Effectiveness of extracellular vesicles derived from hiPSCs in repairing hyperoxia-induced injury in a fetal murine lung explant model.

BACKGROUND: Despite advances in neonatal care, the incidence of Bronchopulmonary Dysplasia (BPD) remains high among preterm infants. Human induced pluripotent stem cells (hiPSCs) have shown promise in repairing injury in animal BPD models. Evidence suggests they exert their effects via paracrine mechanisms. We aim herein to assess the effectiveness of extracellular vesicles (EVs) derived from hiPSCs and their alveolar progenies (diPSCs) in attenuating hyperoxic injury in a preterm lung explant model. METHODS: Murine lung lobes were harvested on embryonic day 17.5 and maintained in air-liquid interface. Following exposure to 95% O2 for 24 h, media was supplemented with 5 × 106 particles/mL of EVs isolated from hiPSCs or diPSCs by size-exclusion chromatography. On day 3, explants were assessed using Hematoxylin-Eosin staining with mean linear intercept (MLI) measurements, immunohistochemistry, VEGFa and antioxidant gene expression. Statistical analysis was conducted using one-way ANOVA and Multiple Comparison Test. EV proteomic profiling was performed, and annotations focused on alveolarization and angiogenesis signaling pathways, as well as anti-inflammatory, anti-oxidant, and regenerative pathways. RESULTS: Exposure of fetal lung explants to hyperoxia induced airspace enlargement, increased MLI, upregulation of anti-oxidants Prdx5 and Nfe2l2 with decreased VEGFa expression. Treatment with hiPSC-EVs improved parenchymal histologic changes. No overt changes in vasculature structure were observed on immunohistochemistry in our in vitro model. However, VEGFa and anti-oxidant genes were upregulated with diPSC-EVs, suggesting a pro-angiogenic and cytoprotective potential. EV proteomic analysis provided new insights in regard to potential pathways influencing lung regeneration. CONCLUSION: This proof-of-concept in vitro study reveals a potential role for hiPSC- and diPSC-EVs in attenuating lung changes associated with prematurity and oxygen exposure. Our findings pave the way for a novel cell free approach to prevent and/or treat BPD, and ultimately reduce the global burden of the disease.

[Impact of hyperoxia on the phenotype of pulmonary artery smooth muscle cells].

Objective: To investigate the influence of varied oxygen (O2) concentration environments on the phenotypic transformation of pulmonary artery smooth muscle cells (PASMC) and the mechanism of pulmonary hypertension. Methods: Primary rat PASMC were isolated and cultured through the process of enzymatic digestion. Following identification, the stable passaged PASMC were subjected to a 6-hour incubation in sealed containers with normal O2 content (group C) and relative O2 content comprising 55% (group H55), 75% (group H75), and 95% (group H95). mRNA and protein expression of α-Actin (α-SMA), smooth muscle 22α (SM22α), osteopontin (OPN), and matrix metalloproteinase-2 (MMP-2) were measured using real-time quantitative PCR and western blot analysis. Results: The H55 group displayed no significant difference from the C group in terms of mRNA and relative protein expression levels for α-SMA, SM22α, OPN, and MMP-2 (all P>0.05). On the other hand, groups H75 and H95 exhibited a reduction in mRNA and relative protein expression of α-SMA and SM22α, along with an increase in mRNA and relative protein expression of OPN and MMP-2 when compared with both the C and H55 groups (all P<0.05). The H95 group showed a higher relative mRNA expression of MMP-2 as compared to the H75 group (P<0.05). Conclusions: Oxygen concentration environments of 75% or higher can serve as the foundation for the pathogenesis of pulmonary hypertension, essentially by inducing a phenotypic transformation in PASMC towards adopting a robust secretory function. This induction is contingent upon the concentration of oxygen present.

Lysine demethylase KDM3A alleviates hyperoxia-induced bronchopulmonary dysplasia in mice by promoting ETS1 expression.

