5-羟色胺—迷走神经通路在食管酸反流致气道高反应性中的作用和机制

发布时间：2018-04-10 08:58

  本文选题：5-羟色胺　切入点：迷走神经　出处：《浙江大学》2013年硕士论文

【摘要】：研究背景： 胃食管反流病(Gastro Esophageal Reflux Disease, GERD)的呼吸系统表现非常广泛,如慢性咳嗽、难治性哮喘、反复肺炎,严重影响患者生活质量。胃食管反流性咳嗽(GER induced cough, GERC)是指与GER相关的以咳嗽为主要表现的呼吸道症候群,常规抑酸治疗效果并不理想,因此寻找有效的GERC治疗药物非常必要,它的发病机制引起广泛关注。其中,食管-支气管反射引起的气道神经源性炎症是其主要的病理生理机制。 反射机制的提出是以食管和气管共同起源于胚胎前肠且具有共同的迷走神经通路为基础的,已有研究表明食管迷走C纤维是主要神经通路。反流时,酸刺激食管内的酸敏感受体促发迷走神经活动,通过食管-支气管反射,使气道感觉神经受到刺激。瞬时感受器电位香草酸受体1(transient receptor potential vanilloid receptor1,TRPV1)在迷走神经C纤维上表达,对辣椒素敏感,也称辣椒素敏感受体(VR1),对酸、温度或炎症介质非常敏感。TRPV1激活后,可开放离子通道,C纤维兴奋后末梢释放神经肽,神经肽主要有神经肽A(NKA)、神经肽B(NKB)、P物质(SP),它们直接刺激咳嗽感受器或通过引起炎症、影响气道敏感性间接引发呼吸道症状。5-羟色胺(5-hydroxytryptamine,5-HT)又称血清素(serotonin),是肠神经系统内的重要神经递质,调节胃肠道感觉、运动、分泌,其在GERD发病机制中的作用也受到广泛关注。研究表明5-HT具有选择性的刺激食管迷走神经传入纤维的特性,这为研究GERC的发病机制提供了新的方向。因此,我们根据已有研究提出如下假设：食管内的酸反流时,如食管黏膜5-HT分泌增加,可激活迷走神经C纤维,通过食管-支气管神经反射通道,引起速激肽的释放,导致气道神经源性炎症,气道反应性增高,而引起咳嗽等呼吸道症状。 目的： 确定5-HT-迷走神经C传入纤维通路在食管酸反流致气道高反应性中的作用及其机制,分析影响5-HT-迷走神经C传入纤维-神经肽分泌途径的药物对食管酸反流致气道高反应性的防治作用,为食管反流所致气道高反应性的治疗提供新的靶点。 方法： 普通级健康雄性白化豚鼠30只(7周龄,体重300g-400g),随机分成：模型组、对照组和空白组。模型组豚鼠经胃管在食管的中下段以18ml/h的速率缓慢灌注0.1mmol/1的HCL溶液(含0.5%胃蛋白酶),持续20min,每天一次,连续14d,以建立GER模型。对照组用蒸馏水代替盐酸灌注；空白组不做任何处理。检测不同浓度乙酰甲胆碱(Mch)对豚鼠肺阻力和肺顺应性的变化,评估气道反应性；采集支气管肺泡灌洗液(BALF)进行白细胞计数和分类计数,并利用酶联免疫吸附法测其中NKA、NKB、SP含量；评估食管、气管和肺组织病理变化；采用高效液相色谱-电化学法测食管组织5-HT和5-HIAA含量；分别利用RT-PCR和Western Blot测定食管TRPV1、5HT4R、SERT的mRNA和蛋白表达的水平。进一步在每次食管酸灌注之前30min分组(每组10只)采用5HT4R拮抗剂GR113808(1μmol/kg)和TRPV1拮抗剂辣椒平(capsazepine,0.5μmol/kg)进行腹腔注射2周,观察其干预效果。 结果： 1.GER动物模型的建立和气道高反应性。当Mch剌激浓度达到0.25mMol/L和0.5mMol/L时,模型组肺阻力增加百分比和肺顺应性减少百分比大于对照组和空白组,气道反应性增高；差异具有统计学意义。模型组远端食管黏膜出现溃疡糜烂,炎症表现明显；气管组织和肺组织炎症明显,主要以嗜酸性粒细胞浸润为主,而对照组和空白组远端食管黏膜结构完好,支气管和肺泡结构正常。模型组BALF中的白细胞总数和嗜酸性粒细胞百分比,及NKA和SP含量均显著高于对照组和空白组。模型组食管组织中5-HT水平显著高于对照组和空白组,而SERT mRNA表达水平明显降低。TRPV1mRNA,5-HT4RmRNA和蛋白表达水平在三组间的差异没有统计学意义；模型组TRPV1蛋白表达水平高于对照组和空白组,但差异无统计学意义。 2.药物干预结果：Mch浓度为0.5mMol/L时,GR113808组和辣椒平组的肺阻力增加百分比和肺顺应性减少百分比小于模型组,气道高反应性得到一定程度缓解。两药物干预组食管黏膜、气管黏膜和肺组织的炎症程度比模型组有所减轻。而GR113808组的BALF中白细胞总数和嗜酸性粒细胞计数均比模型组有所下降,辣椒平组的白细胞总数低于模型组。两组BALF中SP含量均比模型组有所降低。 结论： 豚鼠食管酸灌注模型可以引起食管黏膜损伤、气道神经源性炎症和气道高反应性；食管酸灌注可引起食管组织5-HT升高和SERT的降低,5-HT-迷走神经通路可能在食管酸反流致气道高反应性中发挥作用；5-HT4R拮抗剂GR113808和TRPV1拮抗剂辣椒平可有效降低食管酸反流引起的气道反应性,并减轻肺部组织炎症,5HT4R和TRPV1有望成为治疗GERC的新靶点。
[Abstract]:Research background:
Gastroesophageal reflux disease (Gastro Esophageal Reflux Disease, GERD) of the respiratory system is very extensive, such as chronic cough, refractory asthma, recurrent pneumonia, seriously affect the life quality of patients. Gastroesophageal reflux cough (GER induced, cough, GERC and GER) is related to cough as the main manifestation of respiratory tract the syndrome of conventional acid suppression therapy effect is not ideal, so the search for effective drug therapy for GERC is necessary, its pathogenesis has aroused widespread concern. Among them, the airway neurogenic inflammation caused by esophageal tracheobronchial reflex is the main pathophysiological mechanism.
