Induction of MTHFD2 in Macrophages Inhibits Reactive Oxygen Species-mediated NF-κB Activation and Protects against Inflammatory Responses.

The one-carbon metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is critical for cancer cell proliferation and immune cell phenotypes, but whether it can contribute to macrophage inflammatory responses remains unclear. In this study, we show that MTHFD2 was upregulated by LPS in murine macrophages upon activation of the TLR4-MyD88-IKKα/ß-NF-κB signaling pathway. MTHFD2 significantly attenuated LPS-induced macrophage proinflammatory cytokine production through its enzymatic activity. Notably, ablation of myeloid MTHFD2 rendered mice more sensitive to septic shock and CCl4-induced acute hepatitis. Mechanistically, MTHFD2 restrained IKKα/ß-NF-κB activation and macrophage inflammatory phenotype by scavenging reactive oxygen species through the generation of NADPH. Our study reveals MTHFD2 as a "self-control" mechanism in macrophage-mediated inflammatory responses.

NHH promotes Sepsis-associated Encephalopathy with the expression of AQP4 in astrocytes through the gut-brain Axis.

Sepsis-associated encephalopathy (SAE) is a significant cause of mortality in patients with sepsis. Despite extensive research, its exact cause remains unclear. Our previous research indicated a relationship between non-hepatic hyperammonemia (NHH) and SAE. This study aimed to investigate the relationship between NHH and SAE and the potential mechanisms causing cognitive impairment. In the in vivo experimental results, there were no significant abnormalities in the livers of mice with moderate cecal ligation and perforation (CLP); however, ammonia levels were elevated in the hippocampal tissue and serum. The ELISA study suggest that fecal microbiota transplantation in CLP mice can reduce ammonia levels. Reduction in ammonia levels improved cognitive dysfunction and neurological impairment in CLP mice through behavioral, neuroimaging, and molecular biology studies. Further studies have shown that ammonia enters the brain to regulate the expression of aquaporins-4 (AQP4) in astrocytes, which may be the mechanism underlying brain dysfunction in CLP mice. The results of the in vitro experiments showed that ammonia up-regulated AQP4 expression in astrocytes, resulting in astrocyte damage. The results of this study suggest that ammonia up-regulates astrocyte AQP4 expression through the gut-brain axis, which may be a potential mechanism for the occurrence of SAE.

Molecular hydrogen promotes retinal vascular regeneration and attenuates neovascularization and neuroglial dysfunction in oxygen-induced retinopathy mice.

BACKGROUND: Retinopathy of Prematurity (ROP) is a proliferative retinal vascular disease occurring in the retina of premature infants and is the main cause of childhood blindness. Nowadays anti-VEGF and retinal photocoagulation are mainstream treatments for ROP, but they develop a variety of complications. Hydrogen (H2) is widely considered as a useful neuroprotective and antioxidative therapeutic method for hypoxic-ischemic disease without toxic effects. However, whether H2 provides physiological angiogenesis promotion, neovascularization suppression and glial protection in the progression of ROP is largely unknown.This study aims to investigate the effects of H2 on retinal angiogenesis, neovascularization and neuroglial dysfunction in the retinas of oxygen-induced retinopathy (OIR) mice. METHODS: In this study, mice that were seven days old and either wild-type (WT) or Nrf2-deficient (Nrf2-/-) were exposed to 75% oxygen for 5 days and then returned to normal air conditions. Different stages of hydrogen gas (H2) inhalation were administered. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. To count the number of neovascularization endothelial nuclei, routine HE staining of retinal sections was conducted. Immunohistochemistry was performed using DyLight 594 labeled GSL I-isolectin B4 (IB4), as well as primary antibodies against proliferating cell nuclear antigen (PCNA), glial fibrillary acidic protein (GFAP), and Iba-1. Western blots were used to measure the expression of NF-E2-related factor 2 (Nrf2), vascular endothelial growth factor (VEGF), Notch1, Dll4, and HIF-1α. Additionally, the expression of target genes such as NQO1, HO-1, Notch1, Hey1, Hey2, and Dll4 was measured. Human umbilical vein endothelial cells (HUVECs) treated with H2 under hypoxia were used as an in vitro model. RT-PCR was used to evaluate the mRNA expression of Nrf2, Notch/Dll4, and the target genes. The expression of reactive oxygen species (ROS) was observed using immunofluorescence staining. RESULTS: Our results indicate that 3-4% H2 does not disturb retinal physiological angiogenesis, but ameliorates vaso-obliteration and neovascularization in OIR mice. Moreover, H2 prevents the decreased density and reverses the morphologic and functional changes in retinal astrocytes caused by oxygen-induced injury. In addition, H2 inhalation reduces microglial activation, especially in the area of neovascularization in OIR mice. H2 plays a protective role in vascular regeneration by promoting Nrf2 activation and suppressing the Dll4-induced Notch signaling pathway in vivo. Also, H2 promotes the proliferation of HUVECs under hypoxia by negatively regulating the Dll4/Notch pathway and reducing ROS levels through Nrf2 pathway aligning with our findings in vivo.Moreover, the retinal oxygen-sensing mechanisms (HIF-1α/VEGF) are also involved in hydrogen-mediated retinal revascularization and neovascularization suppression. CONCLUSIONS: Collectively, our results indicate that H2 could be a promising therapeutic agent for POR treatment and that its beneficial effect in human ROP might involve the activation of the Nrf2-Notch axis as well as HIF-1α/VEGF pathways.

