| Purpose:The research was supported by grants from National Natural Science Foundation of China(project number: 81273927)and Significant new drugs creation " the Twelfth Five-Year" plan special science and Technology Major(project number: 2012ZX09303-017),through the clinical research,observing the curative effect of Shegan Qingke tablets treating post influenza cough,using LegendPlex multi factor quantitative analysis method to assay the expression level of 13 kinds of Th cytokines in the serum of the patients with post influenza cough of Shegan Qingke tablets,in order to provide basis for the further animal research and the selection of animal models and indicators.Selection of post infectious cough as an animal model based on the results of clinical trials,using the isoflavone monomer compounds and compatible mixtures with physiological activity in Shegan as the research object,starting from the angle of regulation of airway inflammation of post influenza cough,taking the Inflammatory cells,the cytokines,the inflammatory mediators and their interactions as the starting point,through the animal experiment,from the whole level,the organ level,the cell level and the molecular level,the research was revealing the anti airway inflammation mechanism and the target of the different isoflavone monomer compounds of Shegan and its compatible mixtures and clearing different proportions were making different ways of anti airway inflammation,and the optimum proportion of anti airway inflammation in the compatible mixtures of isoflavone monomer compounds was preliminarily determined,under the conditions of this experiment.Material and method: 1.Clinical researchThrough the design of clinical testing scheme of post influenza cough of clinical adaptation to disease,based on in the discussion through the drug supervision and Administration Bureau of Liaoning province and the approval of the ethics committee of the hospital,a randomized,double-blind,controlled trial was conducted to screen for the best clinical application of Shegan Qingke tablets.The clinical test included 80 patients with post influenza cough(treatment group and positive control group),with the Feining tablet(Astertataricus agents)as the positive control drug,the number of cases was arranged according to the proportion of 1:1.The daytime cough score,the night cough score,the sputum expectoration,the sputum volume,the white blood cell count(WBC),the neutrophil(NEUT)and the lymphocyte(LYMPH)percentage were measured before and after treatment.The results were applied comparative t-test to perform the statistical analysis by SPSS 17.0 software.The LegendPlex multi factor quantitative analysis method was used to assay the expression level of 13 kinds of the Th cytokines in the serum of the 36 patients with post influenza cough of Shegan Qingke tablets,providing a basis for further animal experiments to select animal models and test indexes.2.Experimental researchAccording to the results of clinical trials,the results showed that Shegan Qingke tablets had a good effect on the treatment of post influenza cough,the model of post influenza cough was replicated by using the commonly accepted respiratory syn cytial virus infection in guinea pigs.Using randomized control method,all guinea pig s were divided into the blank control group,the model group,the positive control g roup-Chinese medicine Feining tablets(Aster tataricus agents),the tectorigenin group,the irigenin group,the irisflorentin group,the dichotomitin group,the compatibility 1 group,the compatibility 2 group,the compatibility 3 group,the compatibility 4 group,the compatibility 5 group,and the compatibility 6 group,through the way(1)The citric acid was used to induce cough.The number of the coughing(within 5 min an d after 5 min)was recorded in each group of animals after the cough induction,to observe the effect of the citric acid on the coughing number;(2)The influence of t he blood and the alveolar lavage fluid on inflammatory cells was detected by autom atic modular blood and body fluid analyzer;(3)By hematoxylin-eosin(HE)observation of the effect on the lung tissue morphology;(4)Detection of the cytokines in seru m and alveolar lavage fluid by ELISA Ki and detecting the expression of Th1 and Th2 specific transcription factors T-bet and GATA-3 by Western-blot method(WB);(5)T he reversed-phase high performance liquid chromatography gradient elution,electrosp ray ionization tandem mass spectrometry multiple reaction detection mode was usedto detect the content of arachidonic acid(AA)and its three metabolic pathway pro ducts cyclooxygenase(COX)pathway metabolites prostaglandins D2(PGE2),prostagland ins E2(PGD2),thromboxanes B2(TXB2),11-hydroxyeico satetraenoic acid(11HETE),Cyto chrome P(CYP)450 pathway metabolites 5.6-dihydroxyeicosatrienoic acids(5.6-DHET),8.9-dihyDroxyeicosatrie noic