Cells In Bronchoalveolar Lavage Fluid Research Articles

Lipocalin-2 as a prognostic marker in patients with acute exacerbation of idiopathic pulmonary fibrosis

BackgroundLipocalin-2 (LCN2) is a secretory glycoprotein upregulated by oxidative stress; moreover, patients with idiopathic pulmonary fibrosis (IPF) have shown increased LCN2 levels in bronchoalveolar lavage fluid (BALF). This study aimed to determine whether circulatory LCN2 could be a systemic biomarker in patients with IPF and to investigate the role of LCN2 in a bleomycin-induced lung injury mouse model.MethodsWe measured serum LCN2 levels in 99 patients with stable IPF, 27 patients with acute exacerbation (AE) of IPF, 51 patients with chronic hypersensitivity pneumonitis, and 67 healthy controls. Further, LCN2 expression in lung tissue was evaluated in a bleomycin-induced lung injury mouse model, and the role of LCN2 was investigated using LCN2-knockout (LCN2 -/-) mice.ResultsSerum levels of LCN2 were significantly higher in patients with AE-IPF than in the other groups. The multivariate Cox proportional hazards model showed that elevated serum LCN2 level was an independent predictor of poor survival in patients with AE-IPF. In the bleomycin-induced lung injury mouse model, a higher dose of bleomycin resulted in higher LCN2 levels and shorter survival. Bleomycin-treated LCN2 -/- mice exhibited increased BALF cell and protein levels as well as hydroxyproline content. Moreover, compared with wild-type mice, LCN2-/- mice showed higher levels of circulatory 8-isoprostane as well as lower Nrf-2, GCLC, and NQO1 expression levels in lung tissue following bleomycin administration.ConclusionsOur findings demonstrate that serum LCN2 might be a potential prognostic marker of AE-IPF. Moreover, LCN2 expression levels may reflect the severity of lung injury, and LCN2 may be a protective factor against bleomycin-induced acute lung injury and oxidative stress.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) restores immune regulatory functions of airway macrophages of patients with asthma.

Allergic asthma is characterized by the polarization of Th2 cells and impaired immune regulation. Macrophages occupy the largest proportion of airway immune cells. This study aims to discover the mechanism that hinders the immune regulatory functions of airway macrophages. In this study, macrophages were isolated from cells in bronchoalveolar lavage fluids (BALF) collected from asthma patients and normal control (NC) subjects. The results indicated that macrophages occupied the largest portion of the cellular components in BALF. The frequency of IL-10+ macrophage was significantly lower in asthma patients than in NC subjects. The expression of IL-10 in macrophages of BALF was associated with the levels of asthma-related parameters. The immune-suppressive functions of BALF M0 cells were defective in asthma patients. The inducibility of IL-10 expression was impaired in BALF macrophages of asthma patients, which could be restored by exposing to CpG. In conclusion, the induction of IL-10 in macrophages of BALF in asthma patients was impaired, and it could be restored by exposure to CpG.

Concordance between In Vitro and In Vivo Relative Toxic Potencies of Diesel Exhaust Particles from Different Biodiesel Blends.

Diesel exhaust particles (DEPs) contribute to air pollution exposure-related adverse health impacts. Here, we examined in vitro, and in vivo toxicities of DEPs from a Caterpillar C11 heavy-duty diesel engine emissions using ultra-low-sulfur diesel (ULSD) and biodiesel blends (20% v/v) of canola (B20C), soy (B20S), or tallow-waste fry oil (B20T) in ULSD. The in vitro effects of DEPs (DEPULSD, DEPB20C, DEPB20S, and DEPB20T) in exposed mouse monocyte/macrophage cells (J774A.1) were examined by analyzing the cellular cytotoxicity endpoints (CTB, LDH, and ATP) and secreted proteins. The in vivo effects were assessed in BALB/c mice (n = 6/group) exposed to DEPs (250 µg), carbon black (CB), or saline via intratracheal instillation 24 h post-exposure. Bronchoalveolar lavage fluid (BALF) cell counts, cytokines, lung/heart mRNA, and plasma markers were examined. In vitro cytotoxic potencies (e.g., ATP) and secreted TNF-α were positively correlated (p < 0.05) with in vivo inflammatory potency (BALF cytokines, lung/heart mRNA, and plasma markers). Overall, DEPULSD and DEPB20C appeared to be more potent compared to DEPB20S and DEPB20T. These findings suggested that biodiesel blend-derived DEP potencies can be influenced by biodiesel sources, and inflammatory process- was one of the potential underlying toxicity mechanisms. These observations were consistent across in vitro and in vivo exposures, and this work adds value to the health risk analysis of cleaner fuel alternatives.

