Flavors To Long Tang Experimental Study, The Role Of Mechanisms Of Asthmatic Guinea Pigs

1 ObjectiveBronchial Asthma (abbreviation asthma) is a kind of common respiratory system disease. It is a chronic airway allergic reaction inflammation which cause by the Eosinophil (EOS), the adipose cell, the T-lymphocyte cell and so on. And asthma bring out Airway hyperreactivity (AHR) to patient, thus cause patient's airway remodeling. In resent 20 years, the morbidity and mortality of asthma is increasing in the world. It serious demised people's health and survival quality. Therefore, it has an important significance to reach about pathogenesis of asthma and the preventing and controlling method to it.This project uses histamine challenge and egg albumin (OVA) to allergize and aggress to duplicate the model of asthma in guinea pigs. we approach this subject from the effect of the Wuweidilong Decoction playing on trachea smooth muscle of guinea pigs model in vivo and in vitro, the quantity of EOS in BALF, histomorphology of lung, and LTB₄, IFN-γ, IL-4 in serum. We want to approach the mechanism of action on spasmolysis from allergy, airway inflammation and airway remodeling, so as to exploit available drug provide evidence.2 Material and Method2.1 Material2.1.1 animal: guinea pigs, about 200-300g, both male and female, provided by experimental animal center of Tongji medical college of Huazhong university of science and technology. The experiment animal licence NO: syxk(Hubei) 2004-0028.2.1.2. Medical preparations: Wuweidilong decoction: Ephedra Herba Ephedrae 5g, Lumbricus 5g, Herba Houttuyniae 10g, Radix Bupleuri. 10g, Semen Lepidii and Semen Descurainia 10g, Rhizoma Belamcanda 10g, Schisandra chinensis (Turcz.) Baill. 10g, Asarum L. 5g, honey comb 5g, Radix Glycyrrhiza 10g. Xiaoqinglong decoction: Herba Ephedrae 6g, Radix Paeoniae Alba6g, Asarum L. 6g, Rhizoma Zingiberis 6g, Radix Glycyrrhizae Preparata 6g, Ramulus Cinnarnomi 6g, Rhizoma Pinelliae Preparata 6g, Schisandra chinensis (Turcz.) Baill. 6g.The two prescriptions decocted three times and permixied together to filter. The filtrate was concentrated in water-bath that the crude drug content of Wuweidilong Decoction was 2.88g/ml, and the Xiaoqinglong Decoction was 0.86 g/ml. Reserve at 4℃. Using the water to dilute it when its needed. Wuweidilong decoction large dose group is 2.88g/ml, and the medium dose group is 1.44g/ml, the small dose group is 0.72g/ml.2.1.3 Chief reagentAcetylcholine chloride, Military and Medical Science Institute, science and technology depart. Batch Number. 20010708:Histamine Phosphate, shanghai Lizhu DongFeng Biotechnology Ltd. Batch Number. 030811;Aminophylline, Yangzhou Zhongbao Pharmaceutic Ltd. Batch Number. 0403082;Isoprenaline Hydrochloride, Shanghai Hefeng Pharmaceutic Ltd. Batch Number. 20000702;OVA, made by Sigma Company (USA), encasedby Wuhan Life Technology Ltd. Batch Number 200410008;Aluminum hydroxide, Tianjin GuangFu Exquisite Chemical Industry Research Intitute, Batch Number. 20041229;Dexamethasone sodlum phosphate injection, Hubei Tianyao Medical Ltd. Batch Number. 20041112;Guinea pig LTB₄ reagent box, Rapid Bio Lab. California (USA). Batch Number. 02040602, Encased by Shanghai Biology reagent Ltd.;Guinea pig IFN-γreagent box, Rapid Bio Lab. California (USA). Batch Number. 03260502, Encased by Shanghai Biology reagent Ltd.;Guinea pig IL-4 reagent box, Rapid Bio Lab. California (USA). Batch Number. 02040602, Encased by Shanghai Biology reagent Ltd.2.2 Methods2.2.1 Animals models duplication2.2.1.1 Histamine challenge to cause asthma of guinea pig: utend experiment one.2.2.1.2 OVA allergizing: intraperitoneal injection: 10% OVA (contained 10% aluminium hydroxide) normal sodium 1.0ml. 1%OVA normal sodium misted spray to breathe in at the 15d, 30s a time. And misted spray a time every other day in 8d continuous. Utend experiment two-six.2.2.2 animals groupings and examination target 2.2.2.1 Experiment one: To observe the influence of Wuweidilong Decoction after guinea pigs breathed in histamine and induced to asthma. 