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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.

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 (H 2 )-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)-1β 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-1β 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.

Objective: To investigate the key role of nuclear factor E2-related factor 2 (Nrf2) in the treatment of lung injury in sepsis mice by regulating Nrf2/heme oxygenase-1 (HO-1)/high mobility group protein B1 (HMGB1) pathway., Methods: 120 male wild type (WT) and 120 Nrf2 knockout (Nrf2-KO) ICR mice were randomly divided into Sham group, H 2 control group (Sham+H 2 group), cecal ligation and puncture (CLP) induced sepsis model group (CLP group) and H 2 intervention group (CLP+H 2 group), with 30 mice in each group. The sepsis model was reproduced by CLP. The same operation was done in Sham group and Sham+H 2 group except CLP. The mice in Sham+H 2 group and CLP+H 2 group were challenged by 2% H 2 for 1 hour at 1 hour and 6 hours after operation respectively, while the mice in Sham group and CLP group only inhaled air. Twenty mice in each group were collected to observe the 7-day survival. The other mice were sacrificed at 24 hours after the reproduction of model, and the lung tissues were harvested. The activities of superoxide dismutase (SOD) and catalase (CAT) and malondialdehyde (MDA) contents were determined by enzyme-linked immunosorbent assay (ELISA). The expressions of HO-1 and HMGB1 were determined by Western Blot, and the positive expression of HO-1 was also detected by immunofluorescence., Results: Compared with Sham groups, the 7-day survival rates of WT and Nrf2-KO mice in CLP groups were significantly lowered [WT: 0% (0/20) vs. 100% (20/20), Nrf2-KO: 0% (0/20) vs. 100% (0/20), both P < 0.05]; the 7-day survival rates of CLP+H 2 group in WT mice were significantly higher than those of CLP group [40% (8/20) vs. 0% (0/20), P < 0.05], but there was no significant difference between CLP+H 2 group and CLP group in Nrf2-KO mice [0% (0/20) vs. 0% (0/200), P > 0.05]. In WT mice, compared with Sham group, the activities of SOD and CAT in lung tissue of CLP group were decreased significantly [SOD (kU/g): 131.30±28.21 vs. 251.00±22.84, CAT (kU/g): 13.43±1.52 vs. 20.76±1.63, both P < 0.01], the MDA content, the expressions of HO-1 and HMGB1 were increased significantly [MDA (μmol/g): 6.26±1.18 vs. 4.16±0.58, HO-1/β-actin: 0.160±0.045 vs. 0.023±0.005, HMGB1/β-actin: 0.656±0.055 vs. 0.005±0.001, all P < 0.05]. Compared with CLP group, the activities of SOD, CAT and HO-1 expression in lung tissue of CLP+H 2 group were significantly increased [SOD (kU/g): 220.32±35.06 vs. 131.30±28.21, CAT (kU/g): 18.95±2.49 vs. 13.43±1.52, HO-1/β-actin: 0.376±0.025 vs. 0.160±0.045, all P < 0.01], while the MDA contents and HMGB1 expressions were significantly decreased [MDA (μmol/g): 4.26±0.75 vs. 6.26±1.18, HMGB1/β-actin: 0.343±0.040 vs. 0.656±0.055, both P < 0.05]. In Nrf2-KO mice, compared with Sham group, the activity of CAT in CLP group was significantly lowered (kU/g: 12.28±1.49 vs. 19.11±1.53, P < 0.01), MDA contents and the expressions of HO-1 and HMGB1 were significantly increased [MDA (μmol/g): 6.85±0.54 vs. 4.59±0.50, HO-1/β-actin: 0.063±0.005 vs. 0.021±0.003, HMGB1/β-actin: 0.713±0.035 vs. 0.005±0.001, all P < 0.01], while there was no significant difference in SOD activity (kU/g: 114.19±9.94 vs. 135.75±28.10, P > 0.05). There was no significant difference in above parameters between CLP+H 2 group and CLP group., Conclusions: H 2 inhibits lung injury in septic mice through Nrf2/HO-1/HMGB1 pathway. Nrf2 plays a major role in the treatment of septic lung injury by H 2 .

