Lung Epithelial Injury Research Articles

Silence of MLK3 alleviates lipopolysaccharide-induced lung epithelial cell injury via inhibiting p53-mediated ferroptosis.

Acute lung injury (ALI) is characterized with a high rate of morbidity and mortality. The injury and apoptosis of lung epithelial cells play crucial roles in the progression of ALI. Mixed lineage kinase 3 (MLK3) has been reported to be involved in the regulation of cellular biological functions, such as cell proliferation, apoptosis and ferroptosis. However, the effect of MLK3 exerted on ALI has not been reported. Here, LPS-stimulated MLE12 pulmonary epithelial cells were used as an in vitro model for ALI. In this research, LPS elevated the expression of MLK3 in MLE12 cells. The silence of MLK3 alleviated LPS-induced cell injury. Notably, LPS promoted ferroptosis through enhancing GSH depletion and the productions of MDA and iron, which was attenuated by MLK3 knockdown. Moreover, the silence of MLK3 inhibited p53 expression in LPS-induced cells along with a decrease in the expressions of p21 and Bax, while overexpressing p53 reversed these effects of MLK3 silence. Meanwhile, p53 overexpression reversed the positive effects of MLK3 knockdown on LPS-induced cell ferroptosis and injury. Together, our results confirmed that the silence of MLK3 alleviated LPS-induced lung epithelial cell injury by inhibiting p53-mediated ferroptosis.

Lidocaine attenuates hypoxia/reoxygenation‑induced inflammation, apoptosis and ferroptosis in lung epithelial cells by regulating the p38 MAPK pathway

Lung ischemia-reperfusion (I/R) injury poses a serious threat to human health, worldwide. The current study aimed to determine the role of lidocaine in A549 cells, in addition to the involvement of the p38 MAPK pathway. Oxygen deprivation/reoxygenation-induced A549 cells were utilized to simulate I/R injury in vitro. Cell viability and apoptosis were detected using MTT and TUNEL assays, respectively. The levels of IL-6, IL-8, TNF-α, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase, iron and reactive oxygen species (ROS) were measured using corresponding commercial kits. The corresponding protein expression levels were also measured using western blotting. Moreover, a monolayer cell paracellular permeability assay was performed to determine the permeability of A549 cells. The results demonstrated that, whilst lidocaine had no influence on untreated A549 cells, it significantly increased the viability of hypoxia/reoxygenation (H/R)-induced A549 cells. A549 cell apoptosis and the release of inflammatory cytokines in the H/R group were decreased after the addition of lidocaine. When compared with the H/R group, increased MDA level and decreased SOD level were observed in H/R-induced A549 cells following lidocaine treatment. In addition, the permeability of H/R-induced A549 cells was markedly decreased following lidocaine treatment. Compared with the H/R group, the expression levels of tight junction and ferroptosis-related proteins were significantly upregulated by lidocaine, whereas the expression of transferrin was downregulated. However, p79350, an agonist of p38, reversed the effects of lidocaine on H/R-induced A549 cells. In conclusion, lidocaine exerted a protective role in HR-induced lung epithelial cell injury, which may serve as a potential agent for the treatment of patients with lung I/R injury.

Knockdown of circRNA Paralemmin 2 Ameliorates Lipopolysaccharide-induced Murine Lung Epithelial Cell Injury by Sponging miR-330-5p to Reduce ROCK2 Expression

ABSTRACT Previous data have reported the high expression of circRNA paralemmin 2 (circPALM2) in mice with acute lung injury (ALI). However, the role of circPALM2 in ALI pathogenesis remains unclear. The study aims to reveal the function of circPALM2 in ALI and the underlying mechanism. C57BL/6 J mice and murine lung epithelial-12 (MLE-12) cells were treated with lipopolysaccharide (LPS) to simulate ALI mouse and ALI cell models, respectively. Lung injury score and lung wet-to-dry ratio assays were used to evaluate the ALI mouse model. Quantitative real-time polymerase chain reaction and Western blot assays were implemented to analyze the expressions of circPALM2, microRNA-330-5p (miR-330-5p), rho-associated coiled-coil containing protein kinase 2 (ROCK2), and apoptosis-related markers. Cell viability, apoptosis, and the production of inflammatory cytokines were investigated by cell counting kit-8, flow cytometry, and enzyme-linked immunosorbent assays. The expressions of circPALM2 and ROCK2 were significantly increased, while miR-330-5p was decreased in ALI mice and LPS-induced MLE-12 cells compared with controls. LPS treatment inhibited cell viability but induced apoptosis, inflammatory cytokine production, and oxidative stress; however, these effects were attenuated after the combination of circPALM2 knockdown and LPS. CircPALM2 regulated LPS-caused MLE-12 cell damage by targeting miR-330-5p. Additionally, ROCK2, a target gene of miR-330-5p, participated in LPS-induced MLE-12 cell injury. Further, circPALM2 activated ROCK2 by associating with miR-330-5p. CircPALM2 modulated LPS-caused murine lung epithelial cell injury by the miR-330-5p/ROCK2 pathway, providing a therapeutic target for ALI.

