| According to "A Cancer Journal.for Clinicians",there will be an estimated 18.1 million new cancer incidences and 9.6 million cancer deaths worldwide in 2018.Compared with other countries,China's cancer incidence and mortality are the world's highest.Among the 18.1 million new cancer cases,China accounted for 3.804 million;of the 9.6 million cancer deaths,China accounted for 2.296 million.Although chemotherapy widely used internationally can effectively kill tumor cells,it also seriously damages the normal cells of the body.However,polysaccharide is a biologically active substance that can kill tumor cells and increase the body's immune function.Therefore,polysaccharide has become one of the hot topics in domestic and foreign research.Sorghum silk smut(Sporisorium reilianum)is a medicinal plant pathogenic fungus with a variety of biological activities.Its active ingredients such as melanin and polysaccharides have also been shown to have anti-tumor,anti-viral,anti-radiation,anti-oxidation,and anti-infective effects.In this study,neutral polysaccharide fractions(WM-N)and acid polysaccharide fractions(WM-A)were isolated and purified from Sporisorium reilianum fruit bodies.However,the acid polysaccharide fraction yield and sugar content are extremely low and difficult to purify.In this paper,the structure and biological activity of neutral polysaccharide of homogeneity fraction was studied,including the following aspects:(1)In this study,crude polysaccharides(WM)were extracted from sorghum silk smut fruit bodies by water extraction and alcohol precipitation,and obtained after separation and purification by DEAE-cellulose ion exchange column chromatography,membrane separation,and alcohol precipitation purification.It is graded equally and named WM-NP-60.High performance gel permeation chromatography(HPGPC)was used to determine the molecular weight.Monosaccharide composition was analyzed by high performance liquid chromatography(HPLC).The molecular weight of WM-NP-60 was 15.6 kDa and consisted of Glc(93.3%)and Gal(6.7%).UV spectrum showed no protein or nucleic acid in the polysaccharide fraction WM-NP-60.WM-NP-60 has a right optical rotation of+0.049.The structure of pyranose was demonstrated by FT-IR analysis.The characterization of polysaccharide WM-NP-60 was analyzed by means of Periodate oxidation,Smith degradation,1D/2D NMR and Methylation.In our study,it was speculated that the main chain of WM-NP-60 was composed of ?-1,6-D-Glcp and its side chains were ?-1,3-D-Glcp.The side chains bound to the main chain with glycosyl groups at the C-3 positions.Gal might be attached to the backbone as a side chain or bound to the linear ?-1,3-D-Glcp side chain.(2)Antioxidant effect of polysaccharide WM-NP-60 shows that polysaccharide WM-NP-60 has significant scavenging effects on DPPH radicals,hydroxyl radicals(·OH),and superoxide anions(O2-·),Hydrogen peroxide(H2O2),the ability to chelate Fe2+,and the ability to inhibit self-oxidation of 1,2,3-phentriol,as well as excellent reducing power,indicating that it has good antioxidant activity.(3)The polysaccharide fraction WM-NP-60 can inhibit the proliferation of HepG2,SGC7901 and HCT116 cells by MTT assay,showing time and concentration dependence,and the most significant inhibition effect on HCT116 cells.The polysaccharide fraction WM-NP-60 can block HepG2,SGC7901 and HCT116 cell cycle in G1 phase,thereby inhibiting tumor cell proliferation and inducing apoptosis.An important feature of apoptosis in phosphatidylserine exposure is conveniently and directly detected by fluorescence microscopy.The morphological characteristics of HepG2,SGC7901 and HCT116 cells are observed by transmission electron microscopy(TEM).The typical apoptotic bodies were found by TEM.(4)All the proteins were identified in WM-NP-60-treated HCT116 by TMT and 2D-LC-MSMS and 369 proteins were identified as differentially expressed proteins(DEPs),the expressions of 240 proteins were up-regulated,and the expressions of 129 proteins were down-regulated.The corresponding bioinformatics analysis was presented,and it was found that KEGG pathway was enriched to 15 significantly different metabolic pathways(P<0.05).The focal adhesion pathway and TGF-? signaling pathway were related to cell cycle and apoptosis.PPI analysis showed that six differentially expressed proteins were on key nodes.qRT-PCR and Western blot experiments found that the polysaccharide WM-NP-60 up-regulates the expression of TGF?R1,p107,and DP1,and down-regulated THBS1 in the TGF-? signaling pathway,thereby WM-NP-60 can block the cell cycle in G1 phase and inhibit the proliferation of HCT116 cells and induce apoptosis in HCT116 cells.In addition,the polysaccharide WM-NP-60 can up-regulate ITGA7 and Rap1 genes in the Focal adhesion signaling pathway,thereby WM-NP-60 may block the cell cycle in G1 phase.In this study,a homogenized polysaccharide WM-NP-60 was obtained from sorghum silk smut fruiting bodies by hot water extraction and ethanol precipitation.The structure of WM-NP-60 was deduced by analyzing the structure characteristics of polysaccharide WM-NP-60.The antioxidant effect of polysaccharide WM-NP-60 and its inhibition on HepG2,SGC7901 and HCT116 cells were demonstrated and apoptosis was induced.The differentially expressed proteins were identified by TMT and 2D-LC-MSMS analysis,and the anti-tumor molecular mechanism of polysaccharide WM-NP-60 was speculated by cell cycle,apoptosis,bioinformatics and related protein verification experiments. |
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