Hydrogen sulphide decreases IL‐1β‐induced activation of fibroblast‐like synoviocytes from patients with osteoarthritis

Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H₂S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H₂S on fibroblast‐like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro‐inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL‐1β and treated with the H₂S donor NaHS. Cellular responses were analysed by ELISA, quantitative real‐time PCR, phospho‐MAPkinase array and Western blotting. Treatment‐induced effects on cellular structure and synovial architecture were investigated in three‐dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL‐1β‐induced secretion of IL‐6, IL‐8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo‐proteinases MMP‐2 and MMP‐14. IL‐1β induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL‐1β‐induced activation of several MAPK whereas it increased phosphorylation of pro‐survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer‐like structure; stimulation with IL‐1β altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H₂S partially antagonizes IL‐1β stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA.     

Effects of Hydrogen Peroxide on Wound Healing in Mice in Relation to Oxidative Damage

It has been established that low concentrations of hydrogen peroxide (H₂O₂) are produced in wounds and is required for optimal healing. Yet at the same time, there is evidence that excessive oxidative damage is correlated with poor-healing wounds. In this paper, we seek to determine whether topical application of H₂O₂ can modulate wound healing and if its effects are related to oxidative damage. Using a C57BL/6 mice excision wound model, H₂O₂ was found to enhance angiogenesis and wound closure at 10 mM but retarded wound closure at 166 mM. The delay in closure was also associated with decreased connective tissue formation, increased MMP-8 and persistent neutrophil infiltration. Wounding was found to increase oxidative lipid damage, as measured by F₂-isoprostanes, and nitrative protein damage, as measured by 3-nitrotyrosine. However H₂O₂ treatment did not significantly increase oxidative and nitrative damage even at concentrations that delay wound healing. Hence the detrimental effects of H₂O₂ may not involve oxidative damage to the target molecules studied.     

Xanthine Oxidoreductase Function Contributes to Normal Wound Healing

Chronic, nonhealing wounds result in patient morbidity and disability. Reactive oxygen species (ROS) and nitric oxide (NO) are both required for normal wound repair, and derangements of these result in impaired healing. Xanthine oxidoreductase (XOR) has the unique capacity to produce both ROS and NO. We hypothesize that XOR contributes to normal wound healing. Cutaneous wounds were created in C57Bl6 mice. XOR was inhibited with dietary tungsten or allopurinol. Topical hydrogen peroxide (H₂O₂, 0.15%) or allopurinol (30 μg) was applied to wounds every other day. Wounds were monitored until closure or collected at d 5 to assess XOR expression and activity, cell proliferation and histology. The effects of XOR, nitrite, H₂O₂ and allopurinol on keratinocyte cell (KC) and endothelial cell (EC) behavior were assessed. We identified XOR expression and activity in the skin and wound edges as well as granulation tissue. Cultured human KCs also expressed XOR. Tungsten significantly inhibited XOR activity and impaired healing with reduced ROS production with reduced angiogenesis and KC proliferation. The expression and activity of other tungsten-sensitive enzymes were minimal in the wound tissues. Oral allopurinol did not reduce XOR activity or alter wound healing but topical allopurinol significantly reduced XOR activity and delayed healing. Topical H₂O₂ restored wound healing in tungsten-fed mice. In vitro, nitrite and H₂O₂ both stimulated KC and EC proliferation and EC migration. These studies demonstrate for the first time that XOR is abundant in wounds and participates in normal wound healing through effects on ROS production.     

EVIDENCE OF A LATENT OXIDATIVE BURST IN RELATION TO WOUND REPAIR IN THE GIANT UNICELLULAR CHLOROPHYTE DASYCLADUS VERMICULARIS1

We investigated the kinetics and composition of the second phase of the wound repair process of Dasycladus vermicularis ([Scropoli] Krasser) using fluorescent probes, chromatography, UV spectroscopy, and histochemistry. Our new evidence supports the hypothesis that the second phase of wound repair (initiated at approximately 35–45 min postinjury) is based on the activation of an oxidative burst that produces micromolar H₂O₂ levels. These results provide evidence of peroxidase activity at the wound site, real‐time measurements of an oxidative burst, and catechol localization in wound plugs. Strong evidence is presented indicating that the biochemical machinery exists for oxidative cross‐linking to ensue in the wound‐healing process of D. vermicularis.     

