| Research purpose: This study uses the Th17/Treg cellular immune imbalance in asthma as a starting point.Through in vivo and in vitro experimental studies,to explore the regulatory effect and mechanism of Yiqi Wenyang Huwei Method on Th17/Treg imbalance in asthmatic rats,Provide reliable experimental basis,enrich the scientific connotation of Yiqi Wenyang Huwei method.Research method: The first chapter Theoretical discussion To collect and sort out the relevant literature on the name,etiology,pathogenesis and treatment of asthma from pre Qin to Ming and Qing Dynasties,and preliminarily summarize the understanding and treatment experience of modern doctors on asthma.This paper expounds the theoretical basis of TCM master Hong Guangxiang’s prevention and treatment of asthma by Yiqi Wenyang Huwei Method;Taking asthma immune imbalance as the breakthrough point,the theory of "weekness of Qi and Yang,Wei Qi deficiency" of traditional Chinese medicine is deeply connected with modern medical immune imbalance mechanism.The second chapter Experimental research Section one In vivo experimental research: Establish asthma rat models,which are divided into Control group(Control),Model group(Model),Low dose Yiqi Wenyang Huwei group(1g/m L),Medium dose Yiqi Wenyang Huwei group(2g/m L),High dose Yiqi Wenyang Huwei group(4g/m L)and the Dexamethasone group(Dex),eight rats in each group were treated at the end of 1 week,2 weeks and 4 weeks after the stimulation.Observation and testing indicators in each period: 1.Observe the general state and behavioral changes of rats during atomization stimulation.2.HE staining of pathological sections of lung tissue.3.Count and classification of inflammatory cells in bronchoalveolar lavage fluid.4.Flow cytometry was used to detect the proportion of Th17 and Treg cells in bronchoalveolar lavage fluid.5.RT-PCR was used to detect the m RNA expressions of TGF-β,IL-6,STAT3,RORγ,IL-17,IL-21,and Treg cell differentiation factors Foxp3,IL-10 in lung tissue.6.Western blot was used to detect the protein levels of Th17 cell differentiation factors TGF-β,IL-6,STAT3,RORγ,IL-17,IL-21,and Treg cell differentiation factors Foxp3,IL-10 in lung tissue.Section two In vitro experimental research: The imbalance model of Th17/Treg cells in vitro was established by inducing factors,and the serum containing Yiqi Wenyang Huwei decoction was given to intervene.The regulatory effect and specific regulatory mechanism of the serum containing Yiqi Wenyang Huwei decoction on Th17 / Treg cell imbalance in vitro were observed.1.Preparation of serum containing Yiqi Wenyang Huwei decoction.2.Primary isolation,culture and identification of CD4+ T cells.3.Cytotoxicity of serum containing Yiqi Wenyang Huwei decoction.4.Construction of Th17/Treg cell imbalance model in vitro: Control group(Control),Model group(Model),Yiqi Wenyang Huwei group(YQWYHW),Dexamethasone group(Dex),except the control group,the other groups were added the same amount of TGF-β,IL-6,IL-23 solution to induce CD4+T cells to differentiate into Th17 cells.5.Effect and mechanism of Yiqi Wenyang Huwei medicated serum on the imbalance of Th17/Treg cells in vitro: the corresponding medicated serum was intervened for 48 hours,and the proportion of Th17 and Treg cells in each group was detected by flow cytometry.RT-PCR and Western blot were used to detect the m RNA and protein expression levels of Th17 cell differentiation factors TGF-β,IL-6,STAT3,RORγ,IL-17,IL-21 and Treg cell differentiation factors Foxp3,IL-10.Research result: The first chapter Theoretical discussion This paper summarizes the evolution of TCM understanding of the name,etiology,pathogenesis and treatment of asthma,and summarizes some modern doctors’ understanding and experience of syndrome differentiation and treatment of asthma.On this basis,this paper expounds the characteristics of TCM master Hong Guangxiang’s asthma pathogenesis view of "weekness of Qi and Yang,Wei Qi deficiency" and the theoretical basis of using the method of Yiqi Wenyang Huwei Method to prevent and treat asthma,deeply connects the immune imbalance mechanism