Inhibition of glutaminase 1 activity reverses airway hyperresponsiveness and decreases IL-1ß+ M1s and IL-17 producing ILC3s in high-fat diet-fed obese mice.

In obesity, disturbed glutamine metabolism contributes to enhanced inflammation by inducing alterations in immune cells. As macrophages and innate lymphoid cells (ILCs) are known to be involved in the pathogenesis of obesity-related asthma, we tested our hypothesis that altered glutamine metabolism may link obesity to airway hyperresponsivenss (AHR), a cardinal feature of asthma, focusing on these innate immune cells. Four-week-old male C57BL/6 mice were fed a high-fat diet (HFD) for 13 wk in the presence or absence of BPTES [Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, a selective inhibitor of glutaminase 1 which converts glutamine to glutamate] and their blood, lung, and adipose tissues were analyzed. We then conducted in vitro experiments using bone marrow-derived macrophages (BMDMs) and mouse alveolar macrophage cell line. Furthermore, we investigated plasma glutamine and glutamate levels in obese and nonobese asthmatics. BPTES treatment prevented HFD-induced AHR and significantly decreased IL-1ß+ classically activated macrophages (M1s) and type 3 ILCs (ILC3s) which increased in the lungs of HFD-fed obese mice. In in vitro experiments, BPTES treatment or glutamine supplement significantly reduced the proportion of IL-1ß+NLRP3+ M1s in lipopolysaccharide-stimulated BMDMs and mouse alveolar macrophage cell line. BPTES treatment also significantly reduced the IL-17 producing ILC3s differentiated from ILCs in naïve mouse lung. In addition, plasma glutamate/glutamine ratios were significantly higher in obese asthmatics compared to nonobese asthmatics. Inhibition of glutaminolysis reverses AHR in HFD-induced obese mice and decreases IL-1ß + NLRP3+ M1s and IL-17 producing ILC3s, which suggests altered glutamine metabolism may have a role in the pathogenesis of obesity-related AHR.

Polysaccharides from Platycodonis Radix ameliorated respiratory syncytial virus-induced epithelial cell apoptosis and inflammation through activation of miR-181a-mediated Hippo and SIRT1 pathways.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis in young children, but there are few safe and effective treatments for this disease. Platycodonis Radix is widely used as an antitussive and expectorant drug for preventing various diseases in lower respiratory tract, in which the polysaccharides are one of the main bioactivity constituents. In this study, the protective effects of the P. Radix polysaccharides (PRP) against RSV-induced bronchiolitis in juvenile mice and RSV-induced apoptosis of epithelial HEp-2 cells were investigated. The results showed that PRP obviously decreased the levels of IL-1ß, IL-4, IL-6, TNF-α, IFN-γ and TSLP in lung tissues, and reduced the number of inflammatory cells in bronchoalveolar lavage fluid (BALF) of RSV-infected mice. Furthermore, it reduced the apoptosis of RSV-infected HEp-2 cells and remarkably inhibited the mRNA expressions of RSV L gene, which indicated that PRP affected transcription and replication of RSV in host cells. Compared with that in RSV-infected group, miR-181a-5p in the PRP-treated group presented the highest relative abundance and its expression was violently reduced by approximately 30%. Mechanistically, PRP had the similar effects as miR-181a-5p antagomir on RSV-induced apoptosis and inflammation in HEp-2 cells via upregulating BCL2, MLL3 and SIRT1, which could be reversed by miR-181a-5p mimic. Therefore, it demonstrated that PRP not only protected against RSV-induced lung inflammation in mice but also inhibited apoptosis of RSV-infected HEp-2 cells via suppressing miR-181a-5p and transcriptionally activating Hippo and SIRT1 pathways.

Nitroalkene fatty acids modulate bile acid metabolism and lung function in obese asthma.