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease among neonates, with increasing morbidity and mortality. This study aims to investigate the effect and mechanism of lysine demethylase 3A (KDM3A) on hyperoxia-induced BPD. Hyperoxia-induced BPD mouse and alveolar epithelial cell models were constructed. The effects of hyperoxia on lung development were evaluated by histological and morphological analysis. The levels of KDM3A, E26 transformation specific-1 (ETS1), H3 lysine 9 dimethylation (H3K9me2), and endoplasmic reticulum (ER) stress-related indexes were quantified by RT-qPCR, Western blot, and IF staining. Cell apoptosis was assessed by flow cytometry and TUNEL staining. Transfection of oe-ETS1, oe-KDM3A, and sh-ETS1 was applied in hyperoxia-induced alveolar epithelial cells to explore the mechanism of the KDM3A/ETS1 axis in hyperoxia-induced apoptosis. KDM3A inhibitor IOX1 was applied to validate the in vivo effect of KDM3A in hyperoxia-induced BPD mice. The results displayed that hyperoxia-induced BPD mice showed reduced body weight, severe destruction of alveolar structure, decreased radial alveolar count (RAC), and increased mean linear intercept (MLI) and mean alveolar diameter (MAD). Further, hyperoxia induction down-regulated ETS1 expression, raised ER stress levels, and increased apoptosis rate in BPD mice and alveolar epithelial cells. However, transfection of oe-ETS1 improved the above changes in hyperoxia-induced alveolar epithelial cells. Moreover, transfection of oe-KDM3A up-regulated ETS1 expression, down-regulated H3K9me2 expression, inhibited ER stress, and reduced apoptosis rate in hyperoxia-induced alveolar epithelial cells. In addition, transfection of sh-ETS1 reversed the inhibitory effect of KDM3A on hyperoxia-induced apoptosis by regulating ER stress. In vivo experiments, KDM3A inhibitor IOX1 intervention further aggravated BPD in newborn mice. In a word, KDM3A alleviated hyperoxia-induced BPD in mice by promoting ETS1 expression.

Rectal temperature after hypoxia-ischemia predicts white matter and cortical pathology in the near-term ferret.

BACKGROUND: Neonatal encephalopathy (NE) remains a common cause of infant morbidity and mortality. Neuropathological corollaries of NE associated with acute hypoxia-ischemia include a central injury pattern involving the basal ganglia and thalamus, which may interfere with thermoregulatory circuits. Spontaneous hypothermia (SH) occurs in both preclinical models and clinical hypoxic-ischemic NE and may provide an early biomarker of injury severity. To determine whether SH predicts the degree of injury in a ferret model of hypoxic-ischemic NE, we investigated whether rectal temperature (RT) 1 h after insult correlated with long-term outcomes. METHODS: Postnatal day (P)17 ferrets were presensitized with Escherichia coli lipopolysaccharide before undergoing hypoxia-ischemia/hyperoxia (HIH): bilateral carotid artery ligation, hypoxia-hyperoxia-hypoxia, and right ligation reversal. One hour later, nesting RTs were measured. RESULTS: Animals exposed to HIH were separated into normothermic (NT; ≥34.4 °C) or spontaneously hypothermic (SH; <34.4 °C) groups. At P42, cortical development, ex vivo MRI, and neuropathology were quantitated. Whole-brain volume and fractional anisotropy in SH brains were significantly decreased compared to control and NT animals. SH brains also had significantly altered gyrification, greater cortical pathology, and increased corpus callosum GFAP staining relative to NT and control brains. CONCLUSION: In near-term-equivalent ferrets, nesting RT 1 h after HIH may predict long-term neuropathological outcomes. IMPACT: High-throughput methods to determine injury severity prior to treatment in animal studies of neonatal brain injury are lacking. In a gyrified animal model of neonatal inflammation-sensitized hypoxic-ischemic brain injury in the ferret, rectal temperature 1 h after hypoxia predicts animals who will have increased cortical pathology and white matter changes on MRI. These changes parallel similar responses in rodents and humans but have not previously been correlated with long-term neuropathological outcomes in gyrified animal models. Endogenous thermoregulatory responses to injury may provide a translational marker of injury severity to help stratify animals to treatment groups or predict outcome in preclinical studies.

The role of oxygen tension in cell fate and regenerative medicine: implications of hypoxia/hyperoxia and free radicals.

