Tracheal Preparations Research Articles

Protective Effects of Resveratrol Against Airway Hyperreactivity, Oxidative Stress, and Lung Inflammation in a Rat Pup Model of Bronchopulmonary Dysplasia

Oxygen therapy provides an important treatment for preterm and low-birth-weight neonates, however, it has been shown that prolonged exposure to high levels of oxygen (hyperoxia) is one of the factors contributing to the development of bronchopulmonary dysplasia (BPD) by inducing lung injury and airway hyperreactivity. There is no effective therapy against the adverse effects of hyperoxia. Therefore, this study was undertaken to test the hypothesis that natural phytoalexin resveratrol will overcome hyperoxia-induced airway hyperreactivity, oxidative stress, and lung inflammation. Newborn rats were exposed to hyperoxia (fraction of inspired oxygen - FiO2>95 % O2) or ambient air (AA) for seven days. Resveratrol was supplemented either in vivo (30 mg·kg-1·day-1) by intraperitoneal administration or in vitro to the tracheal preparations in an organ bath (100 μM). Contractile and relaxant responses were studied in tracheal smooth muscle (TSM) using the in vitro organ bath system. To explain the involvement of nitric oxide in the mechanisms of the protective effect of resveratrol against hyperoxia, a nitric oxide synthase inhibitor – Nω-nitro-L-arginine methyl ester (L-NAME), was administered in some sets of experiments. The superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and the tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) levels in the lungs were determined. Resveratrol significantly reduced contraction and restored the impaired relaxation of hyperoxia-exposed TSM (p<0.001). L-NAME reduced the inhibitory effect of resveratrol on TSM contractility, as well as its promotion relaxant effect (p<0.01). Resveratrol preserved the SOD and GPx activities and decreased the expression of TNF-α and IL-1β in hyperoxic animals. The findings of this study demonstrate the protective effect of resveratrol against hyperoxia-induced airway hyperreactivity and lung damage and suggest that resveratrol might serve as a therapy to prevent the adverse effects of neonatal hyperoxia. Keywords: Bronchopulmonary dysplasia • Hyperoxia • Airway hyperreactivity • Resveratrol • Pro-inflammatory cytokines

New promising agents against COPD and asthma among the amides of 1-oxo-3-phenyl-isochroman-6-carboxylic acid

Background: Bronchodilators, which are compounds that can relax airway smooth muscle, are perhaps the most important component of combination therapy for chronic obstructive pulmonary disease, one of the most common non-communicable diseases in the world, which is the second most lethal disease after cardiovascular disease. Unfortunately, current clinical bronchodilators, whose activity is mediated by their interaction with muscarinic acetylcholine receptors, have side effects (up to myocardial infarction) due to their cross-affinity for different types of these receptors, including those prevalent in the heart muscle. Objectives: The aim of this work is to search/develop compounds — effective bronchodilators capable of selectively inhibiting type 3 muscarinic acetylcholine receptors (M3 receptors), predominantly present in smooth muscles and not characteristic of cardiomyocytes. Materials and Methods: High-throughput virtual screening of a collection of 150,000 compounds was conducted on the spatial structure of the M3 receptor, reconstructed in our previous studies. The effect of substances on contractile activity was investigated using tensometry in isometric mode on multicellular tracheal preparations. Antagonistic activity and type of inhibition were determined against the background of acetylcholine application (concentration range 10-10–10-3 M). To establish the affinity value of the compound-antagonist, the Schild regression equation was used. Results: Based on virtual screening data, a series of compounds — amides of 1-oxo-3-phenyl-iso-chroman-6-carboxylic acid — were selected for biological testing. For two of these compounds (Compounds 1 and 7), the ability to selectively inhibit M3 receptors was demonstrated. Specifically, the affinity value pKB for Compound 1 was 7.28 ± 0.70, with an IC50 of 5.25·10-8 M. A critically important advantage of this compound is its ability, at equal concentrations, to more effectively inhibit signal transmission through M3 receptors compared to ipratropium bromide — a clinical cholinergic receptor inhibitor. Conclusions: The sufficient effectiveness of inhibition and significantly increased selectivity of the studied compounds specifically towards M3 receptors provide strong grounds to consider these compounds as promising precursors of new generation cholinolytic drugs with targeted action on M3-type cholinergic receptors.

