Guolin Wang

PROTECTIVE EFFECTS OF HYDROGEN GAS ON MURINE POLYMICROBIAL SEPSIS VIA REDUCING OXIDATIVE STRESS AND HMGB1 RELEASE

Despite recent advances in antibiotic therapy and intensive care, sepsis is still considered to be the most common cause of death in intensive care units. Excessive production of reactive oxygen species plays an important role in the pathogenesis of sepsis. Recently, it has been suggested that molecular hydrogen (H2) exerts a therapeutic antioxidant activity by selectively reducing hydroxyl radicals (•OH, the most cytotoxic reactive oxygen species) and effectively protects against organ damage induced by I/R. Therefore, we hypothesized that H2 treatment had a beneficial effect on sepsis. In the present study, we found that H2 inhalation starting at 1 and 6 h after cecal ligation and puncture (CLP) or sham operation significantly improved the survival rate of septic mice with moderate or severe CLP in a concentration- and time-dependent manner. Furthermore, moderate or severe CLP mice showed significant multiple organ damage characterized by the increases of lung myeloperoxidase activity, wet-to-dry weight ratio, protein concentration in bronchoalveolar lavage, serum biochemical parameters, and organ histopathologic scores at 24 h after CLP operation, which was significantly attenuated by 2% H2 treatment. In addition, we found that the beneficial effects of H2 treatment on sepsis and sepsis-associated organ damage were associated with the decreased levels of oxidative product, increased activities of antioxidant enzymes, and reduced levels of high-mobility group box 1 in serum and tissue. Thus, H2 inhalation may be an effective therapeutic strategy for patients with sepsis.ABBREVIATIONS-ALI-acute lung injury; ALT-alanine aminotransferase; AST-aspartate aminotransferase; BAL-bronchoalveolar lavage; BUN-blood urea nitrogen; CAT-catalase; CLP-cecal ligation and puncture; Cr-creatinine; H2-hydrogen; H2O2-hydrogen peroxide; HMGB1-high-mobility group box 1; 8-iso-PGF2&agr;-8-iso-prostaglandin F2&agr;; MPO-myeloperoxidase; •OH-hydroxyl radicals; ROS-reactive oxygen species; SOD-superoxide dismutase; W/D-wet-to-dry

Molecular hydrogen ameliorates lipopolysaccharide-induced acute lung injury in mice through reducing inflammation and apoptosis.

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our and other studies have found that hydrogen gas (H2) treatment can ameliorate the lung injury induced by sepsis, ventilator, hyperoxia, and ischemia-reperfusion. However, the molecular mechanisms by which H2 ameliorates lung injury remain unclear. In the current study, we investigated whether H2 or hydrogen-rich saline (HS) could exert protective effects in a mouse model of ALI induced by intratracheal administration of lipopolysaccharide (LPS) via inhibiting the nuclear factor &kgr;B (NF-&kgr;B) signaling pathway–mediated inflammation and apoptosis. Two percent of H2 was inhaled for 1 h beginning at 1 and 6 h after LPS administration, respectively. We found that LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology and histologic scores, wet-to-dry weight ratio, and oxygenation index (PaO2/FIO2), as well as total protein in the bronchoalveolar lavage fluid (BALF), which was attenuated by H2 treatment. Hydrogen gas treatment inhibited LPS-induced pulmonary early and late NF-&kgr;B activation. Moreover, H2 treatment dramatically prevented the LPS-induced pulmonary cell apoptosis in LPS-challenged mice, as reflected by the decrease in TUNEL (deoxynucleotidyl transferase dUTP nick end labeling) staining–positive cells and caspase 3 activity. Furthermore, H2 treatment markedly attenuated LPS-induced lung neutrophil recruitment and inflammation, as evidenced by downregulation of lung myeloperoxidase activity, total cells, and polymorphonuclear neutrophils in BALF, as well as proinflammatory cytokines (tumor necrosis factor &agr;, interleukin 1&bgr;, interleukin 6, and high-mobility group box 1) and chemokines (keratinocyte-derived chemokine, macrophage inflammatory protein [MIP] 1&agr;, MIP-2, and monocyte chemoattractant protein 1) in BALF. In addition, i.p. injection of 10 mL/kg hydrogen-rich saline also significantly attenuated the LPS-induced ALI. Collectively, these results demonstrate that molecular hydrogen treatment ameliorates LPS-induced ALI through reducing lung inflammation and apoptosis, which may be associated with the decreased NF-&kgr;B activity. Hydrogen gas may be useful as a novel therapy to treat ALI. ABBREVIATIONS ALI—acute lung injury; ARDS—acute respiratory distress syndrome; BALF—bronchoalveolar lavage fluid; ELISA—enzyme-linked immunosorbent assay; H2—hydrogen gas; HMGB1—high-mobility group box 1; HS—hydrogen-rich saline; i.t.—intratracheal; KC—keratinocyte-derived chemokine; LPS—lipopolysaccharide; MCP-1—monocyte chemoattractant protein 1; MIP-1&agr;—macrophage inflammatory protein 1&agr;; MIP-2—macrophage inflammatory protein 2; MPO—myeloperoxidase; PBS—phosphate-buffered saline; PMNs—polymorphonuclear neutrophils; TUNEL—deoxynucleotidyl transferase dUTP nick end labeling; W/D—wet-to-dry