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) undergoes the process of pathological event including lung tissue dysfunction, pulmonary edema, and inflammation in sepsis. Autophagy is a cytoprotective process recognized as one of the major pathways for degradation and recycling of cellular constituents. Autophagy as a protective or maladaptive response was still confused in ALI during sepsis. Acute lung injury was performed by cecal ligation and puncture (CLP). Autophagic inducer rapacymin and inhibitor 3-MA and autophagosomal-lysosome fusion inhibitor bafilomucin (Baf) A1 and chloroquine (CQ) were administrated by intraperitoneal injection at 1 h after CLP operation. Microtubule-associated protein light chain 3 II (LC3II), Beclin 1, Rab7, and lysosome-associated membrane protein type 2 (LAMP2) were detected by western blotting. Seven-day survival rate of septic mice was observed. Histologic scores, lung wet-to-dry (W/D) weight ratio, oxygenation index (PaO 2 /FiO 2 ), total cells and polymorphonuclear neutrophils (PMN) in bronchial alveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity and cytokine tumor necrosis factor (TNF)-α, high-mobility group box (HMGB)1, interleukin (IL)-6, IL-10, and monocyte chemotactic protein (MCP)1 were measured after sham or ALI operation. ALI induced the increasing expression of autophagy-related protein LC3II, Beclin 1, Rab7, and LAMP2 in CLP operation. Autophagic inducer rapacymin significantly induced the expression of LC3II, Beclin 1, LAMP2, and Rab7 in mice model of CLP, and inhibitor 3-MA reduced expression of LC3II, Beclin 1, LAMP2, and Rab7 expressions in CLP + RAP mice compared to CLP group. Compared with ALI group, Baf and CQ obviously elevated the level of LC3II and Beclin 1, and reduced the LAMP2 and Rab7 expressions in CLP + Baf group and ALI + CQ group. Compared with CLP group, autophagic inducer rapacymin improved the survival rate, histologic scores, lung wet/dry weight ratio, PaO 2 /FiO 2 , total cells, and PMNS in BALF and MPO activity and cytokines TNF-α, HMGB1, IL-6, IL-10, and MCP1 in CLP + RAP group, but there were exacerbated above indicators in CLP + 3-MA group, CLP + Baf group, and CLP + CQ group. Autophagy activation participated in the pathophysiologic process of sepsis, and alleviated the cytokine excessive release and lung injury in sepsis.

Objective: To investigate the effects of autophagy on N-methy-D-aspartate (NMDA) receptor and its subunit NR2B and behavioral test in a rat model of neuropathic pain (NP)., Methods: Male Sprague-Dawley (SD) rats were divided into sham group, NP group, autophagy inhibitor 3-methyladenine (3-MA) pretreatment group (3-MA+NP group) and autophagy inducer rapamyein (Rap) group (Rap+NP group) by random number table with 22 rats in each group. NP animal model was reproduced by ligating sciatic nerve, while sciatic nerve of the rats in the sham group were only exposed but not ligated. The rats in two pretreatment groups were intraperitoneally challenged with 3-MA 15 mg/kg or Rap 10 mg/kg injection 1 hour before operation. Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before and 1, 3, 7, 14 days after operation in each group. Spinal cord tissues were harvested at 1 day and 7 days after operation for autophagosome observation by electron microscope. The expressions of autophagy protein microtubule-associated protein 1 light chain 3-II (LC3-II), Beclin1, and NMDA, NR2B were determined by Western Blot. The positive expression of LC3 was detected by immunofluorescence., Results: Compared with sham group, the MWT and TWL of rats in NP group were decreased gradually with the prolongation of operation time, the number of autophagosome, the expressions of LC3-II, Beclin1, NMDA, NR2B, and the positive expression of LC3 in spinal cord were significantly increased at 1 day after operation and till 7 days, which indicated that NP led to hyperpathia and autophagy activation. Compared with NP group, MWT was significantly further decreased, TWL was further shortened, the number of autophagosome was decreased, the expressions of LC3-II and Beclin1 in spinal cord were decreased, and NMDA and NR2B expressions were further increased after 3-MA pretreatment, with significant differences at 1 day after operation [MWT (g): 29.4±2.4 vs. 42.5±6.6, TWL (s): 7.2±1.0 vs. 8.8±1.1, LC3-II/β-actin: 0.38±0.03 vs. 0.52±0.07, Beclin1/β-actin: 0.29±0.06 vs. 0.59±0.05, NMDA/β-actin: 0.62±0.06 vs. 0.50±0.06, NR2B/β-actin: 0.57±0.03 vs. 0.46±0.03, all P < 0.05]. Immunofluorescence staining confirmed that the positive expression of LC3 was significantly decreased. Rap pretreatment could increase MWT, TWL and the number of autophagosome, increase LC3-II and Beclin1 expressions in spinal cord, and decrease NMDA and NR2B expressions in NP rats, and significant differences at 1 day after operation were found as compared with those in NP group [MWT (g): 49.4±4.4 vs. 42.5±6.6, TWL (s): 10.5±1.2 vs. 8.8±1.1, LC3-II/β-actin: 0.67±0.09 vs. 0.52±0.07, Beclin1/β-actin: 0.71±0.08 vs. 0.59±0.05, NMDA/β-actin: 0.40±0.05 vs. 0.50±0.06, NR2B/β-actin: 0.34±0.04 vs. 0.46±0.03, all P < 0.05], and immunofluorescence showed that the positive expression of LC3 was increased and lasted for 7 days. It indicated that Rap could increase the activity of autophagy, alleviate the occurrence of hyperalgesia, and reduce the expressions of NMDA receptor and its NR2B subunit., Conclusions: NP could regulate the variety of NMDA/NR2B and hyperalgesia via increasing autophagy.

