RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Ophiocordyceps sinensis (O. sinensis) is a genus of Ascomycete fungus that is endemic to the alpine meadows of the Tibetan Plateau and adjoining Himalayas. It has been used traditionally as a tonic to improve respiratory health in ancient China as well as to promote vitality and longevity. Bioactive components found in O. sinensis such as adenosine, cordycepin, 3-deoxyadenosine, L-arginine and polysaccharides have gained increasing interest in recent years due to their antioxidative and other properties, which include anti-asthmatic, antiviral, immunomodulation and improvement of general health. AIM OF THE STUDY: This study's primary aim was to investigate the effect of a cultivated fruiting body of O. sinensis strain (OCS02®) on airways patency and the secondary focus was to investigate its effect on the lifespan of Caenorhabditis elegans. MATERIALS AND METHODS: A cultivated strain, OCS02®, was employed and the metabolic profile of its cold-water extract (CWE) was analysed through liquid chromatography-mass spectrometry (LC-MS). Organ bath approach was used to investigate the pharmacological properties of OCS02® CWE when applied on airway tissues obtained from adult male Sprague-Dawley rats. The airway relaxation mechanisms of OCS02® CWE were explored using pharmacological tools, where the key regulators in airway relaxation and constriction were investigated. For the longevity study, age-synchronised, pos-1 RNAi-treated wild-type type Caenorhabditis elegans at the L4 stage were utilised for a lifespan assay. RESULTS: Various glycopeptides and amino acids, particularly a high concentration of L-arginine, were identified from the LC-MS analysis. In airway tissues, OCS02® CWE induced a significantly greater concentration-dependent relaxation when compared to salbutamol. The relaxation response was significantly attenuated in the presence of NG-Nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) and several K+ channel blockers. The longevity effect induced by OCS02® CWE (5 mg/mL and above) was observed in C. elegans by at least 17%. CONCLUSIONS: These findings suggest that the airway relaxation mechanisms of OCS02® CWE involved cGMP-dependent and cGMP-independent nitric oxide signalling pathways. This study provides evidence that the cultivated strain of OCS02® exhibits airway relaxation effects which supports the traditional use of its wild O. sinensis in strengthening respiratory health.
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Cuerpos Fructíferos de los Hongos , Músculo Liso , Ratas Sprague-Dawley , Animales , Masculino , Cuerpos Fructíferos de los Hongos/química , Músculo Liso/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Ratas , Tráquea/efectos de los fármacos , Tráquea/metabolismo , Longevidad/efectos de los fármacos , HypocrealesRESUMEN
Pulmonary surfactant serves as a barrier to respiratory epithelium but can also regulate airway smooth muscle (ASM) tone. Surfactant (SF) relaxes contracted ASM, similar to ß2-agonists, anticholinergics, nitric oxide, and prostanoids. The exact mechanism of surfactant relaxation and whether surfactant relaxes hyperresponsive ASM remains unknown. Based on previous research, relaxation requires an intact epithelium and prostanoid synthesis. We sought to examine the mechanisms by which surfactant causes ASM relaxation. Organ bath measurements of isometric tension of ASM of guinea pigs in response to exogenous surfactant revealed that surfactant reduces tension of healthy and hyperresponsive tracheal tissue. The relaxant effect of surfactant was reduced if prostanoid synthesis was inhibited and/or if prostaglandin E2-related EP2 receptors were antagonized. Atomic force microscopy revealed that human ASM cells stiffen during contraction and soften during relaxation. Surfactant softened ASM cells, similarly to the known bronchodilator prostaglandin E2 (PGE2) and the cell softening was abolished when EP4 receptors for PGE2 were antagonized. Elevated levels of PGE2 were found in cultures of normal human bronchial epithelial cells exposed to pulmonary surfactant. We conclude that prostaglandin E2 and its EP2 and EP4 receptors are likely involved in the relaxant effect of pulmonary surfactant in airways.
