CN110279697B - 铁死亡诱导剂在治疗或缓解过敏性气道炎症药物中的应用 - Google Patents
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Abstract
本发明提供了铁死亡诱导剂在未来治疗或缓解过敏性气道炎症药物中应用，所述应用中，铁死亡诱导剂通过靶向诱导嗜酸性粒细胞铁死亡实现对过敏性气道炎症的治疗或缓解。本发明首次在过敏性气道疾病治疗中引入嗜酸性粒细胞铁死亡的概念。我们发现铁死亡诱导剂体外能诱导人及小鼠嗜酸性粒细胞发生铁死亡；通过构建动物模型，发现铁死亡诱导剂可显著缓解小鼠过敏性气道炎症；并且，铁死亡诱导剂青蒿琥酯在保护过敏性气道炎症上和糖皮质激素存在协同调控作用，这对减少激素用量，减轻激素带来的全身副作用具有重大研究意义。本发明为未来临床开发新型药物治疗过敏性气道疾病，乃至全身嗜酸性粒细胞相关性疾病提供了新思路、新靶点。
Description
技术领域
本发明涉及呼吸疾病治疗领域，首次提出靶向诱导嗜酸性粒细胞铁死亡的小分子化合物能有效保护过敏性气道炎症，且其与糖皮质激素在疾病治疗上具有协同调控作用。
背景技术
支气管哮喘(Asthma，简称哮喘)是最为常见的气道慢性炎症性疾病，也是全球发病率增长最快的疾病之一。目前全世界约有3亿哮喘病人，全球因哮喘支出的费用已超过结核和艾滋病的总和。中国约有3千万哮喘患者，是全球哮喘病死率最高的国家之一。随着全球工业化进展以及环境污染的加重，哮喘的发病率和病死率逐年增高，给患者及社会均造成了沉重的负担。因此，探索哮喘潜在的发病机制，并积极开展相关靶向治疗的研究对哮喘的预防和控制具有重要的意义。哮喘是常见的慢性疾病之一,全球约有3亿哮喘患者,而我国约有3000万人罹患哮喘,哮喘的发病率和病死率正逐年上升,严重威胁人类的身心健康,造成严重的经济和社会负担。
一般认为哮喘的发病机制主要涉及TH1/TH2细胞免疫失衡，它是一种以嗜酸性粒细胞(Eosinophil,Eos)、肥大细胞和T淋巴细胞等炎症细胞参与，气道粘液高分泌、气道高反应性(AHR)和气道重构为特征的气道慢性炎症性疾病。抗原经过树突状细胞等呈递后，诱导Naive T细胞分化成为TH2细胞，从而分泌各种TH2型炎症因子如IL-4、IL-5和IL-13，其中IL-5能诱导Eos的分化。大量产生和募集的Eos则进一步分泌各种炎症因子，从而诱导哮喘慢性气道炎症，气道高反应性和粘液高分泌。Eos是哮喘患者气道的主要炎症细胞，其浸润与哮喘严重程度存在密切联系，外周血、痰液、肺泡灌洗液(Bronchoalveolar lavage fluid,BALF)或支气管黏膜活检中发现Eos及其产物，提示炎症反应的持续存在。此外，Eos浸润不仅与气道重构、肺功能的下降及威胁生命的哮喘发作有关，而且与肺总量、胸腔气体容量呈显著正相关。而哮喘气道中的其他炎性细胞如淋巴细胞、巨噬细胞、肥大细胞和中性粒细胞等却与肺容量等指标无关。所有这些证据表明，Eos在哮喘的发生发展中起着重要的作用，诱导Eos死亡将有利于哮喘控制，且不会因细胞死亡而引起局部炎症反应。糖皮质激素作为目前哮喘控制最有效的药物之一，通过快速诱导外周血和气道组织内Eos的凋亡发挥强大的抗炎作用；新型靶向药物IL-5单克隆抗体也是通过降低Eos的产生和存活来治疗严重嗜酸性粒细胞哮喘。因此，开发靶向诱导Eos死亡的药物在哮喘防治中具有极大的应用前景，成为当前哮喘研究的新热点。
发明内容
本发明的目的在于针对现有技术的不足，提供铁死亡诱导剂在未来过敏性气道炎症治疗或缓解药物中的应用。