Oxygen pressure plays an integral role in regulating various aspects of cellular biology. Cell metabolism, proliferation, morphology, senescence, metastasis, and angiogenesis are some instances that are affected by different tensions of oxygen. Hyperoxia or high oxygen concentration, enforces the production of reactive oxygen species (ROS) that disturbs physiological homeostasis, and consequently, in the absence of antioxidants, cells and tissues are directed to an undesired fate. On the other side, hypoxia or low oxygen concentration, impacts cell metabolism and fate strongly through inducing changes in the expression level of specific genes. Thus, understanding the precise mechanism and the extent of the implication of oxygen tension and ROS in biological events is crucial to maintaining the desired cell and tissue function for application in regenerative medicine strategies. Herein, a comprehensive literature review has been performed to find out the impacts of oxygen tensions on the various behaviors of cells or tissues.

Quercetin supplementation attenuates airway hyperreactivity and restores airway relaxation in rat pups exposed to hyperoxia.

Hyperoxia exposure of immature lungs contributes to lung injury and airway hyperreactivity. Up to now, treatments of airway hyperreactivity induced by hyperoxia exposure have been ineffective. The aim of this study was to investigate the effects of quercetin on hyperoxia-induced airway hyperreactivity, impaired relaxation, and lung inflammation. Newborn rats were exposed to hyperoxia (FiO2 > 95%) or ambient air (AA) for seven days. Subgroups were injected with quercetin (10 mg·kg-1·day-1). After exposures, tracheal cylinders were prepared for in vitro wire myography. Contraction to methacholine was measured in the presence or absence of organ bath quercetin and/or Nω-nitro-L-arginine methyl ester (L-NAME). Relaxation responses were evoked in preconstricted tissues using electrical field stimulation (EFS). Lung tumor necrosis factor-alpha (TNF-α) and interleukin-1ß (IL-1ß) levels were measured by enzyme-linked immunosorbent assay (ELISA). A P < 0.05 was considered statistically significant. Contractile responses of tracheal smooth muscle (TSM) of hyperoxic animals were significantly increased compared with AA animals (P < 0.001). Treatment with quercetin significantly reduced contraction in hyperoxic groups compared with hyperoxic control (P < 0.01), but did not have any effect in AA groups. In hyperoxic animals, relaxation of TSM was significantly reduced compared with AA animals (P < 0.001), while supplementation of quercetin restored the lost relaxation in hyperoxic groups. Incubation of preparations in L-NAME significantly reduced the quercetin effects on both contraction and relaxation (P < 0.01). Treatment of hyperoxic animals with quercetin significantly decreased the expression of TNF-α and IL-1ß compared with hyperoxic controls (P < 0.001 and P < 0.01, respectively).The findings of this study demonstrate the protective effect of quercetin on airway hyperreactivity and suggest that quercetin might serve as a novel therapy to prevent and treat neonatal hyperoxia-induced airway hyperreactivity and inflammation.

Comparison of hyperoxia or normoxia resolution of intermittent hypoxia and intermittent hyperoxia episodes on liver histopathology, IGF-1, IGFBP-3, and GHBP in neonatal rats.

OBJECTIVE: Extremely low gestational age neonates requiring oxygen therapy for chronic lung disease experience repeated fluctuations in arterial oxygen saturation, or intermittent hypoxia (IH), during the first few weeks of life. These events are associated with a high risk for reduced growth, hypertension, and insulin resistance in later life. This study tested the hypothesis that IH, or intermittent hyperoxia have similar negative effects on the liver; somatic growth; and liver insulin-like growth factor (IGF)-I, IGF binding protein (BP)-3, and growth hormone binding protein (GHBP), regardless of resolution in normoxia or hyperoxia between episodes. DESIGN: Newborn rats on the first day of life (P0) were exposed to two IH paradigms: 1) hyperoxia (50% O2) with brief hypoxia (12% O2); or 2) normoxia (21% O2) with hypoxia (12% O2); intermittent hypoxia (50% O2/21% O2); hyperoxia only (50% O2); or room air (RA, 21% O2). Pups were euthanized on P14 or placed in RA until P21. Controls remained in RA from P0-P21. Growth, liver histopathology, apoptosis, IGFI, IGFBP-3, and GHBP were assessed. RESULTS: Pathological findings of the liver hepatocytes, including cellular swelling, steatosis, apoptosis, necrosis and focal sinusoid congestion were seen in the IH and intermittent hyperoxia groups, and were particularly severe in the 21-12% O2 group during exposure (P14) with no significant improvements during recovery/reoxygenation (P21). These effects were associated with induction of HIF1α, and reductions in liver IGFI, IGFBP-3, and GHBP. CONCLUSIONS: Exposure to IH or intermittent hyperoxia during the first few weeks of life regardless of resolution in RA or hyperoxia is detrimental to the immature liver. These findings may suggest that interventions to prevent frequent fluctuations in oxygen saturation during early neonatal life remain a high priority.

