Hydrogen sulfide improves intestinal recovery following ischemia by endothelial nitric oxide-dependent mechanisms

Abstract

Hydrogen sulfide (H₂S) is an endogenous gasotransmitter that has vasodilatory properties. It may be a novel therapy for intestinal ischemia-reperfusion (I/R) injury. We hypothesized that 1) H₂S would improve postischemic survival, mesenteric perfusion, mucosal injury, and inflammation compared with vehicle and 2) the benefits of H₂S would be mediated through endothelial nitric oxide. C57BL/6J wild-type and endothelial nitric oxide synthase knockout (eNOS KO) mice were anesthetized, and a midline laparotomy was performed. Intestines were eviscerated, the small bowel mesenteric root identified, and baseline intestinal perfusion was determined using laser Doppler. Intestinal ischemia was established by temporarily occluding the superior mesenteric artery. Following ischemia, the clamp was removed, and the intestines were allowed to recover. Either sodium hydrosulfide (2 nmol/kg or 2 µmol/kg NaHS) in PBS vehicle or vehicle only was injected into the peritoneum. Animals were allowed to recover and were assessed for mesenteric perfusion, mucosal injury, and intestinal cytokines. P values < 0.05 were significant. H₂S improved mesenteric perfusion and mucosal injury scores following I/R injury. However, in the setting of eNOS ablation, there was no improvement in these parameters with H₂S therapy. Application of H₂S also resulted in lower levels of intestinal cytokine production following I/R. Intraperitoneal H₂S therapy can improve mesenteric perfusion, intestinal mucosal injury, and intestinal inflammation following I/R. The benefits of H₂S appear to be mediated through endothelial nitric oxide-dependent pathways.NEW & NOTEWORTHY H₂S is a gaseous mediator that acts as an anti-inflammatory agent contributing to gastrointestinal mucosal defense. It promotes vascular dilation, mucosal repair, and resolution of inflammation following intestinal ischemia and may be exploited as a novel therapeutic agent. It is unclear whether H₂S works through nitric oxide-dependent pathways in the intestine. We appreciate that H₂S was able to improve postischemic recovery of mesenteric perfusion, mucosal integrity, and inflammation. The beneficial effects of H₂S appear to be mediated through endothelial nitric oxide-dependent pathways.

Keywords: endothelial nitric oxide synthase; hydrogen sulfide; inflammation; intestinal ischemia; nitric oxide; perfusion; sodium hydrosulfide.

Fig. 1.

Hydrogen sulfide (H₂S) improves mesenteric perfusion following ischemic injury through endothelial nitric oxide synthase (eNOS)-dependent pathways. Postischemic mesenteric perfusion was significantly increased with both low- and high-range doses of sodium hydrosulfide (NaHS) in wild-type (WT) animals. However, there were no observed improvements in postischemic mesenteric perfusion following treatment in eNOS knockout (KO) animals (*P < 0.05 vs. respective vehicle).

Fig. 2.

Histological examination of small intestine following intestinal ischemia and reperfusion (I/R). A: representative histology slides of each treatment group (hematoxylin and eosin stain, ×20) demonstrating improvements in intestinal histology with NaHS therapy in WT animals but not in eNOS KO animals. B: histological scoring of intestinal specimens (*P < 0.05 vs. respective vehicle).

Fig. 3.

Inflammatory cytokines and growth factors are impacted by H₂S therapy following intestinal I/R. Intestinal levels of IL-6, IL-9, IL-10, VEGF, and fibroblast growth factor 2 (FGF-2) are decreased with H₂S therapy following intestinal ischemia. H₂S decreases in IL-9, IL-10, and VEGF appear to be facilitated by intact eNOS, whereas decreases in IL-6 and FGF-2 may work through H₂S-independent pathways. Hepatocyte growth factor (HGF) does not appear to be affected by H₂S therapy or ablation of eNOS (*P < 0.05 vs. WT vehicle, #P < 0.05 vs. eNOS KO vehicle).

Fig. 4.

Chemokines are impacted by H₂S therapy following intestinal I/R. Intestinal levels of macrophage inflammatory protein 1α (MIP-1α), eotaxin, chemokine ligand 10 (IP-10), MIP-2, granulocyte-colony stimulating factor (G-CSF), and C-X-C ligand 1 (KC) are decreased in ischemic intestines following H₂S therapy. Levels of these factors were not impacted significantly by genetic ablation of eNOS (*P < 0.05 vs. WT vehicle).