| ?objective?It is the purpose to observe the effects of Qingjin Huatan decoction in treatment of ac ute airway inflammation model of airway injury and mucus hypersecretion of rats,and it is revealed that the signaling pathways of P38MAPK/NF-?B play an very important role in r egulating acute airway inflammation and inhibiting the state of mucus hypersecretion in rats through observing changes of acute airway inflammation and the state of mucus hypersecre tion at different time,detecting the related cytokinesand and the expression of mucin of mu cus secretion markers with ELISA(enzyme-linked immunosorbent assay)and examining the r elated protein and the expression of mucin in acute inflammatory pathways with immunohist ochemistry and Western blot,to analyze the possible signaling mechanism of inhibition of a cute airway inflammation and mucus hypersecretion using Qingjin expectorant.The applicati on value and its mechanism of Qingjin expectorant in treatment of acute airway inflammati on disease is presented to provide experimental basis for clinical practice of Qingjin expect orant,More than that,the feasibility and importance of the method of ?Qingfei huatan? as one of the main therapies of acute airway inflammation disease are demonstrated deeply lay ing the foundations of traditional Chinese medicine to the world.?methods?1.Healthy Male SD rats 96,randomly divided into 6 groups: blank group(A group),model group(B group),high dose group of Qingjin Huatan decoction(C group),middle do se group of Qingjin Huatan decoction(D group),low dose group of Qingjin Huatan decocti on(E group),Dexamethasone group(F group),16 rats in each group.Each group is treate d with LPS drip into the airway to establish a rat model of acute airway inflammation,the blank group excepted,while blank group is dripped with saline.2.Qingjin Huatan decoction consisted of 11 Herbs of Huang qin,Zhi zi,Jie geng,Ma i men dong,Zhe bei mu,Ju hong,and so on is made into decoction according to the conc entration of 2.98 g.ml-1 which is the high dose for rats,diluted with water twice times as the middle dose,diluted with water four times as the low dose.3.24 hours after modeling,Dose 1 time a day until execution.Blank group and mode l group are given gavage with normal saline of 0.25 m L.Each group of high dose,middle dose and low dose is given gavage with Qingjin Huatan decoction of different density(hig h dose 7.44 g.kg-1,middle dose 3.72 g.kg-1,low dose 1.86 g.kg-1),one rat is given gavage of 2.5mL each time,Dexamethasone group is given intraperitoneal injection with Dexameth ason Sodium Phosphate Injection of 0.001 g.kg-1.4.To observe total number of BALF cells and classification count,pathological change s,the level of such cytokines as IL-1?,IL-8,and TNF-?,moreover,the expression of mucin Muc5 ac in bronchoalveolar lavage fluid(BALF)with enzyme-linked immunosorbent assay(ELISA)on the fourth day and the seventh day after establishing model to determine the st atus of acute airway inflammation and mucus hypersecretion.5.Investigate the status of expression of mucin MUC5 AC and P38 MAPK in lung tissu e using immunohistochemical method and detect the level of expression of mucin P38 MAP K,I?B? and NF-?BP65 in lung tissue using Western blot method.6.Statistical MethodStatistical software is SPSS17.0,measurement data is described with average±standard deviation(),and the difference between two groups is revealed with one-way ANOVA(analysis of variance).Univariate correlation is analyzed with bivariate Pearson Correlation Analysis.The difference between two groups or two variants is statistically significant when P<0.05 or P<0.01.?results?1.General observation: After model,compared with the blank group,each treatment gr oup of rats show that the activities decrease,slow in reaction,lack lustre in their eyes,dri nk and eat less,hair color fade,there are secretions flowing out of their mouths and nostr ils and the sound of cough and phlegm is audible;compared with the model group,after g iving drugs,the above symptoms and signs of each treatment group of rats are improved,e specially as it is most obvious for high dose group of Qingjin Huatan decoction on 7d,sec ond is middle group and Dexamethasone group.2.Total number of leukocytes and cell sorting in BALF: Compared with the blank gro up,total number of leukocytes of model group increase significantly,and compared with m odel group,the total number of leukocytes of high group,middle group,low group and De xamethason group all decrease significantly at the same time phase(P<0.05 or P<0.01).Esp ecially,the total number of leukocytes of high group is approximately equal to those of the blank on 7d;Cell classification shows that the numbers of fine neutrophils and lymphocyt es of the model group all increase significantly compared with those of the blank,while th e numbers of fine neutrophils and lymphocytes of each group of Qingjin Huatan decoction all decrease,but those of macrophages increase compared with the model,and this situation of high dose group is obvious particularly on 7d,but there is no significant difference in the high group and middle group at other time.This demonstrate that Qingjin Huatan decoc tion and Dexamethason all can depress the increase of leukocytes,fine neutrophils and lym phocytes for acute airway inflammation.3.Pathology change of trachea and lung tissue: As for rats of the blank,its pseudostra tified ciliated columnar epithelium on the bronchial mucosa arranged regularly,alveolar struc ture was clear and complete,alveolar