with the theory of "weekness of Qi and Yang,Wei Qi deficiency" in TCM,and lays a solid theoretical basis for the follow-up experimental research.The second chapter Experimental research Section one Results of in vivo experiments: 1.Effect of Yiqi Wenyang Huwei decoction on the behavior of asthmatic rats: After one week of stimulation,the rats in the model group and each drug treatment group showed asthma like symptoms,and rough hair,loss of luster,loss of appetite,decrease of food intake,like crouching,poor spirit and other phenomena of "Qi Yang weakness and insufficient defense Qi".There was no obvious abnormal performance in the control group.After intervention with Yiqi Wenyang Huwei decoction and dexamethasone respectively,the general state and behavioral changes of rats were improved in varying degrees compared with those of model group.2.Influence of Yiqi Wenyang Huwei decoction on the lung tissue pathology of asthmatic rats: Compared with the control group at the same period,the bronchial wall of the model group was significantly thickened,the lumen was significantly narrowed and deformed,smooth muscle hyperplasia,alveolar tissue and lumen mucosa edema,mucinous secretion increased significantly,bronchial epithelial cells arranged disorderly,and a large number of inflammatory cells infiltration could be seen.Compared with the model group at the same period,the inflammation infiltration of lung tissue in the low,medium and high dose group of Yiqi Wenyang Huwei decoction and the dexamethasone group were improved to different extent,mainly manifested in the reduction of stenosis and smooth muscle thickening of bronchus lumen,and the decrease of inflammatory cell infiltration and mucinous secretion.3.Effect of Yiqi Wenyang Huwei decoction on inflammatory cells in BALF of asthmatic rats: Compared with the control group in the same period,the proportion of eosinophils and lymphocytes in the model group were significantly increased(P<0.01).Compared with the model group in the same period,the proportion of eosinophils and lymphocytes in medium dose group,high dose group of Yiqi Wenyang Huwei decoction and dexamethasone group were significantly reduced(P < 0.05,P < 0.01).4.Effect of Yiqi Wenyang Huwei decoction on the ratio of Th17 cells and Treg cells in asthmatic rats: Compared with the control group in the same period,the proportion of Th17 cells in BALF of the model group were significantly increased(P<0.05),and the proportion of Treg cells was significantly decreased(P<0.05);Compared with the model group in the same period,the proportion of BALF Th17 cells in the medium dose group,high-dose group of Yiqi Wenyang Huwei and dexamethasone group were significantly decreased(P<0.05),and the proportion of Treg cells was significantly increased(P<0.05).5.Effect of Yiqi Wenyang Huwei decoction on Th17 cell and Treg cell differentiation factor gene in lung tissue of asthmatic rats:(1)Effect on Th17 cell differentiation factor gene: Compared with the control group in the same period,the relative m RNA expression level of TGF-β,IL-6,STAT3,RORγ,IL-17 and IL-21 in the model group were significantly increased(P<0.05,P< 0.01).Compared with the model group in the same period,the relative m RNA expression level of TGF-β,IL-6,STAT3,RORγ,IL-17 and IL-21 in the medium and high dose group of Yiqi Wenyang Huwei decoction and dexamethasone group were significantly increased(P<0.05,P<0.01).(2)Effect on Treg cell differentiation factor gene: Compared with the control group in the same period,the relative expression level of Foxp3 and IL-10 m RNA in the model group were significantly reduced(P<0.05,P<0.01).Compared with the model group in the same period,The relative expression level of Foxp3,L-10 m RNA in the medium dose group,high dose group of Yiqi Wenyang Huwei decoction and the dexamethasone group were significantly up-regulated(P<0.05,P<0.01).6.Effect of Yiqi Wenyang Huwei decoction on Th17 cell and Treg cell differentiation factor protein in lung tissue of asthmatic rats:(1)Effect on Th17 cell differentiation factor protein: Compared with the control group in the same period,the relative expression level of TGF-β,IL-6,STAT3,RORγ,IL-17 and IL-21 protein in the model group increased significantly(P<0.05);Compared with the model group in the same period,The relative expression level of TGF-β,IL-6,STAT3,RORγ,IL-17,IL-21 protein in the medium dose group,high dose group of Yiqi Wenyang Huwei decoction and the dexamethasone group were significantly reduced(P<0.05).