| ObjectiveBronchial asthma is a chronic allergic disease caused by various of factors and characterized by reversible airway obstruction, airway hyperreactivity and air tube inflammation.The global morbidity of asthma has been increasing in recent years and become an important issue undermining the health of mankind. Glucocorticosteroid and bronchodilators are applied constantly in clinical treatment of asthma, simutaneously the therapeutic effect of some Chinese traditional medicines including XIAOCHUANNING has been confirmed in animal and clinical trials. Yet it is still an important topic to manufacture Chinese traditional patent medicines by making use of the affluent plant resource which has good therapeutic effct, tiny side effect and widespread source in China. Baicalin and glycyrrhizin are commonly-used main active component of Chinese crude drugs baical skullcap root and Glycyrrhiza uralensis respectively.Both of them have been reported for their effects on resisting asthma. In this study, the effects of complex prescription baiglycyrrhizin injection on the inflammation process of bronchial asthma were estigated and the mechanism was explored initially. A new method was established for clinical therapy of bronchial asthma. Material and MethodIn this study 30 guinea-pigs were used and grouped randomly, one control group and four experimental groups including asthma model group, baicalin group, glycyrrhizin group and complex prescription of baiglycyrrhizin group were designed and 6 guinea-pigs in each group. The experimental groups were all treated with defferent reagents 14 days after sensitizing with OVA. Briefly, in the first day of the experiment, 1ml 10% OVA normal saline solution was intraperitoneal injected to sensitize the guinea-pigs, 14 days later, 1% OVA normal saline solution nebul was given once a day for five days to stimulate asthma. The normal control group was injected normal saline solution in the same way both in the process of sensitizing and stimulating of Guinea-pigs. Guinea-pigs were treated with different reagents 30 min before asthma stimulation. Whether symptoms of asthma of the Guinea-pigs would emerge or not was observed during 360 sec after the first simulation with 1% OVA nebul, and latent period of induced asthma and grade of asthma were recorded. The weight of guinea-pigs of each group was recorded and compared before the first administration and satisfice. 4 hours after the last stimulation, blood samples were collected from abdominal aorta of guinea pigs,and the activity of the NO and NOS, the level of the cytokines IL-4,IL-12,TNF-α,IFN-γin the serum were then detected.We also determined lencocyte count in a little BALF ,the rest BALF was centrifuged at 1500r/min for 10min to precipitate cells which would be stained with Wright-Giemsa method,and then be classified and counted.The tissue of lung will be stained with HE stain so that pathological inflaming changes surrounding bronchus could be observed with light microscope.The expression of T-bet,GATA-3 positive particles surrounding bronchus would be examined by immunohistochemical technique. The results were demonstrated as Mean±standard deviation (x__±s)and analysized with SPSS statistical software. The comparison among samples would need unpaired t-test.Asthma grades were compared with interclassχ2 test. Result1. The effects of complex prescription baiglycyrrhizin injection on weight, asthma grade and latence of induced asthma of experimental asthma guinea pig.1.1 The effects of complex prescription baiglycyrrhizin injection on latence of induced asthma of experimental asthma guinea pig. The latence of induced asthma was markedly shorter of the asthma model group than that of the normal control group(P<0.01);Although there was no statistical significance(P>0.05),it was on an extending tendency in baicalin group and glycyrrhizin group rather than in asthma model group;The latence of induced asthma was remarkably prolonged in complex prescription of baiglycyrrhizin group(P<0.05).1.2 The effects of complex prescription baiglycyrrhizin injection on asthma gradesThe asthma symptom of the normal control group were severer than that of the asthma model group(P<0.05);Although there was no statistical significance ( P>0.05 ) , The asthma symptom of baicalin group and glycyrrhizin group were relieved; The asthma symptom of complex prescription of baiglycyrrhizin group were relieved significantly(P<0.05).1.3 