Total Protein In Bronchoalveolar Lavage Fluid Research Articles

The efficacy of extracellular vesicles for acute lung injury in preclinical animal models: a meta-analysis

BackgroundWith the increasing research on extracellular vesicles (EVs), EVs have received widespread attention as biodiagnostic markers and therapeutic agents for a variety of diseases. Stem cell-derived EVs have also been recognized as a new viable therapy for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). To assess their efficacy, we conducted a meta-analysis of existing preclinical experimental animal models of EVs for ALI treatment.MethodsThe database was systematically interrogated for pertinent data encompassing the period from January 2010 to April 2022 concerning interventions involving extracellular vesicles (EVs) in animal models of acute lung injury (ALI). The lung injury score was selected as the primary outcome measure for statistical analysis. Meta-analyses were executed utilizing RevMan 5.3 and State15.1 software tools.ResultsThe meta-analyses comprised 31 studies, exclusively involving animal models of acute lung injury (ALI), categorized into two cohorts based on the presence or absence of extracellular vesicle (EV) intervention. The statistical outcomes from these two study groups revealed a significant reduction in lung injury scores with the administration of stem and progenitor cell-derived EVs (SMD = -3.63, 95% CI [-4.97, -2.30], P < 0.05). Conversely, non-stem cell-derived EVs were associated with an elevation in lung injury scores (SMD = -4.34, 95% CI [3.04, 5.63], P < 0.05). EVs originating from stem and progenitor cells demonstrated mitigating effects on alveolar neutrophil infiltration, white blood cell counts, total cell counts in bronchoalveolar lavage fluid (BALF), lung wet-to-dry weight ratios (W/D), and total protein in BALF. Furthermore, pro-inflammatory mediators exhibited down-regulation, while anti-inflammatory mediators demonstrated up-regulation. Conversely, non-stem cell-derived EVs exacerbated lung injury.ConclusionIn preclinical animal models of acute lung injury (ALI), the administration of extracellular vesicles (EVs) originating from stem and progenitor cells demonstrably enhances pulmonary function. This ameliorative effect is attributed to the mitigation of pulmonary vascular permeability and the modulation of immune homeostasis, collectively impeding the progression of inflammation. In stark contrast, the utilization of EVs derived from non-stem progenitor cells exacerbates the extent of lung injury. These findings substantiate the potential utility of EVs as a novel therapeutic avenue for addressing acute lung injury.

TRPV4 Regulates the Macrophage Metabolic Response to Limit Sepsis-induced Lung Injury.

Sepsis is a systemic inflammatory response that requires effective macrophage metabolic functions to resolve ongoing inflammation. Previous work showed that the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), mediates macrophage phagocytosis and cytokine production in response to lung infection. Here, we show that TRPV4 regulates glycolysis in a stiffness dependent manner by augmenting macrophage glucose uptake by GLUT1. In addition, TRPV4 is required for lipopolysaccharide (LPS)-induced phagolysosome maturation in a GLUT1-dependent manner. In a cecal slurry mouse model of sepsis, TRPV4 regulates sepsis-induced glycolysis as measured by bronchoalveolar lavage fluid (BALF) lactate and sepsis-induced lung injury as measured by BALF total protein and lung compliance. TRPV4 is necessary for bacterial clearance in the peritoneum to limit sepsis-induced lung injury. Interestingly, BALF lactate is increased in septic patients compared with healthy controls, supporting the relevance of lung cell glycolysis to human sepsis. These data show that macrophage TRPV4 is required for glucose uptake through GLUT1 for effective phagolysosome maturation to limit sepsis-induced lung injury. Our work presents TRPV4 as a potential target to protect the lung from injury in sepsis.

Protein-Protein interactive networks identified in bronchoalveolar lavage of severe compared to nonsevere asthma.

Previous bronchoalveolar lavage fluid (BALF) proteomic analysis has evaluated limited numbers of subjects for only a few proteins of interest, which may differ between asthma and normal controls. Our objective was to examine a more comprehensive inflammatory biomarker panel in quantitative proteomic analysis for a large asthma cohort to identify molecular phenotypes distinguishing severe from nonsevere asthma. Bronchoalveolar lavage fluid from 48 severe and 77 nonsevere adult asthma subjects were assessed for 75 inflammatory proteins, normalized to BALF total protein concentration. Validation of BALF differences was sought through equivalent protein analysis of autologous sputum. Subjects' data, stratified by asthma severity, were analysed by standard statistical tests, principal component analysis and 5 machine learning algorithms. The severe group had lower lung function and greater health care utilization. Significantly increased BALF proteins for severe asthma compared to nonsevere asthma were fibroblast growth factor 2 (FGF2), TGFα, IL1Ra, IL2, IL4, CCL8, CCL13 and CXCL7 and significantly decreased were platelet-derived growth factor a-a dimer (PDGFaa), vascular endothelial growth factor (VEGF), interleukin 5 (IL5), CCL17, CCL22, CXCL9 and CXCL10. Four protein differences were replicated in sputum. FGF2, PDGFaa and CXCL7 were independently identified by 5 machine learning algorithms as the most important variables for discriminating severe and nonsevere asthma. Increased and decreased proteins identified for the severe cluster showed significant protein-protein interactions for chemokine and cytokine signalling, growth factor activity, and eosinophil and neutrophil chemotaxis differing between subjects with severe and nonsevere asthma. These inflammatory protein results confirm altered airway remodelling and cytokine/chemokine activity recruiting leukocytes into the airways of severe compared to nonsevere asthma as important processes even in stable status.

