Hydrogen alleviated cognitive impairment and blood‒brain barrier damage in sepsis-associated encephalopathy by regulating ABC efflux transporters in a PPARα-dependent manner

Hydrogen (H2) can protect against blood‒brain barrier (BBB) damage in sepsis-associated encephalopathy (SAE), but the mechanism is still unclear. We examined whether it is related to PPARα and its regulatory targets, ABC efflux transporters. After injection with DMSO/GW6471 (a PPARα inhibitor), the mice subjected to sham/caecal ligation and puncture (CLP) surgery were treated with H2 for 60 min postoperation. Additionally, bEnd.3 cells were grown in DMSO/GW6471-containing or saline medium with LPS. In addition to the survival rates, cognitive function was assessed using the Y-maze and fear conditioning tests. Brain tissues were stained with TUNEL and Nissl staining. Additionally, inflammatory mediators (TNF-α, IL-6, HMGB1, and IL-1β) were evaluated with ELISA, and PPARα, ZO-1, occludin, VE-cadherin, P-gp, BCRP and MRP2 were detected using Western blotting. BBB destruction was assessed by brain water content and Evans blue (EB) extravasation. Finally, we found that H2 improved survival rates and brain dysfunction and decreased inflammatory cytokines. Furthermore, H2 decreased water content in the brain and EB extravasation and increased ZO-1, occludin, VE-cadherin and ABC efflux transporters regulated by PPARα. Thus, we concluded that H2 decreases BBB permeability to protect against brain dysfunction in sepsis; this effect is mediated by PPARα and its regulation of ABC efflux transporters.

APOA2: New Target for Molecular Hydrogen Therapy in Sepsis-Related Lung Injury Based on Proteomic and Genomic Analysis

Target biomarkers for H2 at both the protein and genome levels are still unclear. In this study, quantitative proteomics acquired from a mouse model were first analyzed. At the same time, functional pathway analysis helped identify functional pathways at the protein level. Then, bioinformatics on mRNA sequencing data were conducted between sepsis and normal mouse models. Differential expressional genes with the closest relationship to disease status and development were identified through module correlation analysis. Then, common biomarkers in proteomics and transcriptomics were extracted as target biomarkers. Through analyzing expression quantitative trait locus (eQTL) and genome-wide association studies (GWAS), colocalization analysis on Apoa2 and sepsis phenotype was conducted by summary-data-based Mendelian randomization (SMR). Then, two-sample and drug-target, syndrome Mendelian randomization (MR) analyses were all conducted using the Twosample R package. For protein level, protein quantitative trait loci (pQTLs) of the target biomarker were also included in MR. Animal experiments helped validate these results. As a result, Apoa2 protein or mRNA was identified as a target biomarker for H2 with a protective, causal relationship with sepsis. HDL and type 2 diabetes were proven to possess causal relationships with sepsis. The agitation and inhibition of Apoa2 were indicated to influence sepsis and related syndromes. In conclusion, we first proposed Apoa2 as a target for H2 treatment.

Effect of Hydrogen on AM Pyroptosis Induced by Severe Burns in Rats

Background: Hydrogen has anti-inflammatory and antioxidant effects and is beneficial to multiple organs. However, its effect on alveolar macrophage (AM) pyroptosis induced by burns is still unclear. The purpose of this research was to study the possible positive effects of hydrogen on burn-induced lung injury and the effects of hydrogen on AM pyroptosis during acute lung injury (ALI) induced by burns. Methods: In this study, histological changes in rat lungs in vivo were evaluated by micro-CT, and histological changes in isolated lungs were evaluated by hematoxylin and eosin (HE) staining. The expressions of leucine rich repeat (LRR) and pyrin domain (PYD) containing protein 3 (NLRP3), caspase-1 and Gasdermin-D (GSDMD) were analyzed by Western blotting. The expression of GSDMD was measured by immunofluorescence to evaluate the levels of lung inflammation and pyroptosis. The level of inflammation was assessed by enzyme-linked immunosorbent assay (ELISA). Pyroptosis was observed by transmission electron microscopy. Results: We observed that severe burn resulted in increased IL-1β and IL-18, overexpression of NLRP3 and caspase-1 proteins, and pyroptosis in rat lung tissues, as demonstrated by GSDMD overexpression and electron microscopy of AMs. We also observed that hydrogen treatment partially reversed the increase in lung tissue density and reduced pulmonary inflammation. Moreover, hydrogen reduced the HE pathological injury score in the lung tissues of severely burned rats. Hydrogen treatment significantly reduced the contents of IL-1β and IL-18 in the lung tissues and decreased the expression of NLRP3, caspase-1 and GSDMD proteins compared with the burn group. Transmission electron microscopy results also showed that the number of AM membrane pores was significantly reduced in the hydrogen treatment group. Conclusions: The results of this study suggest that hydrogen may protect against ALI induced by burn injury by inhibiting pyroptosis of macrophages via NLRP3.

