Ventilator-induced Lung Injury Group Research Articles

Delineation and authentication of ferroptosis genes in ventilator-induced lung injury

BackgroundMechanical ventilation, a critical support strategy for individuals enduring severe respiratory failure and general anesthesia, paradoxically engenders ventilator-induced lung injury (VILI). Ferrostatin-1 mitigates lung injury via ferroptosis inhibition, yet the specific ferroptosis genes contributing significantly to VILI remain obscure.MethodsLeveraging the Gene Expression Omnibus database, we acquired VILI-associated datasets and identified differentially expressed genes (DEGs). To identify the hub genes, we constructed a protein–protein interaction network and used three parameters from CytoHubba. Consequently, we identified hub genes and ferroptosis genes as ferroptosis hub genes for VILI (VFHGs). We conducted enrichment analysis and established receiver operating characteristic (ROC) curves for VFHGs. Subsequently, to confirm the correctness of the VFHGs, control group mice and VILI mouse models, as well as external dataset validation, were established. For further research, a gene-miRNA network was established. Finally, the CIBERSORT algorithm was used to fill the gap in the immune infiltration changes in the lung during VILI.ResultsWe identified 64 DEGs and 4 VFHGs (Il6,Ptgs2,Hmox1 and Atf3) closely related to ferroptosis. ROC curves demonstrated the excellent diagnostic performance of VFHGs in VILI. PCR and external dataset validation of the VILI model demonstrated the accuracy of VFHGs. Subsequently, the gene-miRNA network was successfully established. Ultimately, an Immune cell infiltration analysis associated with VILI was generated.ConclusionsThe results emphasize the importance of 4 VFHGs and their involvement in ferroptosis in VILI, confirming their potential as diagnostic biomarkers for VILI.

Differential urine proteome analysis of a ventilator-induced lung injury rat model by label-free quantitative and parallel reaction monitoring proteomics

Urine is a promising resource for biomarker research. Therefore, the purpose of this study was to investigate potential urinary biomarkers to monitor the disease activity of ventilator-induced lung injury (VILI). In the discovery phase, a label-free data-dependent acquisition (DDA) quantitative proteomics method was used to profile the urinary proteomes of VILI rats. For further validation, the differential proteins were verified by parallel reaction monitoring (PRM)-targeted quantitative proteomics. In total, 727 high-confidence proteins were identified with at least 1 unique peptide (FDR ≤ 1%). Compared to the control group, 110 proteins (65 upregulated, 45 downregulated) were significantly changed in the VILI group (1.5-fold change, P < 0.05). The canonical pathways and protein–protein interaction analyses revealed that the differentially expressed proteins were enriched in multiple functions, including oxidative stress and inflammatory responses. Finally, thirteen proteins were identified as candidate biomarkers for VILI by PRM validation. Among these PRM-validated proteins, AMPN, MEP1B, LYSC1, DPP4 and CYC were previously reported as lung-associated disease biomarkers. SLC31, MEP1A, S15A2, NHRF1, XPP2, GGT1, HEXA, and ATPB were newly discovered in this study. Our results suggest that the urinary proteome might reflect the pathophysiological changes associated with VILI. These differential proteins are potential urinary biomarkers for the activity of VILI.

Transcriptome-Wide Gene Expression in a Murine Model of Ventilator-Induced Lung Injury.

Background Mechanical ventilation could lead to ventilator-induced lung injury (VILI), but its underlying pathogenesis remains largely unknown. In this study, we aimed to determine the genes which were highly correlated with VILI as well as their expressions and interactions by analyzing the differentially expressed genes (DEGs) between the VILI samples and controls. Methods GSE11434 was downloaded from the gene expression omnibus (GEO) database, and DEGs were identified with GEO2R. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using DAVID. Next, we used the STRING tool to construct protein-protein interaction (PPI) network of the DEGs. Then, the hub genes and related modules were identified with the Cytoscape plugins: cytoHubba and MCODE. qRT-PCR was further used to validate the results in the GSE11434 dataset. We also applied gene set enrichment analysis (GSEA) to discern the gene sets that had a significant difference between the VILI group and the control. Hub genes were also subjected to analyses by CyTargetLinker and NetworkAnalyst to predict associated miRNAs and transcription factors (TFs). Besides, we used CIBERSORT to detect the contributions of different types of immune cells in lung tissues of mice in the VILI group. By using DrugBank, small molecular compounds that could potentially interact with hub genes were identified. Results A total of 141 DEGs between the VILI group and the control were identified in GSE11434. Then, seven hub genes were identified and were validated by using qRT-PCR. Those seven hub genes were largely enriched in TLR and JAK-STAT signaling pathways. GSEA showed that VILI-associated genes were also enriched in NOD, antigen presentation, and chemokine pathways. We predicted the miRNAs and TFs associated with hub genes and constructed miRNA-TF-gene regulatory network. An analysis with CIBERSORT showed that the proportion of M0 macrophages and activated mast cells was higher in the VILI group than in the control. Small molecules, like nadroparin and siltuximab, could act as potential drugs for VILI. Conclusion In sum, a number of hub genes associated with VILI were identified and could provide novel insights into the pathogenesis of VILI and potential targets for its treatment.

