| BackgroundAutism spectrum disorder(ASD)refers to a group of neurodevelopmental disorders that start in early childhood.These disorders,whose main symptoms are social communication impairment and repetitive stereotyped behaviors and interests,have a high prevalence and high disability rate.ASD is one of the fastest-growing severe diseases in the world.In 2021,the US Centers for Disease Control and Prevention reported that the prevalence of ASD in 8-year-old children was as high as2.27%.The onset of ASD often occurs in early childhood when there is no self-care ability,and the disease often persists throughout their whole life.The etiology and pathogenesis of the disease are still unclear.So it remains one of the most challenging global health problems to our society.It is generally believed that ASD may be resulted from the the combined effects of genetic factors and early embryonic environmental factors.Vitamin D(VitD)deficiency during pregnancy and early brain development is considered to be one of the environmental risk factors for ASD.High-dose vitamin D supplementation will help improving the core symptoms of ASD,however,the specific mechanism is still unclear.Intensive study on the target and specific mechanism of VitD affecting ASD is important for the prevention,early diagnosis and precise treatment of ASD.Tryptophan(Trp)is an essential amino acid for the human body,which is the precursor of many bioactive substance including 5-hydroxytryptamine(5-HT),kynurenine(KYN),and indole derivatives,the ligands of aryl hydrocarbon receptor(AHR),etc.And it is also the precursor of nicotinamide adenine dinucleotide(NAD~+),an important coenzyme in electron transfer reaction.Trp metabolism has dramatic effects on both the central nervous system and the peripheral tissues and organs.The main pathway of Trp metabolism includes the generation of 5-HT,AHR ligand and KYN.Previous studies have showed that Trp metabolism is closely related to ASD in neurotransmitter,immune disorder and gut-brain axis.5-HT,a Trp metabolite,is closely related to gastrointestinal motility.Therefore,the disorder of Trp metabolism may play a key role in the pathogenesis of ASD and may be closely related to the morbidity of children with ASD consolated with gastrointestinal symptoms.VitD as a kind of steroid hormone,mainly exists in blood in the form of 25(OH)D,1,25(OH)2D is generated after further hydroxylation of 25(OH)D by kidney,then binds to vitamin D receptors,activates downstream signaling pathways,and regulates the transcription of target genes.Studies have shown that VitD regulates Trp in several steps in Trp metabolism,including 5-HT,KYN,and AHR ligand indole derivatives.Therefore,it is pressumed that the effect of VitD on ASD might be through its action on Trp metabolic pathway.In this study,we first retrospectively analyzed the correlation between symptoms,blood amino acid levels,and serum 25(OH)D levels in 659 children with ASD,analyzed the relationship between Trp metabolism and clinical symptoms of ASD and serum VitD level,and explored the possible target of VitD on ASD.On the basis of clinical analysis,this study used valproic acid(VPA)intraperitoneal injection during pregnancy to construct an ASD rat model.The changes of behavior,gastrointestinal motility and Trp metabolism in rats after VitD intervention were observed.Further discussion on the effect of VitD on ASD and whether Trp metabolism can be used as a classification index of ASD subtypes will provide a new idea for the research on the pathogenesis of ASD,provide a theoretical basis for the prevention and improvement of ASD by VitD,and possibly provide a new index and target for the early diagnosis and precise treatment of ASD.Part 1 Relationship between VitD and clinical symptoms and blood amino acids in children with ASDObjective:To discuss the correlation between Trp metabolism and VitD in ASD children by analyzing interaction between blood amino acid levels,ASD symptoms and serum VitD levels in ASD children.Methods:Clinical data from 659 children with ASD were retrospectively analyzed.Serum25(OH)D and blood amino acid levels were determined