Antitumor Effect And Intestinal Bacteria-mediated Mechanism Of Ginseng Polysaccharide Combined With Antibiotics

The anti-tumor effect of antibiotics is related to intestinal flora,and ginseng polysaccharides can regulate intestinal flora.We propose that ginseng polysaccharides combined with antibiotics may play a synergistic antitumor effect by regulating intestinal flora.In this study,by studying the effects of ginseng polysaccharides combined with antibiotics on tumor-bearing mice regarding tumor inhibition rate,intestinal flora,intestinal metabolites and related protein expression,and cytokine levels,the antitumor effect of ginseng polysaccharides combined with antibiotics and the mechanism mediated by intestinal bacteria were preliminarily elucidated.The study is divided into five parts:1.Preparation and quality characterization of ginseng polysaccharidesThe crude ginseng polysaccharide was prepared by water extraction and alcohol precipitation with a yield of 17.4%.The total polysaccharide content in the crude ginseng polysaccharide was 57.21%determined by the phenol sulfuric acid method,the protein content was 2.97%by the coomassie brilliant blue method,and the uronic acid content was 11.71%by m-hydroxybiphenyl method;At the same time,the molecular weight range of ginseng polysaccharides was characterized to be 1.00-33503.68 kDa by HPGPC-ELSD method,and the monosaccharide composition of ginseng polysaccharide was determined to be Man,GlcA,GalA,Rha,Glc,Gal and Ara in a molar ratio of 1.00:7.04:24.06:18.11:728.50:10.50:9.80 by PMP pre-column derivatization HPLC-UV method.2.Study on Antitumor effect of ginseng polysaccharides combined with antibioticsMC38 tumor-bearing C57BL/6 mouse colon cancer models were established by subcutaneously injecting MC38 tumor cells into the abdomen.The mice were divided into five groups:blank control group(Control),antibiotics group(ABX),ginseng polysaccharides combined with antibiotics low(ABX+low),medium(ABX+middle)and high(ABX+high)dose groups.The ginseng polysaccharides combined with antibiotics were evaluated by comparing tumor weight changes and tumor cell apoptosis(TUNEL method)in each group.The results showed that compared with the antibiotic group,ginseng polysaccharides combined with antibiotics treatment could more effectively inhibit the growth of colon cancer MC38 transplantation tumors in mice.The tumor inhibition rates of the antibiotic group and ginseng polysaccharides combined with antibiotics at low,medium and high dose groups were 40%,77%,92%,58%,respectively.And ginseng polysaccharides combined with antibiotics could more effectively induce apoptosis of colon cancer MC38 tumor cells in mice than the antibiotics.3.Effects of ginseng polysaccharides combined with antibiotics treatment on intestinal flora of tumor-bearing miceThe 16S rRNA RT-PCR sequencing technology was used to study the effect of ginseng polysaccharides and antibiotics on the intestinal flora of mice.The results showed that the dyversity of the intestinal flora of mice was significantly reduced after the treatment of antibiotics.However,ginseng polysaccharides could increase the diversity of flora,in particular improve the abundance of some flora,such as bacteroidales s24-7_group,Prevotellaceae UCG-001,etc..A total of 18 bacterial groups with significant changes in species abundance were screened out after treatment with antibiotics and ginseng polysaccharides combined antibiotics.Among them,the relative abundance of Providencia in the middle-dose group of ginseng polysaccharides combined with antibiotics was significantly decreased,and the relative abundances of Eschericia-Shigella,parasutterella,bacteroidales s24-7_group,Prevotellaceae_UCG-001,norank_f_Lachnospiraceae,Lactobacillus and Enterococcus increased.Together with the content change of the screened species,these species were initially divided into two categories that were further correlated with the antitumor effects.It was found that ginseng polysaccharides were associated with the abundance increasing of the bacteria(such as unclassified_f_Enterobacteriaceae,Enterococcus)that benefit to the antitumor effect,whereas the abundance decreasing of the antibiotics induced bacteria(such as Providencia)that have non-beneficial to the antitumor effects,indicating that the ginseng polysaccharides can increase the anticancer effects of antibiotics in two ways.4.Effects of ginseng polysaccharides combined with antibiotics treatment on medium and short-chain fatty acids and endogenous metabolites in tumor-bearing miceThe medium chain fatty acids and short chain fatty acids in mice feces and other endogenous metabolites in blood,urine and feces were analyzed respectively by using the pre-column derivatization UHPLC-QTOF-MS/MS technology and the UHPLC-QTOF-MS/MS based non-targeted metabolomics approach.The results showed that ginseng polysaccharides combined with antibiotics significantly increased the levels of pyruvate,succinic acid,and lactic acid in mice feces,and significantly changed the metabolic profile of serum,urine,and feces in mice.Biomarkers related to antitumor effect were selected and identified,including 28 in serum such as PC(16:1(9Z)/22:5(4Z,7Z,10Z,13Z,16Z)),PE(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/P-18:1(9Z)),25 in urine such as 5,6-Dihydroxyindole,L-Dopa and 15 in feces such as 7-Aminomethyl-7-carbaguanine,4a-Methylzymosterol.The related metabolic pathways mainly include lipid metabolism,primary bile acid metabolism,and tyrosine metabolism.5.Effects of ginseng polysaccharides combined with antibiotics treatment on the expression of related proteins and the level of immune inflammatory factors in tumor-bearing miceELISA,RT-PCR and immunohistochemical methods were used to measure the levels of cytokines in mouse peripheral blood,the relative expression of TLRs mRNA in tumor tissues,and the expression of NF-?? and TNF-? in tumor,spleen and colon tissues,respectively.The results showed that ginseng polysaccharides combined with antibiotics significantly increased the levels of immunoregulatory factors IFN-y,IL-17,and IL-10,and changed the expression of TLRs in tumor tissues.Among them,the relative expression of TLR2,TLR4 and TLR9 increased while that of TLR3,TLR5 and TLR7 decreased.At the same time,the expression of NF-? in spleen and colon increased while that in tumor decreased,exerting an antitumor effect.Therefore,it is speculated that ginseng polysaccharides combined with antibiotics exert the antitumor effect through regulating intestinal bacteria and intestinal metabolites,then activating the immune response and TLRs/NF-?? signaling pathway in tumor tissues,and eventually triggering the NF-??-mediated apoptosis.