Study On The Anti-alzheimer’s Disease Activity Of Purified Polysaccharides From Hericium Erinaceus Fermentation Mycelium

Alzheimer’s disease（AD）is one of the most important forms of dementia,which can lead to a progressive decline in memory and learning.Almost every 3 seconds,one AD patient is newly diagnosed in the world.With the inevitable acceleration of aging in various countries,the burden on society and families will be greatly increased.The pathogenesis of ad is complex and is linked.At present,the treatment strategies around AD focus on improving the abnormal accumulation ofβ-amyloid（Aβ）plaque,promoting the release of acetylcholine（ACh）and blocking glutamate nerve transmission,but there are still many problems such as unstable drug effect and toxic and side effects.It seems that the single target treatment strategy cannot be used to control the course of disease.Therefore,as a potential new therapy,natural drugs acting on multiple targets and systemic have become the research hotspot of AD treatment.Fungi are widely used as a part of the daily diet in many countries because of their rich nutrients.Hericium erinaceus（H.erinaceus）,as a high-protein and low-fat food,with delicious taste,is not easy to get fat but also can nourish and strengthen the body,stomach,and nerves,so it is also a precious traditional Chinese medicine.With the development of the health industry,the demand for rare fungi is increasing.Therefore,the establishment of a systematic fermentation system of H.erinaceus is very helpful for the long-term development of H.erinaceus and its downstream industry.Polysaccharide is the main active component of H.erinaceus.It has been proved that H.erinaceus polysaccharide has antioxidant,immune regulation,gastrointestinal protection and nerve development promoting activities.There is no research on the anti-AD mechanism of H.erinaceus polysaccharide in the APP/PS1 mouse model.Therefore,based on the above theoretical basis,the liquid fermentation mycelia polysaccharide of H.erinaceus was taken as the research object,and its purification method and structural properties were clarified.At the same time,its protective activity against AD was investigated to explore the molecular mechanism.In this study,a submerged fermentation process with high yield mycelium and intracellular polysaccharide as index was established by combining satisfaction function,single factor experiment,screening experiment design and central composite design.The medium formula was:sucrose 30,yeast extract 19.83,tryptone 5,（NH₄）₂SO₄ 5,KH₂PO₄4,Mg SO₄ 2.58,Zn SO₄ 0.02,Ca Cl₂ 0.01 and vitamin B₁ 0.03.The fermentation parameters were as follows:fermentation temperature 26℃,rotation speed 200 r/min,initial p H 6.5,inoculum 5%,fermentation for 6 days.After that,combined with a variety of detection methods,the main active components of H.erinaceus mycelium were identified,including total protein（48.5%）,mannitol（8.90 g/kg）,total flavonoids（1.04g/kg）,total triterpenoids（1.13 g/kg）and 17 amino acids.Compared with wild H.erinaceus,submerged fermentation mycelia of H.erinaceus has similar active components,which provides a basis for future pharmacological rese arch.Then,H.erinaceus extract（HE）was prepared.In the rat model of adrenal chromaffin tumor（PC12）cells,HE induced the neuronal differentiation of PC12 cells by increasing the expression ofβ-tubulin III in the cells,and did not affect the proliferation of PC12 cells.In the model of differentiation PC12 cells induced by L-glutamate（L-Glu）,HE can enhance cell activity and produce anti-apoptosis by inhibiting apoptosis,improving mitochondrial dysfunction,alleviating Ca²⁺overload and inhibiting the accumulation of reactive oxygen species（ROS）.In the model of AD mice induced by D-galactose（D-gal）and Al Cl₃,HE can improve the spatial memory,learning ability and endurance of mice based on safety.HE can improve cholinergy and anti-AD by regulating the content of acetylcholine and ACh transferase（Ch AT）in serum and hypothalamus.To obtain purified polysaccharide with neuroprotective activity,combined with L-Glu-induced mouse hippocampal neuron cell line（HT22）cell injury model,we established a system for screening and purifying H.erinaceus polysaccharide combined with neuroprotective indexes,and finally obtained purified polysaccharide（PHEB）,and analyzed its structure and physicochemical properties.The purity of PHEB was about96.9%,and there was no obvious protein and nucleic acid component in the UV spectrum.Its molecular weight was 36.1 k Da,which is an irregular linear homogeneous polysaccharide.PHEB was mainly composed of six kinds of monosaccharides,and the molar ratio of fucose（Fuc）:Gal:glucose（Glc）:xylose（Xyl）:mannose（Man）:Glucuronic Acid（Glc UA）=2.622:9.372:66.660:1.956:13.514:4.821.Infrared spectrum analysis showed that PHEB contained characteristic peaks of sugars,such as O-H stretching vibration,C-H stretching vibration,C-O symmetric stretching vibration and O-H variable angle vibration.The major glycosidic residues were 6-glc（p）and 3-glc（p）,which indicated that the main chain of the polysaccharide was glucan.All glycosidic bond signals were assigned by nuclear magnetic resonance（NMR）,and finally the main glycosidic bond composition of PHEB was clarified.Finally,the anti-AD effect and molecular mechanism of PHEB with neuroprotective activity were studied in this study using the APP/PS1 mouse model.The results showed that PHEB could improve the spatial memory and learning behavior of mice in the open-field test（OFT）,diving platform and Morris water maze（MWM）;hematoxylin and Eosin（H&E）staining showed that PHEB improved the neuronal damage in the brain of AD mice,and had no side effects on other organs;the results of immunohistochemistry showed that PHEB reduced the deposition of Aβ_1-42,tau over phosphorylation and 4-hydroxynonaldehyde（4-HNE）expression;regulated the cholinergic factors（ACh,Ch AT and acetylchlolinesterase（ACh E））The levels of ROS,malondialdehyde（MDA）,superoxide dismutase（SOD）,catalase（CAT）and glutathione peroxidase（GSH-Px）and Aβ_1-42 were observed in hippocampus,hypothalamus,hypophysis and serum.The potential target and pathway of PHEB anti-AD activity were analyzed by non-standard quantitative proteomics and bioinformatics.52 kinds of protein expression differences were obtained.Combined with Gene Ontology（GO）and Kyoto Encyclopedia of genes and genomics（KEGG）,the significant difference in expression proteins mainly involves Ca²⁺homeostasis,glutamate receptors（Glu Rs）expression,synaptic intima formation and neurodevelopment,and there was a clear interaction relationship.Therefore,the molecular mechanism of PHEB was further studied by Western blotting around Ca²⁺homeostasis.The results showed that the mechanism of PHEB may be the activation of nuclear factor erythroid 2-related factor 2（Nrf2）pathway,which can inhibit the oxidative stress-mediated Ca²⁺overload and The phosphorylation expression of Ca²⁺/calmodulin kinase（Ca MK）II/IV increase,the Ca²⁺homeostasis in neurons was improved,synaptic development and anti-AD effect were improved,which provided the theoretical basis for the development of drugs and health products to improve AD symptoms and cognitive and memory.