天贝汤对支气管哮喘大鼠气道炎症和气道重塑的影响的实验研究
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摘要
目的：支气管哮喘（bronchial asthma，简称哮喘）是以气道炎症、气道重塑、可逆性气道阻塞、气道高反应性为主要特征的由多种因素引起的一种气道慢性炎症性疾病，是由嗜酸性粒细胞、肥大细胞和T淋巴细胞等多种炎症细胞参与的一种呼吸系统疾病。近二十年来，随着工业化和全球污染的加剧，在世界范围内哮喘的发病率和死亡率呈逐年上升的趋势，全球患者约3亿左右，严重威胁着人们的身体健康和生存质量。加之哮喘病程绵长难愈，给社会带来了严重的经济负担。所以，哮喘已成为严重的公共卫生问题而引起了世界各国的极大关注。到目前为止，全身或局部应用糖皮质激素仍然是治疗哮喘的首选，但长期应用副作用明显。中医中药防治哮喘应该受到重视，特别是在减少副作用，预防复发等方面与西药比较有一定的优势。本文还概述了历代中医学家对哮喘的病名、病因病机、辨证施治的认识，对中药及中医药治疗哮喘的现代临床与机理研究作了全面总结。阐述了哮喘的现代流行病学、病因病机等。天贝汤是导师郭振武教授的临床经验方，具有宣肺降气、化痰平喘之功。本课题在建立大鼠支气管哮喘模型的基础上，予天贝汤进行干预，通过对本动物实验的观察，选择与哮喘发病机制相关的多项指标，探讨天贝汤对支气管哮喘大鼠气道炎症和气道重塑的作用和机制，为中医中药临床治疗支气管哮喘提供理论依据。
材料与方法：72只健康雄性SPF级Wistar大鼠，体重240±20g，实验动物许可证号SCXK(京)2009-0004，随机分为正常对照组、哮喘模型组、天贝汤高剂量组（以下简称为高剂量组）、天贝汤中剂量组（以下简称为中剂量组）、天贝汤低剂量组（以下简称为低剂量组）、小青龙对照组（以下简称为小青龙组），每组12只。
天贝汤组成：麻黄、茯苓、清半夏、川贝、天麻等组成，小青龙胶囊组成：麻黄、白芍、细辛、干姜、炙甘草、桂枝、半夏、五味子。将上方加水煎煮三次合并后过滤。低温(4℃)放置备用。天贝汤高、中、低剂量组，给药剂量分别是：高剂量组9ml/kg,中剂量组4.5ml/kg，低剂量组2.25ml/kg，小青龙组给药剂量0.972g/kg。从第15天开始给药，直至处死，每日1次灌胃，连续6周。各组参照吕国平等介绍的方法，分为致敏和激发两步。除正常对照组外，实验第1d和第8d共2次每只大鼠腹腔注射新鲜配制的含10%卵蛋白的生理盐水1ml（内含卵蛋白100mg，氢氧化铝100mg和灭活百日咳杆菌6×109个）致敏，致敏15d后各组灌胃后1小时后将大鼠放入不完全封闭的雾化吸入箱内开始激发，用超声雾化器喷入含1%卵蛋白激发，每次雾化20分钟，每日1次，激发6周，激发后以大鼠烦躁、打喷嚏、咳嗽、抓鼻、大小便失禁、呼吸幅度加深及喘促发作、紫绀等哮喘发作症状，认为哮喘造模成功。正常对照组以生理盐水代替卵蛋白进行腹腔注射及雾化吸入，雾化流量及时间同前。
第一部分天贝汤对支气管哮喘大鼠支气管病理组织学观察末次喷雾激发后18-24h内予乙醚麻醉动物，以固定板固定后打开并暴露胸腔，开胸结扎，剥离肺组织，取出肺组织，于4℃4%多聚甲醛固定，脱水、包埋、切片，HE染色观察。
第二部分各组大鼠体重和被激发时的表现的变化
第三部分天贝汤对支气管哮喘大鼠肺组织核因子-κB（NF-κB）的影响核因子-κB（NF-κB)是一种具有多种功能的核转录因子，具有广泛的生物学活性，不仅参与哮喘的气道炎症反应，还参与哮喘气道重塑的过程。动物分组、模型建立、用药情况及数据统计处理同前。动物麻醉完全后，取肺组织于4%多聚甲醛固定，脱水、包埋、切片，行免疫组织化学ABC法染色。
一抗选用兔抗p52(N F-кB的一个亚单位)多克隆抗体(武汉博士德生物工程公司)稀释度1∶100;用SABC法试剂盒进行操作,操作步骤按试剂盒说明书进行。主要操作步骤如下：石蜡切片常规脱蜡脱水，3%H2O2溶液灭活内源性过氧化物酶，热修复1h。滴加山羊封闭血清，湿盒孵育，加入一抗（1:100稀释）湿盒孵育1h，4℃冰箱内过夜，PBS漂洗，加入生物素化二抗37℃孵育，PBS漂洗5min，3次。⑧滴加ABC复合物37℃孵育30min，PBS漂洗5min，3次。⑦二氨基联苯胺(DAB)光镜下显色5-15min，终止呈色反应。苏木精衬染，常规裱片、封片和观察。计算机图象处理软件半定量测定NF－κB表达。
第四部分天贝汤对支气管哮喘大鼠血清中基质金属蛋白酶-9（MMP-9）、基质金属蛋白酶组织抑制剂-1（TIMP-1）的影响。
气道重塑是慢性哮喘反复发作的重要病理生理基础，它与哮喘肺功能损害及气道高反应性密切相关。细胞外基质(ECM)的沉积和降解的失衡是导致气道壁结构异常、间质增生和肺实质破坏的重要原因，ECM主要由基质金属蛋白酶（MMPs）来降解,其中基质金属蛋白酶-9（MMP-9）是锌依赖性蛋白酶，MMP-9催化集团区含有3个重复的类Ⅱ型纤维连接蛋白结合区，决定了它对明胶和弹力纤维的底物特异性，主要降解Ⅳ型以及Ⅴ型胶原。近来研究表明MMP-9在支气管哮喘患者的气道炎症和气道重塑中发挥重要作用。
动物分组、模型建立、给药情况及数据统计处理同第一部分。动脉血血清中MMP-9、TIMP-1含量检测：采用双抗体夹心法ABC-ELISA法测定MMP-9、TIMP-1含量，具体方法如下。
①建立标准曲线：设标准孔8孔，每孔中加入样品稀释液100μl，第一孔加入标准品100μl，混匀后用加样器吸出100μl，移至第二孔，如此反复行对倍稀释至第七孔，最后，从第七孔中吸出100μl弃去，使之体积为100μl，第八孔为空白对照。
②加样：待测样品孔中每孔加入待测样品100μl。
③将反应板充分混匀后放置37℃120分钟。
④洗板：用洗涤液将反应板充分洗涤4-6次，向滤纸上印干。
⑤每孔中加入第一抗体工作液50μl，将反应板充分混匀后置37℃60分钟。
⑥用洗涤液将反应板充分洗涤4-6次，向滤纸上印干。
⑦每孔加酶标抗体工作液100μl，将反应板置37℃60分钟。
⑧用洗涤液将反应板充分洗涤4-6次。每孔加入底物工作液100μl，置37℃暗处反应5-10分钟。每孔加入1滴终止液混匀，在492nm处测吸光值。
以标准品2000、500、250、125、62、31、0ng/ml之OD值在半对数纸上作图，画出标准曲线，根据样品OD值在该标准曲线上查出MMP-9的含量，TIMP-1方法同MMP-9。第五部分天贝汤对支气管哮喘大鼠肺组织表面相关凋亡基因蛋白Fas和Bcl-2表达的影响
动物分组、模型建立、用药情况及数据统计处理同前。动物完全麻醉后，取肺组织，加入Trizol充分匀浆，提取总RNA，采用RT-PCR法检测各组大鼠肺组织中相关凋亡基因蛋白Fas和Bcl-2表达的变化。
统计学方法：数据采用均数±标准差（—x±s)表示，用SPSS统计软件处理，统计方法采用单因素方差分析及LSD检验。显著差异水平以0.05和0.01为标准。
结果：
1.天贝汤对哮喘大鼠支气管-肺组织病理学的影响