Effect of GHX02 on an Asthma-Rhinitis Mouse Model Induced by Ovalbumin and Diesel Particulate Matter.

Fine dust concentrations come in direct contact with the human respiratory system, thereby reducing lung function and causing respiratory diseases such as asthma and rhinitis. The aim of this study was to evaluate the efficacy of GHX02 (combination of four herbs [Trichosanthes kirilowii, Prunus armeniaca, Coptis japonica, and Scutellaria baicalensis]), a herbal extract with established efficacy against bronchitis and pulmonary disease, in the treatment of asthma accompanied by rhinitis aggravated by fine dust. Therefore, we constructed an asthma-rhinitis mouse model of Balb/c mice challenged with ovalbumin (OVA) and fine diesel particulate matter, which were administered with three concentrations of GHX02. GHX02 significantly inhibited the increase of total cells and immune cells in bronchoalveolar lavage fluid, lung tissue, and nasal ductal lymphoid tissue (NALT). GHX02 also reduced the severity of histological lung injury and the expression of interleukin (IL)-1α and nuclear factor kappa B (NF-κB), which regulate inflammatory responses. The results indicate that GHX02 inhibited the inflammatory immune response in mice. Therefore, this study highlights the potential of GHX02 as a treatment for patients with asthma accompanied by rhinitis. Balb/c mice were challenged with OVA and PM10D, and then treated with three concentration of GHX02. GHX02 significantly inhibited the increase of total cells, immune cells lymphocytes, neutrophils, and macrophages, as well as their expression in lung tissue. GHX02 significantly inhibited the increase of total cells and immune cells in NALT. GHX02 decreased the severity of histological lung injury, expression of IL-1α and NF-κB. This study suggests the probability that GHX02 is effective for asthma patients with rhinitis by inhibiting inflammatory immune response.

Guggulsterone protects against cigarette smoke-induced COPD linked lung inflammation.

Recently, we have shown that guggulsterone is the principal constituent responsible for protective effects of Commiphora wightii against elastase-induced chronic obstructive pulmonary disease (COPD)-linked inflammation/emphysema. Given that cigarette smoke (CS) exposure is a primary risk factor for COPD and beneficial effects of guggulsterone have not been investigated in CS-induced COPD-linked lung inflammation. The present work was designed to validate the potential of guggulsterone in amelioration of COPD-linked lung inflammation by using a CS-based mouse model of the condition. Male BALB/c mice were exposed to 9 cigarettes/day with 1 h interval for 4 days daily. Guggulsterone was administered daily at a dose of 10 mg/kg orally for 4 consecutive days, 1 h before initiation of CS exposure. Mice were subjected to measurement of lung function followed by procurement of bronchoalveolar lavage fluid (BALF)/lung tissue. BALF was analyzed for inflammatory cells and pro-inflammatory cytokines. Lung tissue was subjected to RT-PCR for gene expression analysis. Data showed that CS exposure resulted in a significant increase in total BALF cells, predominantly neutrophils, and macrophages. Interestingly, guggulsterone administration significantly blunted CS-induced inflammation as reflected by reduced neutrophil and macrophage count. Further, the compound inhibited CS-induced gene expression of pro-inflammatory mediators TNF-α/ IL-1β/ G-CSF/and KC in lungs along with the production of pro-inflammatory mediators TNF-α/ IL-1β/ IL-6/ G-CSF/ KC/and MCP-1 in BALF. Further, guggulsterone improved the lung function parameters upon CS exposure. Analysis of mRNA expression of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 suggests that guggulsterone may restore the fine balance between matrix-degrading proteases and its inhibitor in lung tissue upon CS exposure, which may contribute in the development of emphysema at later stages. Overall, our data show that guggulsterone protects against CS-induced COPD-linked lung inflammation by modulating relevant molecular players. Based on the potential effects of guggulsterone in the amelioration of CS-induced lung inflammation, we speculate that guggulsterone might alter chronic CS-induced emphysema.

