Analysis Of Intestinal Flora And Metabonomics Of Hirschsprung’s Disease

Background:Congenital megacolon,also known as Hirschsprung’s disease(HSCR),was first described by the Danish doctor Hirschsprung in 1886.The incidence rate is about 1 in 5000 live births.The cause of HSCR is that there is no ganglion cells in the distal intestinal wall.The pathophysiological mechanism is due to the absence of the enteric nervous system(ENS)and no nerve-mediated propulsion mode.The distal intestine is in a state of stenosis,and the proximal colon is stored,the secondary expansion of the gas and the hypertrophy gradually cause the megacolon lesion.The main symptoms of HSCR include constipation,vomiting,bloating and growth disorders.In untreated children,bloodstream bacterial infections usually die in childhood due to intestinal inflammation(colitis)or intestinal perforation.At present,the pathogenesis of HSCR is not fully understood,but there are more than ten susceptibility genes closely related to the pathogenesis of Hirschsprung’s disease,such as RET,GDNF,ECE1,EDN3,NRTN,SOX10,PHOX2 B,NRG1 and so on.In addition to genomics,consideration of changes in intestinal microenvironment has an impact on the development of ENS.Intestinal flora is a new hotspot of current research.It is not only the most complex and important microbial dynamic system in the body,but also plays a vital role in maintaining the dynamic balance between the body and the external environment.A stable flora environment is conducive to the growth and development of the infant and the establishment of the immune system.There are about 1000 kinds of bacteria in the intestine,the total number is 1 X 1014.Thick-walled bacteria and Bacteroides are the main bacteria in the intestinal flora,accounting for about 90% of the total number of intestinal flora.These microbes that settle in the intestine are interdependent with the human body,and they coexist peacefully with the host,interdependent and mutually restrictive.At present,there are literatures that Bifidobacteria and lactic acid bacteria in patients with Hirschsprung’s disease are lower than normal,and Bacteroides and thick-walled strains are more than normal.The occurrence of Hirschsprung’s disease is a complex process involving multiple genetic mutations and disorders of the intestinal microenvironment.Whether there is intestinal flora change or abnormal metabolites,and may play a role in the pathogenesis of Hirschsprung’s disease,only to solve the above problems,to provide a theoretical basis for understanding the disease.Objects:In this study,intestinal flora and metabolomics analysis were used to investigate the differences in intestinal flora and metabolic pathways in patients with Hirschsprung’s disease,in order to find a signal pathway related to the pathogenesis,and to develop a new direction for the comprehensive diagnosis and treatment of HSCR.Methods:(1)Thirteen children with congenital megacolon treated with neonatal surgery in Tianjin Children’s Hospital from April 2018 to December 2018 and 9mothers with stool and control normal children and their mother group were selected(no digestive diseases and Children who did not use antibiotics within 1 month and their mothers).Patient selection criteria: no glucocorticoid and any antibiotics were used within 30 days before fecal collection;his mother had no drinking habits;she was between the ages of 0 and 15,and the ratio of male to female was balanced.Hirschsprung’s disease was diagnosed by pathological examination.Exclusion criteria:children with a history of acute diarrhea or chronic diarrhea in the past week.The selected patients and their mothers were recorded as group 1(group1,n=13)and group 2(group2,n=9),respectively.The control group selected children and their mothers who did not have digestive system diseases and did not use antibiotics within one month.The children and mothers in the control group were recorded as group 3(group3,n=4)and group 4(group4,n=4),respectively.The clinical data,plasma samples and fecal specimens of the subjects were collected.Fecal DNA,was extracted and the V4-V5 hypervariable region of 16 S r DNA was amplified by PCR.The high throughput sequencing method was used to analyze the data obtained from sequencing,and the distribution structure,diversity and abundance of intestinal flora in congenital children were analyzed by constructing species evolutionary tree,Alpha diversity,Beta diversity and LEf SE.The representative strains which can be used to monitor intestinal microecosystem disorders and meet certain sensitivity and specificity in patients with Hirschsprung’s disease were screened out.(2)Metabonomics uses liquid chromatograph-mass spectrometry(LC-MS)to detect the collected research objects,find metabolic markers,analyze the related metabolic pathways,and further reveal the possible regulatory role and mechanism.Results: 1.From the distribution of the whole flora,the difference between the richness of the two groups of bacteria and the total level of the species was statistically significant(P<0.01).2.The children with HSCR have endocrine system,nervous system,immune system,secondary metabolite product system and transformation and migration,low content,low system regulation ability in16 S function prediction results show that at the level of lever2,and prone to disorder.At the level of lever3,PPAR signaling pathway,adipocytokines,glycophospholipid biosynthesis,sphingolipid(neurolipid)metabolism,linoleic acid metabolism,secondary bile acid biosynthesis,primary bile acid biosynthesis,NOD-receptor signaling pathway Progesterone-mediated oocyte maturation and flavonoid biosynthesis are low,suggesting a lipid metabolism synthesis disorder in children with HSCR,which may be inversely related to the pathogenesis of HSCR.There was no significant difference between the HSCR mother group and the normal mother group,indicating that the genetic association was small.3.Prediction results on differential proteins showed that COG1183,COG1640 and COG0464,etc.,demonstrated that lipid metabolism,carbohydrate metabolism and cell signal transduction disorders in children with HSCR are involved in the pathogenesis of HSCR.4.Comparative analysis of the full metabolic profile of feces in children with HSCR and normal controls,found that linoleic acid metabolism,bile secretion,sphingolipid metabolism,choline metabolism in tumors,phospholipase D signaling pathway,fat digestion and absorption in children with HSCR Significantly different from PPAR signaling pathway,sphingosine,lactosidic,sphingomyelin,sphingosine phosphorylcholine,cholesterol ester,cholesterol,bile compounds,lecithin,prostaglandin alpha 2,phosphatidylcholine,fatty acids,phospholipids C00865),9,10-dihydroxyoctadec-12-enoic acid and 9,10,13-trihydroxyoctyl-11-enoic acid down-regulated;L-glutamic acid,phospholipid(C00416),lysophosphatidylcholine Alkali,tetraethyl,9S-hydroxyoctadecadienoic acid and 13S-hydroxyoctadecadienoic acid are up-regulated.There is a correlation between the pathogenesis of HSCR and lipid metabolism.Conclusions: Based on high-throughput sequencing and metabolomics analysis,disorders of lipid metabolism,carbohydrate metabolism,and cell signaling in children with HSCR may impede the development of the enteric nervous system.On the other hand,both studies have shown differences in PPAR signaling pathways in children.