HO-1/CO Maintains Intestinal Barrier Integrity through NF-κB/MLCK Pathway in Intestinal HO-1-/- Mice

Background Intestinal barrier injury is an important contributor to many diseases. We previously found that heme oxygenase-1 (HO-1) and carbon monoxide (CO) protect the intestinal barrier. This study is aimed at elucidating the molecular mechanisms of HO-1/CO in barrier loss.Materials and Methods We induced gut leakiness by injecting carbon tetrachloride (CCl 4 ) to wildtype or intestinal HO-1-deficient mice. In addition, we administrated tumor necrosis factor- α (TNF- α ) to cells with gain- or loss-of-HO-1 function. The effects of HO-1/CO maintaining intestinal barrier integrity were investigated in vivo and in vitro .Results Cobalt protoporphyrin and CO-releasing molecule-2 alleviated colonic mucosal injury and TNF- α levels; upregulated tight junction (TJ) expression; and inhibited epithelial I κ B- α degradation and phosphorylation, NF- κ B p65 phosphorylation, long MLCK expression, and MLC-2 phosphorylation after administration of CCl 4 . Zinc protoporphyrin completely reversed these effects. These findings were further confirmed in vitro , using Caco-2 cells with gain- or loss-of-HO-1-function after TNF- α . Pretreated with JSH-23 (NF- κ B inhibitor) or ML-7 (long MLCK inhibitor), HO-1 overexpression prevented TNF- α -induced TJ disruption, while HO-1 shRNA promoted TJ damage even in the presence of JSH-23 or ML-7, thus suggesting that HO-1 dependently protected intestinal barrier via the NF- κ B p65/MLCK/p-MLC-2 pathway. Intestinal HO-1-deficient mice further demonstrated the effects of HO-1 in maintaining intestinal barrier integrity and its relative mechanisms. Alleviated hepatic fibrogenesis and serum ALT levels finally confirmed the clinical significance of HO-1/CO repairing barrier loss in liver injury.Conclusion HO-1/CO maintains intestinal barrier integrity through the NF- κ B/MLCK pathway. Therefore, the intestinal HO-1/CO-NF- κ B/MLCK system is a potential therapeutic target for diseases with a leaky gut.