Glutamine inhibits inflammation, oxidative stress, and apoptosis and ameliorates hyperoxic lung injury

Glutamine (Gln) is the most widely acting and abundant amino acid in the body and has anti-inflammatory properties, regulates body metabolism, and improves immune function. However, the mechanism of Gln’s effect on hyperoxic lung injury in neonatal rats is unclear. Therefore, this work focused on examining Gln’s function in lung injury of newborn rats mediated by hyperoxia and the underlying mechanism. We examined body mass and ratio of wet-to-dry lung tissue weights of neonatal rats. Hematoxylin and eosin (HE) staining was performed to examine histopathological alterations of lung tissues. In addition, enzyme-linked immunoassay (ELISA) was conducted to measure pro-inflammatory cytokine levels within bronchoalveolar lavage fluid (BALF). Apoptosis of lung tissues was observed using TUNEL assay. Western blotting was performed for detecting endoplasmic reticulum stress (ERS)-associated protein levels. The results showed that Gln promoted body weight gain, significantly reduced pathological damage and oxidative stress in lung tissue, and improved lung function in neonatal rats. Gln reduced pro-inflammatory cytokine release as well as inflammatory cell production in BALF and inhibited apoptosis in lung tissue cells. Furthermore, we found that Gln could downregulate ERS-associated protein levels (GRP78, Caspase-12, CHOP) and inhibit c-Jun N-terminal kinase (JNK) and inositol-requiring enzyme 1 alpha (IRE1α) phosphorylation. These results in an animal model of bronchopulmonary dysplasia (BPD) suggest that Gln may have a therapeutic effect on BPD by reducing lung inflammation, oxidative stress, and apoptosis and improving lung function; its mechanism of action may be related to the inhibition of the IRE1α/JNK pathway. (AU)

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

BACKGROUND: Supplemental 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. METHODS: Neonatal 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. RESULTS: The 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. CONCLUSIONS: Intranasal administration of L. johnsonii protects against hyperoxia-induced lung injury and modulates the gut microbiota.

METTL3 promotes hyperoxia-induced pyroptosis in neonatal bronchopulmonary dysplasia by inhibiting ATG8-mediated autophagy.

OBJECTIVES: N6-Methyladenosine (m6A) modification plays a vital role in lung disorders. However, the potential of m6A in neonatal Bronchopulmonary Dysplasia (BPD) has not been reported. This study aimed to investigate the roles of METTL3 in BPD. METHODS: BPD models were established by hyperoxia in vivo and in vitro. Histological analysis was determined using HE staining. Gene expression was determined using Western blotting, qRT-PCR, and immunofluorescence. The release of IL-1ß and IL-18 was detected using ELISA. The m6A sites of ATG8 were predicted by SCRAPM and verified by MeRIP assay. The location of GSDMD and ATG8 was determined by FISH assay. The interaction between ATG8 and GSDMD was detected using Coimmunoprecipitation (Co-IP). Cell pyroptosis was determined using flow cytometry and TUNEL assays. RESULTS: METTL3 was overexpressed in BPD, which was accompanied by an increase in m6A levels. Interestingly, METTL3 suppressed hyperoxia-mediated damage and pyroptosis in BEAS-2B cells and promoted cell autophagy. METTL3-mediated m6A modification of ATG8 suppressed its expression and disrupted the interaction between ATG8 and GSDMD. However, autophagy inhibition induced pyroptosis in BEAS-2B cells. In vivo assays showed that METTL3-mediated autophagy inhibition induced a decrease in the radial alveolar count and an increase in the mean linear intercept and promoted cell pyroptosis. CONCLUSION: In conclusion, METTL3-mediated cell pyroptosis promotes BPD by regulating the m6A modification of ATG8. This may provide new insight into the development of BPD.

Oxygen-Induced Retinopathy in the Rat: An Animal Model to Study the Proliferative Retinal Vascular Pathology.

