Hydrogen gas and preservation of intestinal stem cells in mesenteric ischemia and reperfusion

Methods: Three rat groups underwent 60-min mesenteric artery occlusion (ischemia), 60-min reperfusion following 60-min occlusion (reperfusion), or ischemia-reperfusion with the same duration under continuous 3% hydrogen gas inhalation (hydrogen). The distal ileum was harvested. Immunofluorescence staining with caspase-3 and leucine-rich repeat-containing G-protein-coupled 5 (LGR5), a specific marker of intestinal stem cell, was conducted to evaluate the injury location and cell types protected by hydrogen. mRNA expressions of LGR5, olfactomedin 4 (OLFM4), hairy and enhancer of split 1, Jagged 2, and Neurogenic locus notch homolog protein 1 were measured by quantitative polymerase chain reaction. Tissue oxidative stress was analyzed with immunostaining for 8-hydroxy-2'-deoxyguanosine (8-OHdG). Systemic oxidative stress was evaluated by plasma 8-OHdG.

Results: Ischemia damaged the epithelial layer at the tip of the villi, whereas reperfusion induced extensive apoptosis of the cells at the crypt base, which were identified as intestinal stem cells with double immunofluorescence stain. Hydrogen mitigated such apoptosis at the crypt base, and the LGR5 expression of the tissues was higher in the hydrogen group than in the reperfusion group. OLFM4 was also relatively higher in the hydrogen group, whereas other measured RNAs were comparable between the groups. 8-OHdG concentration was high in the reperfusion group, which was reduced by hydrogen, particularly at the crypt base. Serum 8-OHdG concentrations were relatively higher in both reperfusion and hydrogen groups without significance.

Conclusion: This study demonstrated that hydrogen gas inhalation preserves intestinal stem cells and mitigates oxidative stress caused by mesenteric ischemia and reperfusion.