<p>iTRAQ-Based Quantitative Proteomic Analysis of Intestines in Murine Polymicrobial Sepsis with Hydrogen Gas Treatment</p>

Abstract

Objective: Sepsis-associated intestinal injury has a higher morbidity and mortality in patients with sepsis, but there is still no effective treatment. Our research team has proven that inhaling 2% hydrogen gas (H 2 ) can effectively improve sepsis and related organ damage, but the specific molecular mechanism of its role is not clear. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics analysis was used for studying the effect of H 2 on intestinal injury in sepsis. Methods: Male C57BL/6J mice were used to prepare a sepsis model by cecal ligation and puncture (CLP). The 7-day survival rates of mice were measured. 4-kd fluorescein isothiocyanate-conjugated Dextran (FITC-dextran) blood concentration measurement, combined with hematoxylin-eosinstain (HE) staining and Western blotting, was used to study the effect of H 2 on sepsis-related intestinal damage. iTRAQ-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used for studying the proteomics associated with H 2 for the treatment of intestinal injury. Results: H 2 can significantly improve the 7-day survival rates of sepsis mice. The load of blood and peritoneal lavage bacteria was increased, and H 2 treatment can significantly reduce it. CLP mice had significant intestinal damage, and inhalation of 2% hydrogen could significantly reduce this damage. All 4194 proteins were quantified, of which 199 differentially expressed proteins were associated with the positive effect of H 2 on sepsis. Functional enrichment analysis indicated that H 2 may reduce intestinal injury in septic mice through the effects of thyroid hormone synthesis and nitrogen metabolism signaling pathway. Western blot showed that H 2 was reduced by down-regulating the expressions of deleted in malignant brain tumors 1 protein (DMBT1), insulin receptor substrate 2 (IRS2), N-myc downregulated gene 1 (NDRG1) and serum amyloid A-1 protein (SAA1) intestinal damage in sepsis mice. Conclusion: A total of 199 differential proteins were related with H 2 in the intestinal protection of sepsis. H 2 -related differential proteins were notably enriched in the following signaling pathways, including thyroid hormone synthesis signaling pathway, nitrogen metabolism signaling pathways, digestion and absorption signaling pathways (vitamins, proteins and fats). H 2 reduced intestinal injury in septic mice by down-regulating the expressions of SAA1, NDRG1, DMBT1 and IRS2. Keywords: sepsis, intestinal injury, hydrogen gas, proteomics, isobaric tags for relative and absolute quantitation, iTRAQ

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