Molecular hydrogen attenuates sepsis-induced cardiomyopathy in mice by promoting autophagy.

BACKGROUND: Approximately 40 to 60% of patients with sepsis develop sepsis-induced cardiomyopathy (SIC), which is associated with a substantial increase in mortality. We have found that molecular hydrogen (H2) inhalation improved the survival rate and cardiac injury in septic mice. However, the mechanism remains unclear. This study aimed to explore the regulatory mechanism by which hydrogen modulates autophagy and its role in hydrogen protection of SIC. METHODS: Cecal ligation and puncture (CLP) was used to induce sepsis in adult C57BL/6J male mice. The mice were randomly divided into 4 groups: Sham, Sham + 2% hydrogen inhalation (H2), CLP, and CLP + H2 group. The 7-day survival rate was recorded. Myocardial pathological scores were calculated. Myocardial troponin I (cTnI) levels in serum were detected, and the levels of autophagy- and mitophagy-related proteins in myocardial tissue were measured. Another four groups of mice were also studied: CLP, CLP + Bafilomycin A1 (BafA1), CLP + H2, and CLP + H2 + BafA1 group. Mice in the BafA1 group received an intraperitoneal injection of the autophagy inhibitor BafA1 1 mg/kg 1 h after operation. The detection indicators remained the same as before. RESULTS: The survival rate of septic mice treated with H2 was significantly improved, myocardial tissue inflammation was improved, serum cTnI level was decreased, autophagy flux was increased, and mitophagy protein content was decreased (P < 0.05). Compared to the CLP + H2 group, the CLP + H2 + BafA1 group showed a decrease in autophagy level and 7-day survival rate, an increase in myocardial tissue injury and cTnI level, which reversed the protective effect of hydrogen (P < 0.05). CONCLUSION: Hydrogen exerts protective effect against SIC, which may be achieved through the promotion of autophagy and mitophagy.

Spinal microRNA-134-5p targets glutamate receptor ionotropic kainate 3 to modulate opioid induced hyperalgesia in mice.