Toll-interacting protein inhibits transforming growth factor beta signaling in mouse lung fibroblasts.

Variations in the Toll-interacting protein (TOLLIP) gene have been identified in genome-wide association studies to correlate with risk of disease, mortality, and response to N-acetylcysteine therapy in idiopathic pulmonary fibrosis. Although TOLLIP is known to modulate innate immune responses, its relevance in organ fibrogenesis remains unknown. Prior work in the literature suggests TOLLIP dampens transforming growth factor beta (TGFβ) signaling in human cell lines. In this study, we examined the role of TOLLIP in mouse lung fibroblast (MLF) responses to TGFβ and in the bleomycin model of experimental lung fibrosis using Tollip-/- mice. We hypothesize that if TOLLIP negatively regulates TGFβ signaling, then Tollip-/- mouse lung fibroblasts (MLFs) would have enhanced response to TGFβ treatment, and Tollip-/- mice would develop increased fibrosis following bleomycin challenge. Primary MLFs were stimulated with TGFβ (1 ng/mL) for 24 h. RNA was obtained to assess global transcriptional responses by RNA-seq and markers of myofibroblast transition by qPCR. Functional assessment of TGFβ-stimulated MLFs included cell migration by scratch assay, cell proliferation, and matrix invasion through Matrigel. In the invivo model of lung fibrosis, Tollip-/- mice and wild-type (WT) littermates were administered bleomycin intratracheally and assessed for fibrosis. We further examined TGFβ signaling invivo after bleomycin injury by SMAD2, ERK1/2, and TGFβR1 Western blot. In response to TGFβ treatment, both WT and Tollip-/- MLFs exhibited global transcriptional changes consistent with myofibroblast differentiation. However, Tollip-/- MLFs showed greater number of differentially expressed genes compared to WT MLFs and greater upregulation of Acta2 by qPCR. Functionally, Tollip-/- MLFs also exhibited increased migration and Matrigel invasiveness compared to WT. We found evidence of enhanced TGFβ signaling in Tollip-/- through SMAD2 invitro and invivo. Tollip-/- mice experienced lower survival using a standard weight-adjusted dosing without evidence of differences in fibrosis at Day 21. With adjustment of dosing for sex, no differences were observed in fibrosis at Day 21. However, Tollip-/- mice had greater weight loss and increased bronchoalveolar lavage fluid total protein during early resolution at Day 14 compared to WT without evidence of differences in acute lung injury at Day 7, suggesting impaired resolution of lung injury.

Adipose tissue-derived mesenchymal stromal cells attenuate acute lung injury induced by trauma and haemorrhagic shock

BackgroundMesenchymal stromal cells (MSCs) have shown promising therapeutic options for acute lung injury (ALI) caused by multiple factors. Here, we evaluated the therapeutic potential of adipose tissue-derived mesenchymal stromal cells (ADSCs) in trauma and hemorrhagic shock (THS)-induced ALI. MethodsALI model induced by THS was constructed by fractures plus abdominal trauma plus acute hemorrhage plus fluid resuscitation. The ADSCs group rats were generated by injecting 2 × 106 ADSCs at 0 and 1 h after THS. The sham, ALI, and ADSCs group rats were sacrificed at 24 h after resuscitation. The changes in lung histopathology, total protein in bronchoalveolar lavage fluid (BALF), mRNA expression of pro-inflammatory/anti-inflammatory cytokines, antioxidant, and anti-apoptotic indicator, and the activity of Toll-like receptor 4 (TLR4) signaling in lung tissues were evaluated. ResultsAdministration of the ADSCs reversed ALI induced by THS, including lung histopathological changes/scores, and BALF total protein concentration. Additionally, ADSCs therapy also significantly down-regulated mRNA expression of pro-inflammatory TNF-α, IL-1β, and IL-6, up-regulated mRNA expression of anti-inflammatory IL-10, anti-apoptotic molecule Bcl-2, and anti-oxidative molecule HO-1 in THS rats. Furthermore, ADSCs suppressed the expression of TLR4 in lung tissue. ConclusionOur data show that ADSCs administration can exert therapeutic effects on THS-induced ALI in rats and may provide beneficial in preventative strategies for ALI.

