| 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 ratsPart3The 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 asthmaAirway 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 asthmaThe 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 asthmaAfter 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 asthmaCompared 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 bronchialasthmaCompared 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 principle3. 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. |
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