Effects And Functioning Mechanisms Of Pleurotus Eryngii Polysaccharide On Gut Immune Function

As the second industrial cultivated species compared with Flammulina velutipes,Pleurotus eryngii has been considered as a kind of rare edible fungus,which could be utilized in dietary therapy or officinal.Along with the deepening of study focusing on the nutritional value of P.eryngii,increasing attention on the development of functional foods are paid in the present days,from which the P.eryngii polysaccharide has been revealed exerting multiple nutrition and medical functions.Moreover,as research continues on the relationship between gut microbiota and host health level,the biological activities of P.eryngii polysaccharide fraction through its interaction with gut microbiota has not been investigated.Hence,the correlation between the health benefits and gut microbiota alteration induced by P.eryngii polysaccharide,especially the gut immune function was carried out in the present study.The results could provide theoretical foundations of P.eryngii healthy improvement and the related functional mechanisms.More information of the present research was listed below:1.P.eryngii super-fines was utilized for the preparation of a P.eryngii polysaccharide named PEP.With the gavage of PEP on health mice,the gut microbiota structure alteration effects and fermentation characteristics of PEP were investigated.Moreover,the impacts of PEP administration on mice immune response was studied.Results showed that the total concentrations of short chain fatty acids(SCFAs)in mice cecum and colon contents were increased significantly with PEP administration.Specifically,the concentrations of acetic acid,propionic acid,i-butyric acid,n-butyric acid and i-valeric acid in cecum contents,while only the concentrations of i-valeric acid and n-valeric acid in colon contents were enhanced significantly with the administration of PEP(P<0.05).Moreover,PEP gavage resulted in a pH value decreasing and moisture increasing trend of both cecum and colon contents,which could improve the mice fecal volume and displayed a positive effect on mice defecate process.Based on the 16s rDNA high-throughput sequencing,the gut microbiota alteration with PEP administration was revealed,an enhancement effect on beneficial bacteria abundance and reduction effect on harmful bacteria was found in the phylum,class,order and family level.Specifically,the abundance of Firmicutes was increased and Bacteroidetes was decreased significantly in phylum level with the treatment of PEP(P<0.05).And the abundances of Porphyromonadaceae,Rikenellaceae,Bacteroidaceae and Lactobacillaceae were increased,while Lachnospiraceae and Ruminococaceae were decreased significantly by the oral of PEP compared to the control group(P<0.05).In addition,an improvement of PEP gavage on the indexes of liver,spleen and thymus,as well as the concentrations of TNF-?,IFN-?,IL-1,IL-2 and IL-6 in serum,and SlgA in cecum and colon contents were detected,which suggested the positive role of PEP treatment on host immune response function.2.After 6 weeks of PEP administration,C57BL/6 mice in both control and PEP treatment groups were sacrificed to isolate the intestinal and colonic tissues,following with a functional protein profile evaluation study based on isobaric tag for relative and absolute quantification(iTRAQ)proteomics method.Results revealed that PEP supplementation led to a total of 49 and 87 up-expressed differential proteins(DPs),64 and 107 down-expressed DPs compared to control group in small intestine and colon,respectively.And a larger impact on colon was found with the administration of PEP.Based on the analyzation of GO annotations and KEGG pathway annotations,a significant impact difference was found between mice intestine and colon with the treatment of PEP.Specifically,DPs found in intestine were considered mainly related to the digestive and absorption function,while DPs in colon were mainly the involved in the metabolism process.However,some of the DPs discovered in both intestine and colon were annotated to the immune response and the related signal pathways.Focusing on the specific functions of the detected DPs in intestine,we could confirm that the protection effect of PEP on intestinal tissues,especially its activity against the exogenous pathogenic substances stimulation on intestine mucosa,as well as its combination with specific ligands on intestinal membrane,were the main process for the immune response function exertion of PEP on mice intestine.On the other hand,the interaction between PEP and gut microbiota,including SCFAs production and specific bacteria abundance alteration were considered the main process for mice colonic function improvement induced with the administration of PEP.3.PEP was further purified through the combination of DEAE-52 cellulose ion-exchange column and Sephadex G-200 column to get the final purified fractions named WPEP and NPEP.After the purification,the preliminary characteristics of the purified fractions were investigated.Moreover,a cell inflammation model induced with lipopolysaccharide(LPS)was established using RAW264.7 macrophages,and study focusing on the anti-inflammation activities and the related mechanisms of the purified fractions were carried out in the present study.Preliminary characteristic analysis revealed that the molecular weights of WPEP and NPEP were 167 kDa and 274 kDa,respectively.Monosaccharide composition suggested that WPEP and NPEP were mainly consist of xylose,mannose,glucose and galactose with the molar ratio of 21.65:3.13:73.27:1.96 and 1.33:8.82:85.00:4.85,respectively.Moreover,Fourier-transform infrared spectra(FT-IR)found that both the purified fractions were glucan with ? linkages.No cytotoxicity effects were found with the treatment of PEP,WPEP or NPEP in the cell inflammation model,the increasing of the polysaccharide concentration displayed the ability to decrease the NO and PGE2 concentrations in the cell culture supernatant in a dose-dependent manner,as well as IL-6,IL-1?and TNF-?(P<0.05).From which a stronger inhibition effect was found with the pre-treatment of both the two purified fractions compared to PEP,and WPEP displayed the highest anti-inflammation activities.Western blotting was utilized to evaluate the inflammation relate protein expression level in the LPS-induced RAW264.7 macrophages,and results revealed that WPEP and NPEP inhibited the activation of MAPK pathways by inhibiting phosphorylation of p38 and extracellular regulated protein kinases 1/2(ERK1/2).Moreover,WPEP and NPEP also displayed the anti-inflammation activities through the inhibition on NF-?B signaling activation by reducing the nuclear translocation and phosphorylation of p65(P<0.05).4.The purified fraction with the highest anti-inflammation activities in cell inflammation model was utilized to reveal its improvement effect on colitis in mice with pre-treatment of dextran sulfate sodium(DSS).The doses used in the study were 0.2 g/kg body weight and 0.8 g/kg body weight.Results showed that the oral administration could inhibit the colitis symptoms in mice,mainly including the improvement on daily fecal situation,colon length and colon structure damage(P<0.05).Moreover,the WPEP gavage also resulted in a decrease trend on the immune cells ratio,inflammatory cytokines and proinflammatory protein expression level(P<0.05).Specifically,total immune cells ratio,and the ratio of macrophages cells and neutrophils in the total immune cells were all decreased with WPEP pre-treatment in DSS-induced colitis mice.Moreover,concentrations of the inflammatory cytokines,including IL-1?,IL-2,IL-6,TNF-?,INF-y and IL-10 were all reduced in the WPEP gavage group compared to the DSS treatment group,as well as the expression level of proinflammatory proteins named p50,p65,p-p38,TLR4,COX-2 and iNOS in colonic mucosa(P<0.05).At last,16s rDNA high-throughput sequencing was utilized,and a beneficial effect was found with the pre-treatment of WPEP on mice gut microbiota disorder induced by colitis.Particularly,with the gavage concentration increased,the gut microbiota structure in colitis mice was developed toward the healthy mice,mainly including the decreased abundances of Akkermansia muciniphila and Clostridium cocleatum,and the increased abundances of Bifidobacterium pseudolongum,Lactobacillus reuteri,Lactobacillus salivarius and Ruminococcus bromii,which the alteration of the 6 kinds gut microbiota have been proved a deep correlation with the host inflammation response.In summary,the improvement of WPEP on mice colitis were found displayed a strong association with its alteration effect on gut microbiota.