Epithelial sodium channel is involved in H2S-induced acute pulmonary edema

Abstract

Acute pulmonary edema is one of the major outcomes of exposure to high levels of hydrogen sulfide (H₂S). However, the mechanisms involved in H₂S-induced acute pulmonary edema are still poorly understood. Therefore, the present study is designed to evaluate the role of epithelial sodium channel (ENaC) in H₂S-induced acute pulmonary edema. The Sprague–Dawley rats were exposed to sublethal concentrations of inhaled H₂S, then the pulmonary histological and lung epithelial cell injury were evaluated by hematoxylin–eosin staining and electron microscopy, respectively. In addition to morphological investigation, our results also revealed that H₂S exposure significantly decreased the alveolar fluid clearance and increased the lung tissue wet–dry ratio. These changes were demonstrated to be associated with decreased ENaC expression. Furthermore, the extracellular-regulated protein kinases 1/2 pathway was demonstrated to be implicated in H₂S-mediated ENaC expression, because PD98059, an ERK1/2 antagonist, significantly mitigated H₂S-mediated ENaC down-regulation. Therefore, our results show that ENaC might represent a novel pharmacological target for the treatment of acute pulmonary edema induced by H₂S and other hazardous gases.     

Protective effects of puerarin on acute lung and cerebrum injury induced by hypobaric hypoxia via the regulation of aquaporin (AQP) via NF-κB signaling pathway

ObjectiveHypobaric hypoxia, frequently encountered at high altitude, may lead to lung and cerebrum injury. Our study aimed to investigate whether puerarin could exert ameliorative effects on rats exposed to hypobaric hypoxia via regulation of aquaporin (AQP) and NF-κB signaling pathway in lung and cerebrum.Materials and methods40 Sprague Dawley rats were divided into four groups (normal control group, hypobaric hypoxia group, puerarin group and dexamethasone group). Wet/dry ratio, blood gas, pathological changes of lung and cerebrum and spatial memory were observed in each group. Inflammatory cytokines in bronchoalveolar lavage fluid (BALF) were determined with ELISA and expression of AQP1, AQP4, NF-κB signaling pathway in lung and cerebrum with western blotResultsPuerarin showed significant preventative effects on tissue injury and behavioral changes, as evidenced by histopathological findings and Morris water maze. In addition, levels of inflammatory cytokines in BALF decreased in the two preventative groups compared with those of hypobaric hypoxia group. AQP in lung and cerebrum increased under the condition of hypobaric hypoxia while was down regulated in both two preventative groups. NF-κB and IκB was also inhibited by puerarin.ConclusionOur study suggested that lung and cerebrum injury, increased inflammatory cytokines in BALF and increased AQP1, AQP4 and NF-κB signaling pathway occurred under the condition of hypobaric hypoxia. Moreover, puerarin could prevent lung and cerebrum injury of rats exposed to hypobaric hypoxia via down-regulation of inflammatory cytokines, AQP1 and AQP4 expression and NF-κB signaling pathway.     

Hydrogen sulfide protects against bleomycin-induced pulmonary fibrosis in rats by inhibiting NF-κB expression and regulating Th1/Th2 balance

Hydrogen sulfide (H₂S) displays vasodilative, anti-oxidative, anti-inflammatory and cytoprotective activities. The objective of this study was to evaluate the inhibitory effect of H₂S on bleomycin (BLM)-induced pulmonary fibrosis in rats and its possible mechanisms. Fifty-four pathogen-free Male Wistar rats were randomly divided into three groups: control, BLM and H₂S treated groups with 18 rats in each group. Each group was then divided into three subgroups based on time of study (7, 14 and 28 day). Pulmonary fibrosis model was established by a single intratracheal instillation of BLM A₅ (5 mg/kg). While control rats received saline, rats of the treated group simultaneously were administered intraperitoneal injections of NaHS (the H₂S donor, 28 μmol/kg) once daily. BLM induced pulmonary inflammation and fibrosis, increased lung hydroxyproline levels, lung index, total cell counts, neutrophils and eosinophils counts and expression of NF-κB p65 in lung tissue, decreased lymphocytes and macrophages counts. In addition, Th1 response is suppressed as shown by diminished IFN-γ in bronchoalveolar lavage fluid (BALF) after BLM exposure, and enhancement of Th2 response is marked by increased IL-4 in BALF. H₂S administration significantly attenuated these effects. The findings reveal the therapeutic potential of H₂S for BLM-induced pulmonary fibrosis in male rats, which were at least partly due to inhibition NF-κB p65 expression and regulation of Th1/Th2 balance.     

