Study On The Structural Characteristics And Antitumor Activity Of A Novel Polysaccharide Extracted From Rhizopus Nigricans Mediated Burdock Residue Fermentation

Cancer is one of the major diseases that threaten human life and health.At present,chemotherapy is still an important therapys of clinical cancer treatment,but chemotherapy drugs have poor selectivity,serious side effects and easy to produce drug resistance.Therefore,it is of great significance to search for antitumor drugs with remarkable efficacy and low side effects.Polysaccharide,which is one of the important sources of novel antitumor drugs,can play an anti-tumor role by regulating the immune function of the body and/or directly killing tumor cells.Rhizopus nigricans is a filamentous fungus of Rhizopus.It grows rapidly and is easy to culture,can be used for the transformation and extraction of plant polysaccharides.Burdock residue is a by-product of burdock root through inulin extraction,which is rich in cell wall components.In this study,a kind of polysaccharide(RBPS3)with high homogeneity was obtained from burdock residue fermented by Rhizopus nigricans,and its chemical structure was perlimainary analyzed.We also studied the effect of RBPS3 of the immunomodulatory effect in vitro,the attenuation and synergism effect on cyclophosphamide(CTX),and the anti-colon cancer effect.The main research findings are as follows:1.Isolation,purification.and structural characterization of polysaccharide from burdock dregs fermented by Rhizopus stolohiferThe crude polysaccharide(RBPS)was isolated from burdock residue fermented by Rhizopus nigricans,and further purified by anion exchange resin and sephadex resin.RBPS3 was obtained with a molecular weight of 1.41 × 105 Da,monosaccharide composition of GalA:Gal:Rha:Ara:GlcA:Glc:Xyl with the molecular ratio of 31.0:30.1:14.5:14.3:5.5:3.8:0.8.Methylation and NMR analysis indicated that the main chain of RBPS3 was composed of→2,4)-α-L-Rhap-(1→,→4)-α-D-GalAp-(1→and→4)-β-D-Galp-(1→,and formed the homogalacturonan(HG)domain and rhamnogalacturonan 1(RG-1)domain.Arabinan and Galactan are connected to the main chain by O2 of→2,4)-α-L-Rhap-(1→,O-3 of→-3,4)-β-DGalp-(1→,respectively.Atomic force electron microscopy(AFM)and transmission electron microscopy(TEM)showed that RBPS3 formed with multiple sugar chains,and scanning electron microscopy(SEM)showed that the ultrastructure of RBPS3 was banded.2.The immune regulatory activity in vitro and underlying mechanism of RBPS3RBPS3 has immune-enhancing activity in vitro.RBPS3 promoted the cell proliferation of RAW264.7 cell in a dose-dependent manner.Compared with Control,the cell proliferation rate of 100 μg/mL RBPS3 treatment was increased by 17.2%(p<0.01).Cell cycle and apoptosis analysis indicated that RBPS3 increased the proportion of G2/M phase,inhibited cell apoptosis.In the 100 μg/m L RBPS3 treatment,the G2/M phase ratio was increased by 1.7 times,and the apoptosis rate was decreased by 61.0%,compared with Control.RBPS3 enhanced phagocytoic activity,promoted ROS production,actived the secretion of NO and pro-inflammatory cytokines,and induced the polarization of M1-type macrophages.Compared with Control,100μg/mL RBPS3 increased the neutral red phagocytosis rate by 15.7%,and the contents of NO,TNF-α,IL-6 and IL-1β were increased by 5.36,5.41,5.04 and 7.13 times,respectively.Transcriptome analysis showed that 1520 differential genes(297 up-regulated genes and 1223 down-regulated genes)were generated after RAW264.7 cells were treated with 100 μg/mL RBPS3.The main enrichment immunoregulatory-related signaling pathways of differential genes include MAPK signaling pathway,Chemokine signaling pathway,Cytokine-cytokine receptor interaction and TLR signaling pathway.The experiment of colocalization,receptor blocking and Western blot verified that RBPS3 regulated the immune response of RAW264.7 cells mainly via TLR4 receptor recognition and activating the proteins phosphorylation ofM APK/NF-κB signaling pathway.The concentrations of NO and TNF-α induced by 100 μg/mL RBPS3 were decreased by 60.5%and 21.4%under TLR4 blocking,which were not significantly change under