Wound-induced superoxide production and PAL activity decline with potato tuber age and wound healing ability

Wound healing of potato tubers involves the concerted action of several enzymes that facilitate polymerization of phenolics into suberin at the wound site. A decline in the efficiency of healing and resistance to pathogens with advancing tuber age was associated with reduced ability of older tubers to produce superoxide radicals (FRs) in response to wounding. Autophotographs of luminol‐treated longitudinal sections of tissue from 6‐, 18‐ and 30‐month‐old tubers revealed a substantial decline in superoxide production at the wound surface with advancing age. Older tubers were less able to respond to wounding by increasing phenylalanine ammonia lyase (PAL) activity. This enzyme produces t‐cinnamic acid, which constitutes a component of the phenolic domain of suberin, and is normally induced by wounding and/or ethylene. Interestingly, the ability of wounded tissue to oxidize exogenous 1‐aminocyclopropane‐1‐carboxylic acid (ACC) to C₂H₄ also decreased with advancing tuber age. The oxidation of ACC was inhibited by the FR scavenger, n‐propyl gallate (PG), and inhibition was greatest in tissue from younger tubers, reflecting their greater ability to produce superoxide radicals upon wounding. Regardless of tuber age, 1‐aminocyclobutane‐1‐carboxylic acid, an ACC oxidase inhibitor, did not inhibit C₂H₄ generation from exogenous ACC. Hence, C₂H₄ production from ACC by wounded tuber tissue is largely non‐enzymatic and FR‐driven, and thus serves as an indicator of the ability of wounded tissue to produce superoxide. Age‐induced reduction in PAL activity and FR production at the wound surface probably limited the oxidative polymerization of phenolics into suberin during wound periderm formation. The age‐induced loss in ability of wounded tissue to heal and resist pathogens is thus consistent with reduced synthesis and polymerization of phenolic adducts into suberin, a consequence of reduced FR and PAL activity at the wound surface.     

Senescence determines the fate of activated rat pancreatic stellate cells

In chronic pancreatitis (CP), persistent activation of pancreatic stellate cells (PSC) converts wound healing into a pathological process resulting in organ fibrosis. Here, we have analysed senescence as a novel mechanism involved in the termination of PSC activation and tissue repair. PSC senescence was first studied in vitro by establishing long‐term cultures and by applying chemical triggers, using senescence‐associated β‐Galactosidase (SA β‐Gal) as a surrogate marker. Subsequently, susceptibility of PSC to immune cell‐mediated cytolysis was investigated employing cocultures. Using the model of dibutyltin dichloride‐induced CP in rats, appearance of senescent cells was monitored by immunohistochemistry and immunofluorescence, and correlated with the progression of tissue damage and repair, immune cell infiltration and fibrosis. The results indicated that long‐term culture and exposure of PSC to stressors (doxorubicin, H₂O₂ and staurosporine) induced senescence. Senescent PSC highly expressed CDKN1A/p21, mdm2 and interleukin (IL)‐6, but displayed low levels of α‐smooth muscle actin. Senescence increased the susceptibility of PSC to cytolysis. In CP, the number of senescent cells correlated with the severity of inflammation and the extension of fibrosis. Areas staining positive for SA β‐Gal overlapped with regions of fibrosis and dense infiltrates of immune cells. Furthermore, a close physical proximity of immune cells and activated PSC was observed. We conclude that inflammation, PSC activation and cellular senescence are timely coupled processes which take place in the same microenvironment of the inflamed pancreas. Lymphocytes may play a dual‐specific role in pancreatic fibrogenesis, triggering both the initiation of wound healing by activating PSC, and its completion by killing senescent stellate cells.     

