| Alzheimer’s disease(AD)is the most common type of dementia and a degenerative disease of the central nervous system with an insidious onset and unknown pathogenesis.The clinical diagnosis of AD is always made after the onset of dementia symptoms,and most patients are already in the middle or late stages,which makes clinical treatment extremely difficult due to the lack of wonder drugs.Mild cognitive impairment(MCI)is a transitional state between normal aging and dementia,and is the pre or early stage of dementia.It is particularly important to reduce or delay the occurrence of AD if the MCI population can be screened and detected early to identify the main influencing factors and take effective intervention and treatment measures as early as possible.In recent years,the Microbiota-Gut-Brain Axis(MGBA)theoretical system has been widely recognized by scholars at home and abroad,providing new ideas to explore the connection between gut microbiota and brain function.A large number of studies have shown that gut microbiota dysbiosis is closely related to AD;and a few studies have also reported that the MCI population has gut microbiota dysbiosis similar to that of AD in recent years.It has also been suggested that constipation and abnormal gut microbiota are closely related in other researches.In this study,we investigated the main influencing factors of MCI and AD through epidemiological surveys,and then analyzed the differences of gut microbiota among elderly population in an AD group,MCI group and Control group by using 16 S r RNA gene sequencing,and analyzed the differences of metabolites among the three groups with non-targeted metabolomics,and performed correlation analysis of the significantly different gut microbiota and metabolites,so as to provide ideas and basis for further in-depth research on the pathogenesis of AD and MCI.This study is divided into four parts as follows.Part Ⅰ Influencing Factors on MCI among elderly population in Singapore CommunityObjective To explore the influencing factors of MCI among the elderly in the Singapore community and provide a theoretical basis for the formulation of targeted interventions.Methods Based on the Singapore Diet and Healthy Ageing Study Cohort,questionnaire survey and a series of neuropsychological tests and assessments were conducted in a community-based elderly population in the western Jurong area of Singapore.An analysis of variance or χ2 test was performed on the indicators of sociodemographic characteristics,lifestyle behaviors,underlying diseases and diets in the MCI group(119 elderly people)and Control group(632elderly people),the indicators with P less than 0.1 were included in a multiple Logistic regression analysis model so as to screen out the influencing factors of MCI,and then a Bayesian network model of R software was applied to reveal the hypothesized causal relationship between the influencing factors and the MCI.Results(1)Comparison of sociodemographic characteristics: The mean age and proportion of housewives among elderly people in the MCI group were significantly higher than those in the Control group,but the years of education were lower than that in the Control group(all P<0.05),and there was no statistical significance between the two groups in all other aspects(P>0.05).(2)Comparison of lifestyle behaviors: The constipation incidence in later-life was significantly higher than that in the control group(26.7% vs.17.2%,P<0.05),and there was no significant difference in other aspects(all P>0.05).There was no statistical significance in the comparison of lifestyle behaviors in midlife between the two groups(all P>0.05).(3)Comparison of underlying diseases:The incidences of hypertension,hyperlipidemia,diabetes,stroke,and cataract/glaucoma of elderly people in the MCI group were significantly higher than that in the control group(all P<0.05),and there was no statistical significance in all other diseases(all P>0.05).(4)Comparison of diets: the proportion of older adults with the frequency of eating fruits,meat and meat products or eggs ≥4 days per week in the control group was significantly higher than that in the MCI group(all P<0.05),and there was no statistical significance in the frequency of diets in all other aspects(all P>0.05).(5)The results of the multiple Logistic regression analysis showed that long years of schooling and frequency of eating fruits ≥4 days per week were the protective factors for MCI in the elderly in Singapore community;and hypertension,stroke,cataract/glaucoma and constipation were the risk factors.