Flavones isolated from Pseudognaphalium liebmannii with tracheal smooth muscle relaxant properties

The inflorescences of Pseudognaphalium liebmannii are used as folk medicine to treat various respiratory diseases. In this work, we report the isolation of seven known flavones: 5-hydroxy-3,7-dimethoxyflavone 1, 5,8-dihydroxy-3,7-dimethoxyflavone 2, 5,7-dihydroxy-3,8-dimethoxyflavone 3 (gnaphaliin A), 3,5-dihydroxy-7,8-dimethoxyflavone 4 (gnaphaliin B), 3,5-dihydroxy-6,7,8-trimethoxyflavone 5, 3,5,7-trimethoxyflavone 6 and 3-O-methylquercetin 7. All these flavones except 1 and 6 showed a relaxant effect on guinea pig tracheal preparation with EC50 between 69.91 ± 15.32 and 118.72 ± 7.06 µM. Aminophylline (EC50 = 122.03 ± 7.05 µM) was used as a relaxant reference drug. The active flavones shifted the concentration-response curves of forskolin and nitroprusside leftward, and significantly reduced the EC50 values of these drugs. Furthermore, these flavones dose-dependently inhibited phosphodiesterase (PDE) in an in vitro assay. This reveals that the inflorescences of P. liebmannii contain several flavones with relaxant effect on airway smooth muscle and with PDEs inhibition that contribute to supporting the anti-asthmatic traditional use.

Human airway tuft cells influence the mucociliary clearance through cholinergic signalling

BackgroundAirway tuft cells, formerly called brush cells have long been described only morphologically in human airways. More recent RNAseq studies described a chemosensory cell population, which includes tuft cells, by a distinct gene transcription signature. Yet, until which level in the tracheobronchial tree in native human airway epithelium tuft cells occur and if they function as regulators of innate immunity, e.g., by regulating mucociliary clearance, remained largely elusive.MethodsWe performed immunohistochemistry, RT-PCR and immunoblotting analyses for various tuft cell markers to confirm the presence of this cell type in human tracheal samples. Immunohistochemistry was conducted to study the distribution of tuft cells along the intrapulmonary airways in humans. We assessed the influence of bitter substances and the taste transduction pathway on mucociliary clearance in mouse and human tracheal samples by measuring particle transport speed.ResultsTuft cells identified by the expression of their well-established marker POU class 2 homeobox 3 (POU2F3) were present from the trachea to the bronchioles. We identified choline acetyltransferase in POU2F3 expressing cells as well as the transient receptor potential melastatin 5 (TRPM5) channel in a small population of tracheal epithelial cells with morphological appearance of tuft cells. Application of bitter substances, such as denatonium, led to an increase in mucociliary clearance in human tracheal preparations. This was dependent on activation of the TRPM5 channel and involved cholinergic and nitric oxide signalling, indicating a functional role for human tuft cells in the regulation of mucociliary clearance.ConclusionsWe were able to detect tuft cells in the tracheobronchial tree down to the level of the bronchioles. Moreover, taste transduction and cholinergic signalling occur in the same cells and regulate mucociliary clearance. Thus, tuft cells are potentially involved in the regulation of innate immunity in human airways.

Potential biomedical applications of Araucaria araucana as an antispasmodic, bronchodilator, vasodilator, and antiemetic: Involvement of calcium channels

