Cellular Distribution of Secreted Phospholipase A2 in Lungs of IPF Patients and Its Inhibition in Bleomycin-Induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease with a very poor prognosis as it has a 2.5 to 5 years mean survival after proper diagnosis. Even nintedanib and pirfenidone cannot halt the progression, though they slow the progression of IPF. Hence, there is a need to understand the novel pathophysiology. Phospholipase A2 (PLA2) could be the ideal candidate to study in IPF, as they have a role in both inflammation and fibrosis. In the present study, we have shown the expression profile of various secretory Phospholipase A2 (PLA2) isoforms by analyzing publicly available transcriptome data of single cells from the lungs of healthy individuals and IPF patients. Among 11 members of sPLA2, PLA2G2A is found to be increased in the fibroblasts and mesothelial cells while PLA2G5 is found to be increased in the fibroblasts of IPF patients. We identified a subset of fibroblasts expressing high PLA2G2A with moderate expression of PLA2G5 and which are specific to IPF only; we named it as PLA2G2A+ IPF fibroblast. Pathway analysis revealed that these PLA2G2A+ IPF fibroblast have upregulation of both inflammatory and fibrosis-related pathways like the TGF-ß signaling pathway, IL-17 signaling, the arachidonic acid metabolism pathway and ECM-receptor interaction. In addition to this, we found elevated levels of sPLA2-IIA in plasma samples of IPF patients in our cohort. PLA2G3, PLA2G10 and PLA2G12B are found in to be increased in certain epithelial cells of IPF patients. Thus, these findings indicate that these five isoforms have a disease-dominant role along with innate immune roles as these isoforms are found predominantly in structural cells of IPF patients. Further, we have targeted sPLA2 in mice model of bleomycin-induced lung fibrosis by pBPB, a known sPLA2 inhibitor. pBPB treatment attenuated lung fibrosis induced by bleomycin along with a reduction in TGF-ß and deposition of extracellular matrix in lung. Thus, these findings indicate that these sPLA2 isoforms especially PLA2G2A may serve as a therapeutic target in lung fibrosis.

House dust mite allergen causes certain features of steroid resistant asthma in high fat fed obese mice.

Obesity is a high risk factor for diseases such as cardiovascular, metabolic syndrome and asthma. Obese-asthma is another emerging phenotype in asthma which is typically refractive to steroid treatment due to its non-classical features such as non-eosinophilic cellular inflammation. The overall increased morbidity, mortality and economical burden in asthma is mainly due to steroid resistant asthma. In the present study, we used high fat diet induced obese mice which when sensitized with house dust mite (HDM) showed steroid resistant features. While the steroid, dexamethasone (DEX), treatment to high fat fed naïve mice could not reduce the airway hyperresponsiveness (AHR) induced by high fat, DEX treatment to high fat fed allergic mice could not reduce the HDM allergen induced airway remodeling features though it reduced airway inflammation. Further, these HDM induced high fat fed mice with or without DEX treatment had shown the increased activity and expression of arginase as well as the inducible nitric oxide synthase (iNOS) expression. However, DEX treatment had reduced the expressions of high iNOS and arginase I in control chow diet fed mice. Thus, we speculate that the steroid resistance seen in human obese asthmatics could be stemming from altered NO metabolism and its induced airway remodeling and with further investigations, it would encourage new treatments specific to obese-asthma phenotype.

Parabromophenacyl bromide inhibits subepithelial fibrosis by reducing TGF-ß1 in a chronic mouse model of allergic asthma.