肺组织肉眼形态观察:正常对照组:肺组织颜色淡红，肺组织柔软而富有弹性，表面光滑。哮喘模型组:肺组织过度膨胀，部分肺组织颜色变深，呈暗红色，质地稍硬，表面有呈颗粒状的瘀点。天贝汤各剂量组和小青龙组肺组织颜色稍暗，膨胀不明显，无瘀斑瘀点。光镜观察：正常对照组：大鼠支气管内及其周围无明显的炎性细胞浸润，支气管壁完整光滑，细胞排列规整，平滑肌层厚度正常，支气管粘膜规整，管腔内无脱落上皮细胞。模型组：支气管管壁及伴行的动脉周围有大量嗜酸性粒细胞、单核细胞及淋巴细胞为主的炎性细胞浸润，管腔内容物增多，微血管渗漏，可见管腔内炎性分泌物增多，有的小支气管甚至被粘液栓及炎性细胞所堵塞。杯状细胞增生，气道上皮指状增生，平滑肌层增厚，排列紊乱，支气管粘膜水肿增厚，上皮脱落，上皮下纤维化，支气管粘膜皱壁增多、延长。
高剂量组：嗜酸性粒细胞及淋巴细胞等炎性细胞浸润明显减少，几乎消失，水肿明显消失，管壁和平滑肌层厚度接近正常对照组,逐渐恢复，支气管粘膜规整，支气管管腔内偶见脱落上皮细胞及粘液。
中剂量组：嗜酸性粒细胞及淋巴细胞等炎性细胞浸润有所减少，管壁和平滑肌层厚度有所减少，气管管壁结构层次清楚，支气管管腔内偶见脱落上皮细胞及黏液，管腔渗出物逐渐吸收。
低剂量组：支气管周围嗜酸性粒细胞及其他炎性细胞浸润略微减少，管壁和平滑肌层厚度略减少，管腔内偶见脱落上皮细胞及黏液。
小青龙组：黏膜下层见轻度充血水肿及少量炎症细胞，管腔内见炎性渗出物，粘膜增生，支气管腔内少量脱落细胞和粘液。
2.各组大鼠体重和被激发时的表现的变化
体重：激发后各组大鼠体重均增。空白组大鼠体重与其余各组有差异，高于其余各组(p<0.01)；高、中、低剂量组，小青龙组及模型组之间无统计学意义(p>0.05)。被激发时的表现：空白组大鼠表现活泼好动，动作灵敏，毛色光滑，呼吸平稳。模型组大鼠在吸入OVA后不久即出现过敏症状如：躁动不安，搔抓、舔肢体、打喷嚏等，继而大鼠反应迟钝，出现呼吸急促，呼吸困难(明显的腹式呼吸)，伴轻度紫绀，部分大鼠可闻及响亮的呼气相喘鸣音和不规则呼吸。与哮喘模型组比较，高、中剂量组症状减轻，无呼吸急促，无紫绀，无呼气相喘鸣音；低剂量组和小青龙组呼吸稍急促，无紫绀，喘鸣音减轻。在高中低剂量组比较中，高剂量组较低剂量组比较有明显好转，没有呼吸急促和呼吸困难症状，大鼠毛色光泽较好，精神也较好。
3.对哮喘大鼠支气管NF－κB表达的影响
与正常对照组比较，模型组NF－κB表达明显增加，差异具有显著性意义（P<0.01）。与模型组比较，NF－κB在各治疗组阳性表达减少，高剂量组降低最明显（P<0.01），其次为中剂量组、小青龙组、小剂量组；与小青龙组对比，高剂量组NF－κB含量低，二者差异具有显著意义﹙p<0.01﹚。
4.对支气管哮喘大鼠动脉血清中基质金属蛋白酶-9（MMP-9）、组织基质金属蛋白酶抑制剂-1（TIMP-1）的影响。
与正常对照组相比，模型组大鼠MMP-9、TIMP-1含量明显升高，差异具有显著性意义﹙p<0.01﹚，证明哮喘模型建立成功；与模型组比较，各治疗组MMP-9含量均降低，高剂量组降低最明显﹙p<0.01﹚，其次为中剂量组、小青龙组，低剂量组次之。与小青龙组相比，高剂量组含量低，二者差异具有显著意义﹙p<0.01﹚。
5.对支气管哮喘大鼠相关基因蛋白Fas mRNA、Bcl-2mRNA表达的影响。
与正常对照组比较，模型组Bcl-2mRNA表达明显增多(P﹤0.01)，Fas mRNA表达明显减少﹙p<0.05﹚；与模型组比较，各治疗组Bcl-2mRNA表达减少，高剂量组减少最明显﹙p<0.01﹚，其次为中剂量组、小青龙组、小剂量组，与小青龙组比较高、中、低剂量组无统计学意义；与低剂量组比较，高剂量组Bcl-2mRNA表达强度低，二者差异具有显著意义﹙p<0.05﹚。与模型组比较，Fas mRNA在各治疗组表达有所增多,高剂量组增多最明显﹙p<0.05﹚，其次为中剂量组、小青龙组、小剂量组，但均无统计学意义；与小青龙组比较高、中、低剂量组均无统计学意义。与低剂量组比较，高、中剂量组、小青龙组均无统计学意义。
结论：
1.哮喘发病与痰、风等致病因素有关。风盛痰阻、气道挛急是哮喘发作的主要病机，治疗以宣肺降气、化痰平喘及调理脏腑并举。
2.天贝汤具有宣肺降气、化痰平喘及调理脏腑的作用，对哮喘有确切的治疗作用。
3天贝汤治疗哮喘的作用机制
3.1对气道重塑的影响。对支气管-肺组织病理学观察结果显示，模型组支气管粘膜上皮部分脱落，黏膜下层充血水肿，嗜酸性粒细胞等炎细胞浸润，黏膜下平滑肌显著增生肥厚。与正常对照组相比，模型组出现气道重塑。经天贝汤治疗后，气管管壁结构逐渐恢复，炎症明显减轻，水肿明显消失，增生的平滑肌细胞基本恢复正常。提示天贝汤具有抑制哮喘豚鼠气道重塑，从而发挥平喘作用。其机制可能是通过减少气道EOS浸润，减少炎症介质分泌、释放，减轻炎性细胞渗出，抑制杯状细胞和平滑肌细胞增生，从而减轻气道炎症反应，抑制气道重塑。
3.2天贝汤能降低支气管哮喘大鼠肺组织中NF－κB的含量，从而从细胞因子角度解释天贝汤对气道炎症的抑制作用。
3.3天贝汤抑制气道重塑和细胞外基质沉积的作用可能与其降低动脉血清中MMP-9、TIMP-1含量有关。
3.4天贝汤抑制气道炎症和气道重塑的作用可能与其降低肺组织中Bcl-2mRNA表达，增加Fas mRNA表达有关。
4.天贝汤对实验性哮喘大鼠的平喘作用，具有剂量依赖关系，并且部分优于经典方剂小青龙汤，其机制有待于进一步深入研究。天贝汤通过多途径、多层次、多靶点抑制气道炎症和气道重塑，具有广阔的应用前景。
Purpose： Bronchial asthma is a chronic allergic disease-caused by variousfactors（Eosinophil,the adipose cell,the T-lymphocyte cell and so on） andcharacterized by airway inflammation、airway remodeling、reversible airwayobstruction and airway hyperreactivity.In resent20years,with the increaseof global industrialization and pollution,in the world the morbidity andmortality of asthma is gradually increasing,there are nearly3hundredmillion patients on the world.It serious demised people’s health and survivalquality. Together with the long course and the difficulty of cure, bronchialasthma brings heavy economic burden to society.
