Therapy for bronchial asthma by regulating a small G protein as a targeting molecule

本项目旨在确定小G蛋白Rho是否可作为靶向分子用于支气管哮喘的治疗。由于Rho及其效应酶Rho激酶与Ca^<2+>致敏、细胞增殖、细胞迁移和细胞骨架具有功能相关性，因此Rho/Rho激酶通路可能与支气管哮喘的病理生理机制嗜酸性粒细胞浸润、气道高反应性、β-肾上腺素能脱敏和气道重塑有关。用载fura-2的豚鼠气管平滑肌同时测定等长张力和细胞内Ca^<2+> [Ca^<2+>]_i浓度。当β-肾上腺素能受体激动剂异丙肾上腺素累积作用于1 μM甲胆碱预收缩的组织时，异丙肾上腺素可引起甲胆碱诱导的收缩抑制，并呈浓度依赖性地降低[Ca^<2+>]_i。然而，在抑制异丙肾上腺素的作用中，张力的降低大于[Ca^<2+>]_i，这表明β-肾上腺素能作用不仅可以拮抗Ca^<2+>的动员，还可以拮抗Ca^<2+>的致敏。后一过程的缺失导致β-肾上腺素能受体功能障碍。过氧化氢作为氧化应激的指标，随着[Ca^<2+>]_i的升高而产生力。然而，rho激酶抑制剂Y-27632抑制过氧化氢的收缩而不降低[Ca^<2+>]_i，表明氧化应激引起的气流限制与rho激酶的激活有关。暴露于炎症细胞释放的一种生物活性溶血磷脂sphingosine 1-phosphate （S1P）后，甲基苯丙胺诱导的收缩明显增强，但没有升高[Ca^<2+>]_i， Y-27632通过S1P预处理可拮抗甲基苯丙胺增强的反应性。气道高反应性是由rho通过肌球蛋白磷酸酶失活诱导Ca^<2+>致敏介导的。在体外培养的人支气管平滑肌细胞中，辛伐他汀、HMG-CoA还原酶和Y-27632可抑制FBS诱导的细胞增殖和DNA合成。胆固醇合成中甲羟戊酸途径调控的Rho/Rho激酶活性有助于气道重塑相关的细胞增殖。在致敏小鼠中，抗原刺激引起气道嗜酸性粒细胞浸润和对甲胆碱反应的肺阻力增加。Y-27632和法舒地尔（另一种rho激酶抑制剂）可抑制嗜酸性粒细胞增多，增强抗原刺激下的肺抵抗。Rho/Rho激酶途径通过嗜酸性粒细胞募集参与气道炎症。综上所述，抑制Rho/Rho激酶通路可能具有治疗支气管哮喘的潜力。少

This project was designed to determine whether Rho, a small G protein, is useful to therapy for bronchial asthma as a targeting molecule. Since Rho, and it's effector enzyme, Rho-kinase, have functional relevance for Ca^<2+> sensitization, cell proliferation, cell migration, and cytoskeleton, the Rho/Rho-kinase pathway may related to eosinophil infiltration, airway hyperresponsiveness, β-adrenergic desensitization, and airway remodeling which are pathophysiogy of bronchial asthma. Isometric tension and concentrations of intracellular Ca^<2+> [Ca^<2+>]_i were simultaneously measured using fura-2 loaded tracheal smooth muscle in guinea pigs. When isoproterenol, a β-adrenergic receptor agonist, was cumulatively applied to the tissues precontracted with 1 μM methacholine, isoproterenol caused an inhibition of contraction induced by methacholine with reducing [Ca^<2+>]_i in a concentration-dependent manner. However, a reduction in tension was greater than that in [Ca^<2+>]_i in the inhibito … More ry action of isoproterenol, indicating that β-adrenergic action antagonizes not only Ca^<2+> mobilization but also Ca^<2+> sensitization. Loss of the latter process leads to dysfunction of β-adrenergic receptors. Hydrogen peroxide, an indicator of oxidative stress, generated force with elevating [Ca^<2+>]_i. However, Y-27632, an inhibitor of Rho-kinase, inhibited contraction by hydrogen peroxide without reducing [Ca^<2+>]_i, indicating that airflow limitation by oxidative stress is associated with activation of Rho-kinase. After exposure to sphingosine 1-phosphate (S1P), a bioactive lysophospholipid released from inflammatory cells, methacholine-induced contraction was markedly enhanced without elevating [Ca^<2+>]_i, and Y-27632 antagonized the augmented responsiveness to methacholine by pretreatment with S1P. Airway hyperresponsiveness is mediated by Rho-induced Ca^<2+> sensitization via inactivation of myosin phosphatase. In cultured human bronchial smooth muscle cells, cell proliferation and DNA synsethsis induced by FBS was inhibited by simvastatin, a HMG-CoA reductase, and Y-27632. Rho/Rho-kinase activity regulated by the mevalonate pathways in cholesterol synthesis contributes to cell proliferation related to airway remodeling. In sensitize mice, antigen challenges caused eosinophil infiltration to airways and an increase in the lung resistance in response to methacholine. Administration of Y-27632 and fasudil, another inhibitor of Rho-kinase, inhibited eosinophilia and augmented lung resistance by antigens challenges. The Rho/Rho-kinase pathway is involved in the airway inflammation by eosinophil recruitment.In conclusion, inhibiting the Rho/Rho-kinase pathway may have therapeutic potential for bronchial asthma. Less