by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).Serum 25(OH)D≤10ng/m L,10～30 ng/m L,and≥30 ng/m L represent VitD deficiency,insufficiency,and sufficiency,respectively.Symptoms of children with ASD were assessed using the autism behavior checklist(ABC)and the autism rating scale(CARS).The ABC table is divided into five attributes,which are sensory stimuli sensorial(SSS),interaction,I,body and object use(BO),and language(L)and social self-care(SSC).Serum25(OH)D was detected in 659 children with ASD.506 of these children were detected alanine(Ala),aspartic Acid(Asp),glutamate(Glu),methionine(Met),phenylalanine(Phe),tyrosine(Tyr),leucine(Leu),Trp,valine(Val),arginine(Arg),citrulline(Cit),glycine,Gly),ornithine(Orn),glutamine(Gln),histidine(His),serine(Ser),threonine(Thr)and proline(Pro)a total of 18 amino acids.Data statistical analysis was performed by BONC DSS Statistics 25.0.The normality of the data was carried out by Kolmogorov-Smirnov.Analysis of variance was used to compare normal samples.Comparison between non-normal samples was conducted by Kruskal-Wallis H or Mann-Whitney U tests.Correlation analysis was conducted by Pearson test or Spearman test.Data were expressed as Mean±standard deviation(Mean±SD)or Median(interquartile distance)[Median(IQR)].Results:1.Correlation analysis of serum 25(OH)D level and symptoms in children with ASD showed that:There were significant differences in CARS scores among the three groups of children with ASD with different serum 25(OH)D levels(P=0.040).The CARS scores of ASD children in 25(OH)D deficient group were significantly higher than those in insufficient group(P=0.001).The CARS scores of ASD children in 25(OH)D deficient group were significantly higher than those in sufficient group(P=0.009).ABC scores is significantly negatively correlated with serum 25(OH)D level(r=-0.082,P=0.036).2.Correlation analysis of blood amino acid level and serum 25(OH)D level in children with ASD showed that:The levels of Ala,Arg,Gly,Orn,Gln and His in the blood of children with ASD were significantly different under the condition of serum25(OH)D sufficiency,insufficiency and deficiency(P=0.019,P<0.001,P=0.003,P=0.001,P=0.002,P=0.019,P<0.001,P=0.002.P=0.025).These 6 amino acids were significantly negatively correlated with serum 25(OH)D levels(r=-0.115,P=0.009;r=-0.278,P<0.001;r=-0.149,P=0.001;r=-0.167,P<0.001;r=-0.148,P=0.001;r=-0.133,P<0.003).Further stratified analysis showed that there was a significant positive correlation between serum 25(OH)D and Trp level when 25(OH)D≤10 ng/m L(P=0.033).3.Correlation analysis of whole blood amino acid level with ABC and CARS scores in children with ASD showed that:There was a significant positive correlation between blood Leu level and ABC score(r=0.104,P=0.020),a significant negative correlation between Trp level and ABC score(r=-0.096,P=0.030),and a significant negative correlation between Trp level and BO score in ABC(r=-0.105,P=0.022).Among the 142 children with ASD accompanied with gastrointestinal symptoms,ABC scores were significantly negatively correlated with Trp levels(r=-0.221,P=0.008).Conclusions:1.The lower the level of 25(OH)D in children with ASD is,the more obvious the symptoms became.2.The lower the blood Trp level of children with ASD is,the more significant the symptoms of ASD became,mainly manifested in increased physical symptoms.The lower the blood Trp level in children with ASD with gastrointestinal symptoms is,the more significant the symptoms became.3.When serum 25(OH)D was deficient in children with ASD,the level of25(OH)D was positively correlated with the level of Trp.Part 2 Effects of VitD on behavior and Trp metabolism in ASD rats.Objective:To establish a ASD rat model with the intervention of VitD,observe rats’behaviours and Trp metabolism level in different groups,disscuss the effects of VitD on the behavior and Trp metabolism of each group of rats.Methods:SPF reproductive age SD female rats were fed AIN93G diet with different VitD content.The rats were divided into normal control group(CON):VitD content was1000 IU/kg;VitD deficiency group(VDD):VitD content was 0 IU/kg;VPA injection