七氟醚对哮喘小鼠气道炎症的影响及其作用机制研究

发布时间：2019-06-13 07:08

【摘要】：背景哮喘是一种病因复杂的慢性气道炎症性疾病,其发病涉及到多种不同的炎性细胞以及细胞组分,主要临床病理特征包括不同程度的气道慢性炎症,气道高反应性,粘液高分泌以及气道重塑。七氟醚(SEV)是临床常用的吸入麻醉药,已报道七氟醚可以缓解哮喘危重患者的临床症状,其确切机制尚待进一步探讨。水通道蛋白5(AQP5)主要表达于肺组织,是一类在维持肺组织水代谢平衡方面具有重要作用的通道蛋白。研究已表明,气道的炎症状态能够改变肺组织AQP5的表达水平。本研究拟以卵清蛋白(OVA)致敏和激发建立的急性哮喘小鼠为模型,研究七氟醚对哮喘小鼠气道炎症的影响及AQP5在其中的作用。方法1.急性哮喘小鼠模型的建立取24只SPF级C57BL/6小鼠,雌性,体重23~27g,6~8周龄。采用随机数字表法分成3组(n=8):对照组、哮喘组以及七氟醚组。哮喘组于造模第0天开始致敏:10μg的卵清蛋白和1 mg的硫酸铝钾溶于0.5 ml的生理盐水溶液中,注入小鼠腹腔。于造模第14天开始激发:采用超声雾化仪连续7d重复吸入50 ml 1%卵清蛋白生理盐水溶液50 ml,每次吸入0.5h。七氟醚组造模方法同上,每次激发前吸入浓度为3%的七氟醚,吸入时间为1 h。对照组于造模第0天腹腔内注射0.5 ml含有硫酸铝钾1 mg的生理盐水溶液,在第14天开始,连续7天重复雾化吸入0.9%生理盐水50 ml,每天1次,每次0.5h。2.气道炎症反应的检测于造模第21天即末次激发后24 h,行支气管肺泡灌洗,回收灌洗液,计数肺泡灌洗液(BALF)中炎性细胞总数,然后分类计数各种炎性细胞,采用ELISA法测定BALF中炎性介质TNF-α、IL-13和IL-10浓度。取适量肺组织切片,HE染色,PAS染色,观察肺组织炎性细胞的浸润情况、支气管上皮和肺泡结构损伤情况,粘液分泌情况。3.AQP5蛋白表达的检测采用免疫组织化学法,Western blot方法检测肺组织AQP5蛋白表达的水平结果1.急性哮喘小鼠模型的建立。与对照组相比,哮喘组小鼠行为学上出现大小便增多,烦躁不安,呼吸急促等改变。组织病理学及分子生物检测显示,哮喘组小鼠肺组织中肺泡壁结构被破坏,大量的炎性细胞浸润在血管和支气管周围,气管壁增厚明显,上皮细胞坏死,粘液分泌增加;哮喘组小鼠BALF中的炎性细胞总数及各炎性细胞的分类计数,白细胞介素13(IL-13),肿瘤坏死因子α(TNF-α)含量增加(P0.05),白细胞介素10(IL-10)含量增加(P0.05)。2.七氟醚抑制哮喘小鼠的气道炎症。与哮喘组小鼠相比,七氟醚组小鼠肺组织结构破坏明显减轻,炎性细胞的浸润数目及粘液分泌量明显减少,BALF中炎性细胞总数及各炎性细胞的分类计数明显减少,细胞因子TNF-α,IL-13表达水平降低,IL-10表达水平明显增加(P0.05)。3.七氟醚上调哮喘小鼠的肺组织AQP5蛋白表达量。与对照组相比,哮喘组小鼠AQP5蛋白表达量显著减少;与哮喘组相比,七氟醚组小鼠AQP5蛋白的表达量明显增加(P0.05)。结论七氟醚能够抑制哮喘小鼠的气道炎症,其对气道炎症的抑制作用可能是通过上调AQP5蛋白表达量实现。
[Abstract]:Background asthma is a complicated and complicated chronic airway inflammatory disease, which is involved in many different inflammatory cells and cellular components. The main clinical and pathological features include airway chronic inflammation, airway hyperresponsiveness, mucus hypersecretion, and airway remodeling. Sevoflurane (SEV) is a commonly used inhalation anesthetic, and it has been reported that sevoflurane can alleviate the clinical symptoms of critically ill patients with asthma. The exact mechanism of sevoflurane has yet to be further explored. Aquaporin 5 (AQP5) is mainly expressed in lung tissue, and is a kind of channel protein which plays an important role in maintaining the balance of water metabolism of lung tissue. The study has shown that the inflammatory state of the airway can change the expression level of AQP5 in the lung tissue. The effect of sevoflurane on airway inflammation in asthmatic mice and the role of AQP5 were studied in this study. Method 1. The model of acute asthma mice was established in 24 SPF C57BL/6 mice, female, body weight 23-27 g,6-8 weeks old. In the control group, the asthma group and the sevoflurane group were divided into three groups (n = 8). In the asthma group, the sensitization was started on the first day of the model:10. mu. g of ovalbumin and 1 mg of potassium sulfate were dissolved in 0.5 ml of physiological saline solution and injected into the abdominal cavity of the mice. Excitation was started on the 14th day of the model:50 ml of 1% ovalbumin physiological saline solution was taken in 50 ml for 7 days by means of an ultrasonic