Research For Effect Of Th17/Tregcell Homeostasismediated By Vitamin D On Dysregulation Of Gut Microbiotain Autistic Rats

Autism spectrum disorder?ASD?,referred to as ASD,is a neurodevelopmental disorder characterized by social communication disorders,restricted interests or activities and repetitive/stereotypic behavior.It's commonly seen in children before the age of 3.In recent decades,the prevalence of autism has increased gradually.In2014,according to data released by the autism and developmental disabilities monitoring center of the United States,the prevalence of autism is as high as14.7/1000,equivalent to 1 in 68 children.It is indicated from the World Health Organization that autism has become one of the fastest growing serious diseases in the world and is a major public health problem.At present,the etiology of autism is unknown,but it is generally accepted that genetic and environmental factors work together to cause the disease.The treatment is mainly behavioral intervention and special education.There is no specific treatment drug for the core symptoms.It has become an increasingly important issue to search for the pathogenesis of autism and find effective treatment drugs and methods.Recent studies found that children with autism have immune system disorders.In addition,the morbidity rates of autoimmune diseases,allergies and psoriasis in ASD children are significantly higher than in healthy children.In vivo,the concentration of CD8⁺T cells,CD8⁺B cells,products of CD4⁺T cells,and activities of Th17 increase;a variety of cytokines,such as tumor growth factor beta 1,monocyte chemotactic protein 1,insulin-like growth factor binding protein 1 and a variety of interleukin also increase.However,Treg cells are restrained.At the same time,research evidence shows that the prevalence of inflammatory bowel disease and other gastrointestinal disorders in children with autism is higher.There are significant differences between the vulnerable bacteroides,clostridium,clostridium bauxis and other bacteria in the autism group and the healthy population.And the disorder of intestinal flora can regulate the central nervous system from bottom to top through neuroimmune and neuroendocrine mechanisms based on the principle of brain-gut axis.It is worth noting that the cell balance betweenTh17 and Treg may be a key factor in maintaining normal overall immune function of the body and immune barrier function of intestinal canal.Vitamin D is one of the important essential nutrients for human.Its receptors distribute in many important organs.It can not only regulate calcium phosphorus metabolism,but also be closely related to autoimmune diseases,tumors,cardiovascular diseases,and infectious diseases.Vitamin D may serve as a kind of nerve protectant protecting cerebral cortex neurons from excitability toxicity.In addition,vitamin D can also regulate the function of T helper cells?Th cells?and T regulatory cells?Treg cells?,inhibit Th17 cells,and promote the development of Treg cells.A large numbers of studies have confirmed that vitamin D levels in autistic children are lower than healthy children with the matching age.Adequate and standard vitamin D supplementation can improve the core symptoms of autism,but the mechanism of effect is unknown.In conclusion,vitamin D may regulate intestinal flora disorders in autism by affecting Th17/Treg cell balance,thereby improving the development of the nervous system through the brain-gut axis.This study constructed the rats model as the control group,ASD-N group?ASD model without treatment group?,and ASD-D group?ASD model with vitamin D treatment group?.In order to observe effect of Th17/Treg cell homeostasis mediated by vitamin D on dysregulation of gut microbiota in autistic rats,we designed a series of protocols to study the growth and development,autistic behavior,Th17/Treg cells,cytokines,gut microbiota,prefrontal GABA and 5-HT levels,serum GABA and 5-HT levels and colonic 5-HT of three groups of rats.Part?Established VPA ASD rat modelObjective:to establish VPA ASD rat model and prepare for the following experiment.Methods:Wistar female rats and male rats were caged together.When the vaginal pictures