Bronchopulmonary Dysplasia In Rats Research Articles

Nintedanib preserves lung growth and prevents pulmonary hypertension in a hyperoxia-induced lung injury model.

Bronchopulmonary dysplasia (BPD), the chronic lung disease associated with prematurity, is characterized by poor alveolar and vascular growth, interstitial fibrosis, and pulmonary hypertension (PH). Although multifactorial in origin, the pathophysiology of BPD is partly attributed to hyperoxia-induced postnatal injury, resulting in lung fibrosis. Recent work has shown that anti-fibrotic agents, including Nintedanib (NTD), can preserve lung function in adults with idiopathic pulmonary fibrosis. However, NTD is a non-specific tyrosine kinase receptor inhibitor that can potentially have adverse effects on the developing lung, and whether NTD treatment can prevent or worsen risk for BPD and PH is unknown. We hypothesize that NTD treatment will preserve lung growth and function and prevent PH in an experimental model of hyperoxia-induced BPD in rats. Newborn rats were exposed to either hyperoxia (90%) or room air (RA) conditions and received daily treatment of NTD or saline (control) by intraperitoneal (IP) injections (1 mg/kg) for 14 days, beginning on postnatal day 1. At day 14, lung mechanics were measured prior to harvesting lung and cardiac tissue. Lung mechanics, including total respiratory resistance and compliance, were measured using a flexiVent system. Lung tissue was evaluated for radial alveolar counts (RAC), mean linear intercept (MLI), pulmonary vessel density (PVD), and pulmonary vessel wall thickness (PVWT). Right ventricular hypertrophy (RVH) was quantified with cardiac weights using Fulton's index (ratio of right ventricle to the left ventricle plus septum). When compared with RA controls, hyperoxia exposure reduced RAC by 64% (p < 0.01) and PVD by 65% (p < 0.01) and increased MLI by 108% (p < 0.01) and RVH by 118% (p < 0.01). Hyperoxia increased total respiratory resistance by 94% and reduced lung compliance by 75% (p < 0.01 for each). NTD administration restored RAC, MLI, RVH, PVWT and total respiratory resistance to control values and improved PVD and total lung compliance in the hyperoxia-exposed rats. NTD treatment of control animals did not have adverse effects on lung structure or function at 1 mg/kg. When administered at higher doses of 50 mg/kg, NTD significantly reduced alveolar growth in RA controls, suggesting dose-related effects on normal lung structure. We found that NTD treatment preserved lung alveolar and vascular growth, improved lung function, and reduced RVH in experimental BPD in infant rats without apparent adverse effects in control animals. We speculate that although potentially harmful at high doses, NTD may provide a novel therapeutic strategy for prevention of BPD and PH. Anti-fibrotic therapies may be a novel therapeutic strategy for the treatment or prevention of BPD. High-dose anti-fibrotics may have adverse effects on developing lungs, while low-dose anti-fibrotics may treat or prevent BPD. There is very little preclinical and clinical data on the use of anti-fibrotics in the developing lung. Dose timing and duration of anti-fibrotic therapies may be critical for the treatment of neonatal lung disease. Currently, strategies for the prevention and treatment of BPD are lacking, especially in the context of lung fibrosis, so this research has major clinical applicability.

Eclipta prostrata improves alveolar development of bronchopulmonary dysplasia via suppressing the NLRP3 inflammasome in a DLD-dependent manner.

Bronchopulmonary dysplasia (BPD), the most common late morbidity in preterm infants, is characterized by impaired alveolar development caused by persistent lung inflammation. Studies have shown that NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome-mediated inflammation is critically involved in the development of BPD. As a traditional Chinese medicinal herb, Eclipta prostrata (EAP) exhibits potent anti-inflammatory properties. Our study aims to investigate whether EAP could improve the lung development of BPD by suppressing the lung inflammatory response. The BPD rat model was established by intra-amniotic injection of lipopolysaccharide (LPS) and postnatal exposure to hyperoxia. Changes in the NLRP3 inflammasome and pyroptosis were assessed by treatment with EAP. The effect of EAP on the NLRP3 inflammasome was tested in vitro using the THP-1 cell line and primary alveolar macrophages. Proteomics analysis was used to elucidate the mechanism of action of EAP. Histopathological and immunofluorescence results of lung tissues revealed that LPS and hyperoxia induced lung injury and triggered NLRP3 inflammasome activation and pyroptosis in alveolar macrophages. EAP ameliorated BPD lung injury, inhibited NLRP3 inflammasome activation and reduced gasdermin D (GSDMD) expression in alveolar macrophages. EAP downregulated the expression of NLRP3 inflammasome pathway molecules (NLRP3, caspase-1, and IL-1β) and GSDMD in LPS-stimulated THP-1 macrophages and primary alveolar macrophages. In addition, proteomics analysis identified that dihydrolipoamide dehydrogenase (DLD) interacted with EAP. Inhibition of DLD activity abolished the protective effects of EAP. Our study suggested that EAP could attenuate arrest of alveolar development via inhibiting NLRP3 inflammasome in a DLD-dependent way, and could be a potential therapeutic method for BPD.

