Hydrogen-Rich Medium Ameliorates Lipopolysaccharide-Induced Barrier Dysfunction via Rhoa-Mdia1 Signaling in CACO-2 Cells.

Gastrointestinal barrier dysfunction is associated with the severity and prognosis of sepsis. Hydrogen gas (H-2) can ameliorate multiple organ damage in septic animals. Ras homolog gene family member A (RhoA) and mammalian diaphanous-related formin 1 (mDia1) are important to regulate tight junction (TJ) and adherens junction (AJ), both of which determine the integrity of the intestinal barrier. This study was aimed to investigate whether H-2 could modulate lipopolysaccharide (LPS)-stimulated dysfunction of the intestinal barrier and whether RhoA-mDia1 signaling is involved. Caco-2 cells were exposed to different concentrations of LPS (1g/mL-1mg/mL). The permeability of the intestinal barrier was evaluated by transepithelial resistance (TER) and fluorescein-isothiocyanate-dextran flux. Expression and distribution of occludin and E-cadherin were analyzed by Western blot and immunofluorescence. RhoA activity was measured by G-Lisa assay, and mDia1 expression was assessed by Western blot. LPS (100g/mL) decreased TER and increased fluorescein-isothiocyanate-dextran flux, which were alleviated by H-2-rich medium. Also, H-2 down-regulated LPS-induced oxidative stress. Moreover, H-2 improved the down-regulated expression and redistribution of occludin and E-cadherin caused by LPS. Additionally, H-2 alleviated LPS-caused RhoA activation, and the beneficial effects of H-2 on barrier were counteracted by RhoA agonist CN03. Rho inhibitor C3 exoenzyme mitigated LPS-induced barrier breakdown. Furthermore, H-2-rich medium increased mDia1 expression, and mDia1 knockdown abolished protections of H-2 on barrier permeability. mDia1 knockdown eliminated H-2-induced benefits for occludin and E-cadherin. These findings suggest that H-2 improves LPS-induced hyperpermeability of the intestinal barrier and disruptions of TJ and AJ by moderating RhoA-mDia1 signaling.