| The gut flora comprises a complex community of microbial species that are present in our gastrointestinal system and whose variation affects not only various intestinal diseases but also central nervous system disorders such as Alzheimer’s disease(AD),the most common form of dementia and a neurodegenerative disease associated with cognitive impairment and brain accumulation of amyloid Aβ protein.Most notably,the microbiota-gut-brain axis is a bidirectional communication system that is not fully understood but includes neural,immune,endocrine and metabolic pathways.Studies in germ-free animals as well as in animals exposed to pathogenic microbial infections,antibiotics,probiotics or fecal microbiota transplants suggest an interaction of the gut microbiota in host cognition or AD-related pathogenesis.The drugs that have been approved by the FDA for the treatment of AD so far mainly include the acetylcholinesterase inhibitor donepezil hydrochloride and the NMDA-type glutamate receptor antagonist memantine hydrochloride.The discovery of donepezil became a turning point in the development of acetylcholinesterase(ACh E)inhibitors as drugs for the treatment of Alzheimer’s disease.It blocks ACh E thereby enhancing cholinergic activity function and altering cognitive function.Such changes have been demonstrated in numerous animal and human studies,where the loss of cholinergic neurons and the associated decrease in the level of cholinergic neurotransmission have been associated with cognitive impairment in AD patients.In this study,we investigated the pathological characteristics of brain and colon sections altered by gavage of acetylcholinesterase inhibitors through the bi-directional pathway of the gut-brain axis,and finally isolated and screened "star candidates" from the gut microbiota that were highly indicative and significant in the sequencing results Probiotic Akkermansia muciniphila.In this experiment,the APPswe×PS1d E9 double transgenic mouse model was firstly constructed,and the mice were given donepezil hydrochloride at a dose of 3 mg/kg per day by gavage in the same environment,after which the fecal samples were taken and sent to the mice for 16 S rRNA high-throughput sequencing.The results showed that Akkermansia muciniphila spp.were higher in the diseased group than in the normal group,but the abundance trend increased in both groups after gavage of donepezil hydrochloride;similarly,the abundance of Clostridium intestinalis,Mucispirillum,Odoribacter,Streptococcus,and Ruminiclostridiu spp.increased after gavage of donepezil hydrochloride,while the abundance of Bacillus spp.,E.coli Shigella,and Helicobacter decreased after gavage of According to the literature,the changes in the content of the above mentioned different species of intestinal bacteria may be closely related to AD production,intestinal barrier dysfunction,energy metabolism imbalance,chronic inflammation,insulin resistance and aging.Secondly,the index levels of ACh E and superoxide dismutase(SOD)were calculated by spectrophotometric measurements in the four groups,and the enzyme activity of SOD in brain tissue was significantly lower in the model group than in the normal control group,while by gavage of donepezil hydrochloride,the index was higher in the normal gavage group compared to the previous index,and significantly higher in the model gavage group compared to the previous index.The results showed that ACh E was significantly higher in the model group than in the normal control group,while by gavage,both the model group and the normal control group showed a certain degree of decrease and significant difference compared with the previous index by gavage.This indicates that the gavage drug can significantly increase the SOD enzyme activity,enhance the scavenging function of free radicals in the brain to delay aging,and eventually has an indirect correlation with Alzheimer’s disease;the gavage drug can significantly reduce the ACh E enzyme activity in brain tissue,and more interestingly,the drug also inhibits the ACh E enzyme activity in colon tissue.In the brain and colon sections observed by immunohistochemistry and HE staining,β-amyloid deposition was reduced in the cerebral cortex and hippocampus after gavage with appropriate concentrations of donepezil hydrochloride,and more notably,the colonic tissue of mice with AD showed epithelial mucus layer lesions and cellular inflammatory infiltration,and donepezil hydrochloride reversed the development of this colonic pathological feature.Next,the relationship between gut microbes and AD was explored using bioinformatics techniques based on the above in vitro and in vivo experiments using molecular docking Autodock software.The interaction of the small molecule donepezil hydrochloride with ACh E and Akkermansia muciniphila outer membrane protein Amuc＿1100 was observed,and the results showed that both small molecules were in the hydrophobic lumen of the protein macromolecule,and the interaction between small molecules and protein was mainly hydrogen bonding and hydrophobic.Comparison with ACh E as a macromolecule both have hydrogen bonding interactions of less than 3 ? as well as multiple hydrophobic amino acid binding sites and share a common tyrosine(Tyr)hydrophobic binding site,indicating that the two binding macromolecule proteins of the drug are similar to some extent,and the molecular docking level based on which the AD mechanism intermediate enzyme and the intestinal microbial Akkermansia muciniphila flora will establish a relationship bridge.Eventually,the pure strain of Akkermansia muciniphila was selected to be isolated and identified from fecal samples,after enrichment culture,preliminary screening,secondary screening,liquid culture and 16 S rRNA strain identification,observation of the growth morphology of colonies under plate and microscope,and Gram staining,sugar fermentation test,hesperidin hydrolysis test,miren test,indole test,and monosaccharide polysaccharide growth test for the new strain.The results showed that a new Akkermansia muciniphila strain with 99.56% comparative results was finally obtained,which had the following characteristics(1)Single or paired raised orbicular colonies in the plate.(2)Red Gram-negative staining.(3)Different glucose acid production and gas production at different concentrations of the bacterial solution.(4)Akkermansia muciniphila was resistant to 6.5% Na Cl,could grow at p H8.0 broth,and positive growth test at 40℃ and 60℃.(5)Hydrolyzes bile hesperidin;utilizes mucins in the medium and produces other types of proteins for its own growth;positive indole test result;optimal tolerance to glucose,N-acetylgalactosamine and lactose growth.And the growth of A.muciniphila was promoted by donepezil hydrochloride at 0.3 g/L to a certain extent,as verified by in vitro experiments,indicating a positive correlation between donepezil hydrochloride and intestinal flora A.muciniphila. |
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