Airway smooth muscle relaxation induced by 5-HT(2A) receptors: role of Na(+)/K(+)-ATPase pump and Ca(2+)-activated K(+) channels

AIMS: Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. MAIN METHODS: Using organ baths, patch clamp, and intracellular Ca(2+) measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. KEY FINDINGS: A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 muM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 muM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT(1A), 5-HT(1B)/5-HT(1D) and 5-HT(2B) receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 muM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K(+) currents (I(K(Ca))), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. SIGNIFICANCE: During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na(+)/K(+)-ATPase pump, and increasing activity of the high-conductance Ca(2+)-activated K(+) channels, probably via 5-HT(2A) receptors.     

Cellular and molecular mechanisms of acute exposure to sulfur mustard: a systematic review

Context: Mustard gas (e.g. sulfur mustard (SM)) has been used as a chemical agent in several battles and is still a potential worldwide menace. Besides local absorption, particularly in the skin, eyes and lungs, systemic spread of the agent also has detrimental effects on gonads, bone marrow and nervous system. Moreover, chronic exposure of SM to respiratory system causes death. Inducing oxidative stress, and disturbing DNA and tissue repair systems, inflammation and cell death signaling pathways have been introduced as molecular mechanisms of the injury.

Methods: In this systematic review, more than 1200 (2000–2014) articles focusing on gross or molecular pathological reports in the acute phase of the respiratory injury after SM exposure were reviewed, followed by two different layers of gross and molecular pathological data (clinic and laboratory) integrated together in a spatio-temporal order. Role of epithelial, neutrophil and macrophage cells and three signaling pathways of inflammation, oxidative stress and cell death are covered in details.

Results and conclusion: Our results propose a critical role of interleukin-17 producing cells in acute and chronic inflammatory responses.     

Involvement of redox events in caspase activation in zinc-depleted airway epithelial cells

Airway epithelial cells (AEC) contain both pro- and anti-apoptotic factors but little is known about mechanisms regulating apoptosis of these cells. In this study we have examined the localization of pro-caspase-3 and Zn(2+), a cellular regulator of pro-caspase-3, in primary sheep and human AEC. Zn(2+) was concentrated in both cytoplasmic vesicles and ciliary basal bodies, in the vicinity of both pro-caspase-3 and the antioxidant Cu/Zn superoxide dismutase (Cu/Zn SOD). Depletion of intracellular Zn(2+) in sheep AEC, using the membrane permeant Zn(2+) chelator TPEN, increased lipid peroxidation in the apical cell membranes (as assessed by immunofluorescence with anti-hydroxynonenal) as well as increasing activated pro-caspase-3 and apoptosis. There were smaller increases in caspase-2 and -6 but not other caspases. Activation of caspase-3 in TPEN-treated AEC was inhibited strongly by N-acetylcysteine and partially by vitamin C and vitamin E. These findings suggest that cytoplasmic pro-caspase-3 is positioned near the lumenal surface of AEC where it is under the influence of Zn(2+) and other anti-oxidants.     

Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils

Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.     

COPD急性加重期呼气冷凝液中性粒细胞趋化性增加与气道免疫功能障碍的关系

摘要 目的：探讨COPD急性加重期（AECOPD）呼气冷凝液中性粒细胞趋化性增加与气道免疫功能障碍的关系。方法：2018年4月到2021年5月选择在本院诊治的COPD患者88例作为研究对象,包括AECOPD患者48例（急性组）与COPD缓解期患者40例（缓解组）,检测两组中性粒细胞氧化吞噬百分率、呼气冷凝液CCL18与CC16含量、外周血T淋巴细胞亚群水平,同时记录气道总粘性阻力、气道总阻抗、近端气道粘性阻力等指标并进行相关性分析。结果：急性组的中性粒细胞氧化吞噬百分率低于缓解组（P<0.05）。急性组的呼气冷凝液CCL18、CC16含量与气道总粘性阻力、气道总阻抗、近端气道粘性阻力高于缓解组（P<0.05）。急性组的CD4⁺T淋巴细胞比例低于缓解组,CD8⁺T淋巴细胞高于缓解组（P<0.05）。二分类Logistic回归分析结果显示：中性粒细胞氧化吞噬百分率、气道总阻抗、CD4⁺T淋巴细胞比例、呼气冷凝液CCL18为导致AECOPD发生的重要因素（P<0.05）。结论：AECOPD患者伴随有呼气冷凝液CCL18、CC16含量增加与中性粒细胞氧化吞噬百分率降低,还伴随有气道阻力增加与免疫功能降低,中性粒细胞氧化吞噬百分率、气道总阻抗、CD4⁺T淋巴细胞比例、呼气冷凝液CCL18为导致AECOPD发生的重要因素。     

