Study On The Effect Of Tibetan Yogurt On The Cognitive Function And Gut Microbe Of AD Double Transgenic Mice

Background Alzheimer's disease(AD)is an common type of dementia.With progressive cognitive impairment as the clinical syndromes,which can lead to irreversible brain damage as the disease progresses.At present,AD has become one of the important public health problems threatening human health.The most recognized pathological events in AD are amyloid plaques and neurofibrillary tangles.The mechanisms of its pathological features is still unclear.Neuroinflammation is considered to be an basic component of AD pathology.It has been found that activated microglia(MG)play a corel role in neuroinflammation,and inhibition of MG activation can alleviate A? plaque deposition.When MG is over activated,inflammatory mediators such as TNF-?,MIP-1?,IL-1 and other inflammatory mediators are produced,which activate the proinflammatory factor signaling pathway and cause inflammatory response.TLR4/NF-?B is a classic inflammatory pathway,and TLR4 mediates My D88 dependent signaling pathway in inflammatory response.Probiotics are involved in the prevention and treatment of many chronic diseases including AD by regulating intestinal flora.Traditional fermented yogurt in Qinghai Tibetan area is a kind of fermented food produced by local farmers and herdsmen using traditional methods of fermentation.Studies have shown that yak yogurt in Tibetan area has the functions of antioxidant activity,lowering cholesterol reduction and improving,immunity.It is reported that the prevalence of AD in Tibetan population in Hainan,Qinghai Province is significantly lower than that reported at home and abroad.We hypothesized that the low prevalence of AD in Tibetan population is not only related to the genetic background and religious belief,but may also may be related to the change of gut microbes caused by the long-term consumption of traditional yogurt,thus affecting the disease of AD.In order to verify the above hypothesis,this study intends to discuss through the following three parts:Part ? Microbial diversity analysis of traditional fermented yogurt in Qinghai Tibetan areaObjective: The samples of traditional fermented yogurt were collected for microbiological analysis,and the yogurt with relatively abundant lactobacillus was selected for subsequent experiments.Methods: Yogurt samples were collected from herdsmen's families in Tibetan areas with different altitudes(above 2400m),and 16 S ribosomal RNA(r RNA)high-throughput sequencing was used to analyze the structure and diversity of traditional fermented yogurt.Results: A total of 14 qualified samples of traditional fermented yogurt were collected and classified into 16 phylum and 203 genus after high-throughput sequencing.There are some differences in bacterial species in yogurt samples from different regions.The traditional fermented yogurt sample(A-1)from Gonghe County,Hainan Tibetan Autonomous Prefecture,Qinghai Province contain relatively abundant lactobacillus resources and species abundance.Conclusion: There are abundant lactobacillus resources in the traditional fermented yogurt in Qinghai Tibetan area.There are regional differences in bacterial species in traditional fermented yogurt,and the species richness of A-1 yogurt is relatively high.Part ? Effects of traditional fermented yogurt in Tibetan area of Qinghai on cognitive function and related protein expression of APP/PS1 double transgenic AD model miceObjective: In order to find out the mechanism of the influence of traditional Tibetan fermented yogurt on the cognitive function of AD model mice,we screened out yogurt with relatively rich species abundance to intervene AD model mice and analyzed its cognitive function and the expression of related proteins.Methods: Two-month-old AD double-transgenic mice were randomly divided into three groups:(1)model control group(APP/PS1 group;n=12);(2)lactobacillus intervention group(Lac.group;n=12);(3)traditional fermented Yogurt intervention group in Tibetan area(Yogurt section;n=12).The wild type with same background was the normal control group(WT group;n=12).Four groups of mice were given gavage once a day for 140 days.After the intervention,water maze(MWM)and new object recognition experiment(NORT)were used to evaluate the cognitive function of AD model mice.ELISA was used to measure the serum A? level of mice.Immunohistochemistry was used to observe the brain A? level.WB was used to detect the expression of protein A? and the key proteins in the TLR4/My D88/NF-k Bp65 signal pathway in AD model mice.HE staining was used to observe the changes of intestinal structure in different groups of mice.Results: After 140 days of continuous fermented yogurt