Idiopathic Pulmonary Fibrosis: An Update on Pathogenesis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, lethal fibrotic lung disease that occurs primarily in middle-aged and elderly adults. It is a major cause of morbidity and mortality. With an increase in life expectancy, the economic burden of IPF is expected to continuously rise in the near future. Although the exact pathophysiological mechanisms underlying IPF remain not known. Significant progress has been made in our understanding of the pathogenesis of this devastating disease in last decade. The current paradigm assumes that IPF results from sustained or repetitive lung epithelial injury and subsequent activation of fibroblasts and myofibroblast differentiation. Persistent myofibroblast phenotype contributes to excessive deposition of the extracellular matrix (ECM) and aberrant lung repair, leading to tissue scar formation, distortion of the alveolar structure, and irreversible loss of lung function. Treatments of patients with IPF by pirfenidone and nintedanib have shown significant reduction of lung function decline and slowing of disease progression in patients with IPF. However, these drugs do not cure the disease. In this review, we discuss recent advances on the pathogenesis of IPF and highlight the development of novel therapeutic strategies against the disease.

Arctigenin attenuates paraquat-induced human lung epithelial A549 cell injury by suppressing ROS/p38 mitogen-activated protein kinases-mediated apoptosis.

Paraquat (PQ)-induced acute lung injury (ALI) and pulmonary fibrosis are common diseases with high mortality but without effective antidotes in emergency medicine. Our previous study has proved that arctigenin suppressed pulmonary fibrosis induced by PQ. We wondered whether arctigenin could also have a protective effect on PQ-induced ALI. A PQ-induced A549 cell injury model was used, and the effect of arctigenin was determined by a cell counting kit-8 (CCK-8) cell viability assay. In addition, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) staining assays and mitochondrial membrane potential assays were performed to evaluate the level of cell apoptosis. The generation of reactive oxygen species (ROS) was reflected by dihydroethidium (DHE) staining and a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Moreover, immunoblotting studies were used to assess the expression of mitogen-activated protein kinases (MAPKs) and p38 MAPK. Arctigenin attenuated PQ-induced inhibition of A549 cell viability in a dose-dependent manner. Arctigenin also significantly reduced PQ-induced A549 cell apoptosis, as reflected by the TUNEL assay and mitochondrial membrane potential assay, which may result from suppressed ROS/p38 MAPK signaling because we found that arctigenin dramatically suppressed ROS generation and p38 MAPK phosphorylation. Arctigenin could attenuate PQ-induced lung epithelial A549 cell injury in vitro by suppressing ROS/p38 MAPK-mediated cell apoptosis, and arctigenin might be considered a potential candidate drug for PQ-induced ALI.