group(VPA):VitD content was 1000 IU/kg;VPA+high-dose VitD(VPA+VD):VitD content was 1000 IU/kg,and intramuscular VitD 20000 IU/200g before mating.After The serum 25(OH)D was detected by HPLC-MS/MS after feeding 4 weeks.The25(OH)D of VDD group was significantly lower than that of the other three groups.Male and female rats 2:1 cage,so that female rats impregnated,sperm smear found sperm diary for the first day of pregnancy.At 12.5 days of gestation,rats in the VPA+VD group and the VPA group were given intraperitoneal injection of 600 mg/kg VPA solution.CON and VDD groups were injected with the same amount of normal saline.The rats born from the mother rats were used as the experimental group.10 female and 10 male offspring from each group were taken as the model group for follow-up experiments.The offspring of CON group were divided into male control group(CON-m)and female control group(CON-f).The offspring of VDD group were divided into male VitD-deficient group(VDD-m)and female VitD-deficient group(VDD-f).The offspring of VPA group were divided into male ASD group(ASD-m)and female ASD group(ASD-f).The rats of VPA+VD group were given intramuscular injection of20000 IU/200g body weight one week after birth as ASD supplement for high-dose VitD male rats group(ASD+VD-m).The rats in each group were breastfed for 21 days after birth.After 21 days,the VDD-m and VDD-f groups were fed VitD deprived diet(VitD content was 0 IU),and the offspring of the other groups were fed normal VitD content diet.The offspring rat were fed in separate cages after 21 days of birth.On the 28th day after birth,the rats in each group began to undergo 3-box social test,stereotyping test,olfactory behavior test and gastrointestinal dynamics analysis.After the behavioral test,the rats were weighed and anesthetized for abdominal aorta blood collection and serum isolation at-80℃.The colon was removed and part of the colon was stored at-80℃for the subsequent detection of gene m RNA and protein.The other part of the colon was fixed in 4%paraformaldehyde for subsequent morphological examination.Postpartum blood samples were collected from the caudal vein to extract serum for follow-up detection.Serum 25(OH)D levels were determined by HPLC-MS/MS.Serum Trp,KYN and5-HT levels of mother and offspring rats in each group were detected by ELISA.5-HT levels of intestinal tract of offspring rats were detected by immunofluorescence.The levels of Tph,Ahr and Vdr transcription and protein expression were detected by q PCR and Western Blot.Data Statistical analysis was performed by BONC DSS Statistics 25.0.The normality of the data was tested using Kolmogorov-Smirnov.Comparison between normal samples was performed by T test or analysis of variance.Comparison between non-normal samples was conducted using Kruskal-Wallis H or Mann-Whitney U.Correlation analysis was conducted by Pearson test or Spearman test.Data were expressed as Mean±SD or Median(IQR).Results:1.Results of 3-chambered social testComparison of the social behaviors of offspring male rats showed that:In stage 1,the approach time in both VDD-m and ASD-m groups to the empty cage was significantly reduced compared with CON-m group(P=0.001,P=0.002).The frequency that both VDD-m and ASD-m groups approached the empty cage was significantly lower than that in the CON-m group(P<0.001).The approach time to rat1 in both VDD-m and ASD-m groups was significantly shorter than that of CON-m group(P=0.019,P=0.005).The frequency of approaching rat 1 in ASD-m group was significantly lower than that in CON-m group(P=0.017).Compared with the empty cage,the time of approaching rat 1 in CON-m,VDD-m and ASD+VD-m groups was significantly increased(P=0.028,P=0.011,P=0.005).Compared with empty cage,the number of approaching to rat 1 in both VDD-m and ASD+VD-m groups was significantly increased(P=0.008,P=0.007).In stage 2,the approach time in both VDD-m and ASD-m groups to rat 1 was significantly reduced compared with CON-m group(P=0.001,P=0.006).The frequency of approaching rat 1 in VDD-m,ASD-m and ASD+VD-m groups was significantly lower than that in CON-m group(P=0.001,P=0.002,P=0.031).The time to approach