Bile acid profiles are altered in obese individuals with asthma. Thus, we sought to better understand how obesity-related systemic changes contribute to lung pathophysiology. We also test the therapeutic potential of nitro-oleic acid (NO2-OA), a regulator of metabolic and inflammatory signaling pathways, to mitigate allergen and obesity-induced lung function decline in a murine model of asthma. Bile acids were measured in the plasma of healthy subjects and individuals with asthma and serum and lung tissue of mice with and without allergic airway disease (AAD). Lung function, indices of inflammation and hepatic bile acid enzyme expression were measured in obese mice with house dust mite-induced AAD treated with vehicle or NO2-OA. Serum levels of glycocholic acid and glycoursodeoxycholic acid clinically correlate with body mass index and airway hyperreactivity whereas murine levels of ß-muricholic acid and tauro-ß-muricholic acid were significantly increased and positively correlated with impaired lung function in obese mice with AAD. NO2-OA reduced murine bile acid levels by modulating hepatic expression of bile acid synthesis enzymes, with a concomitant reduction in small airway resistance and tissue elastance. Bile acids correlate to body mass index and lung function decline and the signaling actions of nitroalkenes can limit AAD by modulating bile acid metabolism, revealing a potential pharmacologic approach to improving the current standard of care.

A phospholipid-based formulation for the treatment of airway inflammation in chronic respiratory diseases.

Inflammation, the major hallmark of all chronic respiratory diseases is generally managed by inhaled corticosteroids. However, long term high dose treatment can result in significant side effects. Hence, there is a medical need for non-steroidal anti-inflammatory therapies to address airway inflammation. Phospholipids have been shown to reduce inflammation in several inflammatory conditions; however, their clinical translation has been limited to liposomal formulations traditionally used as drug carriers and their biological activity has not been investigated. Here we report the first application of empty liposomes as an anti-inflammatory treatment in airway inflammation. In the current study, liposomes (UTS-001) were prepared from cholesterol and a synthetic phospholipid (DOPC). The formulation was characterised in terms of size, charge, polydispersity index, morphology and stability as colloidal suspension and freeze-dried nanoparticles. Time-dependant uptake of UTS-001 in airway epithelial cells was observed which was inhibited by nystatin demonstrating that the uptake is via the caveolae pathway. In-vitro, in primary nasal epithelial cells, UTS-001 treatment successfully attenuated IL-6 levels following TNF-α stimulation. Consistent with the in-vitro findings, in-vivo, in the ovalbumin model of allergic airway inflammation, UTS-001 significantly reduced total immune cell counts in bronchoalveolar lavage fluid and reduced airway hyperresponsiveness in response to increasing doses of methacholine challenge. Therefore, our results establish UTS-001 as a potential anti-inflammatory treatment that may be useful as a therapeutic for lung inflammatory diseases.

Vitamin A supplement after neonatal Streptococcus pneumoniae pneumonia inhibits the progression of experimental asthma by altering CD4+T cell subsets.

Studies demonstrated that pneumonia can decrease vitamin A productions and vitamin A reduction/deficiency may promote asthma development. Our previous study showed that neonatal Streptococcus pneumoniae (S. pneumoniae) infection promoted asthma development. Whether neonatal S. pneumoniae pneumonia induced asthma was associated with vitamin A levels remains unclear. The aim of this study was to investigate the effects of neonatal S. pneumoniae pneumonia on vitamin A expressions, to explore the effects of vitamin A supplement after neonatal S. pneumoniae pneumonia on adulthood asthma development. Non-lethal S. pneumoniae pneumonia was established by intranasal inoculation of neonatal (1-week-old) female BALB/c mice with D39. S. pneumoniae pneumonia mice were supplemented with or without all-trans retinoic acid 24 hours after infection. Vitamin A concentrations in lung, serum and liver were measured post pneumonia until early adulthood. Four weeks after pneumonia, mice were sensitized and challenged with OVA to induce allergic airway disease (AAD). Twenty-four hours after the final challenge, the lungs and bronchoalveolar lavage fluid (BALF) were collected to assess AAD. We stated that serum vitamin A levels in neonatal S. pneumoniae pneumonia mice were lower than 0.7µmol/L from day 2-7 post infection, while pulmonary vitamin A productions were significantly lower than those in the control mice from day 7-28 post infection. Vitamin A supplement after neonatal S. pneumoniae pneumonia significantly promoted Foxp3+Treg and Th1 productions, decreased Th2 and Th17 cells expressions, alleviated airway hyperresponsiveness (AHR) and inflammatory cells infiltration during AAD. Our data suggest that neonatal S. pneumoniae pneumonia induce serum vitamin A deficiency and long-time lung vitamin A reduction, vitamin A supplement after neonatal S. pneumoniae pneumonia inhibit the progression of asthma by altering CD4+T cell subsets.

Cordyceps polysaccharide ameliorates airway inflammation in an ovalbumin-induced mouse model of asthma via TGF-ß1/Smad signaling pathway.