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Dinoprostona , Relajación Muscular , Músculo Liso , Surfactantes Pulmonares , Tráquea , Cobayas , Animales , Humanos , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Músculo Liso/metabolismo , Relajación Muscular/efectos de los fármacos , Dinoprostona/farmacología , Dinoprostona/metabolismo , Surfactantes Pulmonares/metabolismo , Surfactantes Pulmonares/farmacología , Tráquea/efectos de los fármacos , Tráquea/fisiología , Tráquea/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Células Cultivadas , Subtipo EP4 de Receptores de Prostaglandina E/metabolismoRESUMEN
BACKGROUND/AIM: Cigarette smoke has been shown to induce a phenotype in humans known as "acquired cystic fibrosis". This occurs because the cystic fibrosis transmembrane conductance regulator (CFTR) functions are impaired systemically due to the deleterious effects of smoke components. Elucidation of cigarette smoke effects on the tracheal epithelium is important. The aim of this study was to develop an ex vivo sheep tracheal model to investigate tracheal ion function. In this model, the epithelial sodium channel (ENaC) is inhibited after exposure to cigarette smoke extract (CSE) as a proof of principle. MATERIALS AND METHODS: Tracheas were isolated from healthy sheep and the tracheal epithelium was surgically excised. Tissues were mounted in Ussing chambers and the short circuit current (Isc) was measured after incubation with 5% CSE in PBS or PBS alone for 30 min. The function of ENaC was investigated by the addition of amiloride (10-5M) apically. Western blot analysis was performed to assess differences in ENaC quantity after CSE exposure. Some specimens were stained with H&E for detection of histological alterations. RESULTS: The amiloride effect on normal epithelium led to a significant decrease in Isc [ΔI=33±5.92 µA/cm2; p<0.001 versus control experiments (ΔI=1.44±0.71 µA/cm2)]. After incubation with CSE, ENaC Isc was significantly reduced (ΔI=14.80±1.96 µA/cm2; p<0.001). No differences in αENaC expression were observed between CSE-exposed and normal tracheal epithelium. Histological images post CSE incubation revealed decreases in the height of the epithelium, with basal cell hyperplasia and loss of ciliated cells. CONCLUSION: Reduced ENaC inhibition by amiloride after CSE incubation could be due to alterations in the tracheal epithelium.
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Canales Epiteliales de Sodio , Tráquea , Animales , Canales Epiteliales de Sodio/metabolismo , Ovinos , Tráquea/metabolismo , Tráquea/efectos de los fármacos , Tráquea/patología , Proyectos Piloto , Humo/efectos adversos , Amilorida/farmacología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/patología , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Epitelio/patologíaRESUMEN
BACKGROUND: Mycoplasma gallisepticum (MG) has long been a pathogenic microorganism threatening the global poultry industry. Previous studies have demonstrated that the mechanism by which quercetin (QUE) inhibits the colonization of MG in chicks differs from that of antibiotics. However, the molecular mechanism by which QUE facilitates the clearance of MG remains unclear. PURPOSE: The aim of this study was to investigate the molecular mechanism of MG clearance by QUE, with the expectation of providing new options for the treatment of MG. METHODS: A model of MG infection in chicks and MG-induced M1 polarization in HD-11 cells were established. The mechanism of QUE clearance of MG was investigated by evaluating the relationship between tracheal mucosal barrier integrity, antibody levels, Th1/Th2 immune balance and macrophage metabolism and M1/M2 polarization balance. Furthermore, network pharmacology and molecular docking techniques were employed to explore the potential molecular pathways connecting QUE, M2 polarization, and fatty acid oxidation (FAO). RESULTS: The findings indicate that QUE remodels tracheal mucosal barrier function by regulating tight junctions and secretory immunoglobulin A (sIgA) expression levels. This process entails the regulatory function of QUE on the Th1/Th2 immune imbalance that is induced by MG infection in the tracheal mucosa. Moreover, QUE intervention impeded the M1 polarization of HD-11 cells induced by MG infection, while simultaneously promoting M2 polarization through the induction of FAO. Conversely, inhibitors of the FAO pathway impede this effect. The results of computer network analysis suggest that QUE may induce FAO via the PI3K/AKT pathway to promote M2 polarization. Notably, inhibition of the PI3K/AKT pathway was found to effectively inhibit M2 polarization in HD-11 cells, while having a limited effect on FAO. CONCLUSIONS: QUE promotes M2 polarization of HD-11 cells to enhance Th2 immune response through FAO and PI3K/AKT pathways, thereby restoring tracheal mucosal barrier function and ultimately inhibiting MG colonization.
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Pollos , Infecciones por Mycoplasma , Mycoplasma gallisepticum , Enfermedades de las Aves de Corral , Quercetina , Células Th2 , Animales , Quercetina/farmacología , Mycoplasma gallisepticum/efectos de los fármacos , Infecciones por Mycoplasma/tratamiento farmacológico , Infecciones por Mycoplasma/inmunología , Enfermedades de las Aves de Corral/tratamiento farmacológico , Enfermedades de las Aves de Corral/inmunología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/inmunología , Tráquea/efectos de los fármacos , Simulación del Acoplamiento Molecular , Uniones Estrechas/efectos de los fármacos , Inmunoglobulina A Secretora/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Células TH1/efectos de los fármacos , Células TH1/inmunología , Macrófagos/efectos de los fármacos , Ácidos GrasosRESUMEN
Single cilia, 100 nm in diameter and 10 µm in length, were isolated from mouse tracheae with Triton X-100 (0.02%) treatment, and the effects of pH on ciliary beating were examined by measuring the ciliary beat frequency (CBF) and the ciliary bend distance (CBD-an index of amplitude) using a high-speed video microscope (250 fps). ATP (2.5 mM) plus 8Br-cAMP (10 µM) reactivated the CBF and CBD in the isolated cilia, similar to the cilia of in vivo tracheae. In the reactivated isolated cilia, an elevation in pH from 7.0 to 8.0 increased the CBF from 3 to 15 Hz and the CBD from 0.6 to 1.5 µm. The pH elevation also increased the velocity of the effective stroke; however, it did not increase the recovery stroke, and, moreover, it decreased the intervals between beats. This indicates that H+ (pHi) directly acts on the axonemal machinery to regulate CBF and CBD. In isolated cilia priorly treated with 1 µM PKI-amide (a PKA inhibitor), 8Br-cAMP did not increase the CBF or CBD in the ATP-stimulated isolated cilia. pH modulates the PKA signal, which enhances the axonemal beating generated by the ATP-activated inner and outer dyneins.
