Study On The Anti-colon Cancer Mechanism Of Total Flavonoids Of Tetrastigma Hemsleyanum

Objective To investigate the mechanism of total flavonoids of Tetrastigma hemsleyanum on the tumor growth in HCT-116 xenografts and the regulation on intestinal microflora and metabolites,so as to provide experimental basis for the clinical application of T.hemsleyanum.Methods 1.The components of total flavonoids of T.hemsleyanum were analyzed by UPLCESI-QTOF-MS and HPLC.2.The cell viability of HCT-116 and HT-29 cells were determined by CCK-8 assay.3.The proliferation ability of colon cancer cell HCT-116 was determined by colony formation assay.4.Flow cytometry was used to analyze the apoptosis of colon cancer cell HCT-116.5.Western blot and qRT-PCR were used to detect the protein and gene expression levels of PI3K/AKT/mTOR signaling pathway.6.HCT-116 xenograft nude mice model was established to detect the effect of total flavonoids of T.hemsleyanum on colon cancer progression in vivo.7.Molecular docking was performed to confirm the interaction between the active components of the total flavonoids of T.hemsleyanum with PI3K and mTOR proteins.8.Intestinal microbiota community was analyzed by 16S rRNA sequencing.9.Fecal metabolites was analyzed based on untargeted LC-MS metabolomics.Results 1.Total flavonoids of T.hemsleyanum could inhibit the growth of HCT-116 and HT-29 cells in a dose-dependent manner with IC50 value of 105.60 μg/mL and 140.80μg/mL,respectively.2.Total flavonoids of T.hemsleyanum could inhibit the colony formation of HCT116 cells and induce cell apoptosis.3.Intragastric administration of total flavonoids of T.hemsleyanum delayed tumor growth in HCT-116 xenograft mice,but had no effect on body weight,organ pathology,organ ratio and blood routine levels.4.Total flavonoids of T.hemsleyanum inhibited the expression of mTOR,PI3K,AKT,and p-AKT in vitro and in vivo,and decreased the mRNA levels of PIK3CA,AKT and mTOR.5.Total flavonoids of T.hemsleyanum reversed the activation of p-PI3K,p-Akt induced by 740Y-P,and p-mTOR induced by MHY1485.6.The eight active compounds in the total flavonoids of T.hemsleyanum closely bind to the active amino acid sites of PI3K and mTOR proteins.7.Total flavonoids of T.hemsleyanum maintained the homeostasis of the intestinal microbial community in tumor-bearing nude mice,and increased the abundance of probiotic o_Bifidobacteriales,f_Bifidobacteriaceae,g_Bifidobacterium and s_Bifidobacterium_pseudolongum.8.Total flavonoids of T.hemsleyanum decreased "harmful" bacteria p_Bacteroidota,p_Firmicutesand g_Alistipes richness.9.Total flavonoids of T.hemsleyanum restored the levels of fecal metabolites including 23-trans-p-coumaroyloxytormentic acid,4-hydroxy-3-(16-methylheptadecyl)-2Hpyran-2-one and MG(22:4(7Z,10Z,13Z,16Z)/0:0/0:0),8-hydroxy-14,16-hentriacontanedione and MG(0:0/20:2(11Z,14Z)/0:0).10.The KEGG pathway was enriched in tryptophan metabolism,vitamin B6 metabolism,linoleic acid metabolism and secondary bile acid biosynthesis after total flavonoids of T.hemsleyanum treatment.11.There was a close correlation between gut microbiota,fecal metabolites,and tumor weight after total flavonoids of T.hemsleyanum treatment.Conclusion Total flavonoids of T.hemsleyanum delayed the development of colon cancer by inhibiting the PI3K/AKT/mTOR signaling pathway and modulated the homeostasis of microbial community structure and fecal metabolites.