Allergic asthma is a chronic inflammatory disease characterized by airflow obstruction, airway hyperresponsiveness (AHR), airway inflammation, and mucus overproduction. Cordyceps polysaccharide (CPS) is one of the main bioactive compounds of Cordyceps militarisis, a traditional Chinese medicine. In this study, we established a mouse model of asthma using ovalbumin (OVA) challenge and evaluated the potential regulatory effect of CPS (25, 50, and 100 mg/kg) on asthmatic mice. These results showed that the asthmatic mice treated with CPS suppressed the secretion of eotaxin, IL-4, IL-5, IL-13, and IFN-γ in the blood and bronchoalveolar lavage fluid (BALF), and decreased serum IgE levels compared to the vehicle-treated mice. CPS also alleviated inflammatory cell infiltration, goblet cell hyperplasia, and the increases of inflammatory cells in the mouse model of asthma. In addition, OVA-induced AHR was inhibited by CPS treatment. Further analyses of protein expression revealed that CPS inhibited the activation of transforming growth factor ß1 (TGF-ß1)/Smad pathway in mice with asthma. These findings indicated that CPS might serve as a potential therapeutic agent for the management of allergic asthma.

A combination of LCPUFA ameliorates airway inflammation in asthmatic mice by promoting pro-resolving effects and reducing adverse effects of EPA.

Lipid mediators derived from omega (n)-3 and n-6 long-chain polyunsaturated fatty acids (LCPUFA) play key roles in bronchoconstriction, airway inflammation, and resolution processes in asthma. This study compared the effects of dietary supplementation with either a combination of LCPUFAs or eicosapentaenoic acid (EPA) alone to investigate whether the combination has superior beneficial effects on the outcome of asthmatic mice. Mice were sensitized with house dust mite (HDM) extract, and subsequently supplemented with either a combination of LCPUFAs or EPA alone in a recall asthma model. After the final HDM and LCPUFA administration, airway hyperresponsiveness (AHR), bronchoalveolar lavages, and lung histochemistry were examined. Lipid mediator profiles were determined by liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). The LCPUFA combination reduced AHR, eosinophilic inflammation, and inflammatory cytokines (IL-5, IFN-γ, and IL-6) in asthmatic mice, whereas EPA enhanced inflammation. The combination of LCPUFAs was more potent in downregulating EPA-derived LTB5 and LTC5 and in supporting DHA-derived RvD1 and RvD4 (2.22-fold and 2.58-fold higher levels) than EPA alone. Ex vivo experiments showed that LTB5 contributes to granulocytes' migration and M1-polarization in monocytes. Consequently, the LCPUFA combination ameliorated airway inflammation by inhibiting adverse effects of EPA and promoting pro-resolving effects supporting the lipid mediator-dependent resolution program.

Upregulation of airway smooth muscle calcium-sensing receptor by low-molecular-weight hyaluronan.

We investigated the mechanisms involved in the development of airway hyperresponsiveness (AHR) following exposure of mice to halogens. Male mice (C57BL/6; 20-25 g) exposed to either bromine (Br2) or Cl2 (600 or 400 ppm, respectively, for 30 min) developed AHR 24 h after exposure. Nifedipine (5 mg/kg body wt; an L-type calcium channel blocker), administered subcutaneously after Br2 or Cl2 exposure, produced higher AHR compared with Br2 or Cl2 alone. In contrast, diltiazem (5 mg/kg body wt; a nondihydropyridine L-type calcium channel blocker) decreased AHR to control (air) values. Exposure of immortalized human airway smooth muscle cells (hASMC) to Br2 resulted in membrane potential depolarization (Vm Air: 62 ± 3 mV; 3 h post Br2:-45 ± 5 mV; means ± 1 SE; P < 0.001), increased intracellular [Ca2+]i, and increased expression of the calcium-sensing receptor (Ca-SR) protein. Treatment of hASMC with a siRNA against Ca-SR significantly inhibited the Br2 and nifedipine-induced Vm depolarization and [Ca2+]i increase. Intranasal administration of an antagonist to Ca-SR in mice postexposure to Br2 reversed the effects of Br2 and nifedipine on AHR. Incubation of hASMC with low-molecular-weight hyaluronan (LMW-HA), generated by exposing high-molecular-weight hyaluronan (HMW-HA) to Br2, caused Vm depolarization, [Ca2+]i increase, and Ca-SR expression to a similar extent as exposure to Br2 and Cl2. The addition of HMW-HA to cells or mice exposed to Br2, Cl2, or LMW-HA reversed these effects in vitro and improved AHR in vivo. We conclude that detrimental effects of halogen exposure on AHR are mediated via activation of the Ca-SR by LMW-HA.

