Hyperoxia-induced Lung Injury Research Articles

BCL6 attenuates hyperoxia-induced lung injury by inhibiting NLRP3-mediated inflammation in fetal mouse

Background The transcriptional repressor B-cell lymphoma 6 (BCL6) has been reported to inhibit inflammation. So far, experimental evidence for the role of BCL6 in bronchopulmonary dysplasia (BPD) is lacking. Our study investigated the roles of BCL6 in the progression of BPD and its downstream mechanisms. Methods Hyperoxia or lipopolysaccharide (LPS) was used to mimic the BPD mouse model. To investigate the effects of BCL6 on BPD, recombination adeno-associated virus serotype 9 expressing BCL6 (rAAV9-BCL6) and BCL6 inhibitor FX1 were administered in mice. The pulmonary pathological changes, inflammatory chemokines and NLRP3-related protein were observed. Meanwhile, BCL6 overexpression plasmid was used in human pulmonary microvascular endothelial cells (HPMECs). Cell proliferation, apoptosis, and NLRP3-related protein were detected. Results Either hyperoxia or LPS suppressed pulmonary BCL6 mRNA expression. rAAV9-BCL6 administration significantly inhibited hyperoxia-induced NLRP3 upregulation and inflammation, attenuated alveolar simplification and dysregulated angiogenesis in BPD mice, which were characterized by decreased mean linear intercept, increased radical alveolar count and alveoli numbers, and the upregulated CD31 expression. Meanwhile, BCL6 overexpression promoted proliferation and angiogenesis, inhibited apoptosis and inflammation in hyperoxia-stimulated HPMECs. Moreover, administration of BCL6 inhibitor FX1 arrested growth and development. FX1-treated BPD mice exhibited exacerbation of alveolar pathological changes and pulmonary vessel permeability, with upregulated mRNA levels of pro-inflammatory cytokines and pro-fibrogenic factors. Furthermore, both rAAV9-BCL6 and FX1 administration exerted a long-lasting effect on hyperoxia-induced lung injury (≥4 wk). Conclusions BCL6 inhibits NLRP3-mediated inflammation, attenuates alveolar simplification and dysregulated pulmonary vessel development in hyperoxia-induced BPD mice. Hence, BCL6 may be a target in treating BPD and neonatal diseases.

Gpnmb silencing protects against hyperoxia-induced acute lung injury by inhibition of mitochondrial-mediated apoptosis.

Background: Hyperoxia-induced acute lung injury (HALI) is a complication to ventilation in patients with respiratory failure, which can lead to acute inflammatory lung injury and chronic lung disease. The aim of this study was to integrate bioinformatics analysis to identify key genes associated with HALI and validate their role in H2O2-induced cell injury model.Methods: Integrated bioinformatics analysis was performed to screen vital genes involved in hyperoxia-induced lung injury (HLI). CCK-8 and flow cytometry assays were performed to assess cell viability and apoptosis. Western blotting was performed to assess protein expression.Results: In this study, glycoprotein non-metastatic melanoma protein B (Gpnmb) was identified as a key gene in HLI by integrated bioinformatics analysis of 4 Gene Expression Omnibus (GEO) datasets (GSE97804, GSE51039, GSE76301 and GSE87350). Knockdown of Gpnmb increased cell viability and decreased apoptosis in H2O2-treated MLE-12 cells, suggesting that Gpnmb was a proapoptotic gene during HALI. Western blotting results showed that knockdown of Gpnmb reduced the expression of Bcl-2 associated X (BAX) and cleaved-caspase 3, and increased the expression of Bcl-2 in H2O2 treated MLE-12 cells. Furthermore, Gpnmb knockdown could significantly reduce reactive oxygen species (ROS) generation and improve the mitochondrial membrane potential.Conclusion: The present study showed that knockdown of Gpnmb may protect against HLI by repressing mitochondrial-mediated apoptosis.

Deciphering regulatory patterns in a mouse model of hyperoxia-induced acute lung injury.

