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

本课题旨在研究小G蛋白Rho作为一种靶向分子对支气管哮喘的治疗作用。由于Rho及其效应酶Rho激酶在Ca^2+增敏、细胞增殖、细胞迁移和细胞骨架等方面具有重要作用，因此Rho/Rho激酶通路可能与嗜酸性粒细胞浸润、气道高反应性、β-肾上腺素能脱敏和气道重塑等支气管哮喘的病理生理过程有关。用Fura-2负载的豚鼠气管平滑肌同时测定了等长张力和细胞内Ca^<2 +> [Ca^<2+>]_i。将β-肾上腺素能受体激动剂异丙肾上腺素（isoproterenol）累积作用于1 μM乙酰甲胆碱预收缩的组织，异丙肾上腺素可抑制乙酰甲胆碱引起的收缩，并呈浓度依赖性地降低[Ca^2+] i。然而，在抑制剂中，张力的降低大于[Ca^<2+>]_i的降低。 ...更多信息 异丙肾上腺素的作用，表明β-肾上腺素能作用不仅拮抗Ca^2+动员，而且拮抗Ca^2+敏化。后一过程的丧失导致β-肾上腺素能受体功能障碍。过氧化氢是氧化应激的指示剂，随着[Ca^<2+>] i的升高而产生力.然而，Rho激酶抑制剂Y-27632可抑制过氧化氢引起的收缩而不降低[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。1-磷酸鞘氨醇（S1 P）可明显增强乙酰甲胆碱引起的收缩反应，但不升高[Ca^<2+] i，Y-27632可拮抗S1 P预处理引起的乙酰甲胆碱引起的收缩反应。Rho诱导的Ca^<2+>敏化通过肌球蛋白磷酸酶失活介导气道高反应性。在培养的人支气管平滑肌细胞中，FBS诱导的细胞增殖和DNA合成被辛伐他汀、HMG-CoA还原酶和Y-27632抑制。胆固醇合成中由甲羟戊酸途径调节的Rho/Rho激酶活性有助于与气道重塑相关的细胞增殖。在致敏小鼠中，抗原攻击引起嗜酸性粒细胞浸润到气道，并增加肺对乙酰甲胆碱的抵抗力。给予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，并通过抗原激发增强肺抵抗。Rho/Rho激酶通路通过嗜酸性粒细胞募集参与气道炎症，抑制Rho/Rho激酶通路可能对支气管哮喘有治疗作用。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