Acaciain peptidase: The first South American pollen peptidase potentially involved in respiratory allergy.

Acacia caven (Mol.) Molina pollen causes pollinosis in South America. The aim of this work was to isolate, purify, and characterize the proteolytic enzymes of A. caven pollen, and study their influence on allergy. A series of chromatographic steps were applied to purify the proteolytic extract of A. caven pollen. The purified fraction was partially characterized, and then it was assayed on airway bioactive peptides (substance P, vasoactive intestinal peptide, and bradykinin), and peptide degradation was visualized by direct protein sequencing. The cellular detachment of an airway-derived epithelial cell line (A-549) was measured by methylene blue binding assay. The degradation of proteins from intercellular junctions (occludin, claudin, and E-cadherin) was visualized by Western blot. A 75-kDa peptidase, named acaciain peptidase, was purified and classified as a serine peptidase. Acaciain peptidase degraded bioactive peptides involved in the maintenance and recovery of the bronchomotor tone; it caused cellular detachment of A-549 cell line, and degradation of intercellular junction proteins. Acaciain peptidase can alter the integrity of the epithelium barrier, causing cell permeability, increasing the allergic sensitization and exacerbating the overall bronchoconstrictive effect detected in asthmatic lungs. This novel serine peptidase constitutes a relevant therapeutic target in the treatment of allergic disorders.

DFSG, a novel herbal cocktail with anti-asthma activity, suppressed MUC5AC in A549 cells and alleviated allergic airway hypersensitivity and inflammatory cell infiltration in a chronic asthma mouse model.

AIM OF THE STUDY: To develop a novel anti-asthma drug. DFSG is a novel herbal cocktail composed of 4 types of herbal medicines. This study explored whether DFSG has the potential to attenuate asthma symptom severity and aimed to determine the immunomodulatory mechanism of DFSG using a chronic asthmatic mouse model induced by repeated challenges with Dermatogoides pteronyssinus (Der p). MATERIALS AND METHODS: BALB/c mice were intratracheally inoculated with Der p (50 µl, 1 mg/ml) once a week for 5 weeks. In addition, 30 min before Der p challenge, the mice were orally administered 1x DFSG (1 g/kg) or 1/2x DFSG (0.5 g/kg). Three days after the final challenge, the mice were sacrificed to evaluate inflammatory cell infiltration, lung histological features, blood total IgE, and cytokine levels in pulmonary alveolar lavage fluid. Furthermore, 30 min after the addition of DFSG, caffeic acid, p-coumaric acid or chlorogenic acid to A549 cells, 10 ng/ml IL-1ß was added to evaluate the effect of the drug on mucin 5AC (MUC5AC) gene expression after stimulation of A549 cells by IL-1ß. RESULTS: DFSG significantly reduced Der p-induced airway hyperresponsiveness, bronchial inflammatory cell infiltration, and total IgE and IgG1 serum levels. Furthermore, DFSG significantly inhibited TH2 cytokines and increased the expression of TH1 cytokines. In addition, immunohistochemical staining demonstrated that DFSG inhibited MUC5AC expression in the bronchial epithelial cells. DFSG and a mixture of caffeic acid, p-coumaric acid, and chlorogenic acid inhibited MUC5AC gene expression in A549 cells after stimulation with IL-1ß. CONCLUSION: These results suggest that by regulating TH1 and TH2 cytokines and MUC5AC expression, DFSG exhibits anti-airway inflammatory cell infiltration and anti-hyperresponsiveness activity and inhibits specific immunity in a chronic asthmatic mouse model. Therefore, DFSG has potential for development into a drug for chronic asthma treatment.

Comparing the Protection Imparted by Different Fraction Extracts of Garlic (Allium sativum L.) against Der p-Induced Allergic Airway Inflammation in Mice.