Hydrogen gas (H2) exerts a beneficial effect against traumatic brain injury (TBI). microRNA-21 (miR-21) is one of the most highly expressed members of small non-coding microRNA family in mammalian cells. miR-21 can improve the neurological outcome after TBI. In the present study, we investigated whether H2 treatment could improve the neurological outcome after TBI via increasing miR-21 expression. TBI was induced by controlled cortical impact in rats. H2 treatment was given by exposure to 2% H2 from 30 min to 5 h after TBI operation. Here, we found that H2 treatment significantly increased the expression of miR-21 in brain from 6 h to 3 d after TBI. The level of miR-21 expression in brain was significantly decreased after intracerebroventricular infusion of miR-21 antagomir in TBI-challenged rats with or without H2 treatment. Moreover, we found that H2 treatment conferred a better neurological outcome after TBI by improving neurological dysfunction, alleviating brain edema as well as decreasing lesion volume and blood-brain barrier permeability, which were significantly prevented by miR-21 antagomir. Furthermore, intracerebroventricular infusion of miR-21 agomir increased the level of miR-21 expression and decreased the lesion volume after TBI. In addition, H2 treatment decreased the levels of oxidative products (malondialdehyde and 8-iso-prostaglandin F2α) and increased the activities of endogenous antioxidant enzymes (superoxide dismutase and catalase) in brain after TBI, which were prevented by miR-21 antagomir. Taken together, these data indicate that H2 treatment improves the neurological outcome after TBI via increasing miR-21 expression.

Background: Noninvasive evaluation of muscle perfusion using near-infrared spectroscopy (NIRS) coupled with a vascular occlusion test (VOT) may provide an early and simple marker of altered perfusion and microcirculatory function in sepsis., Objective: The aim of the study was to compare the time-course of NIRS-derived variables with systemic measures of perfusion in an experimental model of peritonitis., Methods: Peritonitis was induced in eight anesthetized, mechanically ventilated, adult sheep (24-34 kg), by injecting autologous feces into the peritoneal cavity. Animals were followed until death or for a maximum of 30 h. Muscle tissue oxygen saturation (StO2) was determined using NIRS on the right posterior leg and arterial VOTs were performed by intermittent intra-aortic balloon inflation. Microdialysis was used to measure muscle lactate and pyruvate levels., Results: Muscle StO2 was significantly lower than baseline values from 8 h after sepsis induction, but with considerable intersubject variability. The NIRS VOT ascending (Asc) slope decreased to values <120%/min in most animals from 12 h after sepsis induction. Muscle lactate/pyruvate ratios were higher than baseline from 16 h after sepsis induction. Mixed venous oxygen saturation (SvO2) decreased to <70% and blood lactate levels increased to >2 mmol/L in most of the animals only 24 and 28 h after sepsis induction, respectively. Muscle NIRS StO2 correlated strongly with femoral venous oxygen saturation (r = 0.820) and moderately with SvO2 (r = 0.436)., Conclusions: The muscle NIRS Asc slope after a VOT is altered earlier than global markers of tissue hypoperfusion during sepsis. This simple noninvasive test can detect early changes in peripheral perfusion in sepsis.

Background: Neuropathic pain is a chronic and intractable pain, with very few effective analgesics. It involves an impaired cell autophagy process. Hydrogen-rich saline (HRS) reportedly reduces allodynia and hyperalgesia in a neuropathic pain model; however, it is unknown whether these effects involve autophagy induction., Methods: We investigated the relationship between HRS and cell autophagy in a neuropathic pain model generated by chronic constriction injury (CCI) in Sprague-Dawley rats. Rats received an intraperitoneal injection of HRS (10 mL/kg daily, from 1 day before until 14 days after CCI), 3MA (autophagy inhibitor), 2ME2 (HIF-1 α inhibitor), or EDHB (HIF-1 α agonist). The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were tested 1 day before and 1, 3, 7, 10, and 14 days after the operation. HIF-1 α and cell autophagy markers in the spinal cord were evaluated by western blotting and real-time PCR assays at 14 days after CCI. Autophagosomes with double membranes were identified by transmission electron microscopy., Results: CCI caused behavioral hypersensitivity to mechanical and thermal stimulation in the hind-paw of the injured side. HRS improved MWT and TWL, activated autophagy, and increased autophagosomes and autolysosomes in CCI rats. 3-MA aggravated hyperalgesia and allodynia and suppressed autophagy, while EDHB attenuated hyperalgesia and activated the autophagy procedure and the HIF-1 α downstream target gene BNIP3. HIF-1 α inhibitors reversed the regulatory effects of HRS on autophagy in CCI rats at 14 days after spinal cord injury., Conclusion: HRS reduced mechanical hyperalgesia and activation of cell autophagy in neuropathic pain through a HIF1-dependent 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.

Objectives: Optimizing oxygen delivery is an important part of the hemodynamic resuscitation of septic shock, but concerns have been raised over the potentially deleterious effects of hyperoxia. We evaluated the impact of hyperoxia on hemodynamics, the microcirculation, and cerebral and renal metabolism in an ovine model of septic shock., Design: Randomized animal study., Setting: University hospital animal research laboratory., Subjects: Fourteen adult female sheep., Interventions: After induction of fecal peritonitis, sheep were randomized to ventilation with an FIO2 of 100% (n = 7) or an FIO2 adjusted to maintain PaO2 between 90 and 120 mm Hg (n = 7, control). All animals were fluid resuscitated and observed until death., Measurements and Main Results: In addition to hemodynamic measurements, we assessed the sublingual microcirculation, renal and cerebral microdialysis and microvascular perfusion, and brain tissue oxygen pressure. Hyperoxic animals initially had a higher mean arterial pressure than control animals. After onset of shock, hyperoxia blunted the decrease in stroke volume index observed in the control group. Hyperoxia was associated with a higher sublingual microcirculatory flow over time, with higher cerebral perfusion and brain tissue oxygen pressure and with a lower cerebral lactate-to-pyruvate ratio than in control animals. Hyperoxia was also associated with preserved renal microvascular perfusion, lower renal lactate-to-pyruvate ratio, and higher PaO2/FIO2 ratio., Conclusions: In this acute peritonitis model, hyperoxia induced during resuscitation provided better hemodynamics and peripheral microvascular flow and better preserved cerebral metabolism, renal function, and gas exchange. These observations are reassuring with recent concerns about excessive oxygen therapy in acute diseases.

