Hydrogen Sulfide Protects Against Necrotizing Enterocolitis Through a Critical Residue on Endothelial Nitric Oxide Synthase

Background: Necrotizing enterocolitis (NEC) is a devastating intra-abdominal disease that occurs in premature infants, resulting in significant morbidity and mortality. Hydrogen Sulfide (H2S) is a gasotransmitter that has been shown to protect against NEC in a murine model, however the mechanism is not fully known. In vitro studies have suggested that H2S modifies endothelial nitric oxide synthase (eNOS) at a particular cysteine residue (Cys440) through S-sulfhydration, leading to increased nitric oxide production and vasodilation. Our hypothesis is that when Cys440 on eNOS is mutated, the beneficial effects of GYY4137 (a long acting H2S donor) therapy will be abated.

Methods: We obtained a novel mouse with a point mutation at the 440 cysteine residue on eNOS. After IACUC approval (#19122), NEC was induced in wild type (WT) and point mutation (Cys440G) mice. On post-natal day 5 (p5), pups were separated from their mothers and placed in satellite housing. The pups were gavage fed hyperosmolar formula containing LPS and stressed with intermittent hypoxia and hypothermia. The treatment groups received daily intraperitoneal (IP) injections of GYY4137 (50mg/kg) while the control groups did not. Weight gain, clinical sickness scores, and intestinal perfusion using laser doppler imaging were assessed. On p9, the pups were euthanized and intestinal macroscopic appearance was scored. Ordinal data is expressed as median and inter-quartile range (IQR) and continuous data expressed as mean and standard error of the mean. Data was tested for normality and compared using t-tests or Mann-Whitney with p<0.05 considered significant.

Results: Weight gain was similar between all four groups (p=0.2279). Compared to their respective control group, clinical scores were significantly improved in the WT group treated with IP GYY [WT NEC = 3(IQR=2-3); WT NEC + GYY= 1 (IQR=1-2), p=0.0029] but there was no difference seen in the Cys440G group [Cys440G NEC = 3 (IQR=3-4); Cys440G NEC + GYY = 3 (IQR=2.25-3.75), p=0.45]. Median intestinal macroscopic score was 2 (IQR=2-3) in the WT control group and 1 (IQR 0.25-1) in the WT + GYY group (p=0.0055). In the Cys440G mice, there was no improvement in intestinal appearance between the control vs. GYY groups [Cys440G NEC = 2 (IQR= 1-3); Cys440G NEC + GYY = 2 (IQR=1.25-2.75), p=0.99]. Intestinal perfusion was significantly improved by IP GYY in the WT group (p=0.0016) but not in the Cys440G group (0.08).

Conclusion: Cysteine 440 is a critical residue on eNOS and is an essential locus for H2S S-sulfhydration. Without this residue, the H2S donor GYY4137 is unable to provide protection against necrotizing enterocolitis.