The Metabolomics Characteristics Of COPD Combined With T2DM Patients And Their Correlation With Changes Of Th17/Treg Research

Background:Chronic obstructive pulmonary disease(COPD)is a common and prevalent disease that seriously endangers human health.Currently,it is the fourth leading cause of death in the world,and is expected to rise to no.3 by 2020.COPD not only affects the respiratory system,but also affects all organs and systems from multiple links.COPD combined with Type 2 Diabetes mellitus(T2DM)is a common clinical comorbidity,about 10%T2DM patients combine with COPD,and the prevalence of COPD patients with diabetes is as high as 18.7%.Patients with COPD combined with T2DM have severe clinical symptoms and poor prognosis.COPD and T2DM are two different diseases,but recent studies have shown that there is a close pathophysiological link between them,which leads to the disease in the process of occurrence and development of immune disorders and metabolic disorder interaction jointly to promote the complexity of the condition,therefore,the in-depth study of relevant mechanism has important clinical significance.Metabolomics technology,as one of the molecular monitoring technology,can effectively monitor through high-throughput analysis of human body during the process of transition from health to disease states,and on the whole intensively analyze the pathophysiology of disease.The interdependence of immune response and metabolic regulation is the core of the body to maintain steady state.In recent years,as the concept of immunometabolism has been proposed,it has been considered that the independent immune and metabolomics are linked,thus providing a new perspective for the elucidation of the complex mechanism of disease.COPD as a kind of inflammatory lung diseases,immunity plays an important role in its pathogenesis,and systemic chronic low-grade inflammation is a common characteristic of COPD and T2DM,meanwhile there exists pathological state of metabolic disorders.With the development of the disease,COPD may be associated with the risk of T2DM,but the relevant immune cell changes and molecular mechanisms are still being studied.Thl7 and Treg cells are the CD4+T lymphocyte subsets found in recent years.The balance between them is of great significance for the body to maintain the immune homeostasis and maintain normal function of the organ.Based on this clue,this paper carried out the analysis of serum metabolomics in patients with COPD combined with T2DM;the changes of Th17/Treg in peripheral blood of patients with COPD combined with T2DM and their correlation with metabolic disorders;the functional changes of Th17 and Treg in patients with COPD combined with T2DM and their correlation with metabolic disorders.Objectives:This study intends to use the metabolomics method to detect the small molecular metabolites in serum,to explore changes of metabolites and metabolic pathways in patients with COPD combined with T2DM,and to detect Th17 and Treg cells,and the expression of associated transcription factors and cytokines in COPD combined T2DM patients' peripheral blood.Through the new perspective of immunometabolism to intensively study pathological physiology and pathogenesis of COPD combined T2DM,so as to find new diagnostic methods and therapeutic targets for clinic to provide favorable basis.Methods:1.32 COPD combined with T2DM patients,36 COPD patients,35 type 2 diabetes patients and 30 healthy subjects were as research objects at our hospital from March 2016 to September 2017.Demographic and clinical baseline data were collected,including lung function index,blood glucose,and glycosylated hemoglobin,etc.2.The 1H-NMR spectrum of serum samples were determined by NMR spectrometer,and the baseline,phase adjustment and section integration were performed after Fourier transform of all the spectra.Using SIMCA-P+11.0 software,the pattern recognition analysis was carried out through principal component analysis,partial least squares discriminant analysis and orthogonal partial least-squares discriminant analysis.One-way analysis of variance was used to find the differential metabolites in each group.Based on Kyoto Encyclopedia of Genes and Genomes Pathway Database(KEGG),the related metabolic pathways were explained.3.The number of Th 17 and Treg cells in peripheral blood was detected by flow cytometry.The ratio of Th17 and Treg cells to CD4+T cells and Th17/Treg ratio were calculated respectively.Using one-way analysis of variance to analyze the data,and further analyze the influence of small molecular metabolites detected by 1H-NMR metabolomics on the balance of Th17/Treg.4.Real-time fluorescence quantitative PCR was used to detect the expression levels of Th17 and Treg cell specific transcription factor ROR-?t and Foxp3 mRNA in peripheral blood of each group,and to use one-way analysis of variance to carry out data statistics.5.The levels of inflammatory cytokines in Th17 and Treg cells were detected by ELISA.To use one-way analysis of variance to carry out data statistics,furthermore,the effects of small molecular metabolites on Th17/Treg function were further analyzed.Results:1.After multivariate statistical analysis of the metabolites obtained through the 1H-NMR analysis,we found significant differences in serum metabolites among the four groups.Compared with healthy control group,leucine,isoleucine,valine,L-alanine,proline,methyl histidine,triglycerides,LDL,acetoacetic acid,acetone,succinic acid,D-galactose and