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.

Objective: To investigate the effect of combining propofol with hydrogen on organ damage and inflammation of sepsis in cecal ligation and puncture (CLP) mice model., Methods: One hundred and forty male C57BL/6 mice were randomly divided into groups (n = 28): sham group, CLP group, propofol group, H 2 group, and propofol and H 2 group. The sepsis was induced by CLP operation. Mice in sham group did the same operation with ligation and puncture. The mice of propofol group and propofol and H 2 group were given 50 mg/kg propofol through tail vein at 1 hour and 6 hours after CLP and the mice of H2 group and propofol and H 2 group were given 5 mL/kg H 2 -rich saline i.p. at 1 hour and 6 hours after CLP. The survival rates were observed during 7 days in twenty mice of each group. Inferior vena cava blood and part lung, liver and kidney tissue were collected for detection of the concentration of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and high mobility group box 1 (HMGB1) at 24 hours after CLP in the 40 animals left (each n = 8). Then, the rest tissue of lung, liver and kidney tissue were harvested to test histopathology and histological score., Results: The 1, 2, 3, 5, 7 days survival rate of septic mice were 80%, 40%, 20%, 10%, and 0%, respectively. The survival rate of animals increased significantly after propofol or hydrogen-rich treatment, and the combined treatment can further increase survival rate to 90%, 75%, 60%, 55%, and 55%, respectively. Compared with the sham group, inflammatory factors were significantly increased in blood and organ tissues, cell degeneration, necrosis, congestion and inflammatory cell infiltration in lung, liver and kidney, and tissues histological scores were significantly increased. The levels of inflammatory factors were reduced in blood and tissues, cell degeneration, necrosis, congestion and inflammatory cell infiltration were alleviated in lung, liver and kidney, and tissues histological scores were decreased after propofol or hydrogen-rich treatment compared with CLP group; these indicators were further improved in propofol and H 2 group compared with propofol group or H2 group [2, 3, 5, 7-day survival rate: 75% vs. 60%, 65%; 60% vs. 50%, 50%; 55% vs. 45%, 40%; 55% vs. 40%, 40%; blood TNF-α (ng/L): 367±74 vs. 612±132, 588±117; blood IL-1β (ng/L): 321±68 vs. 502±95, 476±86; blood HMGB1 (μg/L): 4.6±0.9 vs. 7.0±1.4, 6.8±1.3; lung TNF-α (ng/g): 307±70 vs. 512±132, 488±102; lung IL-1β (ng/g): 367±77 vs. 571±108, 466±89; lung HMGB1 (μg/g): 5.1±1.0 vs. 7.8±1.7, 7.1±1.5; liver TNF-α (ng/g): 247±57 vs. 431±112, 389±87; liver IL-1β (ng/g): 267±58 vs. 417±85, 399±76; liver HMGB1 (μg/g): 4.2±1.1 vs. 7.1±1.6, 6.6±1.2; kidney TNF-α (ng/g): 257±41 vs. 480±89, 448±82; kidney IL-1β (ng/g): 258±39 vs. 409±68, 411±66; kidney HMGB1 (μg/g): 3.9±0.7 vs. 6.8±1.2, 5.7±1.0; histological scores: lung: 1.22±0.28 vs. 2.61±0.49, 2.58±0.44; liver: 1.38±0.32 vs. 2.76±0.51, 2.62±0.46; kidney: 1.19±0.25 vs. 2.43±0.41, 2.36±0.40; all P < 0.05]., Conclusions: Both propofol and H 2 can improve the survival rate of sepsis, reduce tissue damage and the release of cytokines, and combined application of the two treatment was better.

Objective: To investigate the role of autophagy in hydrogen-rich saline attenuating post-herpetic neuralgia( PHN) in rats., Methods: A total of 100 male SD rats were randomly divided into the five groups( n = 20) : control group,PHN group,PHN group treated with hydrogen-rich saline( PHN-H2group),PHN group treated with hydrogen-rich saline and3-MA( PHN-H2-3-MA group),PHN group treated with hydrogen-rich saline and rapamycin( PHN-H2-Rap group). PHN models were established by varicella-zoster virus( VZV) inoculation. After modeling,15 mg / kg 3-MA or 10 mg / kg rapamycin were intraperitoneally injected in corresponding rats with PHN once two days for 3 times. Hydrogen-rich saline( 10 m L / kg)was injected intraperitoneally twice a day for 7 consecutive days in PHN-H2 group,PHN-H2-3-MA group and PHN-H2-Rap group after VZV injection. The paw withdrawal thresholds( PWT) of 50 rats were detected at 3,7,14 and 21 days after modeling. Spinal cord enlargements of the other 50 rats were collected to examine tumor necrosis factor α( TNF-α),interleukine 1β( IL-1β) and IL-6 by ELISA and autophagy protein microtubule-associated protein 1 light chain 3( LC3),beclin 1and P62 by Western blotting., Results: Compared with the control group,the rats in the PHN group presented with decreased PWT,increased levels of TNF-α,IL-1β,IL-6,LC3Ⅱ and beclin 1,and down-regulated P62 expression. Compared with PHN group,the rats in the PHN-H2 group and PHN-H2-Rap group showed increased PWT,decreased levels of TNF-α,IL-1β and IL-6,further up-regulated expressions of LC3 and beclin 1 as wel as P62 expression. Compared with PHN-H2 group,the rats in the PHN-H2-3-MA group had reduced PWT,elevated expressions of TNF-α,IL-1β and IL-6,suppressed expressions of LC3 and beclin 1,and enhanced p62 expression., Conclusion: Hydrogen-rich saline attenuated PWT and inhibited the release of cytokines TNF-α,IL-1β,IL-6 in rats with PHN via activating autophagy.

