Cells In BALF Research Articles

Genetic Inactivation of the Phospholipase A 2 Activity of Prdx6 Ameliorates Sepsis-induced Acute Lung Injury

Prdx6 is a multi-functional enzyme that expresses peroxidase, phospholipase A2 (PLA2) and LPC acyltransferase activities. While the three activities of Prdx6 contribute to either the scavenging of lipid hydroperoxides or to the repair of peroxidized membranes, the PLA2 activity of Prdx6 (aiPLA2) is also required for Nox2 activation in pulmonary microvascular cells (PMVECs), alveolar macrophages and polymorphonuclear neutrophils. During sepsis-induced acute lung injury (ALI), Nox2 generates superoxide radicals that contribute to vascular inflammation. To test whether inactivation of aiPLA2 ameliorates sepsis-induced ALI, we used a knock-in mouse model that lacks aiPLA2 activity due to a mutation of a key member of the aiPLA2 catalytic triad (Prdx6-D140A). Prdx6-D140A mice treated IP with LPS exhibited increased survival rates compared to WT mice. Total nucleated cells in BALF, MPO activity, BALF cytokine expression, lung VCAM-1 expression, lung PMN infiltration, lung nitrotyrosine levels and lung permeability increased significantly after 24 h treatment with LPS in WT but not in Prdx6-D140A mice. In PMVECs, LPS treatment increased Prdx6 phosphorylation and aiPLA2 activity by two-fold. Furthermore, phosphorylated Prdx6 was localized at the plasma membrane after LPS treatment. LPS also increased H2O2 generation, lipid peroxidation, protein nitration, NfkB nuclear localization and NLRP3:ASC inflammasome assembly in WT but not in Nox2 null or Prdx6-D140A PMVECs. Similarly, LPS increased intracellular H2O2 generation in WT PMVECs stably-expressing the pHyPer-Cyto sensor but not in cells pre-treated with MJ33, a pharmacological inhibitor of aiPLA2. These results demonstrate that aiPLA2 is needed for LPS-induced, Nox2-driven oxidant generation in PMVECs. Results also show that genetic inactivation of aiPLA2 reduces mortality, lung inflammation and oxidative stress in an animal model of sterile sepsis. Furthermore, these studies suggest that aiPLA2 could be a target for prevention or treatment of sepsis-induced ALI.

5-Aminosalicylic Acid Modulates the Immune Response in Chronic Beryllium Disease Subjects.

Chronic beryllium disease (CBD) is characterized by accumulation of macrophages and beryllium-specific CD4+ T cells that proliferate and produce Th1 cytokines. 5-Amino salicylic acid (5-ASA) is currently used to treat inflammatory bowel disease and has both antioxidant and anti-inflammatory actions. We hypothesized that 5-ASA may be a beneficial therapeutic in CBD. Seventeen CBD patients were randomized 3:1 to receive 5-ASA 500-mg capsules or placebo four times daily for 6weeks orally. Primary study endpoints included changes in beryllium lymphocyte proliferation (BeLPT). Secondary endpoints included changes in bronchoalveolar lavage (BAL) fluid, cells, serum, and blood cell glutathione (GSH) levels, BAL cell TNF-α levels, lung function, and quality of life measures. 5-ASA decreased BAL cell BeLPT by 20% within the 5-ASA treatment group. No significant changes were observed in serum, PBMCs, BALF, or BAL cell GSH levels in either the 5-ASA or placebo treatment group. 5-ASA treatment decreased ex vivo Be-stimulated BAL cell TNF-α levels within the 5-ASA group and when compared to placebo. Significant improvements were noted in quality of life measurements with 5-ASA treatment. 5-ASA's ability to decrease BAL cell BeLPT and Be-stimulated BAL cell TNF-α levels suggests that 5-ASA may impact the beryllium-specific immune response in CBD. 5-ASA use in other non-infectious granulomatous lung diseases, such as sarcoidosis, may prove to be a useful alternative treatment to corticosteroids for those with mild to moderate disease.