atomizer and 0.5 h for each time. The model of the sevoflurane group was the same as the above, and the inhalation time was 1 hour. The control group injected 0.5 ml of the saline solution containing 1 mg of potassium sulfate in the abdominal cavity of the day 0 of the model, and started on the 14th day. The inhalation of 0.9% normal saline (50 ml) was repeated for 7 consecutive days, and once a day, 0.5 h each time. The number of inflammatory cells in BALF was measured by bronchoalveolar lavage, recovery and irrigation, and the total number of inflammatory cells in the alveolar lavage fluid (BALF) was counted, and the inflammatory mediators in BALF were determined by ELISA. IL-13 and IL-10 concentrations. An appropriate amount of the lung tissue sections, HE staining, PAS staining, the infiltration of inflammatory cells in the lung tissue, the damage of the bronchial epithelium and the alveolar structure, and the mucus secretion were observed. The expression of AQP5 in lung tissue was detected by Western blot. Establishment of a mouse model for acute asthma. Compared with the control group, the behavior of the mouse behavior of the asthma group was increased, the restlessness, the shortness of breath, and so on. Histopathology and molecular biological tests showed that the structure of the alveolar wall in the lung tissue of the asthma group was destroyed, and a large amount of inflammatory cells were infiltrated around the blood vessel and the bronchi, the wall of the air tube was increased in thickness, the epithelial cells were necrotic and the secretion of mucus increased. The total number of inflammatory cells in BALF of asthmatic group and the number of inflammatory cells, interleukin-13 (IL-13), tumor necrosis factor (TNF-1) content increased (P0.05), and the content of interleukin-10 (IL-10) was increased (P0.05). Sevoflurane inhibits airway inflammation in asthma mice. Compared with the asthma group, the damage of the lung tissue in the sevoflurane group was significantly reduced, the number of infiltration of the inflammatory cells and the amount of mucus secretion were significantly reduced, the total number of inflammatory cells in the BALF and the number of the inflammatory cells were significantly reduced, and the level of the expression of the cytokines TNF-1 and IL-13 decreased. The level of IL-10 expression was significantly increased (P0.05). The expression of AQP5 in the lung of asthmatic mice was increased by sevoflurane. Compared with the control group, the expression of AQP5 in the asthmatic group was significantly decreased, and the expression of AQP5 in the sevoflurane group was significantly increased compared with the control group (P0.05). Conclusion Sevoflurane can inhibit the airway inflammation of asthmatic mice, and its inhibitory effect on airway inflammation may be achieved by up-regulating the expression of AQP5.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614

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