Inhibiting hydrogen sulfide production in umbilical stem cells reduces their protective effects during experimental necrotizing enterocolitis

Abstract

Introduction: Umbilical mesenchymal stem cells (USC) have been shown to reduce illness in animal models of necrotizing enterocolitis (NEC), possibly through the paracrine release of hydrogen sulfide (H₂S). We hypothesized that animals treated with USCs with inhibited H₂S synthesis would exhibit more severe disease.

Methods: NEC was induced in five-day-old mouse pups by formula feeding and hypoxic and hypothermic stress. Experimental groups received intraperitoneal injection of either saline vehicle or 80,000cells/gram of one of the following cell types: USC, USCs with negative-control siRNA, or USCs with targeted siRNA inhibition of the H₂S-producing enzymes. Pups were monitored by clinical assessment and after euthanasia, intestine and lung histologic injury were scored. Tissue was homogenized, and concentrations of IL-6, IL-10, and VEGF were determined by ELISA. For statistical analysis, p<0.05 was considered significant.

Results: Animals treated with negative-control siRNA USCs were significantly improved compared to vehicle. Clinical sickness scores as well as intestinal and lung histologic injury scores in the targeted siRNA groups were significantly worse when compared to the negative-control siRNA group. IL-6, IL-10, and VEGF had varying patterns of expression in the different groups.

Conclusion: Inhibition of H₂S production in USCs reduces the beneficial effects of these cells during therapy in experimental NEC.

Level of evidence: Animal studies are typically described as "foundational evidence" without a true level assigned.

Type of study: Animal Study.

Keywords: Animal model; Hydrogen sulfide; Intestine; Necrotizing enterocolitis; Umbilical stromal cell.

Figure 1:

A- Representative RT-PCR gel demonstrating >50% decrease after siRNA treatment. Each panel demonstrates an amplification for the specified gene, and for each one, from left to right the lanes are as follows: USCs (no transfection), negative control siRNA cells, CBS siRNA, CTH siRNA, MPST siRNA. B- H₂S production over 24 hours in USCs in normoxia and hypoxia (error bars are smaller than data points). *: p<0.0001 at the time point. C- H₂S production expressed as a fold change from baseline over 24 hours in hypoxic conditions. *: p<0.05 vs. (−) control siRNA

Figure 2:

A- Clinical sickness scores for each group. Higher score indicates a sicker animal. B-Macroscopic injury score for each group. Higher score indicates more severe injury. *: p<0.05 vs. vehicle, #: p<0.05 vs. (−) control siRNA.

Figure 3:

A- Intestinal histologic injury scores for each group. Higher score indicates more significant injury. *: p<0.05 vs. vehicle, #: p<0.05 vs. (−) control siRNA. B- Incidence of each grade of injury corresponding with NEC. Grade 3 and 4 indicate severe NEC. C- Representative images of histologic appearance for each group.

Figure 4:

A- Lung histologic injury scores for each group. Higher score indicates more severe injury. *: p<0.05 vs. vehicle, #: p<0.05 vs. (−) control siRNA . B- Representative images of histologic appearance for each group.