Herbal Polysaccharide Mixture Inhibits Colon Cancer By Regulating Phenotypic Polarization Of Tumor Associated Macrophages

ObjectivesThis subject studies the effect of anti-colon cancer of herbal polysaccharide mixture(Saccharomyces cerevisiae cell wall polysaccharide and houttuynia cordata water extract)by regulating the polarization of tumor-associated macrophages from M2 to M1,which provides experiental basis for its development as a drug for the treatment or adjuvant treatment of tumors.Method1.MTT method to measure cell proliferationDifferent concentrations(10~640 μg/m L)of herbal polysaccharide mixture were used to treat CT26 colon cancer cells and RAW264.7 macrophages for 24 h;and at different target ratios(RAW264.7 cells: CT26 cells),CT26 colon cancer cells,RAW264.7 macrophages were co-cultured for 24 hours to screen the most suitable efficiency-to-target ratio;under the best efficiency-to-target ratio,RAW264.7 macrophages were stimulated by herbal polysaccharide mixtures of different concentrations(10~640 μg/m L),After that,they were co-cultured with CT26 cells for 24 hours;Each group was added with MTT solution for 4 h,and then dimethyl sulfoxide was added.The absorbance value(OD value)of CT26 colon cancer cell proliferation,RAW264.7 macrophage proliferation,and cell proliferation of RAW264.7macrophage co-cultured with CT26 cells were measured at 490 nm.2.Study on the phenotype of tumor-associated macrophages in vitroThe culture supernatant of CT26 colon cancer cells cultured for 24 h was collected,Then RAW264.7 cells were cultured with 40% TCS for 48 h.The tumor associated macrophages(TAMs)model was established by observing the changes of cell morphology and detecting the fluorescence intensity of M1 markers CD80,i NOS and M2 markers CD206,Arg1 in RAW264.7 cells by immunofluorescence.Then the control group(normal DMEM medium),herbal polysaccharide mixture group(320 μg/ml),TCS group(DMEM medium containing40% TCS),TCS + herbal polysaccharide mixture group(40% TCS + 320 μg/ml)were set.The fluorescence intensity of M1 markers CD80,i NOS and M2 markers CD206,Arg1 were detected by immunofluorescence method,and the effect of herbal polysaccharide mixture on phenotype polarization of TCS cultured macrophages was determined.3.Establishment of transplanted tumor mouse model and study of tumor associated macrophage phenotypeTake CT26 cells in the logarithmic growth phase with a density of 5×106cells/m L,and inoculate 0.2 m L/cell in the right armpit.Observe the changes in the armpit every day.After about a week,a soy-sized mass is found,indicating success CT26 xenograft tumor models were established and divided into model group(normal saline),herbal polysaccharide mixture low(20 mg/kg),medium(40 mg/kg),high(80 mg/kg)dose group,and positive control group(5-Fu: 20 mg/kg),combined medication group(herbal polysaccharide mixture(40 mg/kg)+5-Fu(20 mg/kg)).And a blank control group was set up,without tumors,intragastric normal saline.After 2 weeks of administration,the tumor,spleen,and thymus tissues were taken to determine the tumor inhibition rate,spleen index and thymus index.Paraffin sections of the tumor tissues were observed by HE staining.Immunofluorescence method to detect the fluorescence intensity of M1 markers CD80,i NOS and M2 markers CD206,Arg1 in tumor tissues.Western blotting was used to detect the protein expression of M1 markers CD80,i NOS and M2 markers CD206,Arg1 in tumor tissues.Result1.The effect of herbal polysaccharide mixture on CT26 colon cancer cells through RAW264.7 macrophages(1)After the herbal polysaccharide mixture in the concentration range of 10~640 μg/m L was applied to CT26 cells and RAW264.7 macrophages,the OD value was measured by MTT method,and the difference was not statistically significant(P>0.05),and no cytotoxicity.(2)Herbal polysaccharide mixtures of different concentrations(10~640 μg/m L)stimulated RAW264.7 macrophages and co-cultured with CT26 cells.The results showed that the inhibition rate of macrophages on CT26 cells was significantly increased(P<0.05),combined with 5-Fu inhibited the proliferation of CT26 cells significantly(P<0.01),suggesting that herbal polysaccharide mixture can activate RAW264.7 macrophages to inhibit the proliferation of CT26 colon cancer cells,and combined with 5-Fu has a better effect on inhibiting the proliferation of CT26 cells.2.Herbal polysaccharide mixture(320 μg/m L)regulates the phenotype of tumor-associated macrophages(1)TCS culture conditions induce macrophages to polarize to the M2 phenotypeThe morphology of macrophages cultured in TCS changed from round to irregular with protruding pseudopods,showing a slender spindle shape.The immunofluorescence results showed that the fluorescence intensity of the M2 markers CD206 and Arg1 in the TCS group increased significantly,and the fluorescence intensity of the M1 markers CD80 and i NOS did not change significantly,compared with the control group.The result prompting that TCS mimics the tumor microenvironment to induce the polarization of macrophages into M2 type.(2)The effect of herbal polysaccharide mixture on the phenotypic polarization of macrophages under TCS culture conditionsThe immunofluorescence results showed that the fluorescence intensity of M1 markers CD80,i NOS and M2 markers CD206,Arg1 of the control group RAW264.7 cells were very weak.Compared with the control group,The fluorescence intensity of i NOS and CD80 in the herbal polysaccharide mixture(320 μg/m L)group was significantly enhanced,and CD206 and Arg1 had no obvious changes.There was no significant change in CD80 and i NOS in the TCS group,and CD206 and Arg1 were significantly enhanced.Compared with the TCS group,the fluorescence intensity of CD80 and i NOS in the TCS + herbal polysaccharide mixture group(320 μg/m L)increased,and the fluorescence intensity of CD206 and Arg1 decreased.The results showed that TCS induced polarization of RAW264.7 cells into M2 type;herbal polysaccharide mixture(320 μg/m L)induced polarization of RAW264.7 cells to M1 type,and regulated M2 type macrophages induced by TCS to M1 type.3.Herbal polysaccharide mixture inhibits phenotypic polarization of tumor-related macrophages in vivo and exerts anti-tumor effects(1)The tumor suppressive effect of herbal polysaccharide mixture on CT26tumor-bearing miceThe tumor mass and volume of mice in the herbal polysaccharide mixture low,medium and high dose groups,positive control group,combined medication group decreased to different degrees(P<0.05,P<0.01)compared with the model group.(2)The effect of herbal polysaccharide mixture on the immune organ index of CT26tumor-bearing miceThe index of thymus and spleen in the positive control group was decreased(P<0.05),the index of thymus and spleen in mixture medium-dose group herbal polysaccharide was increased(P<0.05),the thymus index of the blank control group and the high-dose herbal polysaccharide mixture group increased significantly(P<0.01)compared with the model group.The index of spleen and thymus in the combination group were significantly increased(P<0.05)compared with the positive control group.Which show that the herbal polysaccharide mixture can improve the function of the body’s immune organs and enhance the body’s immunity,and combined with 5-Fu had better effect.(3)Observation of tumor histopathologyThe tumor cells in the model group are dense,large in number,disorderly arranged,large and deep stained nuclei,high nucleoplasm ratio,mitotic phenomena,and obvious cell atypia.The tissue cells of the herbal polysaccharide mixture low,medium,and high dose groups,positive control group,and combination medication group were loosely arranged,the intercellular substance increased significantly,and the multinucleated megakaryocytes decreased significantly compared with the model group.(4)Effect of herbal polysaccharide mixture on the immunofluorescence intensity of CD206,CD80,i NOS,Arg1 in tumor-associated macrophagesThe results showed that the fluorescence intensity of the M2 markers CD206 and Arg1 in the model group was strong,and the fluorescence intensity of the M1 markers CD80 and i NOS was weak,indicating that the macrophages of the model group were mostly polarized toward the M2 phenotype during tumor growth.The fluorescence intensity of CD206 and Arg1 in the low,medium and high dose groups of herbal polysaccharide mixture,positive control group,and combination medication group was significantly weakened,and the fluorescence intensity of CD80 and i NOS was significantly increased compared with the model group,and the difference was statistically significant(P<0.05,P<0.01).The results showed that the herbal polysaccharide mixture treatment groups inhibited the polarization of macrophages in tumor tissues to M2,and regulated the polarization of M2 to M1.(5)Effect of herbal polysaccharide mixture on the protein expression of CD206,CD80,i NOS,Arg1 in tumor-associated macrophagesThe results of WB showed that the expression of M2 marker CD206 and Arg1 protein in the herbal polysaccharide mixture low,medium and high dose groups,positive control group,combined medication group decreased to varying degrees compared with the model group,and the expression of M1 marker CD80 and i NOS protein increased to varying degrees.The difference was statistically significant(P<0.05,P<0.01).Results show that herbal polysaccharide mixture can inhibit the polarization of macrophages in tumor tissues to M2,and regulate the polarization of M2 to M1.Conclusions1.Herbal polysaccharide mixture cannot directly inhibit the proliferation of CT26 colon cancer cells in vitro,but can inhibit the proliferation of CT26 cells by activating macrophages.2.Herbal polysaccharide mixture can regulate the polarization of tumor-associated macrophages from M2 to M1 in vitro.3.Herbal polysaccharide mixture can inhibit the growth of transplanted tumors in CT26 colon cancer mice,increase the index of spleen and thymus,and inhibit the growth of colon cancer by regulating the polarization of tumor-associated macrophages in the body from M2 to M1.In summary,herbal polysaccharide mixture can regulate the phenotypic polarization of tumor-associated macrophages to inhibit the growth of colon cancer.