Hydrogen sulfide and translational medicine

Hydrogen sulfide (H₂S) along with carbon monoxide and nitric oxide is an important signaling molecule that has undergone large numbers of fundamental investigations. H₂S is involved in various physiological activities associated with the regulation of homeostasis, vascular contractility, pro- and anti-inflammatory activities, as well as pro- and anti-apoptotic activities etc. However, the actions of H₂S are influenced by its concentration, reaction time, and cell/disease types. Therefore, H₂S is a signaling molecule without definite effect. The use of existing H₂S donors is limited because of the instant release and short lifetime of H₂S. Thus, translational medicine involving the sustained and controlled release of H₂S is of great value for both scientific and clinical uses. H₂S donation can be manipulated by different ways, including where H₂S is given, how H₂S is donated, or the specific structures of H₂S-releasing drugs and H₂S donor molecules. This review briefly summarizes recent progress in research on the physiological and pathological functions of H₂S and H₂S-releasing drugs, and suggests hope for future investigations.     

Protective effect of diallyl disulfide against cerulein-induced acute pancreatitis and associated lung injury in mice

Garlic (Allium sativum) – derived organosulfur compound diallyl disulfide (DADS) possesses antioxidant, anti-inflammatory and anti-cancer effects. This study was aimed to investigate the anti-inflammatory role and the underlying molecular mechanisms of DADS in cerulein-induced acute pancreatitis (AP) and associated lung injury. Administration of DADS significantly attenuated the severity of pancreatic and pulmonary inflammation by inhibiting cerulein induced serum amylase, myeloperoxidase activity (MPO) and histological changes in pancreas and lung. Furthermore, the anti-inflammatory effect of DADS was associated with the decrease in tumor necrosis factor (TNF)-α,cystathionine-γ-lyase (CSE), preprotachykinin A (PPTA), neurokinin-1-receptor (NK1R) expression and hydrogen sulfide (H₂S) production in both pancreas and lung. In addition, DADS reduced caerulein-induced I-κB degradation and subsequent translocation of NF-κB in the pancreas and lung. These results show for the first time that in AP, DADS exhibits an anti-inflammatory effect by inhibiting CSE/H₂S and SP/NK1R signaling and NF-кB pathway.     

Vasoprotective effects of neurocompensatory response to balloon injury during diabetes involve the improvement of Mas signaling by TGFβ1 activation

Balloon injury in diabetic rats triggers a sensory neurocompensatory response that restores the blood flow in contralateral carotid. These vasoprotective effects result from H₂O₂-mediated relaxation that counteracts AT₁-dependent contractile hyperreactivity. The most important mechanism from the renin–angiotensin-system in counteracting AT₁-mediated effects is that one is mediated by Mas receptors. Thus, we hypothesized that the vasoprotective effects of balloon neurocompensation in diabetic rats could result from the improvement of Mas signaling by H₂O₂-mediated sensory mechanisms. NK₁ receptors are sensory components whose activation could lead to H₂O₂ generation upon TGFβ₁ release and ALK5-mediated Nox4 upregulation. Based on this, we aimed to investigate: (1) the role of the TGFβ₁/ALK5–Nox4–H₂O₂ pathway on modulating Mas signaling in diabetic rat contralateral carotid; and (2) the contribution of Mas signaling in the control of local blood flow. Our results showed that balloon neurocompensation restored diabetic rat contralateral carotid flow by improving Mas signaling through NK₁-mediated TGFβ₁ release. TGFβ₁/ALK5 activation enhanced Nox4 expression and Nox4-driven generation of H₂O₂. In turn, H₂O₂ enhanced the local Mas-mediated relaxation. Since restenosis impairs diabetic rat ipsilateral carotid flow, the restoration of diabetic rat contralateral carotid flow may prevent further damages in cerebral irrigation by carotid pathways after angioplasty during diabetes.     