Protective effect of forsythiaside A on OVA-induced asthma in mice

Forsythiaside A (FSA), an active constituent isolated from the Chinese medicinal herb Forsythia suspensa, has been known to have anti-inflammatory effect. However, the effect of FSA on allergic airway inflammation remains unclear. The aim of this study was to investigate the effect of FSA on OVA-induced asthma in mice. Mice model of asthma was induced by OVA. OVA-induced airway hyperresponsiveness (AHR) and inflammatory cells in BALF were detected. The production of IgE, IL-4, IL-5, IFN-γ, and IL-13 were detected by ELISA. The effects of FSA on Nrf2 and NF-κB signaling pathways were detected by western blot analysis. The results showed that treatment of FSA significantly attenuated OVA-induced lung histopathological changes. FSA inhibited OVA-induced AHR and inflammatory cells in BALF. OVA-induced IgE, IL-4, IL-5, and IL-13 production were also inhibited by FSA. Western blot analysis showed that treatment of FSA inhibited OVA-induced NF-κB activation. Treatment of FSA dose-dependently up-regulated the expression of Nrf2 and HO-1. In addition, we found that FSA up-regulated the expression of Nrf2 and HO-1 both in A549 cells and MS-H cells. Taken together, FSA suppressed inflammatory responses in OVA-induced asthma through activating Nrf2/HO-1 signaling pathway. FSA may be a promising potential preventive agent for asthma treatment.

Artesunate Protects LPS-Induced Acute Lung Injury by Inhibiting TLR4 Expression and Inducing Nrf2 Activation.

Artesunate, a derivative of artemisinin, has been reported to have anti-inflammatory property. However, few studies showed the protective effects of artesunate on lung injury. In this study, we aimed to investigate the effects of artesunate on LPS-induced lung injury in mice. The mice were treated with artesunate 1h before or after LPS treatment. The effects of artesunate on lung MPO activity and malondialdehyde (MDA) content were detected. The lung wet/dry radio and the numbers of inflammatory cells in BALF were also measured. ELISA was used to evaluate the levels of TNF-α, IL-1β, and IL-6 in BALF. Western blot analysis was adapted to detect TLR4 and Nrf2 signaling pathways. The results showed that artesunate protected against LPS-induced ALI by decreasing the numbers of inflammatory cells, lung edema, MPO activity, and MDA content. Furthermore, artesunate significantly inhibited the levels of TNF-α, IL-1β, and IL-6. Artesunate also inhibited LPS-induced IL-6 and IL-8 production in the A549 cells. In addition, artesunate dose-dependently suppressed LPS-induced TLR4 expression and NF-κB activation. The expression of Nrf2 and HO-1 were also up-regulated by artesunate. The data suggest that artesunate possesses anti-inflammatory and anti-oxidant properties against LPS-induced ALI via inhibiting TLR4 signaling pathway and activating Nrf2 signaling pathway.

Bosutinib Therapy Ameliorates Lung Inflammation and Fibrosis in Experimental Silicosis.

Silicosis is an occupational lung disease for which no effective therapy exists. We hypothesized that bosutinib, a tyrosine kinase inhibitor, might ameliorate inflammatory responses, attenuate pulmonary fibrosis, and thus improve lung function in experimental silicosis. For this purpose, we investigated the potential efficacy of bosutinib in the treatment of experimental silicosis induced in C57BL/6 mice by intratracheal administration of silica particles. After 15 days, once disease was established, animals were randomly assigned to receive DMSO or bosutinib (1 mg/kg/dose in 0.1 mL 1% DMSO) by oral gavage, twice daily for 14 days. On day 30, lung mechanics and morphometry, total and differential cell count in alveolar septa and granuloma, levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-4, transforming growth factor (TGF)-β, and vascular endothelial growth factor in lung homogenate, M1 and M2 macrophages, total leukocytes, and T cells in BALF, lymph nodes, and thymus, and collagen fiber content in alveolar septa and granuloma were analyzed. In a separate in vitro experiment, RAW264.7 macrophages were exposed to silica particles in the presence or absence of bosutinib. After 24 h, gene expressions of arginase-1, IL-10, IL-12, inducible nitric oxide synthase (iNOS), metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, and caspase-3 were evaluated. In vivo, in silicotic animals, bosutinib, compared to DMSO, decreased: (1) fraction area of collapsed alveoli, (2) size and number of granulomas, and mononuclear cell granuloma infiltration; (3) IL-1β, TNF-α, IFN-γ, and TGF-β levels in lung homogenates, (4) collagen fiber content in lung parenchyma, and (5) viscoelastic pressure and static lung elastance. Bosutinib also reduced M1 cell counts while increasing M2 macrophage population in both lung parenchyma and granulomas. Total leukocyte, regulatory T, CD4+, and CD8+ cell counts in the lung-draining lymph nodes also decreased with bosutinib therapy without affecting thymus cellularity. In vitro, bosutinib led to a decrease in IL-12 and iNOS and increase in IL-10, arginase-1, MMP-9, and TIMP-1. In conclusion, in the current model of silicosis, bosutinib therapy yielded beneficial effects on lung inflammation and remodeling, therefore resulting in lung mechanics improvement. Bosutinib may hold promise for silicosis; however, further studies are required.

