| Part ?Altered gut microbiota in children with autism spectrum disorder IntrodutionAutism spectrum disorder?ASD?is a neurodevelopmental disorder that is characterized by impairments in social interaction and communication,restricted interests and repetitive behaviors.Several studies report a high prevalence of gastrointestinal?GI?symptoms in autistic individuals.Cumulative evidence reveals that the gut microbiota and its metabolites?especially short-chain fatty acids,SCFAs?play an important role in GI disorders and the pathogenesis of ASD.However,the composition of the gut microbiota and its association with behavior symptoms,fecal SCFAs and GI symptoms of autistic children remain largely unknown.Aims:To investigate the composition of the gut microbiota and its correlation with behavior symptoms,fecal SCFAs and GI symptoms in Chinese children with autism spectrum disorder.Methods:1.Thirty autistic?ASD?subjects and twenty neurotypical?NT?volunteers were recruited.2.Gastrointestinal symptoms were assessed following a modified version of the 6-GI Severity Index,including constipation,diarrhea,average stool consistency,stool smell,flatulence and abdominal pain.3.The behavior symptoms in the ASD group were assessed by Autism Treatment Evaluation Checklist?ATEC?.4.Fecal SCFA levels were analyzed by high-performance liquid chromatography?HPLC?.5.16 S rRNA gene sequencing was used to reveal the composition and structure of the gut microbiota.6.Spearman correlation was used to analyze the relationship between the gut microbiome and the fecal SCFAs.7.Spearman correlation was used to analyze the relationship between the gut microbiome and the behavior symptoms in autistic individuals.8.To analyze the gut microbiome associated with GI symptoms in ASD subjects.Results:1.There were no significant differences in age and gender between the two groups.2.The frequency of constipation in ASD subjects was significantly higher than that in neurotypical subjects?P<0.05?.There were no significant differences in diarrhea,abdominal pain,average stool consistency,stool smell,flatulence and the total GI scores between the two groups.3.The ATEC scores in ASD subjects were: Speech/Language Communication?18.14±3.73?,Sociability?27.83±3.84?,Sensory/ Cognitive Awareness?22.97±4.49?,Health/Physical/Behavior?31.45±6.10?.4.The levels of acetic acid and butyrate were significantly decreased in ASD subjects compared with those in NT controls.However,the valeric acid level in ASD children was markedly higher than that in NT participants.?P<0.05?5.The scores of the Shannon and Shannoneven indexes in ASD samples decreased compared with those in NT controls.The results of beta diversity on the OTU data revealed that the overall composition of the ASD gut microbiota was different from that of the NT gut microbiota.At the phylum level,Firmicutes was significantly decreased in ASD compared with that in NT,whereas Acidobacteria was considerably increased in ASD compared with that in NT.Megamonas,Pseudomonas,Enterococcus and Acidobacteria were enriched in ASD subjects,whereas Eubacterium,Peptostreptococcaceae,Streptococcaceae,Ruminococcaceae and LachnospiraceaeNC2004group were enriched in NT subjects at the genus level.6.Acidobacteria and Actinomycetaceae were positively correlated with the fecal level of valeric acid?P<0.05?.Streptococcaceae,Peptostreptococcaceae,Lactobacillaceae and Clostridiaceae1 had a strong positive correlation with the fecal butyrate level?P<0.05?.7.Megamonas was positively correlated with the scores of Speech/Language Communication and Health/Physical/Behavior in ASD subjects.Holdemanella was positively correlated with the Sensory/Cognitive Awareness score in the ASD group?P<0.05?.8.We also found enriched Fusobacterium,Barnesiella,Coprobacter and valeric acid-associated bacteria?Actinomycetaceae?and reduced butyrate-producing taxa in constipated autistic subjects.Conclusions:Constipation is the only GI disorder in ASD children recruited in the present study.The results showed lower concentrations of fecal acetic acid and butyrate and a higher concentration of fecal valeric acid in ASD individuals.We observed shifted gut microbiota?reduced butyrate-producing taxa