Effect Of Vitamin A On Social Function Of Children With Autism Spectrum Disorder And Its Possible Mechanism

PART I EFFECTS OF DIFFERENT DOSES OF VITAMIN A SUPPLEMENTATION ON VITAMIN A NUTRITION,OXT LEVELS AND CORE SYMPTOMS IN ASD CHILDREN WITH VADPurpose:Autism spectrum disorders(ASD)are a group of neurodevelopmental disorders characterized by repeated stereotypes and impaired social function,which are one of the fastest growing diseases in the world.ASD children are often associated with various comorbidities,which can affect rehabilitation and quality of their lives.Among them,nutrient deficiency is a common comorbidity status in children with ASD.Nutritional supplement therapy has been used to correct the lack of nutrients in ASD patients,and some studies have found some nutrient supplements(folic acid,vitamin B,vitamin D,etc.)might improve the core symptoms of ASD. The effect of vitamin A supplement(VAS)on the improvement of ASD symptoms is unclear.It has been reported that ASD children have a high rate of vitamin A deficiency(VAD).The use of WHO recommended dose supplement of VA can improve the VA level of ASD children,but no significant improvement in core symptoms were found.This part of the study using a continuous low-dose VA supplementation method in children with ASD for the first time.We hope to explore the effect of different VAS doses on improvement of VA status,and to explore suitable VAS supplemental program for children with ASD by comparing with the WHO recommended high-dose VAS program.In addition,by analyzing the correlation among changed VA,serum OXT,ASD symptom scale scores before and after VAS in children with ASD,we further explore whether VAS may be one of the treatment methods to improve the core symptoms of children with ASD.Methods:(1)Participants:138 ASD children(118 males,20 females),aged between 3-8 years,were enrolled in the rehabilitation institution by cluster sampling method.All ASD children were professionally diagnosed according to diagnostic and statistical manual of mental disorder(DSM-V)criteria by developmental behavior pediatrician.(2)Baseline investigation:All the children's physical developmental status was measured by physical measurement and Z-score conversion.Furthermore,children completed evaluation of social responsiveness scale(SRS),Autism behavior checklist(ABC),and Childhood autism rating scale(CARS)to comprehensively assess the severity of autism symptoms.(3)Measurement of blood biochemical indicators:2 mL of peripheral venous blood was collected and serum and blood cells were separated.The serum retinol(vitamin A,VA)concentrations were detected by high performance liquid chromatography(HPLC)and serum oxytocin(OXT)concentrations were detected by Enzyme-Linked Immunosorbent Assay(ELISA).The total RNA was extracted from peripheral blood mononuclear cells(PBMC)and reverse transcription-polymerase chain reaction(RT-PCR)was performed to test the relative mRNA expression levels of retinoic acid receptors(RARs,including RAR?,RAR?,and RAR?)and CD38 gene.(4)Vitamin A supplement:All the ASD children were divided into vitamin A normal group(VAN,serum VA higher than 1.05?mol/L)and vitamin A deficiency(VAD,serum VA less than 1.05?mol/L)through VA levels.VAD children were randomly divided into VAD-1 and VAD-2 groups and received different doses of vitamin A supplement(VAS).56 VAN children were treated with behavioral therapy in rehabilitation institutions.46 children with VAD and ASD who were in VAD-1 group were given a single oral dose of 200,000 IU of vitamin A supplement(VAD-VAS1).36 children in the VAD-2 group were supplemented orally with a low-dose vitamin A(3022 IU/day)supplement(VAD-VAS2)for 6months.After 6 months,the physical development index,ASD symptom rating scales,serum VA and OXT concentrations,and RARs and CD38mRNA expression levels of the 138 ASD patients were reevaluated.(5)Statistical analysis:Paired t-test was used to analyze the changes of related indicators before and after intervention in each group of children.Pearson correlation analysis was performed to test the relationships among changed VA levels and other values before and after intervention of all children with ASD.Results:Both VAS treatment doses could significantly improve the serum VA levels in ASD children with VAD(VAS1:P=0.0061,VAS2:P<0.0001).The proportion indicating the serum retinol concentration returned to normal levels(higher than 1.05?mol/L)was 21/46(45.7%)in children treated with VAD-VAS1 and 23/36(63.9%)in children treated with VAD-VAS2.No statistical difference was observed between the two VAS treatment doses(?~2=2.701,P=0.100).The VAD-VAS2 treatment significantly increased serum OXT levels(P=0.0027),decreased the overall score of SRS(P=0.0162),and the mRNA expression levels of RAR?(P=0.0295)and CD38(P=0.0272)genes in PBMC of children with ASD.The VAD-VAS1 treatment showed no significant changes in serum OXT levels,SRS scores,and PBMC-related gene mRNA expression levels.Both of the two supplementary programs have no significant effect on the ABC and CARS scales.There was a significant positive correlation between changed VA level and changed OXT levels(P<0.0001,r=0.3735).The changed retinol concentration was negatively correlated with the changed SRS scale score(P=0.0026,r=-0.2615).No significant correlation was observed between the changed VA level and the changed scores of CARS and ABC scales after intervention.Conclusions:(1)Both VAS doses could significantly improve the VA levels in children with ASD and VAD.In addition,continuous low-dose VAS enhanced RAR?mRNA expression.(2)Continuous low-dose VAS could significantly improve the CD38 mRNA,serum OXT levels and social function in children with ASD,which is promising to be a potential therapeutic strategy for improving both VA levels and core symptoms in children with ASD.Numerous large-scale randomized controlled clinical studies are needed to test this hypothesis.