Ethnopharmacological relevanceSince pre-Columbian era, the resin of Araucaria araucana tree has been used traditionally for the treatment of ulcers and wounds. Araucaria species have also been used to treat inflammation, respiratory problems, viral infections, ulcers, and rheumatoid, cardiovascular, and neurological disorders. Aims and objectiveDue to its popular use, the authors aimed to scrutinize the potential of this plant as an antispasmodic and an antiemetic agent. Furthermore broncho- and vasodilatory effects of this plant was explored to rationalize its folkloric uses. Materials and methodsAraucaria araucana crude extract (Aa.Cr) was evaluated in isolated preparations of rabbit jejunum, trachea, aorta, and atria to investigate the antispasmodic, bronchodilator, and vasodilator effects. The potential mechanistic approaches were compared with the standard drug ‘verapamil’. The antiemetic activity was determined and compared with the standard drug ‘domperidone’ via chick emesis model. ResultsAa.Cr dose-dependently relaxed both spontaneous and K+-induced contractions in the isolated jejunum preparations of rabbits. In concentration–response curves of calcium (Ca++), Aa.Cr also triggered the rightward shift like verapamil. Applying carbachol and phenylephrine (1 μM) and K+ (80 mM) to the isolated tracheal and aortic tissue preparation, respectively, resulted in broncho- and vasodilatory activities, respectively which may be due to the inhibition of Ca++ channels. Aa.Cr inhibited atrial force and spontaneous contractions in the rabbit's right atria. Aa.Cr exhibited significant antiemetic activity (P < 0.001 vs. saline) in dose-dependent (50–150 mg/kg) manner like domperidone. In silico molecular docking was performed to investigate the biological targets of purified components of Aa.Cr which revealed that cadinol dominantly targets β2 receptors to cause bronchodilation, however, eudesmin binds non-specifically to all the selected targets, while secoisolariciresinol mediated high hydrogen bonding with muscarinic receptors (M1 and M3) and Ca++ channels, thus shows the suggested mechanistic pathways of targeted activities. ConclusionsThe results of this study indicates that Aa.Cr may exhibit antispasmodic activity, bronchodilation, and vasodilation by inhibiting voltage-dependent Ca++ channels and release of subcellular calcium. This explains its folkloric use in hypertension, bronchospasms, gastrointestinal spasms, and emesis.

Efficient Euphorbia hirta Phytosomes for in vitro Antiasthmatic Activity

Background: The purpose of this study was to assess the antiasthmatic activity of Euphorbia hirta extract and Euphorbia hirta phytosomes in an in vitro anti-asthmatic model. Materials and Methods: The phytosomes were tested for entrapment efficiency, solubility studies, and in vitro drug release. The in vitro goat tracheal chain preparation paradigm was used to test the Euphorbia hirta extract and Euphorbia hirta phytosomes. Results: Histamine-induced contraction in an isolated goat tracheal chain revealed that both Euphorbia hirta extract and Euphorbia hirta phytosomes decreased histamine’s contractile effect. The entrapment efficiency was observed well at 60°C and a 1:2 ratio of extract: phospholipids. The solubility studies and in vitro drug release studies depicted a rise in the lipophilic nature of extract by formulating it in the form of phytosomes. The antiasthmatic activity of the extract and phytosomes increased as the concentration was raised. Conclusion: The presence of phytochemicals such as flavonoids, glycosides, and saponine may play a role in the antiasthmatic activity observed. Moreover, enhanced lipophilicity might have enabled phytosomes to show a better antiasthmatic profile as compared to extract.

Endothelium-dependent vasorelaxant effects of praeruptorin a in isolated rat thoracic aorta

ABSTRACT Praeruptorin A (PA) is a natural coumarin compound from the roots of Radix Peucedani and is commonly used in the treatment of certain respiratory diseases and hypertension. Although previous studies identified relaxant effects of PA on tracheal and arterial preparations, little is known about its vasodilative effects and underlying mechanisms. Here, an organ bath system and tension recording methods were used to prepare and analyze isolated rat thoracic aorta artery rings. Aorta artery rings were pre-contracted with phenylephrine and then incubated with PA, and the possible mechanism of relaxation was investigated by adding inhibitors of nitric oxide synthase (NG-nitro-L-arginine methyl ester, L-NAME), endothelial nitric oxide synthase (L-NG-nitroarginine, L-NNA), cyclooxygenase (indomethacin), guanylyl cyclase (1 H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one, ODQ), and KCa channels (tetraethylammonium, TEA). Our study showed that PA-induced vasodilation was blocked by L-NAME, L-NNA, and ODQ, while CaCl2-induced vasoconstriction was countered by PA. Thus, PA may exert a vasodilatory effect by influencing the amounts of endothelium-derived relaxing factors through endothelial-dependent NO-cGMP and prostacyclin pathways (such as NO and prostacyclin 2). In the rat thoracic aorta, PA reduces vasoconstriction by inhibiting Ca2+ inflow.

Antidiarrheal and Cardio-Depressant Effects of Himalaiella heteromalla (D.Don) Raab-Straube: In Vitro, In Vivo, and In Silico Studies.