KLF5-mediated pyroptosis of airway epithelial cells leads to airway inflammation in asthmatic mice through the miR-182–5p/TLR4 axis

Asthma is viewed as an airway disease and an inflammatory condition. This study aims to reveal the role of Kruppel-like factor 5 (KLF5)-mediated pyroptosis of airway epithelial cells in airway inflammation in asthma. The asthmatic mouse model was established. The mice were infected with the lentivirus containing sh-KLF5, antagomiR-182–5p, and pc-Toll-like receptor 4 (TLR4). Airway hyperresponsiveness was measured, and the cells in bronchoalveolar lavage fluid (BALF) were sorted and counted. The expression levels of interleukin (IL)-4/IL-13/IL-6/IL-18/IL-1β/NOD-like receptor family pyrin domain containing 3 (NLRP3)/N-gasdermin D (GSDMD-N)/cleaved caspase-1 were detected. The pathological changes in lung tissue were observed. The enrichment of KLF5 in the miR-182–5p promoter region was measured. The binding relationship among KLF5, miR-182–5p, and TLR4 were analyzed. KLF5 was highly expressed in asthmatic mice. Silencing KLF5 improved airway resistance and lung dynamic compliance, reduced the cells in BALF and the expression of IL-4/IL-13/IL-6/NLRP3/GSDMD-N/cleaved caspase-1/IL-18/IL-1β, and alleviated the pathological changes. Mechanistically, KLF5 bonded to the miR-182–5p promoter to inhibit miR-182–5p expression, and miR-182–5p inhibited TLR4. Silencing miR-182–5p or TLR4 overexpression reversed the improvement of silencing KLF5 on airway inflammation and pyroptosis in asthmatic mice. In conclusion, KLF5 inhibited miR-182–5p to promote TLR4 expression, thus aggravating pyroptosis and airway inflammation in asthmatic mice.

Bufei Huoxue capsule attenuates COPD-related inflammation and regulates intestinal microflora, metabolites.

Background: Bufei Huoxue capsule (BFHX) is widely used for the clinical treatment of chronic obstructive pulmonary disease (COPD) in China. Objectives: The aim of this study is to explore the effects on COPD and the underlying mechanism of BFHX. The process and methods: In this study, we established a COPD mouse model through cigarette smoke (CS) exposure in combination with lipopolysaccharide (LPS) intratracheal instillation. Subsequently, BFHX was orally administrated to COPD mice, and their pulmonary function, lung pathology, and lung inflammation, including bronchoalveolar lavage fluid (BALF) cell count and classification and cytokines, were analyzed. In addition, the anti-oxidative stress ability of BFHX was detected by Western blotting, and the bacterial diversity, abundance, and fecal microbiome were examined using 16S rRNA sequencing technology. Outcome: BFHX was shown to improve pulmonary function, suppress lung inflammation, decrease emphysema, and increase anti-oxidative stress, whereas 16S rRNA sequencing indicated that BFHX can dynamically regulate the diversity, composition, and distribution of the intestinal flora microbiome and regulate the lysine degradation and phenylalanine metabolism of COPD mice. These results highlight another treatment option for COPD and provide insights into the mechanism of BFHX.

Altered circular RNA expressions in extracellular vesicles from bronchoalveolar lavage fluids in mice after bacterial infections.

Circular RNAs (circRNAs) are a class of transcripts that often are generated by back-splicing that covalently connects the 3'end of the exon to the 5'end. CircRNAs are more resistant to nuclease and more stable than their linear counterparts. One of the well-recognized roles of circRNAs is the miRNA sponging effects that potentially lead to the regulation of downstream proteins. Despite that circRNAs have been reported to be involved in a wide range of human diseases, including cancers, cardiovascular, and neurological diseases, they have not been studied in inflammatory lung responses. Here, we analyzed the circRNA profiles detected in extracellular vesicles (EVs) obtained from the broncho-alveolar lavage fluids (BALF) in response to LPS or acid instillation in mice. Next, we validated two specific circRNAs in the BALF-EVs and BALF cells in response to endotoxin by RT-qPCR, using specific primers targeting the circular form of RNAs rather than the linear host RNAs. The expression of these selected circRNAs in the BALF inflammatory cells, alveolar macrophages (AMs), neutrophils, and lung tissue were analyzed. We further predicted the potential miRNAs that interact with these circRNAs. Our study is the first report to show that circRNAs are detectable in BALF EVs obtained from mice. The EV-cargo circRNAs are significantly altered by the noxious stimuli. The circRNAs identified using microarrays may be validated by RT-qPCR using primers specific to the circular but not the linear form. Future studies to investigate circRNA expression and function including miRNA sponging in lung inflammation potentially uncover novel strategies to develop diagnostic/therapeutic targets.

Loranthus tanakae Franch. and Sav. Attenuates Respiratory Inflammation Caused by Asian Sand Dust.