Role of the protease-activated receptor-2 (PAR2) in the exacerbation of house dust mite-induced murine allergic lung disease by multi-walled carbon nanotubes

BackgroundPulmonary exposure to multi-walled carbon nanotubes (MWCNTs) has been reported to exert strong pro-inflammatory and pro-fibrotic adjuvant effects in mouse models of allergic lung disease. However, the molecular mechanisms through which MWCNTs exacerbate allergen-induced lung disease remain to be elucidated. We hypothesized that protease-activated receptor 2 (PAR2), a G-protein coupled receptor previously implicated in the pathogenesis of various diseases including pulmonary fibrosis and asthma, may play an important role in the exacerbation of house dust mite (HDM) allergen-induced lung disease by MWCNTs.MethodsWildtype (WT) male C57BL6 mice and Par2 KO mice were exposed to vehicle, MWCNTs, HDM extract, or both via oropharyngeal aspiration 6 times over a period of 3 weeks and were sacrificed 3-days after the final exposure (day 22). Bronchoalveolar lavage fluid (BALF) was harvested to measure changes in inflammatory cells, total protein, and lactate dehydrogenase (LDH). Lung protein and RNA were assayed for pro-inflammatory or profibrotic mediators, and formalin-fixed lung sections were evaluated for histopathology.ResultsIn both WT and Par2 KO mice, co-exposure to MWCNTs synergistically increased lung inflammation assessed by histopathology, and increased BALF cellularity, primarily eosinophils, as well as BALF total protein and LDH in the presence of relatively low doses of HDM extract that alone produced little, if any, lung inflammation. In addition, both WT and par2 KO mice displayed a similar increase in lung Cc1-11 mRNA, which encodes the eosinophil chemokine CCL-11, after co-exposure to MWCNTs and HDM extract. However, Par2 KO mice displayed significantly less airway fibrosis as determined by quantitative morphometry compared to WT mice after co-exposure to MWCNTs and HDM extract. Accordingly, at both protein and mRNA levels, the pro-fibrotic mediator arginase 1 (ARG-1), was downregulated in Par2 KO mice exposed to MWCNTs and HDM. In contrast, phosphorylation of the pro-inflammatory transcription factor NF-κB and the pro-inflammatory cytokine CXCL-1 was increased in Par2 KO mice exposed to MWCNTs and HDM.ConclusionsOur study indicates that PAR2 mediates airway fibrosis but not eosinophilic lung inflammation induced by co-exposure to MWCNTs and HDM allergens.

Gossypin exert lipopolysaccharide induced lung inflammation via alteration of Nrf2/HO-1 and NF-κB signaling pathway.

Acute Lung Injury (ALI) is a critical medical condition that induces the injury into the lung tissue, resulting in decreased the oxygen levels in the circulation and finally causes the respiratory failure. In this study, we try to made effort for scrutinized the preventive effect of gossypin against lipopolysaccharide (LPS) induced lung inflammation and explore the underlying mechanism. LPS (7.5 mg/kg) was used for induction the lung inflammation in the rats and rats were received the oral administration of gossypin (5, 10 and 15 mg/kg). The wet to dry weight lung ratio and lung index were estimated. The bronchoalveolar lavage fluid (BALF) were collected to determination the inflammatory cells, total protein, macrophages and neutrophils. ELISA kits were used for the estimation of antioxidant, inflammatory cytokines, inflammatory parameters, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) parameters. Finally, we used the lung tissue for scrutinize the alteration in the lung histopathology. Gossypin treatment significantly (p < .001) reduced the W/D ratio of lung tissue and lung index. Gossypin significantly (p < .001) decreased the total cells, neutrophils, macrophages and total protein in BALF. It is also altered the level of inflammatory cytokines, antioxidant and inflammatory parameters, respectively. Gossypin improved the level of Nrf2 and HO-1 at dose dependent manner. Gossypin treatment remarkably enhance the ALI severity via balancing the structural integrity of lung tissue, decrease the thickness of the alveolar wall, decline the pulmonary interstitial edema, and number of inflammatory cells in the lung tissue. Gossypin is a promising agent for the treatment of LPS induced lung inflammation via altering Nrf2/HO-1 and NF-κB.

Overexpression of FoxM1 optimizes the therapeutic effect of bone marrow mesenchymal stem cells on acute respiratory distress syndrome