intervention,we found that fermented yogurt,the latency period for AD model to find the platform,the number of times of crossing the platform and the time of staying in the target quadrant in the MWM were shortened(P<0.05),and the recognition index in the new object recognition experiment of AD model mice was increased(P<0.05).The level of A? in serum and brain were decreased(P<0.05).Downregulation of TLR4,My D88,NF-?Bp65,p-I?B-? protein and inhibit the activation of TLR4/My D88/NF-?B signal pathway.The results of HE staining in the four groups showed that fermented yogurt could improve the intestinal mucosal epithelial structure.Conclusion: The intervention of Tibetan fermented yogurt can improved the learning and memory ability and A? deposition in the brain of AD model mice by regulating the expression of proteins related to the inflammatory pathway.The specific mechanism may be through down regulating TLR4,My D88,NF-?Bp65,p-I?B protein levels to reduces the inflammatory response and inhibits the activation of TLR4/ My D88/NF-?B signal pathway,thereby reducing A? deposition in the brain and improving learning and memory ability.Part ? Effects of traditional fermented yogurt in Tibetan area of Qinghai on intestinal microbial of APP/PS1 double transgenic AD model miceObjective: Intestinal flora disorder plays an important role in the pathogenesis of AD.Therefore,based on the results of the first and second part of the experiment,the mechanism of improving AD model mice by regulating intestinal flora of traditional fermented yogurt in Tibetan areas was further explored.Methods: Taking AD model mice and wild mice with the same background as the research objects,fecas samples of four groups of mice(5-8 in each group)were collected before intervention and two time periods after 140 days gavage.Fecal DNA of each group was extracted for high-throughput sequencing of 16 S r RNA.According to the sequencing results,the composition of intestinal flora,relative species abundance and diversity of each group were analyzed.Spearman correlation analysis was conducted through the behavioral data of mice and the OTU clustering results of intestinal microflora.Results: 1.Compared with the wild mice,the abundance and diversity of intestinal microbial were decreased in AD model mice.2.The fecal colonies of AD mice in different treatment groups showed significantly differences in species richness and microbial diversity.The fecal microbial community diversity and abundance of mice in APP/PS1 group were lower than those in yogurt group and Lac.group,and difference was statistically significant(P<0.05).From the overall structure of fecal flora,the relative abundance of Firmicutes in the phylum level,Yogurt group and Lac.group was lower than that in APP/PS1 group,while the relative abundance of Bacteroides was higher than that in APP/PS1 group,with significant difference(P<0.05).At the level of genus,the relative abundance of Mucispirillum,Anaerotruncus and Faecalibacterium in APP/PS1 group was higher than that in Yogurt and Lac.group(P<0.05).The relative abundance of Bacteroides in the APP/PS1 group was lower than that in the Yogurt and Lac.groups(P=0.0005).The relative abundance of Ruminiclostridium in APP/PS1 group was higher than that in Yogurt group(P=0.0198).Desulfovibrio relative abundance did not change significantly in the APP/PS1 and Yogurt groups,but increased significantly in the Lac.groups(P=0.0065).The relative abundance of Lactobacillus and Alistipes did not change significantly in the APP/PS1,Yogurt and Lac.Groups(P=0.99,P>0.05).3.Spearman correlation analysis was performed on intestinal microbial that may be related to cognitive impairment.The results showed that at the genus level,Mucispirillum,Caproiciproducens,Ruminiclostridium,Lactobacillus,Lachnoclostridium,Candidatus_Arthromitus,Candidatus_Saccharimonas were negatively correlated with cognitive function(P<0.05).Erysipelatoclostridium,Enterorhabdus and Muribaculum were positive correlated(P<0.05).Among them,Musipirillum had the strongest correlation(SCC=-0.880).Conclusion: The intestinal microbial community richness and diversity of WT group were higher than that in AD group.After the intervention of traditional fermented yogurt in Tibetan region,the abundance and diversity of intestinal microbial in AD model could be improved.Fermented yogurt from Tibetan region may improve the cognitive function by increasing the abundance and diversity of intestinal microbial in AD model mice,regulating the imbalance of intestinal microbial,and protecting the intestinal mucosal barrier.The cognitive function of mice was significantly correlated with their intestinal flora,and the traditional fermented yogurt had an adjustment effect after intervention.