Genetic basis of murine responses to hyperoxia-induced lung injury

To evaluate the effect of genetic background on oxygen (O₂) toxicity, nine genetically diverse mouse strains (129/SvIm, A/J, BALB/cJ, BTBR+(T)/tf/tf, CAST/Ei, C3H/HeJ, C57BL/6J, DBA/2J, and FVB/NJ) were exposed to more than 99% O₂ for 72 h. Immediately following the hyperoxic challenge, the mouse strains demonstrated distinct pathophysiologic responses. The BALB/cJ and CAST/Ei strains, which were the only strains to demonstrate mortality from the hyperoxic challenges, were also the only strains to display significant neutrophil infiltration into their lower respiratory tract. In addition, the O₂-challenged BALB/cJ and CAST/Ei mice were among six strains (A/J, BALB/cJ, CAST/Ei, BTBR+(T)/tf/tf, DBA/2J, and C3H/HeJ) that had significantly increased interleukin 6 concentrations in the whole lung lavage fluid and were among all but one strain that had large increases in lung permeability compared with air-exposed controls. In contrast, the DBA/2J strain was the only strain not to have any significant alterations in lung permeability following hyperoxic challenge. The expression of the extracellular matrix proteins, including collagens I, III, and IV, fibronectin I, and tenascin C, also varied markedly among the mouse strains, as did the activities of total superoxide dismutase (SOD) and manganese-SOD (Mn-SOD or SOD2). These data suggest that the response to O₂ depends, in part, on the genetic background and that some of the strains analyzed can be used to identify specific loci and genes underlying the response to O₂.     

Adenovirus-mediated transfer of the SOCS-1 gene to mouse lung confers protection against hyperoxic acute lung injury

Suppressor of cytokine signaling-1 (SOCS-1) is a member of the suppressor of cytokine signaling family of proteins and an inhibitor of interleukin-6 (IL-6) signaling. SOCS-1 has been shown to protect cells from cellular damage and apoptosis induced by tumor necrosis factor (TNF), lipopolysaccharide (LPS), and interferon gamma (IL-γ). However, it is not known whether increased SOCS-1 is protective during pulmonary oxidative stress. Therefore, we hypothesized that increased SOCS-1 in the lungs of mice would be protective in the setting of hyperoxic lung injury. We administered SOCS-1 adenovirus (Ad-SOCS-1) intratracheally into the lungs and exposed the mice to 100% O₂. Mice infected with GFP adenovirus (Ad-GFP) were used as controls. Mice treated with Ad-SOCS-1 had enhanced survival in 100% oxygen compared to Ad-GFP-administered mice. After 3 days of hyperoxia, Ad-GFP mice were ill and tachypnic and died after 4 days. In contrast, all Ad-SOCS-1-treated mice survived for at least 6 days in hyperoxia and 80% survived beyond 7 days. Ad-SOCS-1 transfection protected mouse lungs from injury as indicated by lower lung wet/dry weight, alveolar–capillary protein leakage, reduced infiltration of inflammatory cells, and lower content of thiobarbituric acid-reactive substances in lung homogenate. Our results also indicated that Ad-SOCS-1 significantly inhibits hyperoxia-induced ASK-1 (apoptosis signal-regulating kinase 1) expression. Taken together, these findings show that increased expression of adenovirus-mediated SOCS-1 in the lungs of mice significantly protects against hyperoxic lung injury.     

Subcutaneous administration of bovine superoxide dismutase protects lungs from radiation-induced lung injury