BackgroundInjury of alveolar epithelial cells and capillary endothelial cells is crucial in the pathogenesis of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Mesenchymal stem cells (MSCs) are a promising cell source for ALI/ARDS treatment. Overexpression of Fork head box protein M1 (FoxM1) facilitates MSC differentiation into alveolar type II (AT II) cells in vitro. Moreover, FoxM1 has been shown to repair the endothelial barrier. Therefore, this study explored whether overexpression of FoxM1 promotes the therapeutic effect of bone marrow-derived MSCs (BMSCs) on ARDS by differentiation of BMSCs into AT II cells or a paracrine mechanism.MethodsA septic ALI model was established in mice by intraperitoneal administration of lipopolysaccharide. The protective effect of BMSCs-FoxM1 on ALI was explored by detecting pathological variations in the lung, total protein concentration in bronchoalveolar lavage fluid (BALF), wet/dry (W/D) lung weight ratio, oxidative stress levels, cytokine levels, and retention of BMSCs in the lung. In addition, we assessed whether FoxM1 overexpression promoted the therapeutic effect of BMSCs on ALI/ARDS by differentiating into AT II cells using SPC−/− mice. Furthermore, the protective effect of BMSCs-FoxM1 on lipopolysaccharide-induced endothelial cell (EC) injury was explored by detecting EC proliferation, apoptosis, scratch wounds, tube formation, permeability, and oxidative stress, and analyzing whether the Wnt/β-catenin pathway contributes to the regulatory mechanism in vitro using a pathway inhibitor.ResultsCompared with BMSCs-Vector, treatment with BMSCs-FoxM1 significantly decreased the W/D lung weight ratio, total BALF protein level, lung injury score, oxidative stress, and cytokine levels. With the detected track of BMSCs-FoxM1, we observed a low residency rate and short duration of residency in the lung. Notably, SPC was not expressed in SPC−/− mice injected with BMSCs-FoxM1. Furthermore, BMSCs-FoxM1 enhanced EC proliferation, migration, and tube formation; inhibited EC apoptosis and inflammation; and maintained vascular integrity through activation of the Wnt/β-catenin pathway, which was partially reversed by XAV-939.ConclusionOverexpression of FoxM1 enhanced the therapeutic effect of BMSCs on ARDS, possibly through a paracrine mechanism rather than by promoting BMSC differentiation into AT II cells in vivo, and prevented LPS-induced EC barrier disruption partially through activating the Wnt/β-catenin signaling pathway in vitro.

Sappanone A: A natural PDE4 inhibitor with dual anti-inflammatory and antioxidant activities from the heartwood of Caesalpinia sappan L.

Ethnopharmacological relevanceSumu (Lignum sappan), the dry heartwood of Caesalpinia sappan L., is a traditional Chinese medicine used as an analgesic and anti-inflammatory agent. Aim of the studyThe study aspired to discover natural phosphodiesterase 4 (PDE4) inhibitors with dual anti-inflammatory and antioxidant activities from Sumu for the treatment of chronic obstructive pulmonary disease (COPD). Materials and methodsTo accurately and efficiently identify natural PDE4 inhibitors from Sumu, molecular docking and molecular dynamics (MD) analysis methods were used for structure-based virtual screening of a self-built database of primary polyphenols in Sumu. According to the previous studies of Sumu and the free radical scavenging mechanism of polyphenols, the reported antioxidant components from Sumu and the potential antioxidants with the antioxidant pharmacophore of catechol and π-conjugated moieties were selected from the potential PDE4 inhibitors predicted by docking. Sappanone A, a potential PDE4 inhibitor with antioxidant activity from Sumu, was selected, calculated and synthesized to evaluate its dual anti-inflammatory and antioxidant functions in vitro and in vivo studies. Herein sappanone A was assayed for its inhibitory effects against PDE4 enzyme activity, tumor necrosis factor-alpha (TNF-α) production induced by lipopolysaccharide (LPS) in RAW264.7 macrophages and malondialdehyde (MDA) production induced by Fe2+ in mouse lung homogenate; sappanone A was also assayed for its abilities of radical (DPPH) scavenging, reducing Fe3+ and complexing Fe2+ in vitro. Additionally, LPS-induced acute lung injury (ALI) in mice was used to evaluate its anti-inflammatory activity as a PDE4 inhibitor in vivo, and the levels of TNF-α and total protein in bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity in the lung were assayed. ResultsThe present study predicted and validated that sappanone A was a promising PDE4 inhibitor from Sumu with dual anti-inflammation and antioxidant activities from Sumu. In vitro, sappanone A remarkably inhibited PDE4 enzyme activity and reduced TNF-α production induced by LPS in RAW264.7 macrophages and MDA production induced by Fe2+ in mouse lung homogenate. Meanwhile, it showed outstanding abilities of scavenging DPPH radicals, reducing Fe3+ and complexing Fe2+. In vivo, sappanone A (25 mg/kg and 50 mg/kg, i.p., twice daily for 7 days) distinctly prevented LPS-induced ALI in mice by reducing the levels of TNF-α and total protein in BALF and MPO activity in the lung. ConclusionSappanone A is a natural PDE4 inhibitor with dual anti-inflammatory and antioxidant activities from the traditional Chinese medicine Sumu, which may be a promising therapeutic agent to prevent the vicious cycle of COPD inflammation and oxidative stress.

Trimetazidine, a metabolic modulator, attenuates silica-induced pulmonary fibrosis and decreases lactate levels and LDH activity in rats.