HYDROGEN GAS IMPROVES SURVIVAL RATE AND ORGAN DAMAGE IN ZYMOSAN-INDUCED GENERALIZED INFLAMMATION MODEL

Sepsis/multiple organ dysfunction syndrome is the leading cause of death in critically ill patients. Recently, it has been suggested that hydrogen gas (H2) exerts a therapeutic antioxidant activity by selectively reducing hydroxyl radical (•OH, the most cytotoxic reactive oxygen species). We have found that H2 inhalation significantly improved the survival rate and organ damage of septic mice with moderate or severe cecal ligation and puncture. In the present study, we investigated the effects of 2% H2 treatment on survival rate and organ damage in zymosan (ZY)-induced generalized inflammation model. Here, we found that 2% H2 inhalation for 60 min starting at 1 and 6 h after ZY injection, respectively, significantly improved the 14-day survival rate of ZY-challenged mice from 10% to 70%. Furthermore, ZY-challenged mice showed significant multiple organ damage characterized by the increase in serum biochemical parameters (aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and creatinine), as well as lung, liver, and kidney histopathological scores at 24 h after ZY injection, which was significantly attenuated by 2% H2 treatment. In addition, we found that the beneficial effects of H2 treatment on ZY-induced organ damage were associated with the decreased levels of oxidative product, increased activities of antioxidant enzyme, and reduced levels of early and late proinflammatory cytokines in serum and tissues. In conclusion, this study provides evidence that H2 treatment protects against multiple organ damages in ZY-induced generalized inflammation model, suggesting the potential use of H2 as a therapeutic agent in the therapy of conditions associated with inflammation-related multiple organ dysfunction syndrome.ABBREVIATIONS-8-iso-PGF2&agr;-8-iso-prostaglandin F2&agr;; ALT-alanine aminotransferase; AST-aspartate aminotransferase; BUN-blood urea nitrogen; CLP-cecal ligation and puncture; Cr-creatinine; H2-hydrogen; H2O2-hydrogen peroxide; HMGB1-high-mobility group box 1; MODS-multiple organ dysfunction syndrome; NS-normal saline; •OH-hydroxyl radical; ROS-reactive oxygen species; SOD-superoxide dismutase; ZY-zymosan

