Effects Of Fecal Microbiota Of Children With Autism Spectrum Disorder On The Behavior Of Germ-free Mice And Its Possible Mechanisms

Objective: The purpose of this study was to identify the relationship between the intestinal microbiota and its metabolites associated with autism spectrum disorder(ASD)children and ASD-like behavior in germ-free mice,and to preliminarily explore the possible molecular mechanisms of ASD-related intestinal microbiota and their metabolites affecting ASD-like behaviorin germ-free mice.Methods: Five typical developing(TD)children and five children with ASD aged 2-6 years were included as the donors of fecal microbiota transplantation(FMT).Their fecal suspension was transplanted to4-week-old Babl/c germ-free mice respectively,to construct ASDchildren-associated fecal microbiota transplanted mice group(ASD-FMT)and TD children-associated fecal microbiota transplanted mice group(TD-FMT).Three weeks after fecal microbiota transplantation,the behavioral differences between the two groups were compared by olfactory habituation/habituation test,open-field test and three-chamber social test.Serum,cecal contents,colonic and prefrontal cortex tissuesof the mice were collected immediately after the behavioral tests.16 S rRNA sequencing was used to detect the differences in the community structure and distribution of cecal contents between TD-FMT and ASD-FMT groups.High performance liquid chromatograph-tandem mass spectrometry(HPLC-MS/MS)was used to determine the metabonomic differences of cecal contents,serum and prefrontal cortex between the two groups of mice.KEGG database was used for the enrichment analysis of metabolic pathways in cecal content,serum and prefrontal cortex to search for representative metabolic signaling pathways.Furthermore,differential metabolites were identified in the metabolic pathways,and spearman correlation analysis was performed on the levels of differential metabolites and the abundance of intestinal microbiota,so as to obtain the possible intestinal microbiota affecting the metabolism of mice.Finally,the protein expression levels of tryptophan hydroxylase 1(TPH1),serotonin transporter(SERT)and5-hydroxytryptamine 1A receptor(5-HT1AR)in the colon of mice from both groups were detected by Western blotting.And the protein expression levels of tryptophan hydroxylase 2(TPH2),SERT and 5-HT1 AR in the prefrontal cortex of the mice were also detected.Results: Three weeks after fecal microbiota transplantation,mice in the ASD-FMT group spent significantly less time sniffing social odors than mice in the TD-FMT group on olfactory habituation/habituation test(P<0.001),and no significant difference was found between the two groups in the time to sniff non-social odors(water and beer)(all P >0.05).In the open-field test,the time spent in the center zone of the open-field in ASD-FMT group was significantly lower than that in TD-FMT group(P<0.01),and the self-grooming time was significantly higher than that of TD-FMT group(P<0.01).In the three-chamber social test,the time of staying in the stranger mice chamber was significantly lower in ASD-FMT group than that in TD-FMT group(P<0.001),and the time spent in the novel object chamber was significantly higher than that in TD-FMT group(P<0.001).The results of 16 S rRNA of the cecal contents in the two groups of mice showed that,there was no statistical difference in the ? diversity(Shannon,Simpson,ACE and Chao indexes)of the two groups(all P>0.05),however,there were significant differences in community structure and distribution between the two groups.At the phylum level,the abundance of Verrucomicrobia in the ASD-FMT group was significantly decreased(P<0.05),while the abundance of Unidentified＿Bacteria in the ASD-FMT group was significantly increased(P<0.01).At the genus level,compared with TD-FMT group,the abundance of Parabacteroides(P<0.05),Alistipes(P<0.01),Anaerotruncus(P<0.01),and Helicobacter(P<0.01)were significantly increased in ASD-FMT group,while Parasutterella(P<0.05),Akkermansia(P<0.05),Lachnoclostridium(P<0.01)and unidentified＿Erysipelotrichaceae(P<0.01)abundance weresignificantly decreased in ASD-FMT group.Further LEf Se analysis showed that Verrucomicrobia(LDA=4.19,P=0.0117)and Unidentified＿Bacteria(LDA=4.47,P=0.0023)were the key bacteriaphyla that caused significant differences in the community structure of the two groups of mice.Helicobacter(LDA=4.47,P=0.0023),Lachnoclostridium(LDA=4.66,P=0.0033)and Akkermansia(LDA=4.19,P=0.0117)were the key bacteria genusthat caused significant differences in the community structure between the TD-FMT and ASD-FMT groups.The results of untargeted metabonomics analysis showed that the metabolite characteristics of cecal content,serum and prefrontal cortex of mice in ASD-FMT group and TD-FMT group were significantly different.KEGG enrichment analysis indicated that although the differential metabolites between TD-FMT group and ASD-FMT group were involved in multiple metabolic pathways,the differential metabolites between the two groups could be enriched in tryptophan and serotonergic synaptic metabolic signaling pathways,whether in cecum contents,serum or prefrontal cortex of mice.Further analysis showed that,in the cecum of these mice,the levels of Trp-Trp(P<0.001),kynurenic acid(P<0.01)and Indole-3-acetic acid(P<0.05)in the ASD-FMT group were significantly higher than that in TD-FMT group.And the levels of6-hydroxymelatonin(P<0.05),indole-3-lactic acid(P<0.01)and5-methoxy-indoleacetate(P<0.01)in the ASD-FMT group were significantly lower than TD-FMT group.In the serum,the levels of N-acetyl-DL-tryptophan(P<0.05)and 5-hydroxytryptophol(P<0.05)in the ASD-FMT group were significantly higher than that in TD-FMT group.In the prefrontal cortex,however,the level of 5-hydroxytryptophol(P<0.05)was significantly lower in ASD-FMT group than in TD-FMT group.Spearman correlation analysis indicated that o＿Clostridiales,o＿Bacteroidetes,f＿Erysipelotrichaceae and o＿Lactobacillales were the main genera related to tryptophan and serotonin metabolism.Western blotting results showed that the protein expression levels of SERT(P<0.01)and 5-HT1AR(P<0.05)were lower than TD-FMT group,while the protein expression level of TPH1(P<0.05)was higher than that in TD-FMT group.In the prefrontal cortex,the protein expression levels of TPH2(P<0.05)and SERT(P<0.05)were higher than that in TD-FMT group,while there was no difference in protein expression level of 5-HT1AR(P>0.05)between the two groups.Conclusion:(1)Intestinal microbiota of ASD children induced ASD-like behavior in germ-free mice.(2)The intestinal microbiota structure and distribution of germ-free mice were significantly different after fecal microbiota transplantation withthe intestinal microbiota of ASD and TD children.At the same time,intestinal microbes in children with ASD can cause disorders in tryptophan and serotonin metabolism in ASD-FMT mice.(3)Changes in metabolites in the pathways of tryptophan and serotonin metabolism were associated with specific genera of o＿Clostridiales,o＿Bacteroidetes,f＿Erysipelotrichaceae and o＿Lactobacillales.(4)Changes in key proteins involved in serotonin synthesis and transport in the colon and prefrontal cortex of mice in the ASD-FMT group may be the molecular basis of intestinal metabolic disorders and ASD-like behaviors in mice after fecal microbiota transplantation.