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.
Introduction: In our experiments, male wild-type mice were randomly divided into four groups: the sham, SAE, SAE + 2% hydrogen gas inhalation (H2 ), and SAE + hydrogen-rich water (HW) groups. The feces of the mice were collected for 16 S rDNA analysis 24 h after the models were established, and the serum and brain tissue of the mice were collected for nontargeted metabolomics analysis. Aim: Destruction of the intestinal microbiota is a risk factor for sepsis and subsequent organ dysfunction, and up to 70% of severely ill patients with sepsis exhibit varying degrees of sepsis-associated encephalopathy (SAE). The pathogenesis of SAE remains unclear. We aimed to explore the changes in gut microbiota in SAE and the regulatory mechanism of molecular hydrogen. Results: Molecular hydrogen treatment significantly improved the functional outcome of SAE and downregulated inflammatory reactions in both the brain and the gut. In addition, molecular hydrogen treatment improved gut microbiota dysbiosis and partially amended metabolic disorder after SAE. Conclusions: Molecular hydrogen treatment promotes functional outcomes after SAE in mice, which may be attributable to increasing beneficial bacteria, repressing harmful bacteria, and metabolic disorder, and reducing inflammation. Keywords: gut microbiota; hydrogen gas (H2); hydrogen-rich water (HW); molecular hydrogen treatment; sepsis-associated encephalopathy (SAE).
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.
Background: Multiple organ failure (MOF) is the main cause of early death in septic shock. Lungs are among the organs that are affected in MOF, resulting in acute lung injury. Inflammation is an important factor that causes immune cell dysfunction in the pathogenesis of sepsis. Autophagy is involved in the process of inflammation and also occurs in response to cell and tissue injury in several diseases. We previously demonstrated that hydrogen alleviated the inflammation-induced cell injury and organ damage in septic mice. Aim: The focus of the present study was to elucidate whether mitophagy mediates the inflammatory response or oxidative injury in sepsis in vitro and in vivo. Furthermore, we evaluated the role of mitophagy in the protective effects of hydrogen against cell injury or organ dysfunction in sepsis. Method: RAW 264.7 macrophages induced by lipopolysaccharide (LPS) were used as an in vitro model for inflammation, and cecal ligation and puncture (CLP)-induced acute lung injury mice were used as an in vivo model for sepsis. The key protein associated with mitophagy, PTEN-induced putative kinase 1 (PINK1), was knocked down by PINK1 shRNA transfection in RAW 264.7 macrophages or mice. Results: Hydrogen ameliorated cell injury and enhanced mitophagy in macrophages stimulated by LPS. PINK1 was required for the mitigation of the cell impairment in LPS-stimulated macrophages by hydrogen treatment. PINK1 knockdown abrogated the beneficial effects of hydrogen on mitophagy in LPS-stimulated macrophages. Hydrogen inhibited acute lung injury in CLP mice via activation of PINK1-mediated mitophagy. Conclusion: These results suggest that PINK1-mediated mitophagy plays a key role in the protective effects of hydrogen against cell injury in LPS-induced inflammation and CLP-induced acute lung injury.
Objective: Sepsis-associated encephalopathy (SAE) is characterized by diffuse cerebral and central nervous system (CNS) dysfunction. Microglia play a vital role in protecting the brain from neuronal damage, which is closely related to inflammatory responses. The Nrf2 signaling pathway has an impact on microglial and neuronal injury. Here, we mainly explored the molecular mechanism by which H2 regulates neuroinflammation in SAE and the role of Nrf2 in this process. Methods: An in vivo model of SAE was generated by cecal ligation and puncture (CLP). Primary microglia and neurons were cultured to establish an in vitro model. Microglia, neurons and brain tissue were obtained to detect Nrf2 expression, inflammation, cell injury, apoptosis, and microglial polarization. Escape latency, the number of platform crossings and the time spent in the target quadrant were measured to assess cognitive function. Results: H2 attenuated microglial polarization from the M1 to the M2 phenotype, cytokine release and TLR/NF-κb activation and protected neurons from LPS-activated microglia-induced injury via the Nrf2 pathway. SAE activated Nrf2 expression, and H2 further improved Nrf2 expression in SAE mice. H2 alleviated microglial polarization from the M1 to the M2 phenotype and cytokine release in the cerebral cortex and improved neuronal injury or cognitive dysfunction in SAE mice and wild-type mice but not in Nrf2-/- mice. Conclusion: H2 exerts antineuroinflammatory effects associated with TLR4/NF-κB signaling activation and neuroprotective effects by inhibiting the excessive release of proinflammatory cytokines, neuronal loss and apoptosis in vitro and in vivo through the Nrf2 pathway.