Effects Of In Vitro Cultured Gut Microbiota On Autism Spectrum Disorder

Autism spectrum disorder(ASD)is a brain-based neurodevelopmental disorder characterized with behavioral abnormalities in sociability,communication,and/or compulsive activity.The current global population prevalence of ASD is around 1%,which casts great economic and psychological burden on society and individual families.ASD is currently thought to be a result of complex interaction of genetic and environmental factors,between which the gut microbiota plays an important role because its composition and function are closely related with both host gene and environmental factors.The connection between gut microbiota and ASD has been verified in a model mouse with autism.Recent researches on gut microbiota-related disorders have largely driven the interest in the therapy of fecal microbiota transplantation.Last year,a persisted improvement of gut microbiota composition and ASD symptoms of 18 ASD-diagnosed children were observed after Microbiota Transfer Therapy(MTT)in an open-label study.It indicates that the gut microbiota transplant could be a promising treatment protocol for ASD.However,in clinical application,gut microbiota transplantation(GMT)is facing problems of obtaining effective microbiota of transplantation because of high costs of donor selection and heterogeneity of donors’ gut microbiota.In vitro batch culture model is a fast,easy-to-operate and reproducible method to amplify and study fecal microbial communities.The present study takes it as a starting point to preliminarily explore the feasibility of treating ASD with in vitro-cultured gut microbiota as transferred samples.The research includes three parts:modeling ASD mice,treating the ASD model mice with cultured gut microbiota and assessing the treatment effect,and investigating the differences in the microbial communities of the mice among groups.Firstly,we modeled maternal immune activation(MIA)in C57BL/6N mice to induce ASD mice model.Weight and behavioral tests were performed on mice afterwards.The results showed as following:Modeling MIA on mice did not affect the growth status of the mice;the model mice exhibited anxiety-like behavior,stereotyped behavior and impairments in social interaction,suggesting the ASD mice model was successfully constructedSecondly,we cultured gut microbiota via the in vitro batch culture method.ASD model mice were then treated with the original or cultured human gut microbiota by gavage,followed by body weight tests,behavioral tests and chemokine detection of serum and colon tissues of mice in each group.The results showed as following:GMT did not affect the growth of mice;GMT significantly improved the anxiety-like and repetitive behaviors of ASD mice,and a sign of improvement also appeared in impairments in social interaction;both original and cultured gut microbiota significantly improved the serum level of some chemokines in ASD mice,including Eotaxin,GRO-a,MIP-la,MCP-3 and RANTES;the cultured gut microbiota significantly improved the levels of some chemokines in colon tissue including GRO－a,IP-10,MIP-la and MIP-1β.Lastly,we utilized the Illumina Hiseq 2000 platform to sequence the 16S rDNA of the gut microbiota of the four groups of mice,and analyzed the sequencing results by methods including Alpha and Beta diversity analysis,phylum and genus level comparison,and LEfSe analysis.The results showed as following:Both original and cultured gut microbiota significantly change the microbial structure in the gut of ASD mice,and the original GMT significantly improved the diversity gut microbiota of ASD mice;the relative abundance of several dominant microbiota in the gut of ASD mice was changed by the two kinds of GMTs at the level of phylum and genus;both two GMTs improved the relative abundance of some marker microbiota in the gut of ASD mice;specifically,original human gut microbiota increased the relative abundance of Ruminococcaceae,Oscillospira,and decreased the relative abundance of Prevotella other toward their abundance in healthy mice;cultured human gut microbiota decreased S24-7,Prevotella other,Clostridiaceae and Candidatus Arthromitus toward their abundance in healthy mice.Additionally,the Sperman’s rank correlation analysis showed that the relative abundances of Clostridiaceae,Prevotella other and Candidatus Arthromitus was negatively correlated with the serum level of RANTES and Eotaxin.Overall,the present study preliminarily evidences that both cultured and original gut microbiota had improvement effect on ASD.The result provides a basis for the feasibility of obtaining effective transferred samples for GMT by means of in vitro culture,so as to provide solutions for the clinical problem of qualified donor selection and the heterogeneity of gut microbiota of donor.