Hydrogen sulfide inhibits cigarette smoke-induced inflammation and injury in alveolar epithelial cells by suppressing PHD2/HIF-1α/MAPK signaling pathway

Chronic obstructive pulmonary fibrosis (COPD) is a chronic and fatal lung disease with few treatment options. Sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H₂S), was found to alleviate cigarette smoke (CS)-induced emphysema in mice, however, the underlying mechanisms have not yet been clarified. In this study, we investigated its effects on COPD in a CS-induced mouse model in vivo and in cigarette smoke extract (CSE)-stimulated alveolar epithelial A549 cells in vitro. The results showed that NaHS not only relieved emphysema, but also improved pulmonary function in CS-exposed mice. NaHS significantly increased the expressions of tight junction proteins (i.e., ZO-1, Occludin and claudin-1), and reduced apoptosis and secretion of pro-inflammatory cytokines (i.e., TNF-α, IL-6 and IL-1β) in CS-exposed mouse lungs and CSE-incubated A549 cells, indicating H₂S inhibits CS-induced inflammation, injury and apoptosis in alveolar epithelial cells. NaHS also upregulated prolyl hydroxylase (PHD)2, and suppressed hypoxia-inducible factor (HIF)-1α expression in vivo and in vitro, suggesting H₂S inhibits CS-induced activation of PHD2/HIF-1α axis. Moreover, NaHS inhibited CS-induced phosphorylation of ERK, JNK and p38 MAPK in vivo and in vitro, and treatment with their inhibitors reversed CSE-induced ZO-1 expression and inflammation in A549 cells. These results suggest that NaHS may prevent emphysema via the suppression of PHD2/HIF-1α/MAPK signaling pathway, and subsequently inhibition of inflammation, epithelial cell injury and apoptosis, and may be a novel strategy for the treatment of COPD.     

Hydrogen sulfide ameliorates disorders in the parafacial respiratory group region of neonatal rats caused by prenatal cigarette smoke exposure via an antioxidative effect

We previously found that hydrogen sulfide (H₂S) ameliorated the dysfunction of central chemoreception caused by prenatal cigarette smoke exposure (CSE). In the present study, we further explored whether the parafacial respiratory group (pFRG) is involved in the protection of central chemoreception by H₂S against prenatal CSE-induced injury. We found that NaHS, a donor of H₂S, restored the expression of Phox2b, which was downregulated by prenatal CSE, in the pFRG region of neonatal rats. NaHS also relieved the prenatal CSE-induced excitatory synapse disturbance in the pFRG region of neonatal rats. Additionally, NaHS prevented the increase in the malondialdehyde level and suppression of antioxidase activity in the pFRG region of neonatal rats induced by prenatal CSE. Furthermore, NaHS prevented the downregulation of the expression of antioxidases and Nrf2 in the pFRG region of neonatal rats with prenatal CSE. These results suggest that H₂S can protect the pFRG of neonatal rats against prenatal CSE-induced injury via an antioxidative effect.     

Garlic-derived natural polysulfanes as hydrogen sulfide donors: Friend or foe?

In vitro and in vivo studies reported the anti-cancer potential of organosulfur compounds (OSCs) as they trigger biological effects leading to cell cycle arrest with accumulation of cells in G2/M, alteration of the microtubular network, modulation of Bcl-2 family protein expression patterns and changes of the redox status. Despite these well-described effects, no OSC derivative is yet undergoing clinical trials even though their chemistry is well understood as OSCs act as hydrogen sulfide (H₂S) donors. H₂S is a biological mediator, synthesized through cysteine degradation and modulates vasodilation, cytoprotection, inflammation and angiogenesis. It is well accepted that H₂S plays a biphasic pharmacological role: the inhibition of endogenous synthesis of H₂S and paradoxically also the use of H₂S donors to increase H₂S concentration, induce both anti-cancer effects leading therefore to controversial discussions. Altogether, the role of H₂S in the anti-cancer action of OSCs remains poorly understood. In this review, we hypothesize that OSCs act through H₂S signaling pathways in cancer cells, and that a clearer understanding of the mechanism of action of H₂S in OSC-mediated anti-cancer activity is required for further application of these compounds in translational medicine.     