40 guinea Digs were divided into 5 groups randomly: antigen control group, Wuweidilong Decoction large dose group, medium dose group, small dose group, Aminophylline group. 8 animals in each group. To every team, the ip dose of medicine is 3ml, namely Wuweidilong Decoction 22.8g/kg, 14.4g/kg, 7.2g/kg; Aminophylline group, Aminophylline 125g/kg, use the same ancount normal sodium to the original team ip. Detect the delitescence and falling down amount in 6 minutes.2.2.2.2 Experiment two: To observe the influence of Wuweidilong Decoction on allergized guinea pigs about allergic contracting in vitro trachea smooth muscle. 40 valid guinea pigs were divided the same to experiment one. Animal models were made with OVA and were killed 23d later. Trachea section were taken out and traced with vitro trachea installation. The curve of vitro trachea contracting after antigen attacking were noted.2.2.2.3 Experiment three: To observe the influence of Wuweidilong Decoction on asthma guinea pigs about the EOS in BALF and histomorphology of bronchus. 48 valid guinea pigs were divided into 6 groups randomly: normal control group, asthma model group, Dexamethasone (masculine drug) group, Xiaoqinglong Decoction group, Wuweidilong Decoction large dose group and small dose group. 8 animals in each group. Asthma model were duplicated the same to experiment two. From the day spraying to induce asthma, these groups of asthma were treated with corresponding drugs 3ml, once daily and 8d successively. The asthma model group is only causted allergy but not given the medicine: the Dexamethasone group is given Dexamethasone 1mg/kg; the Xiaoqinglong Decoction group is given Xiaoqinglong Decoction of 8.6g/kg; the large dose of Wuweidilong Decoction group is given Wuweidilong Decoction of 28.8g/kg; the small dose of Wuweidilong Decoction group is given Wuweidilong Decoction of 7.2g/mg. The normal control group were treated with normal sodium of the tales doses. Using the sodium pentobarbital of 3% of 30mg/kg to anaesthesia each animal on the 23th day, demeshed carotid artery and got arterial blood, standing 2 hours and then centrifuged 10 min at 3000 r./min. Took out serum and stored in deep freeze refrigerator. Observed general state of the guinea pig during lab process. After anaesthesia and took the blood serum, the killed animals were alveolar washed(BAL) and BALF were reclaimed, dyeing and EOS counting. The histomorphology of lung and bronchus were observed through naked eye and light microscope.2.2.2.4 The influences of LTB₄,IFN-γ,IL-4 in serum of asthma guinea pigs were observed in experiment four, five and six respectively. Animals grouping, animals model and handling were same to experiment three. LTB₄,IFN-γ,IL-4 were detected through kit directions of reagent box.2.3 Statistical methods: The data were indicated with (?)±s, and uses the SPSS10.0 statistics software to process. Apply variance analysis for comparing the mean value among groups. P<0.05 meaned significance difference.3 Results3.1 Experiment one: the action of Wuweidilong Decoction on guinea pigs breathing in the histamine to asthma. There were no obviously difference of latent phase among-the guinea pigs of all groups (P>0.05). and the incubation period after taken are: antigen control group 76±6.3s, Wuweidilong Decoction large dose group 319±28.3s, Wuweidilong Decoction medium dose group 263±271s, Wuweidilong Decoction small dose group 188±19.5s, Aminophlline group 326±30.8s. There are 8, 0, 1, 2, 0 anmals fall down separately in 6 min. Compare to the control group, the incubation period of the large, medium dose group and the Aminophlline group prolongs obviously (p<0.01), and the incubation, period of the small dose group also has a prolongation(p<0.05).3.2 Experiment two: the influence of Wuweidilong Decoction on allergized guinea pigs about allergic contracting in vitro trachea smooth muscle. Each group is attack by 1mg/ml albumin protein after given the medicine. Compared the difference in height of contracted curve in Wuweidilong Decoction large dose group, medium dose group and Aminophlline group with normal control group, P<0.01. Compared the Wuweidilong Decoction small dose group with control group, P<0.05.3.3 Experiment three: the influence of Wuweidilong Decoction on asthma guinea pigs about the EOS in BALF and histomorphology of bronchus. 