| 【Background】With the aggravation of population aging process,the health problem of the aged has become a major public problem in the world,which has been widely valued and actively dealt with by various countries.China is a country with the largest number of elderly population,and also a country facing the most severe challenge of aging.According to the seventh national census,the number of people aged 60 or above reached 264 million,accounting for 18.7 percent of the total population.However,the overall health status of the elderly is not optimistic.Chronic diseases and syndromes of the elderly are the main factors leading to the decline of the health level of the elderly,especially frailty is the most common and most serious syndrome affecting the health quality of the elderly.It is characterized by weakened body strength,endurance and physiological function,which increases the risk of serious adverse outcomes in the elderly,such as falls,disability,comorbidities,hospitalization,onset of acute and chronic diseases and death.The influencing factors of frailty formation are very complex and the mechanism is still unclear,which may be related to endocrine disorders,chronic inflammation and immune system senescence,cellular senescence,impaired energy metabolism,social environment,psychological factors and physical diseases[1,2].And chronic inflammation,immune system,energy metabolism and so on are inseparable from intestinal function.As we all know,the gut is not only an important digestive organ of the human body,but also the largest immune and regulatory organ of the human body.Trillions of bacteria,viruses,fungi and other microorganisms live here,which is closely related to human health.Intestinal flora has been proved to be involved in host nutritional metabolism,immune regulation,barrier function,anti-aging,anti-tumor and other physiological and pathological processes,and plays a crucial role in maintaining human health due to its unique metabolic activities.So,is natural frailty associated with intestinal microecological changes?Can frailty be improved by interfering with gut flora?If possible,what are the regulatory mechanisms?These scientific questions become the exploration direction of this research.【Objective】1.To investigate the prevalence and risk factors of frailty among the elderly in Xi’an.2.To establish a cohort of frail elderly people and analyze the characteristics and correlation between frail elderly people and intestinal flora and metabolites.3.To investigate the effects and possible mechanisms of prebiotics on frail state,intestinal microflora profile and metabolite levels in a randomized,double-blind,controlled study.【Methods】Part ⅠA cross-sectional survey was conducted on the elderly population over 65 years old in 13 communities in Xi’an.The selected elderly were self-care and willing to participate in the survey.Serious illness,inability to take care of themselves and unwillingness to participate were excluded from the survey.General sociodemographic information was collected,including name,age,sex,height,weight,blood pressure,etc.The frail status of all the selected elderly was evaluated by Fried scale,and the incidence and the compliance of five indexes were calculated.The project was approved by the ethics committee of the First Affiliated Hospital of Air Force Military Medical University（KY20192015-f-1）and registered in the U.S.clinical trial registry（Registration No.:NCT03995342）.Part ⅡFrom the nonfrail group,prefrail group and frail group,100 cases were randomly selected in each group,and all the respondents signed the informed consent.At the same time,in order to explore the correlation between dietary structure,intestinal flora and metabolomics,the daily dietary intake of the elderly was investigated and the commonly used scales for the elderly were tested.The body fat rate,muscle mass and visceral fat were measured by human body composition analyzer.16SrDNA gene sequencing was used to detect intestinal flora,and high throughput chromatography-mass spectrometry was used to detect the metabolomics of non targeted flora.Part ⅢTo investigate the effects of prebiotics on senile frail state,intestinal flora profile and metabolite levels,a randomized,double-blind,controlled intervention study were conducted in the prefrail group and frail group（100 each）enrolled in the second study cohort.Each group was divided into placebo control and prebiotic intervention groups with 50 participants in each group.Prebiotics or placebo were identical in packaging,properties and taste.Dosage:15 g,once a day,followed-up was performed 90 days after continuous administration.The subjects were tested again for frail state,living ability,geriatric depression,anxiety,sleep,constipation and other scales.Full-length 16SrDNA gene sequencing was used to detect intestinal microflora,and high-throughput chromatography-mass spectrometry was used to analyze untargeted microflora metabolomics.