Antitumor Activity Of Polysaccharide From Lentinus Edodes And Its Regulation On The Immune System

Cancer is one of the leading causes of death in the world.Polysaccharides exhibit good antitumor activity against a variety of tumors and cancer cell lines with great safety,showing the potential to aid and substitute chemotherapeutic drugs with side effects in modern cancer therapies.Lentinan,the polysaccharide isolated from Lentinus edodes,is one of the very first polysaccharides found to inhibit tumor growth.However,due to the complexity and diversity of macromolecular structure,the antitumor mechanism of polysaccharides is not yet clarified,and polysaccharide from Lentinus edodes as an antineoplastic medicine has not been widely used in clinic.Therefore,based on the antitumor effects of Lentinus edodes polysaccharide,we studied the immunomodulatory activity of polysaccharide and the direct interaction of tumor cells and polysaccharide,and discussed the antitumor mechanism.The main innovations of this thesis include the following points.?1?Six polysaccharides with the same?-1,6-branched?-1,3-glucan structure were extracted from the fruiting bodies of Lentinus edodes mushrooms,demonstrating no influence of sources of mushrooms on the polysaccharide structure or chain conformation,with the ability to inhibit tumor growth safely and restore the damage of immune system caused by tumor burden and chemotherapy drug.?2?Orally,intraperitoneally and intratumorally administrated polysaccharides all inhibited tumor growth by activating the immune system in tumor-bearing mice and promoting the recruitment of T helper cells and neutrophils in tumors.And for the first time,oral polysaccharide was seen accumulating at the tumor site,showing initiative targetability.?3?Oral polysaccharide was proved to be absorbed in the intestine and transmitted to tissues and organs including the tumors through the blood circulation rapidly,not only restoring the CD4⁺T cell level in tumor-bearing mice but also promoting thymic T lymphocytes maturation and differentiation into CD4⁺T cells in healthy mice,thus improving the immunity.?4?For the first time it was demonstrated that polysaccharide interacted with tumor cells,and the receptor that strongly interacted with polysaccharide was Elongation factor 1-alpha 1 highly expressed on the surface of S-180 tumor cells.?5?Oral polysaccharide inhibited LPS-induced acute inflammation as well as DSS/AOM-induced colitis and colorectal cancer in mice.The main contents and conclusions of this thesis are as follows.Six kinds of polysaccharides named AG,AG2,SG,SG2,CG and CG2 were isolated from the fruiting bodies of Lentinus edodes mushrooms planted in autumn,spring and cultivated in greenhouse by NaOH aqueous or hot water extraction.Among them,polysaccharides from the autumn-planting mushrooms had higher yields and purity,polysaccharides from the cultivation-planting mushrooms had the lowest yields and purity,while polysaccharides isolated by NaOH aqueous extraction had higher purity.FT-IR and ¹³C NMR spectra proved that all six polysaccharides shared the same chemical structure of?-1,3-glucan main chain despite different sources and extraction solvents.Light scattering and viscometry showed different molecular weights and chain sizes of six polysaccharides,with the highest molecular weights for polysaccharides from the spring-planting mushrooms and the lowest for polysaccharides from the cultivation-planting mushrooms.Atomic force microscopy images revealed that polysaccharides at the same concentration formed different aggregates in aqueous solution with molecular weight dependence,that is,CG and CG2 with lower molecular weights and more complicated composition formed random aggregates,SG2 with higher molecular weight formed branched short chains,while AG,AG2 and SG with medium molecular weights formed network-like structures.All six polysaccharides inhibited S-180 tumor growth in mice significantly,showing no molecular weight dependence in the range of60×10⁴?171×10⁴.Besides,six polysaccharides activated the immune system and restored the damage of immune system in tumor-bearing mice and chemotherapeutics-treated mice.By oral gavage,intraperitoneal injection and intratumoral injection at a dose of 2mg?kg^-1?day^-1,polysaccharides significantly inhibited tumor growth,especially by intratumoral injection,the inhibition ratio of which reached 77%.In vivo imaging showed that polysaccharide administrated orally or intraperitoneally accumulated in tumors,suggesting certain targetability to the tumor.Immunofluorescence images of tumor tissues showed that polysaccharide activated the immune system of tumor-bearing mice,and promoted the recruitment of T helper cells and neutrophils at the tumor site,increasing their killing activity towards tumor cells thus inhibited tumor growth.Confocal microscopy and fluorescence detection revealed that polysaccharide bound to mouse peritoneal macrophages which expressed Dectin-1 on the cell surface,and passed through the Caco-2 cell monolayer into the other side,demonstrating that oral polysaccharide was absorbed in the intestine and transmitted to tissues and organs including the tumors through the blood circulation rapidly.Flow cytometry analysis of spleen and thymus lymphocyte subsets from tumor-bearing mice showed that oral polysaccharide restored the CD4⁺T cell level which was decreased by tumor burden.Polysaccharide administrated orally also promoted thymic T lymphocytes maturation and differentiation into CD4⁺T cells in healthy mice,promoting immunomodulation.Therefore,after oral administration,polysaccharide entered the body through intestinal absorption,then transmitted to spleen to command immunomodulation.The interaction of FITC-labeled polysaccharides and S-180,MCF-7,HeLa and HepG2 cells was studied by flow cytometry and confocal microscopy.It could be confirmed that polysaccharides interacted with tumor cells and were took up by tumor cells.The protein on S-180 cell membrane which interacted with polysaccharide was captured by biotinylated polysaccharide,concentrated by streptavidin-magnetic beads and analyzed with proteomics by mass spectrum to be Elongation factor 1-alpha 1?eEF1a1?.Western blot analysis revealed that polysaccharide inhibited the expression of eEF1a1 in S-180 cells.It could be confirmed that polysaccharide interacted with eEF1a1 on S-180 cells and inhibited the synthesis of proteins associated with cell proliferation.Meanwhile,polysaccharide retarded the proliferation of tumor cells by inhibiting the expression of eEF1a1.The anti-colitis/colorectal cancer bioactivity of polysaccharide was studied with LPS-stimulated RAW 264.7 cells and DSS-induced colitis in mice.Polysaccharide inhibited LPS-induced inflammatory factor NO secretion through down-regulating the expression of iNOS mRNA and protein,associated with the MAPK and PI3K/Akt signaling pathway.Oral polysaccharide inhibited LPS-induced acute inflammation as well as DSS/AOM induced colitis and colorectal cancer in mice.Polysaccharide significantly alleviated the body weight loss and colon length decrease in mice with colitis and colorectal cancer,restored the severe injuries caused by inflammation and reduced the number and size of colon tumors.In this thesis,a systematic study on the antitumor effect of Lentinus edodes polysaccharide in vivo was carried out.The immunomodulation and interaction with tumor cells of polysaccharide were explored by different methods of administration.It was confirmed that the antitumor effect of polysaccharide came from its regulation of immune cells and direct inhibition and killing of tumor cells.This thesis not only provides new strategies for the treatment of inflammation and cancer,but also comes up with a new scientific basis for understanding the antitumor effect of polysaccharides,showing important academic value and application prospects.