The reflection mechanism is to the trachea and esophagus originate from embryonic foregut and vagus pathway based on common, studies have shown that esophageal vagal C fiber is the main pathway. Reflux, acid sensitive receptor in esophageal acid stimulation by stimulating vagus nerve activity, esophageal tracheobronchial reflex, the airway sensory nerve the stimulation. The transient potential vanilloid receptor 1 (transient receptor potential vanilloid receptor1, TRPV1) C expression in vagal fibers, sensitive to capsaicin, also called capsaicin sensitive receptor (VR1), the acid, temperature or inflammatory mediators is very sensitive to the activation of.TRPV1, can open the ion channel, neuropeptide C fiber endings release after excitation mainly, neuropeptide neuropeptide A (NKA), neuropeptide B (NKB), substance P (SP), which direct stimulation of cough receptors or by causing inflammation, airway sensitivity indirect effect Respiratory symptoms of.5- hydroxytryptamine (5-hydroxytryptamine, 5-HT) also called serotonin (serotonin), is an important neurotransmitter in enteric nervous system, regulating gastrointestinal sensation, movement, secretion, its role in the pathogenesis of GERD has received extensive attention. Studies show that 5-HT has a selective stimulation of esophageal vagal afferent fibers, the provide a new direction for the study of the pathogenesis of GERC. Therefore, we propose a hypothesis based on the previous research: esophageal acid reflux, esophageal mucosa such as increased secretion of 5-HT, can activate vagal nerve fibers by C, esophageal tracheobronchial reflex channels caused by tachykinin release, leading to airway neurogenic inflammation, airway hyperresponsiveness, caused by respiratory symptoms such as cough.
Objective:
To determine the 5-HT- C pathway in the vagal afferent fibers of esophageal acid reflux caused by the effect and mechanism of airway hyperresponsiveness in the prevention of drug effect analysis on the effect of 5-HT- C vagal afferent fibers - neuropeptide secretion pathway on esophageal acid reflux induced airway hyperresponsiveness, provide a new target for the treatment of esophageal reflux caused by airway hyperresponsiveness the.
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