rat 2 in both VDD-m and ASD-m groups was significantly reduced compared with CON-m group(P<0.001).The frequency of approaching rat 2 in both VDD-m and ASD-m groups was significantly lower than that in CON-m group(P<0.001).The frequency of approaching rat 2 in both VDD-m and ASD-m groups was significantly lower than that in CON-m group(P<0.001).Effects of VitD supplementation on Social behavior of male rats with ASD:In the first stage,the ASD+VD-m group had a significantly longer time to approach rat 1than the ASD-m group(P<0.001).The frequency of approaching the empty cage in ASD+VD-m group was also significantly higher than that in ASD-m group(P=0.001).The frequency of approaching rat 1 in ASD+VD-m group was significantly higher than that in ASD-m group(P<0.001).In the second stage,ASD+VD-m significantly increased the time to approach both rat 1 and rat 2 compared with the ASD-m group(P=0.019,P<0.001).The frequency of approaching rat 1 and rat 2 in ASD+VD-m group was also significantly higher than that in ASD-m group(P=0.028,P<0.001).Comparison of social behaviors between male and female offspring rats showed that:CON-f and CON-m group,in the first stage 1,there was no significant difference between CON-f and CON-m groups in the time and frequency of approaching the empty cage and rat 1(P?0.05).The time to approach rat 1 was significantly higher in both groups than in the empty cage(P=0.028,P=0.007).The frequency of approaching rat 1 in CON-f group was significantly higher than that in empty cage(P=0.012).In the second stage,there was no significant difference in the time and frequency of approaching rat 1 and rat 2 between CON-f and CON-m groups(P?0.05).Compared with the VDD-f group,the time of approaching the empty cage and rat 1 in the first stage of the VDD-f group was significantly increased compared with the VDD-m group(P=0.010,P=0.001),the frequency also increased significantly(P=0.023,P=0.003),The time(P=0.011,P=0.005)and frequency(P=0.008,P=0.005)of approaching rat 1 in the two groups were higher than those in the empty cage.In the second stage,the approach time of VDD-f to rat 1 and rat 2 was significantly increased compared with that of VDD-m group(P<0.001).The frequency also increased significantly(P<0.001,P=0.001).In ASD-m and ASD-f groups,the time to approach empty cage and rat 1 in stage 1 was significantly increased in ASD-f group compared with ASD-m group(P=0.049,P=0.001).The frequency also increased significantly(P=0.028,P=0.001).In the second stage,the approach time of rat 1 and rat 2 in ASD-f group was significantly increased compared with that in ASD-m group(P=0.019,P=0.001).The frequency also increased significantly(P=0.034,P=0.001).2.Results of stereotypical behavior detectionComparison of stereotypical behavior of male rats showed that:Self-grooming time of rats in VDD-m and ASD-m groups were significantly higher than those in CON-m group(P=0.035,P<0.001).The self-grooming frequency of ASD-m group was significantly higher than that of CON-m group(P<0.001).The effects of VitD supplementation on social behavior of male offspring rats with ASD were found:The self-grooming time and number of rats in ASD-m group were significantly higher than those in ASD+VD-m group(P<0.001).Comparison of stereotyped behaviors between male and female offspring rats found that:There was no significant difference in self-grooming time between male and female rats in CON group(P?0.05).The number of self-grooming in CON-m group was higher than that in CON-f group(P=0.005).The frequency and time of self-grooming in VDD-m group were significantly higher than those in VDD-f group(P<0.001).The frequency and time of self-grooming in the ASD-m group were significantly higher than those in the ASD-f group(P<0.001).3.Olfactory recognition test resultsComparison of olfactory recognition results of male rats showed that:The olfactory time of rat litter in VDD-m and ASD-m groups was significantly lower than that in CON-m group(P=0.002,P=0.002).Compared with the CON-m group,the ASD-m group had a significantly lower frequency of approaching the rat litter(P=0.016).Compared with CON-m group,the