Garlic (Allium sativum L.) has been used extensively as a food ingredient and medicinally, but the effect on asthmatic airway inflammation has not been studied in detail. We accordingly explored the protective effects exerted by various garlic fraction extracts against airway inflammation with Dermatophagoides pteronyssinus (Der p)-induced allergic asthma in vivo and in vitro. Garlic extraction was realized using n-hexane, dichloromethane, ethylacetate, n-butanol, and water in sequence to obtain different fraction extracts. Mice were orally administered different fractions (80 mg/kg) daily for four weeks. The histological results showed that the water fraction could ameliorate lung-based goblet cell hyperplasia, inflammatory cell infiltration, and mucus hypersecretion. The water fraction extracts decreased IgE and IgG1, and they decreased inflammatory cells as quantified in bronchoalveolar lavage fluid (BALF); however, they increased IgG2a in serum. Moreover, the water fraction extracts increased IFN-γ and IL-12 (both constituting Th1 cytokines) in BALF, but they reduced IL-13, -4, and -5 (all constituting Th2 cytokines), and also inhibited the expression of IL-1ß, IL-6, and TNF-α. The water fraction also inhibited the PI3K/Akt/NF-κB signal pathways in A549 cells. These findings suggest that water fraction extracts of garlic have a clear anti-inflammatory effect on Der p-induced allergic asthma.

Ocimum basilicum affects tracheal responsiveness, lung inflammatory cells and oxidant-antioxidant biomarkers in sensitized rats.

The anti-inflammatory and antioxidant effects of Ocimum basilicum (O. basilicum) was shown previously. In the present study, the effect of O. basilicum on tracheal responsiveness (TR) to methacholine and ovalbumin (OVA), bronchoalveolar lavage fluid (BALF) levels of oxidant-antioxidant biomarkers as well as total and differential white blood cell (WBC) in sensitized rats was examined. Six groups of rats including control (group C), sensitized rats to OVA (group S), S groups treated with three concentrations of O. basilicum (0.75, 1.50, and 3.00 mg/ml) and one concentration of dexamethasone (1.25 µg/ml) (n = 8 for all groups) were studied. TR to methacholine and OVA, total WBC count, percentages of eosinophils, monocytes, neutrophils, and levels of oxidant biomarkers were significantly increased but other measured parameters were significantly decreased in group S compared to group C. TR to methacholine and OVA, percentages of eosinophils, monocytes, neutrophils, and levels of oxidant biomarkers were significantly decreased but lymphocytes and antioxidant biomarkers were significantly increased in S groups treated with dexamethasone and at least two higher concentrations of the extract compared to group S. Total WBC count was also decreased in treated S groups with dexamethasone and high extract concentration. The effect of extract on most measured parameters was significantly lower than dexamethasone treatment. The effects of two higher concentrations of the extract on most variables were significantly higher than the effect of low extract concentration. These results showed the concentration-dependent effect of O. basilicum on tracheal responses, lung inflammatory cells, and oxidant-antioxidant parameters in sensitized rats.

Prophylactic effect of rosmarinic acid on tracheal responsiveness, white blood cell count and oxidative stress markers in lung lavage of sensitized rats.

BACKGROUND: Rosmarinic acid (RA) as an active component of several medicinal plants, has shown anti-inflammatory and anti-oxidant effects. In this study, the effect of RA on tracheal responsiveness (TR), lung inflammatory cells, oxidant biomarkers in sensitized rats were evaluated. METHODS: TR to methacholine and ovalbumin (OVA) as well as total and differential white blood cell (WBC) count and levels of nitrogen dioxide, nitrate, malondialdehyde, thiol, superoxide dismutase, and catalase in bronchoalveolar lavage fluid were measured in control (group C) rats, sensitized animals to OVA and given drinking water alone (group S), S groups receiving drinking water containing three concentrations of RA (0.125, 0.250 and 0.500 mg/mL) and dexamethasone (1.25 µg/mL), (n = 6 in each group). RESULTS: Increased TR to methacholine and OVA, total WBC count, percentages of eosinophils, monocytes, neutrophils and levels of oxidant biomarkers but decreased other measured parameters were observed in group S compared to group C. Percentages of lymphocytes and antioxidant biomarkers were significantly increased but other measured parameters were significantly decreased in S group treated with dexamethasone and in rats treated with the two higher concentrations of RA compared to S group. The effect of RA medium concentration on percentage of eosinophils and RA high concentration on total WBC count and percentages of eosinophils and lymphocytes, were significantly higher than those of dexamethasone. CONCLUSION: These results showed the concentration-dependent effect of RA on tracheal responses, lung inflammatory cells and oxidant-antioxidant parameters which was comparable to that of dexamethasone at used concentrations in sensitized rats.