Abstract

Background. The objective of the present study was to determine whether single administration of the antioxidant enzyme bovine superoxide dismutase (bSOD) after radiation therapy (RT) mitigates development of pulmonary toxicity in rats. Methods. Female F344 rats (n = 60) were divided among six experimental groups: (1) RT, single dose of 21 Gy to the right hemithorax; (2) RT + 5 mg/kg bSOD; (3) RT + 15 mg/kg bSOD; (4) No RT; (5) sham RT + 5 mg/kg bSOD; and (6) sham RT + 15 mg/kg bSOD. A single subcutaneous injection of bSOD (5 or 15 mg/kg) was administered 24 h post-radiation. The effects of bSOD on radiation-induced lung injury were assessed by measurement of body weight, breathing frequency, and histopathological changes. Immunohistochemistry was used to evaluate oxidative stress (8-OHdG⁺, NOX4⁺, nitrotyrosine⁺, and 4HNE⁺ cells), macrophage activation (ED1⁺), and expression of profibrotic transforming growth factor-β or TGF-β in irradiated tissue. Results. Radiation led to an increase in all the evaluated parameters. Treatment with 15 mg/kg bSOD significantly decreased levels of all the evaluated parameters including tissue damage and breathing frequency starting 6 weeks post-radiation. Animals treated with 5 mg/kg bSOD trended toward a suppression of radiation-induced lung damage but did not reach statistical significance. Conclusions. The single application of bSOD (15 mg/kg) ameliorates radiation-induced lung injury through suppression of reactive oxygen species/reactive nitrogen species or ROS/RNS-dependent tissue damage.     

A bioluminescent transgenic mouse model: Real-time in vivo imaging of antioxidant EC-SOD gene expression and regulation by interferon gamma

Extracellular superoxide dismutase (EC-SOD) is the main antioxidant enzyme in the extracellular matrix. We developed transgenic mice to analyze the EC-SOD promoter activity in vivo in real time and to identify the important cis-elements flanking the 5′ region of the murine EC-SOD gene. Using this model, we demonstrated that luciferase reporter activity correlates closely with endogenous EC-SOD expression, although several interesting differences were also observed. Specifically, luciferase activity was detected at the highest levels in testes, aorta and perirenal fat. Reporter expression was regulated by interferon gamma, a finding that is in agreement with published endogenous EC-SOD gene expression studies. Thus, the 5′-flanking region of mouse EC-SOD gene is responsible, at least in part, for cell specific and inducible expression.     

Effect of overexpression of wild-type and mutant Cu/Zn-superoxide dismutases on oxidative stress and cell death induced by hydrogen peroxide, 4-hydroxynonenal or serum deprivation: potentiation of injury by ALS-related mutant superoxide dismutases and protection by Bcl-2

Mutations in Cu/Zn-superoxide dismutase (SOD1) are associated with some cases of familial amyotrophic lateral sclerosis (ALS). We overexpressed Bcl-2, wild-type SOD1 or mutant SOD1s (G37R and G85R) in NT-2 and SK-N-MC cells. Overexpression of Bcl-2 rendered cells more resistant to apoptosis induced by serum withdrawal, H2O2 or 4-hydroxy-2-trans-nonenal (HNE). Overexpression of Bcl-2 had little effect on levels of protein carbonyls, lipid peroxidation, 8-hydroxyguanine (8-OHG) or 3-nitrotyrosine. Serum withdrawal or H2O2 raised levels of protein carbonyls, lipid peroxidation, 8-OHG and 3-nitrotyrosine, changes that were attenuated in cells overexpressing Bcl-2. Overexpression of either SOD1 mutant tended to increase levels of lipid peroxidation, protein carbonyls, and 3-nitrotyrosine and accelerated viability loss induced by serum withdrawal, H2O2 or HNE, accompanied by greater rises in oxidative damage parameters. The effects of mutant SOD1s were attenuated by Bcl-2. By contrast, expression of wild-type SOD1 rendered cells more resistant to loss of viability induced by serum deprivation, HNE or H2O2. The levels of lipid peroxidation in wild-type SOD1 transfectants were elevated. Overexpression of mutant SOD1s makes cells more predisposed to undergo apoptosis in response to several insults. Our cellular systems appear to mimic events in patients with ALS or transgenic mice overexpressing mutant SOD1.     