Oxygen therapy plays a pivotal role in treating critically ill patients in the intensive care unit (ICU). However, excessive oxygen concentrations can precipitate hyperoxia, leading to damage in multiple organs, with a notable effect on the lungs. Hyperoxia condition may lead to hyperoxia-induced acute lung injury (HALI), deemed as a milder form of acute respiratory distress syndrome (ARDS). Given its clinical importance and practical implications, there is a compelling need to investigate the underlying pathogenesis and comprehensively understand the regulatory mechanisms implicated in the development of HALI. In this study, we conducted a mouse model with HALI and performed regulatory mechanism analysis using RNA-seq on both HALI and control group. Comprehensive analysis revealed 727 genes of significant differential expression, including 248 long non-coding RNAs (lncRNAs). Also, alternative splicing events were identified from sequencing results. Notably, we observed up-regulation or abnormal alternative splicing of genes associated with immune response and ferroptosis under hyperoxia conditions. Utilizing weighted gene co-expression network analysis (WGCNA), we ascertained that genes involved in immune response formed a distinct cluster, showcasing an up-regulated pattern in hyperoxia, consistent with previous studies. Furthermore, a competing endogenous RNA (ceRNA) network was constructed, including 78 differentially expressed mRNAs and six differentially expressed lncRNAs, including H19. These findings uncover the intricate interplay of multiple transcriptional regulatory mechanisms specifically tailored to the pulmonary defense against HALI, substantiating the importance of these non-coding RNAs in this disease context. Our results provide new insights into the potential mechanisms and underlying pathogenesis in the development of HALI at the post-transcriptional level. The findings of this study reveal potential regulatory interactions and biological roles of specific lncRNAs and genes, such as H19 and Sox9, encompassing driven gene expression patterns, alternative splicing events, and lncRNA-miRNA-mRNA ceRNA networks. These findings may pave the way for advancing therapeutic strategies and reducing the risk associated with oxygen treatment for patients.

Investigation on the signaling pathways in the mechanism of hyperoxia-induced acute lung injury based on transcriptomics sequencing

To observe and verify the changes of transcriptome in hyperoxia-induced acute lung injury (HALI), and to further clarify the changes of pathways in HALI. Twelve healthy male C57BL/6J mice were randomly divided into normoxia group and HALI group according to the random number table, with 6 mice in each group. The mice in the normoxia group were fed normally in the room, and the mice in the HALI group was exposed to 95% oxygen to reproduce the HALI animal model. After 72 hours of hyperoxia exposure, the lung tissues were taken for transcriptome sequencing, and then Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathway enrichment analysis was performed. The pathological changes of lung tissue were observed under light microscope after hematoxylin-eosin (HE) staining. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to verify the key molecules in the signal pathways closely related to HALI identified by transcriptomics analysis. Transcriptomic analysis showed that hyperoxia induced 537 differentially expressed genes in lung tissue of mice as compared with the normoxia group including 239 up-regulated genes and 298 down-regulated genes. Further KEGG pathway enrichment analysis identified 20 most significantly enriched pathway entries, and the top three pathways were ferroptosis signaling pathway, p53 signaling pathway and glutathione (GSH) metabolism signaling pathway. The related genes in the ferroptosis signaling pathway included the up-regulated gene heme oxygenase-1 (HO-1) and the down-regulated gene solute carrier family 7 member 11 (SLC7A11). The related genes in the p53 signaling pathway included the up-regulated gene tumor suppressor gene p53 and the down-regulated gene murine double minute 2 (MDM2). The related gene in the GSH metabolic signaling pathway was up-regulated gene glutaredoxin 1 (Grx1). The light microscope showed that the pulmonary alveolar structure of the normoxia group was normal. In the HALI group, the pulmonary alveolar septum widened and thickened, and the alveolar cavity shrank or disappeared. RT-RCR and Western blotting confirmed that compared with the normoxia group, the mRNA and protein expressions of HO-1 and p53 in lung tissue of the HALI group were significantly increased [HO-1 mRNA (2-ΔΔCt): 2.16±0.17 vs. 1.00±0.00, HO-1 protein (HO-1/β-actin): 1.05±0.01 vs. 0.79±0.01, p53 mRNA (2-ΔΔCt): 2.52±0.13 vs. 1.00±0.00, p53 protein (p53/β-actin): 1.12±0.02 vs. 0.58±0.03, all P < 0.05], and the mRNA and protein expressions of Grx1, MDM2, SLC7A11 were significantly decreased [Grx1 mRNA (2-ΔΔCt): 0.53±0.05 vs. 1.00±0.00, Grx1 protein (Grx1/β-actin): 0.54±0.03 vs. 0.93±0.01, MDM2 mRNA (2-ΔΔCt): 0.48±0.03 vs. 1.00±0.00, MDM2 protein (MDM2/β-actin): 0.57±0.02 vs. 1.05±0.01, SLC7A11 mRNA (2-ΔΔCt): 0.50±0.06 vs. 1.00±0.00, SLC7A11 protein (SLC7A11/β-actin): 0.72±0.03 vs. 0.98±0.01, all P < 0.05]. HALI is closely related to ferroptosis, p53 and GSH metabolism signaling pathways. Targeting the key targets in ferroptosis, p53 and GSH metabolism signaling pathways may be an important strategy for the prevention and treatment of HALI.

Hyperoxia-Induced Secondary Respiratory Failure in a Systemic Ischaemia-Reperfusion Injury.

