固本止咳中药方对慢阻肺模型小鼠呼吸道黏膜免疫细胞及因子的影响

【作者】 王玥琦；

【导师】 张洪春；

【作者基本信息】 北京中医药大学 ， 中医内科学（专业学位）， 2020， 博士

【摘要】 背景:慢性阻塞性肺疾病是以不完全可逆的气流受限为特征的阻塞性肺病。急慢性炎症交替持续存在,造成气道重构和肺气肿,表现为劳力性呼吸困难等,为患者的日常生活和社会带来沉重负担。中医药治疗慢阻肺不仅可缓解症状,还能减少急性加重,延缓疾病进展,而其机制缺乏深入研究。黏膜免疫具有重要作用,但中医药对呼吸道黏膜免疫影响机制的研究数量少、处于起步阶段。固本止咳中药方是一个针对慢阻肺稳定期的病机特点,具有扶正祛邪之功效的经验方。前期临床研究表明其能够缓解患者的症状、提高肺功能、调节免疫功能。动物实验研究显示其能够减轻慢阻肺模型小鼠气道炎症,改善肺功能和病理损伤,机制有待进一步研究。目的:1.研究固本止咳中药方对呼吸道黏膜免疫中关键细胞γ δT细胞及其细胞因子KGF的影响,探究其对黏膜免疫功能的影响及扶正祛邪的机制。2.研究固本止咳中药方对循环中及黏膜局部炎症因子的影响,探讨其改善患者症状及疾病进展严重程度的机制。3.为黏膜免疫功能与“卫气”之间的联系、为中医药以扶正固本实现“治未病”以及为从调节黏膜免疫入手治疗慢阻肺提供实验依据。方法:按照固本止咳中药方制作干浸膏,配制成高剂量0.55g生药/ml、中剂量0.28g生药/ml、低剂量0.09g生药/ml。将125只SPF级ICR雌性小鼠随机分为5组:空白对照组、模型对照组、固本止咳中药方高、中、低剂量组,每组25只。采用持续香烟烟雾暴露12周(除第1、29、57天)加经鼻腔向呼吸道滴入LPS溶液(第1、29、57天,共3次)的方法建立慢阻肺小鼠模型,而后两对照组用蒸馏水、中药组用固本止咳中药方溶液灌胃,共4周。造模及药物干预过程中观察其一般生理活动,监测体重。灌胃结束后用小动物肺功能仪测量肺功能。肺组织切片HE染色后观察形态学改变及细胞浸润,评价小鼠造模效果;肺组织切片进行免疫组化染色,检测小鼠肺组织中α βTCR和γ δ TCR表达情况、KGF和KGFR分泌情况;参考小肠黏膜上皮细胞间淋巴细胞分离方法,总结出肺组织淋巴细胞分离方法,用流式细胞术分析其中的αβT细胞和γ δ T细胞群;肺组织匀浆后采用Western Blot方法检测其中的KGF和KGFR蛋白,应用ELISA方法检测KGF蛋白含量,并用RT-PCR法检测KGF的mRNA水平;收集小鼠的血清、支气管肺泡灌洗液和肠黏液,用ELISA方法检测其中的炎性因子IL-6和IL-13水平。结果:1.一般生命质量情况:空白对照组小鼠无死亡,皮毛光泽柔顺,呼吸和缓,频率适中,节律均匀,活动正常,体重逐渐增加;慢阻肺模型组小鼠死亡10只,毛发枯暗,部分有脱毛,熏烟时躁动蹿跳,后期多扎堆蜷缩,甚则全身颤抖,呼吸急促,胸腹部起伏明显,时有不规则点头运动,甚者张口呼吸,体重较空白组明显偏低(P<0.001)。高剂量、中剂量固本止咳中药方组分别死亡1只,低剂量固本止咳中药方组死亡4只,造模期间一般情况同慢阻肺模型组,给予中药灌胃后与模型组相比毛发较顺泽,躁动程度有所减轻,呼吸困难可见改善,频率减缓,高剂量、低剂量固本止咳中药方组体重明显高于模型组(P<0.001)。2.肺组织切片HE染色显示空白对照组小鼠气道和肺泡结构正常,纤毛整齐,肺泡形状、大小规整,气道黏膜上皮完整;模型对照组支气管黏膜皱襞形成、有片状脱落,使管腔变窄或闭塞;肺泡壁破坏,肺泡腔不规则扩大,部分融合成肺大泡,气道周围及肺间质可见炎症细胞浸润,符合慢阻肺病理学表现。经固本止咳中药方治疗的三组与模型对照组对比,可见形态学方面的改善,体现在支气管和肺泡结构较完整,管腔狭窄程度减轻,气道黏膜上皮相对完整,纤毛排列规则、脱落减少,肺泡大小比较均匀,肺大泡数量减少,气道管壁周围及肺间质内炎症细胞浸润程度减轻。3.肺功能测量:模型对照组呼吸系统粘性阻力、弹性阻力明显高于空白对照组(P<0.05);呼吸系统动态顺应性明显低于空白对照组(P<0.05)。高、中、低剂量固本止咳中药方组呼吸系统粘性阻力明显低于模型对照组(P<0.05);中剂量固本止咳中药方组弹性阻力明显低于模型对照组(P<0.05)、呼吸系统动态顺应性明显高于模型对照组(P<0.05)。肺组织阻尼模型对照组明显高于空白对照组(P<0.05);高剂量、低剂量中药组明显低于模型对照组(P<0.05)。准静态顺应性高剂量固本止咳中药方组明显高于模型对照组,准静态弹性量高剂量固本止咳中药方组明显低于模型对照组(P<0.05)。4.肺组织免疫组化染色实验中,空白对照组可见α β T细胞、γ δT细胞均少量存在于肺组织中;模型对照组几乎未见γ δT细胞存在;高、中、低剂量固本止咳中药方组,可见较多γ δ T细胞存在于气管附近,较模型组有明显升高,小鼠肺组织α β T/γ δT细胞比例在模型组较空白对照组升高,固本止咳中药方组较模型组呈降低趋势。5.肺组织淋巴细胞流式分析:小鼠γδT细胞比例在模型组较空白对照组有降低的趋势,低剂量固本止咳中药方组较模型组有明显升高(P<0.05)。6.肺组织切片免疫组织化学染色,空白对照组肺组织中有少量KGF表达;模型对照组少于空白对照组;高、中、低剂量固本止咳中药方组KGF表达明显高于模型组,且稍高于空白对照组。7.Western blot检测结果显示,KGF、FGFR2蛋白表达量模型对照组较空白对照组明显降低(P<0.05),中剂量固本止咳中药方组KGF表达较模型对照组明显升高(P<0.05),高剂量固本止咳中药方组、低剂量固本止咳中药方组FGFR2表达较模型对照组明显升高(P<0.05)。8.ELISA检测结果显示,高剂量固本止咳中药方组KGF蛋白含量较模型对照组明显升高(P<0.05)。9.Real Time PCR检测结果显示,KGF蛋白的mRNA含量模型对照组明显低于空白对照组,中、低剂量中药组明显高于模型对照组(P<0.05)。10.小鼠血清中的IL-6含量慢阻肺模型对照组高于空白对照组(P<0.05),高剂量中药组低于模型对照组(P<0.05);血清中的IL-13含量各组间未见明显差异。BALF中的IL-6含量慢阻肺模型对照组高于空白对照组(P<0.05);高剂量中药组与模型组对照组对比无统计学差异,中剂量中药组低于空白对照组、模型对照组以及高剂量中药组(P<0.05),低剂量中药组低于空白对照组、模型对照组以及高剂量中药组(P<0.05);BALF中的IL-13含量慢阻肺模型对照组高于空白对照组(P<0.05),高剂量中药组以及中剂量中药组低于模型组对照组(P<0.05)。肠黏液中的IL-6含量各组间未见明显差异;肠黏液中的IL-13含量慢阻肺模型对照组高于空白对照组(P<0.05),高、中、低剂量中药组低于模型组对照组(P<0.05)。结论:1.持续香烟烟雾暴露加经鼻腔向呼吸道内滴入LPS溶液的方法造成小鼠肺部形态学病理改变以及肺功能改变与慢阻肺的特征相吻合。2.固本止咳中药方由黄芪、淫羊藿、炒白术、百部、黄芩、赤芍、防风组成,其可以提高慢阻肺模型小鼠生命质量及体重。3.固本止咳中药方可以改善慢阻肺模型小鼠肺组织形态学病理变化。4.固本止咳中药方治疗可减轻慢阻肺模型小鼠呼吸道阻塞程度、提高肺组织顺应性。5.固本止咳中药方明显减低慢阻肺模型小鼠血清和BALF中的IL-6含量以及BALF和肠黏液中的IL-13含量,有效控制气道及全身炎症状态,改善气道及肺组织结构损伤。6.固本止咳中药方通过调节慢阻肺模型小鼠肺组织γ δT细胞比例,促进KGF及KGFR的分泌,加快肺组织损伤的修复,起到改善呼吸道黏膜完整性、增强黏膜免疫功能的作用。与固本止咳中药方通过扶正祛邪、补气助阳、健脾益肾、止咳化痰、活血化瘀,从而增强“卫气”的作用彼此呼应。据此推测,呼吸道黏膜免疫功能是“正气卫外”作用的物质基础之一,中医药通过调节黏膜免疫功能,实现“扶正祛邪”的作用,从而达到延缓疾病进展、减少急性发作的目的,为慢阻肺的治疗提供了新的思路。7.根据肠上皮细胞间淋巴细胞提取方法,在本研究中总结并完善的肺组织淋巴细胞提取分离的实验操作方法,也将为呼吸道黏膜免疫的研究提供实验基础。更多还原

