| Background and Purpose:The incidence rate of Colorectal Carcinoma(CRC)is increasing.Evodiamine(Evo)is a natural alkaloid isolated from Evodiamine.Modern pharmacological studies show that evodiamine has good antitumor activity,anti osteoporosis,anti-inflammatory and other effects.Oxaliplatin(L-OHP),as the third generation of new platinum chemotherapeutic drugs,is widely used in the treatment of colon cancer.However,clinical observation shows that it has certain toxic and side effects and drug resistance,which is easy to have a negative impact on the prognosis of patients.In recent years,studies on the antitumor activity of monomers in traditional Chinese medicine have found that compared with western medicine,chemotherapy drugs have fewer adverse reactions,which can improve the body immunity and prolong the survival time of patients.Through the previous experimental research of the research group,it was found that the anti Zuojin Pill and its main component berberine have good anti-tumor activity.For further exploration,we searched for relevant pathways and research factors through enrichment analysis and data mining,and established a nude mouse model bearing tumor.We applied Evodiamine of different concentrations to study its anti-tumor effect on colon cancer and explore the possible mechanism of action,trying to find a more safe and effective chemotherapy drug,providing more objective experimental basis for the clinical application of evodiamine in the treatment of colon cancer.The purpose of this experiment is to observe the inhibitory effect of evodiamine on human colon cancer SW480 cells in nude mice,and its effects on cell cycle、β-Catenin and Cyclin D1,the results of enrichment analysis were used to explore the possible mechanism.Research Methods:Mining colon cancer related signaling pathways by functional enrichment analysis;Human colon cancer SW480 cells were routinely cultured and adjusted to a concentration of 5 before use×10~6pieces/ml of single cell suspension,according to0 2cell·ml-1 was inoculated subcutaneously under the right armpit of 5 nude mice.The growth of tumors was observed every day.When the diameter of the tumor was measured to be more than 1cm,the transplanted tumor samples without ulceration and in good condition were dissected and inoculated directly under the right armpit of the remaining nude mice until the growth volume of the tumor was more than100mm~3,and they were randomly divided into blank control group;positive control group(oxaliplatin group);evodiamine low dose group;evodiamine medium-dose group;evodiamine high-dose group,a total of 5 groups,6 in each group,were given continuously for 3 weeks.During this period,the mental status of tumor bearing nude mice was observed,and the tumor weight and body weight of nude mice were measured and recorded regularly.After the last administration,the blood was collected by removing the eyeball,the subcutaneous transplanted tumor was quickly stripped,the tumor inhibition rate was weighed and calculated,the liver and kidney function was tested by serum biochemical analyzer,the cell cycle percentage of tumor tissue was measured by flow cytometry,the pathological morphology of tumor tissue was observed by HE staining,and the expression ofβ-catenin and cyclin D1on tumor tissue was analyzed by immunohistochemistry.Results:1.Through the enrichment analysis of KEGG signal pathway,it was found that these colon cancer differential genes involve many signal pathways,among which the top 20 signal pathways include Wnt signaling pathway,TNF signaling pathway,NF kappa B signaling pathway,etc.Using C bio portal tool to analyze the genetic information of CNNB1 and CTNNB1,52(8.7%)of 594 samples collected in TCGA(pancancer Atlas)data set had mutations in these two genes2.The average tumor weight of each group was 1.41±0.83g respectively;0.67±0.16g;0.78±0.43g;0.75±0.52g;0.73±0.29g。Compared with the blank control group,the tumor weight of nude mice in oxaliplatin group,evodiamine low-dose group,evodiamine medium-dose group and evodiamine high-dose group was smaller and the difference was statistically significant(P<0.05).Among them,oxaliplatin group had the strongest tumor inhibition effect,and the tumor inhibition rate was52%;The second was evodiamine high-dose group,and the tumor inhibition rate was48%;Furthermore,in the medium-dose group of evodiamine,the tumor inhibition rate was 47%;The low-dose evodiamine group had the weakest inhibitory effect on tumor growth,with a tumor inhibition rate of 45%,but there was no significant difference among the three groups(P>0.05).Compared with oxaliplatin group,the mean tumor weight of evodiamine low-dose group,evodiamine medium-dose group and evodiamine high-dose group was significantly larger,but the difference was not statistically significant(P>0.05).3.There was no significant difference in liver and kidney function between the experimental drug intervention groups and the blank control group(P>0.05),and there was no significant difference between the experimental groups with different concentrations of evodiamine((P>0.05)).4.The results of flow cytometry showed that the percentages of low-dose evodiamine group(52.66±9.25%),medium-dose evodiamine group(63.76±9.21)%and high-dose evodiamine group(54.83±9.32)%in G0/G1 phase were higher than those in oxaliplatin group(49.72±10.10)%,but the difference was not statistically significant(P>0.05);Compared with the blank control group,the percentage of evodiamine medium dose group in G0/G1 phase was higher,and the percentage of evodiamine medium-dose group and evodiamine high dose group in G2/M phase was significantly higher,and the difference was statistically significant(P<0.05).5.Through the routine HE staining of tumor tissue,microscopic observation shows that the tumor cells in the blank control group are closely arranged and in irregular shape,with different sizes,mainly quasi circular,large nucleus,deep staining and obvious heteromorphism.In the intervention group of evodiamine administration,there were flake necrosis and degeneration of tumor tissue,nuclear pyknosis and disintegration of cells,and the image of nuclear division decreased.6.Through the comparative analysis of immunohistochemical staining results,it can be seen that in the blank control groupβ-Catenin and Cyclin D1 showed high expression levels,while the expression levels decreased in oxaliplatin group,evodiamine low-dose group,evodiamine medium dose group and evodiamine high-dose group;Compared with oxaliplatin group,evodiamine low-dose group,evodiamine medium-dose group and evodiamine high-dose group the expression levels ofβ-catenin and Cyclin D1 were high.Conclusion:Through KEGG signal pathway enrichment analysis,it was found that these colon cancer differential genes involved in Wnt signaling pathway.Using C bio portal tool,it was found that CCNB1 and CTNNB1 genes had a high mutation rate in colon cancer.Further experiments showed that evodiamine has obvious anti-tumor effect on nude mice bearing colon cancer SW480,and can inhibit the abnormal expression ofβ-catenin and Cyclin D1,blocked the cell cycle of colon cancer SW480 cells in G2/M phase or G0/G1 phase,and inhibited cell division and proliferation.Its mechanism may be through the regulation of Wnt/β-Catenin signal transduction pathway to play an anti-tumor role. |
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