(6)The causal inference of the Bayesian network model revealed five hypothesized causal association pathways leading to MCI:(1)stroke→MCI;(2)constipation→MCI;(3)years of schooling→frequency of eating fruits→MCI;(4)hypertension→stroke→MCI;and(5)hypertension→cataract/glaucoma→MCI.When stroke is combined with constipation,the conditional probability of causing MCI was the highest,which was 60.00%.Conclusion Constipation,hypertension,stroke and cataract/glaucoma were the risk factors for MCI in the elderly in Singapore,while the frequency of eating fruits≥4 days per week and long years of schooling were the protective factors;and when stroke is combined with constipation,the conditional probability of causing MCI was the highest.Part Ⅱ Influencing Factors of MCI and AD among elderly population in Nanning CommunityObjective Influencing factors of MCI and AD are explored among elderly population in the Nanning community to provide a theoretical basis for making targeted interventions.Methods Resident aged 60 years or older in areas under administration of a community health service center in Nanning,Guangxi Zhuang Autonomous Region were selected,by convenience sampling,as respondents for questionnaire surveys and physical examinations,and a series of neuropsychological tests and assessments.An analysis of variance or χ2 test was performed on the indicators of sociodemographic characteristics,lifestyle behaviors,underlying diseases and diets in the MCI group(131 elderly people)vs Control group(658 elderly people)and in the AD group(36 elderly people)vs Control group(658 elderly people)respectively.The indicators with P less than 0.1 were included in a multiple Logistic regression analysis model so as to screen out the influencing factors of MCI and AD,and then a Bayesian network model of R software was applied to reveal the hypothesized causal relationships between the influencing factors and the MCI and between the influencing factors and the AD.Results(1)Influencing factors of MCI among elderly population in the Nanning community: 1)Comparison of sociodemographic characteristics: the average years of schooling in the MCI group in Nanning were significantly lower than that in the control group,but the proportions of females and unmarried/divorced/widowed elderly population were significantly higher than those in the control group(all P<0.05);and there was no statistical significance between the two groups in all other aspects(all P>0.05).2)Comparison of lifestyle behaviors in later-life: the frequency of participating in physical exercises,social activities and daytime outdoor activities in the MCI group were significantly lower than those in the control group(all P<0.05);there was no statistical significance in all other aspects(all P>0.05).3)Comparison of underlying diseases: stroke,history of head trauma,and constipation incidence among elderly population in the MCI group were significantly higher than those among elderly population in the control group(all P<0.01),and there was no statistical significance in all other diseases(all P>0.05).4)Comparison of diets:the proportion of elderly population who regularly ate fruits(≥4 days per week)in the MCI group was significantly lower than that in the control group(P<0.001).There was no statistical significance in the frequency of diets in all other aspects(all P>0.05).5)Comparison of physical examination indexes: AST and LDL-C in the MCI group were significantly higher than those in the control group,serum creatinine in the MCI group was significantly lower than that in the control group(all P<0.05),and there was no statistical significance in all other indexes(all P>0.05).6)The results of Logistic regression analysis showed that years of schooling and eating fruits≥4 days per week were the protective factors for MCI elders in Nanning.Lack of physical exercises,lack of social activities,stroke,history of head trauma,constipation and LDL-C were the risk factors.7)The causal inference of the Bayesian network model revealed five hypothesized causal association pathways leading to MCI in the elderly in Nanning:(1)stroke→MCI;(2)constipation→MCI;(3)years of schooling→MCI;(4)years of schooling→frequency of eating fruits→constipation→MCI;and(5)years of schooling→physical exercises→stroke→MCI.When the literacy level was illiteracy and the frequency of physical exercises was ≤ 3 days per week,the conditional probability of causing MCI was the highest,which was 70.39%.