olfactory time and frequency of rat litter in ASD-m group were significantly reduced(P=0.007,P=0.014).The time of rats in CON-m,VDD-m,ASD-m and ASD+VD-m groups to approach rat litter was significantly higher than that of clean litter(P=0.005).The number of times of approaching rat litter was also significantly higher than that of cleaning litter(P=0.008,P=0.019,P=0.008,P=0.005).Study on the effect of VitD supplementation on olfactory recognition of male offspring rats with ASD found that:Compared with ASD-m group,the olfactory time and frequency of rat litter in ASD+VD-m group were significantly increased(P<0.001,P=0.015).Compared with ASD-m group,the olfactory time and frequency of clean litter were significantly increased in ASD+VD-m group(P=0.003,P=0.008).Comparison of olfactory recognition results between male and female offspring showed that:There was no significant difference between CON-m and CON-f groups in the time and frequency of approaching litter bedding and clean bedding(P?0.05).Compared with VDD-m group,VDD-f group had significantly increased the time and frequency of approaching the rat litter(P<0.001,P=0.025).The approach time of clean litter in VDD-f was significantly increased compared with that of VDD-m group(P=0.023).Compared with ASD-m group,the time and frequency of approaching the rat litter material in ASD-f group were significantly increased(P<0.001,P=0.023).Compared with ASD-m group,the time and frequency of approaching clean bedding in ASD-f group were significantly increased(P=0.003,P=0.034).4.Results of gastrointestinal transit and colonic emptying timeGastrointestinal transmission time test results:The gastrointestinal transmission time in VDD-m,ASD-m and ASD+VD-m groups was significantly longer than that in CON-m group(P<0.001).The effect of VitD supplementation on the gastrointestinal transit time of ASD male mice showed that the gastrointestinal transit time of ASD+VD-m group was significantly shorter than that of ASD-m group(P=0.001).There was no significant difference in gastrointestinal transit time between CON-m and CON-f groups(P?0.05).The gastrointestinal transit time in VDD-f group was significantly shorter than that in VDD-m group(P=0.005).The gastrointestinal transit time in ASD-f group was significantly shorter than that in ASD-m group(P<0.001).Colon emptying time test results:Comparison of male rats in each group showed that the colonic emptying time of VDD-m,ASD-m and ASD+VD-m groups was significantly longer than that of CON-m group(P<0.001,P<0.001,P=0.001).The study on the effect of VitD supplementation on colon empty-time in male rats with ASD found that:ASD+VD-m group had significantly shorter colon empting time than ASD-m group(P=0.001).There was no significant difference in gastrointestinal transit time between CON-m and CON-f groups(P?0.05).The gastrointestinal transit time in VDD-f group was significantly shorter than that in VDD-m group(P<0.001).The gastrointestinal transit time in ASD-f group was significantly shorter than that in ASD-m group(P<0.001).5.Detection results of serum 25(OH)D and Trp metabolism related products of mother ratsSerum 25(OH)D levels of female rats in each group before pregnancy:The pre-pregnancy serum VitD values of CON,VDD,VPA and VPA+VD groups were 25.9±3.9 ng/m L,9.9±3.3 ng/m L,29.1±5.2 ng/m L and 29.3±4.8 ng/m L,respectively.The VDD group was significantly lower than the other three groups(P<0.001).There were no significant differences in serum 25(OH)D levels among CON,VPA and VPA+VD groups(P>0.05).Mother rats Trp and its metabolites in serum test results:Serum Trp levels in VDD and VPA groups were significantly lower than those in CON group(P<0.001),and serum Trp levels in VPA group were significantly lower than those in VDD group(P<0.001).The serum KYN level of VDD and VPA groups was significantly lower than that of CON group(P<0.001),and the serum KYN level of VPA+VD rats was significantly higher than that of VPA group(P<0.001).Serum KYN/Trp of VPA female rats was significantly higher than that of CON group(P=0.0012).There was no significant difference between KYN/Trp and CON in VDD female rats(P>0.05).The