Experimental Lung Research最新文献

Objective: Asthma development is significantly influenced by the abnormal proliferation and migration of human airway smooth muscle cells (HASMCs). The aim of this study was to investigate the role and molecular mechanisms of the m6A methyltransferase METTL3 in platelet-derived growth factor BB (PDGF-BB)-treated HASMCs. Methods: An in vitro model of HASMCs stimulation was constructed by PDGF-BB. Proliferation, migration, and levels of inflammatory factors of HASMCs were detected by CCK-8, Transwell assay, and ELISA, respectively. m6A modification sites in GAS5 were predicted using WHISTLE. m6A methylation level in GAS5 was detected by MeRIP-qPCR. The RNA stability of GAS5 was assessed by RNA decay assay. Targeted binding sites between miR-29c-3p and GAS5 or VAMP2 were predicted using starBase, and their targeting relationship was verified by dual-luciferase reporter assay. Results: METTL3 levels were decreased in PDGF-BB-induced HASMCs. Enhancing METTL3 expression in HASMCs inhibited PDGF-BB-induced proliferation, migration, and inflammation. METTL3-mediated m6A methylation decreased GAS5 stability, thereby inhibiting GAS5 expression. GAS5 targeted miR-29c-3p and regulated VAMP2 expression. In PDGF-BB-treated HASMCs, enhancing VAMP2 mitigated the effect of METTL3 upregulation. Conclusion: METTL3 inhibits HASMC proliferation, migration, and inflammation in asthma by regulating the GAS5/miR-29c-3p/VAMP2 axis.

Background: µ-calpain is implicated in idiopathic pulmonary fibrosis (IPF), however its role in the aberrant alveolar epithelial type II (AT2) cells differentiation, and its relationship with FoxO3a, an important transcription factor involving in tissue fibrosis, has not been addressed.

Methods: Bleomycin was used to induce pulmonary fibrosis, which was followed by treatment with calpain inhibitor PD150606. The amount of FoxO3a in nuclear fraction and the status of FoxO3a phosphorylation were evaluated. To study the role of calpain in AT2 cell, tdTomato⁺, sftpc-Cre⁺ mice were treated with AAV-FLEX-shCAPN1. The A549 cell was employed to determine the function of FoxO3a and its relationship with calpain. The lung specimen of patients with pulmonary fibrosis were examined with confocal imaging.

Results: Bleomycin caused substantial nuclear accumulation of calpain-1, a catalytic subunit of µ-calpain, and phosphorylation of AKT. This phenomenon was accompanied with a decrease of FoxO3a in the nucleus and an increase of FoxO3a phosphorylation. Furthermore, all these alterations were blocked by calpain inhibitor PD150606. Of note, delivery of AAV-FLEX-shCAPN1 decreased calpain-1 in AT2 cell, and blunted pulmonary fibrosis. TGFβ caused A549 cell phenotypic alterations, indicated by E-cadherin and α-SMA, along with nuclear accumulation of calpain-1 and phosphorylation of AKT and FoxO3a. These effects were attenuated by CAPN1-siRNA and AKT inhibitor LY294002. Similarly, overexpressing FoxO3a mutant blunted cellular phenotypic alterations caused by TGFβ. In addition, overexpressing calpain-1 caused AKT activation, FoxO3a phosphorylation, and especially increased keratin-8 content, a marker of the aberrant alterations of epithelial cells. Finally, confocal imaging revealed co-existence of calpain-1, phosphorylated FoxO3a, and keratin-8 within AT2 cells of IPF patients.

Conclusions: These data provide evidence that nuclear accumulation of µ-calpain is a critical step to elicit the aberrant AT2 cells differentiation and aggravate pulmonary fibrosis, which involves FoxO3a phosphorylation in an AKT-dependent manner.