Trigonella foenum-graecum alleviates airway inflammation of allergic asthma in ovalbumin-induced mouse model

Trigonella foenum-graecum, a member oldest medicinal plant in the fabaceae (legumes) family, is used as a herb, spice, and vegetable, and known for its olfactory, laxative, and galactogogue effects. However, the inhibitory effect of Trigonella foenum-graecum on allergic inflammatory response remains unclear, therefore, we investigated the precise role of Trigonella foenum-graecum in the allergic asthma and revealed the effects of Trigonella foenum-graecum in regulating airway inflammation and its possible mechanism. Allergic asthma was initiated in BALB/c mice by sensitized with OVA emulsified in aluminum on days 1 and 14, then aerosol challenged with OVA on days 27, 28 and 29. Some mice were administered Trigonella foenum-graecum by oral gavage before challenge. Then mice were evaluated for the presence of airway inflammation, production of allergen-specific cytokine response and lung pathology. Trigonella foenum-graecum significantly ameliorated the number of inflammatory cells in BALF and alleviated lung inflammation. It also reduced the collagen deposition and goblet cells. Meanwhile, Trigonella foenum-graecum treatment evidently decreased the high expression of Th2 cytokines and increased the Th1 cytokines in BALF and lung homogenates. Trigonella foenum-graecum showed a significant inhibition of serum IgE and anti-OVA IgG1. In this study, our data suggest that Trigonella foenum-graecum has a significant anti-inflammatory effect and it may prove to be an efficacious therapeutic regent on allergic asthma.

The time-dependent health and biochemical effects in rats exposed to stainless steel welding dust and its soluble form

ABSTRACTWelding processes that generate fumes containing toxic metals, such as hexavalent chromium (Cr(VI)), manganese (Mn), and nickel (Ni), have been implicated in lung injury, inflammation, and lung tumor promotion in animal models. The principal objective of this study was to determine the dynamics of toxic effects of inhalation exposure to morphologically rated welding dust from stainless steel welding and its soluble form in TSE System with a dynamic airflow. We assessed the pulmonary toxicity of welding dust in Wistar rats exposed to 60.0 mg/m3 of respirable-size welding dust (mean diameter 1.17 µm) for 2 weeks (6 h/day, 5 days/week); the aerosols were generated in the nose-only exposure chambers (NOEC). An additional aim included the study of the effect of betaine supplementation on oxidative deterioration in rat lung during 2 weeks of exposure to welding dust or water-soluble dust form. The animals were divided into eight groups (n = 8 per group): control, dust, betaine, betaine + dust, soluble-form dust, soluble-form dust + betaine, saline and saline + betaine groups. Rats were euthanized 1 or 2 weeks after the last exposure for assessment of pulmonary toxicity. Differential cell counts, total protein concentrations and cellular enzyme (lactate dehydrogenase—LDH) activities were determined in bronchoalveolar lavage (BAL) fluid, and corticosterone and thiobarbituric acid reactive substances (TBARS) concentrations were assessed in serum. The increase in polymorphonuclear (PMN) leukocytes in BAL fluid (a cytological index of inflammatory responses of the lung) is believed to reflect pulmonary toxicity of heavy metals. Biomarkers of toxicity assessed in bronchoalveolar fluids indicate that the level of the toxic effect depends mainly on the solubility of studied metal compounds; biomarkers that showed treatment effects included: total cell, neutrophil and lymphocyte counts, total protein concentrations, and cellular enzyme (lactate dehydrogenase) activity. Betaine supplementation at 250 mg/kg/day in all study rats groups attenuated stress indices, and corticosterone and TBARS serum levels, and simultaneously stimulated increase of polymorphonuclear cells in BALF of rats. The study confirmed deleterious effect of transitory metals and particles during experimental inhalation exposure to welding dusts, evidenced in the lungs and brain by increased levels of total protein, higher cellular influx, rise of LDH in BALF, elevated TBARS and increased corticosterone in serum of rats. Our result confirm also the hypothesis about the effect of the welding dusts on the oxidative stress responsible for disturbed systemic homeostasis and impairment of calcium regulation.