Objective: To investigate the protective effects and underlying molecular mechanisms of hydrogen (H2) on high glucose-induced poly (ADP-ribose) polymerase-1 (PARP-1) dependent cell death (PARthanatos) in primary rat Schwann cells., Methods: Cultured primary rat Schwann cells were randomly divided into five groups: blank control group (C group), H2 control group (H2 group), high osmotic control group (M group), high glucose treatment group (HG group), and H2 treatment group (HG+H2 group). The cells in H2 group and HG+H2 group were cultured with saturated hydrogen-rich medium containing 0.6 mmol/L of H2, and those in three control groups were cultured with low sugar DMEM medium containing 5.6 mmol/L of sugar, and the cells in HG and HG+H2 groups were given 44.4 mmol/L of glucose in addition (the medium containing 50 mmol/L of glucose), the cells in C group and H2 group were given the same volume of normal saline, and the cells in M group were given the same volume of mannitol. Cytotoxicity was evaluated using lactate dehydrogenase (LDH) release rate assays after treatment for 48 hours in each group. The contents of peroxynitrite (ONOO(-)) and 8-hydroxy-2-deoxyguanosine (8-OHdG) reflecting oxidative stress injury and DNA damage were detected by enzyme linked immunosorbent assay (ELISA). Poly (ADP-ribose) (PAR) protein expression was analyzed by Western Blot, and immunofluorescence staining was used to determine the nuclear translocation of the apoptosis-inducing factor (AIF)., Results: The cytotoxicity in HG and HG+H2 groups was significantly increased as compared with that of C group [LDH release rate: (61.40±2.89)%, (42.80±2.32)% vs. (9.92±0.38)%, both P < 0.01], the levels of ONOO(-) and 8-OHdG were markedly elevated [ONOO(-) (ng/L): 853.58±51.00, 553.11±38.66 vs. 113.56±14.22; 8-OHdG (ng/L): 1?177.37±60.97, 732.06±54.29 vs. 419.67±28.77, all P < 0.01], and the PAR protein expression was up-regulated (A value: 0.603±0.028, 0.441±0.010 vs. 0.324±0.021, both P < 0.01). The cytotoxicity, the levels of ONOO(-) and 8-OHdG, and PAR expression in HG+H2 group were significantly lower than those of the HG group (all P < 0.01). There were no significant differences in above parameters between H2 group as well as M group and C group. It was shown by immunofluorescence that AIF was expressed in the cytoplasm in C group, H2 group and M group, AIF was expressed in the whole cell in HG group, and the expression in the nucleus was particularly increased. A small amount of AIF expression was found in the nucleus of HG+H2 group, which indicated that high glucose could promote the AIF nuclear translocation, and that hydrogen-rich medium could prevent the process of translocation., Conclusions: High glucose levels could enhance DNA damage that enhance PARthanatos in primary rat Schwann cells. However, H2 can not only reduce DNA damage of injured cells, but also inhibit the special death process, reduce the cell toxicity, all of which have protective effects.