So it attracts much attentions of countries around the world. UP to now, withpartial or total used dexamethasone is still the first choice to the treatmentof asthma，but its side-effects are obvious. Traditional Chinese Medicine hasgiven many useful experiences and theories to the treatment of asthma,so theattention of Traditional Chinese Medicine should be paid more. In recentyears,plenty of studies on clinical and expriments have shown that TraditionalChinese Medicine has significant effect on bronchial asthma,especially has lessside effect and longer theraputic effect than western medicine.This articleoverviewed the etiology, pathology,sydrome differentiation, treatments，discusses the classification and methodology of treatments on bronchial asthmaof Traditional Chinese Medicine doctors,summed up the developments ofTraditional Chinese Medicine in treating bronchial asthma,outlined therealization on eidemiology,physiology,pathology and some the latest developmentof basic research in western medicine. Analyzed the limitation and puzzle ofwestern medicine,prospected the studying direction in the future. Based on thecharacteristics of bronchial asthma, TianBei Decoction is my tutor,s clinicalpreseription,it could diffuse and downbear lung breath，dispel phlegm and clam pant.Based on the theory discussed above，in this research TianBei Decoctionwere given to the rats with duplicating the experimental bronchial asthma ratmodel to reveal the influence of Tianbei Decoction on airway inflammation andairway remodeling and to explore the mechanism of treating asthma with TianbeiDecoction by selecting some parameters closely relative to asthma.It may providetheoretical foundation for the clinic application for the treatment of bronchialasthma.
Material and method：Part1：72male Wistar rats (the weight of240±20gs)， the experiment animal licence NO: SCXK(Jing)2009-0004,the rats wererandomly divided into six groups:Normal control group,Model controlgroup,Tianbei Decoction high dose group(called high-dose group), TianbeiDecoction middle dose group(called middle-dose group), Tianbei Decoction lowdose group (called low-dose group)，xiaoqinglong control group, each group hadtwelve rats.
Medical preparations: Tianbei Decoction: Ephedra Herba Ephedrae,Poriacocos(schw) wolf,Pinellia ternata (Thunb.)Breit,Fritillaria cirrhosaD.Don,Gastrodia elata BI, Xiaoqinglong Decoetion:Ephedra Herba Ephedrae, Radixpaeoniae Alba, Asarum L., Rhizoma Zingiberis, Radix Glycyrrhizae PreParata,Glycyrrhizae PreParata, Ramulus Cinnarnomi, Rhizoma Pinelliae Preparata,Schisandra chinensis(Turez.)Baill. The preseriptions decocted three times andpermixied together to filter.The filtrate was concentrated in water一bath andthe Xiaoqinglong Decoction was0.105g/ml.Reserve at4℃.
To further investigate the mechanism of Tianbei Decoction, we haveestablished a bronchial asthma model,and experimental treatment was applied tothe animals with the Tianbei Decoction. From the beginning of15thday,The ratsin high-dose group，middle-dose group，low-dose group and xiaoqinglong groupwere given Tianbei Decoction and xiaoqinglong through watering stomacheveryday. Asthma model with reference to the introduction of equality LvWays,Divided into two steps sensitized and challenged.The bronchial asthmaticmodel was sensitized by1ml mixture(include10%OVA（egg albumin）100mg, aluminum hydroxide powder100mg，Bacillus Calmette-Guerin Vaccine(60hundred millionbacterial vaccines)) injected into abdominis on the first day and on the eighthday except normal group,12rats in normal group were injected by0.9%sodiumliquid into abdominis at the same time. From the beginning of the fifteenth day,Each group one hour after oral administration，rats were placed in theincompletely closed box using equipment of pulverization,asthma aroused by1%prepared OVA inbreathe20minutes every day from the first time of provocationto execution,12rats in normal group were treated with0.9%sodium liquid instead of1%OVA at the same time in the same way. Each group was given relevantdose medicines:the dose of high-dose group is9ml/kg, the dose of middle-dosegroup is4.5ml/kg, the dose of low-dose group is2.25ml/kg, The dose ofxiaoqinglong control group is0.972g/kg.After6weeks of treatment,all the ratswere anaesthesia and killed, and then we observed the changes of lunghistomorphology of rats of each group. Chest were opend and lung tissue wereobtained for Hematoxylin-Eosin(HE) staining.The tissue were fixed up by4%formaldehyde liquid,Dehydrated、being sliced and observed by HE，electric-microscope was used to observe the thickness change of bronchus wall and smoothmuscls.
Part2Comparison of the experimental rats changes in body weight and the symptomsof experimental rats
Part3The effection of Tianbei Decoction on NF－κB in bronchia-lung tissueof rats with experimental bronchial asthmaNuclear factor kappa B(NF-κB)is an important transcription factor necessaryfor initiating and sustaining inflammatory and immune reactions.It also playsan important role in asthmatic airway remodeling.
The dividing groups and the method of establishing model were same to thepart1, the method of giving medicine and statistics were same to the part1.The lung tissue were take out rapidly, were fixed up by4%formaldehydeliquid、dehydrateddehydrated by different concentration of ethanol, embed withparaffin wax,being sliced up. Expression of NF－κB p52was assessed in lung tissue by immunohistochemistry assays and micromage analysis system.3%H2O2liquid were used to inactivite endogenous peroxidase.Goat antibody were droponto the tissue slice,then1:100IgG antibody were drop onto every tissue slice,being hatched for12h in4℃washed by PBS5min，3times.The number2antibodymarked by biotin were droped onto every tissue slice,being hatched in37℃,washed by PBS5min，3times, ABC mixture were droped onto every tissueslice,being hatched30mins in37℃,washed by PBS3times,5min per time,keepthe colour5-15mins,washed by PBS,hematoxylin were used to colour every tissueslice,shade of colour were used to express content of NF－κB,special softwareand computer were used to observe the shade.Part4The effection of Tianbei Decoction on the levels of MMP-9and TIMP-1inserum of rats with experimental bronchial asthma
Airway remodeling is very important pathological and physiologicalfoundation in recurrent attacks of chronic asthma.It has correlation with injuryof lung function and airway hyperreactivity(AHR).The imbalance of degradationand deposition in extracellular matrix(ECM) is one of the important factorsthat causes structural abnormalities of airway wall, proliferative interstitiumand destruction of lung parenchyma.ECM mainly was degraded by Matrixmetalloproteinase(MMPs),and Matrix metalloproteinase-9(MMP-9)is zinc-bindingproteinase.It contain fibronectin type II–like repeats within their catalyticdomain,resulting in a higher binding affinity to gelatin and elastin,and itmainly degrades type IV and V collagen.Recently,many studies indicate that MMP-9has important function in airway inflammation and airway remodeling in asthmaticpatients.The methods of this study contain as follows:
The dividing groups and the method of establishing model were same to thepart1, the method of giving medicine and statistics were same to the part1.The protein expressions of MMP-9and TIMP-1in serum of rats were examined byenzyme-linked immunosorbent assay (ELISA) double antibody sandwich method.The expressions of MMP-9and TIMP-1was tested according to the kit:Standardcurve were established according to the request,8detection hole were estab- lished,100μl reagent were taked into the first hole,being mixed enough,then100μl mixture were picked-up and taked into the second hole, The second–7thholes were treated as the same way,at last the100μl mixture in7thhole werepicked-up and be through away.The8thhole was kept as a control sampl. Then100μl sampl were taked into detection holes,hatched in37℃for120minutes, washedby PBS for4-6times,clean the PBS by filter paper,50μl number1antibody liquidwere put into every hole, being mixed enough and hatched in37℃for60minutes,Then being washed by PBS for4-6times,Clean the PBS by filter paper.50μl number2antibody liquid were put into every hole,being mixed enough and hatched in37℃for60minutes. Cleaning the PBS by filter paper,100μl reaction liquid wereput into every hole and the reactor being put into a black box in37℃for5-10minutes,reaction stop liquid were taked into every hole and being mixed enough,spectrophotometer was used to detect the number in492nanometer.Part5The effection of Tianbei Decoction on the expression of apoptosis relatedprotein Bcl-2and Fas in bronchia-lung tissue of rats with experimental bronchialasthma.
The dividing groups and the method of establishing model were same to thepart1, the method of giving medicine and statistics were same to the part1.
