Mechanisms for the induction of bronchial smooth muscle hyperresponsiveness in allergic bronchial asthma

过敏性支气管哮喘诱导支气管平滑肌高反应性的机制

基本信息

批准号：
18590079
负责人：
CHIBA Yoshihiko
金额：
$ 2.47万
依托单位：
Hoshi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590079/
关键词：
Allergic Bronchial Asthma Bronchial Smooth Muscle Calcium Sensitization RhoA RNAi Cytokine Interleukin-13 STAT6 RNA干渉 Interleukin-4

项目摘要

Interleukin-13(IL-13) is one of the central mediators for development of airway hyperresponsiveness in asthma. However, its effect on bronchial smooth muscle(BSM) is not well known Recent studies revealed an involvement of RhoA/Rho-kinase in BSM contraction and this pathway has now been proposed as a new target for asthma therapy. To make clear the role of IL-13 on the induction of BSM hyperresponsiveness, effects of IL-13 on contractility and RhoA expression in BSMs were investigated. Male BALB/c mice were sensitized and repeatedly challenged with ovalbumin antigen. Both in vitro(human BSM cells and mouse BSM tissues) and in vivo(intranasal instillation into mouse airways) effects of IL-13 were also tested. In the repeatedly antigen-challenged mice, marked airway inflammation and BSM hyperresponsiveness with an upregulation of IL-13 in bronchoalveolar lavage fluids were observed In cultured human BSM cells, IL-13 caused an activation of signal transducer and activator of transcription … More 6(STAT6) and an upregulation of RhoA : both of them were inhibited by a STAT6 inhibitor leflunomide. In isolated BSM tissues of naive mice, the contractility was significantly enhanced by organ culture in the presence of IL-13. Moreover, in vivo treatment of airways with 1L-13 caused a BSM hyperresponsiveness with an upregulation of RhoA in naive mice. These findings suggest that IL-13/STAT6 signaling is critical for development of antigen-induced BSM hyperresponsiveness and that agents that specifically inhibit this pathway in BSM may provide a novel strategy for treatment of asthma. Next, the effect of a novel STAT6 inhibitor, AS1517499, on the development of antigen-induced BSM hyperresponsiveness was investigated. In cultured human BSM cells, IL-13(100 ng/mL) caused a phosphorylation of STAT6 and an upregulation of RhoA, a monomeric GTPase responsible for Ca^<2+> sensitization of smooth muscle contraction : both events were inhibited by co-incubation with AS 1517499(100 nM). In BALB/c mice that were actively sensitized and repeatedly challenged with ovalbumin antigen, an increased 1L-13 level in bronchoalveolar lavage fluids and a phosphorylation of STAT6 in BSMs were observed after the last antigen challenge. These mice had an augmented BSM contractility to acetylcholine together with an upregulation of RhoA in BSMs. Intraperitoneal injections of AS1517499(10mg/kg) 1h before each ovalbumin exposure inhibited both the antigen-induced upregulation of RhoA and BSM hyperresponsiveness, almost completely. A partial but significant inhibition of antigen-induced production of IL-13 was also found. These findings suggest that M-13, generated by antigen exposure, upregulates RhoA via an activation of STAT6 in BSM cells directly, resulting in an augmented BSM contraction, which is one of the causes of the airway hyperresponsiveness. The inhibitory effects of AS1517499 both on RhoA and IL-13 upregulations might be useful for asthma treatment. Less

白介素13（IL-13）是哮喘中气道高反应性发展的中心介体之一。然而，其对支气管平滑肌（BSM）的影响尚未众所周知，最近的研究表明，RhoA/Rho-kinase在BSM收缩中的参与，现在已提出该途径是哮喘治疗的新靶标。为了阐明IL-13在诱导BSM高反应性诱导的作用，研究了IL-13对BSMS中收缩力和RHOA表达的影响。雄性BALB/C小鼠敏感，并反复用卵蛋白抗原挑战。还测试了IL-13的体外（人BSM细胞和小鼠BSM组织）和体内（对小鼠气道中的鼻内滴注）的影响。在反复抗原挑战的小鼠中，在培养的人BSM细胞中观察到了在支气管肺泡灌洗液中的明显注射和BSM高反应性，IL-13在人体纯净的灌洗液中观察到IL-13，IL-13在培养的人BSM细胞中观察到，IL-13在转录中的信号转录和统计量增加了rhoa的统计信息。 leflunomide。在天真小鼠的分离的BSM组织中，在IL-13存在下，器官培养显着增强了收缩力。此外，用1L-13对气道进行体内治疗导致BSM高反应性，而天真小鼠的RhoA上调。这些发现表明，IL-13/STAT6信号对于发展抗原诱导的BSM高反应性至关重要，并且专门抑制BSM中该途径的药物可能为治疗哮喘提供新的策略。接下来，研究了一种新型STAT6抑制剂AS1517499的影响，对抗原诱导的BSM过度反应性的发展。在培养的人BSM细胞中，IL-13（100 ng/ml）引起了STAT6的磷酸化和RhoA的上调，RhoA是一种负责CA^<2+>平滑肌肌肉敏感性的单体GTPase：两种事件都通过共孵育而抑制为1517499（10017499（100 nm））。在BALB/C小鼠中，积极敏感并反复挑战卵蛋白抗原，在最后一次抗原挑战之后，观察到了支气管肺泡灌洗液中1L-13水平的升高和BSMS中STAT6的磷酸化。这些小鼠对乙酰胆碱具有增强的BSM收缩性，以及BSMS中RhoA的上调。在每次卵巢蛋白暴露之前，腹膜内注射AS1517499（10mg/kg）1H几乎完全完全抑制了抗原诱导的RhoA和BSM超反应的上调，几乎完全完全完全。还发现了抗原诱导的IL-13产生的部分但显着抑制。这些发现表明，通过抗原暴露产生的M-13通过直接在BSM细胞中的STAT6激活上调RhoA，从而导致增强的BSM收缩，这是气道高反应性的原因之一。 AS1517499对RhoA和IL-13上调的抑制作用可能对哮喘治疗有用。较少的