lactic acid,pyruvic acid,and glucose levels were higher,and glycine,lysine,tryptophan,arginine,glutamic acid,glutamine,HDL,choline,taurine,betaine,vitamin D levels were lower in COPD+T2DM group(P<0.05).Compared with COPD group,leucine,isoleucine,valine,L-alanine,proline,triglyceride,LDL,acetoacetic acid,acetone,succinic acid,D-galactose,lactic acid,pyruvate,glucose levels were higher,and glycine,lysine,tryptophan,glutamic acid,glutamine,HDL,choline,taurine,betaine,and vitamin D levels were lower in COPD+T2DM group(P<0.05).Compared with T2DM group,methylhistidine,acetoacetic acid,acetone,lactic acid,pyruvic acid levels were higher,and tryptophan,glutamic acid,glutamine,HDL,taurine levels were lower in COPD+T2DM group(P<0.05).2.Compared with healthy control group,peripheral blood Th17 cells accounted for a higher proportion of CD4+T lymphocytes in COPD group,T2DM group and COPD+T2DM group(P<0.01).Compared with COPD group or T2DM group,peripheral blood Th17 cells accounted for a higher proportion of CD4+T lymphocytes in COPD+T2DM group(P<0.01).There was no significant difference in the ratio of peripheral blood Th17 cells to CD4+T lymphocytes between COPD group and T2DM group(P>0.05).Compared with healthy control group or COPD group,peripheral blood CD4+CD25+Treg accounted for a lower proportion of CD4+T lymphocytes in COPD+T2DM group(P<0.01),and there was no significant difference between COPD+T2DM group and T2DM group(P>0.05).Compared with healthy control group or COPD group,peripheral blood CD4+CD25+Treg accounted for a lower proportion of CD4+T lymphocytes in T2DM group(P<0.01).There was no significant difference in the ratio of peripheral blood CD4+CD25-Treg to CD4+T lymphocytes between COPD group and healthy control group(P>0.05).Compared with healthy control group,the ratio of peripheral blood Th17 to CD4+CD25+Treg was higher in COPD group,T2DM group and COPD+T2DM group(P<0.01).Compared with COPD group or T2DM group,the ratio of peripheral blood Th17 to CD4+CD25+Treg was higher in COPD+T2DM group(P<0.01).There was no significant difference in the ratio of peripheral blood Th 17 to CD4+CD25+Treg between COPD group and T2DM group(P>0.05).3.Compared with healthy control group,COPD group or T2DM group,the expression of RORyt was higher in COPD+T2DM group,the most significant difference was with the health group(P<0.01),compared with the COPD group and T2DM group,the difference was statistically significant(P<0.01).Compared with healthy control group,the expression of RORyt was higher in COPD group or T2DM group(P<0.01).Compared with COPD group or T2DM group,expression of RORyt was higher in COPD+T2DM group,but there was no significant difference(P>0.05).Compared with healthy control group,COPD group or T2DM group,the expression of Foxp3 was lower in COPD+T2DM group(P<0.01).Compared with healthy control group,the expression of Foxp3 was lower in COPD group or T2DM group(P<0.01).There was no significant difference in the expression of Foxp3 between COPD group and T2DM group(P>0.05).4.Compared with healthy control group,COPD group or T2DM group,serum IL-17A,IL-17F,IL-21,IL-23 and IL-6 levels were higher in COPD+T2DM group(P<0.05).Compared with healthy control group,serum IL-17A,IL-17F,IL-21,IL-23 and IL-6 levels were higher in COPD group or T2DM group(P<0.05).Compared with healthy control group,COPD group or T2DM group,serum TGF-?1and IL-10 levels were lower in COPD+T2DM group(P<0.05).Compared with healthy control group,serum TGF-?1and IL-10 levels were lower in COPD group or T2DM group(P<0.05).Conclusions:1.Metabolic disorders in patients with COPD may increase the risk of type 2 diabetes.Metabolic disorders in COPD combined with T2DM patients are more complex and serious than COPD patients.The changes of the metabolic spectrum characteristics partly reflect the abnormal pathophysiological changes of the COPD combined with T2DM patients,which may provide favorable clues for the early diagnosis of the disease.Regulation and improvement of metabolic disorders in COPD combined with T2DM patients may be conducive to early treatment,management and intervention of the disease.2.In COPD combined with T2DM patients,there are abnormal and proportional imbalance of Thl7/Treg in peripheral blood,and the balance is transferred to pro-inflammatory Th17 cells.The expression of Th17 cell specific transcription factor RORyt is up-regulated in COPD combined with T2DM patients,and the expression of Treg cell specific transcription factor Foxp3 is down-regulated.The imbalance of RORyt/Foxp3 may be one of the important causes of Th17/Treg dysfunction.3.In COPD combined with T2DM patients,Th17 cells related proinflammatory cytokines levels elevate,and Treg cells related anti-inflammatory cytokines levels drop,which indicates peripheral blood of Thl7/Treg function imbalance,and the balance is transfered to the proinflammatory direction.It is shown that there is an immune regulation disorder in COPD combined with T2DM patients.The imbalance of Th17/Treg in peripheral blood may be an important 'reason for the development of COPD combined with T2DM,and the balance of Th17/Treg cells may be beneficial to delay the progression of disease.COPD combined with T2DM patients with the metabolic disorder may affect the balance of Th17/Treg and their function,by maintaining metabolic homeostasis or will find the best balance to inhibit excessive inflammatory reaction and not reduce body's immune response to infection,which is conducive to disease control.