Himalaiella heteromalla (D.Don) Raab-Straube is a commonly used remedy against various diseases. Crude extract and fractions of H. heteromalla were investigated for a gastrointestinal, bronchodilator, cardiovascular, and anti-inflammatory activities. H. heteromalla crude extract (Hh.Cr) relaxed spontaneous contractions and K+ (80 mM)-induced contraction in jejunum tissue dose-dependently. The relaxation of K+ (80 mM) indicates the presence of Ca++ channel blocking (CCB) effect, which was further confirmed by constructing calcium response curves (CRCs) as they caused rightward parallel shift of CRCs in a manner comparable to verapamil, so the spasmolytic effect of Hh.Cr was due to its CCB activity. Application of Hh.Cr on CCh (1 µM) and K+ (80 mM)-induced contraction in tracheal preparation resulted in complete relaxation, showing its bronchodilator effect mediated through Ca++ channels and cholinergic antagonist activity. Application of Hh.Cr on aortic preparations exhibited vasorelaxant activity through angiotensin and α-adrenergic receptors blockage. It also showed the cardio suppressant effect with negative chronotropic and inotropic response in paired atrium preparation. Similar effects were observed in in vivo models, i.e., decreased propulsive movement, wet feces, and inhibition of edema formation.

EVALUATION OF ANTI-ASTHMATIC ACTIVITY OF ETHANOLIC EXTRACT OF CYNODON DACTYLON LEAVES

In the present study Cynodon dactylon leaves ethanolic extract and its chloroform and ethyl acetate fractions were used to evaluate the anti-asthmatic activity, using various experimental in vivo and in vitro animal models. Ethanolic extract of Cynodon dactylon and its chloroform and ethyl acetate fractions were evaluated for its anti-asthmatic activity by in vitro models (isolated guinea pig ileum preparation and isolated guinea pig tracheal chain preparation) in vivo models (Histamine and acetylcholine induced bronchospasm in guinea pigs and milk induced eosinophilia in mice model). Ethanolic extract of Cynodon dactylon and its ethyl acetate fraction at the concentration of 10 mg significantly antagonized the effect of histamine induced contraction on isolated guinea pig ileum preparation. Ethyl acetate fraction of Cynodon dactylon showed the maximum spasmolytic activity and significantly extended the latent period of convulsion as compared to standard (Ketotifen fumarate) upon histamine and acetylcholine exposure in a dose dependent manner. The study concluded that, chloroform and ethyl acetate fractions of Cynadon dactylon was shown significant anti-asthmatic activity.

8-Hydroxyquinolin-2(1H)-one analogues as potential β2-agonists: Design, synthesis and activity study.

β2-Agonists that bind to plasmalemmal β2-adrenoceptors causing cAMP accumulation are widely used as bronchodilators in chronic respiratory diseases. Here, we designed and synthesized a group of 8-hydroxyquinolin-2(1H)-one analogues and studied their β2-agonistic activities with a cellular cAMP assay. Compounds B05 and C08 were identified as potent (EC50<20 pM) and selective β2-agonists among the compounds tested. They behaved as partial β2-agonists in non-overexpressed HEK293cells, and possessed rapid smooth muscle relaxant actions and long duration of action in isolated guinea pig tracheal strip preparations. In summary, B05 and C08 are β2-agonists with potential applicability in chronic respiratory diseases.

Echinocandins Accelerate Particle Transport Velocity in the Murine Tracheal Epithelium: Dependency on Intracellular Ca2+ Stores.

ABSTRACTThe mucociliary clearance of lower airways is modulated by different physiologic stimuli and also by pathophysiologic agents like polluting substances or pharmaceutical molecules. In the present investigation, we measured the particle transport velocity (PTV) of mouse tracheae as a surrogate for mucociliary clearance. In mouse tracheal preparations, we detected a sustained increase in the PTV under the application of the echinocandins caspofungin, anidulafungin, and micafungin. In further experiments, we observed the effects of echinocandins on the PTV were dependent on intracellular Ca2+ homeostasis. In Ca2+-free buffer solutions, the amplitude of the echinocandin-evoked rise in the PTV was significantly reduced relative to that in the experiments in Ca2+-containing solutions. Depletion of intracellular Ca2+ stores of the endoplasmic reticulum (ER) by caffeine completely prevented an increase in the PTV with subsequent caspofungin applications. Mitochondrial Ca2+ stores seemed to be unaffected by echinocandin treatment. We also observed no altered generation of reactive oxygen species under the application of echinocandins as probable mediators of the PTV. Consequently, the observed echinocandin effects on the PTV depend upon the Ca2+ influx and Ca2+ contents of the ER. We assume that all three echinocandins act intracellularly on ER Ca2+ stores to activate Ca2+-dependent signal transduction cascades, enhancing the PTV.