Vitamin E Reversed Apoptosis of Cardiomyocytes Induced by Exposure to High Dose Formaldehyde During Mice Pregnancy.

In this study, we investigated the protection effect of Vitamin E (Vit E) on formaldehyde (FA) exposure during pregnancy induced apoptosis of cardiomyocytes, and used an HL-1 cell line to confirmed the findings in vivo.Pregnant mice received different doses of FA (0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 0.1 µg Vit E, or 1.5 mg/kg + 0.1 µg Vit E). TUNEL staining was used to reveal the apoptosis in cardiomyocytes, and SOD, MDA, GSH, Livin, and Caspase-3 in cardiomyocytes were detected by ELISA, RT-PCR, and Western blot. For in vitro study, HL-1 cells were treated with vehicle, 5 µmol/L FA, 25 µmol/L FA, 50 µmol/L FA, 10 mg/L Vit. E, and 50 µmol/L FA+ 10 mg/L Vit E, respectively. CCK-8 assay and flow cytometry were used to evaluate cell vitality and apoptosis. A high dose of FA exposure led to cytotoxicity in both pregnant mice and offspring, as TUNEL staining revealed a significant apoptosis of cardiomyocytes, and the alternation in SOD, GSH, MDA, Livin, and Caspase-3 was found in cardiomyocytes. 0.1 µg Vit. E could reverse high doses of FA exposure induced apoptosis of cardiomyocytes in both pregnant mice and offspring. The in vitro study revealed that FA exposure induced a decrease of cell viability and increased cell apoptosis, as well as oxidative stress in HL-1 cells with alternation in SOD, GSH, MDA, Livin, and Caspase-3.This study revealed a high dose of FA induced oxidative stress and apoptosis of cardiomyocytes in both pregnant mice and offspring, and Vit E supplement during pregnancy reversed the systemic and myocardial toxicity of FA.

Inhibitory effects of a standardized extract of Justicia pectoralis in an experimental rat model of airway hyper-responsiveness.

OBJECTIVE: Justicia pectoralis is a plant useful for the treatment of respiratory diseases. Here, we studied the antiasthmatic properties of a standardized extract of J. pectoralis (Jp). METHODS: Ovalbumin (OVA)-sensitized rats were actively challenged with saline or OVA to study airway hyper-responsiveness after oral treatment with saline or Jp. The ability of Jp to inhibit hyper-reactivity was evaluated in isolated trachea mounted in isolated organ bath chamber. KEY FINDINGS: Using KCl or carbachol as contractile agents, tracheal rings of OVA-challenged rats contracted with higher magnitude than trachea of rats challenged with saline. Such hyper-responsive phenotype of OVA-challenged tissues decreased with Jp administration. In Ca+ -free medium, Jp or its major constituent coumarin inhibited preferentially the contractions induced by Ca2+ addition in tissues of OVA-challenged rats stimulated with KCl or acetylcholine. In tissues depleted of their internal Ca+ stores in the presence of thapsigargin, Jp inhibited the contraction induced by capacitative Ca2+ entry. By gavage, Jp abolished the increase caused by challenge with OVA on the levels of IL-1ß and TNF-α in the bronchoalveolar fluid and also impaired the changes in gene expression of canonical transient receptor proteins. CONCLUSIONS: Jp has antiasthmatic properties in an experimental model that reproduces tracheal hyper-reactivity.

Therapeutic effects of rosmarinic acid on airway responses in a murine model of asthma.

Rosmarinic acid (RA) is an active component of a traditional Chinese herbal medicine. Previously, we reported that RA exerted a strong anti-inflammatory effect in a mouse acute lung injury model. Therefore, we hypothesized that RA might also have potential therapeutic effects in a murine model of asthma. In this study, we aimed to evaluate the anti-asthmatic activity of RA and explored its possible molecular mechanisms of action. Female BALB/c mice that had been sensitized to and challenged with ovalbumin (Ova) were treated with RA (20mg/kg) 1h after challenge. The results showed that RA greatly diminished the number of inflammatory cells and the production of Th2 cytokines in the bronchoalveolar lavage fluid (BALF); significantly reduced the secretion of total IgE, Ova-specific IgE, and eotaxin; and markedly ameliorated airway hyperresponsiveness (AHR) compared with Ova-induced mice. Histological studies further revealed that RA substantially decreased inflammatory cells infiltration and mucus hypersecretion compared with Ova-induced mice. Moreover, our results suggested that the protective effects of RA were mediated by the inhibition of JNK and p38 MAPK phosphorylation and nuclear factor-κB (NF-κB) activation. Furthermore, RA treatment resulted in a significant reduction in the mRNA expression of AMCase, CCL11, CCR3, Ym2 and E-selectin in lung tissue. These findings suggest that RA may effectively delay the development of airway inflammation and could thus be used as a therapy for allergic asthma.

