A Preliminary Study On The Changes Of Gut Microbita Composition And Its Pathogenic Mechanism In Chronic Obstructive Pulmonary Disease

BackgroundIn recent years,the academic circle has found that the disorder of intestinal flora is closely related to a variety of diseases,such as inflammatory enteritis,irritable bowel syndrome,bowel cancer,diabetes,autoimmune encephalomyelitis and other diseases.Many studies have shown that intestinal flora plays an important role in immune regulation,and the imbalance of intestinal flora can break the balance of human immune response.Further studies have shown that Chronic Obstructive Pulmonary Disease(COPD),a chronic inflammatory disease,is closely linked to the intestinal flora.Systemic hypoxia in patients with COPD can lead to intestinal dysfunction and increased intestinal permeability.Intestinal flora and its harmful products can enter the parenteral tissues and blood circulation through the intestinal barrier,further triggering a local intestinal immune response and aggravating inflammation.This drastic change in the intestinal environment may lead to an imbalance of intestinal flora.At present,the previous work of our research group has found that there is indeed an imbalance in the intestinal flora of patients with COPD,and its imbalance is involved in the occurrence and progression of COPD.However,the characteristics of the intestinal microbiota in patients with COPD remain to be determined.At the same time,the pathway through which the unbalanced intestinal microbiota is involved in the progression of chronic obstructive pulmonary disease has not been cleared up,and the impact of the products of intestinal microecology on the intestinal epithelial barrier remains unknown.As COPD usually co-occurs with gastrointestinal diseases,it is suggested that the imbalance of intestinal flora in COPD may be closely related to intestinal health.Research purposeThe objective of this study was to investigate the characteristics of intestinal flora in patients with chronic obstructive pulmonary disease and its involvement in the progression of chronic obstructive pulmonary diseaseResearch methods and resultsPart I: Metagenomic sequencing analysis of intestinal microbial flora in patients with chronic obstructive pulmonary diseaseMethod: We collected stool samples from 80 healthy people and 80 COPD patients(31 GOLD I-II and 49 GOLD III-IV)who met the inclusion criteria for microbiome analysis,characterized the intestinal microbiome through bioinformatics,and determined microbial markers that could be used to construct COPD disease classifiers.Results: Fecal microbial diversity was higher in patients with COPD I or II than in healthy controls,but lower in patients with COPD III or IV.Twenty-one,24,and 11 microbial species,including potential pathogens and pro-inflammatory bacteria,were significantly enriched or decreased in healthy controls,COPD I or II patients.KEGG orthology(KO)gene profile revealed significant differences in intestinal microbial function among the three groups.In addition,intestinal microbiota classification and functional markers were used to distinguish COPD patients from healthy controls,with AUCs of 0.8814 and 0.8479,respectively.Notably,there were differences in the intestinal microbiota classification between healthy individuals and patients with early(I-II)COPD,indicating the value of fecal metagenomic biomarkers in the early diagnosis of COPD(AUC=0.9207).Part two: Study on the effect of intestinal contents on intestinal epithelial cells in patients with chronic obstructive pulmonary diseaseMethod: We collected Stool of 30 healthy people and 30 patients with COPD who met the inclusion criteria.Stool traits were scored according to the Bristol Stool Scale,and then the difference of stool traits between healthy people and COPD patients was compared,and correlation analysis was conducted between stool traits and lung function indexes.Differences in intestinal function between COPD patients and healthy people were detected.Then feces of healthy people and COPD patients were mixed with PBS respectively,and sterile intestinal contents were prepared by centrifugal filtration.Intestinal epithelial cells were cultured through the air-liquid interface using transwell cell plate for 21 days,and the integrity of the intestinal epithelial cell layer model was judged by the transmembrane resistance value.The intestinal epithelial cells were stimulated by PBS,intestinal contents fluid of healthy people and intestinal contents fluid of COPD patients for 2 days,respectively.The permeability changes of intestinal epithelia were detected by transmembrane resistance and FITC-Dextran,and then the expression of tight junction protein was detected by q PCR,immunofluorescence and wb.Finally,the cell activity was detected by cck8 cytotoxicity test.Results: Intestinal dysfunction is common in COPD patients.The permeability of intestinal epithelial cell layer barrier increased significantly under the stimulation of intestinal fecal fluid.After treatment by different groups,the expression of tight junction proteins ZO-1,claudin-1,occludin and e-cadherin did not change significantly.In terms of cytokine detection,there was no significant difference in the expression of TNF-α,IL-6 and IL-8 in the intestinal fecal fluid of COPD patients compared with the control group and the fecal fluid group of healthy people.Conclusions1.The species classification and functional composition of intestinal microorganisms in patients with COPD have unique characteristics,providing preliminary evidence as a diagnostic tool for COPD.2.Intestinal contents of COPD patients have toxic effects on intestinal epithelial cells,and seriously damage the integrity of intestinal epithelium without affecting the expression of tight junction protein,greatly increasing the permeability of intestinal epithelial barrier.