Recent studies revealed that excessive supplemental oxygen, such as inhaled 100% O2, damages various organ functions in post-cardiac arrest (CA) patients. Optimal indicators of supplemental oxygen are therefore important to prevent hyperoxic organ injuries. In this study, we evaluated a hyperoxic pulmonary injury and assessed the association between alveolar-arterial oxygen difference (AaDO2) and a degree of lung oedema. In this study, we focused on the hyperoxia-induced lung injury and its association with changes of gas-exchange parameters in post-CA rats. Rats were resuscitated from 10 min of asphyxial CA and stratified into two groups: those with inhaled 100% O2 (CA-FiO2 1.0) and those with 30% O2 (CA-FiO2 0.3). We prepared a sham surgery group for comparison (sham-FiO2 0.3). After 2h, animals were sacrificed, and the lung wet-to-dry (W/D) weight ratio was measured. We collected blood gas results and measured the ratio of partial pressure arterial oxygen and fraction of inspired oxygen (p/f ratio), and calculated AaDO2. The lung W/D ratio in the CA-FiO2 1.0 group (5.8±0.26) was higher than in the CA-FiO2 0.3 (4.6±0.42) and sham-FiO2 0.3 groups (4.6±0.38, p<0.01). There was a significant difference in AaDO2 between CA-FiO2 1.0 (215±49.3) and, CA-FiO2 0.3 (36.8±32.3), and sham-FiO2 0.3 groups (49.0±20.5, p<0.01). There were also significant changes in pH and blood lactate levels in the early phase among the three groups. AaDO2 showed the strongest correlation with W/D ratio (r=0.9415, p<0.0001), followed by pH (r=-0.5131, p=0.0294) and p/f ratio (r=-0.3861, p=0.1135). Hyperoxic injury might cause the pulmonary oedema after CA. Measuring respiratory quotient (RQ) in rodents enabled an accurate calculation for AaDO2 at a variety level of inhaled O2. Given that AaDO2 measurement is non-invasive, we therefore consider AaDO2 to be a potentially optimal indicator of post-CA hyperoxic pulmonary injury.

Wedelolactone alleviates hyperoxia-induced acute lung injury by regulating ferroptosis

To study whether wedelolactone can reduce hyperoxia-induced acute lung injury (HALI) by regulating ferroptosis, and provide a basic theoretical basis for the drug treatment of HALI. A total of 24 C57BL/6J mice were randomly divided into normal oxygen control group, HALI model group and wedelolactone pretreatment group, with 8 mice in each group. Mice in wedelolactone pretreatment group were treated with wedelolactone 50 mg/kg intraperitoneally for 6 hours, while the other two groups were not given with wedelolactone. After that, the HALI model was established by maintaining the content of carbon dioxide < 0.5% and oxygen > 90% in the molding chamber for 48 hours, and the normal oxygen control group was placed in indoor air. After modeling, the mice were sacrificed and lung tissues were collected. The lung histopathological changes were observed under light microscope and pathological scores were performed to calculate the ratio of lung wet/dry mass (W/D). The levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1β), superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) in lung tissues of mice in each group were determined. The protein expression of glutathione peroxidase 4 (GPX4) in lung tissue was detected by Western blotting. Under light microscope, the alveolar structure of HALI model group was destroyed, and a large number of neutrophils infiltrated the alveolar and interstitial lung, and the interstitial lung was thickened. The pathological score of lung injury (score: 0.75±0.02 vs. 0.11±0.01) and the ratio of lung W/D (6.23±0.34 vs. 3.68±0.23) were significantly higher than those in the normal oxygen control group (both P < 0.05). Wedelolactone pretreated mice had clear alveolar cavity and lower neutrophil infiltration and interstitial thickness than HALI group. Pathological scores (score: 0.43±0.02 vs. 0.75±0.02) and W/D ratio (4.56±0.12 vs. 6.23±0.34) were significantly lower than HALI group (both P < 0.05). Compared with the normal oxygen control group, the levels of SOD (kU/g: 26.41±4.25 vs. 78.64±3.95) and GSH (mol/g: 4.51±0.33 vs. 12.53±1.25) in HALI group were significantly decreased, while the levels of MDA (mmol/g: 54.23±4.58 vs. 9.65±1.96), TNF-α (μg/L: 96.32±3.67 vs. 11.65±2.03), IL-6 (ng/L: 163.35±5.89 vs. 20.56±3.63) and IL-1β (μg/L: 72.34±4.64 vs. 15.64±2.47) were significantly increased, and the protein expression of GPX4 (GPX4/β-actin: 0.44±0.02 vs. 1.00±0.09) was significantly decreased (all P < 0.05). Compared with the HALI group, the levels of SOD (kU/g: 53.28±3.69 vs. 26.41±4.25) and GSH (mol/g: 6.73±0.97 vs. 12.53±1.25) were significantly higher in the wedelolactone pretreatment group, and the levels of MDA (mmol/g: 25.36±1.98 vs. 54.23±4.58), TNF-α (μg/L: 40.25±4.13 vs. 96.32±3.67), IL-6 (ng/L: 78.32±4.65 vs. 163.35±5.89), and IL-1β (μg/L: 30.65±3.65 vs. 72.34±4.64) were significantly lower (all P < 0.05), and protein expression of GPX4 was significantly higher (GPX4/β-actin: 0.68±0.04 vs. 0.44±0.02, P < 0.05). Wedelolactone attenuates HALI injury by regulating ferroptosis.

