Stress In Acute Lung Injury Research Articles

Knockdown of LincRNACOX2 Alleviates Oxidative Stress in Pathophysiology of Acute Lung Injury

Acute lung injury (ALI) has a complicated etiology that involves oxidative stress and inflammation. The role of lncRNACox2 (lincCOX2) in ALI regulation remains unclear. In this study, the ALI model of mice and MLE-12 cell was induced by LPS. To investigate the expression of lncRNACox2 in these ALI models, we employed a nanomagnetic bead-based RNA extraction method for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. This enabled us to determine the levels of lncRNACox2 expression and evaluate knockdown efficiency. Moreover, we also examined lung tissue histopathology using H&E staining. Cell survival and apoptosis rates were evaluated through CCK-8 and flow cytometry, respectively. The concentration of inflammatory factors was measured using ELISA. Additionally, the concentration (8-OHdG and MDA) and enzymatic activity (CAT, GSH-Px, and SOD) of oxidative stress related factors were measured by biochemical method. The western blot was performed to present the key proteins expression level in Nrf2/ARE signaling pathway in cytoplasm and nucleoprotein. The ALI mouse model was successfully established. The histopathology change and inflammatory cells were observed by H&E staining in LPS treated groups. The expression of lincCOX2 was up-regulated in ALI tissue. LPS induced more cell death in ALI, and the knockdown of lincCOX2 improved the cell survival and suppressed the apoptosis in ALI cell. Furthermore, In addition, downregulation of lincCOX2 attenuated inflammation and oxidative stress in lung cells in ALI. The concentration of 8-OHdG and MDA were highest in the LPS group while reduced by the sh-lincCOX2, the activity of CAT, GSH-Px, and SOD was reduced in the LPS induced ALI and increased by the sh-lincCOX2. In ALI, the distribution of Nrf2 protein is transferred from cytoplasm to nucleus. Furthermore, the lincCOX2 regulated oxidative stress via Nrf2/ARE signaling pathway in ALI. Overall, downregulation of lincRNACOX2 alleviates oxidative stress in ALI via Nrf2/ARE Pathway. This study suggests that lincCOX2 may be a potential target for the treatment of ALI.

Protective Effects of Atractylodis lancea Rhizoma on Lipopolysaccharide-Induced Acute Lung Injury via TLR4/NF-κB and Keap1/Nrf2 Signaling Pathways In Vitro and In Vivo.

Acute lung injury (ALI) is a syndrome caused by an excessive inflammatory response characterized by intractable hypoxemia both inside and outside the lung, for which effective therapeutic drugs are lacking. Atractylodis rhizoma, a traditional Chinese medicine, has excellent anti-inflammatory and antiviral properties in addition to protecting the integrity of the cellular barrier. However, few studies of Atractylodis rhizoma for the treatment of ALI have been published, and its mechanism of action remains unclear. In the present study, the chemical composition of the ethanolic extract of Atractylodis rhizoma (EEAR) was initially clarified by high performance liquid chromatography (HPLC), after which it was studied in vivo using a lipopolysaccharide (LPS)-induced ALI rat model. Treatment with EEAR significantly reduced the lung wet/dry (W/D) ratio, neutrophil infiltration, and malondialdehyde (MDA) and myeloperoxidase (MPO) formation, and enhanced superoxide dismutase (SOD) and glutathione (GSH) depletion in rats with ALI, thereby improving lung barrier function and effectively reducing lung injury. In addition, EEAR significantly reduced histopathological changes, decreased the expression of inflammatory factors (such as tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β), inducible nitric oxide synthase (INOS), and cyclooxygenase-2 (COX-2)), and inhibited the activation of the NF-κB signaling pathway, thus reducing inflammation. In addition, EEAR was found to also reduce oxidative stress in ALI by upregulating the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream proteins heme oxygenase-1 (HO-1) and NADPH quinone acceptor oxidoreductase 1 (NQO-1). EEAR also reduced LPS-induced inflammatory factor expression in THP-1 cells in vitro by inhibition of the NF-κB signaling pathway, and reduced damage from lipopolysaccharide (LPS)-induced oxidative stress in THP-1 cells by promoting the expression of Nrf2 and its downstream targets HO-1 and NQO-1, the molecular mechanism of which was consistent with in vivo observations. Therefore, we conclude that EEAR attenuates oxidative stress and inflammatory responses via TLR4/NF-κB and Keap1/Nrf2 signaling pathways to alleviate LPS-induced ALI, suggesting that Atractylodis rhizoma is a potential drug candidate for the treatment of ALI.