【Results】Part Ⅰ1.A total of 1693 elderly people over 65 years old in Xi’an were screened for frail state.The results showed that the total prevalence of prefrail was 41.6%（53.4%in men and 33.0%in women,F=71.189,P<0.001）,and the prevalence in males was higher than that in females（P<0.05）.The overall prevalence of frail was 16.8%（16.7%in men and 16.9%in women,F=0.019,P=0.892）,with no difference between men and women（P>0.05）.2.Compared with the nonfrail group,the elderly in the prefrail group and frail group were older,with more significant weight loss and physical activity decline,lower grip strength,slower walking speed,and more significant fatigue.The changes of the above indexes in the frail group were more significant than those in the prefrail group（P<0.001）,and the systolic blood pressure in the prefrial group and frial group was higher than that in the nonfrail group（P<0.01）.Part Ⅱ1.There were significant differences among the nonfrail group,prefrail group and frail group in average age,number of children,number of comorbidities,surgical history,exercise frequency and fatigue degree（P<0.05）.High exercise frequency was a protective factor and was negatively correlated with frailty,while other factors were positively correlated（P<0.05）.2.Compared with the nonfrail group,the frail group’s daily living ability and sleep quality decreased（P=0.019 and P<0.001）,visceral fat was also significantly decreased（P=0.004）.3.The intestinal flora of the elderly was mainly Firmicutes,Proteobacteria,Bacteroidetes and Actinobacteria.The relative abundance of Firmicutes decreased and that of Bacteroidetes increased with the increase of frailty（P<0.01）.4.There were significant differences inαandβdiversity of intestinal microflora in nonfrail group,prefrail group and frail group（P<0.05）,indicating that the change of frailty intestinal microflora was a gradual process.The main bacteria such as Bacteroides vulgatus,Ruminococcus bicirculans,Alistipes onderdonkii,Bacteroides Ovatus,Bacteroides Fragilis,Bacteroides Caccae,Bacteroides thetaiotaomicron,Bacteroides Plebeius,etc.increased significantly,while Ruminococcus bromii,Lactobacillus Ruminis,Anaerostipes hadrus,Eubacterium Hallii,Bifidobacterium adolescentis,etc.were relatively reduced.5.Non-target metabolomics was used to detect the differential metabolites among groups.A total of 664 metabolites were detected,including 435 positive and 229 negative ion metabolites.There were 143 differential metabolites between nonfrail group and prefrail group,including 104 positive ion metabolites and 39 negative ion metabolites（P<0.05）.The results showed that compared with the nonfrail group,the downregulated positive metabolites in the prefrail group mainly included deoxycholic acid,kaempferol and phosphoethanolamine,while the up-regulated metabolites mainly included dimethylaminopurine,valine,ethanolamine and tryptophan.The downregulated anion metabolites mainly included palmitic acid and hexyl dodecate,while the up-regulated metabolites mainly included cresol,proline,serine and dihydrothymine.There were 50 differential metabolites between nonfrail group and frail group,including 32 positive ion metabolites and 18 negative ion metabolites（P<0.05）.Compared with the nonfrail group,the down-regulated positive metabolites in frail group mainly included aminocyclopropane carboxylic acid,deoxycholic acid,niacin and thymine,and the up-regulated metabolites mainly included arbutin.The downregulated anion metabolites mainly included phenylpyruvate and ketogenic amino acids,while the up-regulated metabolites mainly included deoxyribose and isocitrate.There were 192 different metabolites between prefrail group and frail group,including 124 positive and 68 negative ion metabolites（P<0.05）.Compared with the prefrail group,the down-regulated positive metabolites in frail group mainly included guanine,dimethylaminopurine,ethanolamine and tryptophan,while the up-regulated metabolites mainly included arbutin,ethanolamine phosphate and normehalamine.The down-regulated anion metabolites mainly included cresol,phenylpyruvate and proline,while the up-regulated metabolites mainly included isocitrate and benzoic acid.6.KEGG pathway enrichment analysis showed that compared with nonfrail group,protein digestion and absorption,amino acid biosynthesis,ABC transport,mineral absorption,alanine tyrosine and tryptophan biosynthesis and other differential metabolic pathways were upregulated in the prefrail group.The pyrimidine metabolism,pentose glucuronic acid conversion and unsaturated fatty acid synthesis pathways were down-regulated in the frail group compared with the nonfrail group.Compared with the prefrail group,protein digestion and absorption,amino acid biosynthesis,ABC transport and other differential metabolic pathways were down-regulated in frail groupPart Ⅲ1.Follow up after 3 months of intervention,compared with the placebo group,the prebiotic mixture group can significantly improve the frailty state（frail and prefrail）,reduce the fatigue state of the elderly in the prefrail group,and improve the walking speed of the elderly in the frail group（P<0.05）.Compared before and after the intervention,prebiotic mixture can increase the body fat rate and body moisture rate of the elderly in the prefrail group,while placebo group can reduce the muscle mass of the elderly in the prefrail group;prebiotic mixture can increase the walking speed,improve grip strength and improve constipation of the elderly in the frail group,while placebo group can increase the body fat rate and reduce the body moisture rate of the elderly in the frail group（P<0.05）.2.There was no significant change inαdiversity of microflora between the prebiotics mixture group and the placebo group after intervention（P>0.05）,but there was a significant change inβdiversity（P<0.05）.In the prefrail group,the prebiotics mixture intervention group was dominated by Bifidobacteria youth,while the placebo group was dominated by Faecalibacterium.In the frail group,the prebiotic mixture intervention group was dominated by Bifidobacterium pseudocatenulatum,Escherichia coli,Veronococci,Enterobacteriaceae,Negativicutes,etc.,while the placebo group had no significant change.3.Based on the bacterial results of 16S gene sequencing,Tax4Fun information analysis software was used to predict the functional abundance of the genome.The data showed that:in the prefrail group,compared with the placebo group,prebiotics mixture group of DNA repair enzyme associated with recombinant protein,amino acids related enzymes,extracellular,metabolic functions such as ribosome access raised significantly（P<0.05）,bacterial two-component