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Adenosina Trifosfato , Cilios , AMP Cíclico , Tráquea , Animales , Cilios/efectos de los fármacos , Cilios/metabolismo , Adenosina Trifosfato/metabolismo , Concentración de Iones de Hidrógeno , Tráquea/metabolismo , Tráquea/efectos de los fármacos , Ratones , AMP Cíclico/metabolismo , MasculinoRESUMEN
OBJECTIVES: This study evaluated the effect of intratracheal administration of basic fibroblast growth factor (bFGF) on tracheal healing following implantation of a novel layered polyglycolic acid (PGA) material to replace a critical-size defect in rat trachea. METHODS: A critical-size defect in the rat cervical trachea was covered with PGA. Distilled water (DW) or 3.125, 6.25, 12.5 or 25 µg bFGF was administered into the trachea for 2 weeks (n = 6 for each of 5 groups). Regenerated areas of cilia, ciliary beat frequency and ciliary transport function (CTF) in the centre of the PGA were measured. To examine potential side effects of intratracheal administration of bFGF, the right lower lobe was pathologically evaluated. RESULTS: All rats survived during the study period. Histological examination showed ciliated epithelization on the PGA material after 2 weeks. Bronchoscopy revealed stenosis due to granulation following administration of high concentrations of bFGF (12.5 and 25 µg). Compared with the DW group, groups administered 3.125, 6.25, 12.5 and 25 µg bFGF had significantly larger areas of regenerated cilia (15.2%, 27.0%, 41.3%, 33.1% and 31.0%, respectively; P = 0.00143), improved ciliary beat frequency (7.10, 8.18, 10.10, 9.50 and 9.50 Hz, respectively), and improved CTS (6.40, 9.54, 16.89, 16.41 and 14.29 µm/sec, respectively). Pathological examination of the right lower lobe revealed pulmonary fibrosis and hyperplasia with high concentrations of bFGF (12.5 and 25 µg). CONCLUSIONS: Intratracheal administration of bFGF effectively promoted tracheal regeneration at an optimal dose of 6.25 µg following implantation of an artificial trachea.
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Factor 2 de Crecimiento de Fibroblastos , Tráquea , Cicatrización de Heridas , Animales , Tráquea/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Factor 2 de Crecimiento de Fibroblastos/farmacología , Ratas , Masculino , Cicatrización de Heridas/efectos de los fármacos , Ratas Sprague-Dawley , Órganos Artificiales , Cilios/efectos de los fármacosRESUMEN
OBJECTIVES: Our objective was to explore the safety and efficacy of a graphene oxide-loaded rapamycin-coated self-expandable metallic airway stent (GO@RAPA-SEMS) in a rabbit model. METHODS: The dip coating method was used to develop a GO@RAPA-SEMS and a poly(lactic-co-glycolic)-acid loaded rapamycin-coated self-expandable metallic airway stent (PLGA@RAPA-SEMS). The surface structure was evaluated using a scanning electronic microscope. The in vitro drug-release profiles of the 2 stents were explored and compared. In the animal study, a total of 45 rabbits were randomly divided into 3 groups and underwent 3 kinds of stent placements. Computed tomography was performed to evaluate the degree of stenosis at 1, 2 and 3 months after the stent operation. Five rabbits in each group were sacrificed after the computed tomography scan. The stented trachea and blood were collected for further pathological analysis and laboratory testing. RESULTS: The in vitro drug-release study revealed that GO@RAPA-SEMS exhibited a sudden release on the first day and maintained a certain release rate on the 14th day. The PLGA@RAPA-SEMS exhibited a longer sustained release time. All 45 rabbits underwent successful stent placement. Pathological results indicated that the granulation tissue thickness in the GO@RAPA-SEMS group was less than that in the PLGA@RAPA-SEMS group. The TUNEL and hypoxia-inducible factor-1α staining results support the fact that the granulation inhibition effect in the GO@RAPA-SEMS group was greater than that in the PLGA@RAPA-SEMS group. CONCLUSIONS: GO@RAPA-SEMS effectively inhibited stent-related granulation tissue hyperplasia.