Diabetic retinopathy (DR) is one of the leading causes of vision loss worldwide. There are numerous animal models available for developing new ocular therapeutics and drug screening and to investigate the pathological processes involved in DR. Among those animal models, the oxygen-induced retinopathy (OIR) model, though originally developed as a model for retinopathy of prematurity, has also been used to investigate angiogenesis in proliferative DR with the phenomenon of ischemic avascular zones and pre-retinal neovascularization it demonstrated. Briefly, neonatal rodents are exposed to hyperoxia to induce vaso-obliteration. Upon removal from hyperoxia, hypoxia develops in the retina that eventually results in neovascularization. The OIR model is mostly used in small rodents such as mice and rats. Here, we describe a detailed experimental protocol of rat OIR model and the subsequent assessment of abnormal vasculature. By illustrating the vasculoprotective and anti-angiogenic activities of the treatment, OIR model might advance to a new platform for investigating novel ocular therapeutic strategies for DR.

High-mobility group box-1 peptide ameliorates bronchopulmonary dysplasia by suppressing inflammation and fibrosis in a mouse model.

BACKGROUND: This study aimed to examine the effect of the HMGB1 peptide on Bronchopulmonary dysplasia (BPD)-related lung injury in a mouse model. RESULTS: HMGB1 peptide ameliorates lung injury by suppressing the release of inflammatory cytokines and decreasing soluble collagen levels in the lungs. Single-cell RNA sequencing showed that the peptide suppressed the hyperoxia-induced inflammatory signature in macrophages and the fibrotic signature in fibroblasts. These changes in the transcriptome were confirmed using protein assays. CONCLUSION: Systemic administration of HMGB1 peptide exerts anti-inflammatory and anti-fibrotic effects in a mouse model of BPD. This study provides a foundation for the development of new and effective therapies for BPD.

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-1ß. Erythromycin intervention caused a further increase in GSH expression and a decrease in TNF-α and IL-1ß expression. Conclusion: GSH, TNF-α and IL-1ß 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.

Location-specific pathology analysis of the monopodial pulmonary vasculature in a rabbit model of bronchopulmonary dysplasia-A pilot study.

The mammalian pulmonary vasculature consists of functionally and morphologically heterogeneous compartments. When comparing sets of lungs, for example, in disease models or therapeutic interventions, local changes may be masked by the overall heterogeneity of the organ structure. Therefore, alterations taking place only in a sub-compartment may not be detectable by global analysis. In the monopodial lung, the characterization of distinct vessel groups is difficult, due to the asymmetrical branching pattern. In this pilot study, a previously established method to classify segments of the monopodial pulmonary arterial tree into homogeneous groups was employed. To test its suitability for experimental settings, the method was applied to a hyperoxia (HYX, ≥95% oxygen) rabbit model of bronchopulmonary dysplasia and a normoxic control group (NOX, 21% oxygen). The method allowed the identification of morphological differences between the HYX and the NOX groups. Globally visible differences in lumen diameter were pinpointed to specific lung regions. Furthermore, local changes of wall dimension and cell layers in single compartments, that would not have been identifiable in an unfocused analysis of the whole dataset, were found. In conclusion, the described method achieves a higher precision in morphological studies of lung disease models, compared to a common, global analysis approach.

Glutamine inhibits inflammation, oxidative stress, and apoptosis and ameliorates hyperoxic lung injury.

Glutamine (Gln) is the most widely acting and abundant amino acid in the body and has anti-inflammatory properties, regulates body metabolism, and improves immune function. However, the mechanism of Gln's effect on hyperoxic lung injury in neonatal rats is unclear. Therefore, this work focused on examining Gln's function in lung injury of newborn rats mediated by hyperoxia and the underlying mechanism. We examined body mass and ratio of wet-to-dry lung tissue weights of neonatal rats. Hematoxylin and eosin (HE) staining was performed to examine histopathological alterations of lung tissues. In addition, enzyme-linked immunoassay (ELISA) was conducted to measure pro-inflammatory cytokine levels within bronchoalveolar lavage fluid (BALF). Apoptosis of lung tissues was observed using TUNEL assay. Western blotting was performed for detecting endoplasmic reticulum stress (ERS)-associated protein levels. The results showed that Gln promoted body weight gain, significantly reduced pathological damage and oxidative stress in lung tissue, and improved lung function in neonatal rats. Gln reduced pro-inflammatory cytokine release as well as inflammatory cell production in BALF and inhibited apoptosis in lung tissue cells. Furthermore, we found that Gln could downregulate ERS-associated protein levels (GRP78, Caspase-12, CHOP) and inhibit c-Jun N-terminal kinase (JNK) and inositol-requiring enzyme 1 alpha (IRE1α) phosphorylation. These results in an animal model of bronchopulmonary dysplasia (BPD) suggest that Gln may have a therapeutic effect on BPD by reducing lung inflammation, oxidative stress, and apoptosis and improving lung function; its mechanism of action may be related to the inhibition of the IRE1α/JNK pathway.