124 - Inactivation of the PLA2 Activity of Prdx6 Ameliorates Sepsis-Induced Acute Lung Injury

Prdx6 is a multi-functional protein that expresses peroxidase, phospholipase A2 (PLA2) and LPC acyltransferase activities. While the three activities of Prdx6 contribute to either the scavenging of lipid hydroperoxides or to the repair of peroxidized membranes, the PLA2 activity of Prdx6 (aiPLA2) is also required for NOX2 activation in pulmonary microvascular cells (PMVECs), alveolar macrophages and polymorphonuclear neutrophils. During sepsis-induced acute lung injury (ALI), NOX2 is activated generating superoxide radicals that contribute to vascular inflammation. To test whether inactivation of aiPLA2 ameliorates sepsis-induced ALI, we used a knockin mouse model (Prdx6-D140A) that lacks aiPLA2 activity due to a mutation of a key member of the PLA2 catalytic triad. Prdx6-D140A mice treated IP with LPS (10 mg/Kg) exhibited increased survival rates compared to WT mice. Total nucleated cells in BALF, VCAM-1 expression, and lung permeability increased after 24 h treatment with LPS in WT but not in Prdx6-D140A knockin mice. In vitro, intracellular hydrogen peroxide generation increased after LPS treatment (1 ug/mL for 16h) in PMVECs stably-expressing the HyPer-Cyto sensor. Increased H2O2 generation was prevented by pre-treatment with MJ33, a pharmacological inhibitor of aiPLA2 activity. These results show that aiPLA2 is needed for LPS-induced oxidant generation in PMVECs and that genetic inactivation aiPLA2 reduces LPS-induced inflammation, increased vascular permeability, and mortality in an animal model suggesting that aiPLA2 could be a target for prevention or treatment of sepsis-induced ALI.

Therapeutic effect of methyl salicylate 2-O-β-d-lactoside on LPS-induced acute lung injury by inhibiting TAK1/NF-kappaB phosphorylation and NLRP3 expression

Acute lung injury (ALI), characterized by pulmonary edema and inflammatory cell infiltration, is a common syndrome of acute hypoxemic respiratory failure. Methyl salicylate 2-O-β-d-lactoside (MSL), a natural derivative of salicylate extracted from Gaultheria yunnanensis (Franch.) Rehder, was reported to have potent anti-inflammatory effects on the progression of collagen or adjuvant-induced arthritis in vivo and in vitro. The aim of this study is to investigate the therapeutic effect of MSL on lipopolysaccharide (LPS)-induced acute lung injury and reveal underlying molecular mechanisms. Our results showed that MSL significantly ameliorated pulmonary edema and histological severities, and inhibited IL-6 and IL-1β production in LPS-induced ALI mice. MSL also reduced MPO activity in lung tissues and the number of inflammatory cells in BALF. Moreover, we found that MSL significantly inhibited LPS-induced TAK1 and NF-κB p65 phosphorylation, as well as the expression of NLRP3 protein in lung tissues. Furthermore, MSL significantly inhibited LPS-induced TAK1 and NF-κB p65 phosphorylation in Raw264.7 cells. In addition, MSL significantly inhibited nuclear translocation of NF-κB p65 in cells treated with LPS in vitro. Taken together, our results suggested that MSL exhibited a therapeutic effect on LPS-induced ALI by inhibiting TAK1/NF-κB phosphorylation and NLRP3 expression.

Therapeutic Effects of Resveratrol in a Mouse Model of LPS and Cigarette Smoke-Induced COPD.

This study was designed to examine whether resveratrol exerts the protective effects on LPS and cigarette smoke (LC)-induced COPD in a murine model. In lung histopathological studies, H&E, Masson's trichrome, and AB-PAS staining were performed. The cytokines (IL-6, IL-17, TGF-β, and TNF-α) and inflammatory cells in BALF were determined. The Beclin1 level in the lungs of mouse was analyzed. Compared with the LC-induced mouse, the level of inflammatory cytokines (IL-17, IL-6, TNF-α, and TGF-β) of the BALF in the resveratrol + cigarette smoke-treated mouse had obviously decreased. Histological examination of the lung tissue revealed that the resveratrol treatment attenuated the fibrotic response and mucus hypersecretion. In addition, resveratrol inhibited the expression of the Beclin1 protein in mouse lungs. The presented findings collectively suggest that resveratrol has a therapeutic effect on mouse LC-induced COPD, and its mechanism of action might be related to reducing the production of the Beclin1 protein.