The protective effect of natural medicines against excessive inflammation and oxidative stress in acute lung injury by regulating the Nrf2 signaling pathway.

Acute lung injury (ALI) is a common critical disease of the respiratory system that progresses into acute respiratory distress syndrome (ARDS), with high mortality, mainly related to pulmonary oxidative stress imbalance and severe inflammation. However, there are no clear and effective treatment strategies at present. Nuclear factor erythroid 2-related factor 2(Nrf2) is a transcription factor that interacts with multiple signaling pathways and regulates the activity of multiple oxidases (NOX, NOS, XO, CYP) related to inflammation and apoptosis, and exhibits antioxidant and anti-inflammatory roles in ALI. Recently, several studies have reported that the active ingredients of natural medicines show protective effects on ALI via the Nrf2 signaling pathway. In addition, they are cheap, naturally available, and possess minimal toxicity, thereby having good clinical research and application value. Herein, we summarized various studies on the protective effects of natural pharmaceutical components such as polyphenols, flavonoids, terpenoids, alkaloids, and polysaccharides on ALI through the Nrf2 signaling pathway and demonstrated existing gaps as well as future perspectives.

Effects of different fluid management on lung and kidney during pressure-controlled and pressure-support ventilation in experimental acute lung injury.

Optimal fluid management is critical during mechanical ventilation to mitigate lung damage. Under normovolemia and protective ventilation, pulmonary tensile stress during pressure‐support ventilation (PSV) results in comparable lung protection to compressive stress during pressure‐controlled ventilation (PCV) in experimental acute lung injury (ALI). It is not yet known whether tensile stress can lead to comparable protection to compressive stress in ALI under a liberal fluid strategy (LF). A conservative fluid strategy (CF) was compared with LF during PSV and PCV on lungs and kidneys in an established model of ALI. Twenty‐eight male Wistar rats received endotoxin intratracheally. After 24 h, they were treated with CF (minimum volume of Ringer's lactate to maintain normovolemia and mean arterial pressure ≥70 mmHg) or LF (~4 times higher than CF) combined with PSV or PCV (VT = 6 ml/kg, PEEP = 3 cmH2O) for 1 h. Nonventilated animals (n = 4) were used for molecular biology analyses. CF‐PSV compared with LF‐PSV: (1) decreased the diffuse alveolar damage score (10 [7.8–12] vs. 25 [23–31.5], p = 0.006), mainly due to edema in axial and alveolar parenchyma; (2) increased birefringence for occludin and claudin‐4 in lung tissue and expression of zonula‐occludens‐1 and metalloproteinase‐9 in lung. LF compared with CF reduced neutrophil gelatinase‐associated lipocalin and interleukin‐6 expression in the kidneys in PSV and PCV. In conclusion, CF compared with LF combined with PSV yielded less lung epithelial cell damage in the current model of ALI. However, LF compared with CF resulted in less kidney injury markers, regardless of the ventilatory strategy.

Protective effects of the suppressed NF-κB/TLR4 signaling pathway on oxidative stress of lung tissue in rat with acute lung injury.

The pathogenesis of acute lung injury (ALI) is characterized by lung inflammation and lung oxidative stress. The study was conducted in order to investigate the effect toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) exhibited on oxidative stress in ALI. After the rats had been assigned into different groups, arterial blood, white blood cell (WBC), lung permeability index (LPI), wet/dry (W/D) ratio, TLR4 and NF-κB expression and superoxide dismutase (SOD), myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) were examined. Afterward, the correlation between the levels of TLR4 and NF-κB was determined. Decreased levels of PaO2 , SOD, MPO, and GSH accompanied by increased levels of PaCO2 , WBC number, LPI and W/D ratio, MDA and ROS, as well as TLR4 and NF-κB expressions in the ALI, ALI + NF-κB inhibitor, and ALI + phosphate buffer saline groups were found. Inhibition of NF-κB resulted in increased PaO2 and decreased PaCO2 levels, WBC number, and LPI and W/D ratio. Decreased expression of NF-κB increased SOD, GSH, and MPO, but decreased MDA and ROS. We also found that NF-κB inhibition resulted in the improvement of ALI in rats. TLR4 and NF-κB expressions were negatively correlated with levels of SOD, MPO, and GSH, and positively correlated with MDA and ROS levels. In summary, our findings provided evidence that inhibition of the TLR4/NF-κB signaling pathway decreases oxidative stress, thereby improving ALI. As a result, NF-κB signaling pathway has shown potential as a therapeutic target in ALI therapy.