Background: There are environment-dependent pro-inflammatory and anti-inflammatory pathways during exposure to high altitudes. Although inhibiting the inflammatory pathway can alleviate high altitude pulmonary edema (HAPE), it is currently unclear whether inflammation is the cause of edema or the result of edema in HAPE-afflicted patients. Natural killer T (NKT) cells are a subset of T cells that play an important role in a variety of lung diseases. However, its specific role in HAPE remains unclear. Methods: HAPE rat model was established under hypobaric hypoxia condition. To investigate the role of NKT cells in HAPE, phenotypic and functional changes of NKT cells and their subpopulations were analyzed by flow cytometry. To further investigate the effect of TNF-α on NKT cells, rats were given intraperitoneal injection of TNF-α, and then, NKT cells were characterized by flow cytometry. Subsequently, the levels of TNF-α in the lungs and spleens of rats were detected by ELISA, and HAPE rats were treated with curcumin. Results: Compared with normal control, the ratio of TNF-α and IL-10 secreted by lung NKT cells was decreased in HAPE rats induced by hypoxia. Further analysis showed that the mean fluorescence intensity (MFI) of TNF-α in NKT cells did not change significantly, but the MFI of IL-10 increased significantly. Moreover, the MFI of IL-10 produced by TNF-α-treated rat lung NKT cells was higher, which was completely different from spleen NKT cells. ELISA experiments indicated that TNF-α was enriched in the lung tissue of rats regardless of hypoxia, and the level of TNF-α in lung tissue was upregulated after hypoxia. Furthermore, when HAPE rats were treated with curcumin, the MFI of IL-10 in the NKT cell subsets decreased. Conclusions: NKT cells produce high levels of IL-10, which inhibits the production of lung inflammation in HAPE rats. With the increase of TNF-α level, the inhibitory effect of lung NKT cells on inflammation was further enhanced. When the level of TNF-α decreases, the anti-inflammatory effect of NKT cells also weakens accordingly. Hence, NKT cells play a protective role in HAPE rat lungs.

Background: Recent studies have shown that fine particulate matter (PM2.5) exposure is a key harmful risk factor for chronic obstructive pulmonary disease (COPD) and PM2.5-associated ferroptosis plays an important role during the process of airway oxidative stress. Our preliminary study revealed that PM2.5 reduces the expression of phosphorylated glycogen synthase kinase (GSK)-3β in airway epithelial cells, the overactivity of the GSK-3β/Nuclear Factor erythroid 2-Related Factor 2 (NRF2) pathway is related to ferroptosis. Accordingly, we explored whether PM2.5 could induce ferroptosis in airway epithelial cells and promote the development of COPD via the GSK-3β/NRF2 pathway. Methods: The effect of GSK-3β/NRF2-mediated ferroptosis was assessed using an in vivo model of 20 μg/μl PM2.5-induced COPD by tracheal infusion and 50 μg/ml PM2.5-exposed airway epithelial cells in vitro. Then we performed qRT-PCR to detect mRNA expression; Western blotting, immunofluorescence and immunohistochemical staining to detect protein expression; flow cytometry and spectrophotometry to measure the levels of intracellular lipid peroxidation; small animal spirometry to examine the lung function in mouse, and hematoxylin and eosin (H&E) staining to measure the average alveolar septa in mouse lung sections. Results: We found that PM2.5 decreased the ferroptosis marker mRNA expression of NRF2, SLC7A11 and GPX4, and also decreased the protein expression of p-GSK-3β, NRF2, SLC7A11 and FTH-1, increased the protein expression of NCOA4, then increased the level of lipid peroxidation and MDA in human airway epithelial cells. Further, PM2.5 reduced the expression of p-GSK-3β, NRF2, SLC7A11 and GPX4 in the lungs, subsequently induced lung injury and impaired lung function of mice. Treatment with ferroptosis inhibitors FER-1 and GSK-3β inhibitor TDZD-8 reversed this effect. Conclusion: Our findings suggested that PM2.5 induced ferroptosis of airway epithelial cells, contributing to airway oxidative stress via the GSK-3β/NRF2 signaling pathway in vivo and in vitro, which could be a therapeutic target for PM2.5-induced COPD.

Background: Lung injury induced by Klebsiella pneumoniae infection presents a significant challenge, with complex molecular mechanisms driving tissue damage and immune dysregulation. This study aimed to establish a zebrafish model of K. pneumoniae-induced lung injury to explore the underlying molecular mechanisms involved in tissue damage, immune responses, and development.

Methods: A zebrafish model was developed by injecting K. pneumoniae into the swim bladder at 96 h post-fertilization (hpf). The immune response, including neutrophil migration and cytokine secretion, was assessed through histological analysis and quantitative measures. Transcriptomic analysis was performed to evaluate gene expression changes related to lung development, immune regulation, and metabolism. The role of the TGF-β signaling pathway in immune response and tissue repair was investigated using the TGF-β inhibitor SB 431542.