Antitussive activity of the Schisandra chinensis fruit polysaccharide (SCFP-1) in guinea pigs models

Ethnopharmacological relevanceSchisandra chinensis (Turcz.) Baill. (S. chinensis), locally known as “Wuweizi”, has been used in the treatment of chronic cough as prescription medications of Traditional Chinese Medicine for thousands of years. However, the components of antitussive activity of S. chinensis and the mechanism are poorly understood. Aim of the studyThis study aims to investigate the antitussive activity of polysaccharides extracted from S. chinensis. Materials and methodsS. chinensis fruit polysaccharide-1 (SCFP-1) was extracted by 95% ethanol and distilled water successively, and then the water extraction was isolated with chromatographic columns. The preliminary characterization of SCFP-1 was analyzed by gel permeation chromatography (GPC), gas chromatography–mass spectrometry (GC–MS) and some other recognized chemical methods. Antitussive potential of SCFP-1 was estimated at dose of 250, 500, and 1000mg/kg respectively by peroral administration in a guinea pigs model with cough hypersensitivity induced by cigarette smoke (Chronic cough model) or acute cough guinea model induced by citric acid (Acute cough model). Also, the time-dependent antitussive effect of SCFP-1 were evaluated with acute cough model, and compared with codeine. ResultsThe molecular of SCFP-1 was 3.18×104Da, mainly being composed of glucose and arabinose (66.5% and 29.4%, respectively). Peroral administration of SCFP-1 at 250, 500, and 1000mg/kg showed remarkable suppressive effects respectively on cough in both of chronic cough model and acute cough model. Meanwhile, inflammatory cell in BALF and some typical characteristics of nonspecific airway inflammation in animals exposed to CS was significantly attenuated after pretreatment with SCFP-1. The cough suppression of SCFP-1 (500 mg/kg) stablly lasted during the whole 5 h of time-dependent experiment, while no positive effect was observed after 300 min of oral administration of codeine. ConclusionsSCFP-1 is one of the antitussive components of S. chinensis.

Altered miRNA expression in pulmonary sarcoidosis

BackgroundmiRNAs control important cellular functions including angiogenesis/angiostasis or fibrosis and reveal altered expression during pathological processes in the lung.MethodsThe aim of the study was to investigate the expression of selected miRNAs (miR-let7f, miR-15b, miR-16, miR-20a, miR-27b, miR-128a, miR-130a, miR-192 miR-221, miR-222) in patients with pulmonary sarcoidosis (n = 94) and controls (n = 50). The expression was assessed by q-PCR in BALF cells and peripheral blood lymphocytes (PB lymphocytes). For statistical analysis, the Kruskal–Wallis test, Mann–Whitney U- test, Neuman–Keuls’ multiple comparison test, and Spearman’s rank correlation were used.ResultsIn BALF cells, significantly higher expression of miR-192 and miR-221 and lower expression of miR-15b were found in patients than controls. MiR-27b, miR-192 and miR-221 expression was significantly higher in patients without parenchymal involvement (stages I) than those at stages II-IV. Patients with acute disease demonstrated significantly higher miR-27b, miR-192 and miR-221 expression than those with insidious onset. For PB lymphocytes, patients demonstrated significantly greater miR-15b, miR-27b, miR-192, miR-221 and miR-222 expression, but lower miR-let7f and miR-130a expression, than controls. Stage I patients demonstrated significantly higher miR-16 and miR-15b expression than those in stages II-IV, and patients with the acute form demonstrated higher miR-130a and miR-15b expression. In BALF cells, miR-16 and miR-20a expression was significantly higher in patients with lung volume restriction, and miR-let7f was higher in the PB lymphocytes in patients with obturation. Several correlations were observed between the pattern of miRNA expression, lung function parameters and selected laboratory markers.ConclusionThe obtained results suggest that the studied miRNAs play a role in the pathogenesis of sarcoidosis, and that some of them might have negative prognostic value.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-016-0266-6) contains supplementary material, which is available to authorized users.

Dendritic cells inversely regulate airway inflammation in cigarette smoke-exposed mice.