PC (16:0/14:0) ameliorates hyperoxia-induced bronchopulmonary dysplasia by upregulating claudin-1 and promoting alveolar type II cell repair

Bronchopulmonary dysplasia (BPD) remains a significant challenge in neonatal care, the pathogenesis of which potentially involves altered lipid metabolism. Given the critical role of lipids in lung development and the injury response, we hypothesized that specific lipid species could serve as therapeutic agents in BPD. This study aimed to investigate the role of the lipid Phosphatidylcholine (PC) (16:0/14:0) in modulating BPD pathology and to elucidate its underlying mechanisms of action. Our approach integrated in vitro and in vivo methodologies to assess the effects of PC (16:0/14:0) on the histopathology, cellular proliferation, apoptosis, and molecular markers in lung tissue. In a hyperoxia-induced BPD rat model, we observed a reduction in alveolar number and an enlargement in alveolar size, which were ameliorated by PC (16:0/14:0) treatment. Correspondingly, in BPD cell models, PC (16:0/14:0) intervention led to increased cell viability, enhanced proliferation, reduced apoptosis, and elevated surfactant protein C (SPC) expression. RNA sequencing revealed significant gene expression differences between BPD and PC (16:0/14:0) treated groups, with a particular focus on Cldn1 (encoding claudin 1), which was significantly enriched in our analysis. Our findings suggest that PC (16:0/14:0) might protect against hyperoxia-induced alveolar type II cell damage by upregulating CLDN1 expression, potentially serving as a novel therapeutic target for BPD. This study not only advances our understanding of the role of lipids in BPD pathogenesis, but also highlights the significance of PC (16:0/14:0) in the prevention and treatment of BPD, offering new avenues for future research and therapeutic development.

Untargeted lipidomics of bronchopulmonary dysplasia induced by hyperoxia exposure in rats.

Bronchopulmonary dysplasia (BPD), characterized by impaired lung development, remains a leading cause of morbidity and mortality in premature infants. The synthesis and metabolism of lipids play a critical role in normal lung development, such as dipalmitoylphosphatidylcholine, a key component of pulmonary surfactant (PS). Therefore, we conducted a lipidomics study of rat lung tissue to explore the changes of pulmonary lipid composition in the progression of BPD disease. In this study, we exposed neonatal Sprague-Dawley (SD) rats to hyperoxia for 14 days. After hyperoxia exposure, the lung tissues of rats were analyzed pathologically, and untargeted lipidomics was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hematoxylin-eosin (H&E) staining showed that the alveoli enlarged, the number of alveoli decreased and the pulmonary surfactant-associated protein D (SFTPD) decreased in hyperoxia-exposed rats. A total of 620 pulmonary lipids were detected by LC-MS/MS, covering 27 lipid categories. The most common lipids were triacylglycerol (TAG), followed by phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Compared with those rats exposed to normoxic conditions, the lipid levels in the lungs of rats exposed to hyperoxia for 14 days generally decreased, with the levels of TAG and PC decreasing most significantly. In short, our results provide a clue for finding therapeutic targets and biomarkers of a BPD rat model lung liposome.