showed sperm,this day were recorded as the first day of pregnancy.The rats were divided into two groups at 12.5 days of gestation.The control group was given a single intraperitoneal injection of 100?L physiological saline,and the model group was given a single intraperitoneal injection of VPA?600mg/kg?.After birth,10 male offspring of the control group were selected as the control group.Ten male offspring of the model group were selected as model group?model group?.Two groups of rats were weighed on day 7,12,21,35 and 42 after birth in sequence.On day12¹6 after birth,eye opening was recorded.On day 8,10,12 and 16 after birth,swimming assessment was recorded.On day 21,32 and 42after birth,self-grooming test was records.On day 30 afterbirth,olfactory habituation was recorded.On day 42 after birth,social interaction test was recorded.Results:Compared with the control group,in the model group,weight significantly reduced?p<0.001?,eye opening time delayed?p<0.01?,swimming performance decreased?p<0.05?,self-grooming time prolonged?p<0.05?,olfactory sensitivity to other rats decreased?p<0.05?,and social interaction time decreased?p<0.05?.Conclusion:Intraperitoneal injection of sodium valproate at 12.5d of gestation in Wistar female rats can lead to the characteristic symptoms of autism in their male offspring.Stable animal models of autism was successfully replicated.Part?Effect of vitamin D on treatment of autistic ratsObjective:to observe the effects of vitamin D on autistic rats.Methods:The modeling method was the same as part?.There were three groups as the control group,ASD-N group and ASD-D group.The ASD-N group and the control group received a single intramuscular injection of 100?Lphysiological saline on day12after birth,and the ASD-D group received a single intramuscular injection of 80,000IU/kg vitamin D₃ onday12after birth.Three groups of rats were weighed on day 7,12,21,35 and 42 after birth.On day12¹6 after birth,eye opening was recorded.On day 8,10,12 and 16 after birth,swimming assessment was recorded.On day 21,32and 42 after birth,self-grooming test was records.On day 30 after birth,olfactory habituation was recorded.On day 42 after birth,social interaction test was recorded.On day 45after birth,serum 25?OH?D₃was determined by high performance liquid chromatography?HPLC?.Results:compared with the ASD-N group,the weight significantly gained?p<0.001?,eye opening time didn't delay?p<0.01?,swimming performance was better in the ASD-D group?p<0.01?.All were closed to the control group.Compared with the ASD-N group,the self-grooming time was shortened?p<0.05?,the olfactory sensitivity to other rats was increased?p<0.001?,and the performance of social interaction was increased in the ASD-D group?p<0.01?.Serum 25?OH?D₃ level in the ASD-N group was significantly lower than the control group?p<0.05?,and the serum 25?OH?D₃ level was negatively correlated with the self-grooming time onday42 after birth?p<0.05?.Serum 25?OH?D₃ level in ASD-D group was significantly higher than ASD-N group?p<0.01?.Conclusion:Vitamin D can promote the growth and development of ASD rats and improve ASD-like behavior.Repetitive behaviors in late development of ASD rats were aggravated by decreased vitamin D levels.Part?Studying the mechanism of vitamin D in the treatment of autistic ratsObjective:to investigate the effect of vitamin D treatment on Th17/Treg cell balance,gut microbiota,prefrontal GABA and 5-HT levels,serum GABA and 5-HT levels and colonic 5-HT levels in autistic rats.Methods:The modeling method and grouping method were the same as part?.In three groups,the proportion of Treg cells and Th17 cells were tested by flow cytometry,serum IL-17,IL-6 and IL-23levels,prefrontal and serum GABA and 5-HT levels and colonic 5-HT levels were detected by ELISA method.Gut microbiota of the three groups of rats was analyzed by high throughput sequencing.Results:compared with the control group,the proportion of Th17 cells increased?p<0.05?,the proportion of Treg cells decreased?p<0.005?,and the levels of IL-17,IL-6 and IL-23 significantly increased in the ASD-N group?all p<0.05?.Compared with the ASD-N group,the proportion of Th17 cells decreased?p<0.05?,the