Asian sand dust (ASD), generally produced in East Asia, including China, Japan, and Korea, directly leads to the development of pulmonary disease and exacerbates underlying pulmonary diseases. Loranthus tanakae Franch. and Sav. is a traditional herbal medicine applied to improve various inflammatory conditions. Here, we evaluated the curative properties of L. tanakae ethanol extract (LTE) against pulmonary inflammation caused by ASD. Additionally, to investigate the mechanism of action of LTE, we performed network pharmacological analysis. ASD was administrated on day 1, 3, and 5 by intranasal instillation, and LTE was orally administered for 6 days. Administration of LTE significantly decreased inflammatory cytokines and the number of inflammatory cells in bronchoalveolar lavage fluid, which was accompanied by a decrease in inflammatory cell accumulation in pulmonary tissue. Administration of LTE decreased the expression of cyclooxygenase2 and matrix metalloproteinase-9 in mice exposed to ASD with the decline in p65 phosphorylation. Additionally, administration of LTE significantly elevated hemeoxygenase (HO)-1 expression in the pulmonary tissue of mice exposed to ASD. These results were consistent with the data of network pharmacological analysis. This experiment showed that LTE attenuated pulmonary inflammation caused by ASD via inhibition of NF-κB and elevation of HO-1. Therefore, LTE may have potential as a therapeutic agent to treat pulmonary inflammation caused by ASD.

Role of macrophage bioenergetics in N-acetylcysteine-mediated mitigation of lung injury and oxidative stress induced by nitrogen mustard

Nitrogen mustard (NM) is a toxic vesicant that causes acute injury to the respiratory tract. This is accompanied by an accumulation of activated macrophages in the lung and oxidative stress which have been implicated in tissue injury. In these studies, we analyzed the effects of N-acetylcysteine (NAC), an inhibitor of oxidative stress and inflammation on NM-induced lung injury, macrophage activation and bioenergetics. Treatment of rats with NAC (150 mg/kg, i.p., daily) beginning 30 min after administration of NM (0.125 mg/kg, i.t.) reduced histopathologic alterations in the lung including alveolar interstitial thickening, blood vessel hemorrhage, fibrin deposition, alveolar inflammation, and bronchiolization of alveolar walls within 3 d of exposure; damage to the alveolar-epithelial barrier, measured by bronchoalveolar lavage fluid protein and cells, was also reduced by NAC, along with oxidative stress as measured by heme oxygenase (HO)-1 and Ym-1 expression in the lung. Treatment of rats with NAC attenuated the accumulation of macrophages in the lung expressing proinflammatory genes including Ptgs2, Nos2, Il-6 and Il-12; macrophages expressing inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)α protein were also reduced in histologic sections. Conversely, NAC had no effect on macrophages expressing the anti-inflammatory proteins arginase-1 or mannose receptor, or on NM-induced increases in matrix metalloproteinase (MMP)-9 or proliferating cell nuclear antigen (PCNA), markers of tissue repair. Following NM exposure, lung macrophage basal and maximal glycolytic activity increased, while basal respiration decreased indicating greater reliance on glycolysis to generate ATP. NAC increased both glycolysis and oxidative phosphorylation. Additionally, in macrophages from both control and NM treated animals, NAC treatment resulted in increased S-nitrosylation of ATP synthase, protecting the enzyme from oxidative damage. Taken together, these data suggest that alterations in NM-induced macrophage activation and bioenergetics contribute to the efficacy of NAC in mitigating lung injury.

Mesenchymal Stem Cell-Derived Extracellular Vesicle Therapy for Asthma in Murine Models: A Systematic Review and Meta-analysis.

Asthma is a common disease, and among the most predominant causes of the years lived with disability. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as a promising avenue for asthma management. The objective of this study is to perform a systematic review and meta-analysis of pre-clinical studies investigating the therapeutic use of MSC-EVs in murine models of asthma. A systematic search of electronic databases was performed. Meta-analyses were conducted on broncho-alveolar lavage fluid (BALF) cells and cytokines, as well as airway hyper-responsiveness Penh values and histological staining scores to determine the efficacy of MSC-EVs-based therapy, comparing treated rodents with untreated ones. BALF IL-4, BALF total cells, and BALF eosinophils were chosen as the primary outcomes, while airway hyper-responsiveness Penh values, BALF cytokines excluding IL-4, and histological staining scores were chosen as secondary outcomes. A total of 19 eligible studies were included in the current systematic review, with 9 assessing BALF IL-4, 11 assessing BALF total cells, and 10 assessing BALF eosinophils. Pooled Hedges' g(p-value) for each outcome was - 4.407 (< 0.001), -4.976 (< 0.001), and - 4.071 (< 0.001), showing that MSC-EVs therapy inhibits asthma pathology. Changes in secondary outcomes also indicated a reduction in inflammation, goblet cell hyperplasia, and airway hyper-responsiveness. Subgroup analyses did not reveal significant disparities between the type of rodents and administration routes, and meta-regressions were only significant for MSC-EVs source and dose in the IL-4 meta-analysis, and for administration frequency and time from the last challenge to sacrifice in the BALF total cell meta-analysis. This review highlights the current pre-clinical evidence of MSC-EVs therapy for asthma and finds its application ameliorates multiple aspects of asthma's pathology. We further underline the importance of MSC-EVs source, dose, administration frequency, and timing on the therapeutic effect and warrant further investigation and clinical translation to assess the best treatment regimen and to gauge the efficacy of EV therapy in human asthma cases.

