Nicotine, Airway Smooth Muscle and Asthma

摘要 虽然电子烟被宣传为哮喘患者吸烟的更安全的替代品，但有越来越多的证据表明 他们的肺部毒性，需要更好地了解呼吸道尼古丁生物学：一项临床相关研究 未得到满足的需求，也是这笔赠款的主要目标。在哮喘中，炎症增强了气道平滑肌(ASM) Ca~(2+)([Ca~(2+)]Cyt)和收缩(气道高反应性；AHR)，以及涉及ASM的重构 扩散。鉴于吸入尼古丁可以直接影响ASM，尼古丁对哮喘ASM的作用是 关键，但对尼古丁受体(NAChR)的表达及其在人类ASM或哮喘中的作用知之甚少。 人类ASM的初步数据显示，功能性α7nAChRs A)随着炎症和哮喘的增加而增加， B)增强ASM[Ca~(2+)]Cyt/收缩能力；c)增强内质网应激、线粒体分裂和呼吸作用；以及 D)激活促增殖途径。在哮喘的混合过敏原小鼠模型中观察到了这样的效果 暴露于急性或慢性吸入尼古丁，但被α7抑制和α7 KO小鼠钝化。因此，我们的 假设尼古丁通过ASMα7促进哮喘患者的肾上腺皮质激素释放和重塑。我们的目标是： 目的1：探讨α-7在炎症和哮喘中的表达及其调控机制。 目的2：在非哮喘患者和哮喘患者的人类ASM中，确定α7参与的机制 在AHR的背景下，尼古丁增强[Ca~(2+)]细胞和收缩能力。目的3：非哮喘患者的人体ASM VS哮喘患者，以确定α7促进尼古丁促进细胞增殖的机制。 目的4：在过敏性哮喘小鼠模型中，确定α7在尼古丁吸入对过敏性哮喘的影响中的作用。 和改建。研究使用成人ASM对非哮喘患者和轻中度哮喘患者暴露于 与系统性红斑狼疮和哮喘相关的细胞因子(肿瘤坏死因子α、IL-6、IL-13)，有/没有尼古丁。目标1探索表情 和α7nAChR的定位、调节伴侣以及α7在 炎症/哮喘(如MAPK、PI3/AKT、NFκB、STATS)。使用以下工具测试α7作为渠道的功能 电生理学。目的2探讨急性和慢性尼古丁对[Ca~(2+)]细胞调节(内流，SR Ca~(2+))的影响 释放)和收缩(牵引力、器官浴缸)。目标3探讨尼古丁对ASM增殖的影响，以及 内质网应激，线粒体分裂(Drp1，Fis1)与融合(Mfn1/2，OPA1)和呼吸(3a)的作用，以及 细胞因子相关的增殖途径(3b)。在这些目标中，α7的作用是使用广泛的与亚单位- 特定的激动剂、拮抗剂或siRNA。体外结果被整合在Aim 4中，使用成年小鼠模型 有或没有急性或慢性吸入尼古丁的哮喘。α-7抑制剂(MG624)的降压作用及其机制 检测血管内皮细胞中α7 KO的表达。评估气道反应性、重塑和ASM生化的研究 改变。临床意义在于确定ASMα7为缓解AHR和重塑的新靶点 与哮喘和尼古丁有关。

ABSTRACT While e-cigarettes are advertised as safer alternatives to smoking for asthmatics, there is increasing evidence of their pulmonary toxicity, necessitating better understanding of airway nicotine biology: a clinically-relevant unmet need, and major goal of this grant. In asthma, inflammation enhances airway smooth muscle (ASM) Ca2+ ([Ca2+]cyt) and contractility (airway hyperreactivity; AHR), along with remodeling involving ASM proliferation. Given that inhaled nicotine can directly influence ASM, nicotine effects on asthmatic ASM are critical, but little is known about nicotinic receptor (nAChR) expression, its role in human ASM, or in asthma. Preliminary data in human ASM show functional α7 nAChRs that A) increase with inflammation and asthma, B) enhance ASM [Ca2+]cyt/contractility; C) enhance ER stress and mitochondrial fission, and respiration, and D) activate pro-proliferative pathways. Such effects are observed in a mixed-allergen mouse model of asthma exposed to acute or chronic inhaled nicotine, but blunted by α7 inhibition and in α7 KO mice. Thus, our hypothesis is that nicotine acts via ASM α7 to promote AHR and remodeling in asthma. Our Aims are: Aim 1: : In human ASM, determine mechanisms of α7 expression and regulation in inflammation and asthma. Aim 2: In human ASM of non-asthmatics vs. asthmatics, to determine mechanisms by which α7 contributes to nicotine enhancement of [Ca2+]cyt and contractility in the context of AHR. Aim 3: In human ASM of non-asthmatics vs. asthmatics, to determine mechanisms by which α7 contributes to nicotine enhancement of cell proliferation. Aim 4: In a mouse model of allergic asthma, to determine the role of α7 in effects of inhaled nicotine on AHR and remodeling. Studies use adult human ASM of non-asthmatics vs. mild-moderate asthmatics exposed to cytokines relevant to ASM and to asthma (TNFα, IL-6, IL-13), with/without nicotine. Aim 1 explores expression and localization of α7 nAChR, regulatory chaperones, and mechanisms by which α7 is increased in inflammation/asthma (e.g. MAPKs, PI3/Akt, NFκB, Stats). α7 functionality as a channel is tested using electrophysiology. Aim 2 explores acute vs. chronic nicotine effects on [Ca2+]cyt regulation (influx, SR Ca2+ release) and contractility (traction force, organ bath). Aim 3 explores nicotine effects on ASM proliferation, and the roles of ER stress, mitochondrial fission (Drp1, Fis1) vs. fusion (Mfn1/2, Opa1) and respiration (3a), and of cytokine-associated proliferative pathways (3b). In these Aims, role of α7 is determined using broad vs. subunit- specific agonists, antagonists or siRNAs. In vitro results are integrated in Aim 4 using the adult mouse model of asthma with/without acute vs. chronic inhaled nicotine. Alleviating effects of α7 inhibitor (MG624) and effects of α7 KO in smooth muscle are tested. Studies assess airway reactivity, remodeling, and ASM biochemical changes. Clinical significance lies in identifying ASM α7 as a novel target for alleviating AHR and remodeling of asthma as well as with nicotine.