Morphometric analysis of inflammation in bronchial biopsies following exposure to inhaled diesel exhaust and allergen challenge in atopic subjects.

BACKGROUND: Allergen exposure and air pollution are two risk factors for asthma development and airway inflammation that have been examined extensively in isolation. The impact of combined allergen and diesel exhaust exposure has received considerably less attention. Diesel exhaust (DE) is a major contributor to ambient particulate matter (PM) air pollution, which can act as an adjuvant to immune responses and augment allergic inflammation. We aimed to clarify whether DE increases allergen-induced inflammation and cellular immune response in the airways of atopic human subjects. METHODS: Twelve atopic subjects were exposed to DE 300 µg.m(-3) or filtered air for 2 h in a blinded crossover study design with a four-week washout period between arms. One hour following either filtered air or DE exposure, subjects were exposed to allergen or saline (vehicle control) via segmental challenge. Forty-eight hours post-allergen or control exposure, bronchial biopsies were collected. The study design generated 4 different conditions: filtered air + saline (FAS), DE + saline (DES), filtered air + allergen (FAA) and DE + allergen (DEA). Biopsies sections were immunostained for tryptase, eosinophil cationic protein (ECP), neutrophil elastase (NE), CD138, CD4 and interleukin (IL)-4. The percent positivity of positive cells were quantified in the bronchial submucosa. RESULTS: The percent positivity for tryptase expression and ECP expression remained unchanged in the bronchial submucosa in all conditions. CD4 % positive staining in DEA (0.311 ± 0.060) was elevated relative to FAS (0.087 ± 0.018; p = 0.035). IL-4% positive staining in DEA (0.548 ± 0.143) was elevated relative to FAS (0.127 ± 0.062; p = 0.034). CD138 % positive staining in DEA (0.120 ± 0.031) was elevated relative to FAS (0.017 ± 0.006; p = 0.015), DES (0.044 ± 0.024; p = 0.040), and FAA (0.044 ± 0.008; p = 0.037). CD138% positive staining in FAA (0.044 ± 0.008) was elevated relative to FAS (0.017 ± 0.006; p = 0.049). NE percent positive staining in DEA (0.224 ± 0.047) was elevated relative to FAS (0.045 ± 0.014; p = 0.031). CONCLUSIONS: In vivo allergen and DE co-exposure results in elevated CD4, IL-4, CD138 and NE in the respiratory submucosa of atopic subjects, while eosinophils and mast cells are not changed. TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01792232.

Facilitation of Allergic Sensitization and Allergic Airway Inflammation by Pollen-Induced Innate Neutrophil Recruitment.

Neutrophil recruitment is a hallmark of rapid innate immune responses. Exposure of airways of naive mice to pollens rapidly induces neutrophil recruitment. The innate mechanisms that regulate pollen-induced neutrophil recruitment and the contribution of this neutrophilic response to subsequent induction of allergic sensitization and inflammation need to be elucidated. Here we show that ragweed pollen extract (RWPE) challenge in naive mice induces C-X-C motif ligand (CXCL) chemokine synthesis, which stimulates chemokine (C-X-C motif) receptor 2 (CXCR2)-dependent recruitment of neutrophils into the airways. Deletion of Toll-like receptor 4 (TLR4) abolishes CXCL chemokine secretion and neutrophil recruitment induced by a single RWPE challenge and inhibits induction of allergic sensitization and airway inflammation after repeated exposures to RWPE. Forced induction of CXCL chemokine secretion and neutrophil recruitment in mice lacking TLR4 also reconstitutes the ability of multiple challenges of RWPE to induce allergic airway inflammation. Blocking RWPE-induced neutrophil recruitment in wild-type mice by administration of a CXCR2 inhibitor inhibits the ability of repeated exposures to RWPE to stimulate allergic sensitization and airway inflammation. Administration of neutrophils derived from naive donor mice into the airways of Tlr4 knockout recipient mice after each repeated RWPE challenge reconstitutes allergic sensitization and inflammation in these mice. Together these observations indicate that pollen-induced recruitment of neutrophils is TLR4 and CXCR2 dependent and that recruitment of neutrophils is a critical rate-limiting event that stimulates induction of allergic sensitization and airway inflammation. Inhibiting pollen-induced recruitment of neutrophils, such as by administration of CXCR2 antagonists, may be a novel strategy to prevent initiation of pollen-induced allergic airway inflammation.