All the rats were anaesthesia, put the lung tissue and Trizol into centri-fuge tube, the white cotton wool on the botton of centrifuge tube was the RNA.The mRNA expression of apoptosis related protein Bcl-2and Fas were measuredby reverse transcription-polymerase chain reaction(RT-PCR)．Statistics:All the count date were presented by means±standard deviation（——x±s), The single factor analysis of variance was adoptted when comparing amongmany groups;and LSD Test was adoptted when comparing with each other, and wasstatisticed using spss statistical software processing.
Results：
Part1The effection of Tianbei Decoction on the changes of bronchial andpulmonary histopathology of rats with experimental bronchial asthma
The appearance of lung:normal control group:it was carmoisine,soft, stretchable and smooth. Model control group:it was hyperdistension, darken insome of the tissue and appeared madderred， slightly stiff，and there were somepeteches on the surface with granulo. Tianbei Decoction and xiaoqinglong givinggroups were better than model control group.
Observing the pathology slice of lung and bronchial in asthma rats byHE staining, compared with the normal control group, the results showed that thebronchus wall thickening and basement membrane hyperplasia,smooth musclehyperplasia in model control group increased distinctly,changes of bronchialand pulmonary histopathology in each group were typical,it suggests thatbronchial asthmatic rats model were successful.Model control group showed thatairway smooth muscle spasm and infiltration of inflammatory cells,such aslymphocytes、EOS that was an important effector cell in asthma were notablyincreased,and showed epithelial cells with finger-like hyperplasia, increasedluminal contents and subepithelial fibrosis. Compared with model control group,the thickness of bronchus wall and bronchus smooth muscle in high-dose group、middle-dose group and low-dose group have improved distinctly. Comparedcomprehensively, high-dose group was better than other groups. High-dose group:structure of trachea tubal wall recovered gradually, inflammation lessened andhydrops vanished obviously. inflammation exudates in lumens decreased obvious.Bronchus tubal wall were normal on the whole，epithelium mueosae repairedgradually,hydrops of subnatant mucosa lessened obviously，lumens recovered andinflammatory exudates. Middle-dose group group:The layer of traehea weredistinctness,damaged epithelium mucosae cell recovered gradually，but therewere some inflammatory exudates. Bronchus tubal wall were integrated，hypertrophic smooth muscle endothelial cell recovered and exudatum in lumenswere absorbed gradually. Xiaoqinglong group:The chalice cell of tracheaeithelium mucosae accrementited，subnatant mucosa appeared1ightly engorgedand hydrops and a small quantity of inflammatory cell.There were someinflammmatory exudates and bit of caduceus endothelial cell in lumens，mucosalhyperplasia and slight pachynsis could been seen also. Part2weight: After Provocations the rats are by weight.There was differenceof weight between the normal control group and other groups before and afterprovocation,the weight of control group was higher than that of rats in othergroups(p<0.01).There was no statistical significance among the groups(high-dosegroup,middle-dose group and low-dose group, xiaoqinglong control group and modelcontrol group)(p>0.05).
symptoms of experimental rats: Normal rats were full of vigor and had promptresponse, smooth and clean fur and steady breath.Shortly after provocation ofOVA, rats in model control group came forth symptom of some allergic symptoms,such as restless,scratching,licking limbs,sneezing et.al;furthermore, theywere in tachypnea or dyspnea(visible abdominal breathing)with slight cyanosisand without movement.Some rats in model control group sounded wheeze duringexpiratory phase and breathed irregularly.Compared to model control group,symptom of rats in high-dose group and in middle-dose group decreased, eliminatedthe symptoms of the tachypnea or dyspnea and cyanosis,there was no wheeze,someof the rats in low-dose group and in xiaoqinglong control group were slightlyshortness of breath,no cyanosis,wheezing sounded slightly during expiratoryphase. Compared with low-dose group, symptoms of high-dose group improved moresignificantly there were no tachynea or dyspnea,the fur of the rats was moresmooth and clean with more active spirit.Part3The effection of Tianbei Decoction on NF－κB in bronchia-lung tissueof rats with experimental bronchial asthma
After intervention with Tianbei Decoction,comparing with normalcontrol group，immunohistochemistry assays suggested that NF－κB from modelcontrol group were significantly higher（P<0.01）; Compared with model controlgroup, NF－κB in each treatment groups were decresed（p<0.01）. The NF－κBin high-dose group、middle-dose group and low-dose group were decreaseddistinctly than those in model group. Compared with xiaoqinglong control group,the NF－κB in high-dose group were decresed（p<0.01）Part4The effection of Tianbei Decoction on the serum levels of MMP-9and TIMP-1 in serum of rats with experimental bronchial asthma
Compared with the normal control group, the content and the relative valueof MMP-9and TIMP-1in model control group increased distinctly（p<0.01）, itsuggests that model establishment were successfuly. MMP-9and TIMP-1in eachtreatment groups were decrease than those in model group.Compared with the modelcontrol group,MMP-9and TIMP-1in high-dose group、middle-dose group andxiaoqinglong control group、low-dose group were decreased distinctly. high-dosegroup surpassed xiaoqinglong control group (p<0.01). Detection of serum levelsof MMP-9and TIMP-1is of a certain significance for guiding the clinicaldiagnosis and treatment in patients with bronchial asthma.Part5The effection of Tianbei Decoction on the expression of apoptosis relatedprotein Bcl-2and Fas in bronchia-lung tissue of rats with experimental bronchialasthma
Compared with the normal control group, expression of Fas mRNA from modelcontrol group was significanfly lower(P<0.05)；Expression of Bcl-2mRNA wassignificanfly higher from model control group (P<0.01), indicate that modelestablishment was successful. Compared with the model control group, expressionof Fas mRNA in high-dose group、middle-dose group and xiaoqinglong control group、low-dose group were increased. Expression of Bcl-2mRNA in high-dose group、middle-dose group and xiaoqinglong control group、low-dose group were decreaseddistinctly.
Conclusion：
1.1t is feasible to duplicate asthma model of wistar rats by means of egg albuminto allergize and attack.
2The attack of asthma is closely related with some elements such as phlegmwind et al.Profuse wind,stagnation of Phlegm,and air conduction urgent are theprincipal causes of the occurrence of asthma. Paying equal attention to diffuseand downbear lung breath，dispell phlegm, clam pant and regulate entrail istreating principle
3. Tianbei Decoction has the function of diffusing and downbearing lung breath， dispelling phlegm, clamming pant and regulating entrail to relieve asthma，it is good to treat asthma.
4.The mechanism of action of Tianbei Decoction on treating asthma.
4.1The influence on airway remodeling:From the bronchial and pulmonaryhistopathology of rats,we could see, the partial fal1ing of epithelium mucosaeof bronchus, congestion and hydrops on subnatant mucosa，infiltrating of EOSgranulocyte，significant accrementition and thickening with smooth musclesubnatant tomucosa in model control group.Compared with normal control group，there appeared airway remodeling in asthma model control group. After beingtreated with Tianbei Decoction，inflammation was relieved,hydrops vanishedobviously,hyperplastic endothelial cell of smooth muscle reverted gradually.Ithints that the Tianbei Decoction could decrease infiltration of in flammatorycells and inhibit the thickening and smooth muscle hyperplasia, then restrainthe airway remodeling aroused by asthma.
4.2Tianbei Decoction could decrease the content of NF－κB, then inhibit airwayinflammation by asthma.
4.3Tianbei Decoction could decrease on the serum levels of MMP-9and TIMP-1,suggest that Tianbei Decoction may play a role in airway inflammation andremodeling of extracelluarm atrix in asthma. The mechanism of reducing airwayremodeling may has correlation with its adjustment of the imbalance of MMP-9and TIMP-1.
4.4Tianbei Decoction could decrease expression of Bcl-2mRNA and increaseexpression of Fas mRNA, which is one of the reasons that decrease the numberof Eos in asthma, suggest that Bcl-2and Fas might be involved in the pathogenesisof asthma. Tianbei Decoction could inhibit apoptosis.
