the Caco-2 cell model. NaB reduced Trans-Epithelial Electrical Resistance (TEER) and increased lucifer yellow paracellular flux in a dose-dependent manner on Caco-2 monolayers, effects that persisted after NaB withdrawal. TEER reductions induced by NaB occurred under hyperosmotic conditions (430.5 ± 4.5 mOsm/kg). In contrast treatment with mannitol, a non-metabolizable osmotic agent used to mimic the osmotic effect, only caused a transient decrease in TEER. NaB was actively transported into cells, with increased mRNA expression of butyrate transporters MCT-1 and SMCT-1. However, NaB did not act as an energy substrate, as evidenced by decreased ATP levels and unchanged cellular reductase activity. Instead, NaB elevated Caspase-3 activity, induced stress markers (p21, HSP-70), and stimulated cytokine production (IL-6, IL-8, TNF). Most importantly, treatment with dexamethasone, a commonly used steroid, restored TEER and reduced stress and inflammation markers in Caco-2 monolayers disrupted by 75 mM NaB. Therefore, NaB treated Caco-2 monolayers are a suitable in vitro model for gut barrier dysfunction and inflammation such as Crohn's disease or necrotizing enterocolitis.