Effect Of Fecal Microbiota Transplantation On Alzheimer’s Disease Mice And Its Molecular Mechanism

BacgroundAlzheimer disease(AD),one of the common neurodegenerative diseases,is one of the major diseases that threaten human health.In recent years,the pathogenesis and treatments of AD have made some progresses in research,but there are no specific therapy strategies.With the development of microecology,it has been found that the gut microbiota is a co-evolving microbiome with the organism.Its changes will affect the host’s’health.The relationship between the host flora and the host is mutually beneficial and symbiosis.The gut microbiota called"super organism",plays an important role in the mutual response of the hosts’intestinal and central nervous system.Therefore,brain and gut axis theory is proposed.Many nervous system diseases and mental illnesses have showed great changes in the microbiome,suggesting that we may seek new strategies for the treatment of some neurodegenerative diseases by changing the composition of the microflora in the gut.It is reported that probiotics and prebiotics have effects on the regulation and treatment of neurological diseases in the early stage of the disease.Fecal microbiota transplantation(FMT)can regulate the community structure of intestinal microflora.In this study,on the basis of the differential analysis of intestinal microbiota between normal mice and AD mice,normal mice were used as donors for AD mice transplanted with fecal bacteria to determine whether pathological characteristics of AD mice can be relieved by the inoculation of fecal bacteria.,improve learning and memory skills,and explore its underlying molecular mechanisms.ObjectiveIn this study,16S rDNA high-throughput sequencing was used to analyze the differences of intestinal microflora between wild-type mice and AD mice,and using the method of fecal bacteria transplantation study the effects of change gut microbiota on AD mice.With a view to starting from the intestinal microflora,it provides new ideas and methods for prevention and treatment of AD.MethodsPart I:Differences of Intestinal Microorganism between Normal and AD MiceWild type mice were labelled as WT group(n=5)and APP695V717I transgenic mice were App~+group(n=5).Mice feces of each group were collected and analyzed the differences in species composition and community structure using high-throughput sequencing.Part II:Effect of Fecal Microbiota Transplantation on AD Mice1.Animal grouping and fecal transplantation:The identification and feeding of APP695V717I transgenic AD mice were randomly selected and divided into App~-group(n=5),App~+group(n=5)and AD mouse were FMT group.In the FMT group(n=5),the mice were transplanted for 4 weeks and the weight changes of the mice were measured every week.After the intervention,Morris water maze test was employed in each group of mice.2.Changes of intestinal microbiota in AD mice after transplantation of fecal bacteria:Feces of three groups of mice in wild group mice,AD group and fecal microbiota transplantation group were harvested in a sterile environment and sequenced to analyze fecal bacterial translocation.Differences in species composition and community structure of intestinal microorganisms in AD mice.3.Effects of fecal microbiological transplantation on AD mice:Congo red staining was used to observe the changes of amyloidβ-protein(Aβ)deposition in mouse brain;Immunofluorescence was used to detect the expression of APP protein in hippocampus of mice;Western-blot and RT-PCR were used to detect senescence-associated proteins(p16,p21,p53,sirt1,sirt2,pcna)and inflammatory factors(IL-10,IL-1β,TNF-α),neurotrophic factor(NT3,BDNF,NGF)and APP protein expression at the protein and mRNA levels;ELISA was employed to observe serum SOD activity and MDA content.ResultsPart I:Differences of Intestinal Microorganism between Normal and AD MiceSequencing results showed that compared with normal mice,the intestinal flora diversity of AD mice was significantly reduced,and there were differences in relative abundance at each classification level.The LEfSe analysis showed that there was a significant difference in biomarkers between the WT and AD groups at each level of microbial classification.Especially,the four groups of Bacteroidales,Ruminococcus,Firmicutes and Prevotellaceae are species of microorganisms in the WT group;Verrucomicrobiaceae,Akkermansia,Peptostreptococcaceae,Romboutsia,Lactobacillus-animalis,Lactobacillales,and Bacilli are specific microbial species in AD mice.Part II:Effect of Fecal Bacteria Transplantation on AD Mice1.In the WT,AD,and FMT samples,at the phylum level,the relative abundance of Firmicutes in the FMT group was significantly lower than that in the AD group,and was close to that of the WT group;at the class level,the FMT group was compared with the AD group.The relative abundances of Erysipelotrichia,Clostridia and Bacteroidia increased while the relative abundances of Verrucomicrobia,Clostridia and Bacteroidia decreased.At the order level,Bacteroidales in the FMT group was decreased obviously.The abundance of Erysipelotrichaceae in the FMT group was increased significantly at the level of family level.In the intestine of AD mice,the number of Allobaculum increased,while Ruminococcus decreased remarkably.There was no Romboutsia in WT group,and there was no relative abundance of Romboutsia in the FMT.However,in the AD group,certain abundances of Romboutsia were seen;at the species level,the relative abundance of Lactobacillus-animalis was reduced to almost zero in the FMT group.2.Compared with mice in App~+group,FMT group showed increased learning and memory in behavior(P<0.05).3.The activity of SOD in the serum of AD mice after transplantation of fecal bacteria increased,but the relative expressions of anti-inflammatory cytokines IL-10 in hippocampus were elevated,the relative expression levels of proinflammatory cytokines such as IL-1βand TNFαdecreased(P<0.05.The relative expressions of aging-related proteins and genes p16,p21,etc.decreased(P<0.05),the relative expression levels of neurotrophic factors BDNF,NT3,NGF,etc.increased(P<0.05),and the deposition of APP proteins and Aβamyloid protein decreased(P<0.05).Conclusion1.Compared with wild-type mice,the intestinal microbial diversity of AD mice was significantly reduced,and there was a significant difference in species abundance.2.After the transplantation of fecal bacteria,the species abundance of intestinal microbiota in the FMT group was changed greatly and similar to that in the WT group.Meanwhile,the transplantation of fecal bacteria made the cognitive function of AD mice improved in behavior,increased the ability of anti-oxidative damage,reduced the level of inflammatory reactions to a certain extent,promoted the survival of neurons,and reduced nerve apoptosis,slowed the aging process of AD mice,reduced the expression of APP protein in AD mice,and then reduced the Aβamyloid deposition,to some extent,alleviated the pathological features of Alzheimer’s disease.