The Effect And Mechanism Of Frailty-associated Dysbacteriosis On Intestinal Barrier Function

Objectives: To compare and analyze the diversity and composition of fecal microbiota between the frailty group and the control group to evaluate the characteristic spectrum of intestinal microbiota;Assess the intestinal barrier function of the frailty and control groups;FMT method was used to verify the causal relationship between frailty-related intestinal microbiota disorder and intestinal barrier function damage,and fecal metabonomics was used as a starting point to explore its potential mechanism.Methods: 1.16 Sr RNA high-throughput sequencing technology was used to compare and analyze the diversity and composition structure of fecal microbiota in the frailty and control groups;The levels of serum inflammatory factors IL-6 and HMGB1 were detected by enzyme linked immunosorbent assay(ELISA).2.Alcian blue periodic acid Schiff(ABPAS)staining was used to observe the number of goblet cells(GC)and mucus secretion in frailty and control colon tissues;Immunofluorescence(IF)was used to detect the expression and distribution of MUC2 in colon tissue;Immunohistochemistry(IHC)was used to detect the expression and distribution of TJs proteins Claudin-1,Occludin and ZO-1 in colon tissue.3.The experimental animals were divided into three groups: frailty fecal bacteria receptor group(F-FMT),control fecal bacteria receptor group(NF-FMT)and blank control group(Control).The intestinal microbiota of the donor was established in the intestinal tract of recipient mice by the FMT method,and the reconstruction effect of intestinal microbiota was evaluated by 16 Sr RNA high-throughput sequencing.Small animal in vivo imaging technique was used to detect the intestinal permeability of recipient mice;AB-PAS staining was used to observe the number of GC and mucus secretion in colon tissue of mice;IF method was used to detect the expression and distribution of mucin MUC2 in colon tissue;IHC method and Western bolt(WB)method were used to detect the expression and distribution of TJs proteins Claudin-1,Occludin and ZO-1 in colon tissue;The ultrastructure of the colon of recipient mice was observed by transmission electron microscope;Detection of serum level of IL-6 and Tumor necrosis factor-α(TNF-α)in recipient mice by ELISA;Liquid chromatography mass spectrometry(LC-MS)was used to analyze the metabonomics of fecal samples from recipient mice.Results: 1.There was no significant difference in intestinal microbiota richness and species evenness between the frailty and control groups.Still,there was a considerable difference in the composition and structure of the microbiota.At the genus level,the dominant genera in the frailty group include Parabacteroides,Akkermansia,Klebsiella,Bifidobacterium,Lactobacillus,Pyramidobacter,Alistipes and Dysgonomonas.The dominant genera in the control group included Faecalibacterium,Roseburia,Prevotella,Megamonas,Blautia,Phascolarctobacterium,Megasphaera and Haemophilus;Compared with the frailty group,the interaction between genera in the control group was more common;Firmicutes of the healthy control group is more likely to form a positive symbiotic relationship;Escherichia/Shigella,Pyramidobacter,Aistipes and Akkermansia were positively correlated with serum IL-6 in the frailty group,while Faecalibacterium,Prevotella and Roseburia were negatively correlated with serum IL-6 in the control group.Alistipes were positively correlated with serum HMGB1;Based on 16 different bacterial genera,random forest(RF)was used to build the prediction model,and the area under the curve(AUC)value of the ROC curve was 97.2%.2.2.Compared with the control group,the number of GC in the colon tissue of the weak group decreased,the disappearance of mucus increased,and the number of inflammatory cells in the lamina propria increased.The fluorescence intensity of MUC2 in colon tissue of deficiency syndrome group was significantly lower than that of control group.The average optical density(MOD)of Claudin-1,Ocludin and ZO-1 protein in colon tissue of frailty group was significantly lower than that of control group.3.The fecal microbiota of the donor was successfully colonized in the intestinal tract of the recipient mice.In vivo imaging of small animals showed that compared with control group and NF-FMT group,the functional integrity of intestinal barrier in F-FMT group was damaged,and the fluorescence was diffused.Compared with control group and NF-FMT,in F-FMT group,the inflammatory cells in the lamina propria of colonic mucosa increased,the number of GC decreased,the secretion of mucin MUC2 decreased,and the expression levels of TJs proteins Claudin-1,Occludin and ZO-1 decreased significantly.The results of transmission electron microscope showed that the microvilli of IECs in the colon of F-FMT mice were sparse,irregular and of different length,the intercellular gap structure between IECs was widened,and the intercellular junction complex and desmosome structure were damaged;Compared with the control and NF-FMT groups,the serum level of inflammatory factors IL-6 and TNF-α increased significantly.The frailty-related intestinal microbiota leads to significant changes in metabolites.The metabolic pathways affected by different metabolites in F-FMT group and NF-FMT group include purine metabolism,arginine biosynthesis and biosynthesis of unsaturated fatty acids.Conclusions: There is no significant difference in the richness and species evenness of the intestinal microbiota between the frailty and the healthy control group,but there is a significant difference in the composition and structure of the microbiota.Some genera were correlated with serum inflammatory biomarkers.The prediction model based on different genera can accurately distinguish frailty and healthy controls.In the frailty group,the inflammatory infiltration of colon tissue increased,the number of GC decreased,the secretion of mucin MUC2 decreased,and the expression levels of TJs proteins Claudin-1,Occludin and ZO-1 decreased.The changes of intestinal microbiota related to frailty can increase intestinal permeability,reduce the number of GC in colon tissue,reduce the expression of mucin MUC2 and TJs protein,and promote systemic inflammation.At the same time,it also leads to the change of fecal metabolites and affects the metabolic pathway.Therefore,the dysfunction of intestinal microbiota associated with frailty leads to the destruction of intestinal barrier function and chronic inflammation.This process involves metabolic disorders and may be a key step in the occurrence of frailty.