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Hyperoxic lung injury is a major concern in critically ill patients who receive high concentrations of oxygen to treat lung diseases. Successful abrogation of hyperoxic lung injury would have a huge impact on respiratory and critical care medicine. Hydrogen can be administered as a therapeutic medical gas. We recently demonstrated that inhaled hydrogen reduced transplant-induced lung injury and induced heme oxygenase (HO)-1. To determine whether hydrogen could reduce hyperoxic lung injury and investigate the underlying mechanisms, we randomly assigned rats to 4 experimental groups and administered the following gas mixtures for 60 hours: 98% oxygen (hyperoxia), 2% nitrogen; 98% oxygen (hyperoxia), 2% hydrogen; 98% balanced air (normoxia), 2% nitrogen; and 98% balanced air (normoxia), 2% hydrogen. We examined lung function by blood gas analysis, extent of lung injury, and expression of HO-1. We also investigated the role of NF-E2-related factor (Nrf) 2, which regulates HO-1 expression, by examining the expression of Nrf2-dependent genes and the ability of hydrogen to reduce hyperoxic lung injury in Nrf2-deficient mice. Hydrogen treatment during exposure to hyperoxia significantly improved blood oxygenation, reduced inflammatory events, and induced HO-1 expression. Hydrogen did not mitigate hyperoxic lung injury or induce HO-1 in Nrf2-deficient mice. These findings indicate that hydrogen gas can ameliorate hyperoxic lung injury through induction of Nrf2-dependent genes, such as HO-1. The findings suggest a potentially novel and applicable solution to hyperoxic lung injury, and provide new insight into the molecular mechanisms and actions of hydrogen.
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... Molecular hydrogen inhibits the TLR4-mediated inflammatory pathway, improving hyperglycemia in rats with type 2 diabetes mellitus (Ming et al. 2020). It also regulates Nrf2 pathways, inhibiting oxidative stressinduced inflammatory lung disease in rats (Kawamura et al. 2013). ...
... An animal study found that HRS ameliorated HILI by reducing oxidative stress and inflammatory cascades, inhibiting apoptosis in lung tissues . The mechanism involved the Nrf2/HO-1-dependent pathway, prolonging survival against lethal hyperoxia in rats (Kawamura et al. 2013). Another study found that molecular hydrogen could reduce HILI-related ERS by increasing a master regulator of ERS-silent information regulator type-1 (SIRT1) expression in rats (Sun et al. 2017). ...
... Buchholz et al. (2008),Kawamura et al. (2013),Xiao et al. (2013),Ren et al. (2014),Chen et al. (2015b),Liu et al. (2015a),Shi et al. (2015),Xie et al. (2015),Zhai et al. (2015),Tian et al. (2017), Ning et al. (2018), Yu et al. (2019), Zou et al. (2019), Ming et al. (2020), Yang et al. (2020), Qiu et al. (2021) Anti-oxidation Directly scavenges ·OH and ONOO − ; activates Nrf-2/ HO-1; upregulates the expression of SOD, CAT, GSH-Px, downregulates NADPH oxidase; hormesis through increasing ROS Ohsawa et al. (2007), Yu and Zheng (2012), Shinbo et al. (2013), Hirayama et al. (2018), Zhao et al. (2019) Modulates autophagy Promotes autophagy when autophagy is insufficient; inhibits autophagy when excessive autophagy disrupts cell homeostasis Zhang et al. (2015), Yao et al. (2019), Chen et al. (2020a), Zhuang et al. (2020), Qiu et al. (2021) Modulates cell death Antiapoptosis: inhibiting Bax, caspase-3, and caspase-8, upregulating Bcl-xl and Bcl-2 Inhibits pyroptosis by inhibiting oxidative stress, NLRP3 and mitoK ATP /ERK1/2/p38 MAPK signaling pathways Cai et al. (2008), Kawamura et al. (2010), Du et al. (2016), Nie et al. (2021), Zhang et al. (2021) ...