3.3.1 Generally, guinea pigs of asthma model group appears to be less action, slow reaction, loss of pelage lustre, increse of breath times, difficulty of breath. No obvious change in each pharmaceutical group.3.3.2 The counting of EOS in BALF: EOS in BALF in asthma model group increased obviously. Compared Wuweidilong Decoction large dose group, small dose group, Dexamethasone group, Xiaoqinglong Decoction with asthma model group, it was different obviously in Eos counting in BALF (p<0.01). Compared Wuweidilong Decoction large dose group and Dexamethasone group with normal control group in EOS, it was not statistically significant (p>0.05)3.3.3 The appearance of lung: normal control group: it was carmoisine, soft, stretchable and smooth, asthma model group: it was hyperdistension, darken in some of the tissue and appeared madder red, slightly stiff, and there were some peteches on the surface with granulo. Drugs giving groups were better than asthma model group.3.3.4 Observing with light microscope: asthma model group: bronchus epithelium mucosae amoticed focally, mucosa subnatantly congested and dropsied. Eosinophile granulocyte infiltrated focusly, and we could see bit of lymphocyte cell, histomonocyte, and plasma cell. There were some mucus and exudatum in intracavitary. Dexamethasone group: The tubal wall of windpipe was nomal, damaged epithelium mucosae recovered gradually, subnatant mucosa inflammatory edema mitigated and lumens were clean on the whole. Structure of Bronchus lumens were integrity, damaged epithelium mucosae recovered gradually, lumens were integrity and no inflammatory exudates. Xiaoqinglong Decoction group: The chalice cell of trachea epithelium mucosae accrementited, subnatant mucosa appeared lightly engorged and hydrops and a small quantity of inflammatory cell. There were some inflammatory exudates and bit of caduceus endothelial cell in lumens, mucosal hyperplasia and light pachynsis could been seen also. Wuweidilong Decoction large dose group: structure of trachea tubal wall recovered gradually, inflammation lessened and hydrops vanished obviously. Inflammatory exudates in lumens decreased obviously. Bronchus tubal wall were normal on the whole, epithelium mucosae repaired gradually, hydrops of subnatant mucosa lessened obviously, lumens recovered and Inflammatory exudates. Wuweidilong Decoction small dose group: The layer of trachea were distinctness, damaged epithelium mucosae cell recovered gradually, but there were some inflammatory exudates. Bronchus tubal wall were integrated, hypertrophic smooth muscle endothelial cell recovered and exudatum in lumens were absorbed gradually.3.4 Experiment four: the influence of Wuweidilong Decoction on asthma guinea pig serum LTB₄. Compared all groups with asthma model group on guinea pig serum LTB₄,there were all highly significant statistical significance (p<0.01). But compared all groups with normal control group, there were all no statistical significance (p＞0.05).3.5 Experiment five: the influence of Wuweidilong Decoction on asthma guinea pig serum IFN-γ. Compared Wuweidilong Decoction large dose group, small dose group, Xiaoqinglong Decoction group, Dexamethasone group with asthma model group on IFN-γcontent, there were all highly significant statistical significance (p＜0.01). Compared Wuweidilong Decoction small dose group, Xiaoqinglong Decoction group with Wuweidilong Decoction large dose group, there were statistical significance(p＜0.05); Compared with Dexamethasone group there were statistical significance (p＜0.01); Compared Wuweidilong Decoction large dose group with Dexamethasone group on IFN-γ, there were highly