Sodium‐coupled neutral amino acid transporter SNAT2 counteracts edema formation and reduces autophagy and ER stress in acute lung injury

ObjectiveAcute respiratory distress syndrome (ARDS), the most frequent cause of death in intensive care, is characterized by an excessive inflammatory response, increased vascular permeability, edema formation, and impaired gas exchange. Recent studies have suggested deprivation of amino acids may aggravate lung injury while promoting apoptosis, ER stress, and autophagy. However, RCTs have failed to identify beneficial pharmaco‐nutritional interventions. This may point to a critical role of amino acid transporters in ARDS pathophysiology and its non‐human analogue acute lung injury (ALI). Sodium‐coupled neutral amino acid transporters (SNATs) mediate cellular uptake of amino acids along with Na+ and are known to be upregulated in response to amino acid starvation. Here, we probed for the role of SNATs in a murine model of HCl‐ and LPS‐induced ALI and in a pulmonary epithelial cell culture system.MethodsExpression of SNATs under basal conditions or after amino acid deprivation in epithelial cell lines (NCI‐H441/A549) and in isolated rat type‐I (ATI cells) was determined by PCR and Western blot.For measurement of L‐alanine transport, H441 cells were cultured on Transwell inserts in the presence or absence of amino acids and treated either with HgCl2, which inhibits SNATs, or siRNA (scramble or siSNAT2). Transport of L‐alanine from the upper to the lower compartment was analyzed by ELISA.For in vivo experiments, SNAT2 knockout (slc38a2−/−), heterozygous (slc38a2+/−) and wildtype (slc38a2+/+) mice were anesthetized and HCl or saline was instilled intratracheally. After 2 h of mechanical ventilation, lungs were collected for measurement of wet–to‐dry lung weight ratio (W/D) and protein extraction. In a second model of ALI, mice were anesthetized and LPS (0.4 mg/kg) was given intranasally. After 24 h, mice were euthanized and lungs were collected for protein extraction.ResultsIsolated ATI, H441, and A549 cells showed strong expression of SNAT2 after incubation in amino acid free medium.Under amino acid deprivation, H441 cells showed quantitative L‐alanine transport that was significantly decreased by treatment with HgCl2 or siRNA silencing of SNAT2.Homozygous SNAT2 knockout mice were found to be sublethal and newborn pups typically succumbed to cyanotic dyspnea. In HCl‐induced ALI, W/D was elevated in slc38a2−/− and slc38a2+/− mice as compared to wildtype. Expression of ER stress and autophagy markers was increased in whole protein lysates of heterozygous mice compared to control in both models of ALI.ConclusionSNAT2 counteracts lung edema formation in acid‐induced ALI, presumably by mediating Na+ uptake that drives alveolar fluid clearance. In LPS‐ and HCl‐induced lung injury, SNAT2 reduces ER stress and autophagy, putatively through its role as amino acid transporter. SNAT2 activation may, hence, provide a new multi‐pronged strategy to counteract ALI/ARDS.Support or Funding InformationSupported by a CIHR grant to Dr. W.M. Kuebler

Interactive effects of mechanical ventilation, inhaled nitric oxide and oxidative stress in acute lung injury

To compare conventional mechanical ventilation (CMV) and high-frequency oscillatory ventilation (HFOV), with/without inhaled nitric oxide (iNO), for oxygenation, inflammation, antioxidant/oxidative stress status, and DNA damage in a model of acute lung injury (ALI). Lung injury was induced by tracheal infusion of warm saline. Rabbits were ventilated at FIO2 1.0 and randomly assigned to one of five groups. Overall antioxidant defense/oxidative stress was assessed by total antioxidant performance assay, and DNA damage by comet assay. Ventilatory and hemodynamic parameters were recorded every 30min for 4h. ALI groups showed worse oxygenation than controls after lung injury. After 4h of mechanical ventilation, HFOV groups presented significant improvements in oxygenation. HFOV with and without iNO, and CMV with iNO showed significantly increased antioxidant defense and reduced DNA damage than CMV without iNO. Inhaled nitric oxide did not beneficially affect HFOV in relation to antioxidant defense/oxidative stress and pulmonary DNA damage. Overall, lung injury was reduced using HFOV or CMV with iNO.

