HO‐1/CO Maintains Intestinal Barrier Integrity through NF‐<i>κ</i>B/MLCK Pathway in Intestinal HO‐1<sup>-/-</sup> Mice
Zhenling Zhang, Lijing Zhang, Qiuping Zhang, Bojia Liu, Fang Li, Yi Xin, Zhijun Duan
Abstract
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.