Organoid technology and lung injury mouse models evaluating effects of hydroxychloroquine on lung epithelial regeneration.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) damages lung epithelial stem/progenitor cells. Ideal anti-SARS-CoV-2 drug candidates should be screened to prevent secondary injury to the lungs. Here, we propose that in vitro three-dimensional organoid and lung injury repair mouse models are powerful models for the screening antiviral drugs. Lung epithelial progenitor cells, including airway club cells and alveolar type 2 (AT2) cells, were co-cultured with supportive fibroblast cells in transwell inserts. The organoid model was used to evaluate the possible effects of hydroxychloroquine, which is administered as a symptomatic therapy to the coronavirus disease 2019 (COVID-19) patients, on the function of mouse lung stem/progenitor cells. Hydroxychloroquine was observed to promote the self-renewal of club cells and differentiation of ciliated and goblet cells in vitro. Additionally, it inhibited the self-renewal ability of AT2 cells in vitro. Naphthalene- or bleomycin-induced lung injury repair mouse models were used to investigate the in vivo effects of hydroxychloroquine on the regeneration of club and AT2 cells, respectively. The naphthalene model indicated that the proliferative ability and differentiation potential of club cells were unaffected in the presence of hydroxychloroquine. The bleomycin model suggested that hydroxychloroquine had a limited effect on the proliferation and differentiation abilities of AT2 cells. These findings suggest that hydroxychloroquine has limited effects on the regenerative ability of epithelial stem/progenitor cells. Thus, stem/progenitor cell-derived organoid technology and lung epithelial injury repair mouse models provide a powerful platform for drug screening, which could possibly help end the pandemic.

Surfactant Protein-D: The Potential ARDS Biomarker And Its Role In COVID-19

As of September 2021, the number of patients diagnosed with COVID-19 worldwide has exceeded 221 million; while the Case Fatality Rate in 215 countries and regions is 2.06%. Acute Respiratory Distress Syndrome (ARDS) is the most common complication of COVID-19 and was reported had a 28-day mortality rate of 74%. Pulmonary injury in ARDS is caused by damage to the alveolar epithelium and pulmonary vascular endothelium caused by both direct virulence of SARS-CoV-2 and an inflammatory reaction that mediates neutrophil activation, leading to diffuse alveolar damage, pulmonary edema and surfactant dysfunction. Under inflammatory conditions, such as ARDS, increased alveolar-capillary permeability may result in alveolar Surfactant Protein-D (SP-D) leakage into the circulation, thus SP-D can be used as a specific biomarker of pulmonary epithelial damage and acute lung injury. SP-D is a specific biomarker of lung epithelial injury and has been shown to correlate with the severity and mortality of ARDS (COVID-19 and non COVID-19). Therefore, the use of SP-D as a prognostic biomarker in COVID-19 cases can be considered.

Effect of curcumin on lung epithelial injury and ferroptosis induced by cigarette smoke

Cigarette smoke (CS)-caused ferroptosis was involved in the pathogenesis of COPD, but the role of ferroptosis in lung epithelial injury and inflammation is not clear. Rats were treated with CS or CUR and BEAS-2B cells were exposed to CS extract (CSE), ferrostatin-1 (Fer-1), deferoxamine (DFO), or CUR to detect reactive oxygen species (ROS) accumulation, lipid peroxidation, iron overload, and ferroptosis-related protein, which were the characteristic changes of ferroptosis. Compared with the control group, CSE-treated BEAS-2B cells had more cell death, higher cytotoxicity, and lower cell viability. The infiltration of inflammatory cell around the bronchi in the CS group of rats was more than that in the normal group. Meanwhile, CSE/CS elevated the levels of interleukin-6 and tumor necrosis factor-α in BEAS-2B cells and bronchoalveolar lavage fluid of rats. Besides, accumulative ROS and depleted glutathione was observed in vitro. In BEAS-2B cells and lung tissues of rats, CSE/CS increased malondialdehyde and iron; down-regulated solute carrier family 7, glutathione peroxidase 4, and ferritin heavy chain levels; and up-regulated transferrin receptor level. These changes were rescued by pretreatment of Fer-1 or DFO in vitro, and mitigated by CUR in vitro and in vivo. Collectively, this study reveals that ferroptosis was involved in lung epithelial cell injury and inflammation induced by CS, and CUR may alleviate CS-induced injury, inflammation, and ferroptosis of lung epithelial cell.

Upregulation of tetraspanin 8 may contribute to LPSinduced acute lung injury by activation of the MAPK and NF-κB pathways

Purpose: To investigate the effect of tetraspanin8 (Tspan8, also known as TM4SF3 or CO-029) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the related signaling pathways.Methods: Treatment with LPS was used to induce lung damage in mice and a lung epithelial cell line. The wet-to-dry weight ratio of lung tissue, hematoxylin and eosin (H&E) staining, and quantification of cytokine concentrations were conducted to validate the model. Enzyme-linked immunosorbent assays (ELISA) and quantitative polymerase chain reaction (qPCR) were used to measure levels of tumor necrosis factor alpha, interleukin (IL)-1β, and IL-6. Tspan8 levels were knocked down using shRNAs. Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathway levels were assessed after LPS-induced injury in this cellular model.Results: Levels of Tspan8 were upregulated in the LPS-induced ALI model. Additionally, LPS treatment of mouse lung epithelial cells resulted in Tspan8 upregulation. Tspan8 knockdown alleviated the effects of LPS on lung epithelial injury by inhibiting the upregulation of MAPK and NF-κB signaling pathways.Conclusion: The upregulation of Tspan8 may promote the progression of ALI.