Background: Fentanyl and its analogs are extensively used for pain relief. However, their paradoxically pronociceptive effects often lead to increased opioids consumption and risk of chronic pain. Compared to other synthetic opioids, remifentanil has been strongly linked to acute opioid hyperalgesia after exposure [remifentanil-induced hyperalgesia (RIH)]. The epigenetic regulation of microRNAs (miRNAs) on targeted mRNAs has emerged as an important pathogenesis in pain. The current research aimed at exploring the significance and contributions of miR-134-5p to the development of RIH. Methods: Both the antinociceptive and pronociceptive effects of two commonly used opioids were assessed, and miRNA expression profiles in the spinal dorsal horn (SDH) of mice acutely exposed to remifentanil and remifentanil equianalgesic dose (RED) sufentanil were screened. Next, the candidate miRNA level, cellular distribution, and function were examined by qPCR, fluorescent in situ hybridization (FISH) and Argonaute-2 immunoprecipitation. Furthermore, bioinformatics analysis, luciferase assays, miRNA overexpression, behavioral tests, golgi staining, electron microscopy, whole-cell patch-clamp recording, and immunoblotting were employed to investigate the potential targets and mechanisms underlying RIH. Results: Remifentanil induced significant pronociceptive effects and a distinct miRNA-profile from sufentanil when compared to saline controls. Among top 30 differentially expressed miRNAs spectrum, spinal miR-134-5p was dramatically downregulated in RIH mice but remained comparative in mice subjected to sufentanil. Moreover, Glutamate Receptor Ionotropic Kainate 3 (Grik3) was a target of miR-134-5p. The overexpression of miR-134-5p attenuated the hyperalgesic phenotype, excessive dendritic spine remodeling, excitatory synaptic structural plasticity, and Kainate receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in SDH resulting from remifentanil exposure. Besides, intrathecal injection of selective KA-R antagonist was able to reverse the GRIK3 membrane trafficking and relieved RIH. Conclusion: The miR-134-5p contributes to remifentanil-induced pronociceptive features via directly targeting Grik3 to modulate dendritic spine morphology and synaptic plasticity in spinal neurons.

N6-methyladenosine writer METTL3 accelerates the sepsis-induced myocardial injury by regulating m6A-dependent ferroptosis.

Ferroptosis is an iron-dependent and phospholipid peroxidation-mediated cell death, which has been identified to be involved in sepsis-induced injury. However, the in-depth molecular mechanisms of N6-methyladenosine (m6A) and ferroptosis on sepsis-induced myocardial injury are still unclear. Here, in the septic myocardial injury, m6A methyltransferase METTL3 level and methylation level high-expressed in lipopolysaccharide (LPS)-induced cardiomyocytes (H9C2). Functionally, METTL3 silencing repressed the ferroptosis phenotype induced by LPS. Mechanistically, METTL3-mediated m6A methylation on solute carrier family 7 member 11 (SLC7A11) empowered its mRNA with high methylation level. Moreover, YTHDF2 directly bound to the m6A modification sites of SLC7A11 to mediate the mRNA degradation. The m6A modified SLC7A11 mRNA was recognized by YTHDF2, which promoted the decay of SLC7A11 mRNA, consequently up-regulating ferroptosis in sepsis-induced myocardial injury. Together, these findings establish a role of METTL3 in the ferroptosis of LPS-induced cardiomyocytes, and provide potential therapeutic target to treat the sepsis-induced myocardial injury.

The impact of the new acute respiratory distress syndrome (ARDS) criteria on Berlin criteria ARDS patients: a multicenter cohort study.

OBJECTIVE: The European Society of Intensive Care Medicine (ESICM) recently recommended changes to the criteria of acute respiratory distress syndrome (ARDS), patients with high-flow oxygen were included, however, the effect of these changes remains unclear. Our objectives were to evaluate the performance of these new criteria and to compare the outcomes of patients meeting the new ARDS criteria with those meeting the Berlin ARDS criteria. METHODS: This was a retrospective cohort. The patients admitted to the intensive care unit (ICU) were diagnosed with ARDS. Patients were classified as meeting Berlin criteria ARDS (n = 4279), high-flow nasal oxygen (HFNO) criteria ARDS (n = 559), or new criteria ARDS (n = 4838). RESULTS: In comparison with HFNO criteria ARDS and new criteria ARDS, patients with Berlin criteria ARDS demonstrated lower blood oxygen levels assessed by PaO2/FiO2, SpO2/FiO2, and ROX (SpO2/FiO2/respiratory rate) (p < 0.001); and higher severity of illness assessed by the Sequential Organ Failure Assessment (SOFA) score, Acute Physiology And Chronic Health Evaluations (APACHE II), Simplified Acute Physiology Score (SAPS II) (p < 0.001), (p < 0.001), and longer ICU and hospital stays (p < 0.001). In comparison with the HFNO criteria, patients meeting Berlin criteria ARDS had higher hospital mortality (10.6% vs. 16.9%; p = 0.0082), 28-day mortality (10.6% vs. 16.5%; p = 0.0079), and 90-day mortality (10.7% vs. 17.1%; p = 0.0083). ARDS patients with HFNO did not have severe ARDS; Berlin criteria ARDS patients with severe ARDS had the highest mortality rate (approximately 33%). PaO2/FiO2, SpO2/FiO2, and ROX negatively correlated with the SOFA and APACHE II scores. The SOFA and APACHE II scores had high specificity and sensitivity for prognosis in patients with new criteria ARDS. CONCLUSION: The new criteria of ARDS reduced the severity of illness, length of stay in the ICU, length of hospital stays, and overall mortality. SOFA and APACHE II scores remain important in assessing the prognosis of patients with new criteria ARDS. TRIAL REGISTRATION: Registration number: ChiCTR2200067084.