(2)Influencing factors of AD among the elderly population in the Nanning community: 1)Comparison of sociodemographic characteristics: the mean age and the proportions of elderly population unmarried/divorced/widowed and living alone in the AD group in Nanning were significantly higher than those in the control group,but the years of schooling was lower than those in the control group(all P<0.001),and there was no statistical significance in all other aspects(all P>0.05).2)Comparison of lifestyle behaviors in later-life: the frequency of participating in physical exercises,social activities and mental activities and the proportion of good relationships with family members in the AD group were significantly lower than those in the control group,while the proportion of sleep duration ≥9 hours per night was significantly higher than that in the control group(all P<0.05),and there was no statistical significance in all other aspects(all P>0.05).3)Comparison of underlying diseases: the incidences of hypertension,stroke and constipation among elderly population in the AD group were significantly higher than those in the control group(all P<0.01),and there was no statistical significance in all other diseases(all P>0.05).4)Comparison of diets: the proportion of elderly population with the frequency of eating fruits,garlic,tea,calcium and zinc and other trace element supplements ≤3 days per week in the AD group was significantly higher than that in the control group(all P<0.05),and there was no statistical significance in the frequency of diets in all other aspects(all P>0.05).5)Comparison of physical examination indexes:there was no statistical significance in all physical examination indexes between the two groups(all P>0.05).6)The results of the multiple Logistic regression analysis showed that living alone,lack of physical exercises,stroke and constipation were the risk factors for AD.7)The causal inference of the Bayesian network model revealed five hypothesized causal pathways leading to AD among elderly population in Nanning:(1)stroke→AD;(2)constipation→AD;(3)living alone→AD;(4)physical exercises→stroke→AD;and(5)physical exercises→constipation→AD.When “living alone” and “stroke”coexisted,the conditional probability of causing AD was the highest,which was68.18%.Conclusion(1)Lack of physical exercises,lack of social activities,stroke,history of head trauma,constipation and LDL-C were the risk factors for MCI in the elderly population in Nanning community,whereas frequency of eating fruits ≥4 days per week and long years of schooling were the protective factors;and when “illiteracy” was combined with “frequency of physical exercises ≤3 days per week”,the conditional probability of causing MCI was the highest.(2)Living alone,lack of physical exercises,stroke and constipation were the risk factors for AD in the elderly population in Nanning community;and when “living alone” and “stroke” coexisted,the conditional probability of causing AD was the highest.Part Ⅲ Differences in gut microbiota among elderly population with MCI and AD in Nanning CommunityObjective The differences in the composition and relative abundance of gut microbiota are compared among the elderly population in the MCI group,AD group and Control group,in order to identify the gut microbiota with significant differences in each of the groups.Methods With the elderly population in the Part II of this paper as respondents,30 cases were randomly selected in each group,fresh excretas were collected on an empty stomach in the early morning,and the relationships between the diversity and relative abundance of gut microbiota structure and the MCI or AD were studied by 16 S r RNA gene sequencing,Alpha diversity analysis,Beta diversity analysis,similarity analysis and LEf Se significant difference analysis.Results(1)Comparison of basic conditions: the mean age and constipation incidence in the AD group were significantly higher than those in the MCI group and Control group(all P<0.05);there was no statistical significance in all other aspects(all P>0.05).(2)Alpha diversity analysis: the ACE index and Chao1 index of the AD group were significantly higher than those of the Control group(all P<0.01),but there was no statistical significance in the Shannon index and Simpson index between AD group with Control group(all P>0.05).There was no statistical significance in all the four indexes between AD group with Control group(all P>0.05);and the ACE index,Chao1 index,Shannon index and Simpson index of the MCI group were significantly higher than those of the Control group(all P<0.05).(3)Beta diversity analysis: the composition and relative abundance of gut microbiota among the groups were significantly different at the taxonomic levels of phylum,class,order,family,genus and species(all P<0.05).(4)LEf Se significant difference analysis:compared to the AD group,the relative abundance of Prevotellaceae,unidentified Prevotella,Prevotella Copri and Lachnospiraceae in the Control group were significantly increased.Compared to MCI group,the relative abundance of Lactobacteriaceae and Lactobacillus in AD group were significantly increased,while the relative abundance of Clostridiales,Clostrida,Lachnospiraceae and Faecalibacterium in MCI group were significantly increased.Compared to Control group,the relative abundance of Clostridiales,Clostrida and Ruminococcaceae in MCI group were significantly increased,while the relative abundance of Veillonellaceae,Selenomonadales,Negativicutes,Prevotellaceae,unidentified Prevotellaceae and Prevotella copri were significantly increased in the Control group.