| Part 1 Clinical analysis of 214 infants with cholestatic hepatopathyObjectives:To explore the clinical characteristics and etiology of infantile cholestasis hepatopathy(ICH)so as to assist early diagnosis and treatment.Methods:The basic information of all children with ICH(infant hepatitis syndrome)who were hospitalized in the Department of Pediatrics of the First Affiliated Hospital of Kunming Medical University during the 12 years from January 1,2007 to December 31,2018 were collected.The clinical data were record and the etiology of the primary disease were analyzed.Results:In the past 12 years,214 children diagnosed as ICH were admitted to the Department of Pediatrics of a hospital in Kunming.The population of Yunnan province accounted for 94.4%and the minority population accounted for 21.5%.The disease accounted for 49%of the total number of children hospitalized for hepatobiliary diseases.The average hospitalization age was 72 days and the median hospitalization time was 11 days.ICH of unknow origin,CMV infection and urinary tract infection are the top three causes of ICH in our hospital.In the past five years,the proportion of ICH caused by hereditary metabolic diseases has increased.Of the 214 infants with ICH,11.7%had congenital malformations and 20.1%had respiratory infections.69.6%of the children were getting better when they discharged from hospital.Conclusions:1.The top 5 causes of ICH aomong these 214 infants are:ICH of unknown origin,CMV infection,urinary tract infection,hereditary metabolic disease of unknow origin and biliary atresia.The disease occurs in many ethnic populations.2.ICH is the most common cause of hospitalization for hepatobiliary diseases in pediatric inpatients in our hospital.3.Early identification of the primary cause and early targeted treatment are the key points to the patient's cooperation and the key points to the improvement of condition,genetic metabolism screening and genetic diagnosis should be started as soon as possible.Part 2 Detection and analysis of fecal bile acids in infants with cholestatic hepatopathyObjectives:To detect the contents of fecal primary bile acids CA,CDCA,GCDCA and fecal secondary bile acids DCA,LCA and UDCA of ICH infants.To compare and analyze the characteristics of the changes of fecal bile acid in normal infants and ICH infants before and after treatment.Methods:From November 2014 to November 2015,30 infants diagnosed as ICH in Department of Gastroenterology,Children's Hospital of Kunming Medical University were enrolled.The fecal specimens and liver function test results of these ICH infants were collected before treatment and defined as Non-Treated ICH Group.After 7 days treatment of protecting liver and promoting gallbladder,fecal specimens and liver function tests result of these ICH infants were collected again,defined as Treated ICH group.Compared the results of the two liver function tests before and after treatment,the Treated ICH Group were divided in to two groups:Treated Non-remission ICH Group and Treated Remission ICH Group.Fecal samples from 30 healthy infants were collected as the Health Group.The contents of primary bile acids CA,CDCA,GCDCA and secondary bile acids DCA,LCA and UDCA in the feces of ICH infants and health infants were detected by liquid chromatography-mass spectrometry.The results were analyzed and compared.The long-term prognosis of 30 ICH infants was followed up by telephone.Results:In the comparison of fecal primary bile acids,the contents of CA,CDCA and GCDCA all showed as Non-Treated ICH Group<Health group,Treated ICH Group<Health group,the difference was statistically significant(P<0.05).In the comparison of fecal secondary bile acids,the content of LCA showed as Non-Treated ICH Group<Health group,Treated ICH Group<Health group;the content of UDCA shown as Non-Treated ICH Group<Health group,Non-Treated ICH Group<Treated ICH Group,the difference was statistically significant(P<0.05).The ICH-treated group was further divided into Treated Remission ICH Group and Treated Non-remission ICH Group.The contents of CA in Treated Remission ICH Group and Treated Non-remission ICH Group were both lower than Health Group.The contents of CDCA and LCA showed as Treated Non-remission ICH Group<Health Group.The content of UDCA showed as Non-Treated ICH Group<Treated Remission ICH Group and Treated Non-remission ICH Group,the difference was statistically significant(P<0.05).10 children who's liver function did not remission after 7 days therapy have returned to normal now.Conclusions:1.Fecal bile acid contents in ICH infants was different from that in health infants,which may be caused by the