Study On The Effect Of Kidney-tonifying Method On The Imbalance Of Intestinal Flora In Alzheimer's Disease

Background:Alzheimer’s dlsease is a neurodegenerative disease characterized by progressive loss of memory and other cognitive functions and lacks effective treatments.Current research postulated that AD is closely related to the imbalance of gut microbiota with limited evidence.In the long-term research of our team,we found that the kidney-tonifying method in Traditional Chinese medicine(TCM)can alleviate AD by regulating the metabolism of amyloid-β,inhibiting inflammatory response,reducing phosphorylated tau,protecting neurovascular units.But it is unclear whether the kidney-tonifying method will interfere with AD by regulating the gut microbiota.There have been few research reports on the treatment of AD by regulating the imbalance of gut microbiota with TCM.Objective:To describe the characteristics of the imbalanced gut microbiota in AD and explore the effect and mechanism of the kidney-tonifying method in the gut microbiota of AD.Methods:1.According to the inclusion criteria and exclusion criteria,109 subjects were divided into normal cognitive group(n=29),amnestic mild cognitive impairment group(n=30)and AD group(n=50).Conduct neuropsychological assessments on all subjects.A stool sample was collected for 16srDNA detection of gut microbiota.The analysis was used to compare the structure difference of gut microbiota among three groups and to explore the relationship between the gut microbiota and the cognition scale(MMSE)or function scale(ADL).2.Six-month-old APP/PS1 double transgenic mice were used as AD model mice,and the six-month-old C57 mice were used as the normal control group.16srDNA was used to detect the gut microbiota of the mice.The difference in the composition and structure of gut microbiota about two group mice were analyzed by Alpha analysis,partial least squares discriminant analysis,and Lefse analysis.3.Six-month-old APP/PS1 double transgenic mice were randomly divided into 3 groups:the model group(n=8),the donepezil group(n=8)and the kidney-tonifying group(n=8).Meanwhile,six-month-old C57 mice were used as the normal control group.All the mice were given intervention for 3 months.(1)The gut microbiota of mice before and after the intervention was detected by 16srDNA to explore the effect of the Chinese herbal of kidney-tonifying method on the composition and structure of gut microbiota in mice.(2)Behavioral abnormalities or improvements of mice were tested using the Morris water maze Changes in pathophysiology and morphological structure of the hippocampus and ileum were observed by histopathological examination.This part of the study was to evaluate the effect of kidney method on behaviors of APP/PS1 double transgenic mice and the structure of the hippocampus or ileum.(3)Meanwhile,Elisa was used to evaluating the effect of kidney-tonifying method on peripheral inflammation in AD mice by determining the levels of IL-1β,IL-6,IL-10,and TNF-α.(4)The immunoassay was used to determine the levels of LPS in the hippocampus paraffin section and the ileum paraffin section.Western blot was used to determine the protein levels of TLR4,MyD88,NFκB-p65,MD2 in the hippocampus and ileum.Aimed to explore the effect of the herbal on the LPS-TLR4-MyD88-NFKB-p65 pathwayResults:1.In the aspect of gut microbiota research,the microbial community in AD was significantly changed.(1)The dominant gut microorganisms of the AD group,aMCI group,and NC group were Firmicutes,Bacteroidetes,and Proteobacteria.Compared to the NC group,the abundance of Firmicutes and some subordinate microbiota were reduced in the AD group and aMCI group.The Lefse analysis showed that Campylobacteraceae and Pseudomonadales which belong to the Proteobacteria were the main functionality microbiota in AD patients.(2)When further correlation analysis was performed,it was found that the Firmicutes and the Proteobacteria were related to the disease stage.When analyzed in the level of class,order or family,it was found that the abundance of some microbiota belong to the Proteobacteria was negatively correlated with cognitive function(MMSE).The higher abundance of bacteria,the lower MMSE score.The abundance level of some microbiota belong to the Proteobacteria are positively correlated with the function(ADL).The higher abundance of bacteria,the higher the ADL score.(3)The character with the lower relative abundance of Firmicutes and Clostridiales,a higher relative abundance of Bacteroidetes and Bacteroidales had shown in APP/PS1 double transgenic mice.In addition,the imbalance of gut microbiota in APP/PS1 double transgenic mice became more serious as they grew.The increasing relative abundance of Pseudomonadales and reducing relative abundance of Candidatus-Saccharibacteria and Verrucomicrobia emerged as the mice grew2.The kidney-tonifying method can regulate the imbalance of gut microbiota in the APP/PS1 double transgenic mice.(1)The kidney-tonifying method increases the relative abundance of Verrucomicrobia in order to promote the balance of the gut microbiota.(2)The kidney-tonifying method was shown to alleviate cognitive impairment tested by the Morris water maze.(3)In addition,the kidney-tonifying method can effectively improve the morphological structure of intestinal tract,increase the permeability of the intestinal barrier,reduce the expression of LPS in the hippocampus and ileum tissue,and reduce the level of IL-1β,IL-6 and TNF-α,increase the level of IL-10,but have few effects on the TLR4-MyD88-NFKB signaling pathway.Conclusions:The gut microbiota imbalanced were existed in AD patients and AD model mice which are related to levels change of Firmicutes,Bacteroidetes,Proteobacteria and some of their subordinate microbiota.The kidney-tonifying method can regulate the imbalance of gut microbiota in AD model mice by increases the relative abundance of Verrucomicrobia.Furthermore,it also can protect intestinal mucosa structure,reduce inflammatory factor levels and partially improve the cognitive dysfunction in AD model mice.