Lysine demethylase KDM5B down-regulates SIRT3-mediated mitochondrial glucose and lipid metabolism in diabetic neuropathy.

BACKGROUND: Diabetic peripheral neuropathy (DPN) is a common neurological complication of diabetes mellitus without efficient interventions. Both lysine demethylase 5B (KDM5B) and sirtuin-3 (SIRT3) have been found to regulate islet function and glucose homeostasis. KDM5B was predicted to bind to the SIRT3 promoter by bioinformatics. Here, we investigated whether KDM5B affected DPN development via modulating SIRT3. METHODS: The db/db mice and high glucose-stimulated Schwann cells (RSC96) were used as in vivo and in vitro models of DPN, respectively. Glucose level, glucose and insulin tolerance of mice were measured. Neurological function was evaluated by motor nerve conduction velocity (MNCV), tactile allodynia assay and thermal sensitivity assay. Adenosine triphosphate level, oxygen consumption rate, extracellular acidification rate, ß-oxidation rate, acetyl-CoA level, acetylation levels and activities of long-chain acyl CoA dehydrogenase (LCAD) and pyruvate dehydrogenase (PDH) were detected. Methyl thiazolyl tetrazolium assay was adopted to determine cell viability. Reactive oxygen species (ROS) production was detected by MitoSox staining. Western blotting for measuring target protein levels. Molecular mechanisms were investigated by co-immunoprecipitine (Co-IP), chromatin immunoprecipitation (ChIP) and luciferase reporter assay. RESULTS: KDM5B was up-regulated, while SIRT3 was down-regulated in DPN models. SIRT3 overexpression or AMPK activation ameliorated mitochondrial metabolism dysfunction and ROS overproduction during DPN. KDM5B overexpression triggered mitochondrial metabolism disorder and oxidative stress via directly transcriptional inhibiting SIRT3 expression by demethylating H3K4me3 or indirectly repressing AMPK pathway-regulated SIRT3 expression. CONCLUSION: KDM5B contributes to DPN via regulating SIRT3-mediated mitochondrial glucose and lipid metabolism. KDM5B inhibition may be an effective intervention for DPN.

RBM3 is associated with acute lung injury in septic mice and patients via the NF-κB/NLRP3 pathway.

Sepsis refers to host response disorders caused by infection, leading to life-threatening organ dysfunction. RNA-binding motif protein 3 (RBM3) is an important cold-shock protein that is upregulated in response to mild hypothermia or hypoxia. In this study, we aimed to investigate whether RBM3 is involved in sepsis-associated acute lung injury (ALI). Intraperitoneal injection of LPS (10 mg/kg) was performed in wild type (WT) and RBM3 knockout (KO, RBM3-/-) mice to establish an in vivo sepsis model. An NLRP3 inflammasome inhibitor, MCC950 (50 mg/kg), was injected intraperitoneally 30 min before LPS treatment. Serum, lung tissues, and BALF were collected 24 h later for further analysis. In addition, we also collected serum from sepsis patients and healthy volunteers to detect their RBM3 expression. The results showed that the expression of RBM3 in the lung tissues of LPS-induced sepsis mice and the serum of patients with sepsis was significantly increased and positively correlated with disease severity. In addition, RBM3 knockout (KO) mice had a low survival rate, and RBM3 KO mice had more severe lung damage, inflammation, lung cell apoptosis, and oxidative stress than WT mice. LPS treatment significantly increased the levels of nucleotide binding and oligomerization domain-like receptor family 3 (NLRP3) inflammasomes and mononuclear cell nuclear factor-κB (NF-κB) in the lung tissues of RBM3 KO mice. However, these levels were only slightly elevated in WT mice. Interestingly, MCC950 improved LPS-induced acute lung injury in WT and RBM3 KO mice but inhibited the expression of NLRP3, caspase-1, and IL-1ß. In conclusion, RBM3 was overexpressed in sepsis patients and LPS-induced mice. RBM3 gene deficiency aggravated sepsis-associated ALI through the NF-κB/NLRP3 pathway.