Mechanisms of Lung Injury Induced by SARS-CoV-2 Infection.

The lung is the major target organ of SARS-CoV-2 infection, which causes COVID-19. Here, we outline the multistep mechanisms of lung epithelial and endothelial injury induced by SARS-CoV-2: direct viral infection, chemokine/cytokine-mediated damage, and immune cell-mediated lung injury. Finally, we discuss the recent progress in terms of antiviral therapeutics as well as the development of anti-inflammatory or immunomodulatory therapeutic approaches. This review also provides a systematic overview of the models for studying SARS-CoV-2 infection and discusses how an understanding of mechanisms of lung injury will help identify potential targets for future drug development to mitigate lung injury.

Dynamic Expression of Transient Receptor Potential Vanilloid-3 and Integrated Signaling with Growth Factor Pathways during Lung Epithelial Wound Repair following Wood Smoke Particle and Other Forms of Lung Cell Injury.

Prior studies revealed increased expression of the transient receptor potential vanilloid-3 (TRPV3) ion channel after wood smoke particulate matter (WSPM) treatment of human bronchial epithelial cells (HBECs). TRPV3 attenuated pathologic endoplasmic reticulum stress and cytotoxicity mediated by transient receptor potential ankyrin-1. Here, the basis for how TRPV3 expression is regulated by cell injury and the effects this has on HBEC physiology and WSPM-induced airway remodeling in mice was investigated. TRPV3 mRNA was rapidly increased in HBECs treated with WSPM and after monolayer damage caused by tryptic disruption, scratch wounding, and cell passaging. TRPV3 mRNA abundance varied with time, and stimulated expression occurred independent of new protein synthesis. Overexpression of TRPV3 in HBECs reduced cell migration and wound repair while enhancing cell adhesion. This phenotype correlated with disrupted mRNA expression of ligands of the epidermal growth factor, tumor growth factor-β, and frizzled receptors. Accordingly, delayed wound repair by TRPV3 overexpressing cells was reversed by growth factor supplementation. In normal HBECs, TRPV3 upregulation was triggered by exogenous growth factor supplementation and was attenuated by inhibitors of growth factor receptor signaling. In mice, subacute oropharyngeal instillation with WSPM also promoted TRPV3 mRNA expression and epithelial remodeling, which was attenuated by TRPV3 antagonist pre- and cotreatment. This latter effect may be the consequence of antagonist-induced TRPV3 expression. These findings provide insights into the roles of TRPV3 in lung epithelial cells under basal and dynamic states, as well as highlight potential roles for TRPV3 ligands in modulating epithelial damage/repair. SIGNIFICANCE STATEMENT: Coordinated epithelial repair is essential for the maintenance of the airways, with deficiencies and exaggerated repair associated with adverse consequences to respiratory health. This study shows that TRPV3, an ion channel, is involved in coordinating repair through integrated repair signaling pathways, wherein TRPV3 expression is upregulated immediately after injury and returns to basal levels as cells complete the repair process. TRPV3 may be a novel target for understanding and/or treating conditions in which airway/lung epithelial repair is not properly orchestrated.

Protective effect of taurine on sepsis‑induced lung injury via inhibiting the p38/MAPK signaling pathway