Results: Infection with K. pneumoniae induced rapid neutrophil migration and the secretion of inflammatory cytokines such as IL-6, IL-1β, TNF-α, and TNF-β, similar to immune responses seen in mouse models. Transcriptomic analysis revealed significant alterations in genes involved in lung development, immune responses, and metabolic pathways, underscoring the broad impact of infection on physiological regulation. The TGF-β signaling pathway was found to play a dual role: it promoted immune cell recruitment and cytokine secretion but suppressed developmental genes, delaying tissue repair. Treatment with SB 431542 reduced neutrophil aggregation, lowered cytokine levels, and restored gene expression related to development and repair.

Conclusions: This zebrafish model effectively mimics K. pneumoniae-induced lung injury, offering valuable insights into the molecular mechanisms of tissue damage and immune dysregulation. Targeting the TGF-β signaling pathway holds therapeutic potential for reducing inflammation and promoting tissue repair, providing a foundation for the development of new treatment strategies for lung infections.

Background: Neurogenic pulmonary edema (NPE) is a severe complication of subarachnoid hemorrhage that aggravates pulmonary microvascular endothelial barrier dysfunction. In this study, we aimed to explore the role of TRPV4 in NPE progression. Method: An NPE rat model was established through the endovascular perforation technique for the collection of NPE serum and pulmonary microvascular endothelial cells (PMVECs). PMVECs were incubated with NPE serum, the FITC-dextran extravasation was applied for permeability analysis, and the cell apoptosis was measured by flow cytometry. TRPV4 subcellular localization was detected by immunofluorescent staining. Finally, we performed the co-immunoprecipitation for AIP4 and TRPV4 binding association analysis. Results: NPE serum incubation promoted PMVECs apoptosis and barrier dysfunction. The TRPV4 level and p38 signaling were activated in PMVECs treated with NPE serum. However, these phenomena were reversed by TRPV4 inhibition. AIP4 promoted TRPV4 ubiquitination and led to the transfer of TRPV4 from the cell membrane to the cytoplasm. Overall, AIP4 ubiquitinated TRPV4, leading to p38 signaling inhibition, thereby blocking PMVECs apoptosis and barrier dysfunction under the NPE serum. Conclusion: TRPV4 is ubiquitinated by API4 and transferred to the cytoplasm, enhancing p38 signaling to promote PMVECs apoptosis and barrier dysfunction under NPE serum conditions.

Background: Increased proliferation and migration of abnormal airway smooth muscle cells (ASMCs) are significantly associated with asthma. This study aimed to investigate the effects of methyltransferase-like 14 (METTL14), YTH domain-containing family Protein 1 (YTHDF1), and polypyrimidine tract-binding protein 1 (PTBP1) on platelet-derived growth factor-BB (PDGF-BB)-treated ASMCs.

Methods: ASMCs were treated with PDGF-BB to mimic cell remodeling. A cell counting kit-8 (CCK-8) assay was performed to detect cell viability. Cell proliferation was detected by 5-Ethynyl-2'-deoxyuridine (EdU) assay. The migration and invasion of cells were measured by wound healing assay and transwell assay. Interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) were evaluated using ELISA kits. The oxidative stress markers reactive oxygen species (ROS) and malondialdehyde (MDA) levels were evaluated using corresponding kits. RT-qPCR and western blotting were utilized to assess mRNA and protein expression. The m6A level was determined using methylated RNA immunoprecipitation (MeRIP) assay. RNA Immunoprecipitation (RIP) assay was used to evaluate the binding of METTL14 or YTHDF1 to PTBP1 mRNA. The binding of METTL14 to PTBP1 was quantified by dual-luciferase assay.

Results: PDGF-BB treatment promoted ASMCs proliferation, migration, invasion, secretion of IL-1β and TNF-α, increased MDA and ROS levels, and promoted macrophage polarization. Knockdown of PTBP1 attenuated PDGF-BB-induced proliferation, migration, invasion, inflammation, oxidative stress, and macrophage polarization in ASMCs. METTL14/YTHDF1 facilitated the m6A methylation modification of PTBP1. Elevated PTBP1 expression nullified the influence of increased METTL14 expression on PDGF-BB-stimulated ASMCs. METTL14 influenced the expression of nuclear factor kappa B (NF-κB) pathway-associated proteins via PTBP1.

