The intestine plays an important role in the pathogenesis of sepsis. Hydrogen gas (H2), which has anti-oxidative, anti-inflammatory, and anti-apoptotic effects, can be effectively used to treat septic mice. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive master switch that regulates the expression of antioxidant and protective enzymes. This study investigated the effects of 2% H2 on intestinal injuries and the underlying mechanisms in a mouse model of severe sepsis. Male Nrf2 knockout mice (Nrf2-KO) and wild-type (WT) mice were randomized into four groups: sham, sham+H2, cecal ligation and puncture (CLP), and CLP+H2. The survival rate was observed and recorded within 7 days, and pro-inflammatory cytokines (TNF-α, IL-6, HMGB1), anti-inflammatory cytokine (IL-10), antioxidant enzymes (superoxide dismutase, and catalase ), and oxidative products (MDA, 8-iso-PGF2α) were detected in the serum and intestine using an enzyme-linked immunosorbent assay. In addition, the protein and mRNA levels of heme oxygenase-1 (HO-1) and high mobility group box 1 (HMGB1) were measured by Western blotting and quantitative PCR, respectively. Immunofluorescence and immunohistochemistry were used to measure HMGB1 and HO-1 release into the intestine, respectively. The results showed that therapy with 2% H2 increased the survival rate, alleviated the injuries caused by oxidative stress and inflammation, reduced HMGB1 levels but increased HO-1 levels in WT septic mice, but not in Nrf2-KO mice. These data demonstrate that 2% H2 inhalation may be a promising therapeutic strategy for intestinal injuries caused by severe sepsis through the regulation of HO-1 and HMGB1 release. In addition, Nrf2 plays a key role in the protective effects of H2 against intestinal damage in this disease.
Primary damage or dysfunction of the nervous system may cause or initiate neuropathic pain. However, it has been difficult to establish an effective treatment for neuropathic pain, as the mechanisms responsible for its pathology remain largely unknown. Autophagy is closely associated with the pathological process of neurodegenerative diseases, neuropathic injury and cancer, among others. The aim of the present study was to examine the changes in the autophagy-lysosomal pathway and discuss the effects of autophagy on allodynia, hyperalgesia and astrocyte activation in neuropathic pain. A neuropathic pain model was induced by chronic constriction injury (CCI) in rats. Inducers and inhibitors of autophagy and lysosomes were used to assess autophagy, allodynia, hyperalgesia and astrocyte activity. Neuropathic pain was found to induce an increase in the levels of the autophagy-related proteins, LC3II and Beclin 1 and, and in those of the lysosomal proteins, lysosomal-associated membrane protein type 2 (LAMP2) and Ras-related protein Rab-7a (RAB7), whereas p62 levels were found to decrease from day 1 to 14 following CCI. The autophagy inducer, rapamycin, further increased the LC3II, Beclin 1, lysosomal-associated membrane protein 2 (LAMP2) and Ras-related protein Rab-7a (RAB7) expression levels, and decreased the p62 expression levels, which were accompanied by alleviation of allodynia, hyperalgesia and astrocyte activation in the rats subjected to CCI; the autophagy inhibitor, 3-methyladenine, reversed these effects. The use of the lysosomal inhibitors, bafilomycin and chloroquine, resulted in the accumulation of LC3II and Beclin 1, a decrease in the levels of LAMP2 and RAB7, and the exacerbation of allodynia, hyperalgesia and astrocyte activation in rats with neuropathic pain. On the whole, the findings of this study indicate that neuropathic pain activates autophagy, which alleviates mechanical and thermal hyperalgesia and suppresses astrocyte activity. Therefore, neuropathic pain induced by CCI in rats appears to be mediated via the autophagy-lysosomal pathway.
Sepsis-associated acute lung injury (ALI), which carries a high morbidity and mortality in patients, has no effective therapeutic strategies to date. Our group has already reported that hydrogen gas (H2) exerts a protective effect against sepsis in mice. However, the molecular mechanisms underlying H2 treatment are not fully understood. This study investigated the effects of H2 on lung injuries in septic mice through the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis. Male ICR mice used in this study were subjected to cecal ligation and puncture (CLP) or sham operation. And 2% H2 was inhaled for 1 h beginning at 1 and 6 h after sham or CLP operation. The iTRAQ-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was preformed to investigate lung proteomics. Sepsis-challenged animals had decreased survival rate, as well as had increased bacterial burden in blood, peritoneal lavage, and lung sample, which were significantly ameliorated by H2 treatment. Moreover, a total of 4,472 proteins were quantified, and 192 differentially expressed proteins were related to the protective mechanism of H2 against sepsis. Functional enrichment analysis showed that H2-related differential proteins could be related to muscle contraction, oxygen transport, protein synthesis, collagen barrier membranes, cell adhesion, and coagulation function. These proteins were significantly enriched in four signaling pathways, and two of which are associated with coagulation. In addition, H2 alleviates ALI in septic mice through downregulating the expression of Sema 7A, OTULIN, and MAP3K1 as well as upregulating the expression of Transferrin. Thus, our findings provide an insight into the mechanism of H2 treatment in sepsis by proteomic approach, which may be helpful to the clinic application of H2 in patients with sepsis.
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.
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