decrease of bile acid excretion and the change of gut bacteria microflora during the disease.2.Short-term(7 days)treatment does not represent the long-term prognosis of ICH infants.Part 3 Detection and analysis of fecal short chain fatty acids in infants with cholestatic hepatopathyObjectives:To detect the contents of fecal acetic acid,propionic acid,butyric acid,isobutyric acid and isovaleric acid of ICH infants.To compare and analyze the characteristics of the changes of fecal short chain fatty acids in normal infants and ICH infants before and after treatment.Methods:The case selection criteria and grouping criteria were the same as the part 2.Gas chromatography was used to detect the content of short chain fatty acids including acetic acid,propionic acid,butyric acid,isobutyric acid and isovaleric acid in ICH infants and health infants.The results were analyzed and compared.Results:The contents of carbohydrate-related metabolites acetic acid,propionic acid and butyric acid were compared,it showed that there is no significant difference among the Health Group,Non-Treated ICH Group and Treated ICH Group.The contents of protein-related metabolites isobutyric acid and isovaleric acid were compared,it showed that the content of fecal isobutyric acid in the Non-Treated ICH Group and Treated ICH Group was significantly lower than that in the Health Group,and the content of fecal Isovaleric acid in the Non-Treated ICH Group was significantly lower than that in the Health Group(P<0.05).The contents were compared again after the ICH-treated group was further divided into Treated Remission ICH Group and Treated Non-remission ICH Group.It showed that the contents of acetic acid,propionic acid and butyric acid has no significant difference among four groups(P>0.05),the content of isobutyric acid showed that Treated Non-remission ICH Group<Health Group(P<0.05).Conclusions:1.There is no significant change in intestinal carbohydrate metabolism in ICH infant.Abnormal protein metabolism in the intestine was found in ICH infant,it may cause by gut bacteria microflora dysbiosis.2.Fecal isobutyric acid may be a potential indicator of whether the disease is improving.Part 4 Detection and analysis of gut bacteri microflora in infants with cholestatic hepatopathyObjectives:To explore the composition and diversity of gut bacteria microflora in infant with ICH before and after treatment as well as remission and non-remission,and to find microbial biomarkers related to the occurrence and development of ICH,so as to provide theoretical basis for developing more.accurate and non-invasive diagnostic methods.Methods:The case selection criteria and grouping criteria were as same as the part 2.16S rDNA analysis was used to detect the composition and diversity of intestinal microflora in infant with ICH before and after treatment as well as remission and non-remission.49 successfully sequenced samples were named as "All Cases Included Group" for Alpha diversity analysis and differentially enriched gut bacteria microflora.The samples that were completely paired before and after treatment were named as "Paired Study Group",the Alpha diversity and Beta diversity of the gut bacteria microflora of this group were analyzed.The fecal bile acids and fecal short chain fatty acids corresponding to 38 samples were compared and analyzed.The random forest algorithm and Spearman correlation analysis were used to find the OTUs associated with ICH and related to bile acid metabolism.Then,16s rRNA gene representative sequences of OTUs were blasted against the Microbial Whole-Genome Database in NCBI.Then,the matched genomes was submitted to KEGG,and was mapped to the secondary BA biosynthesis pathway(ko00121),in order to ildentify the gut bacteria microflora which contains the bile salt hydrolase(BSH),a key enzyme in bile acid metabolism,and to search for candidate microbial biomarkers for the diagnosis of ICH.Results:In 49 cases of "All Cases Included Group",the values of Richness,Chao and ACE in three groups showed as Treated ICH group<Health Group,Treated ICH group<Non-Treated ICH group,the difference was statistically significant(P<0.05),there was no significant difference in Shannon and Simpson index between the three groups.The abundance of Firmicutes in the Non-Treated ICH Group was higher than that in the Health Group.The abundance of Bacteroidetes in the Non-Treated ICH Group was lower than that in the Health Group.The abundance of Candidatus Saccharibacteria was higher in the Non-Treated ICH Group than in the Health Group and Treated ICH Group.The abundance of Cyanobacteria/Chloroplast was higher in Treated ICH Group than