Pulmonary fibrosis has been recently linked to metabolic dysregulation. Silica-induced pulmonary fibrosis in rats was employed by the current study to explore the effects of trimetazidine (a metabolic modulator-antianginal drug; TMZ) on silica-induced pulmonary fibrosis. Pulmonary fibrosis was induced by intranasal instillation of silica (50 mg/100 µl/rat) in TMZ versus vehicle-treated rats. Body weights of rats, weights of lungs, and wet-to-dry lung weights were determined. Various parameters were also measured in serum, bronchoalveolar lavage fluid (BALF) in addition to lung tissue homogenates. Moreover, histopathological examination of sectioned lungs for lesion score and distribution and histochemical detection of myeloperoxidase (MPO) in lung tissues were also performed. No significant differences were observed in body weight gains, lung coefficients, lung weights, and wet-to-dry lung weight in silica versus control rats. Elevated lactate levels in serum and lung homogenates were significantly attenuated by TMZ. In addition, lactate dehydrogenase activity, transforming growth factor-β, and total proteins in BALF were significantly normalized with TMZ. Moreover, TMZ significantly increased reduced glutathione and adenosine triphosphate levels and decreased nitrate/nitrite and hydroxyproline content in lungs of silica-treated rats. Histopathological examination of lungs revealed more than 56% reduction in lesion score and distribution by TMZ. MPO expression in lungs of silica-treated rats was also significantly attenuated by TMZ. TMZ attenuates silica-induced pulmonary fibrosis, an effect that could be mediated by suppressing anaerobic glycolysis-induced excessive lactate production. Regulation of oxidative stress could also play a role in TMZ-promoted protective effects.

Diacetyl Vapor Inhalation Induces Mixed, Granulocytic Lung Inflammation with Increased CD4+CD25+ T Cells in the Rat.

Diacetyl (DA) is a highly reactive alpha diketone associated with flavoring-related lung disease. In rodents, acute DA vapor exposure can initiate an airway-centric, inflammatory response. However, this immune response has yet to be fully characterized in the context of flavoring-related lung disease progression. The following studies were designed to characterize the different T cell populations within the lung following repetitive DA vapor exposures. Sprague-Dawley rats were exposed to 200 parts-per-million DA vapor for 5 consecutive days × 6 h/day. Lung tissue and bronchoalveolar lavage fluid (BALF) were analyzed for changes in histology by H&E and Trichrome stain, T cell markers by flow cytometry, total BALF cell counts and differentials, BALF IL17a and total protein immediately, 1 and 2 weeks post-exposure. Lung histology and BALF cell composition demonstrated mixed, granulocytic lung inflammation with bronchial lymphoid aggregates at all time points in DA-exposed lungs compared to air controls. While no significant change was seen in percent lung CD3+, CD4+, or CD8+ T cells, a significant increase in lung CD4+CD25+ T cells developed at 1 week that persisted at 2 weeks post-exposure. Further characterization of this CD4+CD25+ T cell population identified Foxp3+ T cells at 1 week that failed to persist at 2 weeks. Conversely, BALF IL-17a increased significantly at 2 weeks in DA-exposed rats compared to air controls. Lung CD4+CD25+ T cells and BALF IL17a correlated directly with BALF total protein and inversely with rat oxygen saturations. Repetitive DA vapor exposure at occupationally relevant concentrations induced mixed, granulocytic lung inflammation with increased CD4+CD25+ T cells in the rat lung.

Pulmonary delivery of the broad-spectrum matrix metalloproteinase inhibitor marimastat diminishes multiwalled carbon nanotube-induced circulating bioactivity without reducing pulmonary inflammation

BackgroundMultiwalled carbon nanotubes (MWCNT) are an increasingly utilized engineered nanomaterial that pose the potential for significant risk of exposure-related health outcomes. The mechanism(s) underlying MWCNT-induced toxicity to extrapulmonary sites are still being defined. MWCNT-induced serum-borne bioactivity appears to dysregulate systemic endothelial cell function. The serum compositional changes after MWCNT exposure have been identified as a surge of fragmented endogenous peptides, likely derived from matrix metalloproteinase (MMP) activity. In the present study, we utilize a broad-spectrum MMP inhibitor, Marimastat, along with a previously described oropharyngeal aspiration model of MWCNT administration to investigate the role of MMPs in MWCNT-derived serum peptide generation and endothelial bioactivity.ResultsC57BL/6 mice were treated with Marimastat or vehicle by oropharyngeal aspiration 1 h prior to MWCNT treatment. Pulmonary neutrophil infiltration and total bronchoalveolar lavage fluid protein increased independent of MMP blockade. The lung cytokine profile similarly increased following MWCNT exposure for major inflammatory markers (IL-1β, IL-6, and TNF-α), with minimal impact from MMP inhibition. However, serum peptidomic analysis revealed differential peptide compositional profiles, with MMP blockade abrogating MWCNT-derived serum peptide fragments. The serum, in turn, exhibited differential potency in terms of inflammatory bioactivity when incubated with primary murine cerebrovascular endothelial cells. Serum from MWCNT-treated mice led to inflammatory responses in endothelial cells that were significantly blunted with serum from Marimastat-treated mice.ConclusionsThus, MWCNT exposure induced pulmonary inflammation that was largely independent of MMP activity but generated circulating bioactive peptides through predominantly MMP-dependent pathways. This MWCNT-induced lung-derived bioactivity caused pathological consequences of endothelial inflammation and barrier disruption.

