心理应激哮喘大鼠模型的建立及调肝理肺法作用机制研究

发布时间：2018-06-26 02:09

本文选题：心理应激哮喘大鼠模型 + 肝主疏泄　；参考：《北京中医药大学》2017年博士论文

【摘要】：研究目的哮喘是典型的心身疾病,心理应激在疾病过程中起着重要作用。既往研究显示,情志可引起神经-内分泌-免疫失衡,HPA轴是其主要调节途径之一。心理应激条件下,哮喘气道炎症明显增强,对激素反应性下降;其作用机制可能与糖皮质激素受体表达及功能下调,激素敏感性下降有关。本研究模拟临床情志致病特点,建立心理应激哮喘大鼠模型,通过相关指标检测,揭示心理应激哮喘大鼠HPA轴功能变化情况及其与气道炎症反应的相关性;并通过哮喘宁颗粒对心理应激哮喘大鼠进行干预,观察模型大鼠宏观表征、行为学以及血清、灌洗液、肺组织相关指标变化,探索调肝理肺法对心理应激哮喘大鼠HPA轴及气道炎症的调控作用,阐明调肝理肺法的作用机理和作用环节。研究方法本研究在前期工作基础上,将雄性SD大鼠随机分为正常组、应激组、哮喘组、应激哮喘组、西药组和中药组。并模拟临床情志致病特点,采用卵蛋白混合致敏联合束缚制动的方法,建立心理应激哮喘大鼠模型,从宏观表征、行为学、肺通气功能等客观指标进行模型评价;并通过检测血清、灌洗液内CRH、ACTH、CORT水平,肺组织GR表达及气道炎症因子含量,深入研究模型大鼠HPA轴功能的变化及其与气道炎症反应的相关性。并用哮喘宁颗粒对心理应激哮喘大鼠进行干预,与其余各组相比较,观察血清及BALF、肺组织中相关指标浓度、阳性细胞表达及蛋白表达的变化情况,研究调肝理肺法对心理应激哮喘大鼠HPA轴功能及免疫炎症失衡的调节作用。研究结果1)采用OVA混合致敏联合束缚制动成功建立心理应激哮喘大鼠模型。结果显示应激哮喘组大鼠表现为兴趣减退、毛发晦暗无光泽、便溏、饮食量减少、体重增加缓慢;激发过程中有抓咬口鼻、体腹部皮肤,呛咳、喷嚏及喉中哮鸣音等表现。与正常组相比较,应激哮喘组水平运动、垂直运动得分明显下降(P0.05,P0.01);蔗糖水偏好程度、悬吊实验挣扎次数下降(P0.01);悬吊实验中不动时间明显增加(P0.01);肺通气功能 PEF、FEF25-75%、FEV0.3/FVC 下降(P0.05,P0.01,P0.01),气道阻力Ri、Re明显升高(P0.05,P0.01);静脉血嗜酸粒细胞升高(P0.01);肺组织炎症细胞浸润明显。与应激组或哮喘组相比,应激性哮喘组的症状、体征和相关指标均明显恶化。2)与正常组比较,应激哮喘组大鼠血清、BALF内CRH、ACTH、CORT及IgE、IL-4、IL-17含量明显增高(P0.01),IFN-γ含量明显下降(P0.01)。肺组织中GR阳性表达及含量明显下降(P0.01);而IL-4、IL-17阳性细胞表达及含量增高明显(P0.01);IFN-γ阳性细胞表达明显减低(P0.01),而肺组织IFN-γ含量轻度减低,差异无统计学意义(P0.05)。与应激组或哮喘组相比,应激性哮喘组的HPA轴及气道炎症相关指标均有不同程度加重。相关性研究显示,CORT与IL-4、IL-17、IgE呈正相关,与IFN-y呈负相关;而肺组织中GR水平与之相反,即与IFN-y含量呈正相关,而与IL-4、IL-17等呈负相关。而CORT含量与心理应激哮喘大鼠行为学指标相关性研究发现,CORT含量与糖水消耗量、糖水比例等呈负相关,而肺组织GR含量与糖水消耗量、糖水比例、翻动次数及水平垂直运动呈正相关;但与静止时间呈负相关,相关性较弱。3)哮喘宁颗粒治疗后,中药组大鼠前期活动减少,反应迟缓,毛发晦暗,后期情绪改善,较为活跃,饮食量增加,对周围事物兴趣增高;激发过程中呼吸频率增快,腹肌收缩加深、加快,时有搔抓头面、体腹部毛发。与模型组相比较,中药组大鼠水平运动、垂直运动得分明显增加(P0.01);蔗糖水偏好程度、悬吊实验挣扎次数明显增高(P0.01,P0.01);悬吊实验不动时间明显降低(P0.01);肺通气功能中药组大鼠PEF、FEF25-75%、FEV0.3/FVC轻度增高;而Ri、Re减低,差异有显著统计意义(P0.01);静脉血嗜酸粒细胞明显降低(P0.01);肺组织中炎症细胞浸润明显减少。中药组大鼠血清、BALF内CRH、ACTH、CORT含量较应激哮喘组明显下降(P0.01),而肺组织GR阳性表达明显增高(P0.01),其含量轻度升高,差异无统计学差异(P0.05)。而血清、BALF及支气管肺组织IL-4、IL-17含量及阳性细胞表达明显下降(P0.01);肺组织IFN-γ阳性细胞表达明显降低(P0.01),而含量略有降低,差异无统计学意义(P0.05)。相关性研究结果显示CORT含量与IFN-γ呈负相关,而与IgE、IL-4、IL-17等炎症指标呈正相关;而肺组织GR含量与IFN-γ水平呈正相关,而与IL-4、IL-17等炎症指标呈负相关。而CORT含量与大鼠行为学相关性研究发现,CORT含量与糖水消耗量、糖水比例、翻动次数及垂直运动次数呈负相关;而与静止时间呈正相关,但相关性均较弱,无统计学差异(P0.05)。研究结论1)采用OVA混合致敏联合束缚制动成功建立心理应激哮喘大鼠模型,符合支气管哮喘肝失疏泄、肝肺失和证候学特征。2)心理应激通过诱导哮喘HPA轴功能失调,GR表达下调,降低CORT抗炎作用;诱导Th2、Th17免疫偏移,抑制Th1炎症反应,引起Th1/Th2免疫失衡、气道免疫炎症增强,从而促进哮喘发生、发展。3)调肝理肺法可显著改善哮喘心理应激哮喘大鼠焦虑、抑郁程度;同时降低心理应激哮喘大鼠血清、支气管肺泡灌洗液HPA轴相关指标,增加肺组织GR表达,调节Th细胞平衡,降低气道炎症;提示调肝理肺法可能通过调控HPA轴功能,减轻气道炎症反应,从而实现对心理应激哮喘的治疗作用。
[Abstract]:Research objective asthma is a typical psychosomatic disease. Psychological stress plays an important role in the process of disease. Previous studies have shown that emotion can cause neuroendocrine immune imbalance, and the HPA axis is one of its main regulating pathways. The expression of corticosteroid receptor and the downregulation of function and the decrease of hormone sensitivity. This study simulated the pathogenic characteristics of clinical emotion, established the model of psychological stress asthmatic rats, and detected the changes of the HPA axis function and the correlation with the airway inflammation reaction in the psychological stress asthmatic rats. The rats of stress asthma were intervened to observe the changes of macro characterization, behavior and serum, serum, lavage fluid and lung tissue related indexes in the model rats, and explore the regulating effect of regulating liver and lung method on the HPA axis and airway inflammation in the rats with psychological stress asthma. On the base of this, male SD rats were randomly divided into normal group, stress group, asthma group, stress asthma group, western medicine group and traditional