ROS-mediated MAPK activation aggravates hyperoxia-induced acute lung injury by promoting apoptosis of type II alveolar epithelial cells via the STAT3/miR-21–5p axis

Inhibition of type II alveolar epithelial (AE-II) cell apoptosis is a critical way to cure hyperoxia-induced acute lung injury (HALI). It has been reported that miR-21–5p could reduce H2O2-induced apoptosis in AE-II cells. However, the upstream molecular mechanism remains unclear. Herein, we established a cellular model of HALI by exposing AE-II cells to H2O2 treatment. It was shown that miR-21–5p alleviated H2O2-induced apoptosis in AE-II cells. ROS inhibition decreased apoptosis of H2O2-evoked AE-II cells via increasing miR-21–5p expression. In addition, ROS induced MAPK and STAT3 phosphorylation in H2O2-treated AE-II cells. MAPK inactivation reduces H2O2-triggered AE-II cell apoptosis. MAPK activation inhibits miR-21–5p expression by promoting STAT3 phosphorylation in H2O2-challenged AE-II cells. Furthermore, STAT3 activation eliminated MAPK deactivation-mediated inhibition on the apoptosis of AE-II cells under H2O2 condition. In conclusion, ROS-mediated MAPK activation promoted H2O2-triggered AE-II cell apoptosis by inhibiting miR-21–5p expression via STAT3 phosphorylation, providing novel targets for HALI treatment.

Irisin alleviates hyperoxia-induced bronchopulmonary dysplasia through activation of Nrf2/HO-1 pathway

BackgroundBronchopulmonary dysplasia (BPD) is a common pulmonary injury among premature infants, which is often caused by hyperoxia exposure. Irisin is a novel hormone-like myokine derived mainly from skeletal muscles as well as adipose tissues. Many studies have indicated that Irisin exert a variety of properties against hyperoxia-induced inflammation and oxidative stress (OS). We aimed to evaluate the effects of irisin on hyperoxia-induced lung injury explore the underlying mechanisms. MethodsBPD model was established after exposing newborn mouse to 85% oxygen. BPD mouse received continuous intraperitoneal injection of irisin at a dose of 25 μg/kg/day. Lung tissues were collected for histological examination at 7 and 14 days after birth. The alveolarization and alveolar vascularization of each animal was assessed. Levels of oxidative stress indicators, and the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in lung tissues were detected at 14 days after birth. ResultsHyperoxia exposure induced a markedly alveolar simplification and a disrupted alveolar angiogenesis, which was ameliorated by irisin treatment. The hyperoxia-induced increase in these oxidative stress indicators was significantly reversed by irisin treatment. The Nrf2/HO-1 pathway is inducted in the hyperoxia-induced BPD mouse model, which is further activated by irisin treatment. ConclusionOur results demonstrated the beneficial effects of irisin in reducing the OS, enhancing alveolarization, and promoting vascular development through activation of Nrf2/HO-1 axis in a hyperoxia-induced experimental model of BPD.

Recombinant CXCL17 Treatment Alleviates Hyperoxia-Induced Lung Apoptosis and Inflammation In Vivo and Vitro by Activating the AKT Pathway: A Possible Therapeutic Approach for Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD), caused by hyperoxia exposure, is the most common complication affecting preterm infants. The C-X-C motif chemokine ligand 17 (CXCL17) belongs to the chemokine family that plays important roles in various processes, but the function in BPD is unknown. Elevated serum CXCL17 levels were observed in human premature infants with hyperoxia-induced lung injury, suggesting that CXCL17 might be involved in BPD. To further validate our speculation, studies were conducted in a hyperoxia-induced lung injury mouse model and primary murine alveolar epithelial cells Type II (T2AEC) cells exposed to hyperoxia. RT-qPCR and western blot were used to validate CXCL17 expression in newborn mice. Hyperoxia exposure-induced lung injury was determined by assessing the lung wet-weight/dry-weight ratio and histological changes. Oxidative stress and inflammatory factors were examined by ELISA assay and RT-qPCR. Reactive oxygen species (ROS) level was evaluated by DHE staining. Apoptosis was assessed by TUNEL staining and western blot. The results showed that hyperoxia exposure increased CXCL17 levels in newborn mice pups. Hyperoxia exposure increased lung wet-weight/dry-weight ratio, increased alveolar diameter and enlarged alveoli, and reduced surfactant protein C expression. However, recombinant CXCL17 (rCXCL17) treatment alleviated hyperoxia-induced lung injury. rCXCL17 treatment inhibited hyperoxia-induced inflammation, oxidative stress, and apoptosis in neonatal mice. These results were further verified in T2AEC cells. Additionally, rCXCL17 treatment activated the AKT pathway, which is a protective pathway in BPD. Collectively, rCXCL17 alleviates hyperoxia-induced lung injury in neonatal mice by activating the AKT pathway, indicating that CXCL17 may be a promising target for BPD therapy.