Mining the key genes for ventilator-induced lung injury using co-expression network analysis.

Mechanical ventilation is extensively adopted in general anesthesia and respiratory failure management, but it can also induce ventilator-induced lung injury (VILI). Therefore, it is of great urgency to explore the mechanisms involved in the VILI pathogenesis, which might contribute to its future prevention and treatment. Four microarray datasets from the GEO database were selected in our investigation, and were subjected to the Weighted Gene Co-Expression Network Analysis (WGCNA) to identify the VILI-correlated gene modules. The limma package in R software was used to identify the differentially expressed genes (DEGs) between the VILI and control groups. WGCNA was constructed by merging the GSE9314, GSE9368, GSE11434 and GSE11662 datasets. A total of 49 co-expression network modules were determined as associated with VILI. The intersected genes between hub genes screened from DEGs for VILI and those identified using WGCNA were as follows: Tlr2, Hmox1, Serpine1, Mmp9, Il6, Il1b, Ptgs2, Fos and Atf3, which were determined to be key genes for VILI. Those key genes were validated by GSE86229 and quantitative PCR (qPCR) experiment to have significantly statistical difference in their expression between the VILI and control groups. In a nutshell, nine key genes with expression differences in VILI were screened by WGCNA by integrating multiple datasets.

Ginsenoside ameliorated ventilator-induced lung injury in rats

BackgroundGinsenosides have antioxidant and anti-inflammatory features. This study aimed to evaluate the biologic effects of ginsenoside Rb2 pretreatment on ventilator-induced lung injury (VILI) in rats.MethodsRats were divided into four groups with 12 rats per group: control; low tidal volume (TV), TV of 6 mL/kg, VILI, TV of 20 mL/kg, positive end-expiratory pressure of 5 cm H2O, and respiratory rate of 60 breaths per minute for 3 h at an inspiratory oxygen fraction of 0.21; and ginsenosides, treated the same as the VILI group but with 20 mg/kg intraperitoneal ginsenoside pretreatment. Morphology was observed with a microscope to confirm the VILI model. Wet-to-dry weight ratios, protein concentrations, and pro-inflammatory cytokines in the bronchoalveolar lavage fluid were measured. RNA sequencing of the lung tissues was conducted to analyze gene expression.ResultsHigh TV histologically induced VILI with alveolar edema and infiltration of inflammatory cells. Ginsenosides pretreatment significantly reduced the histologic lung injury score compared to the VILI group. Wet-to-dry weight ratios, malondialdehyde, and TNF-α in bronchoalveolar lavage fluid were significantly higher in the VILI group and ginsenoside pretreatment mitigated these effects. In the immunohistochemistry assay, ginsenoside pretreatment attenuated the TNF-α upregulation induced by VILI. We identified 823 genes differentially presented in the VILI group compared to the control group. Of the 823 genes, only 13 genes (Arrdc2, Cygb, Exnef, Lcn2, Mroh7, Nsf, Rexo2, Srp9, Tead3, Ephb6, Mvd, Sytl4, and Ube2l6) recovered to control levels in the ginsenoside group.ConclusionsGinsenosides inhibited the inflammatory and oxidative stress response in VILI. Further studies are required on the 13 genes, including LCN2.

Lipoxin A4 Reduces Ventilator-Induced Lung Injury in Rats with Large-Volume Mechanical Ventilation.

