Protective Effects And Mechanisms Of Amanita Caesarea In Experimental Alzheimers Disease Animal Model

Amanita caesarea is a type of edible fungi of the Amanita genus,which grows in Asia,Europe,Garzêcounty of Sichuan province?China?at an elevation of 3,000 m.It has been studied for many years;however,most studies have focused on improving the growth environment,evaluating of metal ion content or component analysis.Reports have claimed that the polysaccahrides from Amanita caesarea has the potential of anti-oxidative under ABTS+test,but the biological function has not been elucidated.In the present study,we took a variety of methods to detect the proportion of some components in Amanita caesarea.The results showed that Amanita caesarea is rich in many kinds of amino acids,fatty acids,proteins,trace elements and sugars,especially potassium and selenium,and the content of poisonous metals is relatively low.It also laid a foundation for Amanita caesarea to become a drug for Alzheimer's disease?AD?.The neuro-protective effects of A.caesarea water extracts?AC?were determined in L-glutamic acid?L-Glu?induced HT22 cell apoptosis model,and D-galactose?D-gal?and AlCl₃ developed experimental Alzheimer's disease?AD?mouse model.In25 mM of L-Glu-damaged HT22 cells,3-h pretreatment with AC strongly improved cell viability,reduced the proportion of the apoptotic cells,restored mitochondrial function,inhibited the over-production of intracellular reactive oxygen species?ROS?and Ca²⁺,and suppressed the high expression levels of cleaved-caspase-3 and Bax.Compared with L-Glu only exposed HT22 cells,AC enhanced the phosphorylation activities of protein kinase B?Akt?and mammalian target of rapamycin?mTOR?.In the experimental AD mouse,28-day AC administration at doses of 250,500 and 1000mg·kg^-1·day^-1 strongly enhanced the vertical movements and locomotor activities,increased endurance time in rotarod test and decreased escape latency time in Morris water maze test.AC alleviated deposition of amyloid beta?A??in the brain regions and improved the central cholinergic system function indicated by its increasing acetylcholine?Ach?and choline acetyltransferase?ChAT?concentration,reducing acetylcholine esterase?AchE?levels.Moreover,AC reduced the levels of ROS and enhanced the levels of superoxide dismutase?SOD?in brain regions of AD mice.Taken together,our data provided experimental evidence that A.caesarea may serve as potential food for treating or preventing neurodegenerative diseases.We obtained the polysaccharides from AC to further explore the component that could function.The proteins in polysaccharide aqueous solution were removed by Sevage reagent and gradient alcohol precipitation was carried out.We found that a 3-h pre-treatment of ACPS prior to L-glutamic acid?L-Glu?co-exposure reversed the decreased cell viability,inhibited apoptosis,suppressed the accumulation of intracellular reactive oxygen species and restored mitochondrial membrane potential in HT22 cells.Compared to L-Glu-exposed cells,ACPS enhanced the nuclear levels of NF-E2p45-related factor 2?Nrf 2?,reduced the cytoplasmic levels of Nrf 2 and cytochrome C,suppressed the expression of Kelch-like ECH-associated protein 1,and enhanced the expression of heme oxygenase-1,superoxide dismutase 1 and cysteine ligase catalytic subunit.In a D-galactose and aluminum trichloride A)mouse model,42-day administration of ACPS improved the abnormal behaviors.ACPS suppressed the deposition of?-amyloid peptide in the brain and ameliorated oxidative stress via modulating the levels of related enzymes.ACPS improved the functioning of the central cholinergic system,as indicated by an increase in acetylcholine and choline acetyltransferase concentrations,and reduced acetylcholine esterase levels in the serum,hypothalamus and cerebral cortex.On the other hand,ACPS also ameliorated the mitochondrial function in AD mice by corrected the abnormal expression of mitochondria function-related proteins.Our data suggest that ACPS may be a promising candidate for the treatment of AD.In order to explore the specific active components in antioxidant and AD protection,we further purified the polysaccharides.The polysaccharides were separated by DEAE-52 to get ACPS-1.The relative molecular weight of 33500 Da was obtained by high performance liquid phase analysis.The main functional groups were-OH,-C-H,-C=O,-COOH,-C-O-C and pyran sugar.Sephadex G200and G75 gel were used to further purify polysaccharides according to molecular weight,and purified polysaccharide ACPS-2.The purified polysaccharides were applied to 8-month-old APP/PS 1 mutation mice for 6 weeks.The results showed that ACPS-2 significantly improved AD-like behavior,reduced the expression of A?and p-Tau in the brain,restored the abnormal expression of cholinergic neurotransmitters and improved the serum,hypothalamus,pituitary and levels.Furthermore,proteomic analysis was carried out to screen differentially expressed proteins in tissues.The bioavailability,protein function and subcellular structure of these proteins were analyzed by GO enrichment.KEGG pathway analysis was used to screen the signal pathways in the cells enriched by differentially expressed proteins,which could serve as the primary screening target for ACPS-2.In a word,the antioxidant activity and the protective effect on AD animals of the aqueous extract of Amanita caesarea were preliminarily analyzed in this paper.The crude polysaccharides in water extract were extracted,which acted on animals and cells,and the main protective components were preliminarily determined,and the antioxidant activity of Amanita caesarea polysaccharides was found as follows.The crude polysaccharides were purified and were used in APP/PS-1 gene mutation animal models which is closer to human AD patients,then the mutation mice also obtained the results of alleviating AD symptoms.Therefore,our data suggest that ACPS may be a promising candidate for the treatment of AD.