Sigma-1 receptor regulates the endoplasmic reticulum stress pathway in the protective mechanism of dexmedetomidine against hyperoxia-induced lung injury

Perioperative hyperoxia therapy is of great significance to save the lives of patients, but little is known about the possible mechanisms that induce hyperoxia-induced acute lung injury (HALI) and the measures for clinical prevention and treatment. In this experiment, the models were established with a feeding chamber with automatic regulation of oxygen concentration. The results showed that with the increase in inhaled oxygen concentration and the prolongation of exposure time, the severity of lung injury also increases significantly, reaching the diagnostic indication of HALI after 48 h of inhaling 95 % oxygen concentration. Subsequently, according to the dynamic changes of apoptosis in lung specimens, and the expression changes in Sig-1R-regulated ER stress pathway proteins (Sig-1R, GRP78, p-PERK, ATF6, IRE1, Caspase-12, ATF4, CHOP, Caspase-3 and p-JNK), it was confirmed that the Sig-1R-regulated ER stress signaling pathway was involved in the occurrence of HALI. To explore the preventive and therapeutic effects of routine clinical medication on HALI during the perioperative period, our research group selected dexmedetomidine (Dex) with lung protection. The experimental results revealed that Dex partially reversed the changes in the expression levels of Sig-1R-regulated ER stress pathway proteins. These results preliminarily confirmed that Dex may inhibit apoptosis induced by high oxygen concentration through the Sig-1R-regulated ER stress signaling pathway, thus playing a protective role in HALI.

Effects of celecoxib on AQP-1, NF-κB and apoptosis in lung tissue of neonatal rats with hyperoxia-induced lung injury.

To analyze the effects of AQP-1, NF-κB and apoptosis in lung tissue of neonatal rats with hyperoxia-induced lung injury (HILI).162 neonatal SD rats were divided into control group (COG), model group (MOG) and celecoxib group (CEG), 54 rats in each group. Each group was subdivided into 1st, 5th and 10th-day groups with 18 rats in each group. The neonatal rat model of HILI was established. Nine rats in each group were randomly selected on the 3rd, 6th and 12th day respectively. The level of oxidative stress (OS) in bronchoalveolar lavage fluid (BALF), the pathological changes of lung tissue, the ratio of lung wet weight to dry weight (W/D), the expression of inflammatory factors in lung tissue, the l AQP-1 and NF-κB level and apoptosis in lung tissue were tested. In comparison with COG, the level of MDA in BALF, the ratio of lung W/D, the expression level of IL-6, TNF- α, NF- κB and the number of apoptotic cells in lung tissue were significantly increased, while the level of SOD in BALF and AQP-1 in lung tissue notably decreased in the MOG (P<0.05). The level of malondialdehyde (MDA) in BALF, the ratio of lung W/D, IL-6, TNF-α, NF-κB and plenty of apoptotic cells in lung tissue in CEG were notably decreased than MOG(P<0.05), while the level of SOD in BALF and AQP-1 in lung tissue were raised in CEG. In the COG, the lung tissue structure was complete, the arrangement was neat, the alveolar cavity was clear, and there was no inflammatory infiltration. With the extension of time, the inflammatory infiltration of lung tissue in the MOG gradually increased, the plenty of red blood cells gradually increased, and the size of the alveolar cavity varied; compared with the MOG, the inflammatory infiltration in the CEG decreased and the plenty of red blood cells decreased. Celecoxib can improve oxidative injury, inhibit inflammation, up-regulate AQP-1 and down-regulate NF-κB, inhibit apoptosis, and have a certain protective effect on HILI in neonatal rats.

Regulating NLRP3 Inflammasome–Induced Pyroptosis via Nrf2: TBHQ Limits Hyperoxia-Induced Lung Injury in a Mouse Model of Bronchopulmonary Dysplasia