Ozone mediators effect on “in vitro” scratch wound closure

The beneficial effect of low doses of ozone on wound healing has been well documented and attributed mainly to its bactericidal and pro-oxidant properties. Because ozone itself does not penetrate the cells but immediately reacts with polyunsaturated fatty acids, its effects are the results of oxidative mediators. Among the molecule produces by the interaction of ozone with biological systems, there are HNE and H₂O₂. At today, the cellular mechanisms accounting for the positive effects of mild ozonization on wound closure are still largely unexplored. The aim of the present study was to evaluate the effect of different non-toxic doses of ozonated saline ranging from 2 to 300?μM, in an in vitro wound scratch model by the use of human keratinocytes. The results showed that ozonated saline is able to improve in vitro wound healing by stimulating cell proliferation as measured by BrdU assay and PCNA protein levels. In order to better elucidate the molecules that play the main role in the beneficial effect of ozonated saline in wound healing, HNE and H₂O₂ were used alone or in combination to mimic ozonated saline effect. Surprisingly, keratinocytes treated with different doses of HNE and H₂O₂ did not significantly improve the wound closure, while the combination of the two compounds was able to improve wound closure. In addition, Nrf2 pathways were also activated as determined by its translocation to the nucleus and the increased HO1 gene expression. The present work suggests that ozonated saline effect on wound closure is the results of the combination of more molecules among which HNE and H₂O₂ play a key role.     

Role of the cystathionine γ lyase/hydrogen sulfide pathway in human melanoma progression

In humans, two main metabolic enzymes synthesize hydrogen sulfide (H₂S): cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). A third enzyme, 3‐mercaptopyruvate sulfurtransferase (3‐MST), synthesizes H₂S in the presence of the substrate 3‐mercaptopyruvate (3‐MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non‐lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H₂S donors, the most active of which was diallyl trisulfide (DATS). The main pro‐apoptotic mechanisms involved were suppression of nuclear factor‐κB activity and inhibition of AKT and extracellular signal‐regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l ‐cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l ‐cysteine/CSE/H₂S pathway is involved in melanoma progression.     

The Responses of Pro‐ and Antioxidative Systems to Cold‐hardening and Pathogenesis Differ in Triticale (x Triticosecale Wittm.) Seedlings Susceptible or Resistant to Pink Snow Mould (Microdochium nivale Fr., Samuels & Hallett)

Two winter triticale (x Triticosecale Wittm.) cultivars, Magnat (susceptible to pink snow mould) and Hewo (relatively resistant), were used in a model system to test the effect of prehardening and different cold‐hardening regimes on pro‐ and antioxidative activity in seedling leaves. The concentration of hydrogen peroxide and the activity of total superoxide dismutase, catalase, peroxidase and ascorbic peroxidase were analysed spectrophotometrically. As there has been no previous analysis of the pro/antioxidative reaction of cereals to Microdochium nivale infection has been undertaken to‐date, this is the first in the series describing our results. We confirmed that both exposure to abiotic stress of low temperature and subsequent low light intensity, as well as biotic stress of M. nivale infection, change the pro‐ and antioxidative activity in model plants. Genotypes differed substantially in their hydrogen peroxide content: susceptible cv. Magnat generally showed higher levels during all the experiments. This result can lead to the conclusion that cv. Magnat is also more susceptible to low temperature and low light intensity than cv. Hewo. Simultaneous measurements of antioxidative activity indicated that the increased activity of catalases and peroxidases and the consequent lower H₂O₂ level are correlated with a higher resistance to low temperature, low light intensity and pink snow mould in triticale seedlings. The higher H₂O₂ level observed in the susceptible line is likely to be derived from the imbalance of reactive oxygen species production and consumption in this genotype under stress conditions.     

Extremely low‐frequency electromagnetic fields accelerates wound healing modulating MMP‐9 and inflammatory cytokines

Objectives

In our previous reports, we have demonstrated that extremely low‐frequency electromagnetic fields (ELF‐EMF) exposure enhances the proliferation of keratinocyte. The present study aimed to clarify effects of ELF‐EMF on wound healing and molecular mechanisms involved, using a scratch in vitro model.

Materials and methods

The wounded monolayer cultures of human immortalized keratinocytes (HaCaT), at different ELF‐EMF and Sham exposure times were monitored under an inverted microscope. The production and expression of IL‐1β, TNF‐α, IL‐18 and IL‐18BP were measured by enzyme‐linked immunosorbent assay and quantitative real‐time PCR. The activity and the expression of matrix metalloproteinases (MMP)‐2/9 was evaluated by zymography and Western blot analysis, respectively. Signal transduction proteins expression (Akt and ERK) was measured by Western blot.