Ventilator-induced lung injury (VILI) is a severe and inevitable complication in patients who require mechanical ventilation (MV) for respiratory support. Lipoxin A4 is an endogenous anti-inflammatory and antioxidant mediator. The present study determined the effects of lipoxin A4 on VILI. Twenty-four rats were randomized to the sham, VILI, and lipoxin A4 (LX4) groups. The rats in the VILI and LX4 groups received large-volume MV for 4 hours to simulate VILI. Capillary permeability was evaluated using the PaO2/FiO2 ratio, lung wet/dry weight ratio, and protein level in the lung. VILI-induced inflammation was assessed by measuring cytokines in serum and lung tissue, the expression and activity of NF-κB, and phosphorylated myosin light chain. The oxidative stress response, lung tissue injury, and apoptosis in lung tissue were also estimated, and the expression of apoptotic proteins was examined. MV worsened all of the indices compared to the sham group. Compared to the VILI group, the LX4 group showed significantly improved alveolar-capillary permeability (increased PaO2/FiO2 and decreased wet/dry weight ratios and protein levels), ameliorated histological injury, and reduced local and systemic inflammation (downregulated proinflammatory factors and NF-κB expression and activity). Lipoxin A4 notably inhibited the oxidative stress response and apoptosis and balanced apoptotic protein levels in lung tissue. Lipoxin A4 protects against VILI via anti-inflammatory, antioxidant, and antiapoptotic effects.

Dexmedetomidine reduces ventilator-induced lung injury via ERK1/2 pathway activation.

Mechanical ventilation (MV) can contribute to ventilator-induced lung injury (VILI); dexmedetomidine (Dex) treatment attenuates MV-related pulmonary inflammation, but the mechanisms remain unclear. Therefore, the present study aimed to explore the protective effect and the possible molecular mechanisms of Dex in a VILI rodent model. Adult male Sprague-Dawley rats were randomly assigned to one of seven groups (n=24 rats/group). Rats were euthanized after 4 h of continuous MV, and pathological changes, lung wet/dry (W/D) weight ratio, the levels of inflammatory cytokines (IL-1β, TNF-α and IL-6) in the bronchoalveolar lavage fluid (BALF), and the expression levels of Bcl-2 homologous antagonist/killer (Bak), Bcl-2, pro-caspase-3, cleaved caspase-3 and the phosphorylation of ERK1/2 in the lung tissues were measured. Propidium iodide uptake and TUNEL staining were used to detect epithelial cell death. The Dex pretreatment group exhibited fewer pathological changes, lower W/D ratios and lower expression levels of inflammatory cytokines in BALF compared with the VILI group. Dex significantly attenuated the ratio of Bak/Bcl-2, cleaved caspase-3 expression levels and epithelial cell death, and increased the expression of phosphorylated ERK1/2. The protective effects of Dex could be partially reversed by PD98059, which is a mitogen-activated protein kinase (upstream of ERK1/2) inhibitor. Overall, dexmedetomidine was found to reduce the inflammatory response and epithelial cell death caused by VILI, via the activation of the ERK1/2 signaling pathway.

Effect of low-level laser therapy on the inflammatory response in an experimental model of ventilator-induced lung injury.

The effect of low-level laser therapy (LLLT) on an experimental model of ventilator-induced lung injury (VILI) was evaluated in this study. 24 adult Wistar rats were randomized into four groups: protective mechanical ventilation (PMV), PMV + laser, VILI and VILI + laser. The animals of the PMV and VILI groups were ventilated with tidal volumes of 6 and 35 ml kg−1, respectively, for 90 minutes. After the first 60 minutes of ventilation, the animals in the laser groups were irradiated (808 nm, 100 mW power density, 20 J cm−2 energy density, continuous emission mode, and exposure time of 5 s) and after 30 minutes of irradiation, the animals were euthanized. Lung samples were removed for morphological analysis, bronchoalveolar lavage (BAL) and real time quantitative polynucleotide chain reaction (RT-qPCR). The VILI group showed a greater acute lung injury (ALI) score with an increase in neutrophil infiltration, higher neutrophil count in the BAL fluid and greater cytokine mRNA expression compared to the PMV groups (p < 0.05). The VILI ± laser group when compared to the VILI group showed a lower ALI score (0.35 ± 0.08 vs. 0.54 ± 0.13, p < 0.05), alveolar neutrophil infiltration (7.00 ± 5.73 vs. 21.50 ± 9.52, p < 0.05), total cell count (1.90 ± 0.71 vs. 4.09 ± 0.96 × 105, p < 0.05) and neutrophil count in the BAL fluid (0.60 ± 0.37 vs. 2.28 ± 0.48 × 105, p < 0.05). Moreover, LLLT induced a decrease in pro-inflammatory and an increase of anti-inflammatory mRNA levels compared to the VILI group (p < 0.05). In conclusion, LLLT was found to reduce the inflammatory response in an experimental model of VILI.