Conclusion: The m6A methylation of PTBP1, mediated by METTL14/YTHDF1, played a critical role in modulating the functional behavior of ASMCs induced by PDGF-BB during the progression of asthma.

In recent years, with the increasing severity of air pollution and environmental degradation, research on lung-related diseases has become more intensive. Lung organoids, as 3D in vitro culture models, can simulate the local microenvironment and physiological functions of lung tissue and are widely used in studies on the development and mechanisms of lung-related diseases. However, the precise application of lung organoids is still in the developmental stage, particularly regarding the screening and validation of stable housekeeping genes in lung organoids, which remains unclear. This study utilized human/mouse-derived lung organoids and rat lung tissue as research subjects. By establishing physiological, traditional cigarette, and heated cigarette exposure models and combining BestKeeper, GeNorm, and NormFinder software, the expression stability of various housekeeping genes under different research subjects and exposure models was analyzed to identify stable housekeeping genes for lung-related research. The results showed that in human/mouse-derived lung organoids and rat lung tissue, the baseline expression levels of housekeeping genes were generally high. Among them, GAPDH exhibited the highest expression stability and was least affected by exposure environments, followed by β-actin, RPS16, and RPL19, while 18s showed relatively poor stability. Furthermore, when using a stable single housekeeping gene (e.g., GAPDH) for relative quantification of target gene expression, the experimental results were more significant. When GAPDH and β-actin were used as combined housekeeping genes for target gene quantification, the changes in target gene expression were more pronounced, with stronger statistical significance. In conclusion, this study provides stable single housekeeping genes (GAPDH) and combined housekeeping genes (GAPDH + β-actin) for lung organoid research, contributing to further advancements in the study of lung health.

Study aim: Acute respiratory distress syndrome (ARDS) is a critical disease of high mortality. Recent studies have confirmed that metabolic alterations and mitochondrial dysfunction is in involved in the progression of various pulmonary diseases. Moreover, significantly altered metabolite abundances are important in determining the severity of ARDS. Therefore, this study aims to illuminate the pulmonary metabolic profile, investigate the mitochondrial features of ARDS via the integration of metabolomic and transcriptomic analyses, elucidate the pathogenetic mechanism of ARDS.

Methods: Metabolomic data from ARDS patients were downloaded and reanalyzed. Then Mice were randomly allocated into one of three groups as follows: the sham group; the LPS-2 day group (L2); and the LPS-4 day group (L4). All the mice in LPS group were anesthetized and received an intratracheal instillation of LPS. The sham group mice received only sterile saline. Pulmonary metabolic profiles were measured by integrating metabolomic analyses with transcriptomic analyses, and mitochondrial features in the mouse lungs were investigated via integrative -omics, mitochondrial ultrastructural detection and mitochondrial dynamics quantification.

Results: Inflamed lungs induce global metabolic perturbations that limit fatty acid oxidation, facilitate glucose consumption, accelerate amino acid metabolism and anaplerotic flux in the TCA cycle. In addition, impaired energetics followed by mitochondrial morphology alteration and mitochondrial dynamics imbalance are also validated in lung of ARDS.

Conclusions: Global metabolic imbalance and substantial mitochondrial ultrastructural remodeling, characterized by a reduction in cristae density with significant activation of mitochondrial fission processes, have been verified to be pathogenic mechanisms in the lungs of ARDS patients.

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal disease. Current clinically approved treatments slow disease progression but are not curative. Thus, there is a critical need to better define the pathogenic mechanisms of IPF and develop novel approaches to treat this devastating lung condition. Immune dysregulation of both the innate and adaptive immune systems, accompanied by fibrosis, constitutes a key hallmark of IPF. IPF is generally considered to be a fibroproliferative disorder rather than an immune condition because, historically, immunomodulatory therapies have failed to produce significant clinical effect. This lack of response is frustrating given that there is evidence of immune dysfunction in IPF and highlights the need to clarify the role of immune cells and inflammatory pathways in IPF. There is increasing evidence that the extracellular matrix (ECM) directs cell fate and function, and we propose that ECM remodeling and immune dysfunction in IPF generate a self-perpetuating fibrotic circuit that is refractory to classical anti-inflammatory agents. Understanding the relationship between ECM and immune dysfunction in IPF pathogenesis could help identify novel therapeutic approaches for this devastating disease.