Molsidomine decreases hyperoxia-induced lung injury in neonatal rats.

BACKGROUND: The study's objective is to evaluate if Molsidomine (MOL), an anti-oxidant, anti-inflammatory, and anti-apoptotic drug, is effective in treating hyperoxic lung injury (HLI). METHODS: The study consisted of four groups of neonatal rats characterized as the Control, Control+MOL, HLI, HLI + MOL groups. Near the end of the study, the lung tissue of the rats were evaluated with respect to apoptosis, histopathological damage, anti-oxidant and oxidant capacity as well as degree of inflammation. RESULTS: Compared to the HLI group, malondialdehyde and total oxidant status levels in lung tissue were notably reduced in the HLI + MOL group. Furthermore, mean superoxide dismutase, glutathione peroxidase, and glutathione activities/levels in lung tissue were significantly higher in the HLI + MOL group as compared to the HLI group. Tumor necrosis factor-α and interleukin-1ß elevations associated with hyperoxia were significantly reduced following MOL treatment. Median histopathological damage and mean alveolar macrophage numbers were found to be higher in the HLI and HLI + MOL groups when compared to the Control and Control+MOL groups. Both values were increased in the HLI group when compared to the HLI + MOL group. CONCLUSIONS: Our research is the first to demonstrate that bronchopulmonary dysplasia may be prevented through the protective characteristics of MOL, an anti-inflammatory, anti-oxidant, and anti-apoptotic drug. IMPACT: Molsidomine prophylaxis significantly decreased the level of oxidative stress markers. Molsidomine administration restored the activities of antioxidant enzymes. Molsidomine prophylaxis significantly reduced the levels of inflammatory cytokines. Molsidomine may provide a new and promising therapy for BPD in the future. Molsidomine prophylaxis decreased lung damage and macrophage infiltration in the tissue.

SIRT3 improves alveolar epithelial cell damage caused by bronchopulmonary dysplasia through deacetylation of FOXO1.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a serious and long-term lung condition commonly observed in premature babies. Sirtuin 3 (SIRT3) has been reported to reduce pulmonary injury and pulmonary fibrosis. OBJECTIVE: The present study investigated the specific role of SIRT3 in BPD by establishing hyperoxia-induced BPD rat and cell models. Hematoxylin and eosin staining was used to observe pathological changes in lung tissues. MATERIALS AND METHODS: The expression levels of SIRT3 and forkhead box protein O1 (FOXO1), as well as its acetylation levels, were detected in hyperoxia-induced lung tissues and cells by Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Levels of reactive oxygen species, superoxide dismutase, and malondialdehyde were assessed by using biochemical kits. Following SIRT3 overexpression, the levels of inflammatory cytokines were assessed by RT-qPCR. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) and Western blot analysis. Upon FOXO1 knockout, cell inflammation, oxidative stress and apoptosis were evaluated again. RESULTS: Compared to the control group, the SIRT3 and FOXO1 expression levels were decreased and the FOXO1 acetylation levels were increased in hyperoxia-induced lung tissues and cells. In addition, SIRT3 reduced hyperoxia-induced inflammation, oxidative stress, and apoptosis in A549 cells, and inhibited FOXO1 acetylation to activate FOXO1. However, FOXO1 knockdown reversed the effects of SIRT3 overexpression in hyperoxia-induced A549 cells. CONCLUSION: SIRT3 relieved alveolar epithelial cell damage caused by BPD via deacetylation of FOXO1, suggesting that SIRT3 could be a therapeutic target in BPD.

Oxygen toxicity causes cyclic damage by destabilizing specific Fe-S cluster-containing protein complexes.