Intervention with ICOSL Antibodies Alleviates Inflammatory Infiltrations in Mice with Neutrophilic Asthma.

Neutrophilic asthma is characterized by the predominant infiltration of neutrophils in airway inflammation. To explore the therapeutic potential of an antibody against the inducible T cell co-stimulator ligand (ICOSL) in a mouse model of neutrophilic asthma. Female BALB/c mice were randomly assigned to different groups. They were then injected with ovalbumin (OVA)/lipopolysaccharides (LPS) to induce neutrophilic asthma. The mice were then treated with either anti-ICOSL (the I group), control IgG (the G group), or no treatment (the N group). Additionally, a control group of mice received vehicle PBS and was labeled as the C group (n=6 per group). One day after the last allergen exposure, cytokine levels were measured in plasma and bronchoalveolar lavage fluid (BALF) using ELISA. After analyzing and categorizing BALF cells, the lung tissues were examined histologically and immunohistochemically. Administering anti-ICOSL resulted in a significant decrease in the total number of inflammatory infiltrates and neutrophils found in BALF. Moreover, it led to a decrease in the levels of interleukin (IL)-6, IL-13, and IL-17 in both BALF and plasma. Additionally, there was an increase in IFN-γ levels in the BALF of asthmatic mice (p<0.05 for all). Treatment with anti-ICOSL also reduced lung interstitial inflammation, mucus secretion, and ICOSL expression in asthmatic mice. The treatment of anti-ICOSL effectively improved lung interstitial inflammation and mucus secretion in mice with neutrophilic asthma by restoring the balance of Th1/Th2/Th17 responses. These findings indicate that blocking the ICOS/ICOSL signaling could be an effective way to manage neutrophilic asthma.

Dectin-1 aggravates neutrophil inflammation through caspase-11/4-mediated macrophage pyroptosis in asthma

BackgroundThe pattern recognition receptor Dectin-1 was initially discovered to play a pivotal role in mediating pulmonary antifungal immunity and promoting neutrophil-driven inflammation. Recent studies have revealed that Dectin-1 is overexpressed in asthma, but the specific mechanism remains elusive. Additionally, Dectin-1 has been implicated in promoting pyroptosis, a hallmark of severe asthma airway inflammation. Nevertheless, the involvement of the non-classical pyroptosis signal caspase-11/4 and its upstream regulatory mechanisms in asthma has not been completely explored.MethodsHouse dust mite (HDM)-induced mice was treated with Dectin-1 agonist Curdlan, Dectin-1 inhibitor Laminarin, and caspase-11 inhibitor wedelolactone separately. Subsequently, inflammatory cells in bronchoalveolar lavage fluid (BALF) were analyzed. Western blotting was performed to measure the protein expression of caspase-11 and gasdermin D (GSDMD). Cell pyroptosis and the expression of chemokine were detected in vitro. The correlation between Dectin-1 expression, pyroptosis factors and neutrophils in the induced sputum of asthma patients was analyzed.ResultsCurdlan appeared to exacerbate neutrophil airway inflammation in asthmatic mice, whereas wedelolactone effectively alleviated airway inflammation aggravated by Curdlan. Moreover, Curdlan enhanced the release of caspase-11 activation fragments and N-terminal fragments of gasdermin D (GSDMD-N) stimulated by HDM both in vivo or in vitro. In mouse alveolar macrophages (MH-S cells), Curdlan/HDM stimulation resulted in vacuolar degeneration and elevated lactate dehydrogenase (LDH) release. In addition, there was an upregulation of neutrophil chemokines CXCL1, CXCL3, CXCL5 and their receptor CXCR2, which was suppressed by wedelolactone. In asthma patients, a positive correlation was observed between the expression of Dectin-1 on macrophages and caspase-4 (the human homology of caspase-11), and the proportion of neutrophils in induced sputum.ConclusionDectin-1 activation in asthma induced caspase-11/4 mediated macrophage pyroptosis, which subsequently stimulated the secretion of chemokines, leading to the exacerbation of airway neutrophil inflammation.Graphical

Direct vagus nerve stimulation: A new tool to control allergic airway inflammation through α7 nicotinic acetylcholine receptor.