Echinacea complex--chemical view and anti-asthmatic profile.

ETHNOPHARMACOLOGICAL RELEVANCE: Echinacea purpurea (L.) Moench is one of the mostly used herbs in the traditional medicine for the treatment of respiratory diseases. Modern interest in Echinacea is directed to its immunomodulatory activity. Recent studies have shown that secretion of asthma-related cytokines in the bronchial epithelial cells can be reversed by Echinacea preparations. AIM OF THE STUDY: To examine the pharmacodynamics profile of Echinacea active principles, a complex has been isolated from its flowers by alkaline extraction and has been tested using an animal model of allergic asthma. MATERIAL AND METHODS: The structural features of Echinacea purpurea complex was determined using chemical and spectroscopic methods. Allergic inflammation of the airways was induced by repetitive exposure of guinea pigs to ovalbumin. Echinacea complex was then administered 14 days in 50mg/kg b.w. daily dose perorally. Bronchodilatory effect was verified as decrease in the specific airway resistance (sRaw) in vivo and by reduced contraction amplitude (mN) of tracheal and pulmonary smooth muscle to cumulative concentrations of acetylcholine and histamine in vitro. The impact on mucociliary clearance evaluated measurement of ciliary beat frequency (CBF) in vitro using LabVIEW™ Software. Anti-inflammatory effect of Echinacea complex was verified by changes in exhaled NO levels and by Bio-Plex® assay of Th2 cytokine concentrations (IL-4, IL-5, IL-13 and TNF-alpha) in serum and bronchoalveolar lavage fluid (BALF). RESULTS: Chemical and spectroscopic studies confirmed the presence of carbohydrates, phenolic compounds and proteins, as well as the dominance of rhamnogalacturonan and arabinogalactan moieties in Echinacea complex. The significant decrease in sRaw values and suppressed histamine and acetylcholine-induced contractile amplitude of isolated airways smooth muscle that were similar to effects of control drug salbutamol confirmed Echinacea complex bronchodilatory activity. The anti-inflammatory effect was comparable with that of control agent budesonide and was verified as significantly reduced exhaled NO levels and concentration of Th2 cytokines in serum and BALF. The values of CBF were changed only insignificantly on long-term administration of Echinacea complex suggested its minimal negative impact on mucociliary clearance. CONCLUSION: Pharmacodynamic studies have confirmed significant bronchodilatory and anti-inflammatory effects of Echinacea complex that was similar to effects of classic synthetic drugs. Thus, results provide a scientific basis for the application of this herb in traditional medicine as a supplementary treatment of allergic disorders of the airways, such as asthma.

Anti-inflammatory activities of Physalis alkekengi var. franchetii extract through the inhibition of MMP-9 and AP-1 activation.

Physalis alkekengi has been traditionally used for the treatment of coughs, middle ear infections, and sore throats in Korea, Europe, and China. It exhibits a variety of pharmacological activities such as anti-inflammatory, anti-oxidant, and anti-cancer effects. The anti-inflammatory effects of the P. alkekengi methanol extract (PA) and its molecular mechanisms have not yet been fully investigated. In the present study, the chromatogram of PA was established by UPLC analysis. The anti-inflammatory effects of PA were also investigated using murine microphage cell lines, RAW 264.7 cells, and a murine model of OVA induced asthma. In LPS-stimulated RAW264.7 cells, PA reduced the MMP-9 expression with decreases in the production of nitric oxide, inteleukin-6, and tumor necrosis factor-α. Furthermore, PA suppressed the phosphorylation of MAPKs, which resulted in the inhibition of AP-1 activation. These effects of PA were consistent with the results of the in vivo experiment. PA-treated mice significantly inhibited inflammatory cell counts and cytokine production in bronchoalveolar lavage fluids and airway-hyperresponsiveness in OVA-induced asthmatic mice. PA treated mice also showed a marked inhibition of inducible nitric oxide synthase and MMP-9 expression. In conclusion, our results suggest that PA may be a valuable therapeutic material in treating various inflammatory diseases, including allergic asthma.