5The funetion of spasmolysis to conquer asthma with Tianbei Decoction on asthmaexperimental wistar rats is dose-dependent，and it outstrips the traditionalprescription of xiaoqinglong Decoetion in some aspcets.It is worthy for us tostudy profoundly.
To sum up,so it is obviously that Tianbei Decoction could resistant airway inflammation and airway remodeling in many ways.The bronchial asthmatherapy,on one hand,can promote the expression of Fas and restrain the expressionof Bcl-2,which facilitate EOS apotosis.On the other hand,it can decrease thelevel of corrolative cytokine NF－κB, MMP-9and TIMP-1.In a word, TianbeiDecoction may inhibit airway inflammation and airway remodeling.Meanwhile ourresearch confirms from the point of experiment that the effect of TraditionalChinese Medicine on bronchial asthma is a integrative regulative role relatedto many targets.
材料与方法：72只健康雄性SPF级Wistar大鼠，体重240±20g，实验动物许可证号SCXK(京)2009-0004，随机分为正常对照组、哮喘模型组、天贝汤高剂量组（以下简称为高剂量组）、天贝汤中剂量组（以下简称为中剂量组）、天贝汤低剂量组（以下简称为低剂量组）、小青龙对照组（以下简称为小青龙组），每组12只。
天贝汤组成：麻黄、茯苓、清半夏、川贝、天麻等组成，小青龙胶囊组成：麻黄、白芍、细辛、干姜、炙甘草、桂枝、半夏、五味子。将上方加水煎煮三次合并后过滤。低温(4℃)放置备用。天贝汤高、中、低剂量组，给药剂量分别是：高剂量组9ml/kg,中剂量组4.5ml/kg，低剂量组2.25ml/kg，小青龙组给药剂量0.972g/kg。从第15天开始给药，直至处死，每日1次灌胃，连续6周。各组参照吕国平等介绍的方法，分为致敏和激发两步。除正常对照组外，实验第1d和第8d共2次每只大鼠腹腔注射新鲜配制的含10%卵蛋白的生理盐水1ml（内含卵蛋白100mg，氢氧化铝100mg和灭活百日咳杆菌6×109个）致敏，致敏15d后各组灌胃后1小时后将大鼠放入不完全封闭的雾化吸入箱内开始激发，用超声雾化器喷入含1%卵蛋白激发，每次雾化20分钟，每日1次，激发6周，激发后以大鼠烦躁、打喷嚏、咳嗽、抓鼻、大小便失禁、呼吸幅度加深及喘促发作、紫绀等哮喘发作症状，认为哮喘造模成功。正常对照组以生理盐水代替卵蛋白进行腹腔注射及雾化吸入，雾化流量及时间同前。
第一部分天贝汤对支气管哮喘大鼠支气管病理组织学观察末次喷雾激发后18-24h内予乙醚麻醉动物，以固定板固定后打开并暴露胸腔，开胸结扎，剥离肺组织，取出肺组织，于4℃4%多聚甲醛固定，脱水、包埋、切片，HE染色观察。
第二部分各组大鼠体重和被激发时的表现的变化
第三部分天贝汤对支气管哮喘大鼠肺组织核因子-κB（NF-κB）的影响核因子-κB（NF-κB)是一种具有多种功能的核转录因子，具有广泛的生物学活性，不仅参与哮喘的气道炎症反应，还参与哮喘气道重塑的过程。动物分组、模型建立、用药情况及数据统计处理同前。动物麻醉完全后，取肺组织于4%多聚甲醛固定，脱水、包埋、切片，行免疫组织化学ABC法染色。
一抗选用兔抗p52(N F-кB的一个亚单位)多克隆抗体(武汉博士德生物工程公司)稀释度1∶100;用SABC法试剂盒进行操作,操作步骤按试剂盒说明书进行。主要操作步骤如下：石蜡切片常规脱蜡脱水，3%H2O2溶液灭活内源性过氧化物酶，热修复1h。滴加山羊封闭血清，湿盒孵育，加入一抗（1:100稀释）湿盒孵育1h，4℃冰箱内过夜，PBS漂洗，加入生物素化二抗37℃孵育，PBS漂洗5min，3次。⑧滴加ABC复合物37℃孵育30min，PBS漂洗5min，3次。⑦二氨基联苯胺(DAB)光镜下显色5-15min，终止呈色反应。苏木精衬染，常规裱片、封片和观察。计算机图象处理软件半定量测定NF－κB表达。
第四部分天贝汤对支气管哮喘大鼠血清中基质金属蛋白酶-9（MMP-9）、基质金属蛋白酶组织抑制剂-1（TIMP-1）的影响。
气道重塑是慢性哮喘反复发作的重要病理生理基础，它与哮喘肺功能损害及气道高反应性密切相关。细胞外基质(ECM)的沉积和降解的失衡是导致气道壁结构异常、间质增生和肺实质破坏的重要原因，ECM主要由基质金属蛋白酶（MMPs）来降解,其中基质金属蛋白酶-9（MMP-9）是锌依赖性蛋白酶，MMP-9催化集团区含有3个重复的类Ⅱ型纤维连接蛋白结合区，决定了它对明胶和弹力纤维的底物特异性，主要降解Ⅳ型以及Ⅴ型胶原。近来研究表明MMP-9在支气管哮喘患者的气道炎症和气道重塑中发挥重要作用。
动物分组、模型建立、给药情况及数据统计处理同第一部分。动脉血血清中MMP-9、TIMP-1含量检测：采用双抗体夹心法ABC-ELISA法测定MMP-9、TIMP-1含量，具体方法如下。
①建立标准曲线：设标准孔8孔，每孔中加入样品稀释液100μl，第一孔加入标准品100μl，混匀后用加样器吸出100μl，移至第二孔，如此反复行对倍稀释至第七孔，最后，从第七孔中吸出100μl弃去，使之体积为100μl，第八孔为空白对照。
②加样：待测样品孔中每孔加入待测样品100μl。
③将反应板充分混匀后放置37℃120分钟。
④洗板：用洗涤液将反应板充分洗涤4-6次，向滤纸上印干。
⑤每孔中加入第一抗体工作液50μl，将反应板充分混匀后置37℃60分钟。
⑥用洗涤液将反应板充分洗涤4-6次，向滤纸上印干。
⑦每孔加酶标抗体工作液100μl，将反应板置37℃60分钟。
⑧用洗涤液将反应板充分洗涤4-6次。每孔加入底物工作液100μl，置37℃暗处反应5-10分钟。每孔加入1滴终止液混匀，在492nm处测吸光值。
以标准品2000、500、250、125、62、31、0ng/ml之OD值在半对数纸上作图，画出标准曲线，根据样品OD值在该标准曲线上查出MMP-9的含量，TIMP-1方法同MMP-9。第五部分天贝汤对支气管哮喘大鼠肺组织表面相关凋亡基因蛋白Fas和Bcl-2表达的影响
动物分组、模型建立、用药情况及数据统计处理同前。动物完全麻醉后，取肺组织，加入Trizol充分匀浆，提取总RNA，采用RT-PCR法检测各组大鼠肺组织中相关凋亡基因蛋白Fas和Bcl-2表达的变化。
统计学方法：数据采用均数±标准差（—x±s)表示，用SPSS统计软件处理，统计方法采用单因素方差分析及LSD检验。显著差异水平以0.05和0.01为标准。
结果：
1.天贝汤对哮喘大鼠支气管-肺组织病理学的影响
肺组织肉眼形态观察:正常对照组:肺组织颜色淡红，肺组织柔软而富有弹性，表面光滑。哮喘模型组:肺组织过度膨胀，部分肺组织颜色变深，呈暗红色，质地稍硬，表面有呈颗粒状的瘀点。天贝汤各剂量组和小青龙组肺组织颜色稍暗，膨胀不明显，无瘀斑瘀点。光镜观察：正常对照组：大鼠支气管内及其周围无明显的炎性细胞浸润，支气管壁完整光滑，细胞排列规整，平滑肌层厚度正常，支气管粘膜规整，管腔内无脱落上皮细胞。模型组：支气管管壁及伴行的动脉周围有大量嗜酸性粒细胞、单核细胞及淋巴细胞为主的炎性细胞浸润，管腔内容物增多，微血管渗漏，可见管腔内炎性分泌物增多，有的小支气管甚至被粘液栓及炎性细胞所堵塞。杯状细胞增生，气道上皮指状增生，平滑肌层增厚，排列紊乱，支气管粘膜水肿增厚，上皮脱落，上皮下纤维化，支气管粘膜皱壁增多、延长。
高剂量组：嗜酸性粒细胞及淋巴细胞等炎性细胞浸润明显减少，几乎消失，水肿明显消失，管壁和平滑肌层厚度接近正常对照组,逐渐恢复，支气管粘膜规整，支气管管腔内偶见脱落上皮细胞及粘液。
中剂量组：嗜酸性粒细胞及淋巴细胞等炎性细胞浸润有所减少，管壁和平滑肌层厚度有所减少，气管管壁结构层次清楚，支气管管腔内偶见脱落上皮细胞及黏液，管腔渗出物逐渐吸收。
低剂量组：支气管周围嗜酸性粒细胞及其他炎性细胞浸润略微减少，管壁和平滑肌层厚度略减少，管腔内偶见脱落上皮细胞及黏液。
小青龙组：黏膜下层见轻度充血水肿及少量炎症细胞，管腔内见炎性渗出物，粘膜增生，支气管腔内少量脱落细胞和粘液。
2.各组大鼠体重和被激发时的表现的变化