Nuclear factor e2–related factor 2 (Nrf2) plays a key role in cellular resistance to oxidative stress injury. Oxidative stress injury, caused by Nrf2 imbalance, results in increased pyroptosis, DNA damage, and inflammatory activation, which may lead to the arrest of alveolar development and bronchopulmonary dysplasia (BPD) in premature infants under hyperoxic conditions. We established a BPD mouse model to investigate the effects of tert-butylhydroquinone (TBHQ), an Nrf2 activator, on oxidative stress injury, pyroptosis, NLRP3 inflammasome activation, and alveolar development. TBHQ reduced abnormal cell death in the lung tissue of BPD mice and restored the number and normal structure of the alveoli. TBHQ administration activated the Nrf2/heme oxygenase-1 (HO-1) signaling pathway, resulting in the decrease in the following: reactive oxygen species (ROS), activation of the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome, and IL-18 and IL-1β expression and activation, as well as inhibition of pyroptosis. In contrast, after Nrf2 gene knockout in BPD mice, there was more severe oxidative stress injury and cell death in the lungs, there were TUNEL + and NLRP3 + co-positive cells in the alveoli, the pyroptosis was significantly increased, and the development of alveoli was significantly blocked. We demonstrated that TBHQ may promote alveolar development by enhancing Nrf2-induced antioxidation in the lung tissue of BPD mice and that the decrease in the NLRP3 inflammasome and pyroptosis caused by Nrf2 activation may be the underlying mechanism. These results suggest that TBHQ is a promising treatment for lung injury in premature infants with hyperoxia.

Intranasal administration of Lactobacillus johnsonii attenuates hyperoxia-induced lung injury by modulating gut microbiota in neonatal mice

BackgroundSupplemental oxygen impairs lung development in newborn infants with respiratory distress. Lactobacillus johnsonii supplementation attenuates respiratory viral infection in mice and exhibits anti-inflammatory effects. This study investigated the protective effects of intranasal administration of L. johnsonii on lung development in hyperoxia-exposed neonatal mice.MethodsNeonatal C57BL/6N mice were reared in either room air (RA) or hyperoxia condition (85% O2). From postnatal days 0 to 6, they were administered intranasal 10 μL L. johnsonii at a dose of 1 × 105 colony-forming units. Control mice received an equal volume of normal saline (NS). We evaluated the following four study groups: RA + NS, RA + probiotic, O2 + NS, and O2 + probiotic. On postnatal day 7, lung and intestinal microbiota were sampled from the left lung and lower gastrointestinal tract, respectively. The right lung of each mouse was harvested for Western blot, cytokine, and histology analyses.ResultsThe O2 + NS group exhibited significantly lower body weight and vascular density and significantly higher mean linear intercept (MLI) and lung cytokine levels compared with the RA + NS and RA + probiotic groups. At the genus level of the gut microbiota, the O2 + NS group exhibited significantly higher Staphylococcus and Enterobacter abundance and significantly lower Lactobacillus abundance compared with the RA + NS and RA + probiotic groups. Intranasal L. johnsonii treatment increased the vascular density, decreased the MLI and cytokine levels, and restored the gut microbiota in hyperoxia-exposed neonatal mice.ConclusionsIntranasal administration of L. johnsonii protects against hyperoxia-induced lung injury and modulates the gut microbiota.

Cryptotanshinone affects HFL-1 cells proliferation by inhibiting cytokines secretion in RAW264.7 cells and ameliorates inflammation and fibrosis in newborn rats with hyperoxia induced lung injury.

Objective: Bronchopulmonary dysplasia (BPD) is a common complication of prematurity and has no specific treatment option. Moreover, inflammation and fibrosis play a vital role in the development of BPD. Thus, this study aimed to explore the role of the anti-inflammatory and anti-fibrotic drug cryptotanshinone (CTS) in the treatment of inflammation and fibrosis in BPD. Methods: In vivo, Sprague-Dawley rats (male) were divided into air, hyperoxia and CTS groups with different dose interventions (7.5, 15, and 30mg/kg). A BPD rat model was induced by continuous inhalation of hyperoxia (95%) for 7days, during which different doses of CTS were injected intraperitoneally. Furthermore, histological examination, hydroxyproline content measurement, Western blot and real-time quantitative polymerase chain reaction were used to detect the levels of inflammation and fibrosis in the tissues. RAW264.7 cells exposed to 95% oxygen were collected and co-cultured with fibroblasts to determine the expression levels of α-SMA, collagen-Ⅰ and MMPs. The levels of pro-inflammatory cytokines such as TNF-α, IL-6 and pro-fibrotic factor TGF-β1 in the supernatants were measured using enzyme-linked immunosorbent assay. Results: Haematoxylin and eosin staining revealed that CTS reduced the inflammatory response in rat lungs. Masson staining revealed that CTS alleviated the level of pulmonary fibrosis. CTS also reduced the levels of TNF-α, IL-6 and TGF-β1 along with the expression of the fibrosis marker α-SMA in lung tissue. Similarly, in vitro analysis revealed that CTS decreased the levels of TNF-α, IL-6 and TGF-β1 expressed in RAW 264.7 cells, and reduced α-SMA, collagen-Ⅰ, MMPs concentrations in HFL-1 cells co-cultured with the supernatant of RAW264.7 cells after hyperoxia. Conclusion: CTS can attenuate the hyperoxia-induced inflammatory response and the level of fibrosis by regulating the levels of inflammatory factors and fibrotic factor TGF-β1 expressed by macrophages, thereby highlighting the therapeutic potential of CTS in the treatment of BPD.