KYN/Trp of VPA+VD female rats was significantly lower than that of VPA group(P=0.001).Serum 5-HT level in VDD and VPA groups was significantly lower than that in CON group(P<0.001),and serum 5-HT level in VPA+VD group was significantly higher than that in VPA group(P<0.001).6.Serum VitD and Trp metabolites in offspring ratsThe results of serum 25(OH)D in the offspring rats were as follows:CON-m group 41.93±21.58 ng/m L;VDD-m group 6.57±0.77 ng/m L;ASD-m group 34.09±14.45 ng/m L;ASD+VD-m group 35.63±16.90 ng/m L;CON-f group 17.59±4.57ng/m L;VDD-f group 5.90±4.84 ng/m L;ASD-f group 55.68±17.58 ng/m L.Comparison of 25(OH)D levels among male offspring showed that:The level of25(OH)D in VDD-m group was significantly lower than that in CON-m group(P<0.001).The 25(OH)D level in CON-f group was significantly lower than that in CON-m group(P<0.001).VDD-m and VDD-f group rat serum 25(OH)D levels had no significant difference(P?0.05).Serum 25(OH)D level in ASD-f group was significantly higher than that in ASD-m group(P=0.002).Results of Serum Trp and its metabolites in offspring rats:Serum Trp of offspring rats:Compared with CON-m group,the serum Trp level of VDD-m was significantly decreased(P=0.026).The study on the effect of VitD supplementation on male offspring rats with ASD found that there was no significant difference in serum Trp between ASD-m and ASD+VD-m groups(P?0.05).There were no significant differences in serum Trp levels of male and female offspring rats in CON,VDD and ASD groups(P>0.05).Serum KYN of offspring rats:There was no significant difference in serum KYN levels between VDD-m,ASD-m,ASD+VD-m and CON-m groups(P?0.05).There were no significant differences in serum KYN levels between male and female rats in CON,VDD and ASD groups(P>0.05).Serum KYN/Trp:Serum KYN/Trp of VDD-m group is significant different from CON-m group(P=0.015).There were no significant differences in serum KYN/Trp between ASD-m、ASD+VD-m groups and CON-m group(P?0.05).There were no significant differences in serum KYN/Trp between ASD-m and ASD+VD-m group(P?0.05).There were no significant differences in serum KYN/Trp between VDD-m and VDD-f group(P?0.05);There were no significant differences in serum KYN/Trp between ASD-m and ASD-f group(P?0.05).Serum 5-HT of offspring rats:The serum 5-HT level of CON-m,VDD-m,ASD-m and ASD+VD-m was significantly different(P<0.001).The serum 5-HT of VDD-m group was significantly higher than CON-m group(P<0.001).The serum 5-HT level of ASD+VD-m rats was significantly higher than that of ASD-m group(P<0.001).There was no significant difference in serum level of 5-HT in VDD-f group and VDD-m group(P?0.05).The 5-HT level in ASD-f group was significantly higher than that in ASD-m group(P=0.001).7.Intestinal section detection of offspring ratsHE staining results in the intestinal tract of rats in each groupThe intestinal morphology of CON-m,CON-f,VDD-f and ASD-f groups was completely normal.The intestinal tissue morphology of VDD-m,ASD-m and ASD+VD-m groups was roughly normal,with irregular villi arrangement and a few epithelial cells shedding,which was most obvious in ASD-m group,and no other obvious lesions were observed.Intestinal 5-HT immunofluorescence detection in offspring ratsThe percentage of 5-HT in intestinal tissue of rats in each group was detected by immunofluorescence method,and the results were as follows(%):CON-m group 11.70±0.35,VDD-m group 3.59±1.25,ASD-m group 1.46±0.82,ASD+VD-m group 4.98±1.58,CON-f group 11.17±3.62,VDD-f group 9.04±1.41,ASD-f group 12.44±2.78.The intestinal 5-HT levels in VDD-m and ASD-m groups were significantly lower than those in CON-m group(P<0.001).The intestinal 5-HT level of rats in ASD+VD-m group was significantly higher than that in ASD-m group(P=0.005).Intestinal 5-HT in VDD-m group was significantly lower than that in VDD-f group(P=0.005),and intestinal 5-HT in ASD-m group was significantly lower than that in ASD-f group(P<0.001).8.Transcription and protein expression of genes related to intestinal VitD and Trp metabolismVDR:Intestinal Vdr m RNA levels in VDD-m and ASD-m groups were significantly decreased compared with CON-m