Asthma is characterized by airway inflammation, mucus hypersecretion, and airway hyperresponsiveness. The use of nicotinic agents to mimic the cholinergic anti-inflammatory pathway (CAP) controls experimental asthma. Yet, the effects of vagus nerve stimulation (VNS)-induced CAP on allergic inflammation remain unknown. BALB/c mice were sensitized and challenged with house dust mite (HDM) extract and treated with active VNS (5Hz, 0.5ms, 0.05-1mA). Bronchoalveolar lavage (BAL) fluid was assessed for total and differential cell counts and cytokine levels. Lungs were examined by histopathology and electron microscopy. In the HDM mouse asthma model, VNS at intensities equal to or above 0.1mA (VNS 0.1) but not sham VNS reduced BAL fluid differential cell counts and alveolar macrophages expressing α7 nicotinic receptors (α7nAChR), goblet cell hyperplasia, and collagen deposition. Besides, VNS 0.1 also abated HDM-induced elevation of type 2 cytokines IL-4 and IL-5 and was found to block the phosphorylation of transcription factor STAT6 and expression level of IRF4 in total lung lysates. Finally, VNS 0.1 abrogated methacholine-induced hyperresponsiveness in asthma mice. Prior administration of α-bungarotoxin, a specific inhibitor of α7nAChR, but not propranolol, a specific inhibitor of β2-adrenoceptors, abolished the therapeutic effects of VNS 0.1. Our data revealed the protective effects of VNS on various clinical features in allergic airway inflammation model. VNS, a clinically approved therapy for depression and epilepsy, appears to be a promising new strategy for controlling allergic asthma.

Metabolomics combined with network pharmacology reveals anti-asthmatic effects of Nepeta bracteata on allergic asthma rats

ObjectiveTo investigate the mechanisms that underlie the anti-asthmatic effects of Nepeta bracteata (DBJJ, Dabao Jingjie in Chinese) in rats by integrating metabolomics and network pharmacology. MethodsIn this study, the rat model of asthma was induced by using ovalbumin (OVA), and subsequently, the rats were treated with a decoction of N. bracteata. Pathological changes in lung tissue were observed, and the quantification of eosinophils (EOS) and white blood cells (WBC) in bronchoalveolar lavage fluid (BALF) was performed. Furthermore, the serum levels of asthma-related factors induced by OVA were assessed. The proton nuclear magnetic resonance (1H NMR) spectroscopy serum metabolomics method was utilized to identify differential metabolites and their associated metabolic pathways. Moreover, UPLC-QE-MS/MS combined with network pharmacology was employed to predict the core targets and pathways of DBJJ in its action against asthma. Furthermore, the anti-asthmatic properties of DBJJ were investigated using an integrated approach of metabolomics and network pharmacology. Subsequently, the findings were validated through molecular docking and Western blotting analysis of the key targets. ResultsThe findings indicated that the administration of DBJJ effectively alleviated OVA-induced lung histopathological changes and decreased the number of EOS and WBC in BALF. Additionally, DBJJ inhibited the OVA-induced elevation of TNF-α, IL-18, Ig-E, EOS, IL-1β, MDA, VEGF-A, and TGF-β1. A total of 21 biomarkers and 10 pathways were found by metabolomics analysis. The 29 compounds were identified by UPLC-QE-MS/MS and 13 active components were screened by oral availability and Caco-2 cell permeability, the 120 targets and 173 KEGG pathways were predicted. The integration of metabolomics and network pharmacological analysis revealed that DBJJ's primary constituents, including ferulic acid and ursolic acid, exerted their effects on four targets, namely DAO and NOS2, as well as their associated metabolites and pathways. The active constituents of DBJJ demonstrated a high binding affinity towards DAO and NOS2. Furthermore, DBJJ was observed to decrease the protein expression and phosphorylation levels of NOS2, MAPK, and STAT3. ConclusionThe administration of DBJJ demonstrates notable anti-asthma properties in rats with allergic asthma. This effect can be attributed to the modulation of various targets, including NOS2, MAPK, and STAT3, by primary constituents such as ferulic acid and ursolic acid.