Glycine protection of PC-12 cells against injury by ATP-depletion

A distinctive mechanism of cell injury during ATP depletion involves the loss of cellular glycine. The current study examined whether provision of glycine during ATP depletion can prevent injury in PC-12 cells, a cell line with neuronal property. In addition, we have examined the role played by glycine receptors in cytoprotective effects of the amino acid. It was shown that ATP depletion led to plasma membrane damage in PC-12 cells, which was ameliorated by 0.25–5mM glycine. Cytoprotective activity of glycine was shared by alanine, but not by glutamate or -aminobutyric acid (GABA). Of interest, strychnine, an antagonist of glycine receptor, was also protective. The results, while suggesting the involvement of glycine receptor in cytoprotection, indicate that chloride channel activity of the receptor is dispensable. Such a scenario is further supported by the observation that removal of extracellular chloride did not affect ATP depletion–induced cell injury or its prevention by glycine. In short, this study has provided the first evidence for glycine protection of cells with neuronal properties. Cytoprotection may involve the glycine receptor; however, it can be dissociated from its channel activity.     

Nitric oxide mediates lung injury induced by ischemia-reperfusion in rats

Nitric oxide (NO) has been reported to play a role in lung injury (LI) induced by ischemia-reperfusion (I/R). However, controversy exists as to the potential beneficial or detrimental effect of NO. In the present study, an in situ, perfused rat lung model was used to study the possible role of NO in the LI induced by I/R. The filtration coefficient (Kfc), lung weight gain (LWG), protein concentration in the bronchoalveolar lavage (PCBAL), and pulmonary arterial pressure (PAP) were measured to evaluate the degree of pulmonary hypertension and LI. I/R resulted in increased Kfc, LWG, and PCBAL. These changes were exacerbated by inhalation of NO (20-30 ppm) or 4 mM L-arginine, an NO precursor. The permeability increase and LI caused by I/R could be blocked by exposure to 5 mM N omega-nitro-L-arginine methyl ester (L-NAME; a nonspecific NO synthase inhibitor), and this protective effect of L-NAME was reversed with NO inhalation. Inhaled NO prevented the increase in PAP caused by I/R, while L-arginine had no such effect. L-NAME tended to diminish the I/R-induced elevation in PAP, but the suppression was not statistically significant when compared to the values in the I/R group. These results indicate that I/R increases Kfc and promotes alveolar edema by stimulating endogenous NO synthesis. Exogenous NO, either generated from L-arginine or delivered into the airway, is apparently also injurious to the lung following I/R.     

Protective role of ascorbic acid against lipid peroxidation and myocardial injury

Ascorbic acid (AH₂) is a potential scavenger of superoxide radical and singlet oxygen. In the guinea pig, marginal AH₂ deficiency results in intracellular oxidative damage in the cardiac tissue as evidenced by lipid peroxidation, formation of fluorescent pigment and loss of structural integrity of the microsomal membranes. The oxidative damage does not occur due to lack of enzymatic scavengers of reactive oxygen species such as superoxide dismutase, catalase and glutathione peroxidase. Also, glutathione transferase activity is not decreased in AH₂ deficiency. Lipid peroxidation, fluorescent pigment formation and protein modification disappear after AH₂ therapy. These results, if extra-polated to human beings, would indicate that chronic subclinical AH₂ deficiency may result in progressive oxidative damage which in the long run may lead to permanent degenerative diseases in the heart.     

Thiobarbituric acid-reactive material content and enzymatic protection against peroxidative damage during the course of cryogenic rabbit brain edema

The relationship between free radicals reactions and the cell detoxifying system was investigated during the development of brain edema following a cryogenic lesion in the rabbit cerebral cortex. The amount of TBA-reactive material present six hours after freezing was less than in the controls, then increased at 48 and 96 hours. The activity of superoxide dismutase (SOD) decreased 6 hours post-injury; at the same time, we observed a stimulation of catalase activity. The glutathione peroxidase activity (GSH-Px) rose 96 hours post-lesion. The decrease of TBA-reactive products could result from an elimination rate that exceeds generation.     

