Autism spectrum disorders (ASD) is a severe neurodevelopmental disorder. ASD has brought a heavy economy and health burden for social and families. So far clinical level does not have effective treatment for the disorder. According to conservative estimation, the prevalence of ASD in China is about 1%，and keeps increasing year by year. The ASD patients not only show behavioral symptoms, but also have many typical biological symptoms which were omitted by clinical doctors for long time, such as abnormal immune function, gastrointestinal disorders and multiple allergies.Within three decades, people have been tracing the disease genes, but the sharp increase of the disorder does not correspond with the population genetic law of Hardy-Weinberg Equilibrium, that means the disorder is more influenced by environmental factors instead of genes. In fact, gut bacteria play more important role in neural system development and occurrence of ASD which is known to be closely associated to the unbalanced of gut microorganism and the abnormality of gut- brain axis. Since the brain development accompanied with gut-brain development simultaneously, any factor that affect their gut microbes could increase the risk of ASD during the critical period of individual development.
It is well known that gut microbes affect the brain and behavior through their metabolites, immune, neuroendocrine, and vagus pathway. Therapies with the gut bacteria as a target intervention for ASD is becoming a new hotspot, which includes dietary intervention, drug intervention, fecal microbiome transplantation, and probiotics intervention. Among the applications, probiotics is more concerned with its effectiveness and safety.
The present study aims to understand the interaction of the gut bacteria and ASD, and supplies two special commensal probiotic strains on the patients with ASD, to compare the biological and psychological change of the patients.
1 .Establish gut microbiota database of Chinese healthy children and ASD children,analyze the gut microbiota composition of ASD children, and try to treat ASD through gut microbiota regulation.
2. Compare the gut microbiota of healthy children and that of ASD children, identify the gut microbiota abnormalities of ASD children, and find reliable ASD related bacteria biomarkers.
3. Detect the changes of biochemical indicators related to microbiota-gut-brain axis,analyze the differences of biochemical indicators between healthy and ASD children,explore the effects of gut microbiota in ASD, identify the biochemical indicators closely related with ASD, and hope to provide reference for the clinical diagnosis and intervention of ASD.
4. Analyze the questionnaires filled out by the guardians of ASD children, compare the changes of behavioral and gastrointestinal symptoms before and after NS lactobacillus intervention, and evaluate the improvements after intervention.
5. Study the effectiveness and possibly mechanism of NS lactobacillus intervention which include Lactobacillus helveticus NS8 and lactobacillus fermentium NS9.
Materials and methods:
1 .Collect the questionnaires, stool samples and blood samples of ASD children.
2. Evaluate the behavioral and cognitive symptoms of ASD children using the Autism Treatment Evaluation Checklist (ATEC).
3. Evaluate the gastrointestinal symptoms of ASD children using the Gastrointestinal Severity Index (GSI).
4. Analyze the microbiota of stool samples using 16s rRNA and ITS 1 high-throughput sequencing technique, compare the microbiota composition between ASD children and healthy control children, and evaluate the effects of NS lactobacillus intervention on microbiota.
5. Compare the immune indicators, neurotransmitters and hormones related to microbiota-gut-brain axis between ASD children and healthy children, explore the differences and the influence of NS lactobacillus intervention, and reveal the effectiveness and mechanism the intervention.
1 .ASD children not only presented obvious cognitive and behavioral symptoms,accompanied with a variety of gastrointestinal symptoms, but also they showed abnormal gut microbiota including bacteria and fungi and abnormal biochemical indicators including higher Complement C3 levels and lower GABA and Glu levels in serum.
2. NS lactobacillus intervention significantly improved gastrointestinal symptoms and ASD-like symptoms. After three months of intervention, the gastrointestinal symptoms of ASD children decreased (total GSI score significantly reduced). For example, the stool consistency improved, the stool smell alleviated, the flatulence alleviated, unexplained daytime irritability behaviors reduced, and nighttime awaking times decreased. NS intervention also significantly alleviated the severity of ASD (total ATEC scores decreased and all subscales scores reduced). For example, the language communication, social interaction and sensory/cognitive awareness increased, and health/physical/behavior improved after intervention.
3. NS lactobacillus intervention significantly improved the microbial imbalance of ASD children.
4. NS lactobacillus intervention significantly improved the biochemical abnormalities of ASD children. After three months of intervention, the serum Complement C3 content of ASD children increased significantly, while GABA content of that decreased significantly.
5. The improvement in physiology and behavior of NS lactobacillus intervention was correlated with the regulation of gut microbiota. For example, the Complement C3 content was negatively correlated with the relative abundance of Romboutsia and Terrisporobacter, while the GABA content was positively correlated with the relative abundance of Romboutsia and Terrisporobacter.
In summary, there were significant differences in gut bacteria and fungi between ASD and healthy control children. Abnormal gut microbiota can induce ASD through the immunological pathway and neuroendocrine pathway of microbiota-gut-brain axis.NS lactobacillus intervention reduced the gut microbiota abnormalities of ASD children, and alleviated ASD through immunological pathway and neuroendocrine pathway.
The present study implemented NS lactobacillus combination intervention to the so-called incurable disease ASD for the first time. The study collected and analyzed the behavioral, physiological, biochemical, and microbiota data before and after intervention. The study firstly presented that NS lactobacillus intervention corrected the microbiota abnormalities of ASD children, improved the immune and neuroendocrine function, and alleviated the behavioral, cognitive and gastrointestinal symptoms of ASD children.