Lactobacillus plantarum L168 improves hyperoxia-induced pulmonary inflammation and hypoalveolarization in a rat model of bronchopulmonary dysplasia

Alteration of gut microbiota can affect chronic lung diseases, such as asthma and chronic obstructive pulmonary disease, through abnormal immune and inflammatory responses. Previous studies have shown a feasible connection between gut microbiota and bronchopulmonary dysplasia (BPD) in preterm infants. However, whether BPD can be ameliorated by restoring the gut microbiota remains unclear. In preterm infants with BPD, we found variance in the diversity and structure of gut microbiota. Similarly, BPD rats showed gut dysbiosis, characterized by a deficiency of Lactobacillus, which was abundant in normal rats. We therefore explored the effect and potential mechanism of action of a probiotic strain, Lactobacillus plantarum L168, in improving BPD. The BPD rats were treated with L. plantarum L168 by gavage for 2 weeks, and the effect was evaluated by lung histopathology, lung function, and serum inflammatory markers. Subsequently, we observed reduced lung injury and improved lung development in BPD rats exposed to L. plantarum L168. Further evaluation revealed that L. plantarum L168 improved intestinal permeability in BPD rats. Serum metabolomics showed altered inflammation-associated metabolites following L. plantarum L168 intervention, notably a marked increase in anti-inflammatory metabolites. In agreement with the metabolites analysis, RNA-seq analysis of the intestine and lung showed that inflammation and immune-related genes were down-regulated. Based on the information from RNA-seq, we validated that L. plantarum L168 might improve BPD relating to down-regulation of TLR4 /NF-κB /CCL4 pathway. Together, our findings suggest the potential of L. plantarum L168 to provide probiotic-based therapeutic strategies for BPD.

Nesfatin-1 alleviates hyperoxia-induced bronchopulmonary dysplasia (BPD) via the nuclear factor-κB (NF-κB) p65 signaling pathway.

Bronchopulmonary dysplasia (BPD) is a chronic respiratory disease in newborns, which severely influences the health of infants and lacks effective clinical treatment strategies. The pathogenesis of BPD is correlated to enhanced inflammation and activated oxidative stress (OS). The application of antioxidants and anti-inflammatory treatment could be hot spots for BPD treatment. Nesfatin-1, a peptide with a suppressive property against inflammation, was tested herein for its potential therapeutic value in BPD. Neonatal SD rats were stimulated with hyperoxia, followed by being intraperitoneally administered with 20 μg/kg/day Nesfatin-1 for 2 weeks. Decreased RAC value in lung tissues, increased wet weight/dry weight (W/D) pulmonary ratio and bronchoalveolar lavage fluid (BALF) proteins, elevated cytokine release in BALF, increased malondialdehyde (MDA) content, and declined superoxide dismutase (SOD) activity were observed in BPD rats, all of which were sharply mitigated by Nesfatin-1. Rat epithelial type II cells (AECIIs) were handled with hyperoxia, and then cultured with 1 and 10 nM Nesfatin-1. Reduced cell viability, elevated lactate dehydrogenase production, elevated cytokine secretion, elevated MDA content, and decreased SOD activity were observed in hyperoxia-handled AECIIs, all of which were markedly alleviated by Nesfatin-1. Furthermore, activated nuclear factor-κB (NF-κB) signaling observed in both BPD rats and hyperoxia-handled AECIIs were notably repressed by Nesfatin-1. Collectively, Nesfatin-1 alleviated hyperoxia-triggered BPD by repressing inflammation and OS via the NF-κB signaling pathway.

Itaconic acid regulation of TFEB-mediated autophagy flux alleviates hyperoxia-induced bronchopulmonary dysplasia

BackgroundPremature infants often require oxygen supplementation, which can elicit bronchopulmonary dysplasia (BPD) and lead to mitochondrial dysfunction. Mitochondria play important roles in lung development, in both normal metabolism and apoptosis. Enhancing our comprehension of the underlying mechanisms in BPD development can facilitate the effective treatments. MethodsPlasma samples from BPD and non-BPD infants were collected at 36 weeks post-menstrual age and used for metabolomic analysis. Based on hyperoxia-induced animal and cell models, changes in mitophagy and apoptosis were evaluated following treatment with itaconic acid (ITA). Finally, the mechanism of action of ITA in lung development was comprehensively demonstrated through rescue strategies and administration of corresponding inhibitors. ResultsAn imbalance in the tricarboxylic acid (TCA) cycle significantly affected lung development, with ITA serving as a significant metabolic marker for the outcomes of lung development. ITA improved the morphological changes in BPD rats, promoted SP-C expression, and inhibited the degree of alveolar type II epithelial cells (AEC II) apoptosis. Mechanistically, ITA mainly promotes the nuclear translocation of transcription factor EB (TFEB) to facilitate dysfunctional mitochondrial clearance and reduces apoptosis in AEC II cells by regulating autophagic flux. ConclusionThe metabolic imbalance in the TCA cycle is closely related to lung development. ITA can improve lung development by regulating autophagic flux and promote the nuclear translocation of TFEB, implying its potential therapeutic utility in the treatment of BPD.