本发明的目的是通过如下技术方案实现的：铁死亡诱导剂在研发过敏性气道炎症治疗或缓解药物中的应用，所述应用中，铁死亡诱导剂通过靶向诱导嗜酸性粒细胞铁死亡实现对过敏性气道炎症的治疗或缓解。
进一步地，所述的铁死亡诱导剂包括：Erastin、RSL3和青蒿琥酯(Artesunate,ART)。
本发明的有益效果是：本发明应用的铁死亡诱导剂能有效缓解过敏性气道炎症，且其在疾病治疗中和糖皮质激素存在协同作用，可显著减少激素用量，减轻激素带来的全身副作用，可作为未来过敏性气道炎症治疗或缓解药物研发的新方向。
附图说明
图1为铁死亡诱导剂干预后哮喘患者外周血嗜酸性粒细胞存活图；
图2为铁死亡诱导剂干预后小鼠外周血嗜酸性粒细胞存活图；
图3为铁死亡诱导剂干预后小鼠外周血嗜酸性粒细胞电镜图；
图4为经典的OVA过敏性气道炎症疾病小鼠模型构建示意图；
图5为应用铁死亡诱导剂后小鼠BALF细胞分类计数图；
图6为应用铁死亡诱导剂后小鼠肺组织H&E染色病理图；
图7为应用铁死亡诱导剂后实时荧光定量PCR检测小鼠肺组织TH2相关炎症因子表达图；
图8为ART和地塞米松联用小鼠BALF细胞分类计数图；
图9为ART和地塞米松联用实时荧光定量PCR检测小鼠肺组织TH2相关炎症因子表达图。
具体实施方式
本发明提出了铁死亡诱导剂在制备过敏性气道炎症治疗或缓解药物中的应用，所述应用中，铁死亡诱导剂通过靶向诱导嗜酸性粒细胞铁死亡实现对过敏性气道炎症的治疗或缓解。
铁死亡(Ferroptosis)是由2012年Dixon等首次发现铁依赖的脂质过氧化物蓄积为特征的新型细胞死亡模式。这种全新的细胞死亡形式在发生机制上与已知的凋亡、坏死、自噬等细胞死亡形式有显著差异，主要表现为线粒体固缩，脂质双分子膜密度增加，线粒体嵴减少或消失。当细胞内还原性物质，如谷胱甘肽(Glutathione)耗竭，谷胱甘肽过氧化物酶4(Glutathione peroxidase 4,GPX4)活性降低，导致脂质过氧化物蓄积，或者二价铁离子通过类似Fenton反应的方式产生大量ROS，从而促使细胞发生铁死亡。已知的铁死亡诱导剂(Ferroptosis inducing agents，FINs)包括Erastin、RSL3和青蒿琥酯(Artesunate,ART)等，其作用分子靶点和通路不尽相同。
下面结合附图和实施例对本发明进一步说明；
实施例1：体外实验证实各类铁死亡诱导剂能有效诱导人及小鼠嗜酸性粒细胞死亡。
分离哮喘患者外周血白细胞体外培养，分别干预铁死亡诱导剂Erastin、RSL3和ART，24小时后，其嗜酸性粒细胞存活情况如图1所示，可以看出铁死亡诱导剂体外能诱导哮喘患者外周血嗜酸性粒细胞死亡，且呈浓度依赖性。
进一步地，分离NJ.1638小鼠外周血嗜酸性粒细胞体外培养，分别干预铁死亡诱导剂Erastin、RSL3和ART，24小时后流式分析细胞存活情况，结果如图2所示，表明铁死亡诱导剂体外能诱导小鼠嗜酸性粒细胞死亡，且呈浓度依赖性。而且，收集的细胞通过电镜观察发现铁死亡诱导剂干预嗜酸性粒细胞后，细胞主要表现为线粒体破坏，线粒体嵴消失，未观察到细胞膜出泡、染色质断裂等其他形态变化，与文献报道铁死亡形态改变吻合，说明铁死亡诱导剂可以诱导嗜酸性粒细胞铁死亡，结果如图3所示。
实施例2：利用野生型小鼠构建经典的过敏性气道疾病模型，腹腔给予铁死亡诱导剂，观察其对小鼠过敏性气道炎症的调控作用。
构建经典的卵清蛋白(Ovalbumin，OVA)过敏性气道炎症疾病小鼠模型：如图4所示，第0、14天腹腔注射(i.p)致敏野生型小鼠，并于第24、25、26天1.2％OVA雾化激发，第27天检测。
