Antitumor Effect And Mechanism Of Polysaccharide From Lachnum Sp.

Previously,our research group have analyzed the structure of the exopolysaccharide from Lachnum YM130,and found the antitumor effect of LEP130.However,mechanism of the antitumor effect of LEP130 is still unclear.This paper studied the antitumor effect of LEP130 and its synergistic effect with chemotherapeutic drugs,and the possible mechanism was preliminarily explored.The main results were as follows:1)In vitro experiments were conducted to investigate the inhibitory effect of LEP130 combined with 5-fluorouracil(5-FU)on Hep G2 cells.The results of MTT experiment showed that combination treatment of LEP130 and 5-FU had a significant inhibitory effect on the activity of Hep G2 cells,which was stronger than that of LEP130 or 5-FU alone,and there was a significant synergistic effect between LEP130 and 5-FU.However,LEP130 showed no significant effect on human skin fibroblast HSF and human normal hepatocyte LO2,which indicated that LEP130 was safe and non-toxic to normal cells.Results of Western blot etc.showed that LEP130 reduced the expression of thymidylate synthetase and dihydropyrimidine dehydrogenase,thus inhibiting the degradation and deactivation of 5-FU.In addition,LEP130 inhibited Ras/Raf/MEK/ERK and PI3K/Akt/m TOR mediated cell survival pathway,enhanced the sensitivity of Hep G2 cells to 5-FU,and prevented drug resistance generation.Moreover,LEP130 inhibited Nrf2 mediated antioxidant defense system,increased the production of ROS,destroyed mitochondrial membrane potential and activated mitochondrial apoptosis pathway.What's more,LEP130 activated p53,suppressed the expression of NF-?B and its downstream target protein,and led to cell cycle arrest in S phase.It was also found that LEP130 inhibited DNA base excision repair and mismatch repair,and thereby enhanced the effect of 5-FU.Therefore,LEP130 and 5-FU exerted a synergistic effect on inhibiting Hep G2 cells in vitro in various ways.2)The inhibitory effect of LEP130 and cyclophosphamide(CTX)on H22 tumor growth was studied in H22 tumor-bearing mice.The results showed that LEP130 activated Fas/Fas L mediated death receptor pathway,increased the expression of related proteins(such as caspase 3,caspase 8,PARP1,etc.),and activated mitochondrial apoptosis pathway through t-Bid.LEP130 also reduced angiogenesis by inhibiting the expression of angiogenesis related proteins(such as VEGF,b FGF,MMPs,etc.).In addition,LEP130 improved the spleen index and thymus index of H22 tumor-bearing mice,promoted the infiltration of T cells,NK cells and macrophages into tumor tissues,increased the content of immune factors(IFN-?,IL-1?,IL-2 and TNF-?)in serum and tumor tissues,and then activated the anti-tumor immune system to inhibit tumor growth.In addition,it was found that LEP130 inhibited the expression of Stat3 and NF-?B-mediated cell survival proteins(such as Bcl-2,Bcl-XL,survivin,etc.)and prevented the multidrug resistance of H22 tumor cells,thereby enhancing the antitumor effect of CTX.Finally,LEP130 significantly improved liver injury,kidney injury,immunosuppression and other side effects caused by CTX,and promoted the recovery of health.The above results indicated that LEP130 combined with CTX can synergistically inhibit the tumor growth,and LEP130 can alleviate the side effects caused by CTX.3)Through S180 tumor-bearing mouse model,the effect of LEP130 on anti-tumor immune system was studied.In vitro experiments showed that LEP130 could not inhibit the proliferation of S180 cells,while in vivo experiments confirmed that LEP130 significantly inhibited the tumor growth of S180 tumor-bearing mice.The results showed that LEP130 exerted antitumor effect mainly by activating the body's immune system.Gastrointestinal treatment of S180 tumor-bearing mice found that LEP130 induced helper T cells to polarize to Th1 type,and promoted the expression of Th1 cytokines(IL-2 and IFN-?);inhibited helper T cells to Th2 type polarization,and inhibited the expression of Th2 cytokines(IL-4 and IL-10).In addition,the high level of IFN-? caused by LEP130 has been shown to regulate macrophage typing in tumor microenvironment,promoted macrophage polarization to M1 type,and then exerted a tumor suppressive effect.In addition,LEP130 promoted tumor suppression immune cells(such as leukocytes,B cells,NK cells,etc.)and tumor suppressive immunochemokines(such as CXCL9,CXCL10,CXCL13,etc.),inhibited immunosuppressive cells(such as myelogenous suppressive cells,regulatory T cells,etc.),immunosuppressive cell chemokines(such as G-CSF,M-CSF,CCL22,etc.)and immunosuppressive factors(such as TGF-?,IL-10,PEG2,etc.)to improve immunosuppressive tumor microenvironment and enhance the killing effect of the immune system on tumor cells.Finally,results of in vitro experiments showed that LEP130 could directly induce the transformation of primary macrophages to M1 type through TLR4-mediated NF-?B signaling pathway to inhibit S180 cell growth.The above results indicated that LEP130 can activate the anti-tumor immune system to inhibit the growth of tumors in S180 tumor-bearing mice.4)AOM/DSS-induced mouse colon cancer model was used to study the inhibitory effect of LEP130 on colon cancer and its effect on intestinal microbiota.LEP130 significantly alleviated the weight loss and colon length reduction of colon cancer mice caused by AOM/DSS,and effectively inhibited the occurrence of colon tumors.In addition,LEP130 significantly improved the intestinal pathology of colon cancer mice,relieved intestinal damage caused by AOM/DSS,and restored the integrity of the intestinal barrier by increasing the expression of proteins such as ZO-1,occludin,and E-cadherin.LEP130 can also reduce pro-inflammatory factors(TNF-?,IL-6,IL-1?,and IFN-?)in colon tissue of colon cancer mice and inhibit tissue inflammation.Results of bacterial 16 S r RNA high-throughput sequencing technology showed that LEP130 improved the structure and composition of gut microbiota of colon cancer mice,increased microbial abundance and species diversity.LPE130 reduced the relative abundance of(opportunity)pathogenic bacteria and pathogenic bacteria(such as Parabacteroides,Escherichia?Shigella,Desulfovibrio,Helicobacter,Lachnospiraceae?NK4A136?group and Anaeroplasma)and increase the relative abundance of short-chain fatty acid-producing bacteria and probiotics(such as Ruminiclostridium,Lachnospiraceae?incertae?sedis,Prevotellaceae?NK3B31?group,Bifidobacterium and Roseburia,etc.).Results of non-targeted metabolomics experiments showed that LEP130 significantly improved the metabolism of gut microbiota of colon cancer mice,reduced the content of chenodeoxycholic acid,deoxycholic acid and lithocholic acid in the intestine,and increased short-chain fatty acids(acetic acid,propionic acid,n-butyric acid)and ?-tocopherol content,thereby inhibiting the occurrence of colon cancer.In addition,correlation analysis results showed that the regulation of gut microbiota was highly correlated with the changes in metabolites.The above results indicated that LEP130 inhibited the occurrence of colon cancer,and this inhibitory effect may be related to the regulation of the intestinal microbiota and its metabolites.