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.

Spinal NLRP3 inflammasome activation mediates IL-1ß release and contributes to remifentanil-induced postoperative hyperalgesia by regulating NMDA receptor NR1 subunit phosphorylation and GLT-1 expression in rats.

BACKGROUND: Trafficking and activation of N-methyl-D-aspartate (NMDA) receptors play an important role in initiating and maintaining postoperative remifentanil-induced hyperalgesia (RIH). Activation of the NOD-like receptor protein 3 (NLRP3) inflammasome has been linked to the development of inflammatory and neuropathic pain. We hypothesized that activation of NLRP3 inflammasome mediates IL-1ß release and contributes to RIH in rats by increasing NMDA receptor NR1 (NR1) subunit phosphorylation and decreasing glutamate transporter-1 (GLT-1) expression. METHODS: Acute exposure to remifentanil (1.2 µg/kg/min for 60 min) was used to establish RIH in rats. Thermal and mechanical hyperalgesia were tested at baseline (24 h before remifentanil infusion) and 2, 6, 24, and 48 h after remifentanil infusion. The levels of IL-1ß, GLT-1, phosphorylated NR1 (phospho-NR1), and NLRP3 inflammasome activation indicators [NLRP3, Toll-like receptor 4 (TLR4), P2X purinoceptor 7 (P2X7R), and caspase-1] were measured after the last behavioral test. A selective IL-1ß inhibitor (IL-1ß inhibitor antagonist; IL-1ra) or three different selective NLRP3 inflammasome activation inhibitors [(+)-naloxone (a TLR4 inhibitor), A438079 (a P2X7R inhibitor), or ac-YVADcmk (a caspase-1 inhibitor)] were intrathecally administered immediately before remifentanil infusion into rats. RESULTS: Remifentanil induced significant postoperative hyperalgesia, increased IL-1ß and phospho-NR1 levels and activated the NLRP3 inflammasome by increasing TLR4, P2X7R, NLRP3, and caspase-1 expression, but it decreased GLT-1 expression in the L4-L6 spinal cord segments of rats, which was markedly improved by intrathecal administration of IL-1ra, (+)-naloxone, A438079, or ac-YVADcmk. CONCLUSION: NLRP3 inflammasome activation mediates IL-1ß release and contributes to RIH in rats by inducing NMDA receptor NR1 subunit phosphorylation and decreasing GLT-1 expression. Inhibiting the activation of the NLRP3 inflammasome may be an effective treatment for RIH.

Risk stratification of patients with acute respiratory distress syndrome complicated with sepsis using lactate trajectories.