PPARα contributes to the therapeutic effect of hydrogen gas against sepsis-associated encephalopathy with the regulation to the CREB-BDNF signaling pathway and hippocampal neuron plasticity-related gene expression

Sepsis-associated encephalopathy (SAE), a fatal complication of sepsis, contributes to cognitive impairment, high morbidity, and mortality. The molecular mechanism of hydrogen (H2) administration, as a promising strategy for the treatment of SAE, is still unclear. Peroxisome proliferator-activated receptor α (PPARα) is essential for alleviating symptoms and complications of SAE. However, little is known about the role of PPARα in SAE. This study was designed to evaluate the expression of PPARα in SAE and determine whether H2 can alleviate SAE through regulation of the cAMP response element-binding protein (CREB)-brain-derived neurotrophic factor (BDNF) signaling pathway and its downstream proteins via PPARα. After the injection of GW6471 (the PPARα inhibitor) or GW7647 (the PPARα agonist) or saline, C57BL/6 J mice were subjected to cecal ligation and puncture (CLP) or sham operation, then treated with 2% H2 by inhalation for 1 h after the operation. The 7-day survival rate was recorded, and the Y-maze test was used to assess cognitive function. Apoptotic cells were observed by TUNEL staining, and brain tissues were collected for pathological analysis by H&E staining. In addition, the levels of pro-inflammatory and anti-inflammatory cytokines (TNF-α, IL-6, IL-18, HMGB1, and IL-1β) were measured by ELISA and the expression of PPARα, CREB, BDNF and other neurotrophins, postsynaptic density protein of 95 kDa (PSD95) by Western blot. The relationship between PPARα and the CREB-BDNF signaling pathway was explored by coimmunoprecipitation (CO-IP). The results showed the expression of PPARα was decreased in SAE mice and that activation of PPARα in septic mice improved the survival rate and alleviated cognitive dysfunction. Furthermore, PPARα may have exerted anti-inflammatory and anti-apoptotic effects in septic mice. In addition, the GW6471 downregulated the expression of CREB, BDNF and other neurotrophins in SAE mice treated with H2. The expression of PSD95 was also downregulated and upregulated following the expression of PPARα. These results illustrated that H2 alleviates sepsis-induced brain injury in mice through the regulation of neurotrophins and hippocampal plasticity-related genes via PPARα by activating the CREB-BDNF signaling pathway.

Molecular hydrogen attenuates sepsis-induced neuroinflammation through regulation of microglia polarization through an mTOR-autophagy-dependent pathway

Sepsis-associated encephalopathy (SAE) is the cognitive impairment resulting from sepsis and is associated with increased morbidity and mortality. Hydrogen has emerged as a promising therapeutic agent to alleviate SAE. The mechanism, however, remains unclear. This research aimed to determine whether hydrogen alleviates SAE by regulating microglia polarization and whether it is mediated by the mammalian target of rapamycin (mTOR)-autophagy pathway. Septic models were established by cecal ligation and puncture (CLP) performed on mice. The Morris Water Maze was used to evaluate cognitive function. M1/M2 microglia polarization was assessed by immunofluorescence. Inflammatory cytokines were determined by ELISA. Septic cell models were established using BV-2 cells incubated with 1 μg/ml lipopolysaccharide (LPS). M1/M2 microglia polarization was assessed by flow cytometry. Inflammatory cytokines from culture medium supernatant were determined by ELISA, and associated protein expression levels of mTOR-autophagy pathway were assessed by Western blot. Hydrogen inhalation attenuated sepsis-induced cognitive impairment with improved escape latency, time spent in the target platform quadrant and number of times crossing the target platform. In both animal and cell research, hydrogen reduced TNF-α, IL-6 and HMGB1 levels and M1 polarization, but increased IL-10 and TGF-β levels and M2 polarization. Hydrogen treatment decreased the ratio of p-mTOR/mTOR and the expression of p62 and increased the ratio of p-AMPK/AMPK, LC3II/LC3I and the expression of TREM-2 and Beclin-1 in LPS-treated BV-2 cells. MHY1485, an mTOR activator, abolished the protective effects of hydrogen in vitro. Taken together, these results demonstrated that hydrogen attenuated sepsis-induced neuroinflammation by modulating microglia polarization, which was mediated by the mTOR-autophagy signaling pathway.