acids(8.9-DHET),11,12-dihydroxyeicosatrienoic acids(11.12-DHET),14,15-dihydroxyEicosatrienoic acids(14.15-DHET),16-hydroxyeico satetraenoic acid(16HETE),lipoxygenase(LOX)pathway metabolites 5-hydroxyeico satetraenoic acid(5HE TE),Hydroxyeico 12-satetraenoic acid(12HETE),15-hydroxyeico satetraenoic acid(15HET E)in the serum and the alveolar lavage fluid and leukotriene4(LTC4)was detected by ELISA.From the whole level,the organ level,the cell level and the molecular le vel,it was revealing the anti airway inflammation mechanism and the target of the different isoflavone monomer compounds of Shegan;The relationship between the do sage of the monomer and the pharmacological effects of the corresponding model a nimal was correlated,and the correlation between the components of the monomer and the different ways of anti airway inflammation was found through the correlati on analysis;4 kinds of the isoflavone glycosides,such as tectorigenin,irigenin,irisflor entin and dichotomitin,were studied with 4 factors and 6 levels by uniform design,and the dosage of the combination group was associated with the corresponding mo del animal pharmacological effects.By regression analysis,different proportions were determined by different ways.It can resist airway inflammation and get the best do se ratio of anti airway inflammation,under the conditions of this experiment.Results: 1.Clinical research 1.1 The curative effect of Shegan Qingke tablets treating post influenza cough(syndrome of lingering heat)Taking post influenza cough(syndrome of lingering heat)patients as the research object,through collecting,collating and counting the clinical information of the subjects during the visit period,the two groups of cases were statistically treated in the sex,theage,the course of disease and the severity of the disease(the total score of disease,the totalscore of syndromethe,the daytime cough score,the night cough score,the sputum expectoration,the sputum volume,the pulmonary signs and the tongue image).The two groups had no significant difference and had comparability.Through the daytime cough score,the night cough score,the sputum expectoration,the sputum volume,it was suggested that both the Shegan Qingke tablets and the Feining tablets had the effect of relieving cough and eliminating phlegm.It had a good effect on the treatment of post influenza cough(syndrome of lingering heat).By measuring the white blood cell count,the neutrophil and lymphocyte percentage in the blood of the two groups before and after the group,the total number of white blood cells and the percentage of neutrophils and lymphocytes in the two groups did not decrease.1.2 The expression level of 13 kinds of Th cytokines in the serum of the patients with post influenza cough of Shegan Qingke tablets.Using the LegendPlex multi factor quantitative analysis method to assay the expression level of 13 kinds of Th cytokines secreted mainly by follicular cells of Th1,Th2,Th17,Th9,Th22 and T in follicular cells,including interleukin-2,4,5,6,9,10,13,21,22,17 A,17F(IL-2,IL-4,IL-5,IL-6,IL-9,IL-10,IL-13,IL-21,IL-22,IL-17 A,IL-17F),interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α),the content of IL-4(Th2)and the content of Th17A(Th17)after treatment was significantly lower than the before,the content of IFN-γ(Th1)was significantly higher than the before,to correct the drift of Th1/Th2/Th17,so as to reduce airway inflammation,to achieve anti-inflammatory and relieve cough.2.Experimental research2.1 Replicating the animal model of post influenza cough in guinea pigsThe lung tissue of guinea pig virus culture medium showed that the RSV infected model group;the frequency of cough significantly higher than the control group was suggesting the formation of airway hyperresponsiveness;the eosinophil level in the alveolar lavage fluid in model group significantly higher than the control group was causing airway inflammation;the lung tissue HE results showed no substantial model serum pneumonia;the inflammatory cells in the blood of model group had no significant differencethe with the control group,according with the clinical features of post influenza cough,these results showed thatsyncytial virus infection cough model was established successfully.2.2 Effects of the isoflavone monomer compounds of Shegan and its compatible mixtures on the hypersensitivity of the airway of the model animalIn the model group,the number of coughs in 5min was significantly higher than that in the blank group.The drug group,the irisflorentin group,the dichotomitin group,Chinese medicine positive tablets,the compatibility 1 group and the compatibility 2 group were significantly inhibited compared to the model group.2.3 Effects of the isoflavone monomer compounds of Shegan and its compatible mixtures on the inflammatory cells in the model blood and alveolar lavage fluidIn the alveolar lavage fluid of the model group,except