The Association Between Gut-brain Axis Related Markers And Cognitive Function In Residents In Longevity County And Analysis The Relationship Between Trace Elements And Non-alcoholic Fatty Liver Based On Public Database

PartⅠThe association between gut-brain axis related markers and cognitive function in residents in longevity countyObjective In the process of aging,the brain will undergo degenerative changes,which in turn leads to cognitive function decline.The gut-brain axis can not only affect lifespan,but also affect the healthspan,especially the cognitive function.However,the current research on relationship between gut-brain axis and cognitive function is mostly concentrated in model animals and general population,and lack of research evidence from the natural longevity population.Therefore,this study based on the established natural longevity population cohort（Clinical Trials:So Mi LY;NCT04210934）establish a comprehensive evaluation system for serum gut-brain axis related markers,including metabolites,brain-gut peptides and inflammatory factors.The correlations between gut microbiota and target metabolites and cognitive function were evaluated in a cross-sectional design based on fecal metagenomic sequencing and serum non-targeted metabolomics.In addition,a method system was established to clear gut microbiota and fecal microbiota transplantation（FMT）with compound antibiotics,and FMT intervention was performed on mice with human-derived cognitively weak fecal bacteria to evaluate the effects of different FMT methods and intervention time and the changes in the ileum,colon and in vitro fecal gut microbiota of the mice.At the same time,detect the changes of mouse serum,cerebrospinal fluid and gut-brain peptide markers,and preliminarily evaluate the causal effect and driving value of the gut-brain axis and cognitive function.Materials and Methods（1）Population study:this study is based on the Somily study established in Yongfu of Longevity in Guangxi（"Chinese Township of Longevity"recognized by the Chinese Society of Gerontology and Geriatrics）,using cluster sampling and continuous enrollment methods to include the longevity population（age≥90 years）and local control populations,divided into the full analysis set and the longevity subset.In addition,this study also included 100 aging populations of under10 years old,25-45 years old,60-69 years old,70-79 years old and 80-89 years old（each group of 20 people and the ratio of male to female is 1:1）.The recognition function of study population was defined by MMSE scale,and divided study populations into dementia（MMSE≤17）and control group（MMSE>17）,and divided into low（MMSE<12）,medium（12?MMSE<18）and high（MMSE?18）cognitive function groups according to MMSE tertiles.A comprehensive evaluation system of multi-dimensional brain-gut axis of serum metabolites,brain-gut peptides and inflammatory factors was established according to the literature search method.LC-MS/MS（liquid chromatography tandem mass spectrometry）was used to detect serum non-targeted metabolomics of study population,and metabolites related to the brain-gut axis were screened by molecular formula and molecular weight of metabolites.The differences of target metabolites and their linear relationship with MMSE were compared between different cognitive function groups in the complete analysis set and macrobion set,respectively.Furthermore,the aging trend of target metabolites was further verified in the aging population.Metagenomic sequencing was used to detect gut microbiota of feces.Alpha diversity,relative abundance and Beta diversity of gut microbiota at genus level were compared between different cognitive function groups in the full analysis set and longevity subset,respectively,and the relationship between the gut microbiota and serum metabolites was analyzed.（2）Animal study:Establish a compound antibiotic of ampicillin,metronidazole,neomycin sulfate and vancomycin to eliminate the intestinal flora of C57BL/6J mice,and further establish human-derived cognitively weak fecal bacteria FMT by gavage and enema system.The 16sr DNA sequencing method was used to evaluate the changes of the gut microbiota in different intestinal parts of the mice after FMT intervention,and the new object recognition behavior test method was used to evaluate the short-term learning and memory cognitive function changes of the mice.After the appearance of cognitive decline phenotype,the changes of related markers in serum,cerebrospinal fluid and hippocampus in mice were evaluated.Results（1）Establish a comprehensive evaluation system for serum brain-gut axis related markers,of which 8 metabolites include short-chain fatty acids,tryptophan and trimethylamine oxide;17 brain-gut peptides include serotonin,dopamine,neuropeptide Y,etc.;inflammatory factors 6 include IL-1,IL-6,TNF-αand etc.（2）9 target metabolites were selected from the serum metabolomics database.In the full analysis and longevity subset,the metabolite tryptophan level was significantly positively correlated with cognitive function（P=0.026）,while propionic acid was significantly negatively correlated with cognitive function（P=0.022）,independent of gender and age.It was found that glutamate and dopamine showed an upward trend with increasing age,and tryptophan,serotonin andγ-aminobutyric acid showed a downward trend with increasing age.After stratification by gender,dopamine showed an upward trend with increasing age in the male population,while serotonin showed a downward trend;in the female population,dopamine and glutamate showed an upward trend with increasing age,while tryptophan,serotonin andγ-GABA showed a downward trend.