Role of TRPM8 in dorsal root ganglion in nerve injury-induced chronic pain

BackgroundChronic neuropathic pain is an intractable pain with few effective treatments. Moderate cold stimulation can relieve pain, and this may be a novel train of thought for exploring new methods of analgesia. Transient receptor potential melastatin 8 (TRPM8) ion channel has been proposed to be an important molecular sensor for cold. Here we investigate the role of TRPM8 in the mechanism of chronic neuropathic pain using a rat model of chronic constriction injury (CCI) to the sciatic nerve.ResultsMechanical allodynia, cold and thermal hyperalgesia of CCI rats began on the 4th day following surgery and maintained at the peak during the period from the 10th to 14th day after operation. The level of TRPM8 protein in L5 dorsal root ganglion (DRG) ipsilateral to nerve injury was significantly increased on the 4th day after CCI, and reached the peak on the 10th day, and remained elevated on the 14th day following CCI. This time course of the alteration of TRPM8 expression was consistent with that of CCI-induced hyperalgesic response of the operated hind paw. Besides, activation of cold receptor TRPM8 of CCI rats by intrathecal application of menthol resulted in the inhibition of mechanical allodynia and thermal hyperalgesia and the enhancement of cold hyperalgesia. In contrast, downregulation of TRPM8 protein in ipsilateral L5 DRG of CCI rats by intrathecal TRPM8 antisense oligonucleotide attenuated cold hyperalgesia, but it had no effect on CCI-induced mechanical allodynia and thermal hyperalgesia.ConclusionsTRPM8 may play different roles in mechanical allodynia, cold and thermal hyperalgesia that develop after nerve injury, and it is a very promising research direction for the development of new therapies for chronic neuroapthic pain.

The role of phosphoinositide-3-kinase/Akt pathway in propofol-induced postconditioning against focal cerebral ischemia-reperfusion injury in rats.

The aim of this study was to investigate whether propofol could provide postconditioning to ischemic brain injury and the role of phosphoinositide-3-kinase/Akt (PI3K/Akt) pathway in this phenomenon. Rats underwent 2 h of middle cerebral artery occlusion (MCAO) followed by 22 h of reperfusion were randomly divided into nine groups (n=15 each): sham-operated group, MCAO group, propofol 10, 20 and 35 mg x kg(-1) x h(-1) group (propofol 10, 20, 35 mg x kg(-1) x h(-1) infused at the onset of reperfusion for 30 min), wortmannin group (wortmannin 0.6 mg/kg administered 30 min before MCAO), and the other three groups received wortmannin followed by 10, 20 and 35 mg x kg(-1) x h(-1) propofol respectively. Propofol at doses of 10 and 20 mg x kg(-1) x h(-1) significantly reduced infarct volume, decreased neurological deficit scores and attenuated neuron apoptosis compared with MCAO group alone. Increased phosphorylated Akt (P-Akt) was observed in the ischemic penumbra of propofol 10 and 20 mg x kg(-1) x h(-1) group after transient MCAO. The selective PI3K inhibitor, wortmannin partly eliminated the neuroprotective effect and the elevation of P-Akt expression in ischemic penumbra induced by propofol. Propofol at dose of 35 mg x kg(-1) x h(-1) did not affect infarct volume, neurological deficit scores, neuronal apoptosis and the level of P-Akt in transient MCAO rats. Taken together, these results demonstrated that propofol at doses of 10 or 20 mg x kg(-1) x h(-1) infused at the onset of reperfusion for 30 min could provide neuroprotection to transient MCAO rats, and the postconditioning effect induced by propofol partly through maintaining the activity of PI3K/Akt pathway.

Beneficial effects of hydrogen gas against spinal cord ischemia-reperfusion injury in rabbits.