The recruitment and activation of inflammatory cells into the respiratory system is considered a crucial feature in the pathophysiology of chronic obstructive pulmonary disease (COPD). Because dendritic cells (DCs) have a pivotal role in the onset and regulation of immune responses, we investigated the effect of modulating DC subsets on airway inflammation by acute cigarette smoke (CS) exposure. CS-exposed mice (5 days) were treated with fms-like tyrosine kinase 3 ligand (Flt3L) and 120g8 antibody to increase total DC numbers and deplete plasmacytoid DCs (pDCs), respectively. Flt3L treatment decreased the number of inflammatory cells in the bronchoalveolar lavage (BALF) of the smoke-exposed mice and increased these in lung tissue. DC modulation reduced IL-17 and increased IL-10 levels, which may be responsible for the suppression of the BALF cells. Furthermore, depletion of pDCs led to increased infiltration of alveolar macrophages while restricting the presence of CD103(+) DCs. This study suggests that DC subsets may differentially and compartment-dependent influence the inflammation induced by CS. pDC may play a role in preventing the pathogenesis of CS by inhibiting the alveolar macrophage migration to lung and increasing CD103(+) DCs at inflammatory sites to avoid extensive lung tissue damage.

Mechanisms of antimicrobial peptide cathelicidin secreted by non-tumorous cells for lung tumor growth

To investigate the effect of antimicrobial peptide cathelicidin secreted by non-tumorous cells in lung tumor growth. CRAMP(-/-) mice and WT mice were used to establish a lung cancer model via tail vein injection of Lewis lung carcinoma cells (LLC1). Lung was weighted and tumor number on the lung surface was counted. Kaplan-Meier (K-M) survival curve was used to analyze survival rate of mice. Expression of cathelicidin, Ki-67 and CD68 in the tumor tissue was measured by immunohistochemical analysis. BALF cells were stained with Diff Quik and percentages of leukocyte types were determined by light microscopy. Cathelicidin was high expression in inflammatory cells of tumor tissue, whereas weak expression in tumor cells. The lung weight and number of tumor in CRAMP-/- mice were (0.25±0.04)g and (9.60± 2.25), respectively, which were significantly lower than those of WT mice (0.65±0.05) g and (23.40± 2.68). The difference was statistically significant (t=6.07, 3.95, all P<0.05). And Kaplan-Meier survival analysis showed median survival time of CRAMP-/- mice was 49(46-51)d, which was longer than 34(28-39) d of WT mice (χ2=12.00, P<0.05). And the positive rate of Ki-67 tumor cells was significant reduced from (35.80±2.96)% in WT mice to (18.80±2.38)% in CRAMP-/- groups (t=4.48, P<0.05). The total cell number as well as the number of lymphocytes, neutrophils, and macrophages in BALFs of CRAMP-/- mice were (4.72±0.86)×10(4), (0.08±0.02)×10(4), (0.05±0.02)×10(4) and (4.60±0.84)×10(4), respectively, while of WT mice were (16.18±1.61)×10(4), (0.32±0.05)×10(4), (0.20±0.05)×10(4) and (15.66±1.57)×10(4). All of them had significant difference (t=6.28, 4.39, 3.00, 6.20, all P<0.05). In addition, the infiltration of macrophages into lung tumors was decreased in CRAMP-/- mice compared to WT mice, from (15.53±2.28)/high power field to (6.77±3.12)/high power field (t=3.41, P<0.05). Non-tumor cells secreted cathelicidin promotes tumor cell proliferation and lung tumor growth. Recruitment of inflammatory cells such as macrophages into the tumor microenvironment may be the main mechanism of action.

A plant proteinase inhibitor from Crataeva tapia (CrataBL) attenuates elastase-induced pulmonary inflammatory, remodeling, and mechanical alterations in mice

Chronic obstructive pulmonary disease (COPD) can lead to chronic and obstructive bronchitis and emphysema resulting in decreased bronchial lumen size.This study evaluated the effect of CrataBL, a protein isolated from the bark of Crataeva tapia, on lung mechanics, inflammation, and remodeling after elastase-induced pulmonary alterations in mice.The use of CrataBL led to decreased mechanical alterations, alveolar septum disruption (Lm), number of macrophage and neutrophil cells in the BALF, and TNF-α, MMP-9, MMP-12, TIMP-1, eNOS, and iNOS positive cells in the airways and alveolar walls compared to the animals in the ELA group. Moreover, a reduction in MUC-5-positive cells in the airway walls was observed. In conclusion, CrataBL attenuates changes in lung mechanics, inflammation, extracellular lung remodeling, and oxidative stress responses induced by the administration of elastase and decreased the volume fraction of isoprostane, collagen, and elastic fibers in the airways and alveolar walls compared to the animals in the ELA groups. Therefore, CrataBL is a potential therapeutic tool in the treatment of COPD.