Targeting CXCR1 alleviates hyperoxia-induced lung injury through promoting glutamine metabolism

Although increasing evidence suggests potential iatrogenic injury from supplemental oxygen therapy, significant exposure to hyperoxia in critically ill patients is inevitable. This study shows that hyperoxia causes lung injury in a time- and dose-dependent manner. In addition, prolonged inspiration of oxygen at concentrations higher than 80% is found to cause redox imbalance and impair alveolar microvascular structure. Knockout of C-X-C motif chemokine receptor 1 (Cxcr1) inhibits the release of reactive oxygen species (ROS) from neutrophils and synergistically enhances the ability of endothelial cells to eliminate ROS. We also combine transcriptome, proteome, and metabolome analysis and find that CXCR1 knockdown promotes glutamine metabolism and leads to reduced glutathione by upregulating the expression of malic enzyme 1. This preclinical evidence suggests that a conservative oxygen strategy should be recommended and indicates that targeting CXCR1 has the potential to restore redox homeostasis by reducing oxygen toxicity when inspiratory hyperoxia treatment is necessary.

Loss of cytochrome P450 (CYP)1B1 mitigates hyperoxia response in adult mouse lung by reprogramming metabolism and translation

Oxygen supplementation is life saving for premature infants and for COVID-19 patients but can induce long-term pulmonary injury by triggering inflammation, with xenobiotic-metabolizing CYP enzymes playing a critical role. Murine studies showed that CYP1B1 enhances, while CYP1A1 and CYP1A2 protect from, hyperoxic lung injury. In this study we tested the hypothesis that Cyp1b1-null mice would revert hyperoxia-induced transcriptomic changes observed in WT mice at the transcript and pathway level. Wild type (WT) C57BL/6J and Cyp1b1-null mice aged 8–10 weeks were maintained in room air (21% O2) or exposed to hyperoxia (>95% O2) for 48h. Transcriptomic profiling was conducted using the Illumina microarray platform. Hyperoxia exposure led to robust changes in gene expression and in the same direction in WT, Cyp1a1-, Cyp1a2-, and Cyp1b1-null mice, but to different extents for each mouse genotype. At the transcriptome level, all Cyp1-null murine models reversed hyperoxia effects. Gene Set Enrichment Analysis identified 118 hyperoxia-affected pathways mitigated only in Cyp1b1-null mice, including lipid, glutamate, and amino acid metabolism. Cell cycle genes Cdkn1a and Ccnd1 were induced by hyperoxia in both WT and Cyp1b1-null mice but mitigated in Cyp1b1-null O2 compared to WT O2 mice. Hyperoxia gene signatures associated positively with bronchopulmonary dysplasia (BPD), which occurs in premature infants (with supplemental oxygen being one of the risk factors), but only in the Cyp1b1-null mice did the gene profile after hyperoxia exposure show a partial rescue of BPD-associated transcriptome. Our study suggests that CYP1B1 plays a pro-oxidant role in hyperoxia-induced lung injury.

Endothelial progenitor cells derived from embryonic stem cells prevent alveolar simplification in a murine model of bronchopulmonary dysplasia.

Introduction: Vascular remodeling and compromised alveolar development are hallmarks of chronic pulmonary diseases such as bronchopulmonary dysplasia (BPD). Despite advances in neonatal healthcare the number of BPD cases worldwide continues to increase. One approach to overcoming the premature arrest in lung development seen in BPD is to stimulate neonatal angiogenesis via delivery and engraftment of endothelial progenitor cells (EPCs). One such population is resident to the pulmonary microvasculature and expresses both FOXF1 and c-KIT. Previous studies have shown that c-KIT+FOXF1+ EPCs are highly sensitive to elevated levels of oxygen (hyperoxia) and are decreased in premature infants with BPD and hyperoxia-induced BPD mouse models. We hypothesize that restoring EPCs through transplantation of c-KIT+FOXF1+ EPCs derived in vitro from pluripotent embryonic stem cells (ESCs), will stimulate neonatal angiogenesis and alveolarization in mice with hyperoxia-induced lung injury. Methods: Utilizing a novel ESC line with a FOXF1:GFP reporter, we generated ESC-derived c-KIT+FOXF1+ EPCs in vitro. Using a second ESC line which contains FOXF1:GFP and tdTomato transgenes, we differentiated ESCs towards c-KIT+FOXF1+ EPCs and tracked them in vivo after injection into the neonatal circulation of hyperoxia-injured mice. After a recovery period in room air conditions, we analyzed c-KIT+FOXF1+ EPC engraftment and quantified the number of resident and circulating endothelial cells, the size of alveolar spaces, and the capillary density after EPC transplantations. Results and conclusion: Herein, we demonstrate that addition of BMP9 to the directed endothelial differentiation protocol results in very efficient generation of c-KIT+FOXF1+ EPCs from pluripotent ESCs. ESC-derived c-KIT+FOXF1+ EPCs effectively engraft into the pulmonary microvasculature of hyperoxia-injured mice, promote vascular remodeling in alveoli, increase the number of resident and circulating endothelial cells, and improve alveolarization. Altogether, these results provide a proof-of-principle that cell therapy with ESC-derived c-KIT+FOXF1+ EPCs can prevent alveolar simplification in a hyperoxia-induced BPD mouse model.