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

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 (H2) 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 H2 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 H2 on sepsis-related intestinal damage. iTRAQ-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used for studying the proteomics associated with H2 for the treatment of intestinal injury. Results: H2 can significantly improve the 7-day survival rates of sepsis mice. The load of blood and peritoneal lavage bacteria was increased, and H2 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 H2 on sepsis. Functional enrichment analysis indicated that H2 may reduce intestinal injury in septic mice through the effects of thyroid hormone synthesis and nitrogen metabolism signaling pathway. Western blot showed that H2 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 H2 in the intestinal protection of sepsis. H2-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). H2 reduced intestinal injury in septic mice by down-regulating the expressions of SAA1, NDRG1, DMBT1 and IRS2.

Molecular hydrogen alleviates brain injury and cognitive impairment in a chronic sequelae model of murine polymicrobial sepsis

Sepsis-related encephalopathy (SAE), which causes a series of brain injuries and long-term, potentially irreversible cognitive dysfunction, is closely associated with increased morbidity and mortality. Hydrogen (H2) is a new type of medical gas molecule that has been widely used in the treatment of various diseases in recent years. The aim of the present study was to explore the protective effects of H2 inhalation on brain injury and long-term cognitive impairment in an improved chronic septic mouse model. Male C57BL/6J mice were randomized into four groups: Control, Control + H2, SAE and SAE + H2. The SAE and Control models were established by intraperitoneal injection of human stool suspension or saline in mice. H2 (2%) was inhaled for 60 min at 1 h and 6 h after SAE or Control treatment. The survival rates were recorded for 14 days (days 1-14) and the Morris Water Maze was performed for 7 days (days 8-14). To assess the severity of the brain injury, hematoxylin and eosin staining, Nissl staining, Evans blue (EB) extravasation and the wet/dry weight ratio of brain tissue were detected 24 h after SAE or Control treatment. In addition, inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin 6 (IL-6), high-mobility group box 1 (HMGB1), as well as the protein levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), zonula occludens-1 (ZO-1) and Occludin, were measured 6, 12 and 24 h after SAE or Control treatment. The results showed that H2 treatment increased survival rates, mitigated cognitive impairment, reduced hippocampal histological damage, decreased EB and water content, and decreased the levels of TNF-α, IL-6, HMGB1, Nrf2, HO-1, ZO-1 and Occludin, as compared with the SAE group. These data revealed that 2% H2 could suppress brain damage and improve cognitive function in septic mice by inhibiting oxidative stress, inflammatory response and the sepsis-induced blood-brain barrier (BBB) disruption.

Drinking Hydrogen-Rich Water Alleviates Chemotherapy-Induced Neuropathic Pain Through the Regulation of Gut Microbiota

Introduction: Chemotherapy-induced neuropathic pain (CINP) is one of the most common complications of chemotherapeutic drugs which limits the dose and duration of potentially life-saving anticancer treatment and compromises the quality of life of patients. Our previous studies have reported that molecular hydrogen (H2) can be used to prevent and treat various diseases. But the underlying mechanism remains unclear. The aim of the present study was to explore the effects of hydrogen-rich water on gut microbiota in CINP. Methods: All C57BL/6J mice were divided into 4 groups: The group fed with normal drinking water and injected with saline (H2O + Saline), the group fed with normal drinking water and injected with oxaliplatin (H2O + OXA), the group fed with hydrogen-rich water and injected with saline (HW + Saline), and the group fed with hydrogen-rich water and injected with oxaliplatin (HW + OXA). The mechanical paw withdrawal threshold of the mice was tested on days 0, 5, 10, 15 and 20 after hydrogen-rich water treatment. On day 20, feces of mice from different groups were collected for microbial community diversity and structure analysis. The levels of inflammatory cytokines (TNF-α and IL-6), oxidative stress factors (OH- and ONOO-), lipopolysaccharide (LPS) and Toll-like receptor 4 (TLR4) were detected in dorsal root ganglia (DRG), L4-6 spinal cord segments and serum by enzyme-linked immunosorbent assay. The expression of TLR4 in DRG and spinal cords was determined by Western blot. Results: The results illustrated that hydrogen-rich water could alleviate oxaliplatin-induced hyperalgesia, reduce the microbial diversity and alter the structure of gut microbiota, reverse the imbalance of inflammatory cytokines and oxidative stress, and decrease the expression of LPS and TLR4. Conclusion: Hydrogen-rich water may alleviate CINP by affecting the diversity and structure of the gut microbiota, and then the LPS-TLR4 pathway, which provides a direction for further research.