Objective: To investigate the effects of hydrogen-rich medium on lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction of human intestinal epithelial (Caco2) cells., Methods: Caco2 cells (passages 28-35) were purchased from the Cell Bank of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences in Shanghai, China, and they were cultured in Dulbecco minimum essential medium (DMEM) containing 20% fetal bovine serum. These cells were randomly divided into four groups: control group (group A), hydrogen-rich medium group (group B), LPS group (group C) and LPS + hydrogen-rich medium group (group D). Cells were cultured with normal medium in group A and group C or with hydrogen-rich medium in group B and group D. Meanwhile, 1 g/L LPS was simultaneously added into group C and group D, while an equivalent volume of normal saline was added into group A and group B instead. In vitro intestinal epithelial models were reproduced with monolayer filter-grown Caco2 and intestinal epithelium. The trans-epithelial electrical resistance (TEER) in models of each group was measured at different incubation times (0, 3, 6, 12, 24 and 48 hours). Cell viability and cytotoxicity were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release assay, respectively, after incubation for 24 hours. The expression levels of claudin-1 and occludin were respectively determined at 6, 12 and 24 hours of incubation by Western Blot assay. The morphological structure of claudin-1 and occludin was respectively observed after incubation for 24 hours with immunofluorescence staining., Results: There was no statistical significance in variables between group A and group B. Compared with group A, it was shown that TEER was time-dependently decreased in groups C and D after 6 hours. Compared with group C, TEER in group D was increased after 6 hours. Compared with group A, the cell viability was significantly reduced in group C [(67.2±7.9)% vs. (100.0±0.0)%, P < 0.05] and cell injury was obvious [LDH release rate: (38.5±2.1)% vs. (1.2±0.3)%, P < 0.05]; the expression levels of claudin-1 and occludin at 6, 12, 24 hours were significantly down-regulated [claudin-1 (gray value): 0.351±0.079, 0.272±0.075, 0.190±0.049 vs. 0.518±0.030; occludin (gray value): 0.416±0.044, 0.290±0.062, 0.226±0.019 vs. 0.602±0.038, all P < 0.05], and the structure of claudin-1 and occludin were profoundly disrupted. Compared with group C, it was shown that the cell viability was significantly increased in group D [(88.8±7.4)% vs. (67.2±7.9)%, P < 0.05] and cell injury was significantly abated [LDH release rate: (16.4±4.3)% vs. (38.5±2.1)%, P < 0.05]; the expression levels of claudin-1 and occludin were significantly up-regulated at 24 hours [claudin-1 (gray value): 0.428±0.046 vs. 0.190±0.049, occludin (gray value): 0.466±0.071 vs. 0.226±0.019, both P < 0.05]; the disrupted structures of claudin-1 and occludin were partially recovered., Conclusion: Hydrogen-rich medium can effectively attenuate LPS-induced dysfunction of intestinal epithelial barrier in human Caco2 cells by ameliorating cell viability as well as regulating claudin-1 and occludin expression and structure.

Neuropathic pain (NP) is characterized by persistent pain, tactile allodynia, or hyperalgesia. Peripheral nerve injury contributes to rapid progress of inflammatory response and simultaneously generates neuropathic pain. Hydrogen (H2) has anti-inflammation, anti-apoptosis, and anti-oxidative stress effects. Therefore, we hypothesized that H2 treatment could alleviate allodynic and hyperalgesic behaviors and the release of inflammatory factors in rats with neuropathic pain. Peripheral neuropathic pain was established by chronic constriction injury of sciatic nerve in rats. H2 was given twice through intraperitoneal injection at a daily dose of 10 mL/kg during days 1-7 after the operation. Hyperalgesia and allodynia were tested, pro-inflammatory factors of dorsal root ganglia (DRG) and the spinal cord were measured by enzyme-linked immunosorbent assay (ELISA) during days 1-14 after the operation, and heme oxygenase (HO)-1 messenger RNA (mRNA) and protein expression and activities were measured at day 14 after sciatic nerve injury in rats. After Sn (IV) protoporphyrin IX dihydrochloride (SnPP)-IX, hemin, and carbon monoxide-releasing molecule (CORM)-2 had been given for chronic constriction injury (CCI) in rats, the above indicators were assessed. We found that H2 clearly inhibited hyperalgesia and allodynia in neuropathic pain and also attenuated the pro-inflammatory cytokines TNF-α, IL-1β, and high-mobility group box (HMGB) 1. H2 improved HO-1 mRNA and protein expression and activities in the process of pain. SnPP-IX reversed the inhibitory effect of H2 on hyperalgesia and allodynia and on pro-inflammatory cytokines in DRG and the spinal cord. The antinociceptive and anti-inflammatory effects of H2 were involved in the activation of HO-1/CO signaling during neuropathic pain in rats.

Background: We revealed that a high concentration of sevoflurane exacerbated cognitive impairment in aged rats, and the inhibition of GluA2 subunit internalization facilitated neuroprotection after a cerebral ischemic injury. However, the trafficking of GluA2 in POCD and its underlying mechanism are not clear. We thus detected the effects of sevoflurane for different inhalation durations on postoperative cognitive function and investigated the role of GluA2 subunit trafficking in this process., Methods: A rat model of orthopedic surgery was performed with different durations of 1.5 MAC sevoflurane inhalation. Cognitive function was evaluated by manipulating the Y maze and fear conditioning tests for 7 days after experiments. Western blot, ELISA and coimmunoprecipitation were applied to analyze GluA2 internalization, PI3K expression and its activity, as well as alterations to the MEF2-Arc pathway in the hippocampus. Neuron apoptosis and the spine morphology in the hippocampus were also observed., Results: We found that neuron apoptosis and GluA2 internalization increased following surgery and 1.5 MAC sevoflurane inhalation for 2h, possibly due to the decrease of the PI3K-GluA2 complex and PI3K activity in the hippocampus after prolonged 1.5 MAC sevoflurane inhalation. We also observed that the MEF2-Arc pathway contributed to long-term cognitive function, which also impaired the spinal morphology after 1.5 MAC sevoflurane inhalation for 2h., Conclusion: The above results suggest that 1.5 MAC sevoflurane inhalation for 2h potentiated surgery-impaired cognitive function and that the inhibition of PI3K-AMPAR GluA2 as well as activation of the MEF2-Arc signal pathway contributes to different stages of POCD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To evaluate protective effects of inhaled hydrogen gas (H2) on cognitive function in a murine model of sepsis-associated encephalopathy (SAE)., Methods: A total of 84 male ICR mice, weighing 20-25 g, aged 6-8 weeks, were randomly divided into 4 groups of sham, sham+H2, sepsis and sepsis+H2. Sepsis was established by cecal ligation and puncture (CLP). Mice in sham+H2 and sepsis+H2 groups received 2% H2 inhalation for 1 h at 1 h and 6 h after sham operation or CLP operation respectively. The changes of neurological function and neuronal damage in hippocampal CA1 region were observed at 24 h post-operation. The activities of superoxide dismutase (SOD) and catalase (CAT) and the levels of malondialdehyde (MDA) and 8-iso-prostaglandin F2α (8-iso-PGF2α) in sera and hippocampus were detected at 24 h post-operation. The changes of cognitive function were observed by Y-maze test and fear conditional test at days 3 to 14 post-operation., Results: Compared with sham group, the neurological function significantly declined and neurons in hippocampal CA1 region were significantly damaged; the activities of SOD and CAT markedly decreased while the levels of MDA and 8-iso-PGF2α markedly increased in sera and hippocampus; the time in new zone and the percentage of freezing time dramatically decreased at days 3 to 14 post-operation in sepsis group (P < 0.05) . Compared with sepsis group, neurological function significantly improved and damaged neurons in hippocampal CA1 region significantly reduced; the activities of SOD and CAT markedly increased and the levels of MDA and 8-iso-PGF2α markedly decreased in sera and hippocampus; the time in new zone and the percentage of freezing time dramatically increased at days 3 to 14 post-operations in sepsis+H2 group (P < 0.05)., Conclusion: H2 inhalation can significantly alleviate neuronal damage and improve cognitive dysfunction in CLP-induced SAE mice. And it is probably associated with the increased activities of antioxidant enzymes and the reduced levels of oxidative products.