Bone marrow-derived mesenchymal stem cells attenuate phosgene-induced acute lung injury in rats

Abstract

Accidental phosgene exposure could result in acute lung injury (ALI), effective therapy is needed for the patients with phosgene-induced ALI. As a type of cells with therapeutic potential, mesenchymal stem cells (MSCs) have been showed its efficacy in multiple diseases. Here, we assessed the therapeutic potential of MSCs in phosgene-induced ALI and explored the related mechanisms. After isolation and characterization of rat bone marrow MSCs (BMMSCs), we transplanted BMMSCs into the rats exposed to phosgene and observed significant improvement on the lung wet-to-dry ratio and partial oxygen pressure (PaO₂) at 6, 24, 48?h after phosgene exposure. Histological analyses revealed reduced sign of pathological changes in the lungs. Reduced level of pro-inflammatory tumor necrosis factor α and increased level of anti-inflammatory factor interleukin-10 were found in both bronchoalveolar lavage and plasma. Significant increased expression of epithelial cell marker AQP5 and SP-C was also found in the lung tissue. In conclusion, treatment with MSC markedly decreases the severity of phosgene-induced ALI in rats, and these protection effects were closely related to the pulmonary air blood barrier repairment and inflammatory reaction regulation.     

The Protective Effect of Eupatilin against Hydrogen Peroxide-Induced Injury Involving 5-Lipoxygenase in Feline Esophageal Epithelial Cells

In this study, we focused to identify whether eupatilin (5,7-dihydroxy-3'',4'',6-trimethoxyflavone), an extract from Artemisia argyi folium, prevents H₂O₂-induced injury of cultured feline esophageal epithelial cells. Cell viability was measured by the conventional MTT reduction assay. Western blot analysis was performed to investigate the expression of 5-lipoxygenase by H₂O₂ treatment in the absence and presence of inhibitors. When cells were exposed to 600 µM H₂O₂ for 24 hours, cell viability was decreased to 40%. However, when cells were pretreated with 25~150 µM eupatilin for 12 hours, viability was significantly restored in a concentration-dependent manner. H₂O₂-treated cells were shown to express 5-lipoxygenase, whereas the cells pretreated with eupatilin exhibited reduction in the expression of 5-lipoxygenase. The H₂O₂-induced increase of 5-lipoxygenase expression was prevented by SB202190, SP600125, or NAC. We further demonstrated that the level of leukotriene B₄ (LTB₄) was also reduced by eupatilin, SB202190, SP600125, NAC, or nordihydroguaiaretic acid (a lipoxygenase inhibitor) pretreatment. H₂O₂ induced the activation of p38MAPK and JNK, this activation was inhibited by eupatilin. These results indicate that eupatilin may reduce H₂O₂-induced cytotoxicity, and 5-lipoxygenase expression and LTB₄ production by controlling the p38 MAPK and JNK signaling pathways through antioxidative action in feline esophageal epithelial cells.     

Pharmacokinetics and Preventive Effects of Sulfo-Albumin as a Novel Macromolecular Hydrogen Sulfide Prodrug on Carbon Tetrachloride-Induced Hepatic Injury

Hydrogen sulfide (H₂S) has been recently recognized as a gaseous signaling molecule that controls various biological activities. In the present study, we developed sulfo-albumin as a macromolecular H₂S prodrug for therapeutic use, in which multisulfide groups (source of H₂S) were conjugated with bovine serum albumin through a covalent linkage. In an in vitro study on H₂S release in phosphate buffered saline solution, we found that H₂S was released from sulfo-albumin in the presence of 5-mM glutathione but not in its absence. Furthermore, sulfo-albumin was taken up by RAW 264.7 cells, and it released H₂S in cells but not in plasma. These results indicate that H₂S can be selectively released from sulfo-albumin in cells. ¹¹¹In-labeled sulfo-albumin predominantly accumulated in the liver, dependent upon the number of sulfide groups, after intravenous injection in mice. In a carbon tetrachloride-induced acute liver injury mouse model, sulfo-albumin significantly suppressed the increase in plasma aspartate aminotransferase and alanine aminotransferase activities, which are indicators of hepatocyte injury, after intravenous injection. These findings indicate that sulfo-albumin is a promising compound for the treatment of hepatic injuries.     

