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 intestinal barrier. This study aimed to elucidate the molecular mechanisms of HO-1/CO in barrier loss.  Methods: We induced gut-leakiness by injecting carbon tetrachloride (CCl 4 ) to wild type or intestinal HO-1-deficient mice, or administrating tumor necrosis factor-α (TNF-α) to HO-1 overexpression or knockdown cells. The effects of HO-1/CO maintaining intestinal barrier integrity were studied at different levels, including in-vivo, in-vitro, molecular mechanism, gene knockout, and disease verification. Findings: Cobalt protoporphyrin and CO-releasing molecule-2 alleviated colonic mucosal injury and TNF-α levels, upregulated tight junctions (TJs) expression, inhibited epithelial IκB-α degradation and phosphorylation, NF-κB p65 phosphorylation, long-MLCK expression and MLC-2 phosphorylation after CCl4.  Zinc protoporphyrin completely reversed these effects. These findings were confirmed in Caco-2 cells with gain- or loss-of-HO-1-function after TNF-α. Pre-treated with JSH-23 (NF-κB inhibitor) or ML-7 (long-MLCK inhibitor), HO-1 overexpression prevented from TNF-α-induced TJs disruption, while HO-1 shRNA promoted TJs damage even in the presence of JSH-23 or ML-7, suggesting HO-1 dependently protected intestinal barrier via NF-κB p65/MLCK/p-MLC-2 pathway. Using intestinal HO-1-deficient mice further demonstrated the effects of HO-1 in maintaining intestinal barrier integrity and its relative mechanisms. We finally confirmed the clinical significance of HO-1/CO repairing barrier loss in liver injury by alleviated hepatic fibrogenesis and serum ALT levels. Interpretation: HO-1/CO maintains intestinal barrier integrity through NF-κB/MLCK pathway. Therefore, the intestinal HO-1/CO-NF-κB/MLCK system is potential therapeutic targets for diseases with leaky-gut.    Funding: National Natural Science Foundation of China (Grant No. 81670479). Declaration of Interests: All authors declare that they have no conflict of interests Ethics Approval Statement: The animal experiments were carried out in strict accordance with the recommendations in the guide of the Animal Care and Use Committee of Dalian Medical University, and the protocols were approved by the institutional Animal Experimental Ethics Committee (Approval No. AEE18006).