Study On The Mechanism Of Yangyin Yifei Fang In The Treatment Of Chronic Obstructive Pulmonary Disease Patients With Qi And Yin Deficiency At The Stable Stage Based On Metabolomics

Chronic obstructive pulmonary disease(COPD)is a common and frequently occurring disease of respiratory system,which is characterized by persistent airflow limitation,and the airflow limitation is progressive.In recent years,with the aggravation of air pollution,the incidence of the disease is on the rise.The global burden of disease research projects that,to 2020,COPD will rank third,becoming one of the most important diseases cause of death worldwide,pose a significant threat to the health of all mankind,and bring heavy burden to the society,so the effective prevention and treatment has also been gradually put on the agenda.The focus of prevention and treatment of COPD is to prevent or reduce the number of acute attacks,so the treatment of COPD stability stage is particularly important.At present,in addition to the treatment of western medicine for COPD stable period,the traditional Chinese medicine(TCM)treatment of COPD has achieved remarkable results.However,in view of the prescription is compound preparation,each herb may contain a variety of active ingredients,and there may be a chemical reaction between the various pharmaceutical ingredients.The modern pharmacological research methods can only be used to evaluate the single effective components,and can not be used to study the mechanism of the action of the other agents.Therefore,it is urgent to find a research method suitable for the characteristics of TCM.Metabonomics is a new "omics" in addition to genomics,transcriptomics and proteomics,which has been widely applied in the field of life science.It analyzes the physiological and pathological states of organisms by analyzing the overallchanges of endogenous small molecule metabolites with molecular weight less than1000 under external stimulation.It regards an organism as awhole,which is very similar to overall concept of the theory of TCM.By comparing the changes of the metabolites in the body before and after treatment,the possible mechanism of prescription was discussed.The research method abandoned the study on the interaction between various pharmaceutical ingredients,and only for the overall mechanism of prescription,such as provides a new and powerful technique for the study of TCM.This study selected patients of chronic obstructive pulmonary disease(COPD)with qi and yin deficiency at the stable stage as research objects and adopted metabolic technology,to analyze plasma differential metabolites between the observation group and the normal group,to identify potential markers of syndrome lung qi and yin deficiency,so as to obtain objective understanding towards the nature of the syndrome.In addition,changes of plasma differential metabolites after the treatment with YangyinYifei Fang,etc.were observed to study the mechanism and ingredients of the compound drug,so as to provide theoretical basis for the prevention and treatment of the diseaseas as well as clinical treatment.The main content of this paper includes the following three parts:Part one Curative Effect and Influence on Serum Related Factors of Yangyin Yifei Fang on Chronic Obstructive Pulmonary Disease Patients with Lung Qi and Yin Deficiency at the Stable StageObjective:To observe the curative effect of Yangyin Yifei Fang at curing patients with qi and yin deficiency at the stable stage of COPD,and the influence of the serum level of lung injury factors interleukin 8(IL-8),interleukin 17(IL-17),tumor necrosis factor α(TNF-α),leukotrieneB4(LTB4),matrix metalloproteinase9(MMP9),tissue inhibitor of metallo-proteinase-1(TIMP-1),malondialchehyche(MDA),and superoxide Dismutase(SOD).Methods: According with diagnostic criteria,65 patients with qi and yin deficiency at the stable stage of COPD were chosen,and randomly divided into the control group(33 cases)and observation group(32 cases).The control group was given basic western medicine treatments,and the observation group was added with oral administration of Yangyin Yifei Fang,with one dosea day;both were treated for one month.The TCM symptom scores,dyspnea scores(mMRC),6 minutes walking test and the changes in pulmonary function before and after treatment as well as the total effective rates of both groups were compared.Test and compare the levels of IL-8,IL-17,LTB4,TNF-α,MMP9,TIMP-1,MDA,and SOD in both groups before and after treatment.Results: TCM symptoms scores of both groups were lower than those before treatment(P<0.05);after treatment,the scores of shortness of breath,wheezing and spontaneous perspiration susceptibility of the observation group were better than that of the control group(P<0.05).The total effective rates of the observation group and the control group were respectively 87.5% and54.6%,with the treatment group higher than the control group(P<0.05).After treatment,the pulmonary function and 6 minutes walking test of the observation group improved compared with before treatment and was better than that of the control group(P<0.05).The mMRC scores of both groups were lower than those before treatment(P<0.05),there was no significant difference between the two groups(P>0.05).Compared with before treatment,the levels of serum IL-8,IL-17,TNF-α and LTB4 of the observation group after treatment decreased,the difference was statistically significant(P<0.05),and the levels of serum IL-8 and TNF-α of the control group after treatment decreased,the difference was statistically significant(P<0.05).IL-17,TNF-αand LTB4 levels after treatment in observation group were lower than that of control group,the difference was statistically significant(P<0.05).After treatment,the MMP9 of the two groups was lower than that of before treatment(P<0.05),and the observation group was lower than the control group after treatment,the difference was statistically significant(P<0.05).There was no significant difference between the two groups before and after treatment with TIMP1(P>0.05).Compared with before treatment,the level of MDA in the observation group after treatment decreased,and SOD activity increased,the difference was statistically significant(P<0.05),while there were no significant difference of the level of MDA,SOD before and aftertreatment in the control group(P>0.05).Part two Studies of Plasma Metabonomics in Patients with Lung Qi and Yin Deficiency at the Stable Stage of Chronic Obstructive Pulmonary DiseaseObjective: To study the