significant statistical significance also (p＜0.01).3.6 Experiment six: the influence of Wuweidilong Decoction on guinea pig serum IL-4. Compared Wuweidilong Decoction large dose group, small dose group, Dexamethasone group with asthma model group on serum IL-4 content, there were all highly significant statistical significance (p＜0.01). Compared Xiaoqinglong Decoction group with asthma model group on serum IL-4 content, there were statistical significance (p＜0.05). Compared all the drug giving groups with normal control group, there were no statistical significance (p＞0.05). Compared normal control group with asthma model group, there were highly significant statistical significance (p＜0.01).4 Conclusion 4.1 It is feasible to duplicate asthma model of guinea pig that induce air tube allergies by means of egg albumin to allergize and attack.4.2 The attack of asthma is closely related with some elements such as wind, phlegm et al. exogenous wind-evil impairment of lung, and endogenous wind-evil trouble the lung, exogenous wind-evil induce endogenous wind-evil, phlegm and muddy. These are essential causes of the occurrence of asthma. Profuse wind, stagnation of phlegm, and air conduction urgent are the principal pathogenesis of asthma.. Wind, phlegm, pneuma, stagnant blood, deficiency, liver and lung functional disorder are principal pathogenesis feature with period of onset of asthma. Pay equal attention to expelling endogenous wind to relieve convulsion, lower adverse-rising energy, dissipate phlegm and regulating entrails are treating principle.4.3 Wuweidilong Decoction has the function of expelling endogenous wind to relieve convulsion and dissipating phlegm to relieve asthma, it is good in treating asthma.4.4 The mechanism of action of Wuweidilong Decoction on treating asthma.4.4.1 Influence on air tube allergies. Wuweidilong Decoction with different doses can restrain markedly the allergic contract of in vivo and in vitro air tube smooth muscle, and they can extend latent period of asthma on guinea pig. They can also increase obviously the IFN-γand decrease IL-4. The function of Wuweidilong Decoction may be educed by means of increasing IFN-γ, decreasing IL-4, regulating the proportion of correlated cytokine of adjunctive T cell, and then to regulate balance immunologic and to refrain IgE synthesis.4.4.2 Influence on airway inflammation. Wuweidilong Decoction can relieve EOS infiltrating in asthmatic guinea pig's air tube, reduce IL-4 in serum, increase IFN-γ, control indirectly the balance of Th1/Th2, and improve hyperreactive inflammation of air tube. It is proximal or comparative to Dexamethasone in this aspect. And we are hinted that the function of relieving convulsion to conquer asthma is prevalent in improving hyperreactive inflammation of air tube.4.4.3 Influence on airway remodeling. From histomorphology of lung we could see the focal falling of epithelium mucosae of bronchus, congestion and hydrops on subnatant mucosa, focal infiltrating of eosinophile granulocyte, significant accrementition and thickening with smooth muscle subnatant to mucosa in asthma model group. Compared with normal control group, there appeared airway remodeling in asthma model group. After being treated with Wuweidilong Decoction, inflammation was relieved, hydrops vanished obviously, hyperplastic endothelial cell of smooth muscle reverted gradually. It hints that the Wuweidilong Decoction has the function of restraining airway remodeling and then to conquer asthma. It may relieve inflammatory reaction and restrain airway remodeling by means of decreasing EOS infiltrating, reducing mediators of inflammation to secrete and to release, lessening inflammatory exudation and restraining hyperplasy of caliciform cell and smooth muscle cell.4.5 The function of spasmolysis to conquer asthma with Wuweidilong Decoction on asthma guinea pig is dose-dependent, and it outstrips the traditional prescription of Xiaoqinglong Decoction in some aspects. It is worth for us to study profoundly.