| Objective: Autism spectrum disorder(ASD)is a neurodevelopmental disorder that characterized by deficits in social communication and social interaction,and restricted,repetitive patterns of behavior,interests,or activities.Its incidence rate is high,and its etiology is complex.The disorder of neurotransmitters is closely related to the core symptoms of ASD,and is considered to play an important role in the pathogenesis of ASD.In recent years,studies have confirmed that gut microbiota can produce a variety of neurotransmitters that are the same as those in the central nervous system.In addition,ASD symptoms can be effectively improved by adjusting the gut microbiota.Meanwhile,studies have confirmed that exercise is an effective means to improve the symptoms of ASD,and it is an effective method to adjust the gut microbiota.However,whether exercise can improve ASD symptoms and related mechanisms by regulating gut microbiota remains unclear.Therefore,in order to provide a theoretical basis for the mechanism of exercise improving ASD,this study aims to use ASD model rats as the research object,to explore whether exercise can regulate central neurotransmitters through gut microbiota,and then improve the symptoms of ASD rats.Methods: Part One: The offspring of rats which were injected 400mg/kg of valproic acid on the 12.5 days of pregnancy,was VPA model rat,and the offspring of rats which injected saline in same time was the control group rat.The developmental indicators(including exercise performance,sensory perception,body weight,etc.)of offspring rats were observed.Morris water maze test,open field test and three-chamber social test were used to evaluate the autism symptoms of rats.Short-chain fatty acids(SCFA)in feces(including acetic acid [AA],propionic acid [PA],butyric acid [BA],valeric acid [VA],hexanoic acid [HA],isobutyric acid [IBA] and isovaleric acid [IVA])were measured by high performance gas chromatography and mass spectrometry tandem method.Fifty-five neurotransmitters in prefrontal cortex and hippocampus were measured by high performance liquid chromatography and mass spectrometry tandem method.All rats were subjected to developmental observation,and behavioral evaluation was performed on the 23 rd day after birth.After screening,16 rats in each group were selected to collect feces for detection of gut microbiota and short-chain fatty acids,and 5 rats in each group were tested for neurotransmitters.Part Two: After the behavioral assessment(postnatal day 28),the autism model rats were divided into the exercise group and the non-exercise group(Eight in each group).The autistic rats in the exercise group were conducted to 6 weeks of voluntary wheel running(the wheel was placed in a living cage),while the non-exercise group was not conducted to any intervention.The behaviors,gut microbiota,short-chain fatty acids and neurotransmitters in brain tissue of autistic rats were observed after 6 weeks of voluntary exercise,the test method is the same as the part one.Part Three: Autism model rats were randomly divided into fecal microbiota transplantation(FMT)group and sham transplantation(s FMT)group,FMT group were conducted to gut microbiota transplantation from 42 days after birth by gavage with fresh feces suspension(1 ml / 100 g)which came from the exercise autistic rats,and the s FMT group rats were gavage with normal saline,once a day,five days a week.After 4 weeks of fecal microbiota transplantation,the behavioral manifestations of autism rats were observed,and the changes of intestinal microbiota,short-chain fatty acids and neurotransmitters in brain tissue were detected,and compared with the autism model rats in the exercise group.The test method is the same as the first part.Results: Part One: 1.Compared with the normal control group,developmental observation results showed that the eye-opening time,motor ability were significantly delayed and abnormal vestibular sensation and thermal pain in autistic rats.2.Behavioral tests results showed that the learning and memory ability of the autistic rats were impaired,the motor or exploration ability was significantly reduced,and the spontaneous activity was decreased.The social ability and social novelty preference in the ASD group rats were decreased compared with those in the normal group.3.16 S r RNA sequencing results showed that there was no significant difference in α diversity of intestinal microorganisms in the VPA group,but there was significant difference in Β diversity.In the VPA group,the abundance of Bacteroidetes increased,while the abundance of Firmicutes and Clostridia decreased.4.Compared with the normal control group,metabolomics results showed that the contents of acetic acid(p = 0.004),butyric acid(p < 0.001),valeric acid(p < 0.001),isobutyric acid(p < 0.001)and isovaleric acid(p < 0.001)in the feces of the ASD group were significantly decreased,and no caproic acid was detected in the feces of the ASD group.The contents of betaine in the prefrontal lobe were significantly increased(p < 0.001),while the contents of threonine(p < 0.001),kynurenine(p = 0.026),tryptophan(p = 0.037),5-hydroxyindoleacetic acid(p = 0.008)and betaine aldehyde chloride(p = 0.001)were significantly decreased.The contents of betaine in hippocampus were significantly increased(p = 0.002),while the contents of threonine(p = 0.001),kynurenine(p = 0.007),5-hydroxyindoleacetic acid(p = 0.009),indole-3-carboxaldehyde(p = 0.015)and betaine aldehyde chloride(p = 0.023)were significantly decreased.The results of network association analysis showed that the spp.Pseudomonas,Collisella