BACKGROUND: No consensus has been reached on an optimal blood lactate evaluation system although several approaches have been reported in the literature in recent years. A group-based trajectory modeling (GBTM) method could better stratify patients with acute respiratory distress syndrome (ARDS) complicated with sepsis in the intensive care unit (ICU). PATIENTS AND METHODS: 760 patients from the comprehensive ICU of Tianjin Medical University General Hospital with ARDS complicated with sepsis were eligible for analysis. Serial serum lactate levels were measured within 48 h of admission. In addition to the GBTM lactate groups, the initial lactate, peak lactate level, the area under the curve of serial lactate (lactate AUC), and lactate clearance were also considered for comparison. The short- and long-term outcomes were the 30- and 90-day mortality, respectively. RESULTS: Three lactate groups were identified based on GBTM, with group 3 exhibiting the worse short- [hazard ratio (HR) for 30-day mortality: 2.96, 95% confidence interval (CI) 1.79-4.87, P < 0.001] and long term (HR for 90-day mortality: 3.49, 95% CI 2.06-5.89, P < 0.001) outcomes followed by group 2 (HR for 30-day mortality: 2.05, 95% CI 1.48-2.84, P < 0.001 and HR for 90-day mortality: 1.99, 95% CI 1.48-2.67, P < 0.001). GBTM lactate groups exhibited significantly improved diagnostic performance of initial lactate + SOFA scores/APACHE II scores models. Based on the multivariable fractional polynomial interaction (MFPI) approach, GBTM lactate groups could better differentiate high-risk patients than the initial lactate groups in short- and long-term outcomes. CONCLUSIONS: To the best of our knowledge, this is the first report that GBTM-based serial blood lactate evaluations significantly improve the diagnostic capacity of traditional critical care evaluation systems and bring many advantages over previously documented lactate evaluation systems.

Effect of sigh in lateral position on postoperative atelectasis in adults assessed by lung ultrasound: a randomized, controlled trial.

BACKGROUND: Postoperative atelectasis occurs in 90% of patients receiving general anesthesia. Recruitment maneuvers (RMs) are not always effective and frequently associated with barotrauma and hemodynamic instability. It is reported that many natural physiological behaviors interrupted under general anesthesia could prevent atelectasis and restore lung aeration. This study aimed to find out whether a combined physiological recruitment maneuver (CPRM), sigh in lateral position, could reduce postoperative atelectasis using lung ultrasound (LUS). METHODS: We conducted a prospective, randomized, controlled trial in adults with open abdominal surgery under general anesthesia lasting for 2 h or longer. Subjects were randomly allocated to either control group (C-group) or CPRM-group and received volume-controlled ventilation with the same ventilator settings. Patients in CPRM group was ventilated in sequential lateral position, with the addition of periodic sighs to recruit the lung. LUS scores, dynamic compliance (Cdyn), the partial pressure of arterial oxygen (PaO2) and fraction of inspired oxygen (FiO2) ratio (PaO2/FiO2), and other explanatory variables were acquired from each patient before and after recruitment. RESULTS: Seventy patients were included in the analysis. Before recruitment, there was no significant difference in LUS scores, Cdyn and PaO2/FiO2 between CPRM-group and C-group. After recruitment, LUS scores in CPRM-group decreased significantly compared with C-group (6.00 [5.00, 7.00] vs. 8.00 [7.00, 9.00], p = 4.463e-11 < 0.05), while PaO2/FiO2 and Cdyn in CPRM-group increased significantly compared with C-group respectively (377.92 (93.73) vs. 309.19 (92.98), p = 0.008 < 0.05, and 52.00 [47.00, 60.00] vs. 47.70 [41.00, 59.50], p = 6.325e-07 < 0.05). No hemodynamic instability, detectable barotrauma or position-related complications were encountered. CONCLUSIONS: Sigh in lateral position can effectively reduce postoperative atelectasis even without causing severe side effects. Further large-scale studies are necessary to evaluate it's long-term effects on pulmonary complications and hospital length of stay. TRIAL REGISTRATION: ChiCTR1900024379 . Registered 8 July 2019,.

Hydrogen alleviates cell damage and acute lung injury in sepsis via PINK1/Parkin-mediated mitophagy.

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.

Dexmedetomidine attenuates myocardial ischemia-reperfusion injury in vitro by inhibiting NLRP3 Inflammasome activation.

