从肠道菌群角度探讨温通方对过敏性哮喘大鼠的治疗作用

发布时间：2018-01-10 20:21

本文关键词：从肠道菌群角度探讨温通方对过敏性哮喘大鼠的治疗作用　出处：《北京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:通过研究温通方干预过敏性哮喘大鼠模型后的一般状态、气道反应性、血EOS计数、肺组织病理形态学、肠道菌群结构等指标的变化情况,探索过敏性哮喘大鼠模型是否存在肠道菌群失衡、温通方能否通过影响肠道菌群干预过敏性哮喘。方法:将40只大鼠随机分为4组(空白对照组,模型对照组,地塞米松组,温通方组),适应性喂养一周,造模组以OVA/Al(OH)3(1mgOVA+30mgAl(OH)3凝胶)于1ml 0.9%生理盐水中五点注射,同时0.2ml百白破疫苗腹腔注射(第1天和第8天,共2次)致敏大鼠,后用1%OVA生理盐水雾化吸入激发(第15天开始用,每天一次,20min/次,共激发7次)复制过敏性哮喘大鼠模型,造模结束后予每日灌胃一次,共14天。温通方组灌胃量为1.34g/kg的水提液浸膏;地塞米松组灌胃量为0.81mg/kg的地塞米松片剂;其他组灌胃使用等量生理盐水。实验过程中观察大鼠一般情况,灌胃结束后测定大鼠气道反应性(呼气阻力),取外周血测定血EOS计数,取大鼠肺组织进行HE染色分析病理损伤程度,取盲肠内容物进行DNA提取、扩增、16SrDNA测序分析,观察肠道菌群结构的变化。结果:1.造模结束后除空白对照组大鼠外,其余各组大鼠均出现不同程度的喷嚏、焦躁易动、挠鼻、呼吸频促等典型哮喘症状。灌胃结束后,模型对照组哮喘症状最为显著,与模型对照组相比,地塞米松组、温通方组症状显著减轻。2.肺组织HE染色:空白对照组未见炎性细胞浸润,且肺泡、支气管粘膜上皮结构完整清晰,支气管无痉挛;模型对照组、地塞米松组及温通方组均可见肺组织炎性细胞浸润,支气管黏膜上皮细胞排列紊乱、成片脱落,肺泡间隔增厚,支气管痉挛等典型支气管哮喘病理表现,但病理损害轻重程度不同,病变程度从高到低依次为模型对照组、温通方组,地塞米松组、空白对照组。3.气道反应性测定:模型对照组基础呼气阻力、各Ach激发浓度下呼气阻力与空白对照组相比差异有统计学意义(P0.05),温通方组和地塞米松组在Ach激发浓度为50、100ug/kg时呼气阻力较模型对照组均有改善,差异具有统计学意义(P0.05);在各Ach激发浓度下,温通方组和地塞米松组呼气阻力测定结果差异无统计学意义(P0.05)。4.血EOS计数:与空白对照组相比,模型对照组、温通方组、地塞米松组与其差异具有显著统计学意义,(P0.01)与模型对照组相比,地塞米松组、温通方组与其差异具有显著统计学意义(P0.01)与地塞米松组相比,温通方组与其差异具有显著统计学意义(P0.01)。5.肠道菌群结构:α多样性:Chao1指数显示,与模型对照组相比,空白对照组、地塞米松组、温通方组与其差异有统计学意义(P0.05);Shannon指数和Simpson指数显示各组差异无统计学意义(P0.05)。β多样性:地塞米松组肠道菌群结构与其余各组差异最大,温通方组菌群结构最接近空白对照组大鼠。菌群结构差异分析:与空白对照组相比,模型对照组 Akkermansia、Oscillospira、Coprococcus、Phascolarctobacterium、Roseburia、Prevotella 丰度增加(Ratiokb/mx0.5),Dorea 丰度降低(Ratio kb/mx2);与模型对照组相比,地塞米松组Phascolarctobacterium、Prevotella、Dorea 丰度增加(Ratio dex/mx2),Akkermansia、Oscillospira 丰度降低(Ratio dex/mx0.5);与模型对照组相比,温通方组Coprococcus、Phascolarctobacterium 丰度增加(Ratio zy/mx2),Akkermansia、Oscillospira、Roseburia、Prevotella丰度降低(Ratio zy/mx0.5);与空白对照组相比,地塞米松组Akkermansia、Coprococcus、Phascolarctobacterium、Prevotella 丰度增加(Ratio dex/kb2),Lactobacillus丰度降低(Ratiodex/kb0.5);与空白对照组相比,温通方组 Coprococcus、Phascolarctobacterium 丰度增加(Ratiozy/kb2),其余菌属与空白对照组无显著差异(0.5Ratiozy/kb2);与地塞米松组相比,温通方组Coprococcus丰度增加(Ratio dex/zy2),Akkermansia、Phascolarctobacterium、Roseburia、Prevotella、Dorea 丰度降低(Ratio dex/zy0.5)。结论:1.温通方对过敏性哮喘大鼠的一般状况、气道反应性、血EOS计数、肺组织病理产生了有利作用,说明本方具有缓解过敏性哮喘大鼠症状、降低气道反应性、减轻气道炎症的效应。2.过敏性哮喘大鼠模型存在肠道菌群紊乱。治疗后地塞米松组大鼠肠道菌群差异较空白对照组较大,提示其对过敏性哮喘的治疗作用不能从调整肠道菌群结构解释;治疗后的温通方组大鼠模型在症状、指标缓解的同时,肠道菌群结构亦发生了一定改变,较地塞米松组更接近于空白对照组,表明中药汤剂温通方可能在一定程度上通过影响肠道菌群结构缓解了过敏性哮喘的症状和相关指标。
[Abstract]:Objective: general state intervention after the rat model of allergic asthma through the study of the temperature Tongfang, airway reactivity, blood EOS count, lung tissue pathological changes of the intestinal flora structure, etc, to explore the rat model of allergic asthma is the presence of intestinal flora imbalance, temperature Tongfang can pass through the intestinal flora intervention allergic asthma. Methods: 40 rats were randomly divided into 4 groups (blank control group, model control group, dexamethasone group, temperature Tongfang group), fed for one week, the model with OVA/Al (OH) 3 (1mgOVA+30mgAl (OH) 3 gel) in five 1ml 0.9% normal saline injection at the same time, 0.2ml intraperitoneal injection of DPT vaccine (first days and eighth days, a total of 2 times) in sensitized rats, with 1%OVA saline aerosol inhalation (start with, once every fifteenth days, 20min/ times a day, a total of 7 excited) copy the rat model of allergic asthma, after the modeling for daily irrigation A stomach, a total of 14 days. The temperature Tongfang group gavage volume extract 1.34g/kg water; dexamethasone group gavage for dexamethasone tablets 0.81mg/kg; the other group by gavage using saline. Observe the general condition of the rats in the experimental process, the determination of airway reactivity in rats after gavage (expiratory resistance), taking peripheral blood serum EOS count was performed with rat lung tissue HE staining analysis of pathological injury degree, cecal contents were collected for DNA extraction, 16SrDNA amplification, sequencing analysis, observe the changes of intestinal microflora. Results: 1. after the modeling except blank control group rats and other groups the rats showed varying degrees of anxiety, sneezing, nose, breathing frequency and promoting typical asthma symptoms. After gavage, model control group, asthma symptoms were the most significant, compared with model control group, dexamethasone group, Tongfang group significantly reduce the temperature and symptoms of.2. in lung tissue HE staining Color: blank control group, no inflammatory cell infiltration, alveolar and