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Stents Liberadores de Fármacos , Tejido de Granulación , Grafito , Sirolimus , Animales , Conejos , Grafito/administración & dosificación , Sirolimus/administración & dosificación , Sirolimus/farmacología , Tejido de Granulación/efectos de los fármacos , Tejido de Granulación/patología , Hiperplasia/prevención & control , Stents Metálicos Autoexpandibles , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Materiales Biocompatibles Revestidos , Modelos Animales de Enfermedad , Tráquea/efectos de los fármacos , Tráquea/patologíaRESUMEN
Agricultural workers exposed to organic dust from swine concentrated animal feeding operations (CAFOs) have increased chances of contracting chronic lung disease. Mucociliary clearance represents a first line of defense against inhaled dusts, but organic dust extracts (ODEs) from swine barns cause cilia slowing, leading to decreased bacterial clearance and increased lung inflammation. Because nutritional zinc deficiency is associated with chronic lung disease, we examined the role of zinc supplementation in ODE-mediated cilia slowing. Ciliated mouse tracheal epithelial cells were pretreated with 0-10 µg/mL ZinProTM for 1 h, followed by treatment with 5% ODE for 24 h. Cilia beat frequency (CBF) and protein kinase C epsilon (PKCε) activity were assayed. ODE treatment resulted in cilia slowing after 24 h, which was reversed with 0.5 and 1.0 µg/mL ZinPro pre-treatment. No zinc protection was observed at 50 ng/mL, and ciliated cells detached at high concentrations (100 µg/mL). ZinPro alone produced no changes in the baseline CBF and showed no toxicity to the cells at concentrations of up to 10 µg/mL. Pre-treatment with ZinPro inhibited ODE-stimulated PKCε activation in a dose-dependent manner. Based on ZinPro's superior cell permeability compared to zinc salts, it may be therapeutically more effective at reversing ODE-mediated cilia slowing through a PKCε pathway. These data demonstrate that zinc supplementation may support the mucociliary transport apparatus in the protection of CAFO workers against dust-mediated chronic lung disease.
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Cilios , Polvo , Proteína Quinasa C-epsilon , Zinc , Animales , Cilios/efectos de los fármacos , Cilios/metabolismo , Porcinos , Ratones , Zinc/farmacología , Proteína Quinasa C-epsilon/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Tráquea/efectos de los fármacos , Tráquea/metabolismoRESUMEN
Objectives: Nocturia with or without asthma is one of the aging diseases. Desmopressin has been used as a nasal spray for patients who are suffering from nocturia. This study determined the effects of desmopressin on isolated tracheal smooth muscle in vitro. Methods: We evaluated desmopressin's efficiency on isolated rat tracheal smooth muscle. Desmopressin was evaluated for the following effects on tracheal smooth muscle: (1) effect on resting tension; (2) effect on contraction brought on by parasympathetic mimetic 10-6 M methacholine; and (3) effect on electrically produced tracheal smooth muscle contractions. Results: As the concentration grew, desmopressin by itself had no impact on the trachea's baseline tension. Addition of desmopressin at doses of 10-5 M or above elicited a significant relaxation response to 10-6 M methacholine-induced contraction. Desmopressin could also inhibit spike contraction of the trachea induced by electrical field. Conclusion: According to this study, desmopressin at high quantities may prevent the trachea's parasympathetic activity. Due to its ability to block parasympathetic activity and lessen the contraction of the tracheal smooth muscle brought on by methacholine, Desmopressin nasal spray might help nocturia sufferers experience fewer asthma attacks.
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Desamino Arginina Vasopresina , Contracción Muscular , Músculo Liso , Rociadores Nasales , Tráquea , Animales , Tráquea/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Desamino Arginina Vasopresina/farmacología , Desamino Arginina Vasopresina/administración & dosificación , Ratas , Contracción Muscular/efectos de los fármacos , Masculino , Cloruro de Metacolina/administración & dosificación , Cloruro de Metacolina/farmacología , Humanos , Sistema Nervioso Parasimpático/efectos de los fármacosRESUMEN
The mucociliary transport apparatus is critical for maintaining lung health via the coordinated movement of cilia to clear mucus and particulates. A metachronal wave propagates across the epithelium when cilia on adjacent multiciliated cells beat slightly out of phase along the proximal-distal axis of the airways in alignment with anatomically directed mucociliary clearance. We hypothesized that metachrony optimizes mucociliary transport (MCT) and that disruptions of calcium signaling would abolish metachrony and decrease MCT. We imaged bronchi from human explants and ferret tracheae using micro-optical coherence tomography (µOCT) to evaluate airway surface liquid depth (ASL), periciliary liquid depth (PCL), cilia beat frequency (CBF), MCT, and metachrony in situ. We developed statistical models that included covariates of MCT. Ferret tracheae were treated with BAPTA-AM (chelator of intracellular Ca2+), lanthanum chloride (nonpermeable Ca2+ channel competitive antagonist), and repaglinide (inhibitor of calaxin) to test calcium dependence of metachrony. We demonstrated that metachrony contributes to mucociliary transport of human and ferret airways. MCT was augmented in regions of metachrony compared with nonmetachronous regions by 48.1%, P = 0.0009 or 47.5%, P < 0.0020 in humans and ferrets, respectively. PCL and metachrony were independent contributors to MCT rate in humans; ASL, CBF, and metachrony contribute to ferret MCT rates. Metachrony can be disrupted by interference with calcium signaling including intracellular, mechanosensitive channels, and calaxin. Our results support that the presence of metachrony augments MCT in a calcium-dependent mechanism.NEW & NOTEWORTHY We developed a novel imaging-based analysis to detect coordination of ciliary motion and optimal coordination, a process called metachrony. We found that metachrony is key to the optimization of ciliary-mediated mucus transport in both ferret and human tracheal tissue. This process appears to be regulated through calcium-dependent mechanisms. This study demonstrates the capacity to measure a key feature of ciliary coordination that may be important in genetic and acquired disorders of ciliary function.