group(P<0.001),and VDR protein levels were significantly decreased compared with CON-m group(P<0.001).Compared with ASD-m group,the Vdr m RNA level in ASD+VD-m group was significantly increased(P<0.001),and the VDR protein level was also significantly decreased(P<0.001).Compared with VDD-m group,VDR m RNA level and VDR protein level in VDD-f group were significantly increased(P<0.001).Compared with ASD-m group,Vdr m RNA level and VDR protein level in ASD-f group were significantly increased(P<0.001).TPH1:Intestinal Tph1 m RNA levels in VDD-m and ASD-m groups were significantly decreased compared with CON-m group(P<0.001),and TPH1 protein levels were significantly decreased compared with CON-m group(P<0.001).The m RNA level of Tph1 in ASD+VD-m group was significantly higher than that in ASD-m group(P<0.001),and the protein level of TPH1 was also significantly decreased(P<0.001).The m RNA level and protein level of TPH1 in VDD-f group were significantly increased compared with that in VDD-m group(P<0.001).The m RNA level and protein level of TPH1 in ASD-f group were significantly increased compared with those in ASD-m group(P<0.001).TPH2:Intestinal Tph2 m RNA levels in VDD-m and ASD-m groups were significantly decreased compared with CON-m group(P<0.001),and TPH2 protein levels were significantly decreased compared with CON-m group(P<0.001).The m RNA level of Tph2 in ASD+VD-m group was significantly higher than that in ASD-m group(P<0.001),and the protein level of TPH2 was also significantly decreased(P<0.001).The m RNA level and protein level of TPH2 in VDD-f group were significantly increased compared with that in VDD-m group(P<0.001).The m RNA level and protein level of TPH2 in ASD-f group were significantly increased compared with those in ASD-m group(P<0.001).AHR:The m RNA level of intestinal Ahr in VDD-m and ASD-m groups was significantly increased compared with CON-m(P<0.001),and the protein level of AHR was also significantly increased(P<0.001).The m RNA level and protein level of AHR in ASD+VD-m group were significantly decreased compared with those in ASD-m group(P<0.001).The m RNA level and protein level of AHR in VDD-f group were significantly decreased compared with that in VDD-m group(P<0.001).The m RNA level and protein level of AHR in ASD-f group were significantly decreased compared with those in ASD-m group(P<0.001).Conclusions:1.Maternal VitD deficiency during rat pregnancy results in abnormal serum Trp metabolism.Mothers of VPA-induced ASD rats showed similar changes,and VitD supplementation improved Trp metabolism in mothers.2.Continous VitD deficiency during pregnancy and early brain development of female rats can lead to decreased social behavior,increased stereotyped behavior and decreased olfactory ability of male offspring;VitD supplementation during pregnancy and early brain development improved the core symptoms of ASD offspring,including increased social behavior and olfactory recognition ability,and decreased stereotypical behavior.The behavior of female rats was not affected.3.Continuous VitD deficiency during pregnancy and early brain development of female mice resulted in changes of serum Trp-related metabolites in male offspring,which were shown as decreased serum Trp level,increased 5-HT level,and increased KYN/Trp.Changes in serum 5-HT level were also observed in VPA-induced ASD modelrats,which were different from VitD-deficient male rats and showed decreased serum 5-HT level.Supplementation of VitD could increase the level of 5-HT in ASD model rats.4.The gastrointestinal peristalsis ability of VitD-deficient male rats and ASD male rats was decreased,accompanied by changes in intestinal Trp metabolism,including decreased intestinal production of 5-HT,decreased transcriptional expression of 5-HT hydroxylase Tph1/2,decreased transcriptional expression of Vdr and increased transcriptional expression of Ahr.VitD supplementation could improve gastrointestinal peristalsis and restore Trp metabolism of male ASD offspring.The two groups of female rats did not show similar changes;Tph1 and Tph2 showed the same trend. |
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