basophil count,the other inflammatory cells were significantly higher than that in the blank group.The irisflorentin group had significant inhibitory effect on the total number of white cells compared to the model group,the irisflorentin group and the compatibility 1 group had significant inhibitory effect on the lymphocyte compared to the model group,and the compatibility 5 group was significant to the neutrophils compared to the model group.The inhibitory effects of the tectorigenin group,the irigenin group,the irisflorentin group and the compatibility 6 group on the eosinophils were significantly compared to the model group.The neutrophil count of blood in the model group was significantly higher than that in the blank group,and there was no significant inhibition in each drug group compared to the model group.2.4 Effects of the isoflavone monomer compounds of Shegan and its compatible mixtures on the cytokine of Th2/Th1 in the serum and alveolar lavage fluidThe content of IL-4 in model group of alveolar lavage fluid was significantly higher than that of blank group.The content of IFN-γwas significantly lower than that of blank group,and the ratio of IL-4/IFN-γwas significantly higher than that of control group.It was suggested that the inflammatory cytokines related to the alveolar lavage fluid of the model group changed significantly.The compatibility 6 group had a significant down-regulation effect on the content of IL-4 compared to the model group,the tectorigenin group,the irigenin group and the irisflorentin group had a significant up-regulation effect on the content of the IFN-γcompared to the model group,and the tectorigenin group and the irigenin group had a significant down-regulation effect on the ratio of IL-4/IFN-γcomparedto the model group.The serum content of IFN-γin model group was significantly lower than that in control group.The ratio ofIL-4/IFN-γin model group was significantly higher than that in control group.It was suggested that the inflammatory cytokines related to the serum of the model group changed significantly.The drug group in addition to white pigment Belamcanda chinensis could significantly reduce the ratio of IL-4/IFN-γcompared to the model group,the other groups had no obvious effect on cytokine compared to the model group.2.5 Effects of the isoflavone monomer compounds of Shegan and its compatible mixtures on the expression of T-bet,GATA-3 protein levels in lung tissue of animal modelWestern blot method experimental results showed that compared with the normal control group,the expression level of GATA-3 protein in the model group were significantly increased,the expression level of T-bet protein decreased significantly,indicating the protein expression of transcription factors relating to Th cell of the post influenza cough model changing significantly.The drug group,the GATA-3 protein expression level of the dichotomitin group,the compatibility 1 group,the compatibility 5 group,and the compatibility 6 group decreased significantly compared to the model group,the T-bet protein expression level of the tectorigenin group and the irigenin group increased significantly compared to the model group.2.6 Effects of the isoflavone monomer compounds of Shegan and its compatible mixtures on the model animal lung histomorphologyThe model group showed typical airway inflammation in the acute phase,and e ach drug group had different effects on reducing the symptoms of infection compare d to the model group.2.7 Effects of the isoflavone monomer compounds of Shegan and its compatible mixtures on the AA and its metabolites of serum and alveolar lavage fluid in model animalsIn the serum COX pathway,the content of the TXB2 of the COX pathway metabolites and the content of the 11 HETE of the COX pathway metabolites in the model group were significantly higher than those in the blank group.The content of the TXB2 of the COX pathway metabolites in the irigenin group,the irisflorentin group,the compatibility 1 groupand the compatibility 6 group was significantly reduced compared to the model group,the content of the 11 HETE of the COX pathway metabolites in the tectorigenin group,the irigenin group,the irisflorentin group and all the compatibility groups was significantly reduced compared to the model group.In the serum P450 pathway,the content of 5.6-DHET of the P450 pathway metabolites and the content of 8.9-DHET of the P450 pathway metabolites in the samples was partly lower than the detection limit,and no statistics were made.The content of the 16 HETE of the P450 pathway metabolites in the model group was significantly higher than that in the blank group.The content of the 16 HETE of the P450 pathway metabolites in the tectorigenin group,the irisflorentin group,the compatibility 3 group and the compatibility 6 group was significantly reduced compared to the model group.In the serum LOX