Results

The results of wound healing in vitro assay revealed a significant reduction of cell‐free area time‐dependent in ELF‐EMF‐exposed cells compared to Sham condition. Gene expression and release of cytokines analysed were significantly increased in ELF‐EMF‐exposed cells. Our results further showed that ELF‐EMF exposure induced the activity and expressions of MMP‐9. Molecular data showed that effects of ELF‐EMF might be mediated via Akt and ERK signal pathway, as demonstrated using their specific inhibitors.

Conclusions

Our results highlight ability of ELF‐EMF to modulate inflammation mediators and keratinocyte proliferation/migration, playing an important role in wound repair. The ELF‐EMF accelerates wound healing modulating expression of the MMP‐9 via Akt/ERK pathway.

     

Non-Thermal Dielectric Barrier Discharge (DBD) Effects on Proliferation and Differentiation of Human Fibroblasts Are Primary Mediated by Hydrogen Peroxide

The proliferation of fibroblasts and myofibroblast differentiation are crucial in wound healing and wound closure. Impaired wound healing is often correlated with chronic bacterial contamination of the wound area. A new promising approach to overcome wound contamination, particularly infection with antibiotic-resistant pathogens, is the topical treatment with non-thermal “cold” atmospheric plasma (CAP). Dielectric barrier discharge (DBD) devices generate CAP containing active and reactive species, which have antibacterial effects but also may affect treated tissue/cells. Moreover, DBD treatment acidifies wound fluids and leads to an accumulation of hydrogen peroxide (H₂O₂) and nitric oxide products, such as nitrite and nitrate, in the wound. Thus, in this paper, we addressed the question of whether DBD-induced chemical changes may interfere with wound healing-relevant cell parameters such as viability, proliferation and myofibroblast differentiation of primary human fibroblasts. DBD treatment of 250 μl buffered saline (PBS) led to a treatment time-dependent acidification (pH 6.7; 300 s) and coincidently accumulation of nitrite (~300 μM), nitrate (~1 mM) and H₂O₂ (~200 μM). Fibroblast viability was reduced by single DBD treatments (60–300 s; ~77–66%) or exposure to freshly DBD-treated PBS (60–300 s; ~75–55%), accompanied by prolonged proliferation inhibition of the remaining cells. In addition, the total number of myofibroblasts was reduced, whereas in contrast, the myofibroblast frequency was significantly increased 12 days after DBD treatment or exposure to DBD-treated PBS. Control experiments mimicking DBD treatment indicate that plasma-generated H₂O₂ was mainly responsible for the decreased proliferation and differentiation, but not for DBD-induced toxicity. In conclusion, apart from antibacterial effects, DBD/CAP may mediate biological processes, for example, wound healing by accumulation of H₂O₂. Therefore, a clinical DBD treatment must be well-balanced in order to avoid possible unwanted side effects such as a delayed healing process.     

Crystal structures of α‐dioxygenase from Oryza sativa: Insights into substrate binding and activation by hydrogen peroxide

α‐Dioxygenases (α‐DOX) are heme‐containing enzymes found predominantly in plants and fungi, where they generate oxylipins in response to pathogen attack. α‐DOX oxygenate a variety of 14–20 carbon fatty acids containing up to three unsaturated bonds through stereoselective removal of the pro‐R hydrogen from the α‐carbon by a tyrosyl radical generated via the oxidation of the heme moiety by hydrogen peroxide (H₂O₂). We determined the X‐ray crystal structures of wild type α‐DOX from Oryza sativa, the wild type enzyme in complex with H₂O₂, and the catalytically inactive Y379F mutant in complex with the fatty acid palmitic acid (PA). PA binds within the active site cleft of α‐DOX such that the carboxylate forms ionic interactions with His‐311 and Arg‐559. Thr‐316 aids in the positioning of carbon‐2 for hydrogen abstraction. Twenty‐five of the twenty eight contacts made between PA and residues lining the active site occur within the carboxylate and first eight carbons, indicating that interactions within this region of the substrate are responsible for governing selectivity. Comparison of the wild type and H₂O₂ structures provides insight into enzyme activation. The binding of H₂O₂ at the distal face of the heme displaces residues His‐157, Asp‐158, and Trp‐159 ～2.5 Å from their positions in the wild type structure. As a result, the Oδ2 atom of Asp‐158 interacts with the Ca atom in the calcium binding loop, the side chains of Trp‐159 and Trp‐213 reorient, and the guanidinium group of Arg‐559 is repositioned near Tyr‐379, poised to interact with the carboxylate group of the substrate.     