Critical roles of PAI-1 in lipopolysaccharide-induced acute lung injury

PurposePlasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of fibrinolytic systems. The effect of PAI-1 on inflammatory response is still inconsistent. Our study was conducted to investigate its effects on inflammation to clarify the role of PAI-1 in acute lung injury (ALI) induced by lipopolysaccharide (LPS). Material and methodsALI models were established in wild-type (WT) and PAI-1 knockout (KO) mice by LPS intervention for 48 ​h. Lung histopathology, wet-dry ratio, total cell count and TNF-α concentration in bronchoalveolar lavage fluid (BALF), and inflammation related proteins were detected. Flow cytometry was used to sort neutrophils, macrophages, regulatory T cells (Treg) and T helper cell 17 (Th17). RNA sequencing was performed to find differentially expressed genes. Masson staining and immunohistochemistry were used to analyze pulmonary fiber deposition and proliferation. ResultsCompared with ALI (WT) group, the wet-dry ratio, the total number of BALF cells, the concentration of TNF-α in BALF, and the expression of pp65 in the lung tissue was increased in ALI (PAI-1 KO) group, with increased proportion of neutrophils, decreased proportion of macrophages and decreased proportion of Treg/Th17 in the lung tissue. Collagen fiber deposition and PCNA expression were lighter in ALI (PAI-1 KO) group than ALI (WT) group. PPI analysis showed that PAI-1 was closely related to TNF, IL-6, IL-1β, Smad2/3 and mainly concentrated in the complement and coagulation system, TNF-α and IL-17 signaling pathways. ConclusionsPAI-1 KO could aggravate ALI induced by LPS at 48 ​h. PAI-1 may be an important target to improve the prognosis of ALI.

GDF3 Protects Mice against Sepsis-Induced Acute Lung Injury by Suppression of Macrophage Pyroptosis.

Sepsis-induced ALI is marked by physiological, pathological, and biochemical irregularities caused by infection. Growth differentiation factor 3 (GDF3) is closely associated with the inflammatory response. Accumulating evidence has demonstrated a close relationship between GDF3 expression and the severity and prognosis of sepsis. However, the precise mechanism by which GDF3 protects against ALI induced by sepsis is still unclear. Following the intravenous administration of GDF3 in this research, we noted a rise in the survival rate, a decrease in the severity of histopathological damage as evaluated through HE staining, a decline in the count of inflammatory cells in bronchoalveolar lavage fluid (BALF), a reduction in the ratio of lung wet/dry (W/D) weight, and a noteworthy decrease in the levels of pro-inflammatory cytokines in both serum and BALF when compared to septic mice who underwent cecal ligation and puncture (CLP). These collective findings unequivocally indicate the protective effects of GDF3 against sepsis-induced ALI. In addition, the GDF3 group showed a significant reduction in the mRNA expression of Caspase-1 and NLRP3 when compared to the CLP group. Following this, we performed in vitro tests to confirm these discoveries and obtained comparable outcomes, wherein the administration of GDF3 notably decreased the levels of Caspase-1 and NLRP3 mRNA and protein in macrophages in comparison to the LPS group. Furthermore, GDF3 exhibited the capacity to reduce the secretion of inflammatory molecules from macrophages. By illuminating the mechanism by which GDF 3 regulates macrophages, this offers a theoretical basis for preventing and treating sepsis-induced ALI.

Jiawei Yanghe Decoction attenuate allergic airway inflammation by suppressing group 2 innate lymphoid cells responses

Ethnopharmacological relevanceJiawei Yanghe Decoction (JWYHD) is modified Yanghe Decoction (YHD). YHD historically utilized as a potent medicinal solution for addressing chronic inflammatory conditions, holds promising therapeutic potential in the treatment of asthma. However, the mechanisms underlying JWYHD's effects on allergic asthma remain unclear. Aim of the studyTo investigate the therapeutic effect as well as the underlying mechanisms of JWYHD on asthmatic mice. Materials and methodsThe ovalbumin (OVA)-induced mouse model was utilized, followed by the administration of JWYHD to allergic asthmatic mice. Subsequently, inflammatory cells in the bronchoalveolar lavage fluid (BALF) and lung tissues were conducted. The levels of various cytokines including interleukin (IL)-4, IL-5, IL-13, IL-33, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in BALF, as well as the total immunoglobulin E (IgE) content in serum, were assessed. Lung function and tissue pathology examinations were performed to assess the protective impacts of JWYHD. The chemical components of JWYHD and its lung prototype compounds (referred to the chemical components present in JWYHD that were observed in the lung) were explored by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). RNA-seq analysis revealed the regulation mechanisms of JWYHD treating asthma. Furthermore, the effect of JWYHD on type 2 innate lymphoid cells (ILC2s) in asthmatic mice was detected by flow cytometry and Smart-RNA-seq analysis. Then molecular docking analysis was used to show the interaction between identified compounds and key targets. ResultsJWYHD significantly attenuated the airway inflammation of asthmatic mice, reduced the levels of inflammatory cells in BALF, as well the levels of the cytokines IL-4, IL-5, IL-13, IL-33, and TNF-α in BALF and IgE in serum. Airway hyperresponsiveness (AHR) and lung inflammation infiltration were also alleviated by JWYHD. Moreover, RNA-seq analysis revealed that JWYHD attenuated airway inflammation in asthmatic mice via regulating immunity. Flow cytometry confirmed that JWYHD could inhibit ILC2 responses. ILC2 Smart-RNA-seq analysis showed that JWYHD impaired the inflammation reaction-related signaling pathways in ILC2s, and neuropilin-1 (Nrp1), endothelial transcription factor 3 (GATA3) and interleukin 1 receptor like protein 1 (ST2) might be the key targets. The molecular docking analysis investigating the connection between the primary targets and JWYHD's prototype compounds in the lung demonstrated that liquiritin apioside, icariin, glycyrrhizic acid, and uralsaponin B, identified through UPLC-Q-TOF/MS, exhibited significant affinity in binding to the mentioned key targets. ConclusionOur results suggested that the mechanism of JWYHD in treating asthma might be related to limiting ILC2 responses. Our findings provided some pharmacological evidence for the clinical application of JWYHD in the treatment of asthma.