In-vitro and in-vivo Evaluation of Anti-asthmatic Activity of Eugenia jambolana bark

Objectives: The present study designed to evaluate the Antiasthmatic activity of aqueous extract of bark of Eugenia Jambolana (AEEJ) on in vitro and in vivo animal models. Materials and methods: Different in vitro and in vivo animal models was used to study the anti asthmatic activity as isolated goat tracheal chain preparation, Acetylcholine and Histamine induced bronconstriction in guinea pigs, effect of drug extract on histamine release from mast cell was checked by clonidine-induced mast cell degranulation, and milk-induced eosinophilia and leukocytosis. Results: In-vitro study on goat tracheal chain preparation revealed that aqueous extract of Eugenia jambolana (AEEJ)bark exerted antagonistic effect on the histamine induced contraction. (P&lt;0.05) The guinea pigs when exposed to 0.2% histamine aerosol showed signs of progressive dyspnoea leading to convulsions. AEEJ significantly prolonged the latent period of convulsions (PCT) as compared to control following the exposure of histamine (0.2%) aerosol (P&lt;0.01). The observation of present study indicates aqueous extract of Eugenia jambolana shows significant inhibition of milk induced eosinophilia and leukocytosis. Group of animals pretreated with aqueous Eugenia jambolana bark extract showed significant reduction in degranulation of mast cells when challenged with clonidine. The prevention of degranulation process by the aqueous Eugenia jambolana bark extract (P&lt;0.01) indicates a possible stabilizing effect on the mast cells, indicating mast cell stabilizing activity. Conclusions: Thus, AEEJ showed antihistaminic, mast cell stabilizing and protective in guinea pigs against histamine induced PCD, reduced eosinophilia and leukocytosis and hence possesses potential role in the treatment of asthma.

Pharmacological Evaluation of the Bronchorelaxant Effect of Waltheria indica L. (Malvaceae) Extracts on Rat Trachea.

Waltheria indica L. (Malvaceae) is a plant used in Burkina Faso for the treatment of various ailments including asthma. The aim of the study was to evaluate the pharmacological relaxant effect of the leafy stem extracts of Waltheria indica and thereby verify claim of use in treating asthma. Aqueous decoction and hydroalcoholic extracts obtained from the powdered leafy stems were screened for the presence of some phytoconstituents. The in vitro relaxant effect of the two extracts was evaluated on acetylcholine- (ACh 10−5 M) and potassium chloride- (KCl 6 × 10−2 M) induced contractions on rat-isolated tracheal preparations. To examine whether the potassium (K+) channels are involved in the relaxant effect, glibenclamide, an ATP-sensitive potassium channel inhibitor, was used. Moreover, to assess the safety of the extracts, acute oral toxicity was carried out on mice. The phytochemical screening revealed the presence of alkaloids, flavonoids, saponins, steroids, triterpenoids, tannins, and coumarins in the hydroalcoholic extract. Tannins, steroids, triterpenoids, and coumarins were not detected in the aqueous decoction. With respective EC50 values of 1.517 ± 0.002 mg/mL and 1.433 ± 0.001 mg/mL on ACh-and KCl-provoked contractions, the hydroalcoholic extract was found more potent in relaxing the isolated rat tracheal preparations compared to the aqueous decoction. In the presence of glibenclamide, the relaxant effect of the hydroalcoholic extract (EC50 = 0.191 ± 0.002 mg/mL) increased and was higher than that of the aqueous decoction. At dose of 5000 mg/kg of body weight, the extracts did not produce deaths or any significant changes in the general behavior of mice. The results suggest that different mechanisms including modulation of calcium and potassium channels, particularly the ATP-sensitive K+ channels, could be involved in the relaxation effect. These findings could justify the traditional use of W. indica in the management of asthma.

Possible Tracheal Relaxant and Antimicrobial Effects of the Essential Oil of Ethiopian Thyme Species (Thymus serrulatus Hochst. ex Benth.): A Multiple Mechanistic Approach.