Changes in expression of caspase recruitment domain protein 3 during ventilator-induced lung injury in rats

Objective To evaluate the changes in the expression of caspase recruitment domain protein 3 (NLRC3) during ventilator-induced lung injury (VILI) in rats. Methods Twenty-four clean-grade healthy adult male Sprague-Dawley rats, aged 8 weeks, weighing 180-220 g, were divided into 2 groups (n=12 each) according to the random number table method: control group (group C) and group VILI.Both groups underwent tracheotomy and intubation, group C kept spontaneous breathing, and the animals were mechanically ventilated, with tidal volume 20 ml/kg, respiratory rate 80 breaths/min, inspiratory/expiratory ratio 1∶1, fraction of inspired oxygen 21%, and positive end-expiratory pressure 0 in group VILI.The rats were sacrificed 4 h later, bronchoalveolar lavage fluid (BALF) was collected for determination of concentrations of interleukin-1beta (IL-1β)and IL-18 in BALF (by enzyme-linked immunosorbent assay), and lung tissues were obtained for examination of the pathological changes which were scored and for determination of wet to dry weight ratio (W/D ratio) and expression of nucleotide binding oligomerization domain-like receptor family caspase recruitment domain containing 3 (NLRC3), NOD-like receptor pyrin domain containing 3 (NLRP3), ASC and caspase-1 protein and mRNA (by Western blot and real-time polymerase chain reaction). Results Compared with group C, the score of pathological damage to lung tissues, W/D ratio, and concentrations of IL-1β and IL-18 in BALF were significantly increased, the expression of NLRP3, ASC and caspase-1 protein and mRNA in lung tissues was up-regulated, and the expression of NLRC3 protein and mRNA in lung tissues was down-regulated in group VILI (P<0.05). Conclusion The mechanism of VILI may be related to down-regulating NLRC3 expression and activating NLRP3 inflammasomes in rats. Key words: Ventilator-induced lung injury; NLR family, pyrin domain-containing 3 protein

Role of microRNA-125b in ventilator-induced lung injury in mice

Objective To evaluate the role of microRNA-125b (miR-125b) on ventilator-induced lung injury (VILI) in mice. Methods Forty healthy male C57BL/6 male mice, weighing 25-30 g, aged 2-3 months, were divided into 4 groups (n=10 each) using a random number table method: sham operation group (group Sham), group VILI, VILI plus miR-125b negative control group (group VILI+ NC), and VILI plus miR-125b overexpression group (group VILI+ miR-125b agomir). In VILI+ NC and VILI+ miR-125b agomir groups, miR-125b negative control and miR-125b agomir transfection complex 50 μl were intratracheally instilled, respectively, and 48 h later VILI model was established.The animals were mechanically ventilated for 4 h with high tidal volume (40 ml/kg) to induce VILI.Blood samples were obtained from the femoral artery at 4 h of mechanical ventilation for detection of PaO2, then animals were sacrificed, lungs were removed for determination of wet to dry weight ratio (W/D ratio) and for examination of pathological changes (with a light microscope), and lung injury was scored.In VILI+ NC and VILI+ miR-125b agomir groups, bronchoalveolar lavage fluid (BALF) was collected to measure the concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by enzyme-linked immunosorbent assay.The cell apoptosis of lung tissues was measured using TUNEL, apoptosis index was calculated, the caspase-3 expression was detected by Western blot, and the miR-125b expression was detected by real-time polymerase chain reaction. Results Compared with Sham group, PaO2 was significantly decreased, and W/D ratio and lung injury score were increased in VILI, VILI+ NC and VILI+ miR-125b agomir groups, and the expression of miR-125b was down-regulated in VILI and VILI+ NC groups (P<0.05). Compared with VILI group, PaO2 was significantly increased, W/D ratio and lung injury score were decreased, the expression of miR-125b was up-regulated (P<0.05), the pathological changes of lung tissues were significantly attenuated in group VILI+ miR-125b agomir, and no significant change was found in the parameters mentioned above in group VILI+ NC (P<0.05). Compared with group VILI+ NC, the concentrations of TNF-α and IL-6 in BALF and apoptotic index were significantly decreased, and the expression of caspase-3 was down regulated in group VILI+ miR-125b agomir (P<0.05). Conclusion MiR-125b is involved in the endogenous protective mechanism of VILI in mice. Key words: MicroRNAs; Ventilator-induced lung injury

Endothelial Progenitor Cells Attenuate Ventilator-Induced Lung Injury with Large-Volume Ventilation