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Calcio , Cilios , Hurones , Depuración Mucociliar , Depuración Mucociliar/efectos de los fármacos , Animales , Humanos , Cilios/metabolismo , Cilios/efectos de los fármacos , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Tráquea/metabolismo , Tráquea/efectos de los fármacos , Masculino , Bronquios/metabolismo , Bronquios/efectos de los fármacosRESUMEN
The Cupressaceae family includes species considered to be medicinal. Their essential oil is used for headaches, colds, cough, and bronchitis. Cedar trees like Chamaecyparis lawsoniana (C. lawsoniana) are commonly found in urban areas. We investigated whether C. lawsoniana exerts some of its effects by modifying airway smooth muscle (ASM) contractility. The leaves of C. lawsoniana (363 g) were pulverized mechanically, and extracts were obtained by successive maceration 1:10 (w:w) with methanol/CHCl3. Guinea pig tracheal rings were contracted with KCl, tetraethylammonium (TEA), histamine (HIS), or carbachol (Cch) in organ baths. In the Cch experiments, tissues were pre-incubated with D-600, an antagonist of L-type voltage-dependent Ca2+ channels (L-VDCC) before the addition of C. lawsoniana. Interestingly, at different concentrations, C. lawsoniana diminished the tracheal contractions induced by KCl, TEA, HIS, and Cch. In ASM cells, C. lawsoniana significantly diminished L-type Ca2+ currents. ASM cells stimulated with Cch produced a transient Ca2+ peak followed by a sustained plateau maintained by L-VDCC and store-operated Ca2+ channels (SOCC). C. lawsoniana almost abolished this last response. These results show that C. lawsoniana, and its active metabolite quercetin, relax the ASM by inhibiting the L-VDCC and SOCC; further studies must be performed to obtain the complete set of metabolites of the extract and study at length their pharmacological properties.
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Calcio , Chamaecyparis , Contracción Muscular , Músculo Liso , Extractos Vegetales , Quercetina , Tráquea , Animales , Cobayas , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Contracción Muscular/efectos de los fármacos , Quercetina/farmacología , Quercetina/química , Tráquea/efectos de los fármacos , Tráquea/metabolismo , Extractos Vegetales/farmacología , Extractos Vegetales/química , Chamaecyparis/química , Calcio/metabolismo , Masculino , Bloqueadores de los Canales de Calcio/farmacología , Histamina/metabolismo , Canales de Calcio Tipo L/metabolismo , Hojas de la Planta/químicaRESUMEN
Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.
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Adenosina Trifosfato , Adenilil Ciclasas , Relajación Muscular , Músculo Liso , Testosterona , Tráquea , Uridina Trifosfato , Animales , Uridina Trifosfato/farmacología , Uridina Trifosfato/metabolismo , Cobayas , Relajación Muscular/efectos de los fármacos , Masculino , Adenosina Trifosfato/metabolismo , Tráquea/metabolismo , Tráquea/efectos de los fármacos , Testosterona/farmacología , Testosterona/metabolismo , Adenilil Ciclasas/metabolismo , Músculo Liso/metabolismo , Músculo Liso/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/metabolismo , Transducción de Señal/efectos de los fármacos , Receptores Purinérgicos P2/metabolismoRESUMEN
High serum estrogen concentrations are associated with asthma development and severity, suggesting a link between estradiol and airway hyperresponsiveness (AHR). 17ß-estradiol (E2) has non-genomic effects via Ca2+ regulatory mechanisms; however, its effect on the plasma membrane Ca2+-ATPases (PMCA1 and 4) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) is unknown. Hence, in the present study, we aim to demonstrate if E2 favors AHR by increasing intracellular Ca2+ concentrations in guinea pig airway smooth muscle (ASM) through a mechanism involving Ca2+-ATPases. In guinea pig ASM, Ca2+ microfluorometry, muscle contraction, and Western blot were evaluated. Then, we performed molecular docking analysis between the estrogens and Ca2+ ATPases. In tracheal rings, E2 produced AHR to carbachol. In guinea pig myocytes, acute exposure to physiological levels of E2 modified the transient Ca2+ peak induced by caffeine to a Ca2+ plateau. The incubation with PMCA inhibitors (lanthanum and carboxyeosin, CE) partially reversed the E2-induced sustained plateau in the caffeine response. In contrast, cyclopiazonic acid (SERCA inhibitor), U-0126 (an inhibitor of ERK 1/2), and choline chloride did not modify the Ca2+ plateau produced by E2. The mitochondrial uniporter activity and the capacitative Ca2+ entry were unaffected by E2. In guinea pig ASM, Western blot analysis demonstrated PMCA1 and PMCA4 expression. The results from the docking modeling demonstrate that E2 binds to both plasma membrane ATPases. In guinea pig tracheal smooth muscle, inhibiting the PMCA with CE, induced hyperresponsiveness to carbachol. 17ß-estradiol produces hyperresponsiveness by inhibiting the PMCA in the ASM and could be one of the mechanisms responsible for the increase in asthmatic crisis in women.