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Acute lung injury (ALI) and acute respiratory distress syndrome, which is a more severe form of ALI, are life-threatening clinical syndromes observed in critically ill patients. Treatment methods to alleviate the pathogenesis of ALI have improved to a great extent at present. Although the efficacy of these therapies is limited, their relevance has increased remarkably with the ongoing pandemic caused by the novel coronavirus disease 2019 (COVID-19), which causes severe respiratory distress syndrome. Several studies have demonstrated the preventive and therapeutic effects of molecular hydrogen in the various diseases. The biological effects of molecular hydrogen mainly involve anti-inflammation, antioxidation, and autophagy and cell death modulation. This review focuses on the potential therapeutic effects of molecular hydrogen on ALI and its underlying mechanisms and aims to provide a theoretical basis for the clinical treatment of ALI and COVID-19.
... However, subsequent studies revealed a variety of signal-modulating activities, which cannot be readily accounted for by radical scavenging activities. For example, hydrogen activates the Nrf2/Keap1 signaling pathway, which, contrary to radical scavenging, is usually activated by oxidative stress [8][9][10][11][12][13][14][15]. We reported that hydrogen suppresses abnormally activated Wnt/β-catenin signaling by enhancing the activity of β-catenin degradation complex, and hydrogen ameliorates a mouse model of osteoarthritis by suppressing Wnt/β-catenin signaling [16]. ...
... Mitochondria-derived ROS activates the Keap1-Nrf2 antioxidant signaling pathway [43]. Hydrogen has been repeatedly reported to activate the Keap1-Nrf2 pathway in both animal models [10][11][12]15] and cultured cells [8,13,14], indicat-ing that hydrogen exerts its effects by enhancing the antioxidant signaling pathway. We showed that hydrogen did not increase the growth of SH-SY5Y cells (a non-responder). ...
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Molecular hydrogen ameliorates pathological states in a variety of human diseases, animal models, and cell models, but the effects of hydrogen on cancer have been rarely reported. In addition, the molecular mechanisms underlying the effects of hydrogen remain mostly unelucidated. We found that hydrogen enhances proliferation of four out of seven human cancer cell lines (the responders). The proliferation-promoting effects were not correlated with basal levels of cellular reactive oxygen species. Expression profiling of the seven cells showed that the responders have higher gene expression of mitochondrial electron transport chain (ETC) molecules than the non-responders. In addition, the responders have higher mitochondrial mass, higher mitochondrial superoxide, higher mitochondrial membrane potential, and higher mitochondrial spare respiratory capacity than the non-responders. In the responders, hydrogen provoked mitochondrial unfolded protein response (mtUPR). Suppression of cell proliferation by rotenone, an inhibitor of mitochondrial ETC complex I, was rescued by hydrogen in the responders. Hydrogen triggers mtUPR and induces cell proliferation in cancer cells that have high basal and spare mitochondrial ETC activities.
... Recently, a new mechanism for hydrogen's effects has been revealed. Hydrogen works not only through a direct pathway as described above but also through indirect pathways to upregulate antioxidants [9,10]. We reported that the expression of superoxide dismutase enzyme (SOD)1 was upregulated by continuous administration of hydrogen in rat corneal epithelial tissue [11]. ...
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Many studies have demonstrated the therapeutic effects of hydrogen in pathological conditions such as inflammation; however, little is known about its prophylactic effects. The purpose of this study is to investigate the prophylactic effects of hydrogen-rich water instillation in a rat corneal alkali burn model. Hydrogen-rich water (hydrogen group) or physiological saline (vehicle group) was instilled continuously to the normal rat cornea for 5 min. At 6 h after instillation, the cornea was exposed to alkali. The area of corneal epithelial defect (CED) was measured every 6 h until 24 h after alkali exposure. In addition, at 6 and 24 h after injury, histological and immunohistochemical observations were made and real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to investigate superoxide dismutase enzyme (SOD)1, SOD2, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) mRNA expression. CED at 12 h and the number of inflammatory infiltrating cells at 6 h after injury were significantly smaller in the hydrogen group than the vehicle group. Furthermore, SOD1 expression was significantly higher in the hydrogen group than the vehicle group at both 6 and 24 h, and the number of PGC-1α-positive cells was significantly larger in the hydrogen group than the vehicle group at 6 h after injury. In this model, prophylactic instillation of hydrogen-rich water suppressed alkali burn-induced inflammation, likely by upregulating expression of antioxidants such as SOD1 and PGC-1α. Hydrogen has not only therapeutic potential but also prophylactic effects that may suppress corneal scarring following injury and promote wound healing.