Ventilator-induced lung injury (VILI) is a common complication that results from treatment with mechanical ventilation (MV) in acute respiratory distress syndrome (ARDS) patients. The present study investigated the effect of endothelial progenitor cell (EPC) transplantation on VILI. Wistar rats were divided into three groups (n = 8): sham (S), VILI model (V) induced by tidal volume ventilation (17 mL/kg), and VILI plus EPC transplantation (VE) groups. The lung PaO2/FiO2 ratio, pulmonary wet-to-dry (W/D) weight ratio, number of neutrophils, total protein, neutrophil elastase level, and inflammatory cytokines in bronchoalveolar lavage fluid (BALF) and serum were examined. Furthermore, the histological and apoptotic analysis, and lung tissue protein expression analysis of Bax, Bcl-2, cleaved caspase-3, matrix metalloproteinase (MMP)-9, total nuclear factor kappa B (total-NF-κB), phosphorylated NF-κB (phospho-NF-κB) and myosin light chain (MLC) were performed. The ventilation-induced decrease in PaO2/FiO2 ratio, and the increase in W/D ratio and total protein concentration were prevented by the EPC transplantation. The EPC transplantation (VE group) significantly attenuated the VILI-induced increased expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8, MMP-9, phospho-NF-κB and MLC, neutrophil elastase levels and neutrophil counts in BALF. In addition, the anti-inflammatory factor IL-10 increased in the VE group. Furthermore, pulmonary histological injury and apoptosis (TUNEL-positive cells, increase in Bax and cleaved caspase-3) were considerably diminished by the EPC transplantation. The EPC transplantation ameliorated the VILI. The mechanism may be primarily through the improvement of epithelial permeability, inhibition of local and systemic inflammation, and reduction in apoptosis.

Ventilation accelerates lung injury in septic mice

Sepsis is one of the systemic inflammatory responses, and it can cause acute respiratory distress syndrome (ARDS). Mechanical ventilation (MV) is one of the best treatments in patients with sepsis related to ARDS. However, mechanical ventilation itself can cause lung injury through various mechanisms. In this study, we applied mouse cecal ligation and puncture (CLP) model of sepsis with subsequent injurious ventilation to determine if sepsis could affect the development of ventilator-induced lung injury (VILI). To evaluate lung injury caused by sepsis, mild, moderate, and severe CLPs were induced. To evaluate lung injury caused by MV with or without sepsis, tidal volume (7 mL/kg, 10 mL/kg, or 24 mL/kg) was maintained for 3 h with or without septic insult (mild sepsis for 72 h). Lung compliance was then determined and biochemical evaluations were performed. Results showed that lung compliance was lower in the severe sepsis group compared to that in the mild or moderate sepsis group. MV alone decreased lung compliance in a tidal volume dependent manner. Both CLP and VILI groups showed significant decrease in lung compliance and increase in total cell count upon bronchoalveolar lavage fluid. These results confirm that ventilation can affect lung injury in mouse sepsis model.

Potential role of M2 macrophage polarization in ventilator-induced lung fibrosis

Mechanical ventilation (MV) is an essential life-support technique, but it can induce ventilator-induced lung injury (VILI) and subsequent pulmonary fibrosis. The mechanisms underlying this fibrosis are largely unknown. Because excessive polarization of M2 macrophages has increasingly been cited as possible inciting factor for tissue remodeling and organ fibrosis, we here hypothesize it might be involved in the development of pulmonary fibrosis after high tidal volume (VT) MV. In our prospective, randomized, controlled animal study, C57BL/6 mice were randomly placed in either a VILI group or sham group. After ventilation, surviving mice were allowed to recover for 0, 1, 3, 5, 7, or 14 days. 200 mice were involved in our in vivo experiment, and the results calculated here refer only to the surviving mice. The results clearly showed that high-VT MV caused early inflammation and a subsequent fibroproliferative response in mice without pre-existing lung disease. High-VT MV was also found to lead to a dramatic increase in the number of M2 macrophages in mouse bronchoalveolar lavage fluid (BALF) cell and lung tissues. Consistent with the progression of fibrosis, there were far more M2 macrophages at the 5th day after ventilation and remained dominant for 2 weeks. High-VT MV induced epithelial–mesenchymal transition (EMT) on day 7, accompanied by the increased expression of TGF-β1 and p-Smad2/3. In vitro experiments, the co-culture of M2 macrophage and MLE-12 cells resulted in a significant EMT and upregulation of TGF-β1 and p-Smad2/3 in MLE-12 cells. To summarize, our findings suggested the persistent tilt polarization toward M2 macrophages was associated with EMT during the course of ventilator-induced pulmonary fibrosis, which may play its roles through activation of epithelial TGF-β1/Smad2/3 signaling.