Sepsis, a leading cause of acute lung injury (ALI), is characterized by an overwhelming systemic inflammatory response and widespread organ injury, particularly in the lungs. Taurine, an intracellular free amino acid, has been used for the treatment of various diseases, including lung injury; however, the underlying mechanisms are unclear. The present study aimed to investigate the protective effect of taurine on septic ALI and the underlying mechanism. A septic ALI model was established by performing cecal ligation and puncture (CLP) surgery on Sprague Dawley rats. Following successful model establishment, rats were treated with taurine. The results of hematoxylin and eosin, respiratory function detection, malondialdehyde level and superoxide dismutase activity determination and ELSIA demonstrated that taurine significantly alleviated lung injury, restored respiratory function, reduced oxidation and decreased the concentrations of inflammatory factors in CLP-induced septic ALI model rats. In addition, compared with that in the ALI group, western blotting results indicated that taurine ameliorated lung epithelial injury by significantly increasing the expression levels of lung epithelial markers, E-cadherin and occludin. The western blotting results demonstrated that, compared with the control group, the p38/MAPK and NF-κB signaling pathways were significantly activated in CLP-induced septic ALI model rats, but taurine significantly suppressed ALI-mediated signaling pathway activation. To investigate the mechanism underlying taurine in the treatment of septic ALI, CLP-induced septic ALI model rats were treated with an antagonist of the p38/MAPK signaling pathway (SB203580). The effects of SB203580 on CLP-induced septic ALI model rats were similar to those of taurine. SB203580 significantly attenuated sepsis-induced lung injury and increases in IL-1β and TNF-α concentrations in the lung tissue. In addition, SB203580 promoted restoration of the injured lung tissue and respiratory function in CLP-induced septic ALI model rats. The western blotting results indicated that SB203580 significantly decreased the ratios of phosphorylated (p)-p38/p38 and p-p65/065, and increased the protein expression levels of E-cadherin and occludin compared with those in the ALI group. In summary, the present study demonstrated that taurine alleviated sepsis-induced lung injury, which was associated with suppression of the inflammatory response and oxidative stress via inhibiting the p38/MAPK signaling pathway. Therefore, the p38/MAPK signaling pathway may serve as a potential therapeutic target for the treatment of sepsis-induced ALI.

Regulated in Development and DNA Damage Response 1 Knockdown Alleviates Lipopolysaccharide-Induced Acute Lung Injury

Regulated in Development and DNA Damage Response 1 (REDD1) knockdown can reduce the endoplasmic reticulum stress response in liver injury. However, its role on lipopolysaccharide (LPS)-induced acute lung injury (ALI) has not been explored. This study aimed to evaluate the effect of REDD1 on lung epithelial cells induced by LPS. Rt-qPCR and Western blot were used to detect REDD1 expression in 16HBE cells induced by LPS. The interfering REDD1 plasmid was constructed, and CCK8 was used to detect the effect of interference with REDD1 on LPS-induced lung epithelial cell activity. The expression of inflammatory factors was detected by ELISA and the apoptotic level was detected by TUNEL staining. String database was used to predict the combination of REDD1 and EP300 in lung epithelial cells, which was verified by CoIP experiment. An overexpressed plasmid of EP300 was constructed to detect the effects of EP300 on inflammatory factors and apoptosis in REDD1 lung epithelial cells. LPS-induced increased REDD1 expression in lung epithelial cells. Interference with REDD1 inhibits LPS-induced lung epithelial cell activity injury and inflammatory factor expression and inhibits LPS-induced lung epithelial cell apoptosis. After interference with REDD1, the expression of EP300 in LPS-induced lung epithelial cells was inhibited, and the overexpression of EP300 was reversed to promote the production of inflammatory factors and apoptosis. In conclusion, these results demonstrate that REDD1 knockdown alleviates LPS-induced acute lung injury.

Changes in Plasma Soluble Receptor for Advanced Glycation End-Products Are Associated with Survival in Patients with Acute Respiratory Distress Syndrome.

The plasma soluble receptor for advanced glycation end-products (sRAGE) is a marker of lung epithelial injury with prognostic value when measured at baseline in acute respiratory distress syndrome (ARDS). However, whether changes in plasma sRAGE could inform prognosis in ARDS remains unknown. In this secondary analysis of the Lung Imaging for Ventilator Setting in ARDS (LIVE) multicenter randomized controlled trial, which evaluated a personalized ventilation strategy tailored to lung morphology, plasma sRAGE was measured upon study entry (baseline) and on days one, two, three, four and six. The association between changes in plasma sRAGE over time and 90-day survival was evaluated. Higher baseline plasma sRAGE (HR per-one log increment, 1.53; 95% CI, 1.16–2.03; p = 0.003) and an increase in sRAGE over time (HR for each one-log increment in plasma sRAGE per time unit, 1.01; 95% CI, 1.01–1.02; p < 10−3) were both associated with increased 90-day mortality. Each 100-unit increase in the plasma sRAGE level per unit of time increased the risk of death at day 90 by 1% in joint modeling. Plasma sRAGE increased over time when a strategy of maximal alveolar recruitment was applied in patients with focal ARDS. Current findings suggest that the rate of change in plasma sRAGE over time is associated with 90-day survival and could be helpful as a surrogate outcome in ARDS.