Protective effect of the effective part of Andrographis paniculata (Burm.f.) Nees on PM2.5-induced lung injury in rats by modulating the NF-κB pathway

Ethnopharmacological relevanceAndrographis paniculata (Burm.f.) Nees, a traditional Chinese herb, has been widely used in various Asian countries as a treatment for upper respiratory tract infections for centuries. Aim of the studyContinuous inhalation of fine particulate matter (PM2.5) may induce various respiratory diseases. This study elucidated the protective effect of the effective part of Andrographis paniculata (Burm.f.) Nees (AEP) against PM2.5-induced lung injury and detailed the underlying mechanism. Materials and methodsMale Wistar rats were orally administered 0.5% sodium carboxymethylcellulose (CMC-Na), andrographolide (AG) (200 mg/kg) and AEP (100 mg/kg, 200 mg/kg and 400 mg/kg) once a day for 28 days. The rats were intratracheally instilled with PM2.5 suspension (8 mg/kg) every other day beginning on the 24th day for a total of 3 times. On the 29th day, bronchoalveolar lavage fluid (BALF) was collected to analyze the levels of lactate dehydrogenase (LDH), acid phosphatase (ACP), alkaline phosphatase (AKP), total proteins (TP), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6). Hematoxylin & eosin staining was conducted to evaluate the pathological changes in the lung tissues. The protein expression of NF-κB p65 in the lung tissues was analyzed by immunohistochemistry staining. Moreover, the nuclear translocation of NF-κB p65 and the phosphorylation of IκBα were analyzed by western blotting. ResultsPM2.5 exposure caused lung toxicity, which was characterized by pathological injury and increased levels of LDH, ACP, AKP and TP in BALF. Meanwhile, PM2.5 exposure induced lung inflammatory response, including infiltration of inflammatory cells and increased levels of inflammatory factors, such as TNF-α and IL-6 in BALF. AEP treatment significantly ameliorated the PM2.5-induced lung toxicity and the inflammatory response in rats. Moreover, AEP significantly inhibited the PM2.5-induced upregulation of NF-κB p65 protein expression, phosphorylation of IκBα and nuclear translocation of NF-κB p65 in lung tissue. Compared to AG, AEP exhibited a better ability to alleviate PM2.5-induced pathological damage and decrease the TP level in the BALF. ConclusionAEP could be used to improve PM2.5-induced lung injury by modulating the NF-κB pathway, and multicomponent therapy with traditional Chinese medicine may be more effective than single-drug therapy.

Rosiglitazone restores pulmonary surfactant and lipid homeostasis in mice exposed to ozone

Ozone is a ubiquitous urban air pollutant that causes airway inflammation and hyperresponsiveness. Evidence indicates that alterations in lung lipid homeostasis due to dysregulated catabolism by alveolar macrophages and/or disrupted recycling by alveolar epithelial type II cells drives inflammation and chronic lung injury. RNA-seq analysis of alveolar macrophages revealed that exposure of mice to ozone resulted in alterations in inflammatory and lipid metabolism pathways, and down-regulation of PPARγ, a nuclear transcription factor important in controlling lipid metabolism, macrophage activation, and inflammation resolution. In the present studies, we analyzed the role of PPARγ in ozone-induced alterations in lung lipid homeostasis. Female C57BL/6J mice were treated with the PPARγ agonist, rosiglitazone or vehicle control by daily intraperitoneal injection beginning 24 hr prior to exposure to ozone (0.8 ppm, 3 hr) or air; mice were euthanized 72 hr post ozone. Bronchoalveolar lavage fluid (BAL) was collected and analyzed for total protein and phospholipid content, and expression of surfactant proteins. Effects on pulmonary function were assessed using a SciReq Flexivent. Ozone exposure resulted in increases in total protein and phospholipid content in BAL, consistent with lung injury and dysregulation of lipid metabolism. An increase in levels of the hydrophilic collectin, surfactant protein D (SP-D) and a decrease in the hydrophobic surfactant protein B (SP-B) were also observed in BAL after ozone exposure. SP-B and phospholipids regulate pulmonary function by maintaining lung surface tension and hysteresivity. Ozone exposure reduced lung hysteresivity at positive end-expiratory pressures of 1 and 3 cm H20 indicating reduced viscoelasticity of the lungs. Rosiglitazone administration significantly reduced BAL phospholipid content and restored SP-B to control levels. Consistent with these findings, rosiglitazone rescued ozone-induced impairment of lung function. Collectively, these results suggest that ozone exposure disrupts SP-B and phospholipid homeostasis which impairs pulmonary function; PPARγ improves lung function by restoring surfactant and lipid balance.

Protective effect of FOXP3-mediated miR-146b-5p/Robo1/NF-κB system on lipopolysaccharide-induced acute lung injury in mice.