in Health Group and Non-Treated ICH Group(P<0.05).Of the 49 samples,a total of 66 genera were detected,and the dominant abundances in the group were Bacteroide,Escherichia/Shigell,Bifidobacteriu,Streptococcus,Clostridium sensu stricto,Veillonella,Lactobacillus,Enterococcus,a nd Akkermansia.There were significant differences in 20 genera between Health Group and Non-Treated Group,and there were significant differences in 17 genera between Health Group and Treated Group,and there were significant differences in 30 genera between Treated Group and Non-Treated Group,the difference was statistically significant(P<0.05).In 38 cases of "Paired Study Group",db-RDA analysis found that CA,CDCA,DCA,LCA,UDCA,acetic acid,propionic acid,butyric acid and isovaleric acid can affect the distribution of gut bacteria microflora in ICH infants.Random forests found that 34 OTUs were important factors associated with ICH,and Spearman correlation analysis found a significant correlation between fecal bile acids and OTUs,the most meaningful 59 OTUs was chose.The above two parts of OTUs were blasted against the whole genome database of NCBI and the secondary bile acid biosynthesis pathway(ko00121)of KEGG to find the species containing BSH.After the comparison and analysis,Streptococcus,Parabacteroides,Lactobacillus and Blautia are candidate microbial biomarkers which is related to the occurrence and development of the diseases in ICH infants.Conclusions:1.There are different phyla and genera between health infants and ICH infants,gut bacteria microflora of ICH infants are dysbiosis.There is a significant correlation between gut bacteria microflora and fecal bile acids,it showed that gut bacteria microflora and bile acid metabolism were interacted.2.Fecal Streptococcus,Parabacteroides,Lactobacillus and Blautia can be used as candidate microbial biomarkers related to the occurrence and development of the diseases in ICH infants,which can provide a basis for subsequent research on clinical non-invasive diagnostic markers.Part 5 Correlation between gut becteria microflora,becteria microflora related metabolites and liver function in infants with cholestatic hepatopathyObjectives:To analyze the correlation between gut bacteria microflora and fecal short chain fatty acid in ICH infants and between gut bacteria microflora and liver function index.To analyze the correlation between host-flora co-metabolite,fecal bile acids and fecal short chain fatty acids,and the correlation between host-flora co-metabolite and liver function in ICH infants.Methods:The case selection criteria and grouping criteria were as same as the part 2,part 3 and part 4.Spearman correlation analysis was used to analyze the correlation between gut bacteria microflora,fecal short chain fatty acids and liver function indexes.And analyzed the correlation between fecal bile acids and fecal short chain fatty acids,and their correlation with liver function indicators in ICH children.Results:There were significant correlations between 13 genera and fecal short chain fatty acids in Non-Treated ICH Group,and significant correlations between 12 genera and fecal short chain fatty acids in Treated ICH Group.There were significant correlations between 34 genera and liver function indexes in Non-Treated ICH Group,and significant correlations between 13 genera and liver function indexes in Treated ICH Group.Multiple fecal bile acids and short chain fatty acids were significantly correlated in Non-Treated ICH Group and Treated ICH Group,among which fecal isobutyric acid was correlated with all fecal bile acids.There were correlations between fecal CA,CDCA and LCA compare with liver function indexes in Non-Treated ICH Group,and there were correlations between fecal CA,CDCA and GCDCA compare with liver function indexes in the Treated ICH Group.There were correlations between fecal propionic acid,butyric acid,isobutyric acid,isovaleric acid compare with liver function indexes in Non-Treated ICH Group,and there were correlations between isobutyric acid and liver function indexes in Treated ICH Group.Conclusions:1.There was a significant correlation between gut bacteria microflora and liver function in ICH infants.Gut bacteria microflora could be used as a microbial biomarker for ICH diagnosis.2.The contents of fecal CA and CDCA can simultaneously reflect the changes of liver function indexes related to cholestasis in ICH infants.3.The combined application of fecal isobutyric acid content and other indicators reflecting bile acid excretion function may have better clinical significance and application prospects. |
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