| Alzheimer’s disease（AD）is a neurodegenerative disease that poses a serious threat to the health of older people.Over the past few decades,multiple pathological hypotheses were proposed,includingβ-amyloid（Aβ）deposition,neurofibrillary tangles,choline dysfunction,oxidative stress,neuroinflammation,etc.Despite great efforts and investments that have been made targeting one of these AD-related pathologies,there is still no effective drug for this fatal disease.Recently,accumulating evidence implicated that gut microbiota involves in the pathogenesis of AD.The gut microbiota perturbation is often accompanied by impairment of the intestinal barrier,elevated plasma proinflammatory cytokines,microbial metabolites lipopolysaccharides（LPS）,and bacterial amyloid,leading to systemic inflammation and ultimately to impairment of brain function.Given that the emerging role of the intestinal microbiome in AD,drug development/nutritional intervention based on the“microbiota-gut-brain”axis may provide a potential opportunity to regulate AD.Sparassis crispa（S.crispa）is a well-known medical and edible fungus.It has been very popular among consumers due to its favorable taste and nutritional values.Notably,the polysaccharide is the significant primary bioactive components in S.crispa,which has been proven to possess a variety of pharmacological activities.In particular,in vitro and in vivo evidence suggests that polysaccharide of Sparassis crispa（SCP）has been effective in improving AD-related pathological symptoms.Most studies were focused on the extraction process and biological activities,while data on the structural characterization of SCP and the underlying mechanism for its anti-AD activity have been rarely reported.In view of this,the present study aimed to optimize the extraction process to obtain highly active SCP,isolate purified fractions by using column chromatography and further investigate its structural properties.The digestion and fermentation characteristics of SCP-1 were evaluated with simulated in vitro models.Establish an AD-like mouse model to investigate the protective effect and potential mechanisms of SCP on alleviating AD-associated symptoms in terms of“microbial-intestinal-brain”axis.The main contents are as follows:（1）Ultrasonic-assisted enzymatic extraction（UAEE）technology was optimized to extract polysaccharides from SCP by response surface methodology.The physicochemical properties and antioxidant activities of SCPs obtained from UAEE and conventional hot water extraction（HWE）were evaluated.According to the optimal parameters,ultrasonic time,41min;ultrasonic power,300 W;ratio of material to water,1:39 g/mL;and cellulose concentration,0.8%.Under these conditions,the yield of SCPs reached up to 14.63%,which increased by about 68.54%compared with HWE procedure.In terms of physicochemical properties,UAEE methods degraded molecular weights,influenced the microstructures of two SCPs.However,UAEE procedure had no effect on polysaccharide functional groups.Antioxidant assays showed that SCP-UAEE possessed better antioxidant activities（including DPPH,ABTS+,and hydroxyl radical）than SCP-HWE in vitro.Furthermore,pretreatment of SCP-UAEE at a concentration of 200μg/mL significantly attenuated HT22 cell neurotoxicity induced by H2O2,by the means of ameliorating cell viability,reducing extracellular LDH release,and decreasing levels of intracellular ROS.Its protective effect was comparable to that of Trolox（50μmol/L）.（2）The purified fractions were isolated by using DEAE Sepharose Fast Flow and Sepharose CL-6B column.Then the structure characterization of the purified polysaccharide SCP-1 was investigated by determining the molecular weight distribution,monosaccharide component,FT-IR,methylation and GC-MS analysis,and NMR spectroscopy analysis.The results revealed that SCP-1 mainly contained total sugar（98.7%）and no uronic acid and protein were found.SCP-1（Mw 1.368×104 Da）was a heteropolysaccharide mainly comprising glucose,galactose,fucose,and mannose in a molar ratio of 52.10:31.10:15.04:1.76.The main backbone of SCP-1 was predominantly composed of（1→6）-α-D-Galp,（1→6）-β-D-Glcp,（1→3）-β-D-Glcp,（1→2,6）-α-D-Galp and（1→3,6）-β-D-Glcp.The branches,substituted at the O-2 of Gal and O-3 of Glc,contained（1→6）-2-OMe-α-D-Galp,（1→4）-β-D-Glcp,（1→3）-β-D-Glcp,and terminated by T-α-L-Fucp and T-β-D-Glcp.Even though mannose was observed in the monosaccharide composition analysis in the SCP-1,the representative carbon and proton signals in the NMR spectra and sugar moieties in methylation analysis were undetectable due to its low abundance.Therefore,the comprehensive structure of SCP-1 needs to be further determined.