Resveratrol suppresses persistent airway inflammation and hyperresponsivess might partially via nerve growth factor in respiratory syncytial virus-infected mice

Respiratory syncytial virus (RSV) infection is involved in persistent and recurrent wheezing. There are no effective and safe drugs for the sequelae of persistent wheezing after early bronchiolitis. In this study, we investigated the effect of resveratrol on persistent airway inflammation and airway hyperresponsiveness (AHR) induced by RSV infection. RSV-infected mice were sacrificed at serial time points after infection to collect samples and measure the number of inflammatory cells and levels of interferon-gamma (IFN-γ), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF). Airway inflammation, AHR, and the levels of NGF and BDNF were detected after resveratrol administration. Furthermore, Ab-NGF was used to investigate the role of NGF in RSV-induced prolonged airway inflammation and AHR. We found that RSV RNA remained detectable in the lungs of RSV-infected mice on day 60, accompanying persistent airway inflammation and AHR for 60 days. IFN-γ levels in BALF were increased on day 7 but reduced to normal levels by day 14 post-RSV infection, while NGF and BDNF levels gradually increased from day 14 to 60. Furthermore, after resveratrol treatment, the total number of cells in BALF was reduced; the number of inflammatory cells infiltrating the lungs was also lower. Resveratrol attenuated the airway response to methacholine and significantly decreased NGF levels in BALF without affecting BDNF levels. Moreover, airway inflammation and AHR associated with RSV persistence were attenuated after Ab-NGF administration. In all, resveratrol suppresses persistent airway inflammation and AHR might partially through reducing the levels of NGF after RSV infection.

Depressed Hypoxic and Hypercapnic Ventilatory Responses (dHVR and dHCVR) Occur in Mice at the Late Stage of Lethal Avian Influenza A (H5N1) Virus Infection

H5N1 viral infection results in ~60% mortality in patients primarily due to respiratory failure, but the relevant mechanisms are unclear. Lethal H5N1 viral infection produced dHVR and dHCVR without significant changes in body weight (BW), body temperature (BT), arterial blood gases, and pulmonary inflammation 2-3 days post-infection in mice (AJRCCM 2013). Here we tested the development of dHVR/dHCVR and disorders of pulmonary functions, leading to respiratory failure at the late stage of infection. The mice were intranasally inoculated with vehicle, 100 PFU HK483 (lethal strain) or HK486 virus (nonlethal strain). At 1 day before or post-inoculation days (PID) 4, 5, 6 and 7, VE, HVR (7% O2 for 3 min) and HCVR (7% CO2 for 5 min) were measured in awake state followed by collecting arterial blood, BALF, the brain and lung after anesthesia/euthanasia. HK483 and HK486 mice showed a peak viral titer at PID4 that gradually declined thereafter. HVR and HCVR were similar in Ctrl and HK486 mice and were not changed over the infection period. At PID7, HK483 mice were very weak (all died one day later), and exhibited hypoventilation with hypoxemia and hypercapnemia; they also lacked HVR (no response) and HCVR (80%↓) with BW and BT declined by 31% and ~10 ºC, respectively. HK483 but not HK486 significantly decreased lung dry/wet ratio. Total cells in BALF increased in both infected groups, but the increase was more evident in HK483 than HK486 mice. Our data suggest that H5N1 viral infection-induced ventilatory and pulmonary disorders are viral strain-dependent and the gradual deterioration of dHVR and dHCVR may account for the mortality in HK483 mice (Supported by HL119683 and HL107462).

The Severity of Lethal H5N1 Viral Infection‐Induced Changes in Pulmonary Inflammation and Functions Is Age‐Dependent

H5N1 viral infection results in ~60% mortality with the highest rate in the juvenile (10‐19 years old). Our previous study has shown that all Balb/c mice at 4W age die 8‐9 days after lethal H5N1 viral (HK483) infection, in which a remarkable drop of body temperature (BT) is an indication of fatality (AJRCCM 2013). This study was undertaken to test the hypothesis that HK483 infection‐induced changes in pulmonary inflammation and function, and drop of BT and body weight (BW) were greater in juvenile (20W) than that in younger (4W) mice. The two age groups of mice were intranasally inoculated with HK483 virus (100 PFU). After measurement of BW and BT, they were anesthetized for collecting arterial blood and then euthanized for BALF, brain and lung tissue collections on days ‐1, 3, 5, and 7 postinfection. We found that the viral infection similarly increased BALF cells, and gradually decreased BW, BT, lung dry/wet ratios, and Sao2 in the two groups without difference in lung H5N1 viral titers. At the early stage of infection the dominant increases of BALF cell types were neutrophils in 4W but lymphocytes in 20W mice. At late stage, 20W mice, compared to 4W mice, showed less severe BT/BW drops and a two‐day delay in the occurrence of hypoxemia and hypercapnemia (appeared on day 5 postinfection). Our data suggest that the impact of lethal H5N1 viral infection on pulmonary inflammation and function is age‐dependent, i.e., it is worse in younger than juvenile mice, which is somewhat different from the clinical observations in humans (Supported by HL119683).