BACKGROUND: Myocardial ischemia-reperfusion injury (MIRI) is the most common cause of death worldwide. The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the inflammatory response to MIRI. Dexmedetomidine (DEX), a specific agonist of α2-adrenergic receptor, is commonly used for sedation and analgesia in anesthesia and critically ill patients. Several studies have shown that dexmedetomidine has a strong anti-inflammatory effect in many diseases. Here, we investigated whether dexmedetomidine protects against MIRI by inhibiting the activation of the NLRP3 inflammasome in vitro. METHODS: We established an MIRI model in cardiomyocytes (CMs) alone and in coculture with cardiac fibroblasts (CFs) by hypoxia/reoxygenation (H/R) in vitro. The cells were treated with dexmedetomidine with or without MCC950 (a potent selective NLRP3 inhibitor). The beating rate and cell viability of cardiomyocytes, NLRP3 localization, the expression of inflammatory cytokines and NLRP3 inflammasome-related proteins, and the expression of apoptosis-related proteins, including Bcl2 and BAX, were determined. RESULTS: Dexmedetomidine treatment increased the beating rates and viability of cardiomyocytes cocultured with cardiac fibroblasts. The expression of the NLRP3 protein was significantly upregulated in cardiac fibroblasts but not in cardiomyocytes after H/R and was significantly attenuated by dexmedetomidine treatment. Expression of the inflammatory cytokines IL-1ß, IL-18 and TNF-α was significantly increased in cardiac fibroblasts after H/R and was attenuated by dexmedetomidine treatment. NLRP3 inflammasome activation induced the increased expression of cleaved caspase1, mature IL-1ß and IL-18, while dexmedetomidine suppressed H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts. In addition, dexmedetomidine reduced the expression of Bcl2 and BAX in cocultured cardiomyocytes by suppressing H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts. CONCLUSION: Dexmedetomidine treatment can suppress H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts, thereby alleviating MIRI by inhibiting the inflammatory response.

Hydrogen attenuates sepsis-associated encephalopathy by NRF2 mediated NLRP3 pathway inactivation.

OBJECTIVE: Sepsis-associated encephalopathy (SAE) is a major cause of mortality worldwide. Oxidative stress, inflammatory response and apoptosis participate in the pathogenesis of SAE. Nuclear factor erythroid 2-related factor 2 (Nrf2) and nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) pathway is involved in oxidative stress and inflammatory response. We reported that hydrogen gas protected against sepsis in wild-type (WT) but not Nrf2 knockout (KO) mice. Therefore, it is vital to identify the underlying cause of hydrogen gas treatment of sepsis-associated encephalopathy. METHODS: SAE was induced in WT and Nrf2 KO mice by cecal ligation and puncture (CLP). As a NLRP3 inflammasome inhibitor, MCC950 (50 mg/kg) was administered by intraperitoneal (i.p.) injection before operation. Hydrogen gas (H2)-rich saline solution (5 mL/kg) was administered by i.p. injection at 1 h and 6 h after sham and CLP operations. Brain tissue was collected to assess the NLRP3 and Nrf2 pathways by western blotting, reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. RESULTS: SAE increased NLRP3 and Nrf2 expression in microglia. MCC950 inhibited SAE-induced NLRP3 expression, interleukin (IL)-1ß and IL-18 cytokine release, neuronal apoptosis and mitochondrial dysfunction. SAE increased NLRP3 and caspase-1 expression in WT mice compared to Nrf2 KO mice. Hydrogen increased Nrf2 expression and inhibited the SAE-induced expression of NLRP3, caspase-1, cytokines IL-1ß and IL-18, neuronal apoptosis, and mitochondrial dysfunction in WT mice but not Nrf2 KO mice. CONCLUSION: SAE increased NLRP3 and Nrf2 expression in microglia. Hydrogen alleviated inflammation, neuronal apoptosis and mitochondrial dysfunction via inhibiting Nrf2-mediated NLRP3 pathway.

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.

Prospects of molecular hydrogen in perioperative neuroprotection from basic research to clinical application.

PURPOSE OF REVIEW: The current systematic review summarizes recent, basic clinical achievements regarding the neuroprotective effects of molecular hydrogen in distinct central nervous system conditions. RECENT FINDINGS: Perioperative neuroprotection remains a major topic of clinical anesthesia. Various gaseous molecules have previously been explored as a feasible therapeutic option in neurological disorders. Among them, molecular hydrogen, which has emerged as a novel and potential therapy for perioperative neuroprotection, has received much attention. SUMMARY: Fundamental and clinical evidence supports the antioxidant, antiinflammation, antiapoptosis and mitochondrial protective effects of hydrogen in the pathophysiology of nervous system diseases. The clinically preventive and therapeutic effects of hydrogen on different neural diseases, however, remain uncertain, and the lack of support by large randomized controlled trials has delayed its clinical application.