Role of CaN/NFATc4 signaling pathway in ventilator-induced lung injury

Objective To evaluate the role of calcineurin (CaN)/nuclear factor of activated T cell cytoplasmic 4 protein (NFATc4) signaling pathway in inflammatory responses in lung tissues of rats with ventilator-induced lung injury (VILI). Methods Twenty-four clean-grade healthy male Wistar rats, aged 5-8 weeks, weighing 220-250 g, were divided into 3 groups (n=8 each) using a random number table method: control C (group C), VILI group and cyclosporine A plus VILI group (group CsA+ VILI). The animals were anesthetized with pentobarbital and tracheostomized. The rats were mechanically ventilated for 6 h with the tidal volume set at 40 ml/kg and respiratory rate at 40 breaths/min to establish the model of VILI.The rats kept spontaneous breathing in group C. CaN specific inhibitor cyclosporine A 10 mg/kg was intraperitoneally injected at 1 h before ventilation in group CsA+ VILI.Rats were sacrificed immediately after ventilation, lung tissues were obtained and stained with hematoxylin and eosin to evaluate lung injury, broncho-alveolar lavage fluid was collected for determination of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 concentrations by enzyme-linked immunosorbent assay, and the lungs were removed for determination of the wet to dry weight ratio (W/D ratio), expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (by real-time polymerase chain reaction), and expression of calcineurin and NFATc4 in lung tissues (using Western blot). Results Compared with group C, the W/D ratio, lung injury scores and concentrations of IL-1β, IL-6 and TNF-α in BALF were significantly increased, and the expression of CaN, NFATc4, ICAM-1 mRNA and VCAM-1 mRNA was up-regulated in group VILI (P<0.05). Compared with group VILI, the W/D ratio, lung injury scores and concentrations of IL-1β, IL-6 and TNF-α in BALF were significantly decreased, and the expression of CaN, NFATc4, ICAM-1 mRNA and VCAM-1 mRNA was down-regulated in group CsA+ VILI (P<0.05). Conclusion CaN/NFATc4 signaling pathway mediates inflammatory responses in lung tissues of rats with VILI. Key words: Ventilator-induced lung injury; Calcineurin; NFATC transcription factors; Inflammation

Role of H1 receptor in inflammatory response during ventilator-induced lung injury in rats

Objective To evaluate the role of H1 receptor in inflammatory responses during ventilator-induced lung injury (VILI) in rats. Methods Thirty clean-grade healthy male Sprague-Dawley rats, aged 9-10 weeks, weighing 250-300 g, were divided into 3 groups (n=10 each) using a random number table method: control group (group C), VILI group (group V) and H1 receptor antagonist clemastine group (group Cle). The animals were anesthetized and tracheostomized, and the rats in group C kept spontaneous breathing.The rats were mechanically ventilated for 4 h with the tidal volume of 40 ml/kg, respiratory rate 40 breaths/min, inspiratory/expiratory ratio 1∶1, and inspired oxygen fraction ratio 21% in group V. In group Cle, clemastine 0.9 mg/kg was intramuscularly injected at 30 min before anesthesia, the animals were tracheostomized after being anesthetized, and then the rats were mechanically ventilated with the same ventilator settings as group V. The animals were sacrificed at 4 h of ventilation, bronchoalveolar lavage fluid (BALF) was collected for determination of concentrations of total protein, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and the lung specimens were obtained for microscopic examination of pathologic changes and for determination of wet/dry weight ratio (W/D ratio). Results Compared with group C, the concentrations of total protein, IL-6 and TNF-α in BALF and W/D ratio were significantly increased in group V (P<0.05). Compared with group V, the concentrations of total protein, IL-6 and TNF-α in BALF and W/D ratio were significantly decreased in group Cle (P<0.05). The pathologic changes of lung tissues were significantly attenuated in group Cle as compared with group V. Conclusion H1 receptor is involved in the process of inflammatory responses during VILI in rats. Key words: Receptors, histamine H1; Ventilator-induced lung injury; Inflammation

Expression profiling analysis of long noncoding RNAs in a mouse model of ventilator-induced lung injury indicating potential roles in inflammation.