Protective role of ortho-substituted Mn(III) N-alkylpyridylporphyrins against the oxidative injury induced by tert-butylhydroperoxide

Abstract

The present work addresses the role of two ortho-substituted Mn(III) N-alkylpyridylporphyrins, alkyl being ethyl in MnTE-2-PyP⁵⁺ and n-hexyl in MnTnHex-2-PyP⁵⁺, on the protection against the oxidant tert-butylhydroperoxide (TBHP). Their protective role was studied in V79 cells using endpoints of cell viability (MTT and crystal violet assays), intracellular O₂?– generation (dihydroethidium assay) and glutathione status (DTNB and monochlorobimane assays). MnPs per se did not show cytotoxicity (up to 25 μM, 24 h). The exposure to TBHP resulted in a significant decrease in cell viability and in an increase in the intracellular O₂^?– levels. Also, TBHP depleted total and reduced glutathione and increased GSSG. The two MnPs counteracted remarkably the effects of TBHP. Even at low concentrations, both MnPs were protective in terms of cell viability and abrogated the intracellular O₂^?– increase in a significant way. Also, they augmented markedly the total and reduced glutathione contents in TBHP-treated cells, highlighting the multiple mechanisms of protection of these SOD mimics, which at least in part may be ascribed to their electron-donating ability.     

Lung injury and complement activation: Role of neutrophils and xanthine oxidase

Evidence is presented that oxygen products generated from xanthine oxidase (XO) may also be involved in the pathogenesis of neutrophil-mediate lung injury following intravascular activation of complement with cobra venom factor (CVF). CVF injection in rats resulted a rapid increase in plasma of both XO activity (but not xanthine dehydrogenase) and its reaction product, uric acid. These changes were greatly attenuated in allopurinol-treated animals. The apperance of XO activity was paralleled by a raise in plasma of histamine. Prevention of histamine release by pretreatment of rats withy cromolyn abolished both the rise in plasma histamine and the increase in XO activity. Since we have previously shown that histamine can enhance XO activity in vitro and in vivo (Am. J. Pathol. 135:203, 1989), these observations suggest that the increase in plasma XO activity following CVF injection is related to the appearance in plasma of histamine. Accordingly, pretreatment of rats with xanthine oxidase inhibitors (allopurinol, lodoxamine) or prevention of histamine release by pretreatment with cromolyn significantly attenuated development of lung injury following injection of CVF. Our data support the concept that oxygen radicals derived from both neutrophils and XO are playing a role in the CVF-induced acute lung injury.     

乙醇对胃粘膜的损伤及牛磺酸的保护作用

为了探讨乙醇性胃粘膜损伤和牛磺酸对其保护作用的机制,将４０只ＳＤ大鼠分为对照组、乙醇损伤组和牛磺酸保护组,观察其胃粘膜中内皮素（ＥＴ）、一氧化氮合成酶（ＮＯＳ）、生长抑素（ＳＳ）和血管活性肠肽（ＶＩＰ）的变化。结果如下：（１）随着乙醇作用时间延长、粘膜的损伤加重,胃粘膜内ＥＴ含量显著上升,而ＮＯＳ、ＳＳ和ＶＩＰ含量显著下降;（２）牛磺酸可显著减轻乙醇性胃粘膜损伤,抑制ＥＴ释放,增加ＮＯＳ活性和ＳＳ、ＶＩＰ的含量。上述结果表示,ＥＴ、ＮＯＳ、ＳＳ和ＶＩＰ的变化参与了乙醇性胃粘膜损伤的病理生理过程;牛磺酸对乙醇性胃粘膜损伤具有保护作用,可能与其调节胃粘膜内ＥＴ、ＮＯＳ、ＳＳ和ＶＩＰ的释放与活性有关。     

Hyperoxic-induced alterations in lung cell structure and function: Effects on cellular cyclic AMP content and comparison to alterations produced by exogenous cyclic AMP

Hyperoxic exposure in vitro of two lung-derived cell types (the epithelial-derived L2 cells and WI-38 fibroblasts) inhibits cellular replication, produces striking morphologic changes and may result in cell death; these effects have been observed consistently in other cell types. Hyperoxic exposure of L2 cells is associated with an increase in cellular cyclic AMP content (cellular cyclic AMP content 454 ± 115 fmol/μg DNA in cells exposed to p_O2 677 Torr for 96 h compared to 136 ± 17 fmol/μg DNA in air-grown cells). Hyperoxic exposure of WI-38 fibroblasts is not associated with increased cyclic AMP content. Although cultivation of L2 cells in the presence of exogenous dibutyryl cyclic AMP does inhibit replication and produce morphologic alterations, similar effects are produced by sodium butyrate alone. Hyperoxic exposure alters cyclic AMP metabolism in some cell types, but the structural and functional alterations observed in L2 cells and WI-38 fibroblasts following hyperoxic exposure are not produced by changes in cellular cyclic AMP content.     