体重：激发后各组大鼠体重均增。空白组大鼠体重与其余各组有差异，高于其余各组(p<0.01)；高、中、低剂量组，小青龙组及模型组之间无统计学意义(p>0.05)。被激发时的表现：空白组大鼠表现活泼好动，动作灵敏，毛色光滑，呼吸平稳。模型组大鼠在吸入OVA后不久即出现过敏症状如：躁动不安，搔抓、舔肢体、打喷嚏等，继而大鼠反应迟钝，出现呼吸急促，呼吸困难(明显的腹式呼吸)，伴轻度紫绀，部分大鼠可闻及响亮的呼气相喘鸣音和不规则呼吸。与哮喘模型组比较，高、中剂量组症状减轻，无呼吸急促，无紫绀，无呼气相喘鸣音；低剂量组和小青龙组呼吸稍急促，无紫绀，喘鸣音减轻。在高中低剂量组比较中，高剂量组较低剂量组比较有明显好转，没有呼吸急促和呼吸困难症状，大鼠毛色光泽较好，精神也较好。
3.对哮喘大鼠支气管NF－κB表达的影响
与正常对照组比较，模型组NF－κB表达明显增加，差异具有显著性意义（P<0.01）。与模型组比较，NF－κB在各治疗组阳性表达减少，高剂量组降低最明显（P<0.01），其次为中剂量组、小青龙组、小剂量组；与小青龙组对比，高剂量组NF－κB含量低，二者差异具有显著意义﹙p<0.01﹚。
4.对支气管哮喘大鼠动脉血清中基质金属蛋白酶-9（MMP-9）、组织基质金属蛋白酶抑制剂-1（TIMP-1）的影响。
与正常对照组相比，模型组大鼠MMP-9、TIMP-1含量明显升高，差异具有显著性意义﹙p<0.01﹚，证明哮喘模型建立成功；与模型组比较，各治疗组MMP-9含量均降低，高剂量组降低最明显﹙p<0.01﹚，其次为中剂量组、小青龙组，低剂量组次之。与小青龙组相比，高剂量组含量低，二者差异具有显著意义﹙p<0.01﹚。
5.对支气管哮喘大鼠相关基因蛋白Fas mRNA、Bcl-2mRNA表达的影响。
与正常对照组比较，模型组Bcl-2mRNA表达明显增多(P﹤0.01)，Fas mRNA表达明显减少﹙p<0.05﹚；与模型组比较，各治疗组Bcl-2mRNA表达减少，高剂量组减少最明显﹙p<0.01﹚，其次为中剂量组、小青龙组、小剂量组，与小青龙组比较高、中、低剂量组无统计学意义；与低剂量组比较，高剂量组Bcl-2mRNA表达强度低，二者差异具有显著意义﹙p<0.05﹚。与模型组比较，Fas mRNA在各治疗组表达有所增多,高剂量组增多最明显﹙p<0.05﹚，其次为中剂量组、小青龙组、小剂量组，但均无统计学意义；与小青龙组比较高、中、低剂量组均无统计学意义。与低剂量组比较，高、中剂量组、小青龙组均无统计学意义。
结论：
1.哮喘发病与痰、风等致病因素有关。风盛痰阻、气道挛急是哮喘发作的主要病机，治疗以宣肺降气、化痰平喘及调理脏腑并举。
2.天贝汤具有宣肺降气、化痰平喘及调理脏腑的作用，对哮喘有确切的治疗作用。
3天贝汤治疗哮喘的作用机制
3.1对气道重塑的影响。对支气管-肺组织病理学观察结果显示，模型组支气管粘膜上皮部分脱落，黏膜下层充血水肿，嗜酸性粒细胞等炎细胞浸润，黏膜下平滑肌显著增生肥厚。与正常对照组相比，模型组出现气道重塑。经天贝汤治疗后，气管管壁结构逐渐恢复，炎症明显减轻，水肿明显消失，增生的平滑肌细胞基本恢复正常。提示天贝汤具有抑制哮喘豚鼠气道重塑，从而发挥平喘作用。其机制可能是通过减少气道EOS浸润，减少炎症介质分泌、释放，减轻炎性细胞渗出，抑制杯状细胞和平滑肌细胞增生，从而减轻气道炎症反应，抑制气道重塑。
3.2天贝汤能降低支气管哮喘大鼠肺组织中NF－κB的含量，从而从细胞因子角度解释天贝汤对气道炎症的抑制作用。
3.3天贝汤抑制气道重塑和细胞外基质沉积的作用可能与其降低动脉血清中MMP-9、TIMP-1含量有关。
3.4天贝汤抑制气道炎症和气道重塑的作用可能与其降低肺组织中Bcl-2mRNA表达，增加Fas mRNA表达有关。
4.天贝汤对实验性哮喘大鼠的平喘作用，具有剂量依赖关系，并且部分优于经典方剂小青龙汤，其机制有待于进一步深入研究。天贝汤通过多途径、多层次、多靶点抑制气道炎症和气道重塑，具有广阔的应用前景。
Purpose： Bronchial asthma is a chronic allergic disease-caused by variousfactors（Eosinophil,the adipose cell,the T-lymphocyte cell and so on） andcharacterized by airway inflammation、airway remodeling、reversible airwayobstruction and airway hyperreactivity.In resent20years,with the increaseof global industrialization and pollution,in the world the morbidity andmortality of asthma is gradually increasing,there are nearly3hundredmillion patients on the world.It serious demised people’s health and survivalquality. Together with the long course and the difficulty of cure, bronchialasthma brings heavy economic burden to society.
So it attracts much attentions of countries around the world. UP to now, withpartial or total used dexamethasone is still the first choice to the treatmentof asthma，but its side-effects are obvious. Traditional Chinese Medicine hasgiven many useful experiences and theories to the treatment of asthma,so theattention of Traditional Chinese Medicine should be paid more. In recentyears,plenty of studies on clinical and expriments have shown that TraditionalChinese Medicine has significant effect on bronchial asthma,especially has lessside effect and longer theraputic effect than western medicine.This articleoverviewed the etiology, pathology,sydrome differentiation, treatments，discusses the classification and methodology of treatments on bronchial asthmaof Traditional Chinese Medicine doctors,summed up the developments ofTraditional Chinese Medicine in treating bronchial asthma,outlined therealization on eidemiology,physiology,pathology and some the latest developmentof basic research in western medicine. Analyzed the limitation and puzzle ofwestern medicine,prospected the studying direction in the future. Based on thecharacteristics of bronchial asthma, TianBei Decoction is my tutor,s clinicalpreseription,it could diffuse and downbear lung breath，dispel phlegm and clam pant.Based on the theory discussed above，in this research TianBei Decoctionwere given to the rats with duplicating the experimental bronchial asthma ratmodel to reveal the influence of Tianbei Decoction on airway inflammation andairway remodeling and to explore the mechanism of treating asthma with TianbeiDecoction by selecting some parameters closely relative to asthma.It may providetheoretical foundation for the clinic application for the treatment of bronchialasthma.