and Streptococcus had the most extensive regulatory effects on their metabolites and neurotransmitters in the prefrontal cortex.Betaine,kynurenine and threonine,are most affected by the regulation of microorganisms or their metabolites.Part Two: 1.Behavioral tests results showed that 6 weeks of voluntary wheel running increased the duration(exploration)time in the open field center and the preference for social ability and social novelty was significantly improved of autistic rats.2.16 S r RNA sequencing results showed that the 6-weeks voluntary wheel running intervention did not have a significant effect on α diversity and β diversity of gut microbiota.Compared with the normal control group,the abundance of spp Lactobacillus and Limosilactobacillus were significantly increased in the exercise group.However,in the non-exercise group,the abundances of spp.Allobaculum and unidentified Proteobacteria were significantly increased,while those of spp.Lactobacillus and Blautia were significantly decreased.3.Metabolomics results showed that after 6 weeks of voluntary wheel running,butyrate content in the exercise group was significantly higher than that in the non-exercise group.The levels of threonine(p = 0.028),kynurenine(p = 0.011),indole-3-carboxaldehyde(p = 0.009),tryptophan(p = 0.023),3-hydroxytyramine(p = 0.014),gamma-aminobutyric acid(p = 0.013),and 5-methotryptamine(p = 0.007)in the prefrontal cortex were significantly lower in the exercise group than in the non-exercise group,but the levels of epinephrine were significantly higher in the exercise group(p = 0.029).There was no significant difference in the neurotransmitters in the hippocampus between the exercise group and the non-exercise group.Network correlation analysis showed that spp.unidentified Prevotella,Allobaculum and Blautia were the most extensive regulatory effects on SCFAs and neurotransmitters in the prefrontal cortex.However,5-methoxytryptamine,3-hydroxytyramine and γ-aminobutyric acid were the most regulated factors by microorganisms or SCFAs.In the hippocampus,the spp.Unidentified_Prevotella,Blautia and Lactobacillus have the most extensive regulatory effects on metabolites and neurotransmitters in the hippocampus.Kynurenine,5-hydroxyindoleacetic acid and choline were most affected by the regulation of microorganisms and their metabolized SCFAs.Part Three: 1.After 4 weeks fecal microbiota transplantation,there is no significant effect on the water maze performance,but significantly improved their exploratory activities,spontaneous behaviors,social ability and preference for social novelty of autistic rats.2.The fecal microbiota transplantation changed the α diversity of gut microbiota,but had no significant effect on β diversity of gut microbiota.The relative abundance of spp.Unidentified_Christensenellaceae in the transplantation group showed the same trend as that in the exercise group,but the relative abundance of spp.Unidentified_Christensenellaceae was significantly lower than that in the sham transplantation group.3.Metabolomics results showed that compared to the exercise group,there was no significant difference in the contents of acetic acid,propionic acid,butyric acid,valeric acid,hexanoic acid,isobutyric acid and isovaleric acid in the transplantation group(p > 0.05).However,the contents of butyric acid(p = 0.007),valeric acid(p = 0.011),hexanoic acid(p = 0.003),isobutyric acid(p = 0.033)and isovaleric acid(p = 0.031)in feces of sham transplantation group were significantly decreased.Compared with the s FMT group,the serine(p = 0.035)and sarcosine(p = 0.044)were significantly increased,while the kynurenine(p = 0.009),tyrosine(p = 0.017),3-hydroxytyramine(p = 0.004),gamma-aminobutyric acid(p = 0.029)and ethanolamine(p = 0.039)were significantly decreased in the prefrontal cortex.The choline(p = 0.039)and succinate(p = 0.017)significantly increased,while the contents of kynurenine significantly decreased(p = 0.018)in hippocampus.The results of network association analysis showed that the spp.unidentified_Christensenellaceae 、 spp.Lactobacillus 、 spp.Limosilactobacillus 、 and spp.Romboutsia had the most extensive effects on the regulation of metabolites and neurotransmitters in the prefrontal cortex and hippocampus.Kynurenine,betaine aldehyde chloride,glycine,epinephrine and choline,were most regulated by microbial groups or their metabolites-SCFAs.Meanwhile,adrenaline,tyrosine,5-methyltryptamine,choline and 3-hydroxytyramine,were most regulated by microbial groups or their metabolites-SCFAs in the hippocampus.Conclusions: 1.The results of this study verified that VPA exposure during pregnancy could lead to developmental delay and ASD-like behavior changes in SD rats.At the same time,the gut microbiota structure,the contents of acetic acid,butyric acid,valeric acid,isobutyric acid and isovaleric acid in the feces,and the disturbance of neurotransmitter metabolism in the central nervous system under VPA exposure.2.Six weeks of voluntary wheel running can effectively improve the spontaneous activity and social function of autism,and affect gut microbiota.It can increase the level of SCFA in feces,especially butyrate.The changes of central neurotransmitters were associated with the changes of gut microbiota and SCFAs 3.Exercise regulates central neurotransmitters(especially kynurenine,gammaaminobutyric acid,and 3-hydroxytyramine)through gut microbiota and their metabolites-SCFAs,which may be one of the important mechanisms for exercise to improve the behavior of ASD rats. |
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