（3）In the full analysis and longevity subset,we found that there was no significant difference in Chao1 and Shannon indexes of Alpha diversity among different cognitive function groups.In the full analysis and longevity subset,we found that 11 genera including Anaerostipes,Blautia and Dorea were higher abundance in better cognitive function group;whilie 12 genera including Akkermansia,Bilophila,Odoribacter were higher abundance in worse cognitive function group.（4）The Beta diversity of gut microbiota was significant differences between the dementia and control group,and found Akkermansia,Odoribacter,Methanobrevibacter and Parabacteroides were more concentrated in the dementia group,while Anaerostipes,Blautia,Dorea and Lachnospira were more concentrated in control group.（5）In the full analysis set,we found that Blautia and Dorea were significantly positively correlated with tryptophan level,while Roseburia was significantly negatively correlated with propionic acid levels.（6）Successfully established compound antibiotic mice’s own gut microbiota clearance and gavage and enema FMT system.（7）Compared with the antibiotic intervention group,the identification coefficient of the antibiotic intervention group decreased by 19.30%;after two weeks of FMT,compared with the antibiotic intervention group,the identification coefficient of the blank control,gastric gavage and enema groups increased by 14.08%,32.87%and 37.86%,respectively;Four weeks after FMT,compared with the FMT two-week group,the identification coefficients of the gavage and enema groups decreased by 14.36%and 14.60%,respectively.Conclusions（1）This study eatablished a evaluation system of the gut-brain axis,including 8 metabolites such as tryptophan and propionic acid,17 brain-gut peptides such as serotonin and dopamine,and 6 inflammatory factors such as IL-6 and TNF-α.（2）Serum metabolite tryptophan showed a protective factor for cognitive function and showed a downward trend with increasing age,while propionic acid showed a risk factor for cognitive function and showed no significant change trend with increasing age.（3）There was no significant difference in the Alpha diversity between the different cognitive function groups,while the aboundance of 11 genera including Anaerostipes,Blautia and Dorea were higher and 12 genera including Akkermansia,Bilophila and Odoribacter were lower in better recognition function group.（4）Gut microbiota Blautia and Dorea are associated with level of metabolite tryptophan,Roseburia is associated with propionic acid level,and gut microbiota maight effect on the cognitive function by affecting markers of the gut-brain axis.（5）The effects of compound antibiotic intervention and human-derived fecal bacteria FMT on the cognitive function of mice have not reached statistical significance,but a downward trend is still found,indicating that the normal intestinal flora has an effect on cognitive function and human-derived fecal bacteria could cause cognitive decline.In the future,it is necessary to expand the sample size and intervention time to further verify its causal relationship and driving value.PartⅡAnalysis of the relationship between trace elements and non-alcoholic fatty liver based on public databasesObjective Trace elements are closely related to insulin resistance,oxidative stress and inflammatory response,and these factors play an important role in the pathogenesis of non-alcoholic fatty liver disease（NAFLD）.Therefore,this study was based on the National Nutrition and Health Survey（NHANES）public database to analyze the independent relationship between trace elements and NAFLD.Materials and Methods（1）The data of 2011-2012 and 2013-2014 in the NHANES database were selected,and 3,211 study subjects were finally included.（2）The non-invasive index hepatic steatosis index（HSI）and the US fatty liver index（USFLI）were used to diagnose NAFLD.（3）First,by comparing the distribution differences of the trace elements copper,zinc,selenium,lead,cadmium,mercury,iron and calcium between the NAFLD and non-NAFLD groups,selected the significant differences trace element and the main analysis element copper.（4）Next,logistic regression model,smooth curve fitting and restricted cubic spline were used to analyze the relationship between serum copper and HSI,USFLI and NAFLD and its dose-response relationship.And according to gender,age and insulin resistance index HOMA-IR for stratified analysis.Results（1）The trace elements copper,lead,cadmium,mercury,iron and calcium were significant difference between NAFLD and the control group,while trace elements zinc and selenium were no significant difference.（2）Further analysis of serum copper and NAFLD showed that serum copper levels in females were significantly higher than males（female:20.68±4.88μmol/L;Male:16.68±3.30μmol/L;P<0.001）;the risk of NAFLD,as defined by both HSI and USFLI,increased with elevated serum copper level.The smoothing curve fitting analysis found that HSI,USFLI and NAFLD prevalence increased with the copper level elevated.Dose-response analysis of restricted cubic splines showed that the incidence of NAFLD_HSI and NAFLD_USFLI was doubled when serum copper reached 17.63 and 18.06μmol/L.Stratified analysis showed that the association between serum copper and NAFLD was more significant in women and middle-aged people.Conclusions（1）Trace elements copper,lead,cadmium,mercury,iron and calcium might be associated with NAFLD.（2）Excess copper is independently associated with the risk of NAFLD,and this risk was more prominent in subgroup of female and middle-aged.More prospective studies are needed to verify the causal effect of copper on NAFLD.