Urolithin A exerts a protective effect on lipopolysaccharide-induced acute lung injury by regulating HMGB1-mediated MAPK and NF-κB signaling pathways.

Acute lung injury (ALI) is a severe inflammatory disorder that has a high morbidity and mortality rate. Urolithin A (UA) is reported to have anti-inflammatory and anti-oxidative effects in ALI. However, its molecular mechanisms in ALI remain to be explored. Mice and BEAS-2B cells were administrated with lipopolysaccharide (LPS) to mimic the ALI model in vivo and in vitro. Hematoxylin-eosin (HE) staining was used to detect the pathological injury of lung tissues. The levels of proinflammatory cytokines in bronchoalveolar lavage fluid (BALF) and culture supernatant and the levels of oxidative stress markers in lung tissues were measured using ELISA. DCFH-DA probe was used to assess the reactive oxygen species (ROS) level. TUNEL staining and flow cytometry were performed to determine cell apoptosis. The key targets and pathways were confirmed by immunohistochemistry (IHC) and western blot. UA suppressed the pathologic damage, wet/dry weight ratio, and total protein and inflammatory cells in BALF. UA decreased neutrophil infiltration and proinflammatory cytokines production. UA reduced the level of malondialdehyde (MDA) and increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in pulmonary tissues. UA also inhibited cell apoptosis in lung tissues by decreasing Bax expression and increasing Bcl-2 expression. In addition, UA suppressed LPS-induced inflammatory factor production, ROS level, and cell apoptosis in BEAS-2B. Importantly, UA decreased the expression of HMGB1 in LPS-treated mice and BEAS-2B cells. HMGB1 overexpression greatly abrogated the inhibition of UA on inflammation, ROS, and cell apoptosis in LPS-administrated BEAS-2B. Furthermore, UA treatment suppressed the phosphorylated levels of p38, JNK, ERK, and p65 in LPS-administrated mice and BEAS-2B cells. UA alleviated lung inflammation, oxidative stress, and apoptosis in ALI by targeting HMGB1 to inactivate the MAPK/NF-κB signaling, suggesting the potential of UA to treat ALI.

Pulmonary evaluation of whole-body inhalation exposure of polycarbonate (PC) filament 3D printer emissions in rats

ABSTRACT During fused filament fabrication (FFF) 3D printing with polycarbonate (PC) filament, a release of ultrafine particles (UFPs) and volatile organic compounds (VOCs) occurs. This study aimed to determine PC filament printing emission-induced toxicity in rats via whole-body inhalation exposure. Male Sprague Dawley rats were exposed to a single concentration (0.529 mg/m3, 40 nm mean diameter) of the 3D PC filament emissions in a time-course via whole body inhalation for 1, 4, 8, 15, and 30 days (4 hr/day, 4 days/week), and sacrificed 24 hr after the last exposure. Following exposures, rats were assessed for pulmonary and systemic responses. To determine pulmonary injury, total protein and lactate dehydrogenase (LDH) activity, surfactant proteins A and D, total as well as lavage fluid differential cells in bronchoalveolar lavage fluid (BALF) were examined, as well as histopathological analysis of lung and nasal passages was performed. To determine systemic injury, hematological differentials, and blood biomarkers of muscle, metabolic, renal, and hepatic functions were also measured. Results showed that inhalation exposure induced no marked pulmonary or systemic toxicity in rats. In conclusion, inhalation exposure of rats to a low concentration of PC filament emissions produced no significant pulmonary or systemic toxicity.