characteristics of plasma metabonomics of patients with lung qi and yin deficiency at the stable stage of COPD,and find the potential biomarkers;Then analysis the metabolic pathway of these potential biomarkers.Methods: 65 patients with lung qi and yin deficiency syndrome at the stable stage of COPD were chosen as the disease group,while 30 healthy people who came for physical examination were selected as control group.The blood samples were collected from both groups in the fasting state,anticoagulation and centrifugation were used to obtain the plasma.Then,use high performance liquid chromatography-mass spectrometry(HPLC-MS)to detect plasma metabolites between two groups,and use the principal component analysis(PCA)and orthogonal partial least squares-discriminant analysis(OPLS-DA)to identify the different metabolites,then analysis the metabolic pathway of these different metabolites.Results: In the positive ion mode,compared with the control group,there were significant differences of metabolites in disease group.Inosinic acid,guanosine triphosphate,ceramide,glucosylceramide,L-Phenylalanine,histidine,glycine,3-methylhistidine,ascorbic acid,alpha-linolenic acid,tetracosanoic acid,squalene were initially speculated as the potential biomarkers of patients with qi and yin deficiency at the stable stage of COPD.Among these metabolites,inosinic acid,guanosine triphosphate,histidine,glycine,3-methylhistidine,ascorbic acid,alpha-linolenic acid,tetracosanoic acid,squalene showed an downward trend,while others showed upward trend.The main metabolic pathways involved contain nitrogen metabolism,aminoacyl-tRNA biosynthesis,purine metabolism,glutathione metabolism,histidine metabolism,alpha linolenic acid metabolism,phenylalanine metabolism and glycine,serine and threonine metabolism.Part Three Study on the Intervention Mechanism of Prescription in the Treatment of Chronic Obstructive Pulmonary Disease Patie- nts with Lung Qi and Yin Deficiency at the Stable Stage Based on MetabonomicsObjective: Metabonomics technology was adopted to find out the differences in plasma metabolites before and after treatment in the observation group,analysis the metabolic pathways involved,and then speculate the possible mechanism of treatment of Yangyin Yifei Fang combined with conventional western medicines;and the observation group and the control group after treatment were compared to find differences in differential metabolites,and abnormal metabolic pathways were found,so as to identify the advantages of treatment of Yangyin Yifei Fang combined with conventional western medicines compared to the treatment of single conventional western medicines.Methods: 65 cases of patients with lung qi and yin deficiency at the stable stage who met diagnostic standard were randomly divided into the control group(33 cases)and the observation group(32 cases)by adopting random number table method.The control group underwent conventional western medicine treatment,the observation group was administrated with Yangyin Yifei Fang in addition to conventional western medicine treatment,and two groups were treated for a month;and plasma differential metabolites of the observation group before and after treatment,and between the two groups after the treatment were compared,then analysis the abnormal metabolic pathways involved.Results: The observation group presented significant difference in metabolism map after treatment under positive ion mode,and inosinic acid,guanosine triphosphate,glucosylceramide,3-methylhistidine,alpha-linolenic acid,tetracosanoicacid,20-COOH-leukotrieneE4 and lysoPC(6:0)were preliminarily identified as differential metabolites of the observation group after treatment of Yangyin Yifei Fang combined with conventional western medicines.Inosinic acid,guanosine triphosphate,3-methylhistidine,alpha-linolenic acid,tetracosanoic acid increased and the remaining three substances declined.The main pathways may be alpha linolenic acid metabolism,purine metabolism,riboflavin metabolism,histidine metabolism,phospholipid metabolism,etc.The two groups were compared after treatment and five differential metabolites were identified,such as inosinic acid,3-methylhistidine,tetracosanoic acid,20-COOH-leukotrieneE4 and 20-trihydroxy-leukotriene-B4,among which inosinic acid,3-methylhistidine,tetracosanoic acid increased and the other two substances decreased in the observation group.The main metabolic pathways involved may be purine metabolism,histidine metabolism and metabolism of arachidonic acid.Conclusions:1 Yangyin Yifei Fang can improve the symptoms of coughing,wheezing,shortness of breath,spontaneous perspiration and thirsty,reduce mMRC score and improve lung function and 6 minute walking distance,showing curative effect.2 Yangyin Yifei Fang can reduce the levels of serum IL-8,IL-17,TNF-α,LTB4,MMP9 and MDA,and increase the activity of SOD,playing an important role in preventing inflammation,improving protease / antiprotease system and fighting against oxidative stress.3 Under positive ion mode,inosinic acid,guanosine triphosphate(GTP),ceramide,glucosylceramide,L-phenylalanine,histidine,glycine,3-methylhistidine,ascorbic acid,alpha-linolenic acid,tetracosanoic acid and squalene may be marker metabolites of COPD patients with qi and yin deficiency at the stable stage.The main metabolic pathways involved contain aminoacyl-tRNA biosynthesis,nitrogen metabolism,purine metabolism,glutathione metabolism,histidine metabolism,alpha linolenic acid metabolism,phenylalanine metabolism and glycine,serine and threonine metabolism.4 Under positive ion mode,inosinic acid,guanosine triphosphate,glucosylceramide,3-methylhistidine,alpha-linolenic acid,tetracosanoic acid,20-COOH-leukotrieneE4,lysoPC(6:0)were preliminarily identified as differential metabolites of the observation group after treatment of YangyinYifei Fang combined with conventional western medicines.The main pathways may be alpha linolenic acid metabolism,purine metabolism,riboflavin metabolism,histidine metabolism,phospholipid metabolism,etc.5 Two groups after treatment were compared under positive ion mode,initially identifying that inosinic acid,3-methylhistidine,tetracosanoic acid,20-COOH-leukotrieneE4 and 20-trihydroxy-leukotrieneB4 were preliminarily identified as differential metabolites between treatment of Yangyin Yifei Fang combined with conventional western medicines and single western medicines.The main metabolic pathways involved may be purine metabolism,histidine metabolism and metabolism of arachidonic acid.