Biohydrogen production from wheat straw hydrolysate by dark fermentation using extreme thermophilic mixed culture

Hydrolysate was tested as substrate for hydrogen production by extreme thermophilic mixed culture (70°C) in both batch and continuously fed reactors. Hydrogen was produced at hydrolysate concentrations up to 25% (v/v), while no hydrogen was produced at hydrolysate concentration of 30% (v/v), indicating that hydrolysate at high concentrations was inhibiting the hydrogen fermentation process. In addition, the lag phase for hydrogen production was strongly influenced by the hydrolysate concentration, and was prolonged from approximately 11 h at the hydrolysate concentrations below 20% (v/v) to 38 h at the hydrolysate concentration of 25% (v/v). The maximum hydrogen yield as determined in batch assays was 318.4 ± 5.2 mL‐H₂/g‐sugars (14.2 ± 0.2 mmol‐H₂/g‐sugars) at the hydrolysate concentration of 5% (v/v). Continuously fed, and the continuously stirred tank reactor (CSTR), operating at 3 day hydraulic retention time (HRT) and fed with 20% (v/v) hydrolysate could successfully produce hydrogen. The hydrogen yield and production rate were 178.0 ± 10.1 mL‐H₂/g‐sugars (7.9 ± 0.4 mmol H₂/g‐sugars) and 184.0 ± 10.7 mL‐H₂/day L_reactor (8.2 ± 0.5 mmol‐H₂/day L_reactor), respectively, corresponding to 12% of the chemical oxygen demand (COD) from sugars. Additionally, it was found that toxic compounds, furfural and hydroxymethylfurfural (HMF), contained in the hydrolysate were effectively degraded in the CSTR, and their concentrations were reduced from 50 and 28 mg/L, respectively, to undetectable concentrations in the effluent. Phylogenetic analysis of the mixed culture revealed that members involved hydrogen producers in both batch and CSTR reactors were phylogenetically related to the Caldanaerobacter subteraneus, Thermoanaerobacter subteraneus, and Thermoanaerobacterium thermosaccharolyticum. Biotechnol. Bioeng. 2010;105: 899–908. © 2009 Wiley Periodicals, Inc.     

Propranolol impairs the closure of pressure ulcers in mice

Aims

β-Adrenoceptors modulate acute wound healing; however, few studies have shown the effects of β-adrenoceptor blockade on chronic wounds. Therefore, this study investigated the effect of β₁-/β₂-adrenoceptor blockade in wound healing of pressure ulcers.

Main methods

Male mice were daily treated with propranolol (β₁-/β₂-adrenoceptor antagonist) until euthanasia. One day after the beginning of treatment, two cycles of ischemia–reperfusion by external application of two magnetic plates were performed in skin to induce pressure ulcer formation.

Key findings

Propranolol administration reduced keratinocyte migration, transforming growth factor-β protein expression, re-epithelialization, and necrotic tissue loss. Neutrophil number and neutrophil elastase protein expression were increased in propranolol-treated group when compared with control group. Propranolol administration delayed macrophage mobilization and metalloproteinase-12 protein expression and reduced monocyte chemoattractant protein-1 protein expression. Myofibroblastic differentiation, angiogenesis, and wound closure were delayed in the propranolol-treated animals. Propranolol administration increased neo-epidermis thickness, reduced collagen deposition, and enhanced tenascin-C expression resulting in the formation of an immature and disorganized collagenous scar.