BACKGROUND: Our previous study showed that parabromophenacyl bromide (PBPB) inhibits the features of allergic airway inflammation and airway hyperresponsiveness (AHR). However, its effect on airway remodeling, e.g. subepithelial fibrosis in a chronic allergic asthma model, was not investigated. We examined this issue in this study. METHODS: PBPB was administered to mice with an induced chronic asthmatic condition. AHR was estimated at the end of the experiment, followed by euthanasia. Lung sections were stained with hematoxylin and eosin, periodic acid-Schiff and Masson's trichrome to determine airway inflammation, goblet cell metaplasia and subepithelial fibrosis, respectively. Transforming growth factor-ß1 (TGF-ß1) was estimated in lung homogenates. To determine the effect of PBPB on smooth-muscle hyperplasia, immunohistochemistry against α-smooth-muscle actin was performed on the lung sections. RESULTS: Chronic ovalbumin challenges in a mouse model of allergic asthma caused significant subepithelial fibrosis and elevated TGF-ß1, along with significant AHR. PBPB attenuated subepithelial fibrosis with a reduction of lung TGF-ß1, airway inflammation and AHR without affecting goblet cell metaplasia. It also attenuated smooth-muscle hyperplasia with a reduction in the expression of α-smooth-muscle actin in the lungs. CONCLUSION: Our findings indicate that PBPB attenuates some crucial features of airway remodeling such as subepithelial fibrosis and smooth-muscle hyperplasia. These data suggest that PBPB could therefore be a therapeutic drug for chronic asthma.

Medicinal plants useful for treating chronic obstructive pulmonary disease (COPD): current status and future perspectives.

Chronic obstructive pulmonary disease (COPD) is a major global health problem. The current therapies are inadequate and have numerous adverse effects. There is an acute need of potential alternative therapies. Medicinal plants are classical and most widespread form of medication for treating various human ailments throughout the world. For COPD also, various plants are practiced in many countries. We have surveyed the literatures for the medicinal plants which are used in obstructive lung diseases such as bronchitis and emphysema. These plants were segregated into two categories. Firstly, the plants which are used for obstructive lung diseases based on various traditional recommendations only. Secondly, the plants or their formulations which have been studied grossly in COPD patients and animal models for their scientific validation. Further, we have described some known cellular and molecular factors involved in COPD and their modulation by plant-derived compounds. Certain future perspectives have also been discussed.

Inhaled carbenoxolone prevents allergic airway inflammation and airway hyperreactivity in a mouse model of asthma.

BACKGROUND: Asthma is a chronic respiratory disease, which needs a safer medication preferably in inhalation form. In view of this, we have evaluated the effect of inhaled carbenoxolone (CBX), a herbal-derived compound, on asthma in a mouse model. METHODS: Mice were sensitized and challenged with ovalbumin (OVA) to develop certain characteristic features of asthma such as airway hyperreactivity (AHR), airway eosinophilia, lung inflammation and mucus hypersecretion. To evaluate the effect of CBX on the above asthmatic features, CBX (2.5, 5 and 10 mg/ml, 3 ml) or vehicle (water) was given by inhalation. AHR was determined using whole-body plethysmography. Infiltration of eosinophils was estimated by microscopy. Lung inflammation and mucus hypersecretion were assessed using hematoxylin and eosin, and periodic acid-Schiff staining, respectively. Th-2 cytokines, IL-4 and IL-5 were measured in bronchoalveolar lavage (BAL) fluid and IgE in sera. To identify the possible mode of CBX action, we measured corticosterone levels in the BAL fluid and 5-lipoxygenase (5-LO) expression in the lungs. RESULTS: CBX (5 mg/ml) inhalation markedly alleviated AHR (p = 0.0032) and reduced lung inflammation and mucus hypersecretion. Also, it prevented the increase in IL-4 (p = 0.0192), IL-5 (p = 0.0116) and eosinophils (p < 0.0005) in the BAL fluid, and OVA-specific IgE levels (p = 0.00061) in sera. 5-LO expression was also markedly reduced. However, corticosterone levels were not affected. CONCLUSIONS: Inhaled CBX alleviates the asthmatic features in mice and could be a potent nebulized therapy in clinical asthma.

Mepacrine alleviates airway hyperresponsiveness and airway inflammation in a mouse model of asthma.