利用野生型小鼠构建经典的过敏性气道疾病模型，OVA雾化后2小时腹腔分别给予铁死亡诱导剂(Erastin 15mg/kg；RSL3 10mg/kg；ART 20mg/kg)或对应溶剂(生理盐水NS；二甲基亚砜DMSO)，于末次雾化后24小时处理模型。小鼠右肺结扎行左肺灌洗，0.4mL*3次，最终收集BALF约1mL左右。取50μL BALF细胞悬液，加入50μl白细胞计数液，混匀后取10μL加至血细胞计数板(改良Neubauer计数板)并在光学显微镜下计白细胞总数。上述BALF离心(4℃，6000rpm，10min)，得到的细胞沉淀用平衡盐溶液PBS重悬并充分混匀。细胞悬液在玻片上进行甩片(700rpm，2min)，瑞氏-吉姆萨染色后计数各类细胞的百分比，并由细胞总数获得对应嗜酸总数。如图5所示，小鼠肺泡灌洗液细胞分类计数显示，腹腔给予铁死亡诱导剂能明显缓解嗜酸性粒细胞绝对值及百分比，保护过敏性气道疾病。
左肺灌洗后通过气管插管向左肺灌注4％甲醛0.4mL使左肺膨胀(内固定)，结扎气管，眼科剪取下左肺并投入4％甲醛中(外固定)24-48小时。固定的肺组织包埋、切片，行H&E染色。在病理成像分析系统(Olympus)的光学显微镜下观察切片内气道(直径在300μm-100μm之间，长径与短径之比≥0.6的完整气道)炎症，并同等条件下拍照。根据气道周围炎症细胞浸润的程度评0-3分，评分标准如下：0分，气道周围没有炎症细胞浸润；1分，气道周围少许炎症细胞浸润；2分，大部分气道周围有1-5层炎症细胞浸润；3分，大部分气道周围有>5层炎症细胞浸润。炎症评分采用双盲法，由两个观察者独立完成，对每个样本的评分取其平均值。如图6所示，H&E染色显示铁死亡诱导剂能明显减少小鼠肺组织气道及血管周围炎症细胞浸润。研究显示哮喘疾病严重程度跟嗜酸性粒细胞浸润密切相关。
取小鼠右肺组织标本，放入进口的1.5mL Ep管中，加入lmL Trizol/tube，用匀浆机充分匀浆，15s/次，持续3次，室温放置10min。利用氯仿提取RNA并纯化，利用所得RNA通过逆转录(RT)制备cDNA样本，并进行实时荧光定量PCR检测小鼠肺组织TH2相关炎症因子表达。如图7所示，铁死亡诱导剂明显下调小鼠肺组织TH2相关炎症因子Il13和Il25的表达，保护小鼠过敏性气道炎症。
实施例3：利用野生型小鼠构建经典的过敏性气道疾病模型，联合给予铁死亡诱导剂和糖皮质激素，检测二者在小鼠过敏性气道炎症中是否存在协同调控作用。
根据图4构建动物模型，OVA雾化后2小时腹腔联合给予ART和地塞米松(Dexamethasone，DXMS)，检测二者在小鼠过敏性气道炎症中是否存在协同调控作用。图8表明，BALF计数显示ART和地塞米松联用对嗜酸性粒细胞性气道炎症具有协同保护作用；图9表明ART和地塞米松联用协同下调肺组织TH2相关炎症因子Il4和Il13的表达水平。
以上实施例说明铁死亡诱导剂能有效缓解过敏性气道炎症，且其在疾病治疗中和糖皮质激素存在协同作用，可显著减少激素用量，减轻激素带来的全身副作用，可应用在过敏性气道炎症治疗或缓解药物中。
以上实施例用来解释本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明做出的任何修改和改变，都落入本发明的保护范围。
Claims (1)
1.铁死亡诱导剂在制备治疗或缓解过敏性气道炎症药物中的应用，其特征在于，所述铁死亡诱导剂通过靶向诱导嗜酸性粒细胞铁死亡实现对过敏性气道炎症的治疗或缓解；所述的铁死亡诱导剂为Erastin或RSL3。
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