SP-D Serum Levels Reveal Distinct Epithelial Damage in Direct Human ARDS.

Acute respiratory distress syndrome (ARDS) is a heterogeneous syndrome with multiple underlying diseases. Particularly epithelial damage results from direct (e.g., pneumonia) rather than indirect lung injury (e.g., nonpulmonary sepsis), which is more likely associated with endothelial damage. Hence, targeting ARDS patients based on their molecular phenotypes is a promising approach to improve outcome. With regard to distinct inflammatory responses and subsequent lung damage in direct ARDS due to the causing pathogen, we quantified markers of epithelial and endothelial damage and pro-inflammatory cytokines in patients with ARDS triggered by bacterial, viral, and atypical pathogen pneumonia or indirect ARDS. The serum levels of interleukin-6 (IL-6) and interleukin-8 (IL-8), lung epithelial injury markers surfactant protein D (SP-D), and soluble receptor for advanced glycation end-products (sRAGE) as well as endothelial injury marker angiopoietin-2 (Ang-2) from 49 patients with distinct types of ARDS were analyzed by multiplex immunoassay. Epithelial damage marker SP-D was significantly higher in direct ARDS caused by viral and atypical pathogens in contrast to ARDS caused by typical bacterial pneumonia and nonpulmonary sepsis. In contrast, sRAGE levels did not differ due to the causing pathogen. Patients with atypical pathogen pneumonia related ARDS showed significantly lower Ang-2 levels compared to patients with viral and indirect ARDS. Patients with viral and atypical pneumonia related ARDS possessed significantly lower serum IL-6 levels compared to bacterial pneumonia related ARDS and IL-6 levels in atypical pneumonia related ARDS were significantly lower than in indirect ARDS. Current findings report a potential difference in ARDS biomarkers due to the underlying disease and pathogen.

MicroRNA-490-3p inhibits inflammatory responses in LPS-induced acute lung injury of neonatal rats by suppressing the IRAK1/TRAF6 pathway.

Acute lung injury (ALI) is a main reason for neonatal death. Studying the molecular mechanism behind neonatal ALI is critical for the development of therapeutic strategies. The present study explored microRNA (miR)-490-3p-mediated regulatory effects on lipopolysaccharide (LPS)-induced neonatal ALI. Initially, LPS (10 mg/kg body weight) was injected to 3-8 day old neonatal SD rats to induce ALI, and LPS (100 ng/ml) was used to treat lung epithelial cells to construct an ALI model in vitro. Next, miR-490-3p, pro-inflammatory factors (that included IL-1β, IL-6 and TNFα), interleukin 1 receptor associated kinase 1 (IRAK1) and TNF receptor associated factor 6 (TRAF6) mRNA expression levels in lung tissues and epithelial cells were assessed via reverse transcription-quantitative PCR. In addition, miR-490-3p mimics were adopted to construct its overexpressed cell model, and Cell Counting Kit-8 and BrdU assays were conducted to assess cell viability. Furthermore, the miR-490-3p target, IRAK was predicted by bioinformatics analysis and verified via Dual-luciferase reporter gene assay. The results revealed that miR-490-3p was markedly downregulated in an LPS-induced rat ALI model, while IL-1β, IL-6, TNFα, IRAK1 and TRAF6 were all upregulated and negatively correlated with miR-490-3p expression. Moreover, overexpressed miR-490-3p significantly inhibited LPS-induced lung epithelial cell injury and inflammatory response. Mechanistically, miR-490-3p targeted and attenuated IRAK1 expression, which thus inactivated the LPS-mediated TRAF6/NF-κB pathway. Overall, the present study indicated that miR-490-3p overexpression significantly inhibited LPS-induced ALI and inflammatory responses by restricting the IRAK1/TRAF6 pathway.