Asthma is a multifactorial respiratory disease. Though its incidence is increasing rapidly all over the world, the available therapeutic strategies are neither sufficient nor safe for long term use. Mepacrine, a known antimalarial drug, has been shown to possess antioxidant, anti-inflammatory, platelet anti-aggregant, and PLA2 inhibitory activities. However, its possible use in asthma has not been studied yet. The objective of this study was to investigate the anti-asthmatic property of mepacrine using a mouse model of asthma. To accomplish this, male BALB/c mice were sensitized and challenged with ovalbumin and treated with increasing concentrations of mepacrine. Airway hyperresponsiveness (AHR) to methacholine was assessed using unrestrained whole body plethysmography. Mepacrine (1 mg/kg) has shown marked attenuation of AHR. Cytokines such as IL-4, IL-5, IL-13 and IFN-gamma and OVA-specific IgE levels were measured in BAL (bronchoalveloar lavage) fluid and sera, respectively. Mepacrine effectively reduced the rise in IL-4, IL-5, IL-13, and OVA-specific IgE and restored IFN-gamma levels. Mepacrine also significantly prevented the increase of sPLA2 (secretory phospholipase A2) activity in BAL fluid supernatant and Cys-LT (cysteinyl leukotrienes) in lung tissue homogenates of asthmatic mice. In addition, mepacrine treatment reduced BAL fluid eosinophilia and signs of allergic airway inflammation such as perivascular and peribronchial distribution of inflammatory cells. These findings indicate that mepacrine reduces the asthmatic features in ovalbumin induced asthma by acting on PLA2-Cys-LT axis. Thus, it could be useful for the development of better asthma therapy.

Novel approaches for inhibition of mucus hypersecretion in asthma.

Many obstructive airway disorders such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease (COPD) are characterized by mucous metaplasia of the airway epithelium and chronic mucus hypersecretion. Airway occlusion by mucus plugging has been reported in many cases of fatal asthma and conventional mucolytic therapies have been unable to significantly affect mucus-related airway obstruction. Recently, for the first time, direct evidence was presented for improvement of mucus related airway obstruction in a murine model of asthma, highlighting the potential usefulness of therapeutically targeting mucus hypersecretion. We review the emerging targets for inhibition of mucus hypersecretion and discuss some of the recent published scientific literature and patent applications in this field, to provide a framework for further drug discovery in mucus modulation.

Glycyrrhizin alleviates experimental allergic asthma in mice.

Asthma is a chronic respiratory disease, the incidence of which is increasing globally. The existing therapy is inadequate and has many adverse effects. It needs a better therapeutic molecule preferably of natural origin, which has negligible or no adverse effects. In view of this, we evaluated Glycyrrhizin (GRZ), a major constituent of a plant Glycyrrhiza glabra, for its efficacy on asthmatic features in a mouse model of asthma. BALB/c mice were sensitized and challenged with ovalbumin (OVA) to develop the asthmatic features such as airway hyperresponsiveness: allergen induced airway constriction and airway hyperreactivity (AHR) to methacholine (MCh), and pulmonary inflammation. The mice were orally treated with GRZ (2.5, 5, 10 and 20 mg/kg) during or after OVA-sensitization and OVA-challenge to evaluate its protective or reversal effect, respectively on the above asthmatic features. The status of airway hyperresponsiveness was measured by monitoring specific airway conductance (SGaw) using a non-invasive method and the pulmonary inflammation was assessed by haematoxylin and eosin staining of lung sections. Several other parameters associated with asthma such as interleukin (IL)-4, IL-5 interferon-gamma (IFN-gamma), OVA-specific IgE, total IgG(2a) and cortisol were measured by ELISA. GRZ (5 mg/kg) markedly inhibited OVA-induced immediate airway constriction, AHR to MCh (p<0.01), lung inflammation, and infiltration of eosinophils in the peribronchial and perivascular areas. It prevented the reduction of IFN-gamma (p<0.02), and decreased IL-4 (p<0.05), IL-5 (p<0.05) and eosinophils (p<0.0002) in the BAL fluid. Also, it reduced OVA-specific IgE levels (p<0.01) and prevented the reduction of total IgG(2a) (p<0.01) in serum. We have also showed that it has no effect on serum cortisol levels. Our results demonstrate that GRZ alleviates asthmatic features in mice and it could be useful towards developing a better therapeutic molecule in the future.

Basis of rise in intracellular sodium in airway hyperresponsiveness and asthma.