septal thickening did not occur,there was no infiltrati on of inflammatory cells in alveolar and no pulmonary interstitial edema.But for those of t he model,infiltration of inflammatory cells(mainly neutrophils and lymphocytes)and exud ation of large number of red cells appeared on the wall of trachea,in the pulmonary vessel s and in part of alveolar space,epithelial cells shed in tracheal lumen,alveolar septal was t hickening obviously and there was pulmonary interstitial edema.After giving the drug of Q ingjin Huatan decoction,the severity of inflammation lessened,especially for high dose on 7d.Inflammation grading statistics showed that inflammation integral of bronchial and lung tissue for rats of each group with acute airway inflammation induced by LPS increased obviously,compared with the blank.The pathological inflammation score in the treatment group were significantly decreased especially for high dose group on 7d,compared with the mod el group.This demonstrated that Qingjin Huatan decoction and Dexamethasone all could de crease the inflammation integral of bronchial and lung tissue,the epithelial damage and infl ammatory cell infiltration.4.Detecting related cytokines and mucin MUC5 AC with ELISA: the factor of IL-1?,I L-8,TNF-? and MUC5 AC in the BALF of the model increased obviously,compared with the blank(P<0.05 or P<0.01);On the other hand,those of treatment group all decreased o bviously except for low dose group on 4d and especially for high dose group on 7d(P<0.05 or P<0.01).5.Detecting the expression of protein MUC5 AC and P38 MAPK with SP: the expressio n of protein MUC5 AC and P38 MAPK in lung tissue of the model group increased obvious ly,compared with the blank(P<0.05 or P<0.01);On the other hand,those of treatment gro up all decreased obviously except for low dose group on 4d and especially for high dose g roup on 7d(P<0.05 or P<0.01).6.Detecting the expression of protein P38 MAPK,I?B? and NF-?BP65 with Wester n blot: the expression of protein P38 MAPK and NF-?BP65 in lung tissue of the model gr oup increased obviously,but I?B? was opposite,compared with the blank(P<0.05 or P<0.01);On the other hand,P38 MAPK and NF-?BP65 of treatment group all decreased obvi ously,but I?B? was opposite,except for low dose group on 4d and especially for high d ose group on 7d(P<0.05 or P<0.01).7.Correlation analysis showed that there was bvious positive correlation between the e xpression of protein P38 MAPK and total number of leukocytes in BALF,P38 MAPK and T NF-?,P38 MAPK and MUC5 AC,still P38 MAPK and NF-?BP65.?conclusions?1.This study confirmed that in the BALF of rats of acute airway inflammation model induced by LPS,inflammatory cells,the expression of such cell factors as IL-1?,IL-8,TN F-? and mucin MUC5 AC,inflammation scores of bronchial and lung tissue increased obvi ously,which showed the rats model of a cute airway inflammation was established successfu lly.2.Experiment found that in the BALF of treatment group inflammatory cells,the expr ession of such cell factors as IL-1?,IL-8,TNF-? and mucin MUC5 AC,inflammation score s of bronchial and lung tissue decreased obviously after giving the drug of Qingjin Huatan decoction,and difference was significant,compared with model group;the expression of P38 MAPK,NF-?BP65 deceased and I?B? protein content increased obviously,the differe nce was significant.There was obvious positive correlation between the expression of protei n P38 MAPK and total number of leukocytes in BALF,P38 MAPK and TNF-?in BALF,P38 MAPK and NF-?BP65 in lung tissues,which showed that Qingjin Huatan decoction dow n regulated the signal protein of P38 MAPK and NF-?B,inhibited degradation of I?B?thr ough modulating the signal transduction pathway of P38MAPK/ NF-?B mediated by the cell factor of TNF-?.3.Experiment found that in the BALF of treatment group mucin MUC5 AC decreased obviously after giving the drug of Qingjin Huatan decoction,and difference was significant,compared with model group;the positive expression of P38 MAPK and MUC5 AC in lung tissue decreased obviously with SP detecting,the difference was significant.At the same ti me,the expression of P38 MAPK and NF-?BP65 in lung tissue decreased obviously,and I?B?increased with Western blot detecting,the difference was significant,compared with model group.There was obvious positive correlation between the expression of protein P38 MAPK and MUC5 AC in lung tissues on 7d,which showed that Qingjin Huatan decoction can inhibit the airway mucus hypersecretion through modulating the signal transduction path way of P38MAPK/ NF-?B,down regulating P38 MAPK and NF-?B,then decreasing the e xpression of mucin MUC5 AC to improve airway mucus secretion.4.Experiment found that Qingjin Huatan decoction can inhibit the inflammatory signali ng pathway activity,decrease airway inflammatory response,anti mucin hypersecretion,and protect the airway with the dose-dependent manner,through modulating the signal transducti on pathway of P38MAPK/NF-?B mediated by the cell factor.5.There came to a conclusion that the regulatory mechanism of improving the acute a irway inflammation in rats by Qingjin Huatan decoction was here: Qingjin Huatan decoctionàActivate P38 MAPK signal transduction pathwayàDown regulate P38MAPKàInhibit degra dation of I?B? proteinàDown regulate NF-?BP65àInhibit the expression of inflammatio n cells,IL-1?,IL-8,TNF-? and MUC5ACà Biological effectà Inhibit acute airway infla mmation reaction and mucous hypersecretion. |
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