Cellular Senescence Contributes to the Progression of Hyperoxic Bronchopulmonary Dysplasia.

Oxidative stress, inflammation, and endoplasmic reticulum (ER) stress sequentially occur in bronchopulmonary dysplasia (BPD), and all result in DNA damage. When DNA damage becomes irreparable, tumor suppressors increase, followed by apoptosis or senescence. Although cellular senescence contributes to wound healing, its persistence inhibits growth. Therefore, we hypothesized that cellular senescence contributes to BPD progression. Human autopsy lungs were obtained. Sprague-Dawley rat pups exposed to 95% oxygen between Postnatal Day 1 (P1) and P10 were used as the BPD phenotype. N-acetyl-lysyltyrosylcysteine-amide (KYC), tauroursodeoxycholic acid (TUDCA), and Foxo4 dri were administered intraperitoneally to mitigate myeloperoxidase oxidant generation, ER stress, and cellular senescence, respectively. Lungs were examined by histology, transcriptomics, and immunoblotting. Cellular senescence increased in rat and human BPD lungs, as evidenced by increased oxidative DNA damage, tumor suppressors, GL-13 stain, and inflammatory cytokines with decreased cell proliferation and lamin B expression. Cellular senescence-related transcripts in BPD rat lungs were enriched at P10 and P21. Single-cell RNA sequencing showed increased cellular senescence in several cell types, including type 2 alveolar cells. In addition, Foxo4-p53 binding increased in BPD rat lungs. Daily TUDCA or KYC, administered intraperitoneally, effectively decreased cellular senescence, improved alveolar complexity, and partially maintained the numbers of type 2 alveolar cells. Foxo4 dri administered at P4, P6, P8, and P10 led to outcomes similar to TUDCA and KYC. Our data suggest that cellular senescence plays an essential role in BPD after initial inducement by hyperoxia. Reducing myeloperoxidase toxic oxidant production, ER stress, and attenuating cellular senescence are potential therapeutic strategies for halting BPD progression.

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.

MSC-EXO and tempol ameliorate bronchopulmonary dysplasia in newborn rats by activating HIF-1α.

Bronchopulmonary dysplasia (BPD) is a major complication of premature infants and an important cause of morbidity and mortality. This study investigates the effect of the combination of mesenchymal stem cells-derived exosomes (MSC-EXO) and tempol on BPD and analyzes its mechanism. MSC-EXO was extracted by centrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis, and western blot analysis(WB). Tidal volume (TV), minute ventilation (MV), peak inspiratory flow (PIF), and dynamic pulmonary compliance (Cdyn) of rats were measured by BuxCo pulmonary function experimental platform. Hematoxylin-eosin staining was performed to observe the lung morphology and radical alveolar count (RAC)and mean linear intercept (MLI) were assessed. Immunofluorescence (IF) was conducted to detect the expression of CD31 and α-SMA in pulmonary blood vessels. The kits were used to calculate malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (TAOC) concentration in lung tissue. Enzyme linked immunosorbent assaywas applied to detect the levels of IL-1β, IL-17, IL-6, and IFN-γ in bronchoalveolar lavage fluid. In addition, the expressions of HIF-1α, vascular endothelial growth factor(VEGF), p-PI3K, and p-AKT were analyzed by WB and IF. We successfully extracted and identified MSC-EXO. In BPD rats, TV, MV, PIF, and Cdyn decreased, alveoli were simplified, and the number of interalveoli small vessels, blood vessel density decreased. Moreover, RAC, CD31, TAOC, and SOD decreased, and MLI, α-SMA, MDA, IL-1β, IL-17, IL-6, and IFN-γ increased, which was reversed by the combination of MSC-EXO and tempol treatment after combined treatment. In addition, the expression levels of HIF-1α, VEGF, p-PI3K, and p-AKT were increased after combined treatment. Combined treatment could improve lung tissue injury, promote pulmonary vascular remodeling, restore lung function, and inhibit oxidative stress in BPD rats. These effects were achieved through activation of HIF-1α.

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

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. 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. 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. 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. SIRT3 relieved alveolar epithelial cell damage caused by BPD via deacetylation of FOXO1, suggesting that SIRT3 could be a therapeutic target in BPD.