(5)The gut microbiota of both the AD group and MCI group were significantly enriched in some gene functions and significantly decreased in some gene functions.Conclusion(1)The composition and relative abundance of the gut microbiota of the three groups of samples were significantly different at the taxonomic levels of phylum,class,order,family,genus and species.(2)The relative abundance of Lactobacteriaceae and Lactobacillus in AD group were significantly increased;the relative abundance of Clostridiales,Clostrida,Faecalibacterium and Ruminococcaceae in MCI group were significantly increased;and the relative abundance of Veillonellaceae,Selenomonadales,Negativicutes,Prevotellaceae,unidentified Prevotellaceae and Prevotella copri were significantly increased in the Control group.Part Ⅳ Serum Metabolomics among elderly population with MCI and AD in Nanning CommunityObjective The differences of serum metabolites are compared among the elderly people in the MCI group,AD group and Control group,so as to identify the metabolites with significant differences in each of the groups and explore their relationship with the different flora.Methods Serum was extracted from the venous blood collected on an empty stomach among the older adults in the Part III of this paper,the significant different metabolites in the MCI group and AD group were explored by principal component analysis,partial least squares discriminant analysis,total different metabolite clustering analysis,authentication of different metabolites,and different metabolite enrichment pathway analysis,and an association analysis was made among the different metabolites and different flora.Results(1)Different metabolite authentication: a total of 22 different metabolites were obtained in the AD group compared with the Control group,among which d AMP(deoxyadenosine monophosphate)was up-regulated and the rest were down-regulated.A total of 14 different metabolites were obtained in the MCI group compared with the AD group,among which N-acetylalanine,5-hydroxytryptophanic acid,4-methoxycinnamaldehyde,sebacic acid,and(2E)-3-(3,4-dimethoxyphenyl)prop-2-enoic acid were down-regulated and the rest were up-regulated.A total of 11 different metabolites were obtained in the MCI group compared with the Control group,among which anacardic acid,L-homocysteine,Ala-trp,prolyl valine,N-propionyl glycine and cystine were up-regulated,and the rest were down-regulated.(2)Enrichment pathway results of different metabolites based on the KEGG database: different metabolites in the AD group and Control group were closely related to 5 metabolic pathways,including unsaturated fatty acid biosynthesis,purine metabolism,caffeine metabolism,cysteine and methionine metabolism,and endocrine resistance.Different metabolites in the MCI group and AD group were closely related to 10 metabolic pathways,including vitamin digestion and absorption,glutathione metabolism,unsaturated fatty acid biosynthesis,folic acid biosynthesis,propionate metabolism,methyl butyrate metabolism,sulfur metabolism,hypoxia-inducible factor 1 signaling pathway,longevity regulatory pathway,and anti-folic agent resistance.Different metabolites in the MCI group and Control group were closely associated with 7 metabolic pathways,including caffeine metabolism,estrogen signaling pathway,endocrine factors regulating calcium reabsorption,carbohydrate digestion and absorption,steroid hormone biosynthesis,fatty acid biosynthesis,and purine metabolism.(3)Association analysis of the Top 10 different metabolites and the Top 5 different flora enriched in each of comparison groups: all the comparison groups had certain flora and metabolites that were positively or negatively correlated with each other.Conclusion(1)A total of 22 different metabolites were obtained in the AD group compared with the Control group,which were closely related to 5metabolic pathways;a total of 14 different metabolites were obtained in the MCI group compared with AD group,which were closely related to 10 metabolic pathways;and a total of 11 different metabolites were obtained in the MCI group compared with the Control group,which were closely related to 7metabolic pathways.(2)All the comparison groups had certain flora and metabolites that were positively or negatively correlated with each other. |
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