... Hydrogen has been reported to possess antioxidative, anti-inflammation, and antiapoptosis effects [13] and demonstrated as a novel therapy for different diseases such as cerebral, myocardial, hepatic, renal, and intestinal diseases [14][15][16][17][18][19][20]. Especially, hydrogen inhalation can alleviate hypertoxic lung injury in rats [21] and protect mice against cigarette-induced chronic obstructive pulmonary disease (COPD) [22]. Hydrogen has been reported to reduce the cytokine storm and oxidative stress reactions in mice [23,24]. ...
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Acute respiratory distress syndrome (ARDS) causes uncontrolled pulmonary inflammation, resulting in high morbidity and mortality in severe cases. Given the antioxidative effect of molecular hydrogen, some recent studies suggest the potential use of molecular hydrogen as a biomedicine for the treatment of ARDS. In this study, we aimed to explore the protective effects of magnesium hydride (MgH2) on two types of ARDS models and its underlying mechanism in a lipopolysaccharide (LPS)-induced ARDS model of the A549 cell line. The results showed that LPS successfully induced oxidative stress, inflammatory reaction, apoptosis, and barrier breakdown in alveolar epithelial cells (AEC). MgH2 can exert an anti-inflammatory effect by down-regulating the expressions of inflammatory cytokines (IL-1β, IL-6, and TNF-α). In addition, MgH2 decreased oxidative stress by eliminating intracellular ROS, inhibited apoptosis by regulating the expressions of cytochrome c, Bax, and Bcl-2, and suppressed barrier breakdown by up-regulating the expression of ZO-1 and occludin. Mechanistically, the expressions of p-AKT, p-mTOR, p-P65, NLRP3, and cleaved-caspase-1 were decreased after MgH2 treatment, indicating that AKT/mTOR and NF-κB/NLRP3/IL-1β pathways participated in the protective effects of MgH2. Furthermore, the in vivo study also demonstrated that MgH2-treated mice had a better survival rate and weaker pathological damage. All these findings demonstrated that MgH2 could exert an ARDS-protective effect by regulating the AKT/mTOR and NF-κB/NLRP3/IL-1β pathways to suppress LPS-induced inflammatory reaction, oxidative stress injury, apoptosis, and barrier breakdown, which may provide a potential strategy for the prevention and treatment of ARDS.
... In the in vitro H/R experiments of the present study, a decrease in autophagy-associated proteins (Beclin-1 and LC3B) and autophagosomes was observed after H 2 treatment, which suggested that the cell homeostasis mechanisms of H 2 therapy in cardioprotection are associated with the inhibition of autophagy. In previous studies, the anti-apoptotic properties of H 2 have also been demonstrated, with the alleviation of hyperoxia inducing lung epithelial cell apoptosis via the induction of Bcl-2 and the suppression of Bax expression (42)(43)(44). These results revealed a potential mechanism of H 2 -mediated cell fate under stress. ...
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Hydrogen (H2) therapy is a therapeutic strategy using molecular H2. Due to its ability to regulate cell homeostasis, H2 therapy has exhibited marked therapeutic effects on a number of oxidative stress-associated diseases. The present study investigated the effectiveness of H2 therapy in protecting against myocardial injury in a rat model of asphyxial cardiac arrest and cardiopulmonary resuscitation. Rats underwent 10-min asphyxia-induced cardiac arrest (CA) and cardiopulmonary resuscitation (CPR), and were randomly divided into control and H2 therapy groups. After resuscitation, the H2 therapy group was administered room air mixed with 2% H2 gas for respiration. During CA/CPR, the arterial pressure and heart rate were measured every minute. Survival rate, cardiac function, myocardial injury biomarkers creatine kinase-MB and cardiac troponin-T, and histopathological changes were evaluated to determine the protective effects of H2 therapy in CA/CPR. Immunohistochemistry and western blot analysis were used to determine the expression levels of autophagy-associated proteins. In vitro, H9C2 cells were subjected to hypoxia/reoxygenation and H2-rich medium was used in H2 treatment groups. Western blotting and immunofluorescence were used to observe the expression levels of autophagy-associated proteins. Moreover, an adenovirus-monomeric red fluorescent protein-green fluorescent protein-LC3 construct was used to explore the dynamics of autophagy in the H9C2 cells. The results showed that H2 therapy significantly improved post-resuscitation survival and cardiac function. H2 therapy also improved mitochondrial mass and decreased autophagosome numbers in cardiomyocytes after resuscitation. The treatment inhibited autophagy activation, with lower expression levels of autophagy-associated proteins and decreased autophagosome formation in vivo and vitro. In conclusion, H2 gas inhalation after return of spontaneous circulation improved cardiac function via the inhibition of autophagy.
