Evaluate The Immunomodulatory Activity Of A Fungal Protein Extracted From Hericium Erinaceus Based On Gut Microbiota

Objective Evaluating Immunomodulatory activity of a fungal protein extracted from Hericium erinaceus after related cell and animal models was build,and observing the change of gut microbiota on the model mice after tacking HEP.And then,explore the relationship between HEP,gut microbiota and immunity.Methods1.A Box-Behnken design was employed to optimize extraction process of Hericium erinaceus water-soluble protein based on ratio water to raw material,extraction pH,extraction temperature and time.SDS-PAGE electrophoresis was used to determine the composition of HEP according to its molecular size.2.Evaluating the effect of HEP on the proliferation of RAW264.7,and Using LPS(1μg/ml)to make cell inflammation model to evaluate the inhibitory effect of HEP on inflammation.3.Made immunoassay model with cyclophosphamide(80mg/kg)by intraperitoneal injection,high dose and low dose group mice given HEP solution,the concentraction is 200 mg/kg/d and 100 mg/kg/d respectively.Then observed the changes of immune related physiological and pathological indexes in mice.16s rRNA sequencing was carried out to observe the changes of gut microbiota in mouse.4.Half of the model mice were treated with large number of antibiotics after the establishment of ulcerative colitis model mouse with TNBS(0.6%,0.1mL each),intragastric administration with HEP(100 mg/kg/d),Bifidobacterium(about 5 billion bacteria),and HEP3+Bifidobacterium,observe the effects of bifidobacteria on inflammation,and monitored the gut microbiota structure by using 16s rRNA sequencing.Results1.Results showed the optimal extraction conditions for the bioactive compounds were as follows:optimal pH is 9.47,ratio of water to raw material is 50mL/g,the optimal extraction time is 6h,and 37.9 ℃ of extractive temperature,optimal extraction yield is 31.81mg/g.SDS-PAGE electrophoresis shown that HEP has a total of 6 bands,distributed between 37-90KD.2.HEP was applied to RAW264.7 cells and cultured for 48 h,it showed a prophylactic effect on RAW264.7 cell proliferation.With the decrease of HEP concentration from 1.250-0.063mg/ml,RAW 264.7 proliferation rate show a trend of increasing.After the inflammation model was established by using LPS,The expression of TNF-a.IL-1β and IL-6 was significantly increased(p<0.05),with the HEP administration concentration increased from 0.05 mg/mL to 0.2 mg/mL,these inflammatory factors showed a decreasing trend(p<0.05).With the increase in HEP concentration,NO,iNOS and NF-κB secretion decreased.When compared with the model group,the levels of factor secretion were significantly different at the concentration of 0.10mg/mL and 0.20mg/mL(p<0.01).3.The immunoassay model of mouse was established by injected with cyclophosphamide,The thymus index,spleen index,white blood cells and platelet count were significantly decreased(p<0.05).After administration of HEP,the above indicators have significantly improved,The low dose group(100mg/kg/d)showed significant difference(p<0.01).In the model group,the degree of neutrophil phagocytosis and spleen lymphocyte transformation were decreased(p<0.05),when compared with the model group,the neutral red phagocytosis of HEP group was significantly improved.The proliferation rate of spleen lymphocytes in HEP group mice was 175%,which was close to normal group.And the value of CD3＋,CD4+,CD8+,CD28+,CD4+/CD8+,CD28+/CD8+and naive T cell were enhanced.Spleen pathological show that HEP could improve mice spleen atrophy and pathological changes after treated with cyclophosphamide.After cyclophosphamide treatment,the composition of the gut microbiota changed significantly at phylum level,as the increase of Actinobacteria,Tenertcutes and TM17,.After given HEP,Actinobacteria,Tenertcutes,proteobacteria reduced,whereas the Bacteroidetes and Fimicutes increased,And close to the normal group.At the genus level,The high level bacteria in the model group were Lactobacillus,Bacteria,Sphingomonas.The high abundance flora of HEP group was mainly Enterobacteriaceae and Corynebacterium.4.Serum LPS levels were measured,results showed model group and model + antibiotic treatment group were 210%and 240%of the normal group,After administration of HEP and Bifidobacterium,the serum LPS levels were significantly decreased.HEP +Bifidobacterium group effect is more obvious.The colon tissues were seriously damaged of the TNBS combined antibiotics treated groups than those of just induced by TNBS,also the splenic tissues.All the cytokines levels were deviated the normal and TNBS.as some anti-inflammatory cytokines such as GM-CSF,INF-γ,1L-10 and IL-12,1L-17α,1L-4,TNF-α,VGEF-a were secreted significantly variant(p<0.05 or p<0.01).When with the treatment of HEP,Bifidobacterium,and HEP+Bifidobacterium,all the symptoms and parameters were recovered to close the normal,especially the HEP+Bifidobacterium treated group.Conclusion At a certain concentration,the proliferation of RAW264.7 cells could be promoted by HEP.HEP also can alleviate the deterioration of inflammation caused by LPS of RAW264.7 cells,which by decreasing the over-production situation of TNF-a,IL-6,IL-1β,and HEP could down-regulating the expression of iNOS and NF-κB,inhibit the secretion of NO.The physiological and pathological indicators have been improvement,The structure of the dominant bacteria in the intestine of the mice is restored,the beneficial bacteria are increased,the pathogens and the conditional pathogens are reduced after treat with HEP.Antibiotics could damage the structure of gut microbiota,leading to inflammation and ulcers more severe.HEP and Bifidobacterium can relieve TNBS-induced ulcerative colitis,there were better effects by using HEP and Bifidobacterium together,probably because HEP can promote Foxp3 and inhibit the expression of NF-κB p65,keep the gut microbiota structure integrity promote the adjustment of gut microbiota and the survival rate of Bifidobacterium and other probiotics.