BackgroundAs a key transcription factor, forkhead box protein 3 (FOXP3) plays an important role in the development and function of natural cluster of differentiation 4 [CD4 (+)] regulatory T cells (Treg cells). However, the function of FOXP3 in Lipopolysaccharide (LPS)-induced acute lung injury (ALI) through regulating miR-146b-5p is unclear. This research aimed to disclose the regulatory effect of the FOXP3-mediated miR-146b-5p/Roundabout 1 (Robo1)/NF-κB system on LPS-induced ALI in mice.MethodsThe mice were subjected to 5 mg/kg of LPS via intratracheal instillation to induce ALI and generate the ALI model. Mice was divided into five group, including control group, ALI group, ALI + FOXP3 group, the ALI + miR antagomir group and ALI + miR antagomir+ FOXP3 group. Lung tissue injury were detected by hematoxylin and eosin (HE) staining. Lung wet/dry weight ratio, total cells in bronchoalveolar lavage fluid (BALF), total protein in BALF and the polymorphonuclear leukocyte (PMN) in BALF were detected. The levels of tumor necrosis factor-α (TNF-α), Interleukin 6 (IL-6) and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA) kit. The dual-luciferase reporter assay were used to detect the target relationship between FOXP3 and Robo1. Mice was divided into five group, including control group, ALI group, ALI + FOXP3 group, ALI + Robo1 group and ALI + FOXP3+ Robo1 group. The protein levels of FOXP3, Robo1 and p-p65 were detected by western bolt. The mRNA levels of miR-146b-5p and Robo1 were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR).ResultsAlthough protein expression levels of FOXP3 were significantly down-regulated in the ALI model, the increased FOXP3 levels promoted an increase in miR-146b-5p. Compared with the control group, the ALI model group exhibited severe histopathologic injury, such as thickening of the alveolar wall, pulmonary congestion, and decreased alveolar numbers. By mediating the overexpression of miR-146b-5p, FOXP3 also increased alveolar clearance and inhibited inflammatory responses in the ALI model. Importantly, Robo1 is a potential target of miR-146b-5p.ConclusionsFOXP3 could inhibit NF-κB activation, reduce lung pathological damage, and inhibit inflammatory responses by mediating the miR-146b-5p/Robo1/NF-κB system in the ALI model. These results may provide a new potential target for the treatment of ALI disease.

The pulmonary toxicity of carboxylated or aminated multi-walled carbon nanotubes in mice is determined by the prior purification method

BackgroundInhalation of multi-walled carbon nanotubes (MWCNTs) poses a potential risk to human health. In order to safeguard workers and consumers, the toxic properties of MWCNTs need to be identified. Functionalization has been shown to either decrease or increase MWCNT-related pulmonary injury, depending on the type of modification. We, therefore, investigated both acute and chronic pulmonary toxicity of a library of MWCNTs derived from a common pristine parent compound (NC7000).MethodsMWCNTs were thermally or chemically purified and subsequently surface functionalized by carboxylation or amination. To evaluate pulmonary toxicity, male C57BL6 mice were dosed via oropharyngeal aspiration with either 1.6 or 4 mg/kg of each MWCNT type. Mitsui-7 MWCNT was used as a positive control. Necropsy was performed at days 3 and 60 post-exposure to collect bronchoalveolar lavage fluid (BALF) and lungs.ResultsAt day 3 all MWCNTs increased the number of neutrophils in BALF. Chemical purification had a greater effect on pro-inflammatory cytokines (IL-1β, IL-6, CXCL1) in BALF, while thermal purification had a greater effect on pro-fibrotic cytokines (CCL2, OPN, TGF-β1). At day 60, thermally purified, carboxylated MWCNTs had the strongest effect on lymphocyte numbers in BALF. Thermally purified MWCNTs caused the greatest increase in LDH and total protein in BALF. Furthermore, the thermally purified and carboxyl- or amine-functionalized MWCNTs caused the greatest number of granulomatous lesions in the lungs. The physicochemical characteristics mainly associated with increased toxicity of the thermally purified derivatives were decreased surface defects and decreased amorphous content as indicated by Raman spectroscopy.ConclusionsThese data demonstrate that the purification method is an important determinant of lung toxicity induced by carboxyl- and amine-functionalized MWCNTs.

An animal model of transfusion-related acute lung injury and the role of soluble CD40 ligand.

Transfusion-related acute lung injury (TRALI) is a life-threatening complication of transfusion and is one of leading causes of transfusion-associated fatalities. However, the pathogenesis of TRALI is still unclear. Soluble CD40 ligand (sCD40L) is a proinflammatory cytokine that accumulates during blood component storage and is involved in transfusion reactions. The objective of this study was to establish a clinically relevant TRALI animal model and to evaluate the role of sCD40L in TRALI. Rats' red-blood-cell (RBC) suspensions were prepared, and the quality of RBC was evaluated. A trauma-haemorrhage-transfusion strategy was applied to build the animal model. Lung oedema was evaluated by histopathology examination, total bronchoalveolar lavage fluid (BALF) protein concentration, Evans blue dye (EBD) leakage and inflammatory cytokines. The sCD40L concentrations were measured. Storage lesions of RBCs gradually increased over time. Obvious histological evidence of lung injury of rats transfused with a 35-day RBC was observed. The total BALF protein concentration, EBD leakage, inflammatory cytokines concentration were increased significantly in the Day 35 group. The sCD40L concentration increased significantly in the storage RBC suspension over time but was slightly elevated in rat plasma. These findings indicated successful establishment of a TRALI animal model with trauma-haemorrhage-transfusion, in which sCD40L may play a minor role in the development of TRALI.