Material and method：Part1：72male Wistar rats (the weight of240±20gs)， the experiment animal licence NO: SCXK(Jing)2009-0004,the rats wererandomly divided into six groups:Normal control group,Model controlgroup,Tianbei Decoction high dose group(called high-dose group), TianbeiDecoction middle dose group(called middle-dose group), Tianbei Decoction lowdose group (called low-dose group)，xiaoqinglong control group, each group hadtwelve rats.
Medical preparations: Tianbei Decoction: Ephedra Herba Ephedrae,Poriacocos(schw) wolf,Pinellia ternata (Thunb.)Breit,Fritillaria cirrhosaD.Don,Gastrodia elata BI, Xiaoqinglong Decoetion:Ephedra Herba Ephedrae, Radixpaeoniae Alba, Asarum L., Rhizoma Zingiberis, Radix Glycyrrhizae PreParata,Glycyrrhizae PreParata, Ramulus Cinnarnomi, Rhizoma Pinelliae Preparata,Schisandra chinensis(Turez.)Baill. The preseriptions decocted three times andpermixied together to filter.The filtrate was concentrated in water一bath andthe Xiaoqinglong Decoction was0.105g/ml.Reserve at4℃.
To further investigate the mechanism of Tianbei Decoction, we haveestablished a bronchial asthma model,and experimental treatment was applied tothe animals with the Tianbei Decoction. From the beginning of15thday,The ratsin high-dose group，middle-dose group，low-dose group and xiaoqinglong groupwere given Tianbei Decoction and xiaoqinglong through watering stomacheveryday. Asthma model with reference to the introduction of equality LvWays,Divided into two steps sensitized and challenged.The bronchial asthmaticmodel was sensitized by1ml mixture(include10%OVA（egg albumin）100mg, aluminum hydroxide powder100mg，Bacillus Calmette-Guerin Vaccine(60hundred millionbacterial vaccines)) injected into abdominis on the first day and on the eighthday except normal group,12rats in normal group were injected by0.9%sodiumliquid into abdominis at the same time. From the beginning of the fifteenth day,Each group one hour after oral administration，rats were placed in theincompletely closed box using equipment of pulverization,asthma aroused by1%prepared OVA inbreathe20minutes every day from the first time of provocationto execution,12rats in normal group were treated with0.9%sodium liquid instead of1%OVA at the same time in the same way. Each group was given relevantdose medicines:the dose of high-dose group is9ml/kg, the dose of middle-dosegroup is4.5ml/kg, the dose of low-dose group is2.25ml/kg, The dose ofxiaoqinglong control group is0.972g/kg.After6weeks of treatment,all the ratswere anaesthesia and killed, and then we observed the changes of lunghistomorphology of rats of each group. Chest were opend and lung tissue wereobtained for Hematoxylin-Eosin(HE) staining.The tissue were fixed up by4%formaldehyde liquid,Dehydrated、being sliced and observed by HE，electric-microscope was used to observe the thickness change of bronchus wall and smoothmuscls.
Part2Comparison of the experimental rats changes in body weight and the symptomsof experimental rats
Part3The effection of Tianbei Decoction on NF－κB in bronchia-lung tissueof rats with experimental bronchial asthmaNuclear factor kappa B(NF-κB)is an important transcription factor necessaryfor initiating and sustaining inflammatory and immune reactions.It also playsan important role in asthmatic airway remodeling.
The dividing groups and the method of establishing model were same to thepart1, the method of giving medicine and statistics were same to the part1.The lung tissue were take out rapidly, were fixed up by4%formaldehydeliquid、dehydrateddehydrated by different concentration of ethanol, embed withparaffin wax,being sliced up. Expression of NF－κB p52was assessed in lung tissue by immunohistochemistry assays and micromage analysis system.3%H2O2liquid were used to inactivite endogenous peroxidase.Goat antibody were droponto the tissue slice,then1:100IgG antibody were drop onto every tissue slice,being hatched for12h in4℃washed by PBS5min，3times.The number2antibodymarked by biotin were droped onto every tissue slice,being hatched in37℃,washed by PBS5min，3times, ABC mixture were droped onto every tissueslice,being hatched30mins in37℃,washed by PBS3times,5min per time,keepthe colour5-15mins,washed by PBS,hematoxylin were used to colour every tissueslice,shade of colour were used to express content of NF－κB,special softwareand computer were used to observe the shade.Part4The effection of Tianbei Decoction on the levels of MMP-9and TIMP-1inserum of rats with experimental bronchial asthma
Airway remodeling is very important pathological and physiologicalfoundation in recurrent attacks of chronic asthma.It has correlation with injuryof lung function and airway hyperreactivity(AHR).The imbalance of degradationand deposition in extracellular matrix(ECM) is one of the important factorsthat causes structural abnormalities of airway wall, proliferative interstitiumand destruction of lung parenchyma.ECM mainly was degraded by Matrixmetalloproteinase(MMPs),and Matrix metalloproteinase-9(MMP-9)is zinc-bindingproteinase.It contain fibronectin type II–like repeats within their catalyticdomain,resulting in a higher binding affinity to gelatin and elastin,and itmainly degrades type IV and V collagen.Recently,many studies indicate that MMP-9has important function in airway inflammation and airway remodeling in asthmaticpatients.The methods of this study contain as follows:
The dividing groups and the method of establishing model were same to thepart1, the method of giving medicine and statistics were same to the part1.The protein expressions of MMP-9and TIMP-1in serum of rats were examined byenzyme-linked immunosorbent assay (ELISA) double antibody sandwich method.The expressions of MMP-9and TIMP-1was tested according to the kit:Standardcurve were established according to the request,8detection hole were estab- lished,100μl reagent were taked into the first hole,being mixed enough,then100μl mixture were picked-up and taked into the second hole, The second–7thholes were treated as the same way,at last the100μl mixture in7thhole werepicked-up and be through away.The8thhole was kept as a control sampl. Then100μl sampl were taked into detection holes,hatched in37℃for120minutes, washedby PBS for4-6times,clean the PBS by filter paper,50μl number1antibody liquidwere put into every hole, being mixed enough and hatched in37℃for60minutes,Then being washed by PBS for4-6times,Clean the PBS by filter paper.50μl number2antibody liquid were put into every hole,being mixed enough and hatched in37℃for60minutes. Cleaning the PBS by filter paper,100μl reaction liquid wereput into every hole and the reactor being put into a black box in37℃for5-10minutes,reaction stop liquid were taked into every hole and being mixed enough,spectrophotometer was used to detect the number in492nanometer.Part5The effection of Tianbei Decoction on the expression of apoptosis relatedprotein Bcl-2and Fas in bronchia-lung tissue of rats with experimental bronchialasthma.
The dividing groups and the method of establishing model were same to thepart1, the method of giving medicine and statistics were same to the part1.
All the rats were anaesthesia, put the lung tissue and Trizol into centri-fuge tube, the white cotton wool on the botton of centrifuge tube was the RNA.The mRNA expression of apoptosis related protein Bcl-2and Fas were measuredby reverse transcription-polymerase chain reaction(RT-PCR)．Statistics:All the count date were presented by means±standard deviation（——x±s), The single factor analysis of variance was adoptted when comparing amongmany groups;and LSD Test was adoptted when comparing with each other, and wasstatisticed using spss statistical software processing.
Results：
Part1The effection of Tianbei Decoction on the changes of bronchial andpulmonary histopathology of rats with experimental bronchial asthma
The appearance of lung:normal control group:it was carmoisine,soft, stretchable and smooth. Model control group:it was hyperdistension, darken insome of the tissue and appeared madderred， slightly stiff，and there were somepeteches on the surface with granulo. Tianbei Decoction and xiaoqinglong givinggroups were better than model control group.