Chinese medicine group. The characteristics of clinical emotion were simulated and the method of combination of ovalbumin sensitization combined with restraint braking was used to establish the model of psychological stress asthma rats. The model was modeled by the objective indexes of macroscopic characterization, behavior and lung ventilation. By detecting serum, CRH, ACTH, CORT levels, GR expression in lung tissue and the content of airway inflammatory factors in the lung tissue, the changes in the function of HPA axis and the correlation with the airway inflammation were studied in the model rats, and the effects of asthma Ning Granule on the psychological stress asthma rats were intervened, and the serum and BALF were observed in comparison with the other groups. The changes in the correlation index concentration, expression of positive cells and protein expression in the lung tissue, study the regulating effect of liver regulating method on the HPA axis function and the imbalance of immune inflammation in the rats with psychological stress asthma. Results 1) the model of psychological stress asthma was established by OVA mixed sensitization combined with binding brake. The results showed that the stress asthma was stressed. Group rats were characterized by diminished interest, dark hair, loose stool, loose stool, reduced diet, and slow weight gain. In the process of stimulation, there were scratching and snout, abdominal skin, choking, sneezing and laryngeal wheezing. Compared with the normal group, the level of stress in the stress asthmatic group was significantly decreased (P0.05, P0.01); the preference of sucrose water, The time of the suspension experimental struggle decreased (P0.01); the duration of the suspension experiment was significantly increased (P0.01); pulmonary ventilation function PEF, FEF25-75%, FEV0.3/FVC (P0.05, P0.01, P0.01), airway resistance Ri, Re significantly increased (P0.05, P0.01); venous blood eosinophils increased; lung tissue inflammatory cells infiltrated obviously. Compared with the stress group or asthma group, The symptoms, signs and related indexes of the stress asthma group were significantly worse in.2). Compared with the normal group, the serum levels of CRH, ACTH, CORT and IgE in the stress asthmatic group were significantly higher (P0.01), and the IFN- gamma content was significantly decreased (P0.01). The expression and content of GR positive in lung tissue decreased. The expression of IFN- gamma positive cells was significantly decreased (P0.01), and the expression of IFN- gamma in the lung tissue decreased slightly, and the difference was not statistically significant (P0.05). Compared with the stress group or asthma group, the HPA axis and airway inflammation related indexes in the stress asthma group were different. The correlation study showed that CORT and IL-4, IL-17, IgE were positive. The correlation was negatively correlated with IFN-y, while the level of GR in lung tissue was opposite to that of IFN-y, but negatively correlated with IL-4 and IL-17. The correlation between CORT content and behavioral indexes of psychological stress asthma rats showed that the content of CORT was negatively correlated with sugar water consumption and sugar water, while the content of GR and the consumption of sugar and water in lung tissue were related. There was a positive correlation between the proportion of sugar and water, the number of turning times and horizontal vertical movement; but it had a negative correlation with the rest time, and the correlation was weak.3). After the treatment of asthma Ning Granule, the