Recently, hydrogen gas (H₂) is reported to be a new therapeutic agent in organ damage induced by ischemia-reperfusion (I/R). The present study was designed to investigate the beneficial effects of H₂ against spinal cord I/R injury and its associated mechanisms. Spinal cord ischemia was induced by infrarenal aortic occlusion for 20 min in male New Zealand white rabbits. Treatment with 1%, 2% or 4% H₂ inhalation was given from 10 min before reperfusion to 60 min after reperfusion (total 70 min). Here, we found that I/R-challenged animals showed significant spinal cord damage characterized by the decreased numbers of normal motor neurons and hind-limb motor dysfunction, which was significantly improved by 2% and 4 % H₂ treatment. Furthermore, we found that the beneficial effects of H₂ treatment against spinal cord I/R injury were associated with the decreased levels of oxidative products [8-iso-prostaglandin F2α (8-iso-PGF2α) and malondialdehyde (MDA)] and pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and high-mobility group box 1 (HMGB1)], as well as increased activities of antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)] in serum and spinal cord. In addition, H₂ treatment reduced motor neuron apoptosis in the spinal cord of this model. Thus, H₂ inhalation may be an effective therapeutic strategy for spinal cord I/R damage.

Heme oxygenase-1 mediates the anti-inflammatory effect of molecular hydrogen in LPS-stimulated RAW 264.7 macrophages

BACKGROUNDnMolecular hydrogen (H2) as a new medical gas has an anti-inflammatory effect. In the present study, we investigated whether heme oxygenase-1 (HO-1) contributes to the anti-inflammatory effect of H2 in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.nnnMETHODSnRAW 264.7 macrophages were stimulated by LPS (1 μg/mL) with presence or absence of different concentrations of H2. Cell viability and injury were tested by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay and lactate dehydrogenase (LDH) release, respectively. The cell culture supernatants were collected to measure inflammatory cytokines [TNF-α, IL-1β, HMGB1 (high mobility group box-1) and IL-10] at different time points. Moreover, HO-1 protein expression and activity were tested at different time points. In addition, to further identify the role of HO-1 in this process, zinc protoporphyrin (ZnPP)-IX, an HO-1 inhibitor, was used.nnnRESULTSnH2 treatment had no significant influence on cell viability and injury in normally cultured RAW 264.7 macrophages. Moreover, H₂ treatment dose-dependently attenuated the increased levels of pro-inflammatory cytokines (TNF-α, IL-1β, HMGB1), but further increased the level of anti-inflammatory cytokine IL-10 at 3 h, 6 h, 12 h and 24 h after LPS stimulation. Furthermore, H₂ treatment could also dose-dependently increase the HO-1 protein expression and activity at 3 h, 6 h, 12 h and 24 h in LPS-activated macrophages. In addition, blockade of HO-1 activity with ZnPP-IX partly reversed the anti-inflammatory effect of H₂ in LPS-stimulated macrophages.nnnCONCLUSIONSnMolecular hydrogen exerts a regulating role in the release of pro- and anti-inflammatory cytokines in LPS-stimulated macrophages, and this effect is at least partly mediated by HO-1 expression and activation.

Protective effects of hydrogen-rich saline in a rat model of permanent focal cerebral ischemia via reducing oxidative stress and inflammatory cytokines☆

Hydrogen gas (H(2)) as a new medical gas exerts organ-protective effects through regulating oxidative stress, inflammation and apoptosis. In contrast to H(2), hydrogen-rich saline (HS) may be more suitable for clinical application. The present study was designed to investigate whether HS can offer a neuroprotective effect in a rat model of permanent focal cerebral ischemia and what mechanism(s) underlies the effect. Sprague-Dawley rats were subjected to permanent focal cerebral ischemia induced by permanent middle cerebral artery occlusion (pMCAO). Different doses of HS or normal saline were intraperitoneally administered at 5min after pMCAO or sham operation followed by injections at 6h, 12h and 24h. Here, we found that HS treatment significantly reduced infarct volume and improved neurobehavioral outcomes at 24h, 48h and 72h after pMCAO operation in a dose-dependent manner (P<0.05). Moreover, we found that HS treatment dose-dependently increased the activities of endogenous antioxidant enzymes (SOD and CAT) as well as decreased the levels of oxidative products (8-iso-PGF2α and MDA) and inflammatory cytokines (TNF-α and HMGB1) in injured ipsilateral brain tissues at 6h, 12h and 24h after pMCAO operation (P<0.05). Thus, hydrogen-rich saline dose-dependently exerts a neuroprotective effect against permanent focal cerebral ischemia, and its beneficial effect is at least partially mediated by reducing oxidative stress and inflammation. Molecular hydrogen may be an effective therapeutic strategy for stroke patients.

