A hydrogen-sulfide derivative of mesalamine reduces the severity of intestinal and lung injury in necrotizing enterocolitis through endothelial nitric oxide synthase

Abstract

Purpose: Necrotizing enterocolitis (NEC) remains a devastating disease that affects preterm infants. Hydrogen sulfide (H2S) donors have been shown to reduce the severity of NEC, but the optimal compound has yet to be identified. We hypothesized that oral H2S-Mesalamine (ATB-429) would improve outcomes in experimental NEC, and its benefits would be dependent upon endothelial nitric oxide synthase (eNOS) pathways. Methods: NEC was induced in five-day-old wild-type (WT) and eNOS knockout (eNOSKO) pups by formula feeding and stress. Four groups were studied in both WT and eNOSKO mice: (1) breastfed controls, (2) NEC, (3) NEC + 50 mg/kg Mesalamine, (4) NEC + 130 mg/kg ATB-429. Mesalamine and ATB-429 doses were equimolar. Pups were monitored for sickness scores and perfusion to the gut was measured by Laser Doppler Imaging (LDI). After euthanasia, intestine and lung were H&E stained and scored for injury in a blind fashion. TLR4 expression was quantified by western blot and IL-6 expression by ELISA. P <0.05 was significant. Results: Both WT and eNOSKO breastfed controls underwent normal development and demonstrated milder intestinal and pulmonary injury compared to NEC groups. For the WT groups, ATB-429 significantly improved weight gain, reduced clinical sickness score, and improved perfusion compared to the NEC group. Additionally, WT ATB-429 pups had significantly milder intestinal and pulmonary histologic injury when compared to NEC. ATB-429 attenuated the increase in TLR4 and IL-6 expression in the intestine. When the experiment was repeated in eNOSKO pups, ATB-429 offered no benefit in weight gain, sickness scores, perfusion, intestinal injury, pulmonary injury, or decreasing intestinal inflammatory markers. Conclusions: An H2S-derivative of mesalamine improves outcomes in experimental NEC. Protective effects appear to be mediated through eNOS. Further research is warranted to explore whether ATB-429 may be an effective oral therapy to combat NEC.

Chondroitin sulfate supplementation improves clinical outcomes in a murine model of necrotizing enterocolitis

“…Given that the sulfated residues of CS are believed to participate and regulate various biological functions (Anower & Kimata, 2015), as well as previous data from our lab suggesting that multiple other sulfur-derived compounds exerted their beneficial properties through the eNOS pathway Hosfield et al, 2022;Jensen et al, 2017), we hypothesized that the effects of CS supplementation would also be mediated through this same pathway. The CS used for our experiments was bovine-cartilage-derived and mono-sulfated, which was as expected from the literature (Martel-Pelletier et al, 2015;Volpi, 2007).…”

“…Due to the young age and sexual immaturity of the pups at the time of the study (5–9 days old), both male and female pups were used collectively, and sex was not considered a factor during statistical analysis. The eNOS KO mice were validated in our laboratory by PCR in a prior study (Hosfield et al, 2022 ).…”

“…Necrotizing enterocolitis was induced in mice as previously described (Drucker, Jensen, Ferkowicz, & Markel, 2018 ; Drucker, Jensen, Te Winkel, et al, 2018 ; Hosfield et al, 2022 ; Zani et al, 2008 ). In brief, experimental pups were separated from their mothers on postnatal day 5 (p5) and placed in satellite housing in a temperature and humidity‐controlled incubator (32°C and 40% humidity).…”

“…It has been demonstrated that chondroitin sulfate has increased H 2 S in mice endogenously by increasing the relative abundance of sulfate reducing bacteria in the gut (Chen et al, 2022 ; Pichette et al, 2017 ), further providing support for the importance of sulfur‐residue signaling. Additionally, previous studies from our laboratory have shown that multiple sulfur donor compounds exerted their beneficial properties through the endothelial nitric oxide synthase (eNOS) pathway by improving mesenteric perfusion and decreasing intestinal injury (Drucker, Jensen, Ferkowicz, & Markel, 2018 ; Hosfield et al, 2022 ). Endothelial cells in the intestine express endothelial nitric oxide synthase (eNOS).…”

“…Endothelial cells in the intestine express endothelial nitric oxide synthase (eNOS). Sulfur donors, including NaHS and GYY4137 are theorized to interact with this enzyme to promote eNOS dimerization and increase nitric oxide production which acts to improve mesenteric perfusion and prevent intestinal injury (Drucker, Jensen, Ferkowicz, & Markel, 2018 ; Hosfield et al, 2022 ; Jensen et al, 2017 ).…”