The key regulators of inflammation underlying ventilator‐induced lung injury (VILI) remain poorly defined. Long noncoding RNAs (lncRNAs) have been implicated in the inflammatory response of many diseases; however, their roles in VILI remain unclear. We, therefore, performed transcriptome profiling of lncRNA and messenger RNA (mRNA) using RNA sequencing in lungs collected from mice model of VILI and control groups. Gene expression was analyzed through RNA sequencing and quantitative reverse transctiption polymerase chain reaction. A comprehensive bioinformatics analysis was used to characterize the expression profiles and relevant biological functions and for multiple comparisons among the controls and the injury models at different time points. Finally, lncRNA‐mRNA coexpression networks were constructed and dysregulated lncRNAs were analyzed functionally. The mRNA transcript profiling, coexpression network analysis, and functional analysis of altered lncRNAs indicated enrichment in the regulation of immune system/inflammation processes, response to stress, and inflammatory pathways. We identified the lncRNA Gm43181 might be related to lung damage and neutrophil activation via chemokine receptor chemokine (C‐X‐C) receptor 2. In summary, our study provides an identification of aberrant lncRNA alterations involved in inflammation upon VILI, and lncRNA‐mediated regulatory patterns may contribute to VILI inflammation.

Effects of Human Interleukin-10 on Ventilator-Associated Lung Injury in Rats.

Ventilator-induced lung injury (VILI) is one of the most serious complications of mechanical ventilation (MV) and can increase the mortality of patients with acute respiratory distress syndrome (ARDS). This work aimed to test the hypothesis that the anti-inflammatory properties of human interleukin-10 (hIL-10) can reduce VILI. Thirty-six healthy male Sprague-Dawley rats were randomly assigned into three groups (n = 12) as follows: a control group, a VILI group, and a hIL-10 group. Lung function was evaluated by oxygenation index and pulmonary edema, and morphological changes associated with lung injury were assessed by HE staining and quantitative histological lung injury score. Malondialdehyde (MDA) and Superoxide dismutase (SOD) were measured, and the levels of various inflammatory cytokines were assessed in BALF and plasma. The oxygenation index in the VILI group decreased significantly relative to the control group and improved substantially in the hIL-10 group (P < 0.01). Compared to the control group, MDA production was stimulated (P < 0.01), and SOD activity rapidly declined (P < 0.01) in the VILI group. After hIL-10, MDA content was lower than that seen in the VILI group (P < 0.01), and SOD activity was enhanced (P < 0.01). The VILI group had the highest cytokine levels, compared to either the hIL-10 group or the control group (P < 0.05). High tidal volume MV can induce VILI. hIL-10 may regulate the inflammatory response in the lung tissue, improve lung tissue oxygenation, and inhibit oxidative stress, therefore reducing VILI in rats. These experiments reveal a potential new treatment option for VILI.

Heparin-binding protein in ventilator-induced lung injury

BackgroundAlthough mechanical ventilation is often lifesaving, it can also cause injury to the lungs. The lung injury is caused by not only high pressure and mechanical forces but also by inflammatory processes that are not fully understood. Heparin-binding protein (HBP), released by activated granulocytes, has been indicated as a possible mediator of increased vascular permeability in the lung injury associated with trauma and sepsis. We investigated if HBP levels were increased in the bronchoalveolar lavage fluid (BALF) or plasma in a pig model of ventilator-induced lung injury (VILI). We also investigated if HBP was present in BALF from healthy volunteers and in intubated patients in the intensive care unit (ICU).MethodsAnaesthetized pigs were randomized to receive ventilation with either tidal volumes of 8 ml/kg (controls, n = 6) or 20 ml/kg (VILI group, n = 6). Plasma and BALF samples were taken at 0, 1, 2, 4, and 6 h. In humans, HBP levels in BALF were sampled from 16 healthy volunteers and from 10 intubated patients being cared for in the ICU.ResultsPlasma levels of HBP did not differ between pigs in the control and VILI groups. The median HBP levels in BALF were higher in the VILI group after 6 h of ventilation compared to those in the controls (1144 ng/ml (IQR 359–1636 ng/ml) versus 89 ng/ml (IQR 33–191 ng/ml) ng/ml, respectively, p = 0.02).The median HBP level in BALF from healthy volunteers was 0.90 ng/ml (IQR 0.79–1.01 ng/ml) as compared to 1959 ng/ml (IQR 612–3306 ng/ml) from intubated ICU patients (p < 0.001).ConclusionsIn a model of VILI in pigs, levels of HBP in BALF increased over time compared to controls, while plasma levels did not differ between the two groups.HBP in BALF was high in intubated ICU patients in spite of the seemingly non-harmful ventilation, suggesting that inflammation from other causes might increase HBP levels.