and enriched valeric acid-associated bacteria?and its association with constipation in ASD individuals.Modulating the gut microbiota,especially butyrate-producing bacteria,could be a promising strategy in the search for alternatives for the treatment of autism spectrum disorder.Part ? Altered gut micriobiota and intestinal barrier function in two mousemodels of autism spectrum disorder Aims:To investigate the characteristics of the gut microbiota,intestinal barrier function and gastrointestinal motility in two different mouse models of autism spectrum disorder.Methods:1.C57bl/6J mice were paired overnight.Pregnant mice were injected at E12.5 with 600 mg/kg of VPA in sterile saline or saline only intraperitoneally.BTBR mice were paired overnight.At 3 weeks of age,saline,VPA and BTBR offspring across individual litters were weaned into cages of non-littermate offspring of the same treatment group to generate a randomized experimental design.2.Intestinal barrier function at 3 weeks of age: the serum was analyzed for FITCdextran concentration with a fluorescence spectrophotometer;the expression of Trek1,claudin1,claudin3,occludin and ZO-1 in the colon was detected by Western blot?WB?and the expression of IL-6,TNF-? and IFN-? at mRNA level in the colon was detected by quantitative polymerase chain reaction?qPCR?.3.Behavioral testing: Pups were tested for ultrasonic vocalizations at day 8.Adult mice were tested beginning at 6 weeks of age for open field exploration,marble burying,social interaction,adult ultrasonic vocalizations and self grooming,in that order,with at least 5 days between behavioral test.4.GI motility: the fecal water content and the small intestinal transit ratio were detected.5.16 S rRNA gene sequencing was used to reveal the composition and structure of the gut microbiota.6.HPLC was used to detect the concentrations of fecal SCFAs.7.Intestinal barrier function in adult mice: the serum was analyzed for FITCdextran concentration;the expression of Trek1,claudin1,claudin3,occludin and ZO-1 in the colon was detected by WB and the expression of IL-6,TNF-? and IFN-? at mRNA level in the colon was detected by qPCR.Results:1.Compared with control pups,the number of calls and the call duration in both 20-50 kHz and 50-100 kHz decresased significantly in BTBR and VPA pups.Adult mice in BTBR and VPA groups exhibited decreased center entries and center duration,decreased chamber duration?novel mouse?,increased marble buried ratio,increased time on self grooming and decreased number of calls and call duration in both 20-50 kHz and 50-100kHz?P<0.05?.2.Intestinal barrier function in 3-week-old ASD offspring: BTBR and VPA mice displayed increased intestinal permeability,decreased expression of Trek1 and tight junction proteins?TJ,including claudin1,claudin3 and occludin?and increased cytokines?IL-6 and TNF-??at mRNA level in the colon as compared with the control group?P<0.05?.3.The fecal water content in BTBR mice decreased significantly compared with that in control mice?P<0.05?.There was no significant difference in small intestinal transit ratio between the ASD group and the control group?P>0.05?.4.BTBR mice had decreased species richness,diversity and evenness?P<0.05?.The overall composition of BTBR and VPA gut microbiota were different from that of the control gut microbiota.5.Compared with the control group,the levels of acetic acid,butyrate and valeric acid decreased significantly in BTBR mice;the level of propionic acid increased significantly and the level of butyrate decreased significantly in VPA mice?P<0.05?.6.Intestinal barrier function in adult ASD offspring: BTBR and VPA mice displayed increased intestinal permeability,decreased expression of Trek1 and TJ proteins?claudin1,claudin3 and occludin?and increased cytokines?IL-6,TNF-? and IFN-??at mRNA level in the colon as compared with the control group?P<0.05?.Conclusions:BTBR and VPA mice exhibit ASD-related behavioral abnormalities,altered gut microbiota and decreased level of fecal butyrate.BTBR and VPA mice display increased intestinal permeability and proinflammatory cytokines and decreased expression of Trek1 and tight junction proteins in the colon at 3 weeks of age and 10 weeks