Effect Of Vitamin A On Enteric Nervous System And Gastrointestinal Function In Children With Antism And Its Possible Mechanism

PART ? EFFECT OF VITAMIN A ON GASTROINTESTINAL PROBLEMS IN CHILDREN WITH AUTISMObjective: Autism spectrum disorder(ASD)is a neurodevelopmental disorder that is characterized by social deficits,narrow interest and repetitive stereotyped behaviors.The prevalence of ASD has been rising in recent years,and ASD is one of the most serious problems affecting children's health.In addition to core symptoms,ASD is often displayed with comorbidities,and gastrointestinal(GI)comorbidities are common in children with ASD.Vitamin A(VA)is an essential micronutrient that plays an important role in brain development and GI function.However,there is no report yet about the relationship between VA and GI comorbidity in children with ASD.This study was performed to explore the relationship between VA and GI comorbidities in autistic children,and to determine whether VA deficiency(VAD)combined with GI comorbidities exacerbate core symptoms in children with ASD.Methods: A total of 323 children(274 boys and 49 girls)with ASD and 180 age-and gender-matched control children(142 boys and 38 girls)were enrolled in this study.Anthropometric examinations of all children were performed by the children's health care doctors who had received unified training.Symptoms of ASD were assessed with the Child Autism Rating Scale(CARS),the Social Responsiveness Scale(SRS),and the Autism Behavior Checklist(ABC).Caregivers of the children completed questionnaires about general demographic information,medical history,and GI symptoms.Serum retinol levels were detected with high-performance liquid chromatography(HPLC).Results: The weight and height of children with ASD were significantly lower than those of the control group(p< 0.05),but there was no significant difference in body mass index(BMI)between the two groups.The serum retinol concentration of the ASD group was significantly lower than that of the control group(p< 0.01),and children with severe ASD had lower serum retinol levels than those with mild-moderate ASD(p< 0.01).Moreover,the rate of GI symptoms of the ASD group(48.3%)was significantly higher than that of the control group(20.0%)(P < 0.01).The children in the ASD group had significantly higher rates of constipation,diarrhea,abdominal pain,nausea/vomiting,and stool odor,compared to the children in the control group(all Ps< 0.05).The most common GI symptoms in the ASD group were constipation(32.5%).As VA levels between the autistic children with and without different GI symptoms were compared,VA levels in children with total GI symptoms and constipation were considerably lower than that in autistic children without these GI symptoms(all Ps< 0.05).In addition,VA level was associated with CARS,SRS,and ABC scores,whereas GI symptoms were associated with some SRS and ABC scores.There was interaction between VAD and GI symptoms,and VAD coexist with GI comorbidity apparently increased some SRS and ABC scores in children with ASD.Conclusion: Children with ASD have higher rates of VAD and GI symptoms than control children.Low serum retinol levels were associated with GI comorbidities(especially constipation)in children with ASD.The core symptoms are more severe in ASD children with VAD,and ASD children with GI comorbidities also have more serious core symptoms than ASD children without GI comorbidities.VAD comorbid with GI symptoms remarkably aggravates autistic children's core symptoms.VAD seems to be related to ASD with GI comorbidities.PART ? THE EFFECT OF VITAMIN A DEFICIENCY ON ENTERIC NERVOUS SYSTEM AND GASTROINTESTINAL FUNCTION AND ITS POSSIBLE MECHANISM IN A RAT MODEL OF AUTISMObjective: The manifestations of ASD are highly heterogeneous.As many individuals with ASD comorbid with GI problems,ASD with GI problems is considered to be a subtype of ASD.VA plays important roles in the development of both the central nervous system and peripheral nervous system.The first part of this study also found that ASD children with VAD have more serious core symptoms,and VA level is associated with GI comorbidities(especially constipation)in children with ASD.However,the mechanism of that VA is associated with GI comorbidity in ASD children is still unclear.The enteric nervous system(ENS)plays an important role in many GI functions such as GI motility.In this part,we constructed autism rat models with different VA levels to explore the effect of VAD on autismlike behaviors,GI function and development of ENS,and to explore the possible mechanism by which VAD affects the development of the ENS.Methods: Establishment of autism model without VAD: female rats aged 9 weeks were mated with normal males,pregnant rats were randomly assigned to receive an intraperitoneal injection of 600 mg/kg valproic acid(VPA)or the same volume of saline on gestational day 12.5,and the offspring were used as the VPA and CON groups in later experiments.Establishment of autism model without VAD: female rats aged 4 weeks were randomly divided into VA normal(VAN)and VAD groups and were fed a VAN or VAD diet,after 4 weeks,blood samples were taken from the tails of each rat,and serum retinol levels were detected;then,rats with appropriate VA levels(VAN: ?1.05 ?mol/L;VAD: <0.70 ?mol/L)were mated with males,and all the pregnant rats received an intraperitoneal injection of VPA;the pregnant rats in the VAD group were randomly divided into two groups,and the offspring rats were VAD + VPA and VAS + VPA groups respectively,the offspring rats in the VAN group were used as the VAN+VPA group;the VAN+VPA group and VAD+VPA group were fed a VAN diet and a VAD diet,respectively,throughout the study,rats in the VAS+VPA group were fed a VAN diet beginning at birth and received 3RNI of VA supplementation(83.33 IU/day)by oral gavage from postnatal day(PND)1 to PND 7.The behavioral tests,including open field test,three-chamber test,water maze test,and Barnes maze test were conducted in the offspring beginning on PND 43 using ANY-Maze Video Tracking System.GI transit time was tested by giving rats with carmine via intragastric administration.The rats were sacrificed and the serum and colonic tissue were collected when they were 8 weeks old.The