system,methane metabolism,fructose and mannose,porphyrin and chlorophyll metabolism pathway significantly lowered（P<0.05）.In the frail group,pathways of cysteine and methionine metabolism,phenylalanine,tyrosine,and tryptophan biosynthesis,and lipid synthetic proteins were significantly up-regulated in the prebiotics group compared with the placebo group（P<0.05）.4.Untargeted metabolomics results showed that in the prefrail group,there were 13differential metabolites between the placebo group and the prebiotic mixture group,including 10 positive and 3 negative metabolites（P<0.05）.In the prefrail group,compared with the placebo group,glycyrrhetinic acid was the main positive metabolites down-regulated in prebiotic mixture group,and hydroxyphenylglycine,methionine,histidine and alanine were the main up-regulated metabolites;the down-regulated anion metabolites mainly included D-mannose and D-mannitol 1 phosphate（P<0.05）,but no metabolites were significantly up-regulated.In the frail group,there were 177 differential metabolites between the placebo group and the prebiotic mixture group,including 116 positive and 61 negative metabolites（P<0.05）.In the frail group,compared with the placebo group,metformin,phosphocholine and phosphoethanolamine were down-regulated in prebiotic mixture group,and histidine,glucose and riboflavin were up-regulated（P<0.05）;the downregulated anion metabolites mainly included epoxyfatty acid methyl ester,minitol phosphate and cholesteryl sulfate（P<0.05）,while the up-regulated metabolites mainly included methionine,phenylpyruvate and dihydrothymine（P<0.05）.5.KEGG pathway analysis showed that there were statistical differences in the three enrichment pathways between the prefrail prebiotics mixture group and the placebo group,but the number of differentially metabolites included was too small to judge the overall change of all metabolites in this pathway.For the elderly in the prefrail group,compared with the placebo group before the intervention,the overall expression of primary bile acid biosynthesis and cholesterol metabolism after intervention tends to be up-regulated,and the overall expression of apoptotic metabolic pathways tends to be down-regulated;compared with the prebiotic mixture group before intervention,the overall expression of primary bile acid biosynthesis,cholesterol metabolism and other pathways after intervention tended to be up-regulated,and the overall expression of protein digestion and absorption,mineral absorption,ABC transport,aminoacyl tRNA biosynthesis and other pathways tended to be down-regulated.For the elderly in the frailty group,compared with the placebo group after intervention,the overall expression of metabolic pathways such as protein digestion and absorption,amino acid biosynthesis,and ABC transport in the prebiotic mixture group tended to be up-regulated（P<0.05）.6.Analysis the correlation between the differential flora and the differential metabolites showed that:for the elderly in the frail group,compared with the placebo group,after the intervention of prebiotic mixture,Dialister was the core flora,which was significantly positively correlated with indole,L alanine,L tryptophan,indole 3 lactic acid,etc（P<0.05）.Compared with placebo group,the correlation analysis of differential flora and differential metabolites of prebiotic mixture group in the prefrail group did not find the core flora（P>0.05）.【Conclusion】1.The total incidence of prefrail of the elderly over 65 years old in Xi’an community was 41.6%,and the total incidence of frail was 16.8%.Frailty state is closely related to the average age of the elderly,the number of children,the number of comorbidities,surgical history,exercise frequency,fatigue and so on.Active exercise can slow down the occurrence of frailty.2.There are significant differences in the species,abundance and distribution uniformity of intestinal flora between different frail status groups.In the frail group,the intestinal flora are dominated by Bacteroides vulgatus,Ruminococcus bicirculans and Bacteroides fragilis,while the probiotics such as Anaerostipes hadrus,Eubacterium huei and Bifidobacteria are significantly reduced.3.Different frail groups have obvious differences in metabonomics.Compared with group N,group P has different metabolisms such as protein digestion and absorption,amino acid biosynthesis,ABC transport,mineral absorption,alanine tyrosine and tryptophan biosynthesis,etc.The overall channel is up-regulated.Compared with group N,the different metabolic pathways of group F,such as pyrimidine metabolism,pentose glucuronic acid conversion,and unsaturated fatty acid synthesis pathway,are overall down-regulated.Compared with group P,the different metabolic pathways in group F,such as protein digestion and absorption,amino acid biosynthesis,and ABC transport,are overall down-regulated.4.Prebiotic mixture intervention can significantly improve the frail status of the elderly,alleviate the fatigue of the elderly in the prefrial group,as well as body fat percentage and body moisture rate,and improve the pace,grip strength and constipation of the frail elderly.The probiotic Bifidobacterium adolescentis in the prefrail group has been increased,and the Bifidobacterium pseudocatenulatum and Escherichia coli in the frail group have been increased.At the same time,it improves the regulation pathways such as protein digestion and absorption,ABC transport,and aminoacyl tRNA biosynthesis.5.After the intervention of prebiotic mixture in the frail elderly,the alpha diversity of the intestinal flora did not change significantly,but the beta diversity changed significantly.Prebiotic mixture intervention improves the intestinal flora with Dialister as the key flora,thus improving the frail status. |
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