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Calcio , Estradiol , Simulación del Acoplamiento Molecular , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Animales , Cobayas , Estradiol/farmacología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Calcio/metabolismo , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Masculino , Tráquea/efectos de los fármacos , Tráquea/metabolismo , Contracción Muscular/efectos de los fármacos , Hipersensibilidad Respiratoria/inducido químicamente , Hipersensibilidad Respiratoria/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Carbacol/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismoRESUMEN
Hydrogels incorporating natural biopolymer and adhesive substances have extensively been used to develop bioactive drugs and to design cells encapsulating sturdy structure for biomedical applications. However, the conjugation of the adhesive in most hydrogels is insufficient to maintain long-lasting biocompatibility inadequate to accelerate internal organ tissue repair in the essential native cellular microenvironment. The current work elaborates the synthesis of charged choline-catechol ionic liquid (BIL) adhesive and a hydrogel with an electronegative atom rich polyphenol (PU)-laden gelatinmethacryloyl (GelMA) to improve the structural bioactivities for in vivo tracheal repair by inducing swift crosslinking along with durable mechanical and tissue adhesive properties. It was observed that bioactive BIL and PU exhibited potent antioxidant (IC 50 % of 7.91 µg/mL and 24.55 µg/mL) and antibacterial activity against E. coli, P. aeruginosa and S. aureus. The novel integration of photocurable GelMA-BIL-PU revealed outstanding mechanical strength, biodegradability and sustained drug release. The in vitro study showed exceptional cell migration and proliferation in HBECs, while in vivo investigation of the GelMA-BIL-PU hydrogel on a rat's tracheal model revealed remarkable tracheal reconstruction, concurrently reducing tissue inflammation. Furthermore, the optimized GelMA-BIL-PU injectable adhesive bioink blend demonstrated superior MSCs migration and proliferation, which could be a strong candidate for developing stem cell-rich biomaterials to address multiple organ defects.
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Gelatina , Hidrogeles , Células Madre Mesenquimatosas , Metacrilatos , Polifenoles , Tráquea , Tráquea/efectos de los fármacos , Gelatina/química , Polifenoles/farmacología , Polifenoles/química , Animales , Ratas , Metacrilatos/química , Metacrilatos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Hidrogeles/química , Hidrogeles/farmacología , Regeneración/efectos de los fármacos , Humanos , Antibacterianos/farmacología , Antibacterianos/química , Proliferación Celular/efectos de los fármacos , Antioxidantes/farmacología , Antioxidantes/química , Movimiento Celular/efectos de los fármacos , Adhesivos Tisulares/química , Adhesivos Tisulares/farmacología , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacologíaRESUMEN
Goblet cell hyperplasia and increased mucus production are features of airway diseases, including asthma, and excess airway mucus often worsens these conditions. Even steroids are not uniformly effective in mucus production in severe asthma, and new therapeutic options are needed. Seihaito is a Japanese traditional medicine that is used clinically as an antitussive and expectorant. In the present study, we examined the effect of Seihaito on goblet cell differentiation and mucus production. In in vitro studies, using air-liquid interface culture of guinea-pig tracheal epithelial cells, Seihaito inhibited IL-13-induced proliferation of goblet cells and MUC5AC, a major component of mucus production. Seihaito suppressed goblet cell-specific gene expression, without changing ciliary cell-specific genes, suggesting that it inhibits goblet cell differentiation. In addition, Seihaito suppressed MUC5AC expression in cells transfected with SPDEF, a transcription factor activated by IL-13. Furthermore, Seihaito attenuated in vivo goblet cell proliferation and MUC5AC mRNA expression in IL-13-treated mouse lungs. Collectively, these findings demonstrated that Seihaito has an inhibitory effect on goblet cell differentiation and mucus production, which is at least partly due to the inhibition of SPDEF.