Rhubarb alleviates hyperoxia induced lung injury in neonatal rats with bronchopulmonary dysplasia by inhibiting inflammation

Purpose: To investigate the effect of rhubarb on hyperoxia-induced lung injury in neonatal rats with bronchopulmonary dysplasia (BPD), and the underlying mechanism.Methods: Sixty 4-day-old neonatal rats were assigned to air control, BPD, and rhubarb intervention groups, with 20 rats in each group. Immunoblotting was employed to assay NF-κB expression. Levels of malondialdehyde (MDA) and SOD were determined spectrophotometrically, while ELISA was used to measure serum levels of IL-6, IL-8 and TNF-α.Results: The peripheral blood levels of TNF-α, IL-8 and IL-1β were markedly higher in BPD-exposed rats than in the air control rats, while peripheral blood levels of TNF-α, IL-8 and IL-1β were reduced in rhubarb intervention rats, relative to BPD-exposed rats. The activity of SOD was markedly lower in lung tissue of BPD rats than in lung tissue of air control rats, while MDA level was markedly elevated in BPD rats (p &lt; 0.05). There was marked up-regulation of NF-κB p65 expression in BPD-exposed rats, relative to air control rats, but it was markedly lower in rhubarb intervention rats than in hyperoxia model rats (p&lt; 0.05).Conclusion: Rhubarb mitigated hyperoxia-induced inflammation, oxidative stress and lung injury in BPD neonatal rat model by inhibiting oxidative stress and reducing the levels of inflammatory factors.

O-linked N-acetylglucosamine affects mitochondrial homeostasis by regulating Parkin-dependent mitophagy in hyperoxia-injured alveolar type II cells injury

BackgroundThe level of linked N-acetylglucosamine (O-GlcNAc) has been proved to be a sensor of cell state, but its relationship with hyperoxia-induced alveolar type 2 epithelial cells injure and bronchopulmonary dysplasia (BPD) has not been clarified. In this study, we evaluated if these effects ultimately led to functional damage in hyperoxia-induced alveolar cells.MethodsWe treated RLE-6TN cells at 85% hyperoxia for 0, 24 and 48 h with Thiamet G (TG), an OGA inhibitor; OSMI-1 (OS), an OGT inhibitor; or with UDP-GlcNAc, which is involved in synthesis of O-GlcNAc as a donor. The metabolic rerouting, cell viability and apoptosis resulting from the changes in O-GlcNAc glycosyltransferase levels were evaluated in RLE-6TN cells after hyperoxia exposure. We constructed rat Park2 overexpression and knockdown plasmmids for in vitro verification and Co-immunoprecipitation corroborated the binding of Parkin and O-GlcNAc. Finally, we assessed morphological detection in neonatal BPD rats with TG and OS treatment.ResultsWe found a decrease in O-GlcNAc content and levels of its metabolic enzymes in RLE-6TN cells under hyperoxia. However, the inhibition of OGT function with OSMI-1 ameliorated hyperoxia-induced lung epithelial cell injury, enhanced cell metabolism and viability, reduced apoptosis, and accelerated the cell proliferation. Mitochondrial homeostasis was affected by O-GlcNAc and regulated Parkin.ConclusionThe results revealed that the decreased O-GlcNAc levels and increased O-GlcNAcylation of Parkin might cause hyperoxia-induced alveolar type II cells injurys.

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.

Involvement of miR-495 overexpression in the pathogenesis of bronchopulmonary dysplasia in preterm infants via the targeting of NEDD4L-ENaC pathway.