The effects of complex prescription baiglycyrrhizin injection on weight of the asthma guinea pigs.The weight of the asthma model group decreased remarkably more than that of the normal control group(P<0.05); Although there was no statistical significance(P>0.05),it was on an increasing tendency in baicalin group and glycyrrhizin group rather than in asthma model group;The weight of asthma guinea pigs increased remarkably in complex prescription of baiglycyrrhizin group(P<0.05).2. The effects of complex prescription baiglycyrrhizin injection on the related immune-cytokines and the content of NO, the level of NOS in experimental asthma guinea pigs serum2.1 The effects of complex prescription baiglycyrrhizin injection on the content of NO, the level of NOS in experimental asthma guinea pigs serum The content of NO, the level of NOS of the asthma model group increased remarkably more than that of the normal control group(P<0.05); Although there was no statistical significance(P>0.05),it was on an decreasing tendency in baicalin group and glycyrrhizin group rather than in asthma model group;The content of NO, decreased remarkably in complex prescription of baiglycyrrhizin group(P<0.05), Although the level of NOS was on a tendency to decrease, there is no statistical significance(P>0.05).2.2 The effect of complex prescription baiglycyrrhizin injection on the level of immune-cytokines IL-4,IL-12,TNF-α,IFN-γin experimental asthma guinea pigs serumThe level of IL-4,TNF-αof the asthma model group increased remarkably more than that of the normal control group(P<0.01),and The level of IL-12,IFN-γof the asthma model group decreased remarkably more than that of the normal control group(P<0.01); compared with asthma model group,it was quite different in the other groups that in baicalin group the level of IL-4 decreased remarkably (P<0.01)while the level of IFN-γsharply increased(P<0.01),in glycyrrhizin group the level of IL-4 was suppressed obviously (P<0.001) while the level of IL-12,IFN-γwas raised(P<0.001). In complex prescription of baiglycyrrhizin group the effect of complex prescription of baiglycyrrhizin could suppress the level of IL-4 ( P<0.001) as well as the level of TNF-α(P<0.01)and raise the level of IL-12,IFN-γremarkably(P<0.001). which was better than herb monomer.3. The effects of complex prescription baiglycyrrhizin injection on cell count of experimental asthma guinea pigs BALFThe total cellular score, the cell population of EOS and macrophage in BALF of the asthma model group increased remarkably as compared with that of normal control group(P<0.01); while compared with asthma model group, the total cellular score decreased remarkably in baicalin group (P<0.01); in glycyrrhizin group the total cellular score, the cell population of EOS and macrophage was raised obviously (P<0.05～0.001); in complex prescription of baiglycyrrhizin group the total cellular score, the cell population of EOS and macrophage were suppressed remarkably(P<0.01～0.001)and the effect was better than herb monomer.4. The effects of complex prescription baiglycyrrhizin injection on pulmonary histomorphology of experimental asthma guinea pigsIn the asthma model group, the tubal wall and surrounding tissue of the bronchus was infiltrated by a great quantity of inflammatory cells major of which was EOS;the air tube narrowed ,whose epithelium mucosae thickened even exfoliated; mucous plug was found in the bronchus. The pathological appearance above-mentioned demonstrated that the asthma model was established successfully.Although the tissue was infiltrated by some inflammatory cells, in the lungs of baicalin group and glycyrrhizin group, the construction of small airway and small airway remained integrity, and secreta in the bronchus and alveolar space reduced; in the lungs of the complex prescription of baiglycyrrhizin group, the inflammatory cells infiltrating the pulmonary tissue and secreta in the bronchus and alveolar space reduced markedly, the construction of small airway and small airway remained integrity without Airway constriction narrowed and cellula epithelialis exfoliated.5.The effects of complex prescription baiglycyrrhizin injection on immunohistochemistry stain of experimental asthma guinea pigs5.1 The effects of complex prescription baiglycyrrhizin injection on T-bet immunohistochemistry stain of experimental asthma guinea pigsPeribronchial tissue of normal control group presented T-bet-positive which