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Diferenciación Celular , Proliferación Celular , Células Caliciformes , Interleucina-13 , Medicina Kampo , Metaplasia , Mucina 5AC , Moco , Animales , Células Caliciformes/efectos de los fármacos , Células Caliciformes/patología , Células Caliciformes/metabolismo , Interleucina-13/metabolismo , Mucina 5AC/genética , Mucina 5AC/metabolismo , Moco/metabolismo , Diferenciación Celular/efectos de los fármacos , Cobayas , Proliferación Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Células Cultivadas , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Masculino , Expresión Génica/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Ratones , Tráquea/citología , Tráquea/efectos de los fármacos , Tráquea/patología , Tráquea/metabolismoRESUMEN
Eight pig tracheal strips were stimulated to contract with log increments of methacholine from 10-8 to 10-5 M. For each strip, the concentration-response was repeated four times in a randomized order to measure isometric force, isotonic shortening against a load corresponding to either 5 or 10â¯% of a reference force, and average force, stiffness, elastance and resistance over one cycle while the strip length was oscillating sinusoidally by 5â¯% at 0.2â¯Hz. For each readout, the logEC50 was calculated and compared. Isotonic shortening with a 5â¯% load had the lowest logEC50 (-7.13), yielding a greater sensitivity than any other contractile readout (p<0.05). It was followed by isotonic shortening with a 10â¯% load (-6.66), elastance (-6.46), stiffness (-6.46), resistance (-6.38), isometric force (-6.32), and average force (-6.30). Some of these differences were significant. For example, the EC50 with the average force was 44â¯% greater than with the elastance (p=0.001). The methacholine sensitivity is thus affected by the contractile readout being measured.
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Broncoconstrictores , Cloruro de Metacolina , Músculo Liso , Tráquea , Animales , Músculo Liso/fisiología , Músculo Liso/efectos de los fármacos , Cloruro de Metacolina/farmacología , Porcinos , Tráquea/fisiología , Tráquea/efectos de los fármacos , Broncoconstrictores/farmacología , Contracción Muscular/fisiología , Contracción Muscular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Elasticidad/fisiología , Contracción Isométrica/fisiología , Contracción Isométrica/efectos de los fármacosRESUMEN
This study aimed to investigate the effects of intravenous injection of iodine contrast agent on the tracheal diameter and lung volume. In this retrospective study, a total of 221 patients (71.1 ± 12.4 years, 174 males) who underwent vascular dynamic CT examination including chest were included. Unenhanced, arterial phase, and delayed-phase images were scanned. The tracheal luminal diameters at the level of the thoracic inlet and both lung volumes were evaluated by a radiologist using a commercial software, which allows automatic airway and lung segmentation. The tracheal diameter and both lung volumes were compared between the unenhanced vs. arterial and delayed phase using a paired t-test. The Bonferroni correction was performed for multiple group comparisons. The tracheal diameter in the arterial phase (18.6 ± 2.4 mm) was statistically significantly smaller than those in the unenhanced CT (19.1 ± 2.5 mm) (p < 0.001). No statistically significant difference was found in the tracheal diameter between the delayed phase (19.0 ± 2.4 mm) and unenhanced CT (p = 0.077). Both lung volumes in the arterial phase were 4131 ± 1051 mL which was significantly smaller than those in the unenhanced CT (4332 ± 1076 mL) (p < 0.001). No statistically significant difference was found in both lung volumes between the delayed phase (4284 ± 1054 mL) and unenhanced CT (p = 0.068). In conclusion, intravenous infusion of iodine contrast agent transiently decreased the tracheal diameter and both lung volumes.
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Medios de Contraste , Aprendizaje Profundo , Yodo , Pulmón , Tomografía Computarizada por Rayos X , Tráquea , Humanos , Masculino , Femenino , Tráquea/diagnóstico por imagen , Tráquea/anatomía & histología , Tráquea/efectos de los fármacos , Medios de Contraste/administración & dosificación , Anciano , Estudios Retrospectivos , Infusiones Intravenosas , Pulmón/diagnóstico por imagen , Pulmón/efectos de los fármacos , Pulmón/anatomía & histología , Persona de Mediana Edad , Tomografía Computarizada por Rayos X/métodos , Yodo/administración & dosificación , Anciano de 80 o más Años , Mediciones del Volumen Pulmonar/métodos , AlgoritmosRESUMEN
Asthma is a disease characterized by chronic inflammation and hyper responsiveness of airways. We aimed to assess the relaxant potential of phosphodiesterase-4 (PDE4) inhibitors N-sulfonilhidrazonic derivatives on non-asthmatic and asthmatic guinea pig trachea. Firstly, guinea pigs were sensitized and challenged with ovalbumin, and then morphological, and contractile changes were evaluated resulting from asthma, followed by evaluation of relaxant effect of derivatives on guinea pig trachea and the cAMP levels measurement by ELISA. It has been evidenced hypertrophy of airway smooth muscle, inflammatory infiltrate, and vascular abnormalities. Moreover, only sensitized tracheal rings were responsive to OVA. Contractile response to histamine, but not to carbachol, was greater in sensitized animals, however the relaxant response to aminophylline and isoprenaline were the same in non-asthmatics and asthmatics. N-sulfonilhidrazonic derivatives presented equipotent relaxant action independent of epithelium, with exception of LASSBio-1850 that presented a low efficacy (< 50%) and LASSBio-1847 with a 4-fold higher potency on asthmatics. LASSBio-1847 relaxant curve was impaired in the presence of propranolol and potentiated by isoprenaline in both groups. Furthermore, relaxation was potentiated 54- and 4-fold by forskolin in non-asthmatics and asthmatics, respectively. Likewise, LASSBio-1847 potentiated relaxant curve of aminophylline 147- and 4-fold in both groups. The PKA inhibitor H-89 impaired the relaxant potency of the derivative. Finally, LASSBio-1847 increased tracheal intracellular cAMP levels similarly to rolipram, selective PDE4 inhibitor, in both animals. LASSBio-1847 showed to be promising to relax guinea pig trachea from non-sensitized and sensitized guinea pigs by activation of ß2-adrenergic receptors/AC/cAMP pathway.