... Considering that inhalation of hydrogen gas (H 2 ) can provide potential protection against ALI through antioxidant, anti-apoptotic, and anti-inflammatory effects [77][78][79], the above research group further investigated the capacity of [ 99m Tc]duramycin and [ 99m Tc]HMPAO to monitor the efficacy of inhaled H 2 treatment in a rat model of ALI induced by exposure to hyperoxia (98% O 2 ) [80]. Histological studies showed that lung samples from rats with hyperoxia exposure for 48 h were not significantly different from those of normoxic controls in terms of neutrophilic influx, edema, or diffusion barrier thickness while lung samples from rats with hyperoxia exposure for 60 h manifested varying degrees of edema and neutrophilic influx as well as an increase in diffusion barrier thickness compared with normoxic controls (Fig. 6a) [80]. ...
... It can selectively react with strong oxidants and can easily penetrate biological membranes, such as nuclear and mitochondrial membranes, without affecting the metabolic redox reaction (Ohta, 2012(Ohta, , 2015. (2) By stimulating nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates the basal and induces expression of many antioxidant enzymes and the proteasome (Zhang HQ et al., 2015), hydrogen can increase the expression of heme oxygenase-1 (HO-1) (Kawamura et al., 2013;Xie et al., 2020;Yu et al., 2020). It also decreases ·ONOO −related gene expression and production (Shinbo et al., 2013) and increases the activity of the antioxidant enzymes SOD, CAT, and myeloperoxidase (MPO) (Cai et al., 2013). ...
Article
Molecular hydrogen exerts biological effects on nearly all organs. It has anti-oxidative, anti-inflammatory, and anti-aging effects and contributes to the regulation of autophagy and cell death. As the primary organ for gas exchange, the lungs are constantly exposed to various harmful environmental irritants. Short- or long-term exposure to these harmful substances often results in lung injury, causing respiratory and lung diseases. Acute and chronic respiratory diseases have high rates of morbidity and mortality and have become a major public health concern worldwide. For example, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. An increasing number of studies have revealed that hydrogen may protect the lungs from diverse diseases, including acute lung injury, chronic obstructive pulmonary disease, asthma, lung cancer, pulmonary arterial hypertension, and pulmonary fibrosis. In this review, we highlight the multiple functions of hydrogen and the mechanisms underlying its protective effects in various lung diseases, with a focus on its roles in disease pathogenesis and clinical significance.
... Considering that inhalation of hydrogen gas (H 2 ) can provide potential protection against ALI through antioxidant, anti-apoptotic, and anti-inflammatory effects [77][78][79], the above research group further investigated the capacity of [ 99m Tc]duramycin and [ 99m Tc]HMPAO to monitor the efficacy of inhaled H 2 treatment in a rat model of ALI induced by exposure to hyperoxia (98% O 2 ) [80]. Histological studies showed that lung samples from rats with hyperoxia exposure for 48 h were not significantly different from those of normoxic controls in terms of neutrophilic influx, edema, or diffusion barrier thickness while lung samples from rats with hyperoxia exposure for 60 h manifested varying degrees of edema and neutrophilic influx as well as an increase in diffusion barrier thickness compared with normoxic controls (Fig. 6a) [80]. ...