The aim of this study was to investigate the basis of disturbances in sodium transport in asthma and in airway hyperresponsiveness without symptoms of asthma (asymptomatic AHR). We measured the intracellular sodium (Na(i)); activity of Na(+)/K(+)-ATPase in unstimulated cells (resting activity) and in cell homogenate under optimal conditions (maximal activity); and sodium influx, in mixed leukocytes of 15 normal subjects, 12 subjects with asymptomatic AHR, and 26 asthmatics with or without active symptoms. Resting Na(+)/K(+)-ATPase activity was the same as sodium influx, consistent with homeostasis. Compared with normal subjects, those with asymptomatic AHR or asthma with controlled symptoms had a twofold increase in sodium influx and Na(i). Symptomatic asthmatics also had a twofold increase in sodium influx but a fourfold elevation of Na(i). Maximal Na(+)/K(+)-ATPase activity was reduced by half in symptomatic asthmatics compared with normal subjects. The reduction of maximal Na(+)/K(+)-ATPase activity was associated with a significant decrease in ATP turnover per Na(+)/K(+)-ATPase molecule but not number of Na(+)/K(+)-ATPase molecules per cell. In summary, airway hyperresponsiveness with or without asthma is associated with increased sodium influx and Na in leukocytes. Resting activity of Na(+)/K(+)-ATPase is also increased as a compensatory response to the increased sodium influx, but it is achieved at the expense of higher Na(i). Symptomatic asthma is additionally associated with reduction in maximal activity of Na(+)/K(+)-ATPase, resulting in reduced capacity to handle the increase in sodium influx and consequent severe elevations in Na(i).

Para-Bromophenacyl bromide alleviates airway hyperresponsiveness and modulates cytokines, IgE and eosinophil levels in ovalbumin-sensitized and -challenged mice.

Airway hyperresponsiveness, airway eosinophilia and increased IgE levels in serum are the important characteristic features of asthma. We evaluated the potential of para-Bromophenacyl bromide (PBPB), a known phospholipase A(2) inhibitor, on allergen-induced airway hyperresponsiveness in a mouse model. We sensitized and challenged mice with ovalbumin (OVA) to develop airway hyperresponsiveness as assessed by airway constriction and airway hyperreactivity (AHR) to methacholine (MCh) induced by allergen. The mice were orally treated with PBPB (0.1, 1 and 10 mg/kg) during or after OVA-sensitization and OVA-challenge to evaluate its protective or reversal effect on airway constriction and AHR to MCh. Determination of OVA-induced airway constriction and AHR to MCh were performed by measuring specific airway conductance (SGaw) using non-invasive dual-chamber whole body-plethysmography. We observed that PBPB (1 mg/kg) significantly reduced OVA-induced airway constriction and AHR to MCh (p<0.01). PBPB (1 mg/kg) treatment significantly inhibited PLA(2) activity in the BAL fluid. Cytokine analysis of the BAL fluid revealed that PBPB caused an increase in interferon-gamma (IFN-gamma) (p<0.02) and a decrease in interleukin-4 (IL-4) (p<0.05) and interleukin-5 (IL-5) (p<0.05) levels. The OVA-specific serum IgE levels (p<0.01) and the BAL eosinophils (p<0.001) were also reduced significantly. Thus, PBPB is capable of modulating allergen induced cytokine levels and serum IgE levels, and alleviating allergen induced airway hyperresponsiveness and eosinophils in mice. These data suggest that PBPB could be useful in the development of novel agents for the treatment of allergen induced airway hyperresponsiveness.

Curcumin attenuates allergen-induced airway hyperresponsiveness in sensitized guinea pigs.

Anti-asthmatic property of curcumin (diferuloylmethane), a natural product from the rhizomes of Curcuma longa, has been tested in a guinea pig model of airway hyperresponsiveness. We sensitized guinea pigs with ovalbumin (OVA) to develop certain characteristic features of asthma: allergen induced airway constriction and airway hyperreactivity to histamine. Guinea pigs were treated with curcumin during sensitization (to examine its preventive effect) or after developing impaired airways features (to examine its therapeutic effect). Status of airway constriction and airway hyperreactivity were determined by measuring specific airway conductance (SGaw) using a non-invasive technique, constant-volume body plethysmography. Curcumin (20 mg/kg body weight) treatment significantly inhibits OVA-induced airway constriction (p<0.0399) and airway hyperreactivity (p<0.0043). The results demonstrate that curcumin is effective in improving the impaired airways features in the OVA-sensitized guinea pigs.