serum retinol concentration was tested by HPLC.The m RNA expression level of PGP9.5 in colonic tissue were detected by quantitative polymerase chain reaction(q PCR).The protein expression levels of Claudin1,Occludin,Zo-1,RAR?,Ret,and PGP9.5 in colonic tissue were detected by Western blotting.The distribution and expression of RAR?,Ret,PGP9.5 and tuj1 in the colons of rats were detected using immunofluorescence.The interaction between RAR? and the Ret gene was assessed by chromatin immunoprecipitation(Ch IP)analysis.Results: The rats in the VPA group displayed repetitive behaviors and impaired social interaction.The results of water maze test also indicated that the learning and memory ability decreased in the VPA group.The protein expression levels of claudin1 and Occludin that related to intestinal barrier function decreased in the VPA group,and the GI transit time of the VPA group was considerably longer than that of the CON group(all Ps< 0.01).The m RNA and protein expression levels of PGP9.5 in the VPA group were decreased compared with those in the CON group(all Ps< 0.05).And the immunofluorescence results showed that enteric nervous stained by PGP9.5 in the colon was markedly reduced in the VPA group.Serum retinol levels at different time points after birth in the VAD+VPA group were all significantly lower than those in the VAN+VPA groups(all Ps< 0.01).The serum VA levels of VAS+VPA group were similar to that of VAD + VPA group at birth.And at all time points from PND3,serum VA levels of VAS+VPA group were significantly higher than that of VAD + VPA group(all Ps< 0.01),and there was no significant difference between VAS+VPA group and VAN+VPA group(all Ps> 0.05).In the open field test,the rats in the VAD+VPA group spent more time in self-grooming and had a higher defecation frequency than those in the VAN+VPA,but the difference was not significant(all Ps> 0.05).The VAD+VPA group spent significantly less time in the central zone than the VAN+VPA group(P< 0.05).Compared with VAD + VPA group,the VAS+VPA group spent less time in self-grooming and had less defecation frequency,but the difference was not significant(all Ps> 0.05).The time of VAS+VPA group in the central area was increased than VAD + VPA group,but there was no significant difference between VAS+VPA group and VAD + VPA or VAN + VPA group(P> 0.05).In the three-chamber test,the results of social preference test between stranger rat and object showed that the VAD+VPA group spent significantly more time in the object chamber and less time in the stranger rat chamber than the VAN+VPA group(all Ps< 0.05),the VAS+VPA group spent significantly less time in the object chamber and more time in the stranger rat chamber than the VAD+VPA group(all Ps< 0.05),and there was no significant difference in time that spent in each chamber between VAS+ VPA group and VAN+VPA group(P> 0.05).The results of social preference test between stranger rat and familiar rat showed that the VAD+VPA group spent significantly more time in the familiar rat chamber and less time in the stranger rat chamber than the VAN+VPA group(all Ps< 0.01),the VAS+VPA group spent significantly less time in familiar rat chamber and more time in the stranger rat chamber than the VAD+VPA group(all Ps< 0.05),and there was no significant difference in time that spent in each chamber between VAS+ VPA group and VAN+VPA group(P> 0.05).The results of Barnes maze test showed that the time that was used to find the target hole in VAD + VPA group was significantly longer than that in VAN + VPA group on both the first and third days after training,and the number of times that detected the wrong holes in VAD + VPA group was more than that in VAN + VPA group(all Ps< 0.01).Compared with VAD + VPA group,the time that was used to find the target hole in VAS + VPA group was significantly shorter in both tests(all Ps< 0.01).The number of times that detected the wrong holes in VAS + VPA group decreased in both tests compared with VAD + VPA group,but the difference was significant only in the third day after training(P< 0.05).There was no significant difference between VAS + VPA group and VAN + VPA group in the time of finding the target hole and the times of detecting the wrong holes in both tests(all Ps> 0.05).The protein expression levels of claudin1,Occludin and Zo-1 that related to intestinal barrier function in the colonic tissue of VAD+VPA group were significantly lower than that of the VAN+VPA and VAS+VPA groups(all Ps< 0.05).The results of GI transit time test showed that the VAD+VPA group had a significantly higher GI transit time than the VAN+VPA group(P<0.001),and the GI transit time recovered to be similar to that of VAN + VPA group after VA supplementation(P> 0.05).The protein levels of RAR?,Ret and PGP9.5 in the VAD+VPA group were significantly lower than those in the VAN+VPA and VAS+VPA groups(all Ps< 0.05).Besides,the immunofluorescence results showed that RAR?,Ret and the enteric nervous markers PGP9.5 and tuj1 were all visibly reduced in the colons of the VAD+VPA group compared with the colons of the VAN+VPA and VAS+VPA groups.The results of Ch IP showed that RAR? was enriched on the promoter of the Ret gene in colonic tissue and that this enrichment was significantly reduced in the VAD+VPA group compared with the VAN+VPA group(P< 0.01).Conclusion: Intestinal barrier function impaired,GI transit time increased,and ENS development was abnormal in the VPA-induced autism rat model.Gestational VAD exacerbates the autism-related behaviors(especially social deficits),impairment of intestinal barrier function,impaired GI motility,and dysplasia of ENS in autism model rats.Supplementation of VA can effectively ameliorate autism-like behaviors,impairment of intestinal barrier function,impaired GI motility,and ENS dysplasia in an autism rat model with VAD.VAD increased ENS dysplasia in autism model rats might be caused by lower expression of RARa,decreased binding between RARa and the promoter of the Ret gene,and the decreased expression of Ret signal pathway.Thus,we speculate that VAD in autism might lead to impairment of both the brain and ENS.Gestational VAD might cause individuals to be more susceptible to risk factors and aggravate a subtype of ASD with GI comorbidities.