TLR2 and TLR4 co-blocking compared with TLR4 blocking.Compared with the Control,the expression levels of TLR4,MyD88,p-ERK,p-JNK,p-p38 and p-p65 of 100 μg/mL in 100 μg/mL RBPS3 treatment were increased by 1.87,5.09,13.0,1.72,1.53 and 16.2 times under,respectively.3.Attenuation and synergism of RBPS3 on Cyclophosphamide(CTX)Cyclophosphamide(CTX)is a broad-spectrum chemotherapeutic drug,and its side effects mainly include bone marrow suppression,immunosuppression and intestinal mucosal injury,etc.Intraperitoneal injection of CTX inhibits humoral immunity and cellular immunity,that is used to construct immunosuppressive animal models.Gavage administration of RBPS3 increased the contents of white blood cells(WBC),red bloqd cells(RBC),platelets(PLT)and lymphocytes(Lymph),and promoted the levels of the serum immunoglobulin(IgG and IgM)and cytokines(IL-2 and TNF-α)in CTX-induced mice.Compared with Model,the contents of these four kind of cells in 200 mg/kg RBPS3 treatment were increased by 2.03,1.28,1.47 and 1.80 times,respectively.Compared with Model,the level of IgG,IgM,IL-2 and TNF-α in 100 mg/kg RBPS3 treatment were increased by 48.7%,17.9%;90.4%and 31.0%,respectively.RBPS3 improved the spleen index and proliferation activity.of splenic lymphocytes.Compared with Model,spleen index,T lymphocyte and B lymphocyte.proliferation index of 100 mg/kg RBPS3 treatment were increased by 24.3%,28.3%and 12.3%respectively.RBPS3 also protected the intestinal mucosal barrier and intestinal immune function via regulating intestinal tight junction protein(ZO-1,C LDN5 and Occludin),autophagy associated protein(Atg5 and Atg7)and TLR4-mediated MAPK and NF-κB signaling pathway in CTX-induced mice.RBPS3 reduced the abundance of harmful bacteria such as Rikenellaceae and Marinifilaceae,increased the abundance of benefi cial bacteria such as Bacteroidaceae and Akkermansiaceae in CTX-induced mice.RBPS3 inhibited the tumoer growth of S180 tumor bearing mice,and enhance the antitumor effect of CTX on S180 sarcoma.Compared with Model,the tumor growth inhibitory rate in RBPS3 treatment,CTX treatment and drug combination treatment were 66.8%,69.9%,and 84.6%,respectively.RBPS3 significantly reduced the immunosuppressive effect of CTX on serum cytokines and immune organ index of S180 tumorbearing mice.4.The anti colon cancer effect of RBPS3Colorectal cancer is a type of malignant tumor of the digestive system with one of the fastest growing incidence rates.RBPS3 significantly inhibited the cell proliferation and migration,induced cell apoptosis through mitochondrial pathway of HT-29 cells.Compared with Control,the cell viability rate and migration rate of HT-29 cells decreased by 39.9%and 80.2%,cell apoptosis rate increased by 2.85 times in 1000 μg/mL RBPS3 treatment.Azomethane oxide(AOM)combined with sodium dextran sulfate(DSS)induced colitisassociated colorectal cancer(CAC)in mice..RBPS3 inhibited the occurrence and development of induced colorectal cancer.RBPS3 significantly increased colon length,decreased tumor number,inhibited abnormal proliferation of colorectal cells,and alleviated inflammatory cell infiltration in CAC mice.Compared with Model,the colon length increased 1.7 times and tumor numbers decreased from 10.6 to 2.2 in 150 mg/kg RBPS3 treatment.The protection of RBPS3 on colorectal function leaded to the decrease of fecal quality of CAC mice.RBPS3 decreased spleen index and contents of serum TNF-α,IL-6 and IL-1β,which was conducive to alleviating the occurrence and development of colon tumors in CAC mice.Compared with Model,the spleen index in 100 mg/kg RBPS3 treatment was decreased by 19.3%,and the contents of three cytokines in 150 mg/kg RBPS3 treatment were decreased by 39.6%,50.2%and 47.1%,respectively.In summary,a novel polysaccharide(RBPS3)obtained from burdock residue fermented by Rhizopus nigricans.RBPS3 prossesses the immunomodulatory activity,the attenuation and synergism on CTX and significant anti-colorectal cancer activity,providing the theoretical basis and scientific support for the development of antitumor polysaccharide for cancer therapy or adjuvant therapy,and has a wide application prospect.