Upregulation of acid sensing ion channel 1a (ASIC1a) by hydrogen peroxide through the JNK pathway

Oxidative stress is intimately tied to neurodegenerative diseases, including Parkinson’s disease and amyotrophic lateral sclerosis, and acute injuries, such as ischemic stroke and traumatic brain injury. Acid sensing ion channel 1a (ASIC1a), a proton-gated ion channel, has been shown to be involved in the pathogenesis of these diseases. However, whether oxidative stress affects the expression of ASIC1a remains elusive. In the current study, we examined the effect of hydrogen peroxide (H₂O₂), a major reactive oxygen species (ROS), on ASIC1a protein expression and channel function in NS20Y cells and primary cultured mouse cortical neurons. We found that treatment of the cells with H₂O₂ (20 µM) for 6 h or longer increased ASIC1a protein expression and ASIC currents without causing significant cell injury. H₂O₂ incubation activated mitogen-activated protein kinases (MAPKs) pathways, including the extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 pathways. We found that neither inhibition of the MEK/ERK pathway by U0126 nor inhibition of the p38 pathway by SB203580 affected H₂O₂-induced ASIC1a expression, whereas inhibition of the JNK pathway by SP600125 potently decreased ASIC1a expression and abolished the H₂O₂-mediated increase in ASIC1a expression and ASIC currents. Furthermore, we found that H₂O₂ pretreatment increased the sensitivity of ASIC currents to the ASIC1a inhibitor PcTx1, providing additional evidence that H₂O₂ increases the expression of functional ASIC1a channels. Together, our data demonstrate that H₂O₂ increases ASIC1a expression/activation through the JNK signaling pathway, which may provide insight into the pathogenesis of neurological disorders that involve both ROS and activation of ASIC1a.     

Hydrogen sulfide,a gaseous signaling molecule,elongates primary cilia on kidney tubular epithelial cells by activating extracellular signal-regulated kinase

Primary cilia on kidney tubular cells play crucial roles in maintaining structure and physiological function. Emerging evidence indicates that the absence of primary cilia, and their length, are associated with kidney diseases. The length of primary cilia in kidney tubular epithelial cells depends, at least in part, on oxidative stress and extracellular signal-regulated kinase 1/2 (ERK) activation. Hydrogen sulfide (H₂S) is involved in antioxidant systems and the ERK signaling pathway. Therefore, in this study, we investigated the role of H₂S in primary cilia elongation and the downstream pathway. In cultured Madin-Darby Canine Kidney cells, the length of primary cilia gradually increased up to 4 days after the cells were grown to confluent monolayers. In addition, the expression of H₂S-producing enzyme increased concomitantly with primary cilia length. Treatment with NaHS, an exogenous H₂S donor, accelerated the elongation of primary cilia whereas DL-propargylglycine (a cystathionine γ-lyase inhibitor) and hydroxylamine (a cystathionine-β-synthase inhibitor) delayed their elongation. NaHS treatment increased ERK activation and Sec10 and Arl13b protein expression, both of which are involved in cilia formation and elongation. Treatment with U0126, an ERK inhibitor, delayed elongation of primary cilia and blocked the effect of NaHS-mediated primary cilia elongation and Sec10 and Arl13b upregulation. Finally, we also found that H₂S accelerated primary cilia elongation after ischemic kidney injury. These results indicate that H₂S lengthens primary cilia through ERK activation and a consequent increase in Sec10 and Arl13b expression, suggesting that H₂S and its downstream targets could be novel molecular targets for regulating primary cilia.     

Biodegradable calcium sulfide-based nanomodulators for H2S-boosted Ca2+-involved synergistic cascade cancer therapy