（3）The digestion and fermentation characteristics of purified polysaccharide SCP-1 were investigated by the in vitro simulated models.Our results revealed that no noticeable change in the relative Mw,the content of reducing sugar and the release of free monosaccharides during the simulated digestion of SCP-1,suggesting that SCP-1 was stable during in vitro digestion model.The results of the in vitro fermentation experiments showed that the residual total sugar was 53.04±2.56%and the pH in the fermentation solution decreased from the 7.97to 5.17,after SCP-1 was fermented by human fecal microbiota.Moreover,acetic acid,propionic acid and butyric acid,the main metabolites of microbiota,increased from 0.58±0.05,0.22±0.00 and 0.12±0.02 mmol/L to 7.41±0.76,3.60±0.29 and 4.20±0.21 mmol/L,respectively.Additionally,SCP-1 fermentation modified the gut microbiota composition through promoting beneficial genera（Prevotella 9,Dialister,Megamonas,and Megasphaera）and inhibiting proliferation of some harmful bacteria（i.e.,Escherichia/Shigella）.The PICRUSt prediction analysis based on KEGG indicated that SCP-1 significantly increased carbohydrate,energy,and amino acid metabolism.（4）D-galactose（D-gal）/AlCl3-induced AD-like mice was established.The neuroprotective effects and the underlying mechanism for SCP-1 was discussed by combining with the results of cognitive-behavioral tests,histopathological changes,physiological indicators of the brain and serum,gut microbiota composition,as well as intestinal flora metabolites（SCFAs）.The results indicated that SCP-1 could ameliorate cognitive deficits,amyloid genesis,and inhibited neuroinflammation induced by microglia and astrocyte activation.Simultaneously,SCP-1 reshaped the gut microbiota structure,through decreasing the abundance of Escherichia/Shigella and Odoribacter,and boosting the abundance of SCFAs-producing bacteria（Intestinaimonas,[Eubacterium]ventriosum group,Lachnospiraceae＿UCG＿010）.SCP-1 intervention promoted production of SCFAs and maintained intestinal barrier function by reducing serum lipopolysaccharide and increasing the colon expression of tight junction proteins.Furthermore,Western blotting analysis indicated that SCP-1 inhibit neuroinflammation and AD-related pathological symptoms partly via the involvement of TLR4/NF-κB pathway.（5）To explore the influence of SCP-1 on the metabolic perturbations in AD-like mice,the serum metabolomics,quantification of neurotransmitters,and the Spearman’s correlation analysis based on“gut microbiota-metabolites-biochemical indicators”were investigated.According to multivariate statistical analysis and on-line databases of HMDB,a total of 20potential biomarkers associated with AD were identified.Metabolic pathways more significantly affected by SCP-1 intervention mainly included:linoleic acid metabolism,arachidonic acid metabolism,nicotinate and nicotinamide metabolism,sphingolipid metabolism,tryptophan metabolism and biosynthesis of unsaturated fatty acids.In addition,SCP-1 intervention could modulate the levels of acetylcholine（Ach）,γ-aminobutyric acid（GABA）,glutamate（Glu）,aspartate（Asp）and tryptophan（5-HT）in the hippocampus and serum of AD mice to some extent.Spearman’s correlation analysis based on“gut microbiota-differential metabolites-biochemical indicators”showed that SCP-1 may regulate gut microbiota to influence the bile acid metabolism,produce SCFAs,neurotransmitters or their precursors,which enter the brain through the vagus nerve or peripheral circulatory system.These bioactive molecules could play a role in anti-inflammatory and neural signaling in the brain and thus improve cognitive impairment in AD mice. |