Biomarkers for oxidative stress in acute lung injury induced in rabbits submitted to different strategies of mechanical ventilation

Oxidative damage has been said to play an important role in pulmonary injury, which is associated with the development and progression of acute respiratory distress syndrome (ARDS). We aimed to identify biomarkers to determine the oxidative stress in an animal model of acute lung injury (ALI) using two different strategies of mechanical ventilation. Rabbits were ventilated using either conventional mechanical ventilation (CMV) or high-frequency oscillatory ventilation (HFOV). Lung injury was induced by tracheal saline infusion (30 ml/kg, 38°C). In addition, five healthy rabbits were studied for oxidative stress. Isolated lymphocytes from peripheral blood and lung tissue samples were analyzed by alkaline single cell gel electrophoresis (comet assay) to determine DNA damage. Total antioxidant performance (TAP) assay was applied to measure overall antioxidant performance in plasma and lung tissue. HFOV rabbits had similar results to healthy animals, showing significantly higher antioxidant performance and lower DNA damage compared with CMV in lung tissue and plasma. Total antioxidant performance showed a significant positive correlation (r = 0.58; P = 0.0006) in plasma and lung tissue. In addition, comet assay presented a significant positive correlation (r = 0.66; P = 0.007) between cells recovered from target tissue and peripheral blood. Moreover, antioxidant performance was significantly and negatively correlated with DNA damage (r = -0.50; P = 0.002) in lung tissue. This study indicates that both TAP and comet assay identify increased oxidative stress in CMV rabbits compared with HFOV. Antioxidant performance analyzed by TAP and oxidative DNA damage by comet assay, both in plasma, reflects oxidative stress in the target tissue, which warrants further studies in humans.

Endotoxin-induced acute lung injury is dependent upon oxidative response

The aim of the present study was to investigate the involvement of oxidative stress in acute lung injury (ALI) induced by lipopolysaccharide (LPS) and its effects upon cell structure, function and inflammation. In total, 108 male C57BL/6 mice were divided into seven groups: CTR Group (50 µL of saline) administered intratracheally (i.t.), LPS 6 h (10 µg of LPS − i.t.), LPS 12 h (10 µg of LPS − i.t.), LPS 24 h (10 µg of LPS − i.t.), LPS 48 h (10 µg of LPS − i.t.), LPS 24 h (10 µg − i.t.) + NAC 40 mg/kg (gavage) and 24 h LPS (10 µg − i.t.) + NAC 100 mg/kg (gavage). The antioxidant treatment protected the lungs from stress in the first 12 h, but significant oxidative stress induction was observed at the 24-hour time point, and, after 48 h, there was no protection exerted by the antioxidant treatment. NAC (N-acetylcysteine) reversed the elastance parameters, and ΔP1 and ΔP2 compared with 24 h LPS alone. NAC reduced the number of inflammatory cells in histology analysis when compared with the 24 h LPS alone-treated group. NAC also inhibited the transcription of NFκB, IL-6, TNF-α and COX2 usually induced by LPS. Our results suggest that oxidative stress plays an important role in structural, functional and inflammatory responses in the ALI model.

New images, new insights for VILI

prioritization of high-volume vs. low-volume mechanisms of ventilator-induced lung injury (VILI) is an issue that is often discussed and debated in clinical ventilator management. Abundant laboratory studies ([7][1]), as well as several prominent clinical studies ([1][2], [3][3], [14][4], [19][5]),

Treatment with N-methyl- d-aspartate receptor antagonist (MK-801) protects against oxidative stress in lipopolysaccharide-induced acute lung injury in the rat

Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) are common syndromes that affect both clinical and surgical patients. This study describes the effects of a potent and specific N-methyl- d-aspartate receptor antagonist (MK-801) against oxidative stress in acute lung injury induced by intratracheal lipopolysaccharide (LPS) injection. This study was performed using male Wistar rats weighing 200–250 g. Rats were randomly divided into four groups: control with isotonic saline instillation (n = 6); LPS (100 μg/100 g of body weight) treated with saline (n = 6); LPS treated with MK-801 (0.3 mg/kg, intraperitoneally; n = 6); LPS treated with MK-801 (0.3 mg/kg, intratracheally; n = 6). Twelve hours after the LPS instillation, rats were anesthetized and a bronchoalveolar lavage (BAL) was performed in order to determine the alveolar–capillary membrane alterations and the inflammatory infiltrate level. Blood and lung samples were isolated and assayed for oxidative stress variables and histopathologic analysis. The use of MK-801 decreased bronchoalveolar lavage fluid protein, LDH activity and inflammatory cells. Indeed, the treatment with MK-801 significantly attenuated lung oxidative damage and histopathologic alterations after LPS instillation. Our data provide the first experimental demonstration that MK-801 decreases oxidative stress and limits inflammatory response and alveolar disarray in lipopolysaccharide-induced acute lung injury.