A Positive Feed Forward Loop between Wnt/β-Catenin and NOX4 Promotes Silicon Dioxide-Induced Epithelial-Mesenchymal Transition of Lung Epithelial Cells.

Silicosis is a chronic fibrotic lung disease caused by the accumulation of silica dust in the distal lung. Canonical Wnt signaling and NADPH oxidase 4 (NOX4) have been demonstrated to play a crucial role in the pathogenesis of pulmonary fibrosis including silicosis. However, the underlying mechanisms of crosstalk between these two signalings are not fully understood. In the present study, we aimed to explore the interaction of Wnt/β-catenin and NOX4 of human epithelial cells in response to an exposure of silica dust. Results demonstrated an elevated expression of key components of Wnt/β-catenin signaling and NOX4 in the lungs of silicon dioxide- (SiO2-) induced silicosis mice. Furthermore, the activated Wnt/β-catenin and NOX4 signaling are accompanied by an inhibition of cell proliferation, an increase of ROS production and cell apoptosis, and an upregulation of profibrogenic factors in BEAS-2B human lung epithelial cells exposed to SiO2. A mechanistic study further demonstrated that the Wnt3a-mediated activation of canonical Wnt signaling could augment the SiO2-induced NOX4 expression and reactive oxygen species (ROS) production but reduced glutathione (GSH), while Wnt inhibitor DKK1 exhibited an opposite effect to Wnt3a. Vice versa, an overexpression of NOX4 further activated SiO2-induced Wnt/β-catenin signaling and NFE2-related factor 2 (Nrf2) antioxidant response along with a reduction of GSH, whereas the shRNA-mediated knockdown of NOX4 showed an opposite effect to NOX4 overexpression. These results imply a positive feed forward loop between Wnt/β-catenin and NOX4 signaling that may promote epithelial-mesenchymal transition (EMT) of lung epithelial cells in response to an exposure of silica dust, which may thus provide an insight into the profibrogenic role of Wnt/β-catenin and NOX4 crosstalk in lung epithelial cell injury and pathogenesis of silicosis.

The endogenous capacity to produce proinflammatory mediators by the ex vivo human perfused lung

BackgroundThe ex vivo human perfused lung model has enabled optimizing donor lungs for transplantation and delineating mechanisms of lung injury. Perfusate and airspace biomarkers are a proxy of the lung response to experimental conditions. However, there is a lack of studies evaluating biomarker kinetics during perfusion and after exposure to stimuli. In this study, we analyzed the ex vivo-perfused lung response to three key perturbations: exposure to the perfusion circuit, exogenous fresh whole blood, and bacteria.ResultsNinety-nine lungs rejected for transplantation underwent ex vivo perfusion. One hour after reaching experimental conditions, fresh whole blood was added to the perfusate (n = 55). Two hours after reaching target temperature, Streptococcus pneumoniae was added to the perfusate (n = 42) or to the airspaces (n = 17). Perfusate and airspace samples were collected at baseline (once lungs were equilibrated for 1 h, but before blood or bacteria were added) and 4 h later. Interleukin (IL)-6, IL-8, angiopoietin (Ang)-2, and soluble tumor necrosis factor receptor (sTNFR)-1 were quantified. Baseline perfusate and airspace biomarker levels varied significantly, and this was not related to pre-procurement PaO2:FiO2 ratio, cold ischemia time, and baseline alveolar fluid clearance (AFC). After 4 h of ex vivo perfusion, the lung demonstrated a sustained production of proinflammatory mediators. The change in biomarker levels was not influenced by baseline donor lung characteristics (cold ischemia time, baseline AFC) nor was it associated with measures of experimental epithelial (final AFC) or endothelial (percent weight gain) injury. In the presence of exogenous blood, the rise in biomarkers was attenuated. Lungs exposed to intravenous (IV) bacteria relative to control lungs demonstrated a significantly higher rise in perfusate IL-6.ConclusionsThe ex vivo-perfused lung has a marked endogenous capacity to produce inflammatory mediators over the course of short-term perfusion that is not significantly influenced by donor lung characteristics or the presence of exogenous blood, and only minimally affected by the introduction of systemic bacteremia. The lack of association between biomarker change and donor lung cold ischemia time, final alveolar fluid clearance, and experimental percent weight gain suggests that the maintained ability of the human lung to produce biomarkers is not merely a marker of lung epithelial or endothelial injury, but may support the function of the lung as an immune cell reservoir.