of age.C.butyricum modulates intestinal barrier function and behavioralabnormalities in two mouse models of autism spectrum disorder Aims:To explore the potential impact of intestinal barrier function on core ASD behavioral abnormalities,we tested whether C.butyricum treatment impacts tight junction proteins,proinflammatory cytokines,intestinal permeability and ASD-related behaviors.Methods:1.Based on the mouse models used in part ?,cages between the VPA and BTBR groups were selected at random for treatment with C.butyricum or vehicle,intragastric administration,every day for 21 days.2.Behavioral testing: mice were tested beginning at 6 weeks of age for open field exploration,marble burying,social interaction,adult ultrasonic vocalizations and self grooming,in that order,with at least 5 days between behavioral test.3.HPLC was used to detect the concentration of fecal butyrate.4.Intestinal permeability: the serum was analyzed for FITC-dextran concentration with a fluorescence spectrophotometer.5.The total protein and RNA were extracted from the colon tissues.The expression of Trek1,HDAC1,claudin1,claudin3 and occludin at protein and mRNA level was detected by WB and qPCR,respectively.The expression of Trek1 in the colonic mucosa was analyzed by immunohistochemistry?IHC?.6.The concentrations of IL-6,TNF-? and IFN-? in the colon were detected by ELISA.Results:1.Mice treated with C.butyricum displayed increased fecal levels of C.butyricum and butyrate.2.The intestinal permeability in BTBR+C.butyricum and VPA+C.butyricum mice decreased significantly compared with that in BTBR and VPA mice,respectively.3.Mice treated with C.butyricum exhibited increased expression of Trek1,claudin1,claudin3 and occludin,and decreased expression of HDAC1 at protein and mRNA level in the colon.Part ?4.The expression of cytokines?IL-6,TNF-? and IFN-??in the colon in BTBR+C.butyricum and VPA+C.butyricum mice was significantly lower than that in BTBR and VPA mice,respectively.5.Mice treated with C.butyricum exhibited increased center entries,center duration and chamber duration?novel mouse?,dececreased marble buried ratio,decreased time on self grooming and increased number of calls and call duration in both 20-50 kHz and 50-100 kHz.Conclusions:C.butyricum treatment modulates the expression of HDAC1,Trek1,tight junction proteins and proinflammatory cytokines in the colon,enhances the intestinal permeability and ameliorates ASD-related behavioral abnormalities.Part ?C.butyricum modulates intestinal barrier function through Trek1 inmouse model of autism spectrum disorder Aims:To explore the potential target of C.butyricum or sodium butyrate on the intestinal epithelial barrier in ASD mice,we tested whether spadin?Trek1 blocker?impacts tight junction proteins,proinflammatory cytokines and intestinal permeability.Methods:1.Male C57bl/6J mice were intraperitoneally injected spadin in sterile saline or saline only every day for 15 days.The intestinal permeability was detected afterwards.2.BTBR mice were selected at random for treatment with C.butyricum,sodium butyrate or vehicle,intragastric administration,every day for 21 days.The mice of these treatments were selected at random for spadin or vehicle,intraperitoneal injection,every day for 21 days.3.Intestinal permeability: the serum was analyzed for FITC-dextran concentration with a fluorescence spectrophotometer;4.The expression of claudin1,claudin3 and occludin at protein level were detected by WB.5.The concentrations of IL-6,TNF-? and IFN-? in the colon were detected by ELISA.Results:1.Spadin administration increased the intestinal permeability in C57bl/6J mice.2.Spadin administration reversed reduced intestinal permeability induced by C.butyricum and sodium butyrate in BTBR mice.3.Spadin administration reversed higher TJ proteins in the colon induced by C.butyricum and sodium butyrate in BTBR mice.4.Spadin administration reversed lower concentrations of IL-6 and TNF-? in the colon induced by C.butyricum and sodium butyrate in BTBR mice.Conclusions:We have identified TREK-1 as a potential target for modulating the intestinal barrier function in autism spectrum disorder. |
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