Erythromycin Attenuates Hyperoxia Induced Lung Injury by Enhancing GSH Expression and Inhibiting Expression of Inflammatory Cytokines

Introduction: Oxidative stress and inflammation have proven to be key factors contributing to the occurrence of BPD. Erythromycin has been shown to be effective in treating the redox imbalance seen in many non-bacterial infectious chronic inflammatory diseases. Methods: Ninety-six premature rats were randomly divided into air + saline chloride group, air + erythromycin group, hyperoxia + saline chloride group and hyperoxia + erythromycin group. Lung tissue specimens were collected from 8 premature rats in each group on days 1, 7 and 14, respectively. Results: Pulmonary pathological changes in premature rats after hyperoxia exposure were similar to those of BPD. Hyperoxia exposure induced high expression of GSH, TNF-α, and IL-1β. Erythromycin intervention caused a further increase in GSH expression and a decrease in TNF-α and IL-1β expression. Conclusion: GSH, TNF-α and IL-1β are all involved in the development of BPD. Erythromycin may alleviate BPD by enhancing the expression of GSH and inhibiting the release of inflammatory mediators.

Nesfatin-1 alleviates hyperoxia-induced lung injury in newborn mice by inhibiting oxidative stress through regulating SIRT1/PGC-1α pathway

Bronchopulmonary dysplasia (BPD) is a pulmonary disease commonly observed in premature infants and it is reported that oxidative stress is a critical induction factor in BPD and is considered as a promising target for treating BPD. Nesfatin-1 is a brain-gut peptide with inhibitory effects on food intake, which is recently evidenced to show suppressive effect on oxidative stress. The present study aims to explore the therapeutic effect and mechanism of Nesfatin-1 in BPD mice. AECIIs were extracted from newborn rats and exposed to hyperoxia for 24 h, followed by treatment with 5 and 10 nM Nesfatin-1. Declined cell viability, increased apoptotic rate, upregulated Bax, downregulated Bcl-2, increased release of ROS and MDA, and suppressed SOD activity were observed in hyperoxia-treated AECIIs, which were extremely reversed by Nesfatin-1. Newborn rats were exposed to hyperoxia, followed by treated with 10 μg/kg Nesfatin-1 and 20 μg/kg Nesfatin-1. Severe pathological changes, elevated MDA level, and declined SOD activity were observed in lung tissues of BPD mice, which were rescued by Nesfatin-1. Furthermore, the protective effect of Nesfatin-1 on hyperoxia-challenged AECIIs was abolished by silencing SIRT1. Collectively, Nesfatin-1 alleviated hyperoxia-induced lung injury in newborn mice by inhibiting oxidative stress through regulating SIRT1/PGC-1α pathway.

LncRNA gadd7 promotes mitochondrial membrane potential decrease and apoptosis of alveolar type II epithelial cells by positively regulating MFN1 in an in vitro model of hyperoxia-induced acute lung injury.

The mortality and morbidity rates of ovarian cancer (OC) are high, but the underlying mechanisms of OC have not been characterized. In this study, we determined the role of Rho GTPase Activating Protein 30 (ARHGAP30) in OC progression. We measured ARHGAP30 abundance in OC tissue samples and cells using immunohistochemistry (IHC) and RT-qPCR. EdU, transwell, and annexin V/PI apoptosis assays were used to evaluate proliferation, invasiveness, and apoptosis of OC cells, respectively. The results showed that ARHGAP30 was overexpressed in OC tissue samples and cells. Inhibition of ARHGAP30 suppressed growth and metastasis of OC cells, and enhanced apoptosis. Knockdown of ARHGAP30 in OC cells significantly inhibited the PI3K/AKT/mTOR pathway. Treatment with the PI3K/AKT/mTOR pathway inhibitor buparlisib simulated the effects of ARHGAP30 knockdown on growth, invasiveness, and apoptosis of OC cells. Following buparlisib treatment, the expression levels of p-PI3K, p-AKT, and p-mTOR were significantly decreased. Furthermore, buparlisib inhibited the effects of ARHGAP30 upregulation on OC cell growth and invasiveness. In conclusion, ARHGAP30 regulated the PI3K/AKT/mTOR pathway to promote progression of OC.