Effect of methylprednisolone on endoplasmic reticulum stress in rats with ventilator-induced lung injury: the relationship with PI3K/Akt signaling pathway

Objective To evaluate the effect of methylprednisolone on endoplasmic reticulum stress in rats with ventilator-induced lung injury (VILI) and the relationship with phosphatidylinositol 3-kinase/serine-threonine protein kinase (PI3K/Akt) signaling pathway. Methods One hundred clean-grade male Sprague-Dawley rats, aged 4-5 months, weighing 270-320 g, were divided into 5 groups (n=20 each) using a random number table method: control group (C group), VILI group (V group) and different doses of methylprednisolone groups (M1-3 groups). Group C received no mechanical ventilation and kept spontaneous breathing for 4 h. Rats were mechanically ventilated (tidal volume 40 ml/kg, respiratory rate 15-17 breaths/min, inspiratory/expiratory ratio 1∶1, positive end-expiratory pressure 0, fraction of inspired oxygen 21% during OLV) in group V. Methylprednisolone 2, 10 and 30 mg/kg were intravenously injected at 20 min before mechanical ventilation in M1-3 groups, respectively, and the equal volume of normal saline was given in group V. Blood samples and lung tissues were taken at 4 h of ventilation for measurement of the lung permeability index (LPI) and wet/dry lung weight ratio (W/D ratio), for examination of pathological changes, and for determination of apoptosis index (AI) in lung tissues (by TUNEL), expression of Akt, phosphorylated Akt (p-Akt), glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-12 in lung tissues (by Western blot). Injured alveoli rate (IAR) was calculated. Results Compared with group C, the W/D ratio, LPI, IAR and AI were significantly increased, the expression of p-Akt was down-regulated, and the expression of GRP78, CHOP and caspase-12 was up-regulated in V and M1 groups (P 0.05). Compared with group V, the W/D ratio, LPI, IAR and AI were significantly decreased, p-Akt expression was up-regulated, and the expression of GRP78, CHOP and caspase-12 was down-regulated in M2 and M3 groups (P<0.05). Conclusion Methylprednisolone inhibits endoplasmic reticulum stress, thus inhibiting cell apoptosis, and the mechanism is related to activating PI3K/Akt signaling pathway in rats with VILI. Key words: Methylprednisolone; Respiration, artificial; Respiratory distress syndrome, adult; Endoplasmic reticulum; Stress; 1-Phosphatidylinositol 3-kinase; Protein-serine-threonine kinases

Landscape of transcription and long non-coding RNAs reveals new insights into the inflammatory and fibrotic response following ventilator-induced lung injury

BackgroundMechanical ventilation can cause ventilator-induced lung injury (VILI) and lung fibrosis; however, the underlying mechanisms are still not fully understood. RNA sequencing is a powerful means for detecting vitally important protein-coding transcripts and long non-coding RNAs (lncRNAs) on a genome-wide scale, which may be helpful for reducing this knowledge gap.MethodsNinety C57BL/6 mice were subjected to either high tidal volume ventilation or sham operation, and then mice with ventilation were randomly allocated to periods of recovery for 0, 1, 3, 5, 7, 14, 21, or 28 days. Lung histopathology, wet-to-dry weight ratio, hydroxyproline concentration, and transforming growth factor beta 1 (TGF-β1) levels were determined to evaluate the progression of inflammation and fibrosis. To compare sham-operated lungs, and 0- and 7-day post-ventilated lungs, RNA sequencing was used to elucidate the expression patterns, biological processes, and functional pathways involved in inflammation and fibrosis.ResultsA well-defined fibrotic response was most pronounced on day 7 post-ventilation. Pairwise comparisons among the sham and VILI groups showed a total of 1297 differentially expressed transcripts (DETs). Gene Ontology analysis determined that the stimulus response and immune response were the most important factors involved in inflammation and fibrosis, respectively. Kyoto Encyclopedia of Genes and Genomes analysis revealed that mechanistic target of rapamycin (mTOR), Janus kinase-signal transducer and activator of transcription (JAK/STAT), and cyclic adenosine monophosphate (cAMP) signaling were implicated in early inflammation; whereas TGF-β, hypoxia inducible factor-1 (HIF-1), Toll-like receptor (TLR), and kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways were significantly involved in subsequent fibrosis. Additionally, 332 DE lncRNAs were identified and enriched in the processes of cellular and biological regulation. These lncRNAs may potentially regulate fibrosis through signaling pathways such as wingless/integrase-1 (Wnt), HIF-1, and TLR.ConclusionsThis is the first transcriptome study to reveal all of the transcript expression patterns and critical pathways involved in the VILI fibrotic process based on the early inflammatory state, and to show the important DE lncRNAs regulated in inflammation and fibrosis. Together, the results of this study provide novel perspectives into the potential molecular mechanisms underlying VILI and subsequent fibrosis.