Article
Cell death is involved in numerous pathological conditions such as cardiovascular disorders, ischemic stroke and organ transplant rejection, and plays a critical role in the treatment of cancer. Cell death imaging can serve as a noninvasive means to detect the severity of tissue damage, monitor the progression of diseases, and evaluate the effectiveness of treatments, which help to provide prognostic information and guide the formulation of individualized treatment plans. The high abundance of phosphatidylethanolamine (PE), which is predominantly confined to the inner leaflet of the lipid bilayer membrane in healthy mammalian cells, becomes exposed on the cell surface in the early stages of apoptosis or accessible to the extracellular milieu when the cell suffers from necrosis, thus representing an attractive target for cell death imaging. Duramycin is a tetracyclic polypeptide that contains 19 amino acids and can bind to PE with excellent affinity and specificity. Additionally, this peptide has several favorable structural traits including relatively low molecular weight, stability to enzymatic hydrolysis, and ease of conjugation and labeling. All these highlight the potential of duramycin as a candidate ligand for developing PE-specific molecular probes. By far, a couple of duramycin-based molecular probes such as Tc-99 m-, F-18-, or Ga-68-labeled duramycin have been developed to target exposed PE for in vivo noninvasive imaging of cell death in different animal models. In this review article, we describe the state of the art with respect to in vivo imaging of cell death using duramycin-based molecular probes, as validated by immunohistopathology.
Article
Hyperoxia is characterized by pronounced inflammatory responses, pulmonary cell apoptosis, and adverse cardiac remodeling due to an excess supply of oxygen. Hyperoxic episodes are frequent in mechanically ventilated patients and are associated with in-hospital mortality. This study extends the analysis of prior published research by our group as it investigates the influence of age in male and female rodents exposed to hyperoxic conditions. Age is an independent cardiovascular risk factor, often compounded by variables like obesity, diabetes, and a decline in sex hormones and their receptors. This study simulates clinical hyperoxia by subjecting rodents to > 90% of oxygen for 72 hours and compares the changes in cardiac structural and functional parameters with those exposed to normal air. While in both sexes conduction abnormalities with ageing were discernible, aged females owing to their inherent higher baseline QTc, were at a higher risk of developing arrhythmias as compared to age-matched males. Quantitative real-time RT-PCR and western blot analysis reflected altered expression of cardiac potassium channels, resulting in conduction abnormalities in aged female rodents. Unaffected by age and sex, hyperoxia-treated mice had altered body composition, as evidenced by a considerable reduction in body weight. Interestingly, compensatory hypertrophy observed as a protective mechanism in young males was absent in aged males, whereas protection of hearts from hyperoxia-induced cardiac hypertrophy was absent in aged female mice, both of which may be at least in part due to a reduction in sex steroid receptors and the systemic steroid levels. Finally, statistical analysis revealed that hyperoxia had the greatest impact on most of the cardiac parameters, followed by age and then sex. This data established an imperative finding that can change the provision of care for aged individuals admitted to ICU by elucidating the impact of intrinsic aging on hyperoxia-induced cardiac remodeling.
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The metabolite itaconate has both anti-inflammatory and immunomodulatory effects. However, its influence on chronic pain is unclear. Here, we demonstrated that intraperitoneal injection of the itaconate derivative dimethyl itaconate (DI) alleviates chronic pain symptoms, such as allodynia and hyperalgesia, in spinal nerve ligation (SNL) and inflammatory pain models. Moreover, intraperitoneal DI reduced the secretion of inflammatory cytokines (i.e., interleukin-1β, tumour necrosis factor-alpha) in dorsal root ganglion (DRG), spinal cord and hind paw tissues, suppressed the activation of macrophages in DRG and glial cells in the spinal dorsal horn and decreased the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the DRG and spinal cord. DI boosted nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) levels in the DRG and spinal cord of SNL mice. Intraperitoneal administration of the Nrf2 inhibitor ML385 abolished the analgesic effect of DI and decreased the expression of Nrf2 in the DRG and spinal cord. Similarly, administration of DI potently reversed the lipopolysaccharide (LPS)-induced inflammatory effect in microglia. Reduction of endogenous itaconate levels by pretreatment with immune-responsive gene 1 (IRG1) siRNA blocked Nrf2 expression, which impaired the analgesic and anti-inflammatory effects of DI in vitro. Therefore, our findings reveal for the first time that intraperitoneal DI elicits anti-inflammatory effect and sustained chronic pain relief, which may be regarded as a promising therapeutic agent for chronic pain treatment.