(3)VAS may increase the level of CD38,OXT and social function of ASD through RAR?.The specific mechanism needs further study.PART II THE REGULATION EFFECT OF RAR? SIGNAL ON THE CD38,OXT IN HYPOTHALAMUS AND THE AUTISTIC-LIKE BEHAVIORS IN RATSPurpose: The RA-RARs signaling pathway plays an important role in brain development.The abnormality of RA signaling pathway might be related to the pathogenesis of ASD.Besides,some studies suggest that RARs may be transcriptional regulators of CD38 and OXT genes.The first part of this study found that continuously low-dose VAS can improve VA levels,OXT levels,RAR? and CD38 m RNA expression and social function in children with ASD,suggesting that RAR? might regulate CD38 and OXT levels that could lead to the improvement of social function.However,the function of RAR? for CD38 and OXT in the hypothalamus(the brain region which produces OXT)is still unclear.The specific regulation mechanism of RA-RAR? signaling pathway on CD38 and OXT genes needs to be further clarified.In order to explore the regulatory mechanism of RAR? on CD38,OXT and the effects on ASD symptoms,this part of the study changed the RAR? expression signal in rat hypothalamus and detected ASD-related molecules(CD38,OXT)and autistic-like behaviors in rats.Further verification of the transcriptional regulation of RAR? on the CD38 gene and OXT gene was conducted.Method:(1)In vivo study: 2-week-old Sprague Dawley rats received injection of the adeno-associated virus(AAV)containing interfering-RAR? or overexpressing-RAR? vector into the hypothalamus through stereotactic injection system.Rats born in the same litter were randomly divided into 6 groups including control group(Control),PBS injection(PBS),empty virus for overexpressing-RAR? AAV injection(CON-RAR?),overexpressingRAR? injection group(AAV-RAR?),empty virus for interfering-RAR?AAV injection(CON-si-RAR?),and interfering-RAR?AAV injection(si-RAR?).The rats were housed to 8 weeks of age,continuously interfered with different RAR? signal in hypothalamus.The autistic-like behaviors of each group of rats were evaluated using the ANYmaze behavioral video-tracking analysis system,including the olfactory test,the 3-chamberd social test,and the open field test.The rats were sacrificed for collecting the blood and hypothalamic tissue samples after completing the behavioral tests.The serum VA concentration was detected by HPLC,the OXT concentration was detected by ELISA,and the expression of RAR? and CD38 protein in rat hypothalamus was detected by Western Blot.(2)In vitro study: The fetal rats aged E17-18 days were sacrificed to extract the primary neurons in the hypothalamus for in vitro research.The neurons were cultured until the 5th day then were treated with 1?mol/L all-trans retinoic acid(at RA).After 24 hours,neurons were collected for subsequent detections.The expression of RARs(RAR?,RAR?,RAR?)and CD38 protein in primary neurons was detected by Western Blot.The relative expression of RARs(RAR?,RAR?,RAR?)and CD38 m RNA were detected by RT-PCR.The chromate immunoprecipitation(Ch IP-q PCR)and dual-luciferase reporter assay were used to detect the transcriptional regulation activity and binding region of RAR?on the promotors of CD38 and OXT genes.Results:(1)The levels of RAR?(P = 0.0091),CD38(P < 0.0001)protein and serum OXT(P = 0.0004)in the hypothalamus of rats injected with RAR?-interfering virus were significantly lower than those in the control group.There was no significant change in RAR?,CD38 protein expression and serum OXT levels in the hypothalamus of rats injected with over-expressed RAR? virus.There was no significant effect on the serum VA level in the hypothalamic RAR? signal.(2)The number of sniffing times for social odor in si-RAR? rats was significantly decreased(P = 0.0463),and the time spent with strange mice in the three-chambered test was less than that in the control group(P = 0.1892),and the interaction time with objects was increased(P = 0.1304)even though there was no statistical difference.The time spent in self-grooming(P = 0.0031)and the number of selfgrooming(P = 0.0057)increased significantly in the open field test.The duration of interaction with unfamiliar rats of AAV-RAR? rat in threechambered social test was increased(P = 0.0445),and there was no significant change was found in the olfactory test and the open field test.(3)With at RA treatment,the m RNA and protein expression levels of RAR?(P = 0.0049),CD38(P = 0.0003),OXT(P = 0.0266)were significantly increased in the hypothalamic neurons.The binding of RAR? to the predicted binding-site of CD38 gene promoter region was enhanced(P = 0.0167),so as to the OXT gene(P = 0.05).Dual-luciferase reporter gene assay showed that RAR? has transcriptional activity on both CD38 and OXT genes.Electron microscopy showed that the secretion of prominent vesicles in neurons treated with at RA was increased compared with the control group.Conclusion:(1)The hypothalamic injection of si-RAR? adenoassociated virus can reduce the expression of RAR? protein,CD38 protein and serum OXT.And overexpression of RAR? signal has no significant effect on RAR?,CD38 protein and serum OXT level.There was no significant change for serum VA levels in rats no matter the decreases or increases of hypothalamic RAR? signal.(2)Injecting si-RAR? rats may have symptoms of impaired social function,mainly due to impaired olfactory exploration of social odor,accompanied by repeated stereotyped behaviors.However,overexpressed AAV-RAR? increased their interaction time with unfamiliar rats,and no significant changes were observed in other behavioral.(3)At RA can significantly increase the m RNA and protein expression levels of RAR?,CD38 and OXT in hypothalamic neurons.The RAR? has transcriptional activity on CD38 and OXT genes.The transcriptional regulation of RAR? on OXT may increase OXT protein synthesis.And the transcriptional regulation to CD38 may lead to the release of OXT indirectly,which will improve both the synthesis and release of OXT-system.Further experimental verifications of neural function are needed.