Investigation Of Altered Intestinal Flora And Serum Metabolites In Children With Functional Constipation And Protective Effect Of L-Pipecolic Acid On Loperamide-Induced Constipation In Mice

Background: Functional constipation(FC)is a common gastrointestinal disease in children with a high incidence rate,accounting for about 29.6% worldwide.At present,the overall treatment effect at home and abroad is still not satisfied.It not only seriously affects the quality of life of children,but also makes their families bear a huge economic burden.Several studies have confirmed the association between FC and abnormal gut microbiome.The relationship between gut microbiota and gut transit may be bidirectional.Gut flora can regulate colonic motility,secretion,and absorption through metabolic changes in microorganisms,and promote the development of FC,such as bile acids,short-chain fatty acids,serotonin,and methane.Current studies have also found that treatment with probiotics and prebiotics can improve the composition and function of gut microbiota.However,which bacteria and metabolites play a role in FC is still unclear.Objective: This study aims to determine the changes of intestinal bacteria and serum metabolites in children with FC,and to screen out differential flora and differential metabolites.At the same time,a constipation model was established in mice induced by loperamide(Lop),to explore the potential effect of L-pipecolic acid on mice with constipation,and to provide a scientific theoretical basis for the treatment of functionally constipated children.Methods: A total of 26 children with FC and 28 healthy children were recruited as the FC group and the control group,respectively.Stool and serum samples were collected from all enrolled members.Stool samples and serum samples were detected by 16 S r DNA sequencing and UPLC-Q/TOF-MS techniques,respectively.Identify the changes of microbiota and metabolites in children with FC,so as to screen out candidate differential metabolites.At the same time,a loperamide-induced mouse constipation model was constructed,and all mice were randomly divided into control(Con)group,loperamide(Lop)model group and L-pipecolic acid(Lop+L-PA)treatment group(6mice in each group).The mice in the Lop+L-PA group were given L-pipecolic acid(250mg/kg,once a day)and loperamide;the Lop group was given loperamide,and the Con group was given saline.The fecal characteristics and intestinal motility of mice in each group were detected.5-HT levels in serum and 5-HT protein expression in colon tissue were detected by ELISA and immunohistochemistry,respectively;q RT-PCR was used to detect the expression of AQP3 and 5-HT4 R mRNA in each group of mice,so as to explore the potential therapeutic effect and possible mechanism of L-pipecolic acid in constipated mice.Results: Compared with healthy children,the α and β diversity of the gut microbiota of children with FC was significantly reduced;18 differential flora and 45 differential metabolites were identified.These differential metabolites were mainly enriched in fatty acid biosynthesis,lysine degradation and choline metabolism pathways.Additionally,L-pipecolic acid was significantly reduced in children with FC.Through microbiomics and metabolomics correlation analysis,L-pipecolic acid and N6,N6,N6-trimethyl-L-lysine involved in the lysine degradation pathway were related to Ochrobactrum and Phascolarcrobacterium,respectively.In animal models,it was found that L-pipecolic acid improved the fecal water content and intestinal transit function of loperamide-induced constipated mice,and significantly increased the expression levels of 5-HT in serum and colon tissue of constipated mice.Meanwhile,L-pipecolic acid increased the expression level of 5-HT4 R and decreased the expression of AQP3.Conclusion: Gut microbiota and serum metabolites were significantly altered in children with FC compared with healthy children.Among them,the richness of Phascolarctobacterium decreased in children with FC;the content of L-pipecolic acid in serum decreased,and the content of p-cresol and trimethylamine oxide increased significantly.In addition,L-pipecolic acid can effectively relieve Lop-induced constipation in mice,which may be related to increasing the expression of 5-HT and5-HT4 R and down-regulating the expression of AQP3.