was tiny in the Peribronchial tissue of the asthma model group; T-bet expression in Peribronchial tissue was positive and strong positive in the two groups of baicalin and glycyrrhizin and the complex prescription of baiglycyrrhizin group respectively.5.2 The effects of complex prescription baiglycyrrhizin injection on GATA-3-positive particles immunohistochemistry stain of experimental asthma guinea pigsNo GATA-3-positive particles could be expressed in Peribronchial tissue of normal control group; in contrast, a great quantity of GATA-3 positive particles was presented in the Peribronchial tissue of the asthma model group. The amount of GATA-3-positive particles was tiny in Peribronchial tissue in the two groups of baicalin and glycyrrhizin; and the complex prescription of baiglycyrrhizin group presented no GATA-3-positive particles.5.3 The effects of complex prescription baiglycyrrhizin injection on LTs immunohistochemistry stain of experimental asthma guinea pigs No LTs could be detected in Peribronchial tissue of normal control group; in contrast, a great quantity of LTs positive particles were expressed by lympholeukocytes and mononuclear macrophages in the Peribronchial tissue of the asthma model group. The amount of LTs-positive particles was tiny in Peribronchial tissue in the two groups of baicalin and glycyrrhizin;and the complex prescription of baiglycyrrhizin group present no LTs-positive particles.1. The effects of complex prescription baiglycyrrhizin injection on the IL-4,IL-12,TNF-α,IFN-γmRNA expression of experimental asthma guinea pigsIt was different in asthma model group from the normal control group that the relative transcriptional level of IL-4,TNF-αmRNA significantly increased while the relative transcriptional level of IL-12p35,IL-12p40,IFN-γmRNA remarkably decreased.contrastly, the relative transcriptional level of IL-4,TNF-αmRNA was suppressed while the relative transcriptional level of IL-12p35,IL-12p40,IFN-γmRNA was raised in the two groups of baicalin and glycyrrhizin. The effect of complex prescription of baiglycyrrhizin that could suppress the relative transcriptional level of IL-4,TNF-αmRNA remarkably and raise the relative transcriptional level of IL-12p35,IL-12p40,IFN-γmRNA was better than herb monomer.ConclusionBased on the guinea pig bronchial asthma model we built, in this study an investigation was performed on the therapeutic effect of complex prescription of baiglycyrrhizin on asthma and the mechanisms of the effect.1. It has been demonstrated that complex prescription of baiglycyrrhizin could play roles in improving the symptoms of asthma by prolonging the latence of induced asthma of guinea pig, degrading asthma grade,raising the weight of asthma guinea pig.2. Another effect of complex prescription of baiglycyrrhizin has been confirmed that it could suppress the bronchial inflammatory cell infiltration when asthma happens. The evidence is that it could reduce total cellular score,cell population of eosinophile granulocytes and macrophages in BALF remarkably, relieve the pathological change of peribronchial tissue of asthma guinea pigs, reduce the population of eosinophile granulocytes that aggregate in peribronchial tissue, decrease the expression of leukotriene in peribronchial tissue.3. Complex prescription of baiglycyrrhizin could decrease the level of serum NO and NOS, suggesting that the therapeutic effect of complex prescription of baiglycyrrhizin on asthma is relevant to suppressing the over-expression and cytotoxic effect of NO.4. Complex prescription of baiglycyrrhizin could significantly reduce the gene expression of IL-4,TNF in pulmonary tissue and the content of them in serum, increase the gene expression level of IL-12,INF in pulmonary tissue and the content of them in serum, suggesting that complex prescription of baiglycyrrhizin has important significance in suppressing differentiation of Th2, promote differentiation of Th1, correct the disequilibrium of Th1/Th2.5. Complex prescription of baiglycyrrhizin could enhance the expression of T-bet, a specific transcription factor of TH1 and attenuate the expression of GATA-3, a specific transcription factor of Th2 in pulmonary tissue. These suggest that its effect on the changes of the correlated cytokines during asthma may be relevant to the specific regulation of cytokine synthesis and the induction of differentiation from Th2 toTh1. |
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