proportion of Treg cells increased?p<0.05?,and the levels of IL-17,IL-6 and IL-23 decreased significantly in the ASD-D group?all p<0.05?.Observed species index and hannon index of ASD-N group were significantly lower than those of the control group?all p<0.01?,and Simpson index of ASD-N group was significantly higher than those of the control group?p<0.05?.Above index changes of ASD-D group were not significantly different from those of ASD-N group.In the classification level of phylum,compared with the control group,Firmicutes in the ASD-N group increased significantly?p<0.05?,while Bacteroidetes and Proteobacteria decreased significantly?p<0.05,p<0.01?.Compared with ASD-N group,Firmicutes in ASD-D group were significantly reduced?p<0.05?,while Bacteroidetes and Proteobacteria were significantly increased?all P<0.05?.In the classification level of class,compared with the control group,Bacteroidia and Deltaproteobacteria in the ASD-N group decreased significantly?p<0.05,p<0.001?,Verrucomicrobiae in the ASD-D group increased significantly?p<0.05?.Compared with ASD-N group,Bacteroidia,Deltaproteobacteria and Verrucomicrobiae in the ASD-D group were significantly increased?p<0.05,p<0.01,p<0.05?,Bacilli in the ASD-D increased significantly?p<0.01?.In the classification level of order,compared with the control group,Bacteroidia and Desulfovibrionales in the ASD-N group decreased significantly?p<0.05,p<0.001?,Verrucomicrobiales in the ASD-D group increased significantly?p<0.05?.Compared with ASD-N group,Bacteroidia,Desulfovibrionales andVerrucomicrobiales in the ASD-D group were significantly increased?p<0.05,p<0.01,p<0.05?,Lactobacillales in the ASD-D group decreased significantly?p<0.01?.In the classification level of family,compared with the control group,Desulfovibrionaceae in the ASD-N group decreased significantly?p<0.05?,Verrucomicrobiaceae in the ASD-D group increased significantly.Compared with ASD-N group,Lactobacillaceae in the ASD-D decreased significantly?p<0.01?,Verrucomicrobiaceaeand Desulfovibrionaceae in the ASD-D group were significantly increased?p<0.05,p<0.01?.In the classification level of genus,compared with the control group,Ruminococcus,Oscillospira?CF231and others in the ASD-N group decreased significantly?p<0.001,p<0.01,p<0.01,p<0.05?,Akkermansia in the ASD-D group increased significantly?p<0.05?.Compared with ASD-N group,Akkermansiaand and CF231 in the ASD-D group were significantly increased?all p<0.05?,Lactobacillus in ASD-D group decreased significantly?p<0.05?.In the classification level of species,compared with the control group,Lactobacillus_reuteri in the ASD-N group increased significantly?p<0.05?,Lactobacillus_reuteri and others in the ASD-D group decreased significantly?p<0.001,p<0.01?.Compared with ASD-N group,Lactobacillus_reuteri and others in the ASD-D group were significantly decreased?p<0.001,p<0.05?.The level of prefrontal GABA and5-HT in the ASD-N group was significantly lower than the control group?all p<0.05?,and the level of prefrontal GABA and 5-HT in the ASD-D group was significantly higher than the ASD-N group?all p<0.05?.The level of serum GABA and 5-HT in the ASD-N group was significantly higher than the control group?all p<0.01?,and the level of serum GABA and 5-HT in the ASD-D group was significantly lower than the ASD-N group?all p<0.05?.The level of colonic 5-HT in the ASD-N group was significantly higher than the control group?p<0.01?,and the level of colonic 5-HT in the ASD-D group was significantly lower than the ASD-N group?p<0.05?.Conclusion:Vitamin D plays a role in Th17/Treg homeostasis regulation in ASD rats.Vitamin D regulates dysregulation of gut microbiotain in ASD rats,and improvements in Th17/Treg homeostasis may contribute to this regulation.Vitamin D can treat ASD by upregulating the level of prefrontal 5-HT.The decreased relative abundance of Firmicutes may be involved in the down-regulation of serum 5-HT.The incongruity between serum 5-HT and prefrontal 5-HT may be related to the effect of brain gut axis.Vitamin D can treat ASD by up-regulation the level of prefrontal GABA.Whether and how gut microbiota affects the regulation of GABA needs further research.