Significance

β₁-/β₂-Adrenoceptor blockade delays wound healing of ischemia–reperfusion skin injury through the impairment of the re-epithelialization and necrotic tissue loss which compromise wound inflammation, dermal reconstruction, and scar formation.     

A new player in bacterial sulfide‐inducible transcriptional regulation

Although hydrogen sulfide (H₂S) is perhaps best known as a toxic gas, the electron‐rich H₂S functions as an energy source and electron donor for chemolithotrophic and photosynthetic bacteria, via sulfide oxidation, and is a universal substrate for cysteine biosynthesis. These distinct harmful and beneficial roles of H₂S suggest the need to ‘sense’ prevailing concentrations of sulfide and downstream reactive sulfur species (RSS) and regulate the expression of genes mediating sulfide homeostasis. The paper by Li et al. in this issue of Molecular Microbiology adds Cupriavidus FisR to an expanding repertoire of regulatory mechanisms that bacteria have evolved to sense cellular RSS and mitigate their deleterious effects.     

The focal complex of epithelial cells provides a signalling platform for interleukin‐8 induction in response to bacterial pathogens

Bacterial pathogens can induce an inflammatory response from epithelial tissues due to secretion of the pro‐inflammatory chemokine interleukin‐8 (IL‐8). Many bacterial pathogens manipulate components of the focal complex (FC) to induce signalling events in host cells. We examined the interaction of several bacterial pathogens with host cells, including Campylobacter jejuni, to determine if the FC is required for induction of chemokine signalling in response to bacterial pathogens. Our data indicate that secretion of IL‐8 is triggered by C. jejuni, Helicobacter pylori and Salmonella enterica serovar Typhimurium in response to engagement of β₁ integrins. Additionally, we found that the secretion of IL‐8 from C. jejuni infected epithelial cells requires FAK, Src and paxillin, which in turn are necessary for Erk 1/2 recruitment and activation. Targeting the FC component paxillin with siRNA prevented IL‐8 secretion from cells infected with several bacterial pathogens, including C. jejuni, Helicobacter pylori, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio parahaemolyticus. Our findings indicate that maximal IL‐8 secretion from epithelial cells in response to bacterial infection is dependent on the FC. Based on the commonality of the host response to bacterial pathogens, we propose that the FC is a signalling platform for an epithelial cell response to pathogenic organisms.     

Terrein reduces age‐related inflammation induced by oxidative stress through Nrf2/ERK1/2/HO‐1 signalling in aged HDF cells

This study investigated whether multiple bioactivity of terrein such as anti‐inflammatory and anti‐oxidant inhibits age‐related inflammation by promoting an antioxidant response in aged human diploid fibroblast (HDF) cells. HDF cells were cultured serially for in vitro replicative senescence. To create the ageing cell phenotype, intermediate stage (PD31) HDF cells were brought to stress‐induced premature senescence (SIPS) using hydrogen peroxide (H₂O₂). Terrein increased cell viability even with H₂O₂ stress and reduced inflammatory molecules such as intracellular adhesion molecule‐1 (ICAM‐1), cyclooxygenase‐2 (COX‐2), interleukin‐1beta (IL‐1β) and tumour necrosis factor‐alpha (TNF‐α). Terrein reduced also phospho‐extracellular kinase receptor1/2 (p‐EKR1/2) signalling in aged HDF cells. SIPS cells were attenuated for age‐related biological markers including reactive oxygen species (ROS), senescence associated beta‐galactosidase (SA β‐gal.) and the aforementioned inflammatory molecules. Terrein induced the induction of anti‐oxidant molecules, copper/zinc‐superoxide defence (Cu/ZnSOD), manganese superoxide dismutase (MnSOD) and heme oxygenase‐1 (HO‐1) in SIPS cells. Terrein also alleviated reactive oxygen species formation through the Nrf2/HO‐1/p‐ERK1/2 pathway in aged cells. The results indicate that terrein has an alleviative function of age‐related inflammation characterized as an anti‐oxidant. Terrein might be a useful nutraceutical compound for anti‐ageing. Copyright © 2015 John Wiley & Sons, Ltd.     

