Background Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disorder that often leads to fatal outcomes, characterized by the aberrant proliferation of myofibroblasts and excessive deposition of extracellular matrix (ECM) components. Follistatin-like 1 (Fstl1), a secreted glycoprotein regulated by transforming growth factor β1 (TGF-β1), has been found to be markedly elevated in the fibrotic lungs of both patients with IPF and mice subjected to bleomycin-induced injury. Studies have shown that Fstl1 haploinsufficiency protects against bleomycin-induced lung injury, implicating Fstl1 as a potential therapeutic target. Here, we investigated the effect of Fstl1 knockdown using small interfering RNA (siRNA) on pulmonary fibrosis in vivo. Methods We designed four siRNA sequences targeting Fstl1 and evaluated their efficiency in mouse embryonic fibroblasts (MEFs). Of these, si-Fstl1-12 achieved maximal Fstl1 knockdown (∼80%) and significantly inhibited TGF-β1-induced upregulation of Fstl1 and ECM proteins in vitro. We used biodegradable poly (D, L-lactic-co-glycolic acid) (PLGA) nanomaterials as carriers for in vivo delivery. Bleomycin-treated mice were administered PLGA-si-Fstl1-12, and lung tissues were analyzed for Fstl1 expression, fibrosis severity, and collagen deposition. Results si-Fstl1-12 markedly suppressed TGF-β1-induced Fstl1 expression and ECM protein synthesis in MEFs in vitro. Treatment with PLGA-si-Fstl1-12 effectively reduced Fstl1 levels in lung tissue, attenuated interstitial fibrosis, and decreased collagen accumulation in bleomycin-challenged mice. Notably, even low doses of PLGA-si-Fstl1-12 achieved significant therapeutic effects, demonstrating efficient and safe siRNA delivery in vivo. Conclusions Targeted Fstl1 inhibition using siRNA significantly mitigated pulmonary fibrosis in a murine bleomycin model. The successful application of PLGA nanomaterials for siRNA delivery underscores their potential for safe and effective in vivo gene silencing. These findings highlight si-Fstl1 as a promising therapeutic candidate for IPF and support further investigation of RNA-based nanomedicine in fibrotic lung diseases.

Purpose Increased venous admixture, a form of ventilation-perfusion (V/Q) mismatch, is a key contributor to hypoxemia in conditions such as lung trauma, polytrauma, and sepsis. The arterial-to-end-tidal CO2 gradient (PaCO2–EtCO2) has been proposed as a noninvasive marker for V/Q mismatch, but its relationship to venous admixture remains unclear. This study investigates the association between PaCO2, EtCO2, and venous admixture as a marker of impaired gas exchange, in distinct lung injury models in swine. Methods Data from 50 swine with a mean weight 56.2 kg, subjected to lung contusion, polytrauma, behind-armor blunt trauma (BABT), and sepsis were retrospectively analyzed. Arterial blood gases, EtCO2, and venous admixture were measured, and the PaCO2–EtCO2 gradient was calculated. Linear mixed-effects models were used to assess the predictive power of PaCO2, EtCO2, and the PaCO2–EtCO2 gradient for venous admixture. Model performance was evaluated using conditional R2 and root mean squared error (RMSE). Results PaCO2 was the strongest predictor of venous admixture across all subgroups, with R2 values ranging from 0.602 to 0.804 (P < 0.001). The PaCO2–EtCO2 gradient was moderately predictive in lung contusion, BABT, and sepsis but showed no significant association in polytrauma. EtCO2 exhibited weaker predictive performance overall. Conclusion The PaCO2–EtCO2 gradient did not outperform PaCO2 alone in predicting venous admixture and is therefore not a reliable standalone marker. Future studies should explore its integration with additional physiological parameters for improved clinical assessment of ventilation-perfusion mismatch.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with significant morbidity and mortality rates. Mesenchymal stem cells (MSCs) derived from nasal mucosa, known as EMSCs, have demonstrated therapeutic potential in conditions such as liver failure and bone defects. However, investigations focusing on the application of EMSCs in ALI are still lacking. In our study, an ALI model was induced in rats through lipopolysaccharide (LPS) administration, with subsequent intravenous delivery of either saline or EMSCs. Co-culture experiments using transwell systems revealed that EMSCs improved the viability and proliferation of A549 cells, while also suppressing LPS-induced inflammation and apoptosis. Moreover, the administration of EMSCs not only improved pulmonary microvascular permeability and alleviated histopathological damage, but also exerted downregulatory effects on the levels of pro-inflammatory cytokines, including TNFα, IL6, and IL-1β, while concurrently upregulating the expression of anti-inflammatory cytokine IL-10 in both bronchoalveolar lavage fluid (BALF) and plasma. Immunohistochemistry analysis further revealed an elevated expression of proliferation marker Ki67 and anti-apoptotic protein Bcl2, accompanied by a reduction in the expression of pro-apoptotic protein Bax, thus indicating the beneficial outcomes of EMSCs. Collectively, these findings underscore the potential of EMSC-based therapies as promising and effective strategies for the treatment of lung injury.

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.

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.

Objective We aimed to investigate the role and mechanisms of chemokine receptor 1 (CCR1) in airway inflammation in chronic obstructive pulmonary disease (COPD) mice. Methods We established a mouse model of cigarette smoke-induced COPD. A mouse model with CCR1 overexpression or silencing COPD was established by tail vein injection of CCR1 overexpression lentivirus or shRNA-CCR1 lentivirus. Pathological changes in the bronchial mucosa were assessed using hematoxylin and eosin (HE) staining. CCR1 expression and cell apoptosis were detected via immunofluorescence and TUNEL. The levels of chemokine (MIP-1β) and inflammatory factors (IL-6 and TNF-α) in bronchoalveolar lavage fluid were detected using enzyme-linked immunosorbent assay (ELISA). The expression levels of the factors in the CCR1 downstream pathway were detected via RT-qPCR and western blotting. Results Compared with the COPD mice, the bronchial mucosa of the COPD model mice transfected with the vector showed apoptosis, inflammatory cell infiltration, airway remodeling, and emphysema. The COPD model mice exhibited significantly increased expression levels of p-IKK, p-JAK2, STAT3, and p-p65 and chemokine concentrations (MIP-1β, IL-6 and TNF-α) than the control mice (p < 0.05), which were further aggravated by overexpressed-CCR1 lentiviral transfection but inhibited by shRNA-CCR1 lentiviral transfection or BX471 pretreatment (p < 0.05). Conclusion CCR1 aggravates the progression of COPD mice by activating JAK/STAT3/NF-κB signaling. This study has the potential to provide theoretical evidence for the diagnosis and therapeutic strategies of cigarette smoke-induced inflammation in COPD 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.

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.

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.