Correlation Analysis Of Abnormalities In The Gut Microbiome And Metabolome Of Kidney Transplantation Patients Based On 16s Sequencing And LC-MS

Kidney transplantation is the ultimate treatment option for end-stage renal disease.In recent years,with the progress of transplantation technology and the use of new immunosuppressive agents,the short-term survival rate after transplantation has been significantly improved.However,rejection and loss of graft function caused by ischemia-reperfusion injury and the toxicity of immunosuppressants are still the main reasons that threaten the long-term survival of transplant patients and organs.As we all know,from the occurrence of immune response to the appearance of clinical symptoms,various changes in metabolism,organ structure and function have been experienced during the period.Whether these changes can be monitored to enable accurate,rapid,non-invasive diagnosis and timely intervention before the appearance of clinical symptoms(subclinical or early)has always been a difficult problem in the field of transplantation.The emergence of metabolomics has provided multi-channel research perspectives and powerful technical support for such scientific problems.Using metabolomics to find potential biomarkers of kidney transplantation is not only helpful to reveal its pathological mechanism,but also provides a scientific basis for early diagnosis and prognosis after surgery.In addition,studies in recent years have found that the body’s intestinal flora will also undergo tremendous changes in disease states.At the same time,there is a close relationship between the intestinal flora and the body’s metabolism.This mutual influence forms a co-metabolism mechanism.It is of great significance to the physical health of the body.Therefore,we intend to study the synergistic changes between the structure of the intestinal flora of patients after kidney transplantation and the metabolic phenotype of the host,find its specificity compared with healthy people,and explore its role in the pathophysiological mechanism after transplantation.Objective: To use metabolomics detection technology and 16 s sequencing technology to study the differences in metabolite levels and changes in the structure of intestinal flora in transplant patients,and to explore the correlation between the two.Identify key bacterial species and metabolites,and reveal the pathophysiological mechanism of intestinal flora involved in the metabolism of kidney transplant patients through metabolic pathway enrichment analysis and flora function annotation.Methods: In this study,28 kidney transplant patients and 30 healthy people were recruited from the 924 th Hospital of the Joint Logistics Support Force of the Chinese People’s Liberation Army.Serum and stool samples of the patients were collected on the same day,using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLCQ/TOF-MS)technology.The serum and fecal metabolites were identified,and the V3-V4 regions of the 16 S r DNA gene of fecal microorganisms were comprehensively analyzed using Illumina Mi Seq technology to explore the structure of intestinal microbes.Obtain high-quality test data through a series of quality control and data preprocessing,screen differential metabolites and differential flora according to multivariate statistical analysis,and perform functional annotations on metabolites and microorganisms according to the KEGG database to analyze changes in metabolism and microbial levels The impact on the body.Further study the co-metabolism mode of the body and the intestinal flora according to the correlation analysis,as well as the correlation with the clinical phenotype,and analyze the metabolites and the intestinal flora that have a greater impact on the pathophysiological mechanism of the body after transplantation,and their interactions.Results:(1)The sum of the abundances of Firmicutes,Bacteroidetes,Actinobacteria,and Proteobacteria in healthy people and transplant patients exceeded 97%,And the relative abundance of Proteobacteria and Bacteroides changed greatly in transplant patients.And at the genus level,10 significantly different bacterial species were found that can be used as potential biomarkers.(2)We identified 127 significantly different metabolites in the serum(47 were down-regulated in the transplantation group and 80 were up-regulated),and 143 significantly different metabolites were identified in the feces(59 were down-regulated in the transplantation group and 84 were upregulated).(3)Carbohydrate metabolism,lipid metabolism,biodegradation and metabolism of heterologous organisms,metabolism of other amino acids,enhanced membrane transport and signal transduction of the microbial community were found in the transplantation group,while the metabolism of coenzymes and vitamins,and amino acid metabolism,Cell movement and translation are weakened.(4)The metabolic pathways that change greatly in feces are aminoacyl t RNA biosynthesis,phenylalanine metabolism,histidine metabolism,etc.In addition to the above metabolic pathways,the serum also includes lysine degradation,Amino acid metabolism such as aspartic acid metabolism.(5)Correlation analysis shows that in transplant patients,Enterococcus and Ruminococcus([Ruminococcus]＿torques＿group)have an increased influence on body metabolism.(6)Weighted co-expression network analysis divides 127 serum metabolites into 8 modules according to similar expression patterns.A total of 42 metabolites under two modules are clinically significantly correlated with creatinine,uric acid,and urea nitrogen(p<0.05).,r>0.6).It involves amino acid metabolism such as pantothenic acid and coenzyme A biosynthesis and phenylalanine metabolism.Conclusion:(1)Based on multivariate statistical analysis,renal transplantation patients are significantly different from healthy people in terms of serum metabolome,fecal metabolome or intestinal microbiome.(2)Amino acids and their derivatives,peptides,carbohydrates,fatty acids,phospholipids and other lipid molecules are differential metabolites that change significantly in kidney transplant patients.(3)In kidney transplant patients,opportunistic pathogens have increased significantly,while the abundance of intestinal probiotics has decreased,revealing the abnormal structure of the intestinal flora.(4)KEGG pathway analysis showed that the overall abnormality of amino acid metabolism and changes in energy metabolism in kidney transplant patients are similar to the function of intestinal flora,indicating that the intestinal flora may have a regulatory effect on body metabolism.(5)Statistical analysis shows that the relationship between gut microbes and metabolites in transplant patients is closer than that in healthy people.This difference may be caused by enterococci and rumen cocci.Kidney transplant patients have a unique interaction network between gut microbes and metabolites.(6)Through the WGCNA model,42 metabolites related to renal function were identified,involving aminoacyl t RNA biosynthesis,phenylalanine metabolism and other amino acid metabolism,and were significantly related to enterococcus and other differential flora,suggesting abnormal intestinal flora The structure may aggravate poor kidney metabolism.