【Abstract】 Background:Chronic obstructive pulmonary disease(COPD)is an obstructive pulmonary disease characterized by incompletely reversible airflow restriction.Acute and chronic inflammation persists in the respiratory tract and the whole body,which damages the airway structure.Repeated damage and repair process causes airway remodeling and emphysema and leads to airway obstruction and gas retention,which is manifested as exertional dyspnea,bringing heavy burden to the patients’daily life and society.Curative effect of Chinese traditional medicine in the treatment of COPD is considerable and advantaged,the mechanism of which is lack in-depth research.The mucosal immunity plays an important role in a variety of diseases,but the research on the influence of traditional Chinese medicine on the respiratory mucosal immunity is in its infancy.According to the characteristics of pathogenesis of COPD,Gubenzhike recipe has the effect of strengthening the vital qi and dispelling the invading pathogenic factors.Preliminary clinical studies have shown that Gubenzhike recipe can effectively relieve cough,wheeze,sputum and other symptoms,improve lung function and regulate immune function of patients.Animal experiments showed Gubenzhike recipe reduced airway inflammation,improved lung function and pathological injury in the airway,the mechanism of which needs further research.Objective:1.To explore the influence of Gubenzhike recipe on γδT cells and their cytokines KGF in respiratory mucosal immune system and the mechanism of this recipe strengthening vital qi to eliminate pathogens.2.To research the influence of Gubenzhike recipe on the inflammatory factors in circulation and locally in the mucous membrane,and discuss the mechanism of improving the patients’ symptoms and the severity of disease progression.3.To provide experimental basis for a link between mucosal immune function and "wei qi",and for traditional Chinese medicine realizing "treat before desease attack" and provide new ideas for the treatment of COPD.Methods:Astragalus,Epimedium,Rhizoma Atractylodes,Radix Stemonae,Radix Scutellariae,Radix Paeoniae Rubra and Radix Saposhnikoviae were mixed at a ratio of 4:3:2:3:3:2:1.Dry extract containing 3.6g raw herb per milliliter was made in the department of pharmaceutical preparation of China-Japan Friendship Hospital.Then we calculated the dose depending upon the per kg body mass dose conversion coefficient table between animals and adults.The high,medium and low-dose solution was administrated at 3.5g raw herb/kg,1.8g raw herb/kg,0.58g raw herb/kg bodyweight of mouse separately.A total of 125 healthy SPF grade ICR female mice were randomly divided into 5 groups:blank control group,model group,high-dose Gubenzhike recipe group,medium-dose