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Asma , Broncodilatadores , AMP Cíclico , Modelos Animales de Enfermedad , Inhibidores de Fosfodiesterasa 4 , Tráquea , Animales , Cobayas , Inhibidores de Fosfodiesterasa 4/farmacología , Asma/tratamiento farmacológico , Asma/fisiopatología , Tráquea/efectos de los fármacos , Masculino , Broncodilatadores/farmacología , AMP Cíclico/metabolismo , Músculo Liso/efectos de los fármacos , Ovalbúmina , Relajación Muscular/efectos de los fármacos , Aminofilina/farmacologíaRESUMEN
The potential antiviral effects of indole-3-carbinol (I3C), a phytochemical found in Cruciferous vegetables, were investigated. Fibroblasts and epithelial cells were co-cultured on Alvetex® scaffolds, to obtain ad hoc 3D in vitro platforms able to mimic the trachea and intestinal mucosae, which represent the primary structures involved in the coronavirus pathogenesis. The two barriers generated in vitro were treated with various concentrations of I3C for different incubation periods. A protective effect of I3C on both intestinal and trachea models was demonstrated. A significant reduction in the transcription of the two main genes belonging to the Homologous to E6AP C-terminus (HECT)-E3 ligase family members, namely NEDD4 E3 Ubiquitin Protein Ligase (NEDD4) and WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1), which promote virus matrix protein ubiquitination and inhibit viral egression, were detected. These findings indicate I3C potential effect in preventing coronavirus cell egression processes that inhibit viral production. Although further studies are needed to clarify the molecular mechanisms whereby HECT family members control virus life cycle, this work paves the way to the possible therapeutic use of new natural compounds that may reduce the clinical severity of future pandemics.
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Antivirales , Brassicaceae , Coronavirus , Intestinos , Modelos Biológicos , Fitoquímicos , Tráquea , Verduras , Antivirales/farmacología , Brassicaceae/química , Coronavirus/efectos de los fármacos , Coronavirus/metabolismo , Técnicas In Vitro , Intestinos/efectos de los fármacos , Intestinos/metabolismo , Intestinos/virología , Fitoquímicos/farmacología , Tráquea/efectos de los fármacos , Tráquea/metabolismo , Tráquea/virología , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Verduras/química , Proteínas de la Matriz Viral/metabolismo , Reproducibilidad de los Resultados , Porcinos , Animales , Humanos , Técnicas de Cultivo Tridimensional de CélulasRESUMEN
Influenza (IAV) neuraminidase (NA) is a glycoprotein required for the viral exit from the cell. NA requires disulfide bonds for proper function. We have recently demonstrated that protein disulfide isomerase (PDI)A3 is required for oxidative folding of IAV hemagglutinin (HA), and viral propagation. However, it not known whether PDIs are required for NA maturation or if these interactions represent a putative target for the treatment of influenza infection. We sought to determine whether PDIA3 is required for disulfide bonds of NA, its activity, and propagation of the virus. Requirement of disulfides for NA oligomerization and activity were determined using biotin switch and redox assays in WT and PDIA3-/- in A549 cells. A PDI specific inhibitor (LOC14) was utilized to determine the requirement of PDIs in NA activity, IAV burden, and inflammatory response in A549 and primary mouse tracheal epithelial cells. Mice were treated with the inhibitor LOC14 and subsequently examined for IAV burden, NA activity, cytokine, and immune response. IAV-NA interacts with PDIA3 and this interaction is required for NA activity. PDIA3 ablation or inhibition decreased NA activity, viral burden, and inflammatory response in lung epithelial cells. LOC14 treatment significantly attenuated the influenza-induced inflammatory response in mice including the overall viral burden. These results provide evidence for PDIA3 inhibition suppressing NA activity, potentially providing a novel platform for host-targeted antiviral therapies.