Hydrogen and methane production from desugared molasses using a two‐stage thermophilic anaerobic process

Hydrogen and methane production from desugared molasses by a two‐stage thermophilic anaerobic process was investigated in a series of two up‐flow anaerobic sludge blanket (UASB) reactors. The first reactor that was dominated with hydrogen‐producing bacteria of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium aciditolerans could generate a high hydrogen production rate of 5600 mL H₂/day/L, corresponding to a yield of 132 mL H₂/g volatile solid (VS). The effluent from the hydrogen reactor was further converted to methane in the second reactor with the optimal production rate of 3380 mL CH₄/day/L, corresponding to a yield of 239 mL CH₄/g VS. Aceticlastic Methanosarcina mazei was the dominant methanogen in the methanogenesis stage. This work demonstrates that biohydrogen production can be very efficiently coupled with a subsequent step of methane production using desugared molasses. Furthermore, the mixed gas with a volumetric content of 16.5% H_2, 38.7% CO₂, and 44.8% CH₄, containing approximately 15% energy by hydrogen is viable to be bio‐hythane.     

Hydrogen sulfide protects blood–brain barrier integrity following cerebral ischemia

By using two structurally unrelated hydrogen sulfide (H₂S) donors 5‐(4‐methoxyphenyl) ‐3H‐1, 2‐dithiole‐3‐thione (ADT) and sodium hydrosulfide (NaHS), this study investigated if H₂S protected blood–brain barrier (BBB) integrity following middle cerebral artery occlusion (MCAO). ICR mice underwent MCAO and received H₂S donors at 3 h after reperfusion. Infarction, neurological scores, brain edema, Evans blue (EB) extravasation, and tight junction protein expression were examined at 48 h after MCAO. We also investigated if ADT protected BBB integrity by suppressing post‐ischemic inflammation‐induced Matrix Metalloproteimase‐9 (MMP9) and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). ADT increased blood H₂S concentrations, decreased infarction, and improved neurological deficits. Particularly, ADT reduced EB extravasation, brain edema and preserved expression of tight junction proteins in the ischemic brain. NaHS also increased blood H₂S levels and reduced EB extravasation following MCAO. Moreover, ADT inhibited expression of pro‐inflammatory markers induced Nitric Oxide Synthase (iNOS) and IL‐1β while enhanced expression of anti‐inflammatory markers arginase 1 and IL‐10 in the ischemic brain. Accordingly, ADT attenuated ischemia‐induced expression and activity of MMP9. Moreover, ADT reduced NOX‐4 mRNA expression, NOX activity, and inhibited nuclear translocation of Nuclear Factor Kappa‐B (NF‐κB) in the ischemic brain. In conclusion, H₂S donors protected BBB integrity following experimental stroke possibly by acting through NF‐κB inhibition to suppress neuroinflammation induction of MMP9 and NOX4‐derived free radicals.

     

Hydrogen gas reduces chronic intermittent hypoxia-induced hypertension by inhibiting sympathetic nerve activity and increasing vasodilator responses via the antioxidation

Molecular hydrogen is reported to be used medically to ameliorate various systemic pathological conditions. This study aimed to investigate the effect of hydrogen (H₂) gas on hypertension induced by intermittent hypoxia in rats. The adult rats were exposed to chronic intermittent hypoxia (CIH) 8 hours/day for 5 weeks and/or H ₂ gas 2 hours/day. We found that the systolic and diastolic blood pressure (BP) increased significantly in rats exposed to intermittent hypoxia, both of which were markedly attenuated after H treatment. Furthermore, intermittent hypoxia exposure elevated renal sympathetic nerve activity, consistent with plasma norepinephrine. Additionally, H ₂ gas significantly improved CIH-induced abnormal vascular relaxation. Nevertheless, inhalation of H ₂ gas alone did not cause such changes. Moreover, H ₂ gas-treated rats exposed to CIH showed a significant reduction in 8-hydroxy-2 deoxyguanosine content and increases in superoxide dismutase activity, indicating improved oxidative stress. Taken together, these results indicate that H ₂ gas has significant effects on the reduction of BP without any side effects. Mechanistically, inhibition of sympathetic activity and reduction of systemic vascular resistance may participate in this process via the antioxidant activity of H ₂.