Gubenzhike recipe group and low-dose Gubenzhike recipe group,with 25 mice in each group.Mice were exposed to successive periods of cigarette smoke(CS)for 7days/week for 12 weeks(except the days using LPS).The LPS(1.5mg/kg)in distilled water was implemented by instillation through the nasal cavity to respiratory tract on day 1,day29 and day57.Mice’s weight was measured every 4 weeks and their health status was monitored.After the model establishment,an equal volume(0.2ml/day/animal)of distilled water or solution of dry extract of Gubenzhike recipe were administered separately by oral gavage to blank control group,model group,and Gubenzhike recipe groups once a day for 4 weeks.Lung function was measured by a small-animal ventilator.After the pulmonary function test,BALF,serum and intestinal mucus were collected and stored for detection of inflammatory factors IL-6 and IL-13.Morphological changes and cell infiltration were observed in lung tissue sections after HE staining,and compared with the typical pathological changes of COPD to evaluate the quality of model.Immunohistochemical staining was performed on the lung sections to detect the expression of αβTCR and γδTCR,and the secretion of KGF and KGFR in the lung tissues of mice.Based on the method of separation of mucosal epithelial lymphocyte in mice’ small intestinal,we innovatively summarized the method of separation of lymphocytes in mice’s lung tissue.Cells were stained with FITC anti-γδTCR and PE anti-αβTCR,then detected by flow cytometer.After lung tissues of mice were homogenized,KGF and KGFR proteins were detected by Western Blot,the content of KGF protein was detected by ELISA,and the mRNA level of KGF was detected by RT-PCR.Results:1.Gubenzhike recipe increased the survival rate and body weight of mice with COPD.None of the blank control group died.They had smooth fur and gentle breath in moderate frequency and uniform rhythm.Mice of the blank control group behaved normally and gradually gained weight.10 of the COPD model group died.Fur of mice in the model group was dry and dark,some of which lost.Mice moved and jumped restlessly when exposed to cigarette smoke.In the later stage,they often huddled and curled up,somewhile their whole body trembled.They breathed rapidly even with their mouths open.Their chest and abdomen undulated obviously,and they sometimes nodded irregularly.Body weight of mice with COPD was significantly lower than that of the blank control group(P<0.001).High-dose and medium-dose Gubenzhike recipe groups had 1 death,respectively,while the low-dose group had 4.Compared with the model group,mice in three Gubenzhike recipe groups had softer and shinier fur,less agitation,slower respiratory rate,and significantly heavier weight(P<0.001).2.The pathological manifestations of COPD include degeneration,necrosis,and detachment of bronchial mucosa epithelium,with the cilia being shorter and adhered.The trachea cavity becomes narrow because of hypertrophy of goblet cells and mucous cells,retention of abundant mucous,airway wall congestion,and edema.The alveoli atrophy and collapse,the alveolar