Adenoviral augmentation of elafin protects the lung against acute injury mediated by activated neutrophils and bacterial infection

During acute pulmonary infection, tissue injury may be secondary to the effects of bacterial products or to the effects of the host inflammatory response. An attractive strategy for tissue protection in this setting would combine antimicrobial activity with inhibition of human neutrophil elastase (HNE), a key effector of neutrophil-mediated tissue injury. We postulated that genetic augmentation of elafin (an endogenous inhibitor of HNE with intrinsic antimicrobial activity) could protect the lung against acute inflammatory injury without detriment to host defense. A replication-deficient adenovirus encoding elafin cDNA significantly protected A549 cells against the injurious effects of both HNE and whole activated human neutrophils in vitro. Intratracheal replication-deficient adenovirus encoding elafin cDNA significantly protected murine lungs against injury mediated by Pseudomonas aeruginosa in vivo. Genetic augmentation of elafin therefore has the capacity to protect the lung against the injurious effects of both bacterial pathogens resistant to conventional antibiotics and activated neutrophils.     

Hydrogen-rich saline reduces lung injury induced by intestinal ischemia/reperfusion in rats

Objective. Hydrogen has been reported to selectively reduce the hydroxyl radical, the most cytotoxic of reactive oxygen species. In this study we investigated the effects of hydrogen-rich saline on the prevention of lung injury induced by intestinal ischemia/reperfusion (I/R) in rats. Methods. Male Sprague-Dawley rats (n = 30, 200-220 g) were divided randomly into three experimental groups: sham operated, intestinal I/R plus saline treatment (5 ml/kg, i.v.), and intestinal I/R plus hydrogen-rich saline treatment (5 ml/kg, i.v.) groups. Intestinal I/R was produced by 90 min of intestinal ischemia followed by a 4 h of reperfusion. Results. Hydrogen-rich saline treatment decreased the neutrophil infiltration, the lipid membrane peroxidation, NF-κB activation and the pro-inflammatory cytokine interleukin IL-1β and TNF-α in the lung tissues compared with those in saline-treated rat. Conclusion. Hydrogen-rich saline attenuates lung injury induced by intestinal I/R.     

Novel role for non-muscle myosin light chain kinase (MLCK) in hyperoxia-induced recruitment of cytoskeletal proteins, NADPH oxidase activation, and reactive oxygen species generation in lung endothelium

We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47(phox) and cortactin. Here, we demonstrate that the non-muscle ~214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47(phox) that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47(phox) at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47(phox) to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK(-/-) null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.     

Activation of Src-dependent Smad3 signaling mediates the neutrophilic inflammation and oxidative stress in hyperoxia-augmented ventilator-induced lung injury

Background

Mechanical ventilation and concomitant administration of hyperoxia in patients with acute respiratory distress syndrome can damage the alveolar epithelial and capillary endothelial barrier by producing inflammatory cytokines and reactive oxygen species. The Src tyrosine kinase and Smad3 are crucial inflammatory regulators used for ventilator-induced lung injury (VILI). The mechanisms regulating interactions between high-tidal-volume mechanical ventilation, hyperoxia, and acute lung injury (ALI) are unclear. We hypothesized that high-tidal-volume mechanical stretches and hyperoxia augment lung inflammation through upregulation of the Src and Smad3 pathways.