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Molecular hydrogen (H2) functions as an extensive protector against oxidative stress, inflammation and allergic reaction in various biological models and clinical tests; however, its essential mechanisms remain unknown. H2 directly reacts with the strong reactive nitrogen species peroxynitrite (ONOO-) as well as hydroxyl radicals (•OH), but not with nitric oxide radical (NO•). We hypothesized that one of the H2 functions is caused by reducing cellular ONOO-, which is generated by the rapid reaction of NO• with superoxides (•O2-). To verify this hypothesis, we examined whether H2 could restore cytotoxicity and transcriptional alterations induced by ONOO- derived from NO• in chondrocytes. We treated cultured chondrocytes from porcine hindlimb cartilage or from rat meniscus fibrecartilage with a donor of NO•, S-nitroso-N-acetylpenicillamine (SNAP) in the presence or absence of H2. Chondrocyte viability was determined using a LIVE/DEAD Viability/Cytotoxicity Kit. Gene expressions of the matrix proteins of cartilage and the matrix metalloproteinases were analyzed by reverse transcriptase-coupled real-time PCR method. SNAP treatment increased the levels of nitrated proteins. H2 decreased the levels of the nitrated proteins, and suppressed chondrocyte death. It is known that the matrix proteins of cartilage (including aggrecan and type II collagen) and matrix metalloproteinases (such as MMP3 and MMP13) are down- and up-regulated by ONOO-, respectively. H2 restoratively increased the gene expressions of aggrecan and type II collagen in the presence of H2. Conversely, the gene expressions of MMP3 and MMP13 were restoratively down-regulated with H2. Thus, H2 acted to restore transcriptional alterations induced by ONOO-. These results imply that one of the functions of H2 exhibits cytoprotective effects and transcriptional alterations through reducing ONOO-. Moreover, novel pharmacological strategies aimed at selective removal of ONOO- may represent a powerful method for preventive and therapeutic use of H2 for joint diseases.
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Findings in recent years strongly suggest that the stress-inducible gene heme oxygenase (HO)-1 plays an important role in protection against oxidative stress. Although the mechanism(s) by which this protection occurs is poorly understood, we hypothesized that the gaseous molecule carbon monoxide (CO), a major by-product of heme catalysis by HO-1, may provide protection against oxidative stress. We demonstrate here that animals exposed to a low concentration of CO exhibit a marked tolerance to lethal concentrations of hyperoxia in vivo. This increased survival was associated with highly significant attenuation of hyperoxia-induced lung injury as assessed by the volume of pleural effusion, protein accumulation in the airways, and histological analysis. The lungs were completely devoid of lung airway and parenchymal inflammation, fibrin deposition, and pulmonary edema in rats exposed to hyperoxia in the presence of a low concentration of CO. Furthermore, exogenous CO completely protected against hyperoxia-induced lung injury in rats in which endogenous HO enzyme activity was inhibited with tin protoporphyrin, a selective inhibitor of HO. Rats exposed to CO also exhibited a marked attenuation of hyperoxia-induced neutrophil infiltration into the airways and total lung apoptotic index. Taken together, our data demonstrate, for the first time, that CO can be therapeutic against oxidative stress such as hyperoxia and highlight possible mechanism(s) by which CO may mediate these protective effects.
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The heme oxygenase-1 (HO-1) enzyme catalyzes the rate-limiting reaction in the catabolism of heme yielding products with pleiotropic, but ultimately, cytoprotective activities. High levels of HO-1 are frequently detected in various pathological states and generally in states of cellular oxidative stress. Induction of HO-1, regulated at the level of gene transcription, is essential for manifestation of the enzyme's cytoprotective function. Extensive analysis of the mouse gene, and to a lesser extent of the human gene, has identified a common mechanism - the stress response element (StRE)/Nrf2 transcription factor pathway - for gene regulation in response to a diverse array of HO-1 inducers including the substrate heme, various environmental and industrial toxins, and plant-derived polyphenolic compounds. In addition to Nrf2 complexes, numerous dimeric transcription factors bind to the StRE, permitting induction, repression and overall fine-tuning of gene activity. In principle, the multiplicity of StRE binding proteins also provides for a range of pharmaceutical targets for controlled production of the potentially therapeutic HO-1 protein.
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Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All ‘multidomain’ anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for ‘multidomain’ pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among ‘BH3 domain-only’ members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.