septa is destroyed and ruptured,and adjacent alveoli fuse to form large alveoli.A large number of chronic inflammatory cells infiltrate around the trachea and alveoli.There are bronchi remodeling,increased collagen content,and scar formation.Gubenzhike recipe significantly improved the morphology situation of COPD mice lungs.It increased the structural integrity of the bronchi,alveoli,pulmonary mesenchyme,and the orderliness of cilia.Gubenzhike recipe also decreased the stenosis degree of airway,the number of lung bullae,and the number of inflammatory cells infiltrated around airway and pulmonary interstitium,3.Lung function parameters:Compared with that of the blank control group,the Rrs and Ers of the model control group were significantly higher(P<0.05),and the Crs was significantly lower(P<0.05).Compared with that in the model control group,the Rrs of three Gubenzhike recipe groups was significantly lower(P<0.05),the Ers of the medium-dose Gubenzhike recipe group was significantly lower(P<0.05),and the Crs was significantly higher(P<0.05).Compared with G of the model group,that of the blank control group was significantly lower(P<0.05),and that of the high-dose and low-dose Gubenzhike recipe group was significantly lower(P<0.05).Compared with that of the model group,the Est was significantly higher and the Cst was significantly lower in the high-dose Gubenzhike recipe group(P<0.05).4.Immunohistochemical staining:In the blank control group,there were a fewαβT cells and γδT cells in the lung tissue.As for the model group,scattering distribution of αβT cells in the alveolar tissue and hardly any γδT cells were observed.There were significantly more γδT cells around bronchi and rarefied αβT cells in the three Gubenzhike recipe groups.Positive brown cells were counted in three random fields at 400x magnification in each section with immunohistochemical staining of αβTCR and γδTCR.The ratio of αβT cell/γδT cell reduced in lung tissue of COPD model mice and increased by Gubenzhike recipe.5.The lymphocytes in lung tissues were isolated through density gradient centrifugation method and then analysed by flow cytometry.The percentage of γδT lymphocytes in the low-dose Gubenzhike recipe group was significantly higher than that in the model group(P<0.05).Compared with the blank control group,the ratio of αβT/γδT lymphocytes in all other groups increased.That ratio decreased in high-dose and low-dose Gubenzhike recipe groups in contrast to that of the model group.6.Immunohistochemical staining of KGF and KGFR was made,respectively,on the paraffin lung sections.In the blank control group and model group,there were a few brown particles representing little expression of KGF.Large area of brown particles was visible in three Gubenzhike recipe groups,in which KGF expression was significantly higher.The amount of KGFR was more in the model group and three Gubenzhike recipe groups than the blank control group.7.Compared to that of the model group,KGF was significantly higher in the blank control group(P<0.05)and medium-dose Gubenzhike recipe group(P<0.05),and KGFR was