| Objective: To explore the changes of gut bacteria in constipated children affect the expression of glial-derived neurotrophic factor(GDNF)in the intestinal wall by regulating the body’s inflammatory factor levels affect intestinal wall intestinal innate immune lymphocyte 3(ILC3)release interleukins-22(IL-22),and to provide new ideas for the precise diagnosis and treatment of in children with constipation by comparing the changes of gut bacteria in children with different types of constipation and their effects on inflammatory factors,ILC3 and intestinal GDNF.Methods: From December 2020 to December 2022,twenty children with constipation,including 12 males and 8 females,were selected from the Department of Pediatric Surgery in the Second Hospital of Hebei Medical University,with an average age of 3.750±2.381 years old.They were divided into the Hirschsprung’s disease(HD)group and sow transit constipation(STC)group,with 10 cases in each group.Another 10 children who underwent closure surgery after traumatic colostomy were selected as the control group,including7 males and 3 females,with an average age of 3.900±2.807 years.The changes of gut bacteria in children with different types of constipation were analyzed by16 S r RNA sequencing.Kruskal Wallis H test and Mann-Whitney U test were used to compare the differences between the groups.The bacteria species with the largest abundance difference among the three groups were screened.The serum levels of constipation-related inflammatory factors were determined by multi-immunomicrosphere assay.Kruskal Wallis H test and Mann-Whitney U test were used to compare the differences between the groups.Spearman correlation analysis was used to screen the highest inflammatory factors associated with gut bacteria in constipation.The expressions of ILC3,IL-22,and GDNF in each group were detected by immunofluorescence staining.Kruskal Wallis H test and Mann-Whitney U test were used to compare the differences between the groups.Linear regression was used to select the inflammatory factor and the expressions of ILC3,IL-22,and GDNF in the intestinal wall,respectively,through single-factor analysis and multifactor analysis.The aim was to observe and compare how gut bacteria variation regulate the release of inflammatory factors in children with different types of constipation,and then affect GDNF in the intestinal wall through ILC3、IL-22 pathway.Results:1.Analysis of Gut microbiota: The abundance of Enterococcus increased in HD group and STC group,and the abundance of Enterococcus in HD group was higher than that in STC group(P < 0.05),on the contrary,the abundance of Faecalibacterium decreased in HD group and STC group,and the abundance of Faecalibacterium in HD group was lower than that in STC group(P < 0.05).2.Analysis of the inflammatory factors: The most correlated inflammatory factors with constipation were IL-6(H=21.099,P<0.01),IL-1β(H=16.761,P<0.01),IL-8(H=18.418,P<0.01),and IL-12p70(H=17.133,P<0.01).At the genus level,Enterococcus was positively correlated with IL-1β,IL-6,IL-8 and other inflammatory factors.Faecalibacterium was negatively correlated with IL-1β,IL-6,IL-8 and other inflammatory factors.IL-12p70 might be associated with Lactobacillus(R=0.672,P=0.02),Klebsiella(R=0.623,P<0.05),Rothia(R=0.472,P<0.05),Sarcina(R=-0.650,P=0.02),Ruminococcus(R=-0.645,P=0.02),Anaerostipes(R=-0.786,P<0.05).3.Analysis of ILC3,IL-22 and GDNF in the intestinal wall: The optical density of ILC3,IL-22 and GDNF in the intestinal wall of different types of constipation was measured by multiple immunofluorescence method,and the differences of optical density among the three groups were analyzed by Kruskal-Wallis H test.There were significant differences in the levels of ILC3(H=25.836,P<0.001),IL-22(H=24.362,P<0.001)and GDNF(H=25.055,P<0.001)in each group.Mann-Whitney U test showed that ILC3 in control group(0.302±0.039)were higher than that in STC group(0.036±0.050)and that in HD group(0.002±0.001),IL-22 in control group(0.013±0.009)were higher than that in STC group(0.007±0.003)and that in HD group(0.002±0.001),GDNF in control group(0.067±0.017)were higher than that in STC group(0.017±0.021)and that in HD group(0.018±0.001).There were statistically significant between HD group and control group(U=0,P<0.001),STC group and control group(U=0,P<0.001).The difference between HD group and STC group was also statistically significant(U=0,P<0.001).4.Univariate regression analysis showed that IL-6,IL-1β,IL-8 and IL-12p70 were negatively correlated with the levels of ILC3,IL-22 and GDNF in intestinal wall(P<0.001).Multivariate regression analysis showed that IL-6 and IL-1β levels were negatively correlated with the levels of ILC3,IL-22 and GDNF in intestinal wall(P<0.05),but irrelevant with the levels of IL-8 and IL-12p70(P>0.05).Conclusions:1.In children with constipation,the abundance and structure of gut bacteria were changed,and the diversity was decreased.The abundance of Enterococcus increased,while the levels of Faecalibacterium and other probiotics decreased.This change was more serious in children with HD than in children with STC.2.Compared with the control group,the inflammatory microenvironment of children with constipation changes,and the levels of IL-1β,IL-6,IL-8,and IL-12p70 are altered.The levels of inflammatory factors in HD are higher than those in STC.3.Enterococcus was positively correlated with IL-1β,IL-6 and IL-8,while Faecalibacterium was negatively correlated with IL-1β,IL-6 and IL-8.4.The contents of ILC3,IL-22 and GDNF in intestinal wall of children with constipation are different,and negatively correlated with the levels of IL-6 and IL-1β,but irrelevant with the levels of IL-8 and IL-12p70.5.Gut bacteria changes in children with constipation,accompanied with the reduction of beneficial bacteria,may lead to changes in the inflammatory microenvironment of the body,and through regulating ILC3,IL – 22 pathways affect the level of GDNF in intestinal wall.6.Compared with STC children,HD children had more serious changes in gut bacteria and inflammatory factors,and lower ILC3,IL-22,GDNF and other indicators in intestinal wall,so the development and maturity of enteric nerve would more heavily involved. |
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