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Hyperoxia, used therapeutically in the treatment of respiratory insufficiencies, can cause lung injury, probably through the actions of reactive oxygen species. The present studies were designed to test the hypothesis that oxidation of specific proteins would provide useful biomarkers of the onset of tissue injury, and thereby provide clues as to the mechanisms responsible. We exposed adult male Sprague-Dawley rats to room air or to greater than 95% O2 for 60 h and examined proteins in pleural effusion and broncho-alveolar lavage (BAL) fluids, and in lung tissue homogenates and subfractions. Oxidation of protein thiols was assessed by derivatization with monobromobimane, separation by electrophoresis, and visualization of the fluorescent thioether derivatives. Derivatization with 2,4-dinitrophenylhydrazine (DNPH), electrophoresis, and western analysis was employed to assess a different class of oxidative modifications, frequently termed `protein carbonyls'. In addition, we investigated the effects of the 21-aminosteroid U-74389G, 10 mg/kg, given intraperitoneally every 12 h, on biomarkers of protein oxidation and on manifestations of lung injury. Hyperoxia caused lung injury evidenced by pleural effusions, increases in BAL protein concentrations, and pulmonary edema; U-74389G attenuated the first two indices of lung injury, but did not alter edema. Protein thiol status of the fractions studied were not affected notably by hyperoxia, or by the aminosteroid. The formation of DNPH-reactive sites on a limited number of proteins by hyperoxia was observed, and some of these effects were attenuated in the animals given U-74389G. Histological examination of lung tissues showed accumulation of intra-alveolar protein exudates in hyperoxic rats, and a significant attenuation of this effect was observed in the animals treated with U-74389G. In conclusion, studies of shifts in protein thiol status that may be caused by hyperoxia will require increasingly specific methods of analysis, and characterization of the specific DNPH-reactive proteins formed in hyperoxia may provide critical insights into the mechanisms of lung injury. Administration of U-74389G offers some degree of protection against hyperoxia and attenuation of these biomarkers of oxidation, but the precise mechanisms by which this protection is effected will require additional study.
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The induction of phase II detoxifying enzymes is an important defense mechanism against intake of xenobiotics. While this group of enzymes is believed to be under the transcriptional control of antioxidant response elements (AREs), this contention is experimentally unconfirmed. Since the ARE resembles the binding sequence of erythroid transcription factor NF-E2, we investigated the possibility that the phase II enzyme genes might be regulated by transcription factors that also bind to the NF-E2 sequence. The expression profiles of a number of transcription factors suggest that an Nrf2/small Maf heterodimer is the most likely candidate to fulfill this rolein vivo.To directly test these questions, we disrupted the murinenrf2 genein vivo.While the expression of phase II enzymes (e.g., glutathione S-transferase and NAD(P)H: quinone oxidoreductase) was markedly induced by a phenolic antioxidantin vivoin both wild type and heterozygous mutant mice, the induction was largely eliminated in the liver and intestine of homozygousnrf2-mutant mice. Nrf2 was found to bind to the ARE with high affinity only as a heterodimer with a small Maf protein, suggesting that Nrf2/small Maf activates gene expression directly through the ARE. These results demonstrate that Nrf2 is essential for the transcriptional induction of phase II enzymes and the presence of a coordinate transcriptional regulatory mechanism for phase II enzyme genes. Thenrf2-deficient mice may prove to be a very useful model for thein vivoanalysis of chemical carcinogenesis and resistance to anti-cancer drugs.
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By its antioxidant effect, molecular hydrogen gas (H2) was reported to protect organs from tissue damage induced by ischemia reperfusion. To evaluate its anti-inflammatory effects, we established a mouse model of human inflammatory bowel disease (IBD) by supplying mice with water containing (1) dextran sodium sulfate (DSS) (5%), (2) DSS (5%) and H2, or (3) H2 only ad libitum up to 7 days. At day-7, DSS-induced pathogenic outcomes including, loss of body weight, increase of colitis score, pathogenic shortening of colon length, elevated level of IL-12, TNF-α and IL-1β in colon lesion, were significantly suppressed by the addition of H2 to DSS solution. Histological analysis also revealed that the DSS-mediated colonic tissue destruction accompanied by macrophage infiltration was remarkably suppressed by H2. Therefore, the present study indicated that H2 can prevent the development of DSS-induced colitis in mice.