Hydrogen sulfide (H₂S) is the most recently discovered gasotransmitter molecule that activates multiple intracellular signaling pathways and exerts concentration-dependent antitumor effect by interfering with mitochondrial respiration and inhibiting cellular ATP generation. Inspired by the fact that H₂S can also serve as a promoter for intracellular Ca²⁺ influx, tumor-specific nanomodulators (I-CaS@PP) have been constructed by encapsulating calcium sulfide (CaS) and indocyanine green (ICG) into methoxy poly (ethylene glycol)-b-poly (lactide-co-glycolide) (PLGA-PEG). I-CaS@PP can achieve tumor-specific biodegradability with high biocompatibility and pH-responsive H₂S release. The released H₂S can effectively suppress the catalase (CAT) activity and synergize with released Ca²⁺ to facilitate abnormal Ca²⁺ retention in cells, thus leading to mitochondria destruction and amplification of oxidative stress. Mitochondrial dysfunction further contributes to blocking ATP synthesis and downregulating heat shock proteins (HSPs) expression, which is beneficial to overcome the heat endurance of tumor cells and strengthen ICG-induced photothermal performance. Such a H₂S-boosted Ca²⁺-involved tumor-specific therapy exhibits highly effective tumor inhibition effect with almost complete elimination within 14-day treatment, indicating the great prospect of CaS-based nanomodulators as antitumor therapeutics.     

Activation of phospholipase D involved in both injury and survival in A549 alveolar epithelial cells exposed to H2O2

To determine the role of the phospholipase D (PLD) pathway in injury and survival of alveolar epithelial cells, A549 cells were exposed to H₂O₂ (500 μM) which resulted in time-dependent injury and bi-phasic increase of PLD activity at 5 min and at 3 h, respectively. n-Butanol (0.5%) inhibited PLD activation, attenuated cell injury at 5 min of H₂O₂ exposure, but enhanced injury at 3 h of exposure. This activation was inhibited by treatment with catalase (500 units/ml). Exogenous phosphatidic acid mimicked the effects of PLD activation, and diphenyliodonium (NADPH oxidase inhibitor) reversed the decline in cell viability induced by H₂O₂ exposure. Propranolol (phosphatidic acid phospholydrolase inhibitor) and quinacrine (phospholipase A2 inhibitor) had weak effects on H₂O₂-induced PLD activation but reversed H₂O₂-induced injury. We speculate that PLD activation at the initiation of H₂O₂ exposure predominantly results in NAPDH oxidase activation, which mediates A549 cell injury, but turns to mediating cell survival as the H₂O₂ attack continues, which might be mainly due to the accumulation of intracellular phosphatidic acid.     

Hydrogen sulfide alleviates uranium‐induced acute hepatotoxicity in rats: Role of antioxidant and antiapoptotic signaling

As an endogenous gaseous mediator, H₂S exerts antioxidative, antiapoptotic, and cytoprotective effects in livers. This study was designed to investigate the protective role of H₂S against uranium‐induced hepatotoxicity in adult SD male rats after in vivo effect of uranium on endogenous H₂S production was determined in livers. The levels of endogenous H₂S and H₂S‐producing enzymes (CBS and CSE) were measured in liver homogenates from uranium ‐intoxicated rats. In rats injected intraperitoneally (i.p.) with uranyl acetate or NaHS (an H₂S donor) alone or in combination, we examined biochemical parameters to assess liver function, revealed hepatic histopathological alteration, investigated oxidative stress markers, and explored apoptotic signaling in liver homogenates. The results suggest that uranium‐intoxication in rats decreased CBS and CSE protein expression, H₂S synthesis capacity, and endogenous H₂S generation. NaHS administration in uranium‐intoxicated rats produced amelioration in liver biochemical indices and histopathological effects, decreased MDA content, and increased GSH level and antioxidative enzymes activities like SOD, CAT, GPx, and GST. NaHS administration in uranium‐intoxicated rats attenuated uranium‐activated phosphorylation state of JNK. NaHS treatment in uranium‐intoxicated rats increased antiapoptotic Bcl‐2 but decreased pro‐apoptotic Bax, resulting in the rise of Bcl‐2/Bax ratio. NaHS treatment in uranium‐intoxicated rats reduced the apoptosis mediator caspase‐3 and cytochrome c release and elevated ATP contents. Taken together, these data implicate that H₂S can afford protection to rat livers against uranium‐induced adverse effects mediated by up‐regulation of antioxidant and antiapoptotic signaling. The anti‐apoptotic property of H₂S may be involved, at least in part, in inhibiting JNK signaling. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 581–593, 2017.     

Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H₂O₂ across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H₂O₂ release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72 h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p < 0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H₂O₂ release, assessed by Amplex Red, was reduced by about 45% (p < 0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+ 120%, p < 0.05) and loss of mitochondrial membrane potential (− 80%, p < 0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H₂O₂ release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death.     

Beneficial effect of a hydrogen sulphide donor (sodium sulphide) in an ovine model of burn- and smoke-induced acute lung injury

Background and purpose:

The present study investigated whether the pathophysiological changes induced by burn and smoke inhalation are modulated by parenteral administration of Na₂S, a H₂S donor.

Experimental approach:

The study used a total of 16 chronically instrumented, adult female sheep. Na₂S was administered 1 h post injury, as a bolus injection at a dose of 0.5 mg·kg⁻¹ and subsequently, as a continuous infusion at a rate of 0.2 mg·kg⁻¹·h⁻¹ for 24 h. Cardiopulmonary variables (mean arterial and pulmonary arterial blood pressure, cardiac output, ventricular stroke work index, vascular resistance) and arterial and mixed venous blood gases were measured. Lung wet-to-dry ratio and myeloperoxidase content and protein oxidation and nitration were also measured. In addition, lung inducible nitric oxide synthase expression and cytochrome c were measured in lung homogenates via Western blotting and enzyme-linked immunosorbent assay (elisa) respectively.

Key results:

The H₂S donor decreased mortality during the 96 h experimental period, improved pulmonary gas exchange and lowered further increase in inspiratory pressure and fluid accumulation associated with burn- and smoke-induced acute lung injury. Further, the H₂S donor treatment reduced the presence of protein oxidation and 3-nitrotyrosine formation following burn and smoke inhalation injury.

Conclusions and implications:

Parenteral administration of the H₂S donor ameliorated the pulmonary pathophysiological changes associated with burn- and smoke-induced acute lung injury. Based on the effect of H₂S observed in this clinically relevant model of disease, we propose that treatment with H₂S or its donors may represent a potential therapeutic strategy in managing patients with acute lung injury.     

REDUCED LUNG WATER TRANSPORT RATE ASSOCIATED WITH DOWNREGULATION OF AQUAPORIN-1 AND AQUAPORIN-5 IN AGED MICE

• 1 The purpose of the present study was to examine lung water transport properties and the expression and regulation of the alveolar endothelial water channel aquaporin (AQP)‐1 and the epithelial water channel AQP‐5 in aged mouse lung using gene expression analysis and water permeability measurements.
• 2 In aged (20–24‐month‐old) mice, AQP‐1 and AQP‐5 mRNA expression decreased by 55.5 and 50.3%, respectively, compared with that in young (8–10‐week‐old) mice (P < 0.01). In addition, AQP‐1 and AQP‐5 protein expression decreased in aged mice by 36.9 and 44.6%, respectively, compared with that in young mice (P < 0.01).
• 3 The osmotically driven water transport rate between the airspace and capillary compartments was reduced by 31.7% in aged mice compared with young mice (2.8 ± 0.3 vs 4.1 ± 0.3 mg/s, respectively; P < 0.01). The hydrostatically driven lung water accumulation rate in response to a 10 cmH₂O increase in pulmonary artery pressure was also reduced in aged mice by 21.9% compared with young mice (0.32 ± 0.06 vs 0.41 ± 0.04 mg/s, respectively; P < 0.01).
• 4 There was a 62.7% decrease in serum glucocorticoids in aged mice compared with young mice (67.6 ± 26.8 vs 181.3 ± 44.4 nmol/L, respectively; P < 0.01). In vivo administration of dexamethasone (4 mg/kg) for 5 consecutive days to aged mice increased lung AQP‐1 mRNA and protein expression by 2.1 ± 0.1 fold (P < 0.01) and 1.8 ± 0.2 fold (P < 0.01), respectively. Accordingly, osmotically and hydrostatically driven water transport rates increased by 35.6% (P < 0.01) and 31.2% (P < 0.01), respectively.
• 5 The present study provides the first evidence of altered lung water transport associated with downregulation of AQPs in aged lung. Blood glucocorticoid hormone levels are important to maintain normal AQP‐1 expression in the lung microvascular endothelium. Corticosteroid‐induced AQP‐1 upregulation may contribute to the role of corticosteroids in accelerating oedema clearance in aged lung.