pathway,the content of the 12 HETE of the LOX pathway metabolites and the content of the 15 HETE of the LOX pathway metabolites in the model group was significantly higher than that in the blank group.The content of the 12 HETE of the LOX pathway metabolites in the tectorigenin group,the irigenin group,the irisflorentin group,the compatibility 3 group,the compatibility 5 group,and the compatibility 6 group was significantly reduced compared to the model group.The content of the 15 HETE of the LOX pathway metabolites in the tectorigenin group,the irigenin group,the irisflorentin group,the dichotomitin group and the compatibility 6 group was significantly reduced compared to the model group.The content of the LTB4 of the LOX pathway metabolites was not detected in the serum,the content of the LTC4 of the LOX pathway metabolites determination conditions were not suitable.AA in the serum: the content of AA in the model group was significantly higher than that in the blank group.The drug group,the dichotomitin group was significantly lower AA compared to the model group.Only the content of the LTC4,AA and the HETEs products were measured in the alveolar lavage solution.The content of PGs and DHETs products was lower than that of the quantitative limit,which was not detected.In the three metabolic pathways,there was no significant difference in the content of 11 HETE,16HETE,5HETE,12 HETE and 15 HETE in the alveolar lavage fluid of the model group compared with those in the blank group.Thecontent of LTC4 and AA in the alveolar lavage fluid was significantly higher than that in the blank group.The content of the LTC4 in the tectorigenin group,the compatibility 5 group and the compatibility 6 group was significantly reduced compared to the model group.2.8 Correlation analysis between 4 kinds of isoflavone monomer compounds of Shegan and pharmacological effect indexThe correlation analysis between the dose of the monomer and the pharmacological effects was made,and the correlation coefficient was obtained,suggesting that dichotomitin was significantly negatively correlated with the number of coughing in 5 minutes;irigenin was negatively correlated with the monocyte count in the alveolar lavage fluid and was significantly positively correlated with the basophil count in the alveolar lavage fluid;tectorigenin was significantly negatively correlated with the IL-4/IFN-γin the serum;Dichotomitin was significantly positively correlated with the IL-4/IFN-γin the serum;irigenin was significantly negatively correlated with the content of 16 HETE of P450 pathway metabolites in the serum;tectorigenin was significantly negatively correlated with the content of LTC4 of LOX pathway metabolites in the alveolar lavage fluid;dichotomitin was significantly positively correlated with the content of LTC4 of LOX pathway metabolites in the alveolar lavage fluid.2.9 Study on the compatibility of 4 isoflavones monomer of Shegan in anti airway inflammationUsing the uniform design 3.0 software,we choosed the single dependent variable to analyze the multi variable regression analysis model,and got the result of the influence of each component to the single index.It suggested that the compatibility of the 4 isoflavones in different proportions could play its anti airway inflammation through different ways;through the multi dependent variable,the multi variable analysis model was divided into the multi variable analysis model.The proportion of comprehensive best compatibility was as follows:tectorigenin : irigenin : irisflorentin : dichotomitin(36:54:36:12).Conclusion:In clinical studies,Shegan Qingke tablets in the treatment of post influenza cough(syndrome of lingering heat)had significant curative effect,decreased the frequency ofcough and expectoration volume,better than Feining tablets.Th1/Th2/Th17 participated in the airway inflammation of post influenza cough.Shegan Qingke tablets could correct the drift of Th1/Th2/Th17,so as to reduce airway inflammation,to achieve anti-inflammatory and relieve cough,to reveal the targets of anti airway inflammation at molecular level,providing a basis for further animal experiments to select animal models and test indexes.In experimental research,according to the results of clinical trials,the results showed that Shegan Qingke tablets had a good effect on the treatment of post influenza cough,coldusing a guinea pig model of post influenza cough,isoflavone compounds of Shegan with anti airway inflammation of different targets was revealed from the whole,organ,cell and molecular level,and through multi targets synergy,the active component combination could enhance the resistance of airway inflammation effect,getting the optimum proportion of anti airway inflammation in the compatible mixtures of isoflavone monomer compounds under the conditions of this experiment. |
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