early activity of the rats in the Chinese medicine group decreased, the reaction was slow, the hair was dark, the later mood improved, the diet increased, the interest in the surroundings increased, and the frequency of respiration increased during the stimulation process. Quickly, the contraction of the abdominal muscles deepened and accelerated. Compared with the model group, the horizontal movement of the rats in the traditional Chinese medicine group increased significantly (P0.01); the preference degree of the sucrose water, the struggle times of the suspension experiment increased obviously (P0.01, P0.01); the time of the suspension experiment was significantly reduced (P0.01); the lung ventilation function group of Chinese medicine group rats PEF, FEF25-75% and FEV0.3/FVC were slightly higher, while Ri, Re decreased, the difference was statistically significant (P0.01), the eosinophil of venous blood was significantly decreased (P0.01), and the infiltration of inflammatory cells in the lung tissue decreased significantly. The serum of the rats in the Chinese medicine group, CRH, ACTH, CORT in BALF were significantly lower than those in the stress asthma group (P0.01), and the positive expression of lung tissue increased significantly ( P0.01), with a slight increase in content, there was no statistical difference (P0.05), but serum, BALF and bronchopulmonary IL-4, IL-17 content and positive cell expression were significantly decreased (P0.01), and the expression of IFN- gamma positive cells in lung tissue decreased significantly (P0.01), but the content was slightly lower, the difference was not statistically significant (P0.05). The correlation study showed the CORT content. There was a negative correlation with IFN- gamma, but positive correlation with IgE, IL-4, IL-17 and other inflammatory markers, while the GR content in lung tissue was positively correlated with the level of IFN- gamma, but negatively correlated with the inflammatory markers such as IL-4 and IL-17, while the correlation between CORT content and rat behavior found that the CORT content was negatively correlated with sugar water consumption, sugar water ratio, the number of flipping times and the number of vertical movements. There is a positive correlation with the rest time, but the correlation is weak, but the correlation is weak, no statistical difference (P0.05). Conclusion 1) the use of OVA hybrid sensitization combined with binding brake to establish the model of psychological stress asthma rats, in line with bronchial asthma liver loss, liver and lung syndrome.2) psychological stress through the induction of asthma HPA axis dysfunction, GR table Down regulation, reducing the anti-inflammatory effect of CORT, inducing Th2, Th17 immunization, inhibiting Th1 inflammatory reaction, causing Th1/Th2 immune imbalance, enhancing airway immunity and inflammation, promoting the occurrence of asthma and developing.3), regulating liver and lung therapy can significantly improve the anxiety and depression degree of asthmatic asthmatic rats; meanwhile, reduce the serum and bronchi of psychological stress asthmatic rats. The HPA axis related index of alveolar lavage fluid increases the expression of GR in lung tissue, regulates the balance of Th cells and reduces airway inflammation, suggesting that the liver regulating method may reduce airway inflammation by regulating the function of HPA axis, thus achieving the therapeutic effect on psychological stress asthma.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R256.12;R-332

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