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 (CCl4) 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 IκB-α degradation and phosphorylation, NF-κB p65 phosphorylation, long MLCK expression, and MLC-2 phosphorylation after administration of CCl4. 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-κB 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-κB 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-κB/MLCK pathway. Therefore, the intestinal HO-1/CO-NF-κB/MLCK system is a potential therapeutic target for diseases with a leaky gut.
Highlights
In the intestine, the epithelial barrier that regulates the interaction between the luminal material and the interstitium is crucial for maintaining homeostasis
Our findings suggested that the upregulation of heme oxygenase-1 (HO-1) and carbon monoxide (CO) may repair intestinal epithelial barrier injury after CCl4 injection
The results showed that in the presence of JSH-23, HO-1 overexpression significantly reduced NF-κB p65 phosphorylation, long myosin light-chain kinase (MLCK) expression, and myosin light-chain 2 (MLC-2) phosphorylation and markedly increased zonula occludens 1 (ZO-1) and occludin expression in Caco-2 cells transfected with the FUGW-HO-1 plasmid after tumor necrosis factor-α (TNF-α) stimulation, compared to the scrambled control group (Figures 3(a) and 3(b))
Summary
The epithelial barrier that regulates the interaction between the luminal material (e.g., gut microbiome) and the interstitium (e.g., mucosal immune cells) is crucial for maintaining homeostasis. The effects of HO-1/CO maintaining intestinal barrier integrity were investigated in vivo and in vitro. Cobalt protoporphyrin and CO-releasing molecule-2 alleviated colonic mucosal injury and TNF-α levels; upregulated tight junction (TJ) expression; and inhibited epithelial IκB-α degradation and phosphorylation, NF-κB p65 phosphorylation, long MLCK expression, and MLC-2 phosphorylation after administration of CCl4. These findings were further confirmed in vitro, using Caco-2 cells with gain- or loss-of-HO-1-function after TNF-α. 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 confirmed the clinical significance of HO-1/CO repairing barrier loss in liver injury. The intestinal HO-1/CO-NF-κB/MLCK system is a potential therapeutic target for diseases with a leaky gut
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