Bronchopulmonary dysplasia (BPD) is a severe pulmonary complication causing morbidity and mortality in preterm infants. A key histopathological feature of BPD is late lung growth retardation, in which the process of alveolarization is hindered and the mechanism of which is unclear. Emerging evidence indicates that microRNAs (miRNAs) promote the development of BPD via the inhibition of their target genes. MiR-495 has been reported to be involved in various lung diseases. However, the physiological function of miR-495 in BPD has not yet been fully understood. Differentially expressed miRNAs in peripheral blood of patients with BPD were compared with those of normal controls. A dual-luciferase reporter assay was performed to identify the target genes of miR-495. A BPD neonatal rat model was established by injecting lipopolysaccharide (LPS) in the amniotic sac of pregnant rats. The morphology of the lungs was observed using hematoxylin and eosin (HE) staining. The expression of miR-495, neural precursor cell expressed developmentally down-regulated 4-like (NEDD4L), and epithelial Na+ channel (ENaC) was tested using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot analysis, and immunofluorescent (IF) staining. The expression of miR-495 was significantly increased in the peripheral blood samples of premature infants with BPD and verified using qRT-PCR. NEDD4L was proven to be the target gene of miR-495. Additionally, miR-495 expression was also increased in the lungs of rat pups with BPD at postnatal day (P) 3 compared with the control group. qRT-PCR and Western blot results showed that NEDD4L expression was decreased while ENaC expression was increased at the transcriptional and translational levels. IF staining results showed that NEDD4L level was decreased while ENaC level was increased in the LPS-induced BPD rat model, which was consistent with abnormal changes in alveolar structure. The aberrant overexpression of miR-495 may contribute to the development of BPD by targeting NEDD4L-ENaC pathway, implying an imbalance in lung fluid clearance.

Inhibition of miR-21 improves pulmonary vascular responses in bronchopulmonary dysplasia by targeting the DDAH1/ADMA/NO pathway.

miR-21 has been confirmed to be overexpressed in neonatal rat lungs with hyperoxia-mediated bronchopulmonary dysplasia (BPD). The specific function of miR-21 in BPD is still unclear. We established the hyperoxia-induced BPD rat model in vivo and the hyperoxia-induced pulmonary microvascular endothelial cells (PMVECs) model in vitro. Transwell assay was utilized to detect the migratory capability of PMVECs. Tube formation assay was utilized to measure angiogenesis ability. ELISA was utilized to test nitric oxide (NO) production and the intracellular and extracellular Asymmetric Dimethylarginine (ADMA) concentration. Furthermore, the interaction between miR-21 and dimethylarginine dimethylaminohydrolase 1 (DDAH1) was evaluated using luciferase reporter assay. We found that miR-21 expression in PMVECs was increased by hyperoxia stimulation. Inhibition of miR-21 improved the migratory and angiogenic activities of PMVECs and overexpression of miR-21 exerted the opposite effects. Furthermore, knockdown of miR-21 increased NO production and decreased intracellular and extracellular ADMA concentration in hyperoxia-treated PMVECs. Next we proved that miR-21 could bind to DDAH1 and negatively regulate its expression. Rescues assays showed that DDAH1 knockdown reversed the effects of miR-21 depletion on hyperoxia-mediated PMVEC functions, NO production, and ADMA concentration. Importantly, miR-21 downregulation restored alveolarization and vascular density in BPD rats. This study demonstrates that inhibition of miR-21 improves pulmonary vascular responses in BPD by targeting the DDAH1/ADMA/NO pathway.

Role of endoplasmic reticulum stress in impaired neonatal lung growth and bronchopulmonary dysplasia.

Myeloperoxidase (MPO), oxidative stress (OS), and endoplasmic reticulum (ER) stress are increased in the lungs of rat pups raised in hyperoxia, an established model of bronchopulmonary dysplasia (BPD). However, the relationship between OS, MPO, and ER stress has not been examined in hyperoxia rat pups. We treated Sprague-Dawley rat pups with tunicamycin or hyperoxia to determine this relationship. ER stress was detected using immunofluorescence, transcriptomic, proteomic, and electron microscopic analyses. Immunofluorescence observed increased ER stress in the lungs of hyperoxic rat BPD and human BPD. Proteomic and morphometric studies showed that tunicamycin directly increased ER stress of rat lungs and decreased lung complexity with a BPD phenotype. Previously, we showed that hyperoxia initiates a cycle of destruction that we hypothesized starts from increasing OS through MPO accumulation and then increases ER stress to cause BPD. To inhibit ER stress, we used tauroursodeoxycholic acid (TUDCA), a molecular chaperone. To break the cycle of destruction and reduce OS and MPO, we used N-acetyl-lysyltyrosylcysteine amide (KYC). The fact that TUDCA improved lung complexity in tunicamycin- and hyperoxia-treated rat pups supports the idea that ER stress plays a causal role in BPD. Additional support comes from data showing TUDCA decreased lung myeloid cells and MPO levels in the lungs of tunicamycin- and hyperoxia-treated rat pups. These data link OS and MPO to ER stress in the mechanisms mediating BPD. KYC's inhibition of ER stress in the tunicamycin-treated rat pup's lung provides additional support for the idea that MPO-induced ER stress plays a causal role in the BPD phenotype. ER stress appears to expand our proposed cycle of destruction. Our results suggest ER stress evolves from OS and MPO to increase neonatal lung injury and impair growth and development. The encouraging effect of TUDCA indicates that this compound has the potential for treating BPD.

Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Enhance Alveolar Type II Cell Proliferation and Attenuate Lung Inflammation in a Rat Model of Bronchopulmonary Dysplasia.

Although it is known that exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs) alleviate hyperoxic lung injury of bronchopulmonary dysplasia (BPD) in animal models, the role of microvesicles (MVs) derived from hUCMSCs in BPD is poorly defined. Furthermore, antenatal inflammation has been linked to high risk of BPD in preterm infants. The purpose of this study was to explore whether MVs derived from hUCMSCs can preserve lung structure and function in an antenatal lipopolysaccharide- (LPS-) induced BPD rat model and to clarify the underlying mechanism. We demonstrate that antenatal LPS induced alveolar simplification, altered lung function, and dysregulated pulmonary vasculature, which restored by hUCMSCs and MVs treatment. Furthermore, MVs were large vesicles with a diameter of 100-900 nanometers and mostly uptaken by alveolar epithelial type II cells (AT2) and macrophages. Compared with the LPS-exposed group, MVs restored the AT2 cell number and SP-C expression in vivo and promoted the proliferation of AT2 cells in vitro. MVs also restored the level of IL-6 and IL-10 in lung homogenate. Additionally, PTEN/AKT and MAPK pathways were associated with the protection of MVs. Taken together, this study suggests MVs derived from hUCMSCs improve lung architecture and function in an antenatal LPS-induced BPD rat model by promoting AT2 cell proliferation and attenuating lung inflammation; thus, MVs provide a promising therapeutic vehicle for BPD treatment.

Dose-dependent immunomodulatory effects of metformin on human neonatal monocyte-derived macrophages

While the association of inflammation with bronchopulmonary dysplasia (BPD) has long been appreciated, M1 proinflammatory macrophage population is emerging as the key element in driving the BPD inflammatory environment. Previous study suggests that low-dose metformin elicits an anti-inflammatory response, possibly through modulating macrophages, to improve disease outcome in a rat BPD model. To investigate this concept further, we examined the dose-dependent immunomodulatory function of metformin directly on human macrophages derived from cord blood (CB) monocytes. We demonstrate that low-dose metformin promotes expansion of M2 anti-inflammatory macrophages, contrasted with high-dose treatment, which exacerbates inflammation by favoring M1 polarization and restricting M2 phenotype. These findings highlight that metformin hold immunomodulatory ability by regulating macrophage polarization in a dose-dependent manner, and only when applied at low dose, exhibiting potential for beneficial anti-inflammatory adjuvant in BPD setting.

Hypoxic preconditioning improves the survival and pro-angiogenic capacity of transplanted human umbilical cord mesenchymal stem cells via HIF-1α signaling in a rat model of bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is a serious chronic respiratory disease that predominates in the neonatal period. Currently, efficacious and effective specific treatments are lacking. Mesenchymal stem cells (MSCs) transplantation has emerged as a promising option for treating BPD. However, the lower cell survival rate limits its therapeutic efficacy. Hypoxic preconditioning is a direct and effective strategy for promoting MSCs survival, proliferation, and paracrine secretion in the recipient after transplantation, which is greatly important to tissue engineering. We investigated whether hypoxia-pretreated MSCs (HPMSCs) confer superior benefit in an experimental BPD rat model. Neonatal Sprague-Dawley rats were exposed to 80–85% O2 for 14 days. Before tracheal transplantation, the MSCs were pretreated for 48 h with deferoxamine, a chemical hypoxia-mimicking agent. In vitro, the HPMSCs reduced the apoptosis rare, caspase-3 expression, and reactive oxygen species (ROS) generation and promoted proliferation, hypoxia inducible factor-1α (HIF-1α) expression, VEGF secretion, and human umbilical vein endothelial cell tube formation (p < 0.05). In vivo, the HPMSCs restored alveolar structure and lung function, ameliorated pulmonary hypertension, increased vessel density in the BPD rat model (p < 0.05). This work demonstrates for the first time that HPMSCs could have a markedly improved therapeutic effect in BPD, presenting a new potential strategy for the clinical implementation of stem cell biotechnology.