| Colon cancer is a malignant tumor that occurs in the colon.The incidence and mortality of colon cancer in China are higher than the world average.The previous research of our research group has confirmed that the compatibility of Astragali Radix-Curcumae Rhizoma(AC)can inhibit the growth and metastasis of colon cancer by inducing apoptosis of cancer cells,promoting normalization of blood vessels,and regulating the function of the immune system.It has been found that the compatibility of the two drugs can affect the expression of chemokine receptor CXCR4 in the tumor tissue of colon cancer model mice.As researchers have studied the interaction between the gut microbiota and the host extensively,it has been found that the imbalance of the intestinal microecology caused by the disorder of the gut microbiota is an important factor in the occurrence and development of colon cancer.Gut microbiota and their metabolites can affect intestinal permeability and stimulate tumor cells to produce chemokines.The receptor CXCR4 of chemokine SDF-1 is expressed on the surface of colonic epithelial cells.In related study,after treating differentiated HT-29 colon cancer cells with sodium butyrate,the mRNA expression of CXCR4 was completely inhibited.Therefore,the paper proposed that AC may exert anti-tumor effect when entering the blood,and at the same time may affect the intestinal barrier function by improving the changes in the gut microbiota and its metabolites.The mechanism of action may be the activation of SDF-1/CXCR4/NF-κB pathway,so as to achieve the purpose of enhancing the inhibition of colon cancer growth.Due to the structure of different combinations of medicines,the efficacy of the medicines will change under different ratios.Therefore,the paper will first study the effect of different compatibility ratios of AC on colon cancer model mice,and the best anticolon cancer comprehensive effect is optimized.Mechanism research will be conducted after the comparison.This paper is divided into two parts to carry out related research work.1 Theoretical researchThis chapter summarizes the research progress of the anti-colon cancer and the relationship between the gut microbiota and colon cancer with the BuQi-HuoXue drug pair.At the same time,according to the previous research of our research group,the anti-tumor effect of the combination of AC was selected to carry out theoretical discussion,which provides the basis for the design of the paper.2 Experimental research2.1 Study on the intervention effect of different ratios of AC on colon cancer orthotopic transplantation tumor model mice2.1.1 Study on the anti-colon cancer effect of different ratios of ACThe method of surgical modeling was used to establish the BALB/c mice CT26.WT colon cancer orthotopic transplantation model.The multiple effect indicators combined with principal component analysis(PCA)was used to evaluate the intervention effect on colon cancer under different compatibility ratios of AC(1:0,3:1,2:1,3:2,1:1,2:3,1:2,1:3,0:1).The results showed that different ratios of AC inhibited tumor growth and liver metastasis to varying degrees.The ratio of AC at 1:1 had the best inhibitory effect on tumor growth,and the 2:1 group had the best effect on inhibiting tumor liver metastasis and improving weighed loss.AC significantly upregulated the expression of HBP1 in tumor tissues,and significantly down-regulated the expression of Ki67 and AFP;meanwhile,AC significantly up-regulated the expression of Ecadherin in liver tissues,and significantly reduced β-catenin,vimentin and p53 expression.The PCA results showed that the first three groups in the AC group that were closer to the