TGF-β_1对不同阶段哮喘模型大鼠气道平滑肌细胞增殖的作用

目的探讨TGF-β1对不同阶段哮喘大鼠气道平滑肌细胞（ASMCs）增殖的作用。方法建立2周、6周哮喘大鼠模型,分别以1μg/L、10μg/L和100μg/LTGF-β1干预ASMCs生长。采用流式细胞仪、MTT法检测ASMCs增殖情况,观察不同浓度TGF-β1对ASMCs增殖的影响。结果 2周和6周哮喘组ASMCs的S期比例、A值分别为（34.31±1.41）%、（35.96±3.46）%;（0.546±0.005）、（0.559±0.009）与对照组（12.24±2.64）%、（0.289±0.009）比较均显著增高（均P〈0.01）。2周、6周哮喘模型组的各TGF-β1干预组ASMCs的S期比例、A值与各自哮喘组比较均显著升高（均P〈0.01）,10μg/L和100μg/LTGF-β1组比1μg/LTGF-β1组对ASMCs增殖作用明显增加（P〈0.01）,10μg/LTGF-β1组和100μg/LTGF-β1组相比,ASMCs增殖细胞占细胞总数的百分比无明显变化,两种浓度增殖作用无有明显差别（P〉0.05）。而2周和6周哮喘组相比,加入不同浓度TGF-β1干预后的差别不大（P〉0.05）。结论与正常鼠相比,2周和6周哮喘大鼠气道平滑肌细胞增殖明显,处于S期的细胞比例明显增高,6周哮喘大鼠气道平滑肌较2周哮喘大鼠增殖更明显。经TGF-β1干预后,2周和6周哮喘哮喘大鼠气道平滑肌细胞处于S期的细胞比例增加,增殖增强,提示TGF-β1可能在哮喘早期阶段即可促进大鼠气道平滑肌细胞增殖,并促进各阶段哮喘大鼠气道平滑肌细胞持续增殖。     

虚拟导航支气管镜联合EBUS-GS技术在直径≤2 cm的肺外周结节诊断中的应用

摘要 目的：探究虚拟导航支气管镜联合EBUS-GS技术在直径≤2 cm的肺外周结节诊断中的诊断价值。方法：本研究采用回顾性分析的方法,选取2019年5月-2022年5月期间在我院就诊的140例直径≤2 cm的肺外周结节患者作为研究对象,随机分为对照组和实验组,各70例。对照组使用传统支气管镜技术进行诊断,实验组使用虚拟导航支气管镜联合EBUS-GS技术进行诊断,并进行随访实验。记录并比较两组患者的可视率、操作时间、诊断率及并发症发生率等指标。结果：对照组和实验组中的病灶位置比较,两组差异无统计学意义（P>0.05）。实验组与对照组病变大小相比差异无统计学意义（P>0.05）,实验组可视率高于对照组。对照组和实验组的支气管镜操作总时间比较,无明显统计学差异（P>0.05）,探及病灶位置时间与对照组相比,实验组低于对照组（P<0.05）。实验组中恶性病变的诊断率为89.74%,良性肺结节的诊断率为83.87%,而对照组恶性病变的诊断率为60%,良性结节的诊断率为50%,两组间病变性质的良性和恶性诊断率相比较,差异均具有统计学意义（P<0.05）。与对照组相比,实验组不良反应发生率2.86%低于对照组不良反应发生率18.57%,差异具有统计学意义（P<0.05）。结论：虚拟导航支气管镜联合EBUS-GS技术是一种可行且有效的方法,可用于直径≤2 cm的肺外周结节的诊断。该技术既可提高诊断准确性,又可减少不必要的切除手术,对患者具有重要的临床意义。     

TSG-6 Protein Is Crucial for the Development of Pulmonary Hyaluronan Deposition,Eosinophilia, and Airway Hyperresponsiveness in a Murine Model of Asthma

Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses, where HA mediates both protective and pathological responses. By modifying the HA matrix, Tnfip6 (TNF-α-induced protein-6; also known as TSG-6 (TNF-stimulated gene-6)) is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. In this study, we examined the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. Compared with wild-type littermate controls, TSG-6^−/− mice exhibited attenuated inflammation marked by a significant decrease in pulmonary HA concentrations measured in the bronchoalveolar lavage and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular Th2 immunity and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6^−/− mice. Most importantly, contrary to their counterpart wild-type littermates, TSG-6^−/− mice were resistant to the induction of airway hyperresponsiveness and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity but is essential for the robust increase in pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and development of airway hyperresponsiveness. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.     

采用TOPOTA克隆技术构建人CC10质粒并在BEAS-2B细胞中的表达

目的:采用TOPO TA克隆技术构建及鉴定人CC10基因表达载体,并在支气管上皮细胞系BEAS-2B细胞中获得稳定表达,初步探讨CC10在呼吸道上皮细胞炎症反应中的作用。方法:提取人下鼻甲组织的总RNA,逆转录反应生成c DNA,再用PCR方法扩增出含人CC10编码区全长的DNA片段,将PCR产物直接连接到pc DNA3.1/V5-His TOPO TA载体中,转化至大肠杆菌后经筛选鉴定出CC10表达载体,用脂质体法将CC10质粒转染到BEAS-2B,细胞免疫荧光法检测CC10蛋白的表达。随后用促炎细胞因子IL-1β刺激转染空质粒和CC10质粒的BEAS-2B细胞,用实时定量RT-PCR和ELISA检测炎性趋化因子RANTES m RNA和蛋白的表达。结果:目的基因与TOPO TA载体在室温下5 min的连接反应效率91.7%,用酶切法鉴定质粒并测序,人CC10基因成功克隆到真核细胞表达载体pc DNA3.1中,CC10蛋白在BEAS-2B细胞中无表达,但在体外转染CC10质粒后CC10蛋白表达明显增高。转染CC10质粒的BEAS-2B细胞可抑制IL-1β诱导的RANTES m RNA和蛋白的表达。结论:利用TOPO TA克隆技术可高效、快速的构建人CC10基因表达载体,并能够在BEAS-2B细胞中获得稳定表达,CC10蛋白在呼吸道上皮细胞中可发挥抗炎作用。