Combination therapy with molecular hydrogen and hyperoxia in a murine model of polymicrobial sepsis.

ABSTRACT Sepsis is the most common cause of death in intensive care units. Some studies have found that hyperoxia may be beneficial to sepsis. However, the clinical use of hyperoxia is hindered by concerns that it could exacerbate organ injury by increasing free radical formation. Recently, it has been suggested that molecular hydrogen (H2) at low concentration can exert a therapeutic antioxidant activity and effectively protect against sepsis by reducing oxidative stress. Therefore, we hypothesized that combination therapy with H2 and hyperoxia might afford more potent therapeutic strategies for sepsis. In the present study, we found that inhalation of H2 (2%) or hyperoxia (98%) alone improved the 14-day survival rate of septic mice with moderate cecal ligation and puncture (CLP) from 40% to 80% or 70%, respectively. However, combination therapy with H2 and hyperoxia could increase the 14-day survival rate of moderate CLP mice to 100% and improve the 7-day survival rate of severe CLP mice from 0% to 70%. Moreover, moderate CLP mice showed significant organ damage characterized by the increases in lung myeloperoxidase activity, lung wet-to-dry weight ratio, protein concentration in bronchoalveolar lavage, serum biochemical parameters (alanine aminotransferase, aspartate aminotransferase, creatinine, and blood urea nitrogen), and organ histopathological scores (lung, liver, and kidney), as well as the decrease in PaO2/FIO2 ratio at 24 h, which was attenuated by either H2 or hyperoxia alone. However, combination therapy with H2 and hyperoxia had a more beneficial effect against lung, liver, and kidney damage of moderate or severe CLP mice. Furthermore, we found that the beneficial effect of this combination therapy was associated with the decreased levels of oxidative product (8-iso-prostaglandin F2&agr;), increased activities of antioxidant enzymes (superoxide dismutase and catalase) and anti-inflammatory cytokine (interleukin 10), and reduced levels of proinflammatory cytokines (high-mobility group box 1 and tumor necrosis factor &agr;) in serum and tissues. Therefore, combination therapy with H2 and hyperoxia provides enhanced therapeutic efficacy via both antioxidant and anti-inflammatory mechanisms and might be potentially a clinically feasible approach for sepsis.

Sevoflurane postconditioning attenuates cerebral ischemia-reperfusion injury via protein kinase B/nuclear factor-erythroid 2-related factor 2 pathway activation

Oxidative damage plays a critical role in many diseases of the central nervous system. This study was conducted to determine the molecular mechanisms involved in the putative anti‐oxidative effects of sevoflurane against experimental stroke. Focal cerebral ischemia was performed via 1 h of middle cerebral artery occlusion followed by reperfusion. At the onset of reperfusion, rats were subjected to postconditioning with sevoflurane or without sevoflurane for 1 h. Neurological deficit score was assessed at different time points after reperfusion. Cerebral infarct volume, oxidative stress level and the binding activity of Nrf2 to antioxidant response element were assessed, meanwhile the expression of nuclear factor‐erythroid 2‐related factor 2 (Nrf2), quinine oxidoreductase 1 (NQO1), protein kinase B (Akt) and phosphor‐Akt was examined by Western blot at 72 h after reperfusion. Sevoflurane postconditioning administration significantly reduced neurological deficit score, infarct volume and oxidative stress levels, while increased the expression of phosphorylation Akt, NQO1, Nrf2 and the binding activity of Nrf2 to ARE in middle cerebral artery occlusion rats. These neuroprotective effects were all suppressed by LY294002, a selective PI3K blocker. Taken together, these findings provided evidence that sevoflurane postconditioning protects brain against ischemic/reperfusion injury, and this neuroprotective effect involves the Akt/Nrf2 pathway.