Observing the pathology slice of lung and bronchial in asthma rats byHE staining, compared with the normal control group, the results showed that thebronchus wall thickening and basement membrane hyperplasia,smooth musclehyperplasia in model control group increased distinctly,changes of bronchialand pulmonary histopathology in each group were typical,it suggests thatbronchial asthmatic rats model were successful.Model control group showed thatairway smooth muscle spasm and infiltration of inflammatory cells,such aslymphocytes、EOS that was an important effector cell in asthma were notablyincreased,and showed epithelial cells with finger-like hyperplasia, increasedluminal contents and subepithelial fibrosis. Compared with model control group,the thickness of bronchus wall and bronchus smooth muscle in high-dose group、middle-dose group and low-dose group have improved distinctly. Comparedcomprehensively, high-dose group was better than other groups. High-dose group:structure of trachea tubal wall recovered gradually, inflammation lessened andhydrops vanished obviously. inflammation exudates in lumens decreased obvious.Bronchus tubal wall were normal on the whole，epithelium mueosae repairedgradually,hydrops of subnatant mucosa lessened obviously，lumens recovered andinflammatory exudates. Middle-dose group group:The layer of traehea weredistinctness,damaged epithelium mucosae cell recovered gradually，but therewere some inflammatory exudates. Bronchus tubal wall were integrated，hypertrophic smooth muscle endothelial cell recovered and exudatum in lumenswere absorbed gradually. Xiaoqinglong group:The chalice cell of tracheaeithelium mucosae accrementited，subnatant mucosa appeared1ightly engorgedand hydrops and a small quantity of inflammatory cell.There were someinflammmatory exudates and bit of caduceus endothelial cell in lumens，mucosalhyperplasia and slight pachynsis could been seen also. Part2weight: After Provocations the rats are by weight.There was differenceof weight between the normal control group and other groups before and afterprovocation,the weight of control group was higher than that of rats in othergroups(p<0.01).There was no statistical significance among the groups(high-dosegroup,middle-dose group and low-dose group, xiaoqinglong control group and modelcontrol group)(p>0.05).
symptoms of experimental rats: Normal rats were full of vigor and had promptresponse, smooth and clean fur and steady breath.Shortly after provocation ofOVA, rats in model control group came forth symptom of some allergic symptoms,such as restless,scratching,licking limbs,sneezing et.al;furthermore, theywere in tachypnea or dyspnea(visible abdominal breathing)with slight cyanosisand without movement.Some rats in model control group sounded wheeze duringexpiratory phase and breathed irregularly.Compared to model control group,symptom of rats in high-dose group and in middle-dose group decreased, eliminatedthe symptoms of the tachypnea or dyspnea and cyanosis,there was no wheeze,someof the rats in low-dose group and in xiaoqinglong control group were slightlyshortness of breath,no cyanosis,wheezing sounded slightly during expiratoryphase. Compared with low-dose group, symptoms of high-dose group improved moresignificantly there were no tachynea or dyspnea,the fur of the rats was moresmooth and clean with more active spirit.Part3The effection of Tianbei Decoction on NF－κB in bronchia-lung tissueof rats with experimental bronchial asthma
After intervention with Tianbei Decoction,comparing with normalcontrol group，immunohistochemistry assays suggested that NF－κB from modelcontrol group were significantly higher（P<0.01）; Compared with model controlgroup, NF－κB in each treatment groups were decresed（p<0.01）. The NF－κBin high-dose group、middle-dose group and low-dose group were decreaseddistinctly than those in model group. Compared with xiaoqinglong control group,the NF－κB in high-dose group were decresed（p<0.01）Part4The effection of Tianbei Decoction on the serum levels of MMP-9and TIMP-1 in serum of rats with experimental bronchial asthma
Compared with the normal control group, the content and the relative valueof MMP-9and TIMP-1in model control group increased distinctly（p<0.01）, itsuggests that model establishment were successfuly. MMP-9and TIMP-1in eachtreatment groups were decrease than those in model group.Compared with the modelcontrol group,MMP-9and TIMP-1in high-dose group、middle-dose group andxiaoqinglong control group、low-dose group were decreased distinctly. high-dosegroup surpassed xiaoqinglong control group (p<0.01). Detection of serum levelsof MMP-9and TIMP-1is of a certain significance for guiding the clinicaldiagnosis and treatment in patients with bronchial asthma.Part5The effection of Tianbei Decoction on the expression of apoptosis relatedprotein Bcl-2and Fas in bronchia-lung tissue of rats with experimental bronchialasthma
Compared with the normal control group, expression of Fas mRNA from modelcontrol group was significanfly lower(P<0.05)；Expression of Bcl-2mRNA wassignificanfly higher from model control group (P<0.01), indicate that modelestablishment was successful. Compared with the model control group, expressionof Fas mRNA in high-dose group、middle-dose group and xiaoqinglong control group、low-dose group were increased. Expression of Bcl-2mRNA in high-dose group、middle-dose group and xiaoqinglong control group、low-dose group were decreaseddistinctly.
Conclusion：
1.1t is feasible to duplicate asthma model of wistar rats by means of egg albuminto allergize and attack.
2The attack of asthma is closely related with some elements such as phlegmwind et al.Profuse wind,stagnation of Phlegm,and air conduction urgent are theprincipal causes of the occurrence of asthma. Paying equal attention to diffuseand downbear lung breath，dispell phlegm, clam pant and regulate entrail istreating principle
3. Tianbei Decoction has the function of diffusing and downbearing lung breath， dispelling phlegm, clamming pant and regulating entrail to relieve asthma，it is good to treat asthma.
4.The mechanism of action of Tianbei Decoction on treating asthma.
4.1The influence on airway remodeling:From the bronchial and pulmonaryhistopathology of rats,we could see, the partial fal1ing of epithelium mucosaeof bronchus, congestion and hydrops on subnatant mucosa，infiltrating of EOSgranulocyte，significant accrementition and thickening with smooth musclesubnatant tomucosa in model control group.Compared with normal control group，there appeared airway remodeling in asthma model control group. After beingtreated with Tianbei Decoction，inflammation was relieved,hydrops vanishedobviously,hyperplastic endothelial cell of smooth muscle reverted gradually.Ithints that the Tianbei Decoction could decrease infiltration of in flammatorycells and inhibit the thickening and smooth muscle hyperplasia, then restrainthe airway remodeling aroused by asthma.
4.2Tianbei Decoction could decrease the content of NF－κB, then inhibit airwayinflammation by asthma.
4.3Tianbei Decoction could decrease on the serum levels of MMP-9and TIMP-1,suggest that Tianbei Decoction may play a role in airway inflammation andremodeling of extracelluarm atrix in asthma. The mechanism of reducing airwayremodeling may has correlation with its adjustment of the imbalance of MMP-9and TIMP-1.
4.4Tianbei Decoction could decrease expression of Bcl-2mRNA and increaseexpression of Fas mRNA, which is one of the reasons that decrease the numberof Eos in asthma, suggest that Bcl-2and Fas might be involved in the pathogenesisof asthma. Tianbei Decoction could inhibit apoptosis.
5The funetion of spasmolysis to conquer asthma with Tianbei Decoction on asthmaexperimental wistar rats is dose-dependent，and it outstrips the traditionalprescription of xiaoqinglong Decoetion in some aspcets.It is worthy for us tostudy profoundly.
To sum up,so it is obviously that Tianbei Decoction could resistant airway inflammation and airway remodeling in many ways.The bronchial asthmatherapy,on one hand,can promote the expression of Fas and restrain the expressionof Bcl-2,which facilitate EOS apotosis.On the other hand,it can decrease thelevel of corrolative cytokine NF－κB, MMP-9and TIMP-1.In a word, TianbeiDecoction may inhibit airway inflammation and airway remodeling.Meanwhile ourresearch confirms from the point of experiment that the effect of TraditionalChinese Medicine on bronchial asthma is a integrative regulative role relatedto many targets.
引文
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