| Fungal polysaccharides are wildly recognized for their bioactivities in anti-tumor,immuno-modulating, anti-bacteria, anti-virus, anti-hyperglycemia, anti-oxidant, etc. In recentyears, many researches revealed that fungal polysaccharides still owed high bioactivities afteroral administration. However, how the polysaccharides exerted their effects were still poorlyunderstood. Especially, what specific changes occured in the gut where the polysaccharidesdirectly contacted with intestine musical barrier. Lentinula edodes is the most popular edibleand medical mushrooms in the world. With the development of purification and structuredetermination methods, many types of polysaccharides have been obtained from the fruitbody of Lentinula edodes. In our previous study, a new heteropolysaccharide named L2wasseparated from the fruit body of Lentinula edodes by boiling water, followed with traditionalpurification procedure. Based on chemical and physical analyses, L2were mainly consisted ofglucose (87.5%), galactose (9.6%), and arabinose (2.8%) with average molecular weight of26K Da. The polysaccharides L2showed significant and toll-like receptor (TLR)2-involvedimmuno-stimulating activities which exhibited high stability in wide ranges of pH from4.0to10.0and temperature below121℃treatment with RAW264.7cells model.In present studies, the main chain structure characteristics were investigated by NMR.Subsequently, the immuno-modulating properties and mechanism of L2after oraladministration in mice were further investigated. NMR analysis indicated that the mainbackbone chain of L2were β-D-(16)-linked glucose and small proportion of-D-(16)-linked arabinose connected with side chains at C-O3and C-O4inmonosaccharide unit. Oral administration of L2with dosage of40mg/kg day for four weeks,the increased of spleen and thymus indices, natural killer cell (NK cell) activity, andproliferation activity of T cell in splenocytes as well as the increased cytokines of IL-2,IFN-γ,and IL-12in serum were observed in adult C57BL/6mice. Interestingly, the results alsoshowed that L2restored the age-attenuated immune responses by improving the proliferationactivity of lymphocytes in splenocytes and cytokines concentration (IL-2and IL-6) in serum.The gut microbes play a critical role in modulation of immune function. The impacts ofL2on the gut microbial communities were examined by high-throughput pyrosequencingtechnology targeting to16S rRNA gene. In the fecal microbial communities of adult mice, L2induced the increase of observed richness while reduced the-diversity. Specifically, thesignificantly increased Proteobacteria were observed in L2-treated mice, which werecharacterized by the increase of Epsilonproteobacteria at class level. The increased Helicobacteraceae and reduced S24-7were present in fecal microbiota after L2treatment inadult mice. In particular, some OTUs (Operational taxonomic units, OTUs), corresponding toHelicobacter suncus, were found to be significantly increased in L2treated-mice. However,L2partly reversed the age-altered composition of gut microbiota such as the decline of-diversity and evenness and the disproportions of some bacteria groups at phylum, class, andfamily levels in aged mice, such as the skewing ratio of Firmicutes/Bacteroidetes, theincreased proportion of Bacteroidia in fecal microbiota. These changes in fecal microbiotastructure might be related to the immune-stimulating of L2to intestinal immune system andfertilizing properties to gut microbiota. Taken together, L2showed dual-direction regulationactivities to fecal microbiota which depended on the status of host immunity. Specifically,some bacteria in Helicobacter（such as Helicobacter suncus）、Alistipes、and Bacteroidesacidifaciens might played important roles in the L2-induced immune-modulating responses inC57BL/6mice.In adult mice, the microbial communities exhibit distinctly different space distributionalong the intestine. L2treatment reduced the observed richness,-diversity, and evenness ofgut microbiota, especially in the cecum and colon. No obvious changes in richness,-diversity, and evenness were observed except for the increase number of bacterial phylumin the small intestine. However, the relative abundance of Proteobacteria was increasedaccompanying of the decreased of Cyanobacteria in cecum. In colon, the microbialcommunities were shifted to Bacteroidetes-dominated characterized by the increase ofBacteroidetes and Proteobacteria and the decrease of Firmicutes. These results furthersuggested the associations between the immune function and the richness,-diversity, andevenness of gut microbiota. The impacts of L2on gut microbial communities might beattributed to the immune-stimulating to intestinal immune system and utilization by gutmicrobiota.Furthermore, the immuno-stimulating profiles of L2were evaluated in Caco-2cells andCaco-2/RAW264.7co-culture system. Subsequently, the customized RT-PCR array containing112genes were employed to investigate the effects of L2on immune-related gene expressionsin small intestine, cecum and colon. The results showed that the patterns of cytokinessecretion induced by L2were significantly different from that of Zymosan A in vitro,indicating different immuno-modulating profiles might be observed after different structure ofpolysaccharides consumption. Especially, the patterns of cytokines secretion by Caco-2cellswere changed in presence of Gram-negative component, LPS, and RAW264.7macrophagecells, indicating the exsting of complicated interaction among polysaccharides L2, intestinal immune system, and gut microbiota. Interestingly, L2differentially impacted the geneexpressions of small intestine, cecum and colon, in which55,26and25genes were markedlychanged, respectively. Most of differential genes were up-regulated in the small intestinewhile majority of differential genes were down-regulated in the cecum and colon. Specificly,L2up-regulated genes related to chemokines, interferon, interleukins, TNF, and patternrecognition receptors, while down-regulated genes related to TLRs-involved signalingpathway and cellular immunity. These results indicated that the gut microbiota might playedimportant roles in the L2-treated gene expression along the intestine. Especially,3coreregulation networks were identified for small intestine (15-gene sub-network), cecum (5-genesub-network) and colon (10-gene sub-network) with R Spider tool. In small intestine, thedifferential genes were involved in biological processes such as regulation of apoptosis,immune response, protein heterooligomerization, platelet derived growth factor receptorsignaling pathway, intracellular signaling pathway, positive regulation of DNA replication. Incecum, B cell activation and protein heterooligomerization were related to differential genesin sub-network. In colon, biological processes such as immune responses (inflammatoryresponse, T cell activation), LPS mediated signaling pathway, cell proliferation, positiveregulation of NF-kB transcription factor activity were involved in sub-network. These datademonstrated a detailed landscape of tissue-specific gene expressions and core regulationnetworks in response to L2.At last, proteomic analysis on the colon and small intestine tissues of mice fed with L2were applied compared with normal mice. Two-dimensional gel electrophoresis (2-DE)combined with MALDI-TOF-MS were employed to detect and identify the protein expressionprofiles.22proteins in colon tissue and25proteins in small intestine tissues were identifiedshowing significant changes in abundance induced by L2, respectively. Little of thedifferential expressed proteins were overlapped, indicating the tissue-specific effects of L2treatment. Among of the differential expressed proteins,12of them were choosed to validateby Western blot. These differentially expressed proteins were mainly involved inphysiological processes related to cellular component organization, developmental process,cellular process, immune system process, metabolic process, multicellular organismal process,response to stimulus. The results suggested the wide range of biological activities of L2.STRING network analysis demonstrated that21colon proteins were mapped to a7-proteinand a3-protein sub-networks,25small intestine proteins were mapped to a17-proteinnetwork, and all the45altered proteins were integrated into a unified network containing29proteins. This suggested the existence of a concert mechanism, although acting on colon and small intestine separately. These findings facilitated the understanding of regulatorymechanism in response to L2treatment in mice. |
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