Multi-omics Studies Revealed The Involvement Of Th1 Cell Differentiation In The Anti-tumor Effect Of Purified Polysaccharide From Sanghuangporus Vaninii In Colorectal Cancer

Colorectal cancer(CRC),one of the most common malignancies in the world,ranks third in terms of new cases and deaths.In 2021,approximately 149,500 new cases of CRC were investigated,with 52,980 deaths,accounting for approximately 9%of all deaths in the United States.Abnormalities in the abundance of the gut microbiota are considered to be an important etiological factor in the development and progression of CRC,and along with the widespread use of gene sequencing technologies on gut microbiota,an increasing number of bacteria have been identified to be positively associated with the incidence of CRC.Dysbiosis is the cause of the body's reduced ability to fight tumors,especially when probiotics are reduced;as a result,tumor cells have an increased chance of escaping the body's immune system surveillance.Therefore,the gut microbiota may serve as a new target for CRC treatment.The localization and function of immune cells in the colorectal tumor microenvironment is complex and diverse and exhibits profound effects on the clinical outcome of CRC patients.CD8⁺cytotoxic T cells can break down established tumors,and T-helper(Th)1 cell-mediated responses in CRC predict longer survival cycles for patients.Most of the carbohydrates in nature exist in the form of polysaccharides,which are gaining increasing attention as dietary supplements to regulate the health of the organism.Among them,mushroom polysaccharides have been found to have great potential in different fields such as molecular biology,immunology,biotechnology and medicinal chemistry.Sanghuangporus,a traditional medicinal mushroom for more than 2,000 years in Asian countries,has been used as a traditional medicine to consolidate hemostatic channels,relieve abdominal pain and treat diarrhea.Sanghuangporus vaninii has been successfully domesticated and cultivated by Chinese scientists,which belongs to Sanghuangporus.Water-soluble S.vaninii polysaccharides(SVP)has been shown to a regulatory effect on the development of breast cancer,non-small cell lung cancer and Melanoma.However,these studies have been conducted in cells,and so far,the anti-tumor effects and underlying mechanisms of the polysaccharides purified from S.vaninii have not been well exploited in animal models,especially for CRC.In this study,SVPs was isolated and purified,and their physicochemical properties and structural characteristics were analyzed.Besides,the main objective was to determine the antitumor effect of SVP on CRC and the underling mechanisms related to intestinal flora regulation and immunomodulation.It provides a new idea to study the anti-CRC activity of natural polysaccharides as dietary supplements.In this study,the extraction conditions of SVP were first optimized by combining single-factor experiment and Box-Behnken experimental design,and then the optimal extraction conditions were as follows.The water extraction temperature was 80?,the extraction time was 2 h,and the material-to-liquid ratio was 1:20.The polysaccharides were screened for their anti-CRC activity by assessing their effect on cell viability,and resulting in the purified polysaccharide from S.vaninii(SVP-A-1).The ultraviolet spectrum detection and analysis showed that SVP-A-1 did not contain nucleic acid and protein.Its molecular weight is uniformly distributed at approximately 22.5 k Da and consists mainly of six monosaccharides with the following molar mass ratios:fucose(Fuc):galactose(Gal):glucose(Glc):mannose(Man):fructose(Fru):glucuronide(Glc UA)=24.473:42.942:11.892:19.988:0.637:0.068.The infrared spectrum detection and analysis of SVP-A-1 revealed the existence of the characteristic peak of saccharides,absorption peaks for the stretching vibration of-OH,and also for C-H telescopic vibration,C=O telescopic vibration and uronic acid.Methylation analysis revealed that 6-Gal(p)and 2,6-Gal(p)were the most abundant glycosidic bond residues in SVP-A-1.Finally,by nuclear magnetic resonance(NMR)analysis of SVP-A-1,the glycosidic bond signals were attributed to determine the composition of the major glycosidic bonds.The anti-CRC activity of SVP-A-1 was then investigated in vitro using two CRC cell lines(sw480 and Caco-2 cells).SVP-A-1 induces the excessive accumulation of reactive oxygen species(ROS)in CRC cells and mitochondrial oxidative damage,thereby increasing mitochondrial membrane permeability.The release of cytochrome c from the mitochondria is followed by a cascade of Caspase family factors that lead to apoptosis.As a positive feedback regulation,the pro-apoptotic proteins such as Bad,Bid and Bax located in cytoplasm would be transferred to mitochondria following Caspase 8stimulation,promoting the release of cytochrome c,while the level of anti-apoptotic proteins in CRC cells was significantly down-regulated after SVP-A-1 treatment.These results indicate that SVP-A-1 can achieve its anti-CRC activity by mediating mitochondrial apoptosis.Subsequently,the anti-CRC activity of SVP-A-1 was investigated at in vivo using Apc^Min/+mice,where SVP-A-1 reduced the number of tumors and inhibited tumor volume expansion in the colorectum of mice.Moreover,IHC analysis showed that SVP-A-1promoted the number of CD4⁺and CD8⁺T lymphocytes in the spleen and tumor tissues of Apc^Min/+mice,predicting that SVP-A-1 achieves anti-tumor effects by mediating the immunoreaction of T lymphocytes.The multi-omics data obtained were integrated and analyzed to explore the mechanism of the anti-colorectal cancer activity of SVP-A-1 in Apc^Min/+mice by analyzing the gut microbiota structure of mouse cecum contents,metabolites in serum,and protein analysis of tumor tissues.Microbial 16S r RNA gene sequencing in the gut showed that SVP-A-1 upregulated the mean abundance of 10 microorganisms and downregulated the mean abundance of 10 microorganisms at the genus level,which,when combined with functional potential analysis,suggested that significantly different microorganisms were mainly involved in immune system regulation,L-arginine biosynthesis,TCA cycle,and glycolysis pathways.Metabolomic assays of metabolites in serum revealed altered levels of 13 metabolites,with metabolites suggesting significant differences by KEGG annotation mainly involving the L-arginine biosynthesis pathway.Proteomics analysis of tumor tissues screened 36 differentially expressed proteins.GO enrichment and KEGG pathway analysis revealed that the differentially expressed proteins were mainly involved in metabolic processes,immune processes,angiogenesis,endogenous peptide antigen via MHC class I,activation of T cell proliferation,PI3K-Akt signaling pathway,and arginine biosynthesis pathways.Combined multi-omics analysis by establishing a microbial-protein-metabolism co-expression network revealed L-arginine biosynthesis as the linkage node between the groups.It was suggested that SVP-A-1 gavage treatment led to an improved intestinal microbial environment in Apc^Min/+mice and promoted the biosynthesis of L-arginine through the regulation of microbial metabolism,thus promoting the proliferation of activated T cells.Finally,cytokine assays and Western Blotting analysis were used to further investigate the mechanisms surrounding T cell activation and CD4⁺T cell differentiation on SVP-A-1 immunoreactivity.SVP-A-1 led to the activation of DCs;consequently,the activated DCs produce IL-12.IL-12,the main cytokine that drives the differentiation of na(?)ve CD4⁺T cells towards Th1 cells,regulates the Th1 expression profile(IL-2,IL-12,IFN-?and TNF-?),which in turn inhibits the differentiation in the direction of Th2 and Th17.Therefore,SVP-A-1 achieves the anti-tumor effect of inhibiting the development of tumor by regulating immunity,which provides a theoretical basis for the development of polysaccharide of S.vaninii as a dietary supplement against CRC.