Objective: To investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in the protective effects of hydrogen against cerebral dysfunction in a mouse model of sepsis., Methods: Male ICR mice were randomly divided into sham operation group, hydrogen control group, sepsis group and hydrogen treatment group, with 20 in each group. Sepsis model was reproduced by cecal ligation and puncture (CLP). 2% hydrogen inhalation was given for 1 hour at 1 hour and 6 hours after operation in hydrogen treatment group. The brain tissues were obtained at 24 hours after operation. The histopathologic changes and neuron apoptosis in the hippocampus were observed under the microscope. The expressions of nucleus and total Nrf2 in hippocampus were detected by Western Blot. The activities of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and 8-iso-prostaglandin F2α (8-iso-PGF2α) in hippocampus were also detected. The changes of learning and memory abilities were observed by Morris water maze test at day 4 to 9 after operation., Results: Compared with the sham operation and hydrogen control groups, in the sepsis group, the number of normal pyramidal neurons in the hippocampal CA1 region was markedly reduced, the apoptotic index was marked increased, the expressions of nucleus and total Nrf2 were partly increased, the activities of SOD and CAT in the hippocampus were significantly decreased, and the levels of MDA and 8-iso-PGF2α were markedly increased, the escape latency at day 4 to 8 after operation was significantly extended, and there was no difference in swimming speed, the percentage of time in the target quadrant and the times of the platform crossing were significantly decreased on probe day. Compared with the sepsis group, in the hydrogen treatment group, the number of normal pyramidal neurons in the hippocampal CA1 region was markedly increased (67.33 ± 6.89 vs. 42.33 ± 6.02, P<0.01), the apoptotic index was dramatically reduced [(30.00 ± 4.77)% vs. (80.50 ± 6.99)%, P<0.01], the expressions of nucleus and total Nrf2 were significantly increased [nucleus Nrf2 (A value): 5.07 ± 0.35 vs. 3.04 ± 0.34, total Nrf2 (A value): 4.24 ± 0.58 vs. 2.91 ± 0.37, both P<0.01], the activities of SOD and CAT in the hippocampus were significantly increased [SOD (U/mg): 120.96 ± 13.44 vs. 81.16 ± 12.28, CAT (U/mg): 9.11 ± 1.28 vs. 5.64 ± 1.88, both P<0.01], and the levels of MDA and 8-iso-PGF2α were markedly reduced [MDA (nmol/mg): 16.12 ± 1.49 vs. 27.64 ± 1.87, 8-iso-PGF2α (pg/mg): 183.43 ± 13.07 vs. 864.07 ± 49.92, both P<0.01], the escape latency at day 5 to 8 after operation was significantly shortened, and there was no difference in swimming speed, the percentage of time in the target quadrant [(37.06 ± 1.16)% vs. (24.42 ± 1.82)%, P<0.01] and the times of the platform crossing (7.13 ± 0.98 vs. 4.88 ± 0.99, P<0.01) were significantly increased on probe day. There was no statistical difference in above indexes between sham operation group and hydrogen control group., Conclusions: Hydrogen inhalation can ameliorate pathological injury in brain and impairment of learning and memory abilities of septic mice, which may be associated with the up-regulation of Nrf2, the increase of antioxidant enzymes activities and the decrease of oxidative products.