The genus Thymus is traditionally used for the treatment of hyperactive airways complaints. The purpose of the current study is to investigate the potential tracheal relaxant effect and possible mechanism(s) of the essential oil of Thymus serrulatus (TS Oil) in isolated guinea pig tracheal tissues. The essential oil was obtained from the fresh erial parts of Thymus serrulatus, and its phyto-components were identified by GC-MS analysis. Guinea pig tracheal preparations were used for testing the tracheal relaxant effect of TS Oil with the determination of the mechanism(s) involved in this relaxation. GC-MS findings reveal that terpenes, fragrance constituents, saponins, and higher fatty acids are present in TS Oil. In isolated guinea pig trachea, TS Oil inhibited carbachol (CCh, 1 µM) and K+ (80 mM)-induced contractions in a pattern similar to that of dicyclomine. TS Oil, at 0.3 mg/ml, shifted parallel CCh-curves towards the right, followed by a non-parallel shift at higher concentration (1 mg/ml), thus suppressing maximum response in the same manner as produced by dicyclomine. Pretreatment of tissues with TS Oil (1 and 3 mg/ml) also produced a rightward shift of Ca++ concentration-response curves (CRCs) in the same manner as caused by verapamil. Further, TS Oil at low concentrations (0.3 and 1 mg/ml) shifted isoprenaline-induced inhibitory CRCs towards the left and increased cAMP levels in isolated tracheal homogenates similar to papaverine, a phosphodiesterase (PDE) inhibitor. In the antimicrobial assay performed by the agar well diffusion method, TS Oil was found most active against Candida albicans and Staphylococcus aureus where the zone of inhibition measured was 28 mm. Additionally, there was little difference between standard strains of gram-positive and gram-negative bacteria. However, methicillin-resistant S. aureus (MRSA) showed a small zone of inhibition as compared to standard strains (22 mm). From these results, it can be concluded that the essential oil of T. serrulatus has the potential to produce antimicrobial effects while causing tracheal relaxation mediated possibly by anticholinergic effects, Ca++ channel blockade, and PDE inhibition whereas additional mechanism(s) cannot be ruled out.

In the quest of the optimal tissue source (porcine male and female articular, tracheal and auricular cartilage) for the development of collagen sponges for articular cartilage

BackgroundIn the quest of the ideal material for scaffold fabrication for cartilage engineering, the use of collagen type II has been advocated, as it is the major constituent of cartilage tissue. Considering though the high batch-to-batch variability of animal-derived products, it is essential to identify suitable collagen type II sources. MethodsHerein, we ventured to assess the influence of biological sex (male, female) and tissue (articular, tracheal, auricular) on the biophysical, biochemical and biological properties of pepsin extracted porcine collagen type II scaffolds. ResultsArticular cartilage resulted in pure collagen type II preparations, whilst tracheal and auricular cartilage preparations were contaminated with collagen type I. Pore size, porosity and denaturation temperature were not affected (p > 0.05) as a function of tissue and biological sex. Articular cartilage derived sponges exhibited significantly (p < 0.05) higher resistance to enzymatic degradation and biomechanical properties in comparison to tracheal and auricular cartilage sponges. Biological analysis using human adipose derived stem cells revealed no significant (p > 0.05) differences between the groups in cell viability, DNA concentration, metabolic activity and Alcian blue staining. In general, the articular cartilage groups induced the highest (p < 0.05) sulphated glycosaminoglycans synthesis and aggrecan and collagen type II mRNA expression (fold change ≥ 2.0). ConclusionsOur data indicate a tissue memory of collagen type II and indicate that for cartilage engineering, articular cartilage derived collagen type II scaffolds may be more suitable for effective chondrogenesis.

Neuropharmacological Exploration of Standardized Extract of Annona squamosa (L.) Fruit Pulp in Experimental Animals