(2)Effect on Treg cell differentiation factor protein: Compared with the control group in the same period,the expression level of Foxp3 and IL-10 protein in the model group were significantly reduced(P<0.05).Compared with the model group in the same period,The relative expression level of Foxp3 and IL-10 protein in the medium dose group,high dose group of Yiqi Wenyang Huwei decoction and the dexamethasone group were significantly up-regulated(P<0.05).Section two Results of in vitro experiments: 1.Construction of Th17 / Treg cell imbalance model in vitro results: compared with the control group,the proportion of Th17 cells and Th17 / Treg cells in the model group were significantly increased(P<0.01),and the proportion of Treg cells was significantly decreased(P<0.01),indicating the successful construction of Th17 / Treg cell imbalance model in vitro.2.Effect of the serum containing Yiqi Wenyang Huwei decoction on the ratio of Th17 cells and Treg cells in vitro: Compared with the model group,the proportion of the Th17 cells and Th17/Treg ratio of the Yiqi Wenyang Huwei group and the dexamethasone group were significantly reduced(P<0.05,P<0.01),the proportion of Treg cells was increased significantly(P<0.05,P<0.01).3.Effect of the serum containing Yiqi Wenyang Huwei decoction on Th17 cell and Treg cell differentiation factors m RNA in vitro:(1)Effect on Th17 cell differentiation factor m RNA in vitro: Compared with the control group,the relative expression level of TGF-β,IL-6,STAT3,RORγ,and IL-17,IL-21 m RNA in the model group were significant increased(P<0.05,P<0.01);Compared with the model group,the relative expression level of TGF-β,IL-6,STAT3,RORγ,IL-17,IL-21 m RNA were significantly reduced(P<0.05).(2)The effect on Treg cell differentiation factor m RNA in vitro: Compared with the control group,the expression level of Foxp3 and IL-10 m RNA in the model group were significantly reduced(P<0.01);Compared with the model group,the relative expression level of Foxp3 and IL-10 m RNA in the Yiqi Wenyang Huwei group and the dexamethasone group were significantly increased(P<0.05,P<0.01).4.Effect of the serum containing Yiqi Wenyang Huwei decoction on Th17 cell and Treg cell differentiation factors protein in vitro:(1)Effect on Th17 cell differentiation factors protein in vitro: Compared with the control group,the relative expression level of TGF-β,IL-6,STAT3,RORγ,IL-17 and IL-21 protein in the model group increased significantly(P<0.05,P<0.01);Compared with the model group,The relative expression level of TGF-β,IL-6,STAT3,RORγ,IL-17,IL-21 protein in the Yiqi Wenyang Huwei group and dexamethasone group were significantly reduced(P<0.05,P<0.01).(2)Effect on Treg cell differentiation factors protein in vitro:Compared with the control group,the expression level of Foxp3 and IL-10 protein in the model group were significantly reduced(P<0.01);Compared with the model group,the relative expression level of Foxp3 and IL-10 protein in the Yiqi Wenyang Huwei group and dexamethasone group were significantly increased(P<0.05,P<0.01).In conclusion 1.Yiqi Wenyang Huwei method can effectively improve the imbalance of Thl7/Treg in asthmatic rats and Thl7 / Treg imbalance in vitro.2.The mechanism of Yiqi Wenyang Huwei Method in treating asthma may be related to inhibiting IL-6-STAT3 signaling pathway,down-regulation of pro-inflammatory factors IL-17 and IL-21 to inhibit the differentiation and function of Th17 cells.3.The mechanism of Yiqi Wenyang Huwei Method in treating asthma may be related to up regulating the expression of Foxp3 and IL-10 to promote the differentiation and function of Treg cells. |
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