bronchial mucosa, epithelial structure clear, no bronchial spasm; model group, dexamethasone group and temperature Tongfang were found in lung tissue inflammatory cell infiltration, bronchial epithelial cells arranged in disorder, a loss, alveolar septal thickening, bronchial bronchial spasm the pathology of asthma, but the pathological damage degree, lesion degree from high to low was model group, temperature Tongfang group, dexamethasone group,.3. control group determination of airway reactivity: blank model control group based Ach expiratory resistance, stimulate concentration of expiratory resistance compared with the control group had significant difference (P0.05), temperature Tongfang group and dexamethasone group at a concentration of 50100ug/kg of expiratory resistance than the model control group were improved at Ach excitation, the difference was statistically significant (P0.05); in the Ach Stimulate concentration, temperature Tongfang group and dexamethasone group expiratory resistance determination results were not statistically significant (P0.05).4. blood EOS count: compared with the control group, model control group, temperature Tongfang group, dexamethasone group and there was a statistically significant difference (P0.01) compared with model control group, dexamethasone group, was statistically significant the temperature difference and its significance Tongfang group (P0.01) compared with the dexamethasone group, there was a statistically significant difference in temperature Tongfang group and (P0.01).5. intestinal flora: alpha diversity: Chao1 index shows that, compared with the model control group, blank control group, dexamethasone group, was statistically significant difference with the temperature Tongfang group (P0.05); Shannon index and Simpson index showed no significant difference between groups (P0.05). Beta diversity: dexamethasone group, intestinal microflora and the rest of the maximum temperature difference, Tongfang group flora structure Close to the blank control group rats. Analysis of microbial community structure difference: compared with the control group, model control group, Akkermansia, Oscillospira, Coprococcus, Phascolarctobacterium, Roseburia, Prevotella, Dorea (Ratiokb/mx0.5) increased abundance abundance decreased (Ratio kb/mx2); compared with the model group, dexamethasone group, Phascolarctobacterium, Prevotella, increased Dorea abundance (Ratio dex/mx2, Akkermansia, Oscillospira) (Ratio dex/mx0.5); abundance decreased compared with the model group, Coprococcus group Tongfang temperature, increased Phascolarctobacterium abundance (Ratio zy/mx2), Akkermansia, Oscillospira, Roseburia, Prevotella (Ratio zy/mx0.5); abundance decreased compared with the control group, dexamethasone group, Akkermansia, Coprococcus, Phascolarctobacterium, Prevotella abundance increased (Ratio dex/kb2), Lactobacillus (Ratiodex/kb0.5); and reduce the abundance of blank According to the temperature Tongfang group, group Coprococcus, Phascolarctobacterium (Ratiozy/kb2), the increase of the abundance of bacteria had no significant difference with the control group (0.5Ratiozy/kb2); compared with the dexamethasone group, temperature Tongfang group increased Coprococcus abundance (Ratio dex/zy2), Akkermansia, Phascolarctobacterium, Roseburia, Prevotella, Dorea (Ratio dex/zy0.5) abundance decreased. Conclusion: the general condition of 1. temperature Tongfang on rats with allergic asthma, airway reactivity, blood EOS count, lung pathology had a beneficial effect, the party can relieve rat allergic asthma symptoms, airway reactivity, intestinal flora disturbance effect.2. rat model of allergic asthma, reduce airway inflammation. After treatment the difference of intestinal flora of rats in the dexamethasone group group than the control group, suggesting that the effect on the treatment of allergic asthma is not from the adjustment of the intestinal flora structure interpretation The temperature Tongfang after treatment; the rats in the model group in symptom remission index at the same time, the structure of intestinal flora has also undergone some changes, compared with the dexamethasone group close to the blank control group, showed that the decoction temperature Tongfang may to a certain extent affected by the structure of intestinal flora to ease the symptoms of allergic asthma and related indicators.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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