Perioperative Open-lung Approach, Regional Ventilation, and Lung Injury in Cardiac Surgery

In the Protective Ventilation in Cardiac Surgery (PROVECS) randomized, controlled trial, an open-lung ventilation strategy did not improve postoperative respiratory outcomes after on-pump cardiac surgery. In this prespecified subanalysis, the authors aimed to assess the regional distribution of ventilation and plasma biomarkers of lung epithelial and endothelial injury produced by that strategy. Perioperative open-lung ventilation consisted of recruitment maneuvers, positive end-expiratory pressure (PEEP) = 8 cm H2O, and low-tidal volume ventilation including during cardiopulmonary bypass. Control ventilation strategy was a low-PEEP (2 cm H2O) low-tidal volume approach. Electrical impedance tomography was used serially throughout the perioperative period (n = 56) to compute the dorsal fraction of ventilation (defined as the ratio of dorsal tidal impedance variation to global tidal impedance variation). Lung injury was assessed serially using biomarkers of epithelial (soluble form of the receptor for advanced glycation end-products, sRAGE) and endothelial (angiopoietin-2) lung injury (n = 30). Eighty-six patients (age = 64 ± 12 yr; EuroSCORE II = 1.65 ± 1.57%) undergoing elective on-pump cardiac surgery were studied. Induction of general anesthesia was associated with ventral redistribution of tidal volumes and higher dorsal fraction of ventilation in the open-lung than the control strategy (0.38 ± 0.07 vs. 0.30 ± 0.10; P = 0.004). No effect of the open-lung strategy on the dorsal fraction of ventilation was noted at the end of surgery after median sternotomy closure (open-lung = 0.37 ± 0.09 vs. control = 0.34 ± 0.11; P = 0.743) or in extubated patients at postoperative day 2 (open-lung = 0.63 ± 0.18 vs. control = 0.59 ± 0.11; P > 0.999). Open-lung ventilation was associated with increased intraoperative plasma sRAGE (7,677 ± 3,097 pg/ml vs. 6,125 ± 1,400 pg/ml; P = 0.037) and had no effect on angiopoietin-2 (P > 0.999). In cardiac surgery patients, open-lung ventilation provided larger dorsal lung ventilation early during surgery without a maintained benefit as compared with controls at the end of surgery and postoperative day 2 and was associated with higher intraoperative plasma concentration of sRAGE suggesting lung overdistension.

Mitophagy-Mediated mtDNA Release Aggravates Stretching-Induced Inflammation and Lung Epithelial Cell Injury via the TLR9/MyD88/NF-κB Pathway.

BackgroundIn animal models of ventilation-induced lung injury, mitophagy triggers mitochondria damage and the release of mitochondrial (mt) DNA, which activates inflammation. However, the mechanism of this process is unclear.MethodsA model of cyclic stretching (CS)-induced lung epithelial cell injury was established. The genetic intervention of phosphatase and tensin homolog-induced kinase 1 (PINK1) expression via lentivirus transfection was used to identify the relationship between PINK1-mediated mitophagy and mtDNA release in stretching-induced inflammatory response and injury. Pharmacological inhabitation of Toll-like receptor 9 (TLR9) and myeloid differentiation factor 88 (MyD88) expression was performed via their related inhibitors, while pre-treatment of exogenous mtDNA was used to verify the role of mtDNA in stretching-induced inflammatory response and injury.ResultsUsing a cell culture model of CS, we found that knocking down PINK1 in lung epithelial cells reduced mitophagy activation and mtDNA release, leading to milder inflammatory response and injury; conversely, up-regulating PINK1 exacerbated stretching-induced inflammation and injury, and similar effects were observed by upregulating TLR9 to induce expression of MyD88 and nuclear factor-κB (NF-κB)/p65. Down-regulating MyD88 protected lung epithelial cells from stretching injury and decreased NF-κB/p65 expression.ConclusionThese findings suggest that PINK1-dependent mitophagy and associated TLR9 activation is indeed a major factor in stretch-induced cell injury via a mechanism in which released mtDNA activates TLR9 and thereby the MyD88/NF-κB pathway. Inhibiting this process may be a therapeutic approach to prevent inflammation and cell injury in patients on mechanical ventilation.