Oxygen is toxic across all three domains of life. Yet, the underlying molecular mechanisms remain largely unknown. Here, we systematically investigate the major cellular pathways affected by excess molecular oxygen. We find that hyperoxia destabilizes a specific subset of Fe-S cluster (ISC)-containing proteins, resulting in impaired diphthamide synthesis, purine metabolism, nucleotide excision repair, and electron transport chain (ETC) function. Our findings translate to primary human lung cells and a mouse model of pulmonary oxygen toxicity. We demonstrate that the ETC is the most vulnerable to damage, resulting in decreased mitochondrial oxygen consumption. This leads to further tissue hyperoxia and cyclic damage of the additional ISC-containing pathways. In support of this model, primary ETC dysfunction in the Ndufs4 KO mouse model causes lung tissue hyperoxia and dramatically increases sensitivity to hyperoxia-mediated ISC damage. This work has important implications for hyperoxia pathologies, including bronchopulmonary dysplasia, ischemia-reperfusion injury, aging, and mitochondrial disorders.

Hyperoxia exposure upregulates Dvl-1 and activates Wnt/ß-catenin signaling pathway in newborn rat lung.

BACKGROUND: Bronchopulmonary dysplasia is a serious and lifelong pulmonary disease in premature neonates that influences around one-quarter of premature newborns. The wingless-related integration site /ß-catenin signaling pathway, which is abnormally activated in the lungs with pulmonary fibrosis, affects cell differentiation and lung development. METHODS: Newborn rats were subjected to hyperoxia exposure. Histopathological changes to the lungs were evaluated through immunohistochemistry, and the activation of disheveled and Wnt /ß-catenin signaling pathway components was assessed by Western blotting and real-time PCR. The abilities of proliferation, apoptosis and migration were detected by Cell Counting Kit-8, flow cytometry and scratch wound assay, respectively. RESULTS: Contrasting with normoxic lungs, hyperoxia-exposed lungs demonstrated larger alveoli, fewer alveoli and thicker alveolar septa. Superoxide dismutase activity was significantly decreased (7th day: P < 0.05; 14th day: P < 0.01) and malondialdehyde significantly increased (7th day: P < 0.05; 14th day: P < 0.01) after hyperoxia exposure. Protein and mRNA expression levels of ß-catenin, Dvl-1, CTNNBL1 and cyclin D1 were significantly upregulated by hyperoxia exposure on 7th day (P < 0.01) and 14th day (P < 0.01). In hyperoxic conditions, Dvl-l downregulation and Dvl-l downregulation + MSAB treatment significantly increased the proliferation rates, decreased the apoptosis rates and improved the ability of cell migration. In hyperoxic conditions, Dvl-l downregulation could decrease the mRNA expression levels of GSK3ß, ß-catenin, CTNNBL1 and cyclin D1 and decrease the protein relative expression levels of GSK3ß, p-GSK3ß, ß-catenin, CTNNBL1 and cyclin D1. CONCLUSIONS: We confirmed the positive role of Dvl-1 and the Wnt/ß-catenin signaling pathway in promoting BPD in hyperoxia conditions and provided a promising therapeutic target.

PAR2 Overexpression is Involved in the Occurrence of Hyperoxygen-Induced Bronchopulmonary Dysplasia in Rats.

BACKGROUND: Bronchopulmonary dysplasia is a chronic lung disease commonly seen in preterm infants. It is characterized by delayed development of the alveoli and lung fibrosis. Protease-activated receptor 2 (PAR2) is an inflammatory driver that plays a proinflammatory role mainly through the P38 MAPK/NF-κB signaling pathway. METHODS: Newborn rat pups were kept under air or oxygen at >60% concentration. Lung tissues were collected at postnatal days (P) 1, 4, 7, and 10 to observe pathological changes and take measurements. RESULTS: In the hyperoxic group, P4 and P7 rats showed delayed alveolar development, septal thickening, and disturbances in alveolar structure.PAR2, P38 MAPK, NF-κB, and IL-18 expression at P4, P7, and P10 was significantly higher than in the air group. CONCLUSION: PAR2 is involved in lung injury induced by persistent hyperoxia. Activated PAR2 promotes IL-18 overexpression through the P38 MAPK/NF-κB signaling pathway, which may be an important mechanism of PAR2-mediated lung injury in bronchopulmonary dysplasia.