Microarray analysis of regional cellular responses to local mechanical stress in acute lung injury

Human acute lung injury is characterized by heterogeneous tissue involvement, leading to the potential for extremes of mechanical stress and tissue injury when mechanical ventilation, required to support critically ill patients, is employed. Our goal was to establish whether regional cellular responses to these disparate local mechanical conditions could be determined as a novel approach toward understanding the mechanism of development of ventilator-associated lung injury. We utilized cross-species genomic microarrays in a unilateral model of ventilator-associated lung injury in anesthetized dogs to assess regional cellular responses to local mechanical conditions that potentially contribute pathogenic mechanisms of injury. Highly significant regional differences in gene expression were observed between lung apex/base regions as well as between gravitationally dependent/nondependent regions of the base, with 367 and 1,544 genes differentially regulated between these regions, respectively. Major functional groupings of differentially regulated genes included inflammation and immune responses, cell proliferation, adhesion, signaling, and apoptosis. Expression of genes encoding both acute lung injury-associated inflammatory cytokines and protective acute response genes were markedly different in the nondependent compared with the dependent regions of the lung base. We conclude that there are significant differences in the local responses to stress within the lung, and consequently, insights into the cellular responses that contribute to ventilator-associated lung injury development must be sought in the context of the mechanical heterogeneity that characterizes this syndrome.

Mononuclear phagocyte xanthine oxidoreductase contributes to cytokine-induced acute lung injury.

Acute lung injury (ALI) is characterized by increased alveolar cytokines, inflammatory cell infiltration, oxidative stress, and alveolar cell apoptosis. Previous work suggested that xanthine oxidoreductase (XOR) may contribute to oxidative stress in ALI as a product of the vascular endothelial cell. We present evidence that cytokine induced lung inflammation and injury involves activation of XOR in the newly recruited mononuclear phagocytes (MNP). We found that XOR was increased predominantly in the MNP that increase rapidly in the lungs of rats that develop ALI following intratracheal cytokine insufflation. XOR was recovered from the MNP largely converted to its oxygen radical generating, reversible O-form, and alveolar MNP exhibited increased oxidative stress as evidenced by increased nitrotyrosine staining. Cytokine insufflation also increased alveolar cell apoptosis. A functional role for XOR in cytokine-induced inflammation was demonstrated when feeding rats two different XOR inhibitors, tungsten and allopurinol, decreased MNP XOR induction, nitrotyrosine staining, inflammatory cell infiltration, and alveolar cell apoptosis. Transfer of control or allopurinol treated MNP into rat lungs confirmed a specific role for MNP XOR in promoting lung inflammation. These data indicate that XOR can contribute to lung inflammation by its expression and conversion in a highly mobile inflammatory cell population.

Neutrophil elastase and acute lung injury: prospects for sivelestat and other neutrophil elastase inhibitors as therapeutics.

To review the evidence and rationale that suggest that neutrophil elastase (NE) may contribute to the development of acute lung injury (ALI) and the acute respiratory distress syndrome. To review selected preliminary data regarding the effectiveness of NE inhibition in animals, in in vitro models, and in patients with ALI. The published literature and observations provided by Ono Pharmaceutical and Eli Lilly investigators and their colleagues. Taken en toto, the data suggest that NE could contribute to ALI and endothelial cell injury that is relevant to ALI. Moreover, the toxic effects of NE are greatly enhanced by increased oxidative stress, which commonly occurs in patients with ALI. In addition to neutrophils, xanthine oxidase, a constituent of endothelial cells, is a potential source of oxidative stress in ALI; xanthine oxidase-derived oxidants enhance NE toxicity in in vivo, isolated lung, and in vitro endothelial cell test systems. Not surprisingly, endogenous nonoxidatively sensitive NE inhibitors (e.g., eglin C) are more effective in combating the detrimental effects of NE than oxidatively sensitive NE inhibitors (e.g., alpha-1-proteinase inhibitor). In addition, a synthetic NE inhibitor, sivelestat (ONO-5046 and LY544349), is effective in reducing measures of inflammation and injury in multiple animal models of ALI. In a trial of ALI patients with systemic inflammatory response syndrome, conducted in Japan by Ono Pharmaceutical scientists, sivelestat treatment improved the investigator assessment of global improvement and the percentages of patients who were removed from ventilators and transferred out of the intensive care unit. Further study of the role of NE inhibition as a treatment for ALI is warranted. Additional clinical and preclinical studies with sivelestat and various other NE inhibitors should not only clarify the clinical potential of this intervention strategy, but also better define the activities of NE in inflammatory disorders such as ALI and multiple organ failure.