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Caspase-8 and caspase-9 play crucial roles in the extrinsic and intrinsic apoptotic pathways, respectively. The nuclear translocation of apoptosis-inducing factor (AIF) is involved in caspase-independent apoptosis. Microtubule-associated protein 1 light chain 3 (LC3) plays a pivotal role in autophagy. In the present study, we analyzed the expression of cleaved caspase-8 (CC8), cleaved caspase-9 (CC9), AIF, and LC3 in 160 gastrointestinal adenocarcinomas. The nuclear expression of AIF was rare. The expression of CC8 in gastric and colorectal adenocarcinomas did not differ, whereas the percentage of CC9-positive tumors in gastric adenocarcinomas was significantly higher than in colorectal adenocarcinomas. In contrast, the percentage of LC3-positive tumors in gastric adenocarcinomas was significantly lower than in colorectal adenocarcinomas. CC8 and CC9 occasionally co-existed in the same tumor cells in gastric adenocarcinoma. However, LC3-positive tumor cells in colorectal adenocarcinomas were constantly negative for CC8. No correlation was identified between the expression of any markers and clinicopathological parameters. These results suggest that different cell death pathways are activated in a manner that depends upon the primary site and cell type. The extrinsic and intrinsic apoptotic pathways may be mutually regulated in gastric adenocarcinomas. Also, autophagy may function as a cellular guardian to avoid apoptosis in colorectal adenocarcinomas.
Article
Inhaled hydrogen gas exerts antioxidant and anti-inflammatory effects in rat intestinal transplantation. Here, we investigated whether ex vivo donor organ treatment with dissolved hydrogen would prevent intestinal graft injury. Isogeneic intestinal transplantation was performed in Lewis rats with vascular flush, luminal preservation, and cold graft storage in nitrogen-bubbled (SITxN2) or hydrogen-bubbled (SITxH2) preservation solution. Lactated Ringer's solution and 3-hr cold ischemia time were used for mechanistic investigations, whereas survival experiments were performed with University of Wisconsin solution and 6-hr cold ischemia time. During the early phase of ischemia-reperfusion injury, hydrogen-enriched solution significantly preserved mucosal graft morphology, diminished graft malondialdehyde levels demonstrating substantial reduction potential and blunted proinflammatory molecular responses (early growth response gene [EGR-1], interleukin [IL]-6, IL-1ß, and inducible nitric oxide synthase) within the reperfused intestinal graft muscularis. During the late phase of ischemia-reperfusion injury, circulating IL-6 protein and lactate dehydrogenase levels were significantly ameliorated in SITxH2 animals, which were associated with a favorable functional outcome in in vivo liquid gastrointestinal transit and recipient solid gastric emptying of chrome steel balls, and marked prevention of the posttransplant associated suppression of in vitro muscarinic jejunal contractility. Reflecting improved graft preservation, hydrogen preloading of grafts increased recipient survival rates from 41% to 80%. Anti-inflammatory and antiapoptotic heme oxygenase-1 was significantly upregulated in the hydrogen-treated graft muscularis but not mucosa before reperfusion. Graft preloading with hydrogen demonstrated superior morphologic and functional graft protection in rodent intestinal transplantation, ultimately facilitating recipient survival. Antioxidant capacity and muscularis heme oxygenase-1 upregulation are possible protective mechanisms.
Article
Because inhaled hydrogen provides potent anti-inflammatory and antiapoptotic effects against acute lung injury, we hypothesized that treatment of organ donors with inhaled hydrogen during mechanical ventilation would decrease graft injury after lung transplantation. Orthotopic left lung transplants were performed using a fully allogeneic Lewis to Brown Norway rat model. The donors were exposed to mechanical ventilation with 98% oxygen plus 2% nitrogen or 2% hydrogen for 3 h prior to harvest, and the lung grafts underwent 4 h of cold storage in Perfadex (Vitrolife, Göteborg, Sweden). The graft function, histomorphologic changes, and inflammatory reactions were assessed. The combination of mechanical ventilation and prolonged cold ischemia resulted in marked deterioration of gas exchange when the donors were ventilated with 2% nitrogen/98% oxygen, which was accompanied by upregulation of proinflammatory cytokines and proapoptotic molecules. These lung injuries were attenuated significantly by ventilation with 2% hydrogen. Inhaled hydrogen induced heme oxygenase-1, an antioxidant enzyme, in the lung grafts prior to implantation, which might contribute to protective effects afforded by hydrogen. Preloaded hydrogen gas during ventilation prior to organ procurement protected lung grafts effectively from ischemia/reperfusion-induced injury in a rat lung transplantation model.