Methods

Wild-type or Src-deficient C57BL/6 mice, aged between 6 and 8 weeks, were exposed to high-tidal-volume (30 mL/kg) ventilation with room air or hyperoxia for 1–4 h after 2-mg/kg Smad3 inhibitor (SIS3) administration. Nonventilated mice were used as control subjects.

Results

We observed that the addition of hyperoxia to high-tidal-volume mechanical ventilation further induced microvascular permeability, neutrophil infiltration, macrophage inflammatory protein-2 and matrix metalloproteinase-9 (MMP-9) production, malondialdehyde, nicotinamide adenine dinucleotide phosphate oxidase activity, MMP-9 mRNA expression, hypoxemia, and Src and Smad3 activation (P < 0.05). Hyperoxia-induced augmentation of VILI was attenuated in Src-deficient mice and mice with pharmacological inhibition of Smad3 activity by SIS3 (P < 0.05). Mechanical ventilation of Src-deficient mice with hyperoxia further reduced the activation of Smad3.

Conclusions

Our data suggest that hyperoxia-increased high-tidal-volume ventilation-induced ALI partially depends on the Src and Smad3 pathways.     

Glycyrrhizin protection against 3-morpholinosydnonime-induced mitochondrial dysfunction and cell death in lung epithelial cells

The present study was designed to assess the preventive effect of licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid against mitochondrial damage and cell death in lung epithelial cells exposed to 3-morpholinosydnonime, a donor of nitric oxide and superoxide. Treatment of lung epithelial cells with 3-morpholinosydnonime resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species and depletion of GSH. Treatment of glycyrrhizin and 18beta-glycyrrhetinic acid attenuated the 3-morpholinosydnonime-induced mitochondrial damage, formation of reactive oxygen species and GSH depletion and revealed a maximal inhibitory effect at 10 and 1 muM, respectively; beyond these concentrations the inhibitory effect declined. Melatonin, carboxy-PTIO, rutin and uric acid reduced the 3-morpholinosydnonime-induced cell death. The results show that glycyrrhizin and 18beta-glycyrrhetinic acid seem to prevent the toxic effect of 3-morpholinosydnonime against lung epithelial cells by suppressing the mitochondrial permeability transition that leads to the release of cytochrome c and activation of caspase-3. The preventive effect may be ascribed to the inhibitory action on the formation of reactive oxygen species and depletion of GSH. The findings suggest that licorice compounds seem to prevent the nitrogen species-mediated lung cell damage.     

The novel peptide PACAP-TAT with enhanced traversing ability attenuates the severe lung injury induced by repeated smoke inhalation

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic peptide with anti-inflammatory and anti-oxidative effects. In order to increase the efficiency of traversing biological barriers, a novel fusion peptide PACAP-TAT was produced by tagging PACAP at its C-terminus with 11-amino acid TAT protein transduction domain. The results of characteristic assays showed that PACAP-TAT activated PACAP specific receptor PAC1 with the same potency as PACAP and PACAP-TAT crossed blood–brain barrier (BBB), blood–air barrier (BAB) and blood–testis barrier (BTB) with the efficiency about 2.5-fold higher than that of PACAP. Both PACAP-TAT and PACAP were used treat the mice with lung injury induced by repeated smoke inhalation. It was shown that both PACAP-TAT and PACAP decreased the mortality, increased the body weight and inhibited the edema and vascular permeability in the lungs of the mice received repeated smoke inhalation, while PACAP-TAT displayed more marked effects than PACAP. PACAP-TAT decreased myeloperoxidase (MPO) activity, increased catalase (CAT) activity and down-regulated interleukin 6 (IL-6) and malondialdehyde (MDA) levels in the lungs with a significantly higher efficiency than PACAP. The histopathological analysis also showed that PACAP-TAT attenuated the cell filtration and bronchi epithelial hyperplasia more significantly than PACAP. Moreover the leukocyte count in blood and the serum superoxide dismutase (SOD) activity in the mice treated with PACAP-TAT were significantly different from that in mice treated with PACAP (p < 0.05). All these data indicated that PACAP-TAT with increased traversing ability was more effective than PACAP in protecting the mice from the lung injury induced by repeated smoke inhalation.