Regulation of Th17/Treg function contributes to the attenuation of chronic airway inflammation by icariin in ovalbumin-induced murine asthma model

Icariin which is a flavonoid glucoside isolated from Epimedium brevicornu Maxim, has been reported to have anti-osteoporotic, anti-inflammatory and anti-depressant-like activities. In this study, we observed the effect of icariin on airway inflammation of ovalbumin (OVA)-induced murine asthma model and the associated regulatory mode on T-helper (Th)17 and regulatory T (Treg) cell function. Our data revealed that chronic OVA inhalation induced a dramatic increase in airway resistance (RL) and decrease in the lung dynamic compliance (Cdyn), and icariin and DEX treatment caused significant attenuation of such airway hyperresponsiveness (AHR). BALF cell counts demonstrated that icariin and DEX led to a prominent reduction in total leukocyte as well as lymphocyte, eosinophil, neutrophil, basophil and monocyte counts. Histological analysis results indicated that icariin and DEX alleviated the inflammatory cells infiltrating into the peribronchial tissues and goblet cells hyperplasia and mucus hyper-production. Flow cytometry test demonstrated that icariin or DEX administration resulted in a significant percentage reduction in CD4+RORγt+ T cells and elevation of CD4+Foxp3+ T cells in BALF. Furthermore, icariin or DEX caused a significant reduction in IL-6, IL-17 and TGF-β level in BALF. Unfortunately, icariin had no effect on IL-10 level in BALF. Western blot assay found that icariin or DEX suppressed RORγt and promoted Foxp3 expression in the lung tissue. qPCR analysis revealed that icariin and DEX resulted in a notable decrease in RORγt and increase in Foxp3 mRNA expression in isolated spleen CD4+ T cell. In conclusion, our results suggested that icariin was effective in the attenuation of AHR and chronic airway inflammatory changes in OVA-induced murine asthma model, and this effect was associated with regulation of Th17/Treg responses, which indicated that icariin may be used as a potential therapeutic method to treat asthma with Th17/Treg imbalance phenotype.

Effect of Simvastatin on 5-HT and 5-HTT in a Rat Model of Pulmonary Artery Hypertension

Background/Aims: To investigaterole of serotonin (5-HT) and serotonin transporter (5-HTT) in a rat model of cigarette smoke-induced pulmonary artery hypertension (PAH) and the effect of statins on regulating 5HT and 5-HTT. Methods: A rat model of COPD comorbid with PAH was established by cigarette smoke exposure with or without simvastatin administration. The smoking and the simvastatin plus smoking groups were exposed to cigarette smoke daily, and the latter received simvastatin at 5mg/kg, once a day. After 16 weeks of cigarette smoke exposure, body weight and mean pulmonary arterial pressure (mPAP) were measured, bronchoalveolar lavage (BAL) was performed, and lung tissues and blood samples were collected to determine cardiopulmonary pathology, physiological indices, blood levelof 5-HT and expression of 5-HTT in the lung. Results: In addition to alveolar structural damage (COPD-like injury), chronic cigarette smoke exposure lead to pulmonary artery remodeling and PAH as evidenced by significant elevation of mPAP, RVHI, WT%and WA%. Cigarette smoke exposure resulted in significant reduction in animal body weight, and simvastatin significantly prevented smoke-induced weight loss. The number of inflammatory cells in BALF was dramatically increased in smoke exposed rats, and simvastatin dampened the number of leukocytes, neutrophils, lymphocytes, and macrophages. In addition, circulating 5-HTand expression of 5-HTT in the lung were significantly increased in the smoked rats compared to control rats, and it was significantly reduced by simvastatin. Alteration of BALF inflammatory cells, 5-HT and 5-HTT was significantly correlated with changes of mPAP, RVHI, WT% and WA%. Conclusions: Cigarette smoke exposure could result in not only COPD, but also PAH, which may attribute to the alteration of blood 5-HT and lung tissue 5-HTT. Simvastatin could significantly inhibited 5-HT and 5-HTT expression, and by which mechanism, it may protect animals from development of PAH.