| Objective:Based on the pathogenesis of stagnant fluid retention theory,this paper probes into the immunomodulatory mechanism of Kangzhi syrup on Th17/Treg imbalance of cough variant asthma,and provides the basis for further study on the mechanism of Kangzhi syrup and its clinical application.Methods:Ninety SPF male guinea pigs(weight 260±15g)were randomly divided into 6 groups:blank control group,model group,dexamethasone group,Kangzhi syrup high dose group,Kangzhi syrup middle dose group and Kangzhi syrup low dose group,there were 15 guinea pigs in each group.This experiment was started after adaptive feeding for 1 week.Except the blank control group,the other groups were sensitized with OVA and aluminum hydroxide on the first day of the experiment;from the 16th day of the experiment,each group were inhaled OVA solution once every other day,total of 14 days.The drug intervention was performed once a day for 14 days in each group from the 15th day of the experiment.At the end of the experiment,the changes of body weight and cough times of guinea pig.models were observed.Samples were collected within 24 hours after the last atomization inhalation,the changes of cell classification and count in BALF were detected by Wrigh’s stain;we observed inflammatory cells by HE stain;detect the level of IL-6,IL-10,IL-17 and IL-35 in serum and BALF by ELISA;detect the protein and gene expression level of Thl7 and Treg cell transcription factor RORyt and Foxp3 in lung tissue by Western Blot and Real-time PCR technology.Results:1.Body weight change:There was no significant difference in body weigh of guinea pigs before CVA induction(P>0.05).After repeated induction of CVA,the growth and development of guinea pigs were inhibited to varying degrees of inhibition.Compared with the model group,there was significant difference of the weight gain in blank control group and each dose group of Kangzhi syrup(P<0.05);compared with dexamethasone group,the body weight of Kangzhi syrup each dosage group increased obviously;compared with blank control group,the model group and the dexamethasone group increased slowly,there was no significant difference between the two groups(P>0.05).2.Cough times change:Compared with blank control group,the times of cough in the model group increased significantly(P<0.05);compared with the model group,the number of cough in each drug therapy group was significantly reduced(P<0.05);compared with dexamethasone group,there was no significant difference in medium and high dose of Kangzhi syrup group(P>0.05).3.Cell classification and counting in BALF change:Compared with blank control group,the total number of white cells in the model group BALF,EOS and LYM increased in different degrees(P<0.05);EOS absolute count increased significantly(P<0.05);the proportion of MAC decreased,but the difference was not statistically significant(P>0.05).Compared with the model group,the total white blood cells count and the proportion of EOS,LYM in BALF of each drug therapy group were significantly decreased(P<0.05),the proportion of NEU was increased(P<0.05).Compared with dexamethasone group,there was no significant difference in the total number of white blood cells and their classification in medium dose of Kangzhi syrup group(P>0.05).4.Histopathologic HE stain show:In the model group,there were more inflammatory cells in lung tissue,bronchial wall was damaged,and mucosal hyperemia was edema,in the lumen,the epithelial cells shed and the secretion increased,and mucus embolus was formed.Compared with the model group,the pathological changes were obviously alleviated,and the secretion was obviously decreased of the different dosage groups of Kangzhi syrup and dexamethasone group.The dexamethasone group and medium,high dose groups of Kangzhi syrup were improved obvious.5.ELISA test show:Compared with the blank control group,the content of cytokines IL-6 and IL-17 in the model group BALF and serum was significantly increased,the levels of IL-10 and IL-35 reduced significantly(P<0.05);compared with the model group,the level of IL-6 and IL-17 in BALF and serum of dexamethasone group and each dosage group of Kangzhi syrup were decreased significantl and the level of IL-10 and IL-35 were increased significantly(P<0.05);compared with dexamethasone group,there was no significant difference in the middle and high dose groups of Kangzhi syrup(P>0.05).6.Western Blot test results show:Compared with blank control group,the expression of RORyt protein in the lung tissue of the model group was significantly increased,the expression of Foxp3 protein was decreased(P<0.05);compared with the model group,the expression of ROR-yt protein in the lung tissue of dexamethasone group,each dosage group of Kangzhi syrup were decreased significantly and the level of Foxp3 protein were increased significantly(P<0.05);compared with dexamethasone dosage group,there was no significant difference in the middle and high dose groups of Kangzhi syrup(P>0.05).7.Real-time PCR detection results show:Compared with the blank control group,the expression of RORytmRNA increased significantly in the model group and the mRNA expression of Foxp3 decreased significantly(P<0.05);compared with the model group,the level of Foxp3mRNA expression in different doses of kangzhi syrup group and dexamethasone group increased(P<0.05),the level of RORytmRNA expression was significantly decreased(P<0.05);compared with dexamethasone dosage group,there was no significant difference in the middle and high dose groups of Kangzhi syrup(P>0.05).Conclusion:1.Kangzhi syrup can improve the general condition of CVA guinea pigs model,such as body weight,cough times,reduce the total number of WBC and EOS count,reduce submucosal edema and mucus secretion,reduce airway inflammatory infiltration,thus improve respiratory hyperresponsiveness and symptoms.2.Kangzhi syrup maybe decrease the level of IL-6 and IL-17 in BALF and serum of CVA guinea pigs model,increase the level of IL-10 and IL-35,thus inhibit the airway inflammation of CVA and regulate the imbalance of Th17/Treg.3.Kangzhi syrup maybe down-regulate the expression of RORytmRNA and protein,and up-regulate the expression of Foxp3mRNA and protein in lung tissue of CVA guinea pigs model,thus regulate the imbalance of Th17/Treg balance. |
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