Protective Effect of Piceatannol Against Acute Lung Injury Through Protecting the Integrity of Air-Blood Barrier and Modulating the TLR4/NF-κB Signaling Pathway Activation.

Acute lung injury (ALI) is a common and complex inflammatory lung syndrome with higher morbidity and mortality rate. Piceatannol (PIC) has anti-inflammation and anti-oxidant properties. The study was designed to explore the effect and the action mechanisms of PIC on lipopolysaccharide (LPS)-induced ALI. Twenty-four hours after LPS challenge, mice from different treatment groups were euthanized, and the bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected. Then the degree of pulmonary edema, lung pathological changes, myeloperoxidase (MPO) activity, and the production of pro-inflammatory cytokines were detected. Additionally, the messenger RNA (mRNA) expressions associated with cell adhesion molecules and tight junction were analyzed through quantitative real-time (qRT)-PCR, and the TLR4/NF-κB activation was examined by western blot. The results showed that PIC significantly inhibited LPS-induced lung edema, histopathological damage, MPO activity, cell infiltration, and pro-inflammatory cytokines production. Moreover, PIC notably suppressed mRNA expressions associated with inflammation and cell adhesion molecules. Furthermore, PIC also alleviated LPS-induced damage of air-blood barrier through reducing the levels of total proteins in BALF and recovering the expression of occludin and ZO-1 in the lung tissues. We also found that PIC remarkably restrained the LPS-induced TLR4/NF-κB pathway activation in lung tissues. In conclusion, PIC may be potential to treat LPS-induced acute lung injury (ALI) via regulating air-blood barrier and TLR4/NF-κB signaling pathway activation.

Intrapulmonary Cellular-Level Distribution of Inhaled Nanoparticles with Defined Functional Groups and Its Correlations with Protein Corona and Inflammatory Response.

Concerns over the health risks associated with airborne exposure to ultrafine particles [PM0.1, or nanoparticles (NPs)] call for a comprehensive understanding in the interactions of inhaled NPs along their respiratory journey. We prepare a collection of polyethylene glycol-coated gold nanoparticles that bear defined functional groups commonly identified in atmospheric particulates (Au@PEG-X NPs, where X = OCH3, COOH, NH2, OH, or C12H25). Regardless of the functional group, these ∼50 nm NPs remain colloidally stable following aerosolization and incubation in bronchoalveolar lavage fluid (BALF), without pronouncedly crossing the air-blood barrier. The type of BALF proteins adhered onto the NPs is similar, but the composition of protein corona depends on functional group. By subjecting Balb/c mice to inhalation of Au@PEG-X NPs for 6 h, we demonstrate that the intrapulmonary distribution of NPs among the various types of cells (both found in BALF and isolated from the lavaged lung) and the acute inflammatory responses induced by inhalation are sensitive to the functional group of NPs and postinhalation period (0, 24, or 48 h). By evaluating the pairwise correlations between the three variables of "lung-nano" interactions (protein corona, intrapulmonary cellular-level distribution, and inflammatory response), we reveal strong statistical correlations between the (1) fractions of albumin or carbonyl reductase bound to NPs, (2) associations of inhaled NPs to neutrophils in BALF or macrophages in the lavaged lung, and (3) level of total protein in BALF. Our results provide insights into the effect of functional group on lung-nano interactions and health risks associated with inhalation of PM0.1.

Quantum dots and mouse strain influence house dust mite-induced allergic airway disease

Quantum dot nanoparticles (QDs) are engineered nanomaterials (ENMs) that have utility in many industries due to unique optical properties not available in small molecules or bulk materials. QD-induced acute lung inflammation and toxicity in rodent models raise concerns about potential human health risks. Recent studies have also shown that some ENMs can exacerbate allergic airway disease (AAD).In this study, C57BL/6J and A/J mice were exposed to saline, house dust mite (HDM), or a combination of HDM and QDs on day 1 of the sensitization protocol. Mice were then challenged on days 8, 9 and 10 with HDM or saline only.Significant differences in cellular and molecular markers of AAD induced by both HDM and HDM + QD were observed between C57BL/6J and A/J mice. Among A/J mice, HDM + QD co-exposure, but not HDM exposure alone, significantly increased levels of bronchoalveolar lavage fluid (BALF). IL-33 compared to saline controls. BALF total protein levels in both mouse strains were also only significantly increased by HDM + QD co-exposure. In addition, A/J mice had significantly more lung type 2 innate lymphoid cells (ILC2s) cells than C57BL/6J mice. A/J lung ILC2s were inversely correlated with lung glutathione and MHC-IIhigh resident macrophages, and positively correlated with MHC-IIlow resident macrophages.The results from this study suggest that 1) QDs influence HDM-induced AAD by potentiating and/or enhancing select cytokine production; 2) that genetic background modulates the impact of QDs on HDM sensitization; and 3) that potential ILC2 contributions to HDM induced AAD are also likely to be modulated by genetic background.