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

白细胞介素-13（IL-13）是哮喘气道高反应性发生发展的中枢介质之一。然而，其对支气管平滑肌（BSM）的作用尚不清楚。最近的研究表明，RhoA/Rho激酶参与了BSM的收缩，该途径现已被提议作为哮喘治疗的新靶点。为了明确IL-13在诱导BSM高反应性中的作用，研究了IL-13对BSM中收缩性和RhoA表达的影响。用卵清蛋白抗原致敏雄性BALB/c小鼠并反复激发。还测试了IL-13的体外（人BSM细胞和小鼠BSM组织）和体内（鼻内滴入小鼠气道）作用。在反复抗原攻击的小鼠中，观察到显著的气道炎症和BSM高反应性，并在支气管肺泡灌洗液中观察到IL-13的上调。在培养的人BSM细胞中，IL-13引起信号转导和转录激活因子的激活。 关于我们 6（STAT 6）和RhoA的上调：它们都被STAT 6抑制剂来氟米特抑制。在幼稚小鼠的分离的BSM组织中，在IL-13存在下通过器官培养显著增强收缩性。此外，在体内用IL-13处理气道引起BSM高反应性，在幼稚小鼠中RhoA上调。这些研究结果表明，IL-13/STAT 6信号传导是抗原诱导的BSM高反应性的发展的关键，并且特异性抑制BSM中该途径的药物可能为治疗哮喘提供新的策略。接下来，研究了新型STAT 6抑制剂AS 1517499对抗原诱导的BSM高反应性发展的影响。在培养的人BSM细胞中，IL-13（100 ng/mL）引起STAT 6磷酸化和RhoA上调，RhoA是一种负责平滑肌收缩的Ca^2+敏感化的单体GT3：这两个事件都被AS 1517499（100 nM）共同孵育抑制。在主动致敏的BALB/c小鼠中，用卵白蛋白抗原反复激发，在最后一次抗原激发后观察到支气管肺泡灌洗液中IL-13水平升高和BSM中STAT 6磷酸化。这些小鼠具有增强的BSM对乙酰胆碱的收缩性以及BSM中RhoA的上调。在每次卵清蛋白暴露前1小时腹膜内注射AS 1517499（10 mg/kg）几乎完全抑制抗原诱导的RhoA和BSM高反应性上调。这些结果表明，抗原暴露产生的M-13通过直接激活BSM细胞中的STAT 6上调RhoA，导致BSM收缩增强，这是气道高反应性的原因之一。AS 1517499对RhoA和IL-13上调的抑制作用可能对哮喘治疗有用。少

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