significantly higher in the blank control group(P<0.05),high-dose Gubenzhike recipe group(P<0.05),and low-dose Gubenzhike recipe group(P<0.05)in the western blot detection.8.ELISA results showed that KGF of the high-dose Gubenzhike recipe group was significantly higher than that of the model group(P<0.05).9.As for the RT-PCR assays,mRNA level of KGF was significantly higher in the blank control group(P<0.05),medium-dose Gubenzhike recipe group(P<0.05),and low-dose Gubenzhike recipe group(P<0.05)than in the model group.10.The levels of IL-6 and IL-13 in serum,BALF,and intestinal mucus of the mice were determined by ELISA according to the manufacturer’s instructions.The IL-6 level in serum of the blank control group was significantly lower than that of the other four groups(P<0.05),while the content of IL-6 in the high-dose Gubenzhike recipe group was significantly lower than that in the model group(P<0.05).The IL-6 level in BALF of the model control group was significantly higher than that of the blank control group(P<0.05)and the medium-dose and low-dose Gubenzhike recipe groups(P<0.05).There was no significant statistical difference in IL-6 level of intestinal mucus and IL-13 level of serum between the groups.The IL-13 level in BALF of the model group was significantly higher than that of the blank control group(P<0.05)and the high-dose and medium-dose Gubenzhike recipe groups(P<0.05).The IL-13 level in intestinal mucus of the model group was significantly higher than that of the blank control group(P<0.05)and the high-dose,medium-dose,and the low-dose Gubenzhike recipe groups(P<0.05).Conclusion:This study suggested that after successive periods of cigarette smoke exposure and instillation LPS solution through the nasal cavity to respiratory tract,pathological changes of lung tissue and lung function of model mice were in conformity with the characteristic of COPD.Gubenzhike recipe could improve life quality and weight of COPD model mice,improve pathological changes of lung tissue morphology of COPD model mice,reduce the degree of airway obstruction and improve lung tissue compliance.Gubenzhike recipe could reduce IL-6 content in serum and BALF and IL-13 content in BALF and intestinal mucous of COPD model mice,effectively control the airway and systemic inflammatory.Gubenzhike recipe could adjust portion of gamma delta T cells in COPD model mice,promote the secretion of KGF and KGFR,accelerate pulmonary tissue reparation,improve respiratory tract epithelial integrity and enhance the respiratory mucosal immune function,which provided experimental evidence for its effects of reinforcing the"wei qi" by means of strengthening vital qi to eliminate pathogens.By regulating the mucosal immune function,traditional Chinese medicine can achieve the function of"nourishing the healthy and dispelling evil",thus delaying the disease progression and reducing the acute attack,providing a new idea for the treatment of COPD.The experimental method of extraction and isolation of lung lymphocytes will also provide the experimental basis for the study of respiratory mucosal immunity.更多还原