Aims: The present study aimed to investigate the neuroprotective potential of standardized Annona squamosa Linn fruit pulp extract using various in-vitro and in-vivo models. Methodology: Neuroprotective potential of standardized extract was screened against dopamine-induced contraction of isolated rat vas deferens, serotonin-induced contractions of isolated rat fundus, acetylcholine-induced contractions of isolated goat tracheal chain. In-vivo models such as elevated plus maze, light and dark model, force swim test, tail suspension test, lithium-induced head twitches, haloperidol-induced catalepsy, PTZ induced seizure and foot shock-induced aggression were implemented to screen various doses intervals (50-200mg/kg) of extracts in experimental animals. Results: Standardization of extract showed content of polyphenols 65.37 mg/g of GAE, total flavonoid 5.33 mg/g of RE and HPLC fingerprinting of ASP-ME showed identical retention time as that of standard gallic acid, quercetine and rutin, viz 3.830, 5.765 and 3.830 respectively. Inhibition of DPPH radical reflected as 91.32±0.19 % while percent inhibition of RRI of DPPH was observed as 95.99±0.47 at 150 min. ASP-ME significantly inhibited dopamine and serotonin induced contraction on isolated rat vas deferens and rat fundus respectively at log dose (1.3, 2.5) for dopamine and log dose (2.2, 2.5) for serotonin. ASP-ME potentiated ach-induced contractions on goat tracheal chain preparation. Ach alone produces 106.90±4.6 % response, while ASP-ME in presence of Ach potentiates response and produces 141.80±10 % response. The extract demonstrated anxiolytic activity by increasing the time spent in open arms and light zone in elevated plus maze and light dark test respectively. The duration of immobility was significantly decreased in force swim &amp; tail suspension test respectively demonstrating antidepressant activity. Administration of ASP-ME shown antipsychotic effect in dose dependent manner by minimising aggression induced by foot shock (reduced number of flights), while potentiation of catalepsy induced by haloperidol. The extract also exhibited serotonergic system inhibitory effect by significantly reducing head twitches imparted by lithium. The ASP-ME significantly delayed the onset of first myoclonic and clonic spasms induced by PTZ indicating anticonvulsant effects. Extract also shown ability to decrease the behavior facilitated by the serotonergic and dopaminergic coordination, while potentiated the actions produced by GABA. Conclusion: Finding of the study suggests anxiolytic, antidepressant, antipsychotic effects of ASP-ME probably mediated through dopamine D2 and 5-HT receptors, with neuroprotective activity.

Morphological characterization of brush cells in the rat trachea

Brush cells have recently been classified as solitary chemosensory cells. However, tracheal brush cells have not been morphologically and immunohistochemically characterized yet. In the present study, the morphological and immunohistochemical characteristics of tracheal brush cells were analyzed using immunohistochemistry and scanning, and transmission electron microscopies. Brush cells in the tracheal epithelium were barrel-like or columnar in shape and were immunoreactive for villin. Scanning and transmission electron microscopies revealed densely arranged thick microvilli on the apical surface of tracheal brush cells and tubular membranous elements and/or vesicular formations in the supranuclear region. A morphometrical analysis of tracheal whole-mount preparations showed that the density of brush cells was greater in the cranial third and the mucosa on the annular ligament. Double immunofluorescence revealed that the morphology of villin-immunoreactive brush cells was distinct from other non-ciliated cells in the tracheal epithelium, i.e., MUC5AC-immunoreactive mucous cells, SNAP25-immunoreactive neuroendocrine cells, and GNAT3-immunoreactive solitary chemosensory cells. On the other hand, tracheal brush cells were immunoreactive for the marker proteins for intestinal brush cells, CK18, DCLK1, and Cox1; however, these antibodies also recognized cells other than brush cells. Furthermore, immunoreactivity for PKD2L1, a cation channel subunit, was detected in brush cells. The present results demonstrated that tracheal brush cells are independent cell types. These brush cells may be activated by acid and the secretion of prostaglandins. In conclusion, the present study revealed that tracheal brush cells are independent cell types based on the morphological and immunohistochemical characteristics.

Metabolomic fingerprinting of Cissampelos sympodialis Eichler leaf extract and correlation with its spasmolytic activity