The role of PI3K-mediated AMPA receptor changes in post-conditioning of propofol in brain protection.

BACKGROUND: We aimed to study the role of amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) glutamate receptor 2 (GluR2) subunit trafficking, and activity changes in short-term neuroprotection provided by propofol post-conditioning. We also aimed to determine the role of phosphoinositide-3-kinase (PI3K) in the regulation of these processes. METHODS: Rats underwent 1 h of focal cerebral ischemia followed by 23 h of reperfusion were randomly divided into 6 groups (n = 36 per group): sham- operation (S), ischemia-reperfusion (IR), propofol (P group, propofol 20 mg/kg/h at the onset of reperfusion for 2 h after 60 min of occlusion), and LY294002 (PI3K non-selective antagonist) + sham (L + S, LY294002 of 1.5 mg/kg was infused 30 min before sham operation), LY294002+ ischemia-reperfusion (L + IR, LY294002 of 1.5 mg/kg was infused 30 min before middle cerebral artery occlusion), LY294002 + IR + propofol (L + P, LY294002 of 1.5 mg/kg was infused 30 min before middle cerebral artery occlusion and propofol 20 mg/kg/h at the onset of reperfusion for 2 h after 60 min of occlusion). RESULTS: Compared with group IR, rats in group P had significant lower neurologic defect scores and infarct volume. Additionally, consistent with enhanced expression of PI3K-AMPAR GluR2 subunit complex substances in ipsilateral hippocampus, GluR2 subunits showed increased levels in both the plasma and postsynaptic membranes of neurons, while pGluR2 expression was reduced in group P. Furthermore, LY294002, the PI3K non-selective antagonist, blocked those effects. CONCLUSION: These observations demonstrated that propofol post-conditioning revealed acute neuroprotective role against transient MCAO in rats. The short-term neuroprotective effect was contributed by enhanced GluR2 subunits trafficking to membrane and postsynaptic membranes of neurons, as well as down-regulated the expression of pGluR2 in damaged hippocampus. Finally, the above-mentioned protective mechanism might be contributed by increased combination of PI3K to AMPAR GluR2 subunit, thus maintained the expression and activation of AMPAR GluR2 in the ipsilateral hippocampus.

Hemopexin alleviates cognitive dysfunction after focal cerebral ischemia-reperfusion injury in rats.

BACKGROUND: Ischemia-reperfusion (I/R) is a critical pathophysiological basis of cognitive dysfunction caused by ischemia stroke. Heme-oxygenase-1 (HO-1) is the rate-limiting enzyme for the elimination of excessive free heme by combining with hemopexin (HPX), a plasma protein that contributes to eliminating excessive free heme during ischemia stroke. This study aimed to elucidate whether HPX could alleviate cognitive dysfunction in rats subjected to cerebral I/R. METHODS: Rats were randomly divided into five groups: sham, MCAO, Vehicle, HPX and HPX + protoporphyrin IX (ZnPPIX). Cerebral I/R was induced by MCAO. Saline, vehicle, HPX and HPX + ZnPPIX were injected intracerebroventricularly at the moment after reperfusion. Morris water maze (MWM) test was used to detect the learning and cognitive function. Western blot was used to detect the expression of HO-1 in ischemic penumbra. CD31/vWF double labeling immunofluorescence was used to detect the neovascularization in the penumbra hippocampus. The structure and function of blood-brain barrier (BBB) was detected by the permeability of Evans Blue (EB), water content of the brain tissue, the Ang1/Ang2 and VE-cadherin expression. RESULTS: Our study verified that HPX improved the learning and memory capacity. Hemopexin up-regulated HO-1 protein expression, the average vessel density in the penumbra hippocampus and the VE- cadherin expression but decreased the permeability of EB, the water content of brain tissue and the ratio of Ang1/Ang2. The effects were reversed by ZnPPIX, an inhibitor of HO-1. CONCLUSION: HPX can maintain the integrity of the blood-brain barrier and alleviate cognitive dysfunction after cerebral I/R through the HO-1 pathway.