Objective: To investigate the role of Nrf2 on hydrogen treatment for intestinal injury caused by severe sepsis., Methods: 152 male ICR mice were randomly divided into four groups: sham operation group, hydrogen control group, sepsis group, and hydrogen treatment group, each n=38. Sepsis model was reproduced by cecal ligation and puncture (CLP). The mice in sham operation group and hydrogen control group did not receive CLP, and the operative procedure was the same as follows. The mice in hydrogen control group and hydrogen treatment group received 1-hour inhalation of 2% hydrogen 1 hour and 6 hours after sham operation or CLP. Twenty animals in each group were selected and observed for 7-day survival rate.Eighteen animals in each group were selected and sacrificed at 6, 12 and 24 hours after CLP. The intestinal tissues were obtained to determine the expression of Nrf2 and high mobility group B1 (HMGB1) protein by Western Blot, and the expression of Nrf2 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The middle portion of jejunum was obtained to evaluate the degree of septic injury by light microscope after hematoxylin and eosin (HE) staining., Results: There was no statistical significance in variables between sham operation group and hydrogen control group. Compared with sham operation group, the 7-day survival rate was significantly decreased in sepsis group (0 vs. 100%, P<0.05); compared with sepsis group, the 7-day survival rate was significantly increased in hydrogen treatment group (55% vs. 0, P<0.05). Compared with sham operation group, the expression of Nrf2 protein (gray value) and Nrf2 mRNA were up-regulated in sepsis group at 6, 12 and 24 hours after CLP (Nrf2 protein 6 hours: 1.973 ± 0.350 vs. 1.000 ± 0.000, t=4.411, P=0.002; 12 hours: 2.367 ± 0.186 vs. 1.000 ± 0.000, t=10.210, P=0.000; 24 hours: 2.517 ± 0.280 vs. 1.000 ± 0.000, t=9.521, P=0.000; Nrf2 mRNA 6 hours: 1.606 ± 0.271 vs. 1.000 ± 0.000, t=3.631, P=0.002; 12 hours: 1.692 ± 0.399 vs. 1.000 ± 0.000, t=3.233, P=0.005; 24 hours: 1.784 ± 0.341 vs. 1.000 ± 0.000, t=3.894, P=0.001), and it was also the expression of HMGB1 (gray value) at 24 hours after CLP operation (1.507 ± 0.220 vs. 1.000 ± 0.000, t=3.948, P=0.004). Compared with sepsis group, the expression of Nrf2 protein and Nrf2 mRNA in intestines were up-regulated at 6, 12 and 24 hours after CLP in hydrogen treatment group (Nrf2 protein 6 hours: 2.583 ± 0.395 vs. 1.973±0.350, t=2.765, P=0.024; 12 hours: 2.725 ± 0.235 vs. 2.367 ± 0.186, t=2.674, P=0.028; 24 hours: 2.930 ± 0.212 vs. 2.517 ± 0.280, t=2.595, P=0.032; Nrf2 mRNA 6 hours: 2.008 ± 0.400 vs. 1.606±0.271, t= 2.405, P=0.029; 12 hours: 2.188 ± 0.475 vs. 1.692 ± 0.399, t= 2.317, P=0.034; 24 hours: 2.333 ± 0.406 vs. 1.784 ± 0.341, t= 2.728, P=0.015). Compared with sepsis group, the expression of HMGB1 was down-regulated significantly at 24 hours after CLP in hydrogen treatment group (1.147 ± 0.152 vs. 1.507 ± 0.220, t=2.805, P=0.023). HE staining showed that there was significantly aggravated intestinal pathological injury in the mice of sepsis group; compared with sepsis group, the pathology was significantly less marked in hydrogen treatment group., Conclusions: Through activation of Nrf2-antioxidant response element (ARE) pathway, hydrogen may increase the level of Nrf2, which is a kind of protective protein, in the intestine of mice, thus decreases the level of late pro-inflammatory factor, HMGB1, and it may protect the intestinal tissues in septic mice and increase the survival rate significantly.

Cervical radiculopathy is a common disease in clinical practice. However, the symptoms are not confined to the affected spinal cord segment indicated by magnetic resonance imaging (MRI) findings. In the present study, we measured c-Fos and c-Jun expression in ipsilateral and adjacent cervical spinal cord segments following C 7 nerve root rhizotomy, to determine whether there is a neural pathway between adjacent cervical spinal cord segments. Forty-eight adult male Wistar rats were randomly divided into two groups: the C 7 rhizotomy group (rhizotomy group, n=24) and the sham-operated group (sham group, n=24). The right C 7 nerve root was completely cut off in the rhizotomy group, while it was exposed but not cut in the sham group. The expression of c-Fos and c-Jun in cervical spinal cord segments was detected by immunohistochemistry at 2 and 4 h after surgery. We observed that the number of c-Fos- and c-Jun-positive neurons in ipsilateral C 5-7 segments were significantly increased at 2 and 4 h after C 7 nerve root rhizotomy (P<0.05 vs. the sham group). The location of c-Fosand c-Jun-positive neurons in C 5-7 gray matter was similar in the rhizotomy and sham groups, which was mainly in lamina IX of the anterior horn and laminae I-II of the dorsal horn of the spinal cord. However, the number of c-Fos- and c-Jun-positive neurons in the C 5-7 gray matter was significantly reduced at 4 h after surgery compared with the number 2 h after surgery. The location of c-Fos- and c-Jun-positive neurons at 4 h was similar with that at 2 h. Therefore, there may be a neural pathway between ipsilateral adjacent cervical spinal cord segments. This may be one possible explanation as to why the radicular symptoms of cervical radiculopathy are not confined to the affected spinal cord segment shown by MRI.