Ethnopharmacological relevanceCissampelos sympodialis Eichler (Menispermaceae) is popularly used in northeastern Brazil for the treatment of respiratory diseases such as bronchitis and asthma. Despite many pre-clinical pharmacological studies, the compounds mediating the anti-asthma activity of polar extracts of Cissampelos sympodialis leaves have not been definitively identified. Aim of the studyAim of the study: The aim of the study was to investigate the correlation between the bioactivity of polar extracts prepared from the leaves of C. sympodialis and the chemical composition using a 1H-NMR-based metabolomics approach. Material and methodsThe metabolic profile of the leaf polar extract during different phenological stages of the plant was investigated using 1H NMR spectroscopy while simultaneously screening for spasmolytic activity using guinea-pig tracheal preparations. The content of the alkaloids previously implicated in the bioactivity of Cissampelos sympodialis was determined by HPLC. ResultsPCA analysis of the 1H NMR data discriminated the extracts from different plant phenological stages. The contents of the major alkaloids decrease (from 2 ± 0.32 μg/mL for warifteine and 1 ± 0.14 μg/mL for methylwarifteine) to undetectable levels from 90 (CsL90 extract) and 120 (CsL120) days onwards for warifteine and methylwarifteine, respectively. All six extracts relaxed the trachea pre-contracted with carbachol, but the CsF210 extract was more potent (EC50 = 74.6 ± 7.9 μg/mL) compared to both CsL90 extracts and CsL180 in the presence of functional epithelium. PLS regression analysis of 1H-NMR spectral data demonstrated that the spasmolytic activity was better correlated with signals for flavonol derivatives. ConclusionsOur data challenge the idea that warifteine and methylwarifteine mediate the spasmolytic activity of the polar extract of C. sympodialis leaves.

Effect of Boswellia serrata on Rat Trachea Contractility In Vitro

Background: Boswellia serrata (family Burseraceae) has been traditionally used for the treatment of a wide variety of diseases as arthritis, inflammatory bowel diseases, and airway diseases. However, the direct bronchodilator efficacy of Boswellia serrata hasn’t been explored yet. Objective: We aimed at the present study to evaluate the direct effect of Boswellia serrata extract (BSE) on isolated rat tracheal preparations precontracted with either Acetylcholine (ACh) or potassium chloride (KCl). Methods: Tracheal rings were prepared from male Wistar rats (200-250 g). BSE (1-200 μg/ml) was added to tracheal strips precontracted with either ACh or KCl and the response was observed. We also investigated the consequences of epithelial denudation, indomethacin, and N-Nitro-L-arginine on the relaxant effect of BSE as compared to that of the β-adrenoceptor agonist isoprenaline, or the bitter taste receptor (TAS2R) agonist denatonium benzoate. Finally, the possible additive effects of BSE to isoprenaline or denatonium-induced relaxation were evaluated. Results: By using a set of serial dosing and washout experiments with tracheal rings, results showed that exposure to BSE resulted into a significant and concentration-dependent inhibitory effect on airway smooth muscle contractions precontracted with either ACh or KCl. Epithelial denudation, indomethacin, or N-Nitro-L-arginine had no significant effect on the obtained relaxation. Furthermore, BSE potentiated the relaxant effect of isoprenaline on rat trachea. Conclusion: BSE exerts a direct concentration-dependent relaxant effect on precontracted tracheal strips. These results could contribute towards validation of the traditional use of BSE in the treatment of airway diseases.

Bronchodilator effect of apigenin and luteolin, two components of Dracocephalum kotschyi on isolated rabbit trachea

Introduction: Dracocephalum kotschyi is a native Iranian plant with antispasmodic activities on smooth muscles such as ileum and uterus. However, so far antispasmodic effect of D. kotschyi on tracheal smooth muscle has not been reported. Therefore, the objective of this research was to investigate antispasmodic activity of D. kotschyi extract and two of its components luteolin and apigenin on rabbit tracheal contraction in vitro. Methods: Rabbits were euthanized by carbon dioxide and the trachea was dissected and immersed in a Tyrode’s solution. Tracheal rings were prepared and mounted vertically in an organ bath at 37°C and gassed continuously with O2. The tracheal ring preparations were contracted with acetylcholine (ACh) and KCl. The isotonic tension was recorded before and after addition of aminophylline, apigenin, luteolin or flavonoids rich extract of D. kotschyi. Flavonoids rich extract were prepared from D. kotschyi using solvent-solvent fractionation technique. Results: Standard drug aminophylline, prevented tracheal ring preparation contracted with ACh. Cumulative addition of aminophylline also attenuated tonic contraction induced by KCl on tracheal smooth muscle. D. kotschyi extract at concentration ranges of 32-512 μg/mL in a concentration dependent manner inhibited KCl and ACh induced tracheal contraction. Apigenin and luteolin (range 16–512 μg/mL) relaxed KCl and ACh-induced contraction of tracheal smooth muscle in vitro in a concentration-dependent manner. Conclusion: This study demonstrated that D. kotschyi extract is a relaxant of tracheal smooth muscle. The relaxant effect of D. kotschyi extract could be due to its flavonoids component such as apigenin and luteolin.