Anti-colon Cancer Effect Of Curcumae Rhizome And Sparganii Rhizoma Based On Internet Pharmacology And Experimental Study On Normalization Of Tumor Vessel

Objetives:In the research,we explored the mechanism of RCRS in the treatment of colon cancer through network pharmacology.The effects of RC on tumor blood vessels and the effect of RC on colon cancer tumors were studied through in vivo and in vitro experiments.This experiment hopes to provide experimental evidence for the treatment of tumors with TCM for promoting blood circulation and removing blood stasis.Methods:Taking RCRS as the research object and colon cancer as the disease object,using the method of network pharmacology to explore the main active components of RCRS,the potential targets of the treatment of colon cancer and the biological effects of drugs in the disease process.In the in vitro experiment,the HUVEC cell experiment was divided into Control group,HUVEC 50 group and HUVEC 150 group.The HCT116 cell experiment was divided into Control group,HCT116 20 group and HCT116 50 group.The colon cancer cell co-culture group experiment was divided into Co-culture Control group,Co-culture 20 group,Co-culture 50 group,and Co-culture 150 group.The effects of drugs on HUVEC cells,HCT116 cells and co-cultured HUVEC cells were detected by CCK-8,Ed U cell proliferation staining experiments,q PCR method,and WB method.In vivo experiments were conducted to explore and establish a nude mouse model of colon cancer.Nude mice were inoculated subcutaneously with 1.5*106 cells.After tumor formation,20 nude mice were randomly divided into 4 groups:the model group,the oxaliplatin group,the zedoary turmeric oil group,and the zedoary volatile oil-oxaliplatin combination group.The body weight and tumor volume were recorded every three days,and the tumor weight was weighed after the nude mice were sacrificed.Immunofluorescence method was used to detect tumor vascular density（CD31）,cadherin positive rate（D31-VEcadherin）,and pericyte coverage（CD31-PDGFR）.Results:When zedoary turmeric oil interferes with cells,normal HUVEC cells have a greater tolerance to turmeric oil,IC₅₀=950mg/l,and disease model colon cancer cell HCT116 has a lower tolerance to zedoary turmeric oil,IC₅₀=678.5mg/l.Subsequent experiments selected different intervention concentrations according to the tolerance of different cells to zedoary turmeric oil:HUVEC cells have a low concentration of 50 mg/L and a medium concentration of 150 mg/L;HCT116 cells have a low concentration of 20 mg/L and a medium concentration of 50 mg/L.The Ed U cell proliferation experiment proved that compared with the blank group,the intervention of zedoary turmeric oil（50）can significantly reduce the proliferation of HUVEC cells.Zedoary turmeric oil（50,150）can significantly reduce the proliferation of HCT116 cells.The results of q PCR experiments showed that zedoary turmeric oil（50）can reduce the expression of VEGF RNA in HUVEC cells cultured alone.HUVEC cells co-cultured with colon cancer have poor cell adhesion,elongated antennae,and have a different morphology than HUVEC cells cultured alone.The q PCR experiment found that the expression of VEGFa in HUVEC cells under the disease model was significantly higher than that in HUVEC cells cultured alone.The intervention of zedoary turmeric oil（50mg/L and 150mg/L）can to some extent reverse the rapid increase in HUVEC cell VEGFa RNA expression when co-cultured with HCT116.WB experiment results showed that in HUVEC cells cultured separately,treatment with zedoary turmeric oil（50mg/L and 150mg/L）can significantly reduce the expression of VEGFa protein in HUVEC cells.When co-cultured with HCT116,treatment with zedoary turmeric oil（50mg/L and 150mg/L）can also significantly reduce the expression of VEGFa protein in co-cultured HUVEC cells.In in vivo experiments,nude mice were inoculated with different concentrations of HCT116 cells subcutaneously,and within 20 days of the observation period,the amount of cells inoculated in the low concentration group was not enough to make HCT116 cells tumorigenic under the skin.The cell inoculation amount of the high concentration group caused the subcutaneous tumors of nude mice to grow too fast,and the difference within the group was large;the tumor growth rate of the nude mice inoculated with the cell inoculation amount of the medium concentration group was relatively slow and the size was relatively uniform.Choose 1.5*106 cells as the inoculum used in the experiment for further experiments.The results of the weight change of nude mice showed that within 15 days after subcutaneous tumor implantation,the weight of nude mice showed an increasing trend,and after the 15th day,the weight of nude mice in each group showed a downward trend.After the 15th day,the tumor volume of each group was different.Compared with the model group,the tumor volume growth in the RC group and the RC-oxaliplatin combination group was significantly slower.The results of CD31 labeling showed that the number of microangiogenesis in the model group was higher.The number of tumor microangiogenesis in the RC group,RC-oxaliplatin combination group,and oxaliplatin group all decreased to varying degrees,and the reduction in the RC-oxaliplatin combination group was the most obvious.The results of immunofluorescence showed that the pericyte coverage of tumor blood vessels in the model group was low.In the RC group,RC-oxaliplatin combination group,and oxaliplatin group,the coverage of tumor perivascular cells all increased to varying degrees.The cadherin coverage of tumor blood vessels in the model group was lower.The RC group,RC-oxaliplatin combination group,and oxaliplatin group had different degrees of increase in tumor vascular cadherin coverage.Conlusion:Curcumae rhizome has a certain inhibitory effect on the proliferation of HUVEC cells and HCT116 cells,and the inhibitory effect increases with the increase of concentration.Curcumae rhizome can inhibit the synthesis of VEGF RNA and protein expression of HUVEC cells,and can also reverse the over-synthesis of VEGFa m RNA and protein over-expression of HUVEC cells in a co-culture system under pathological conditions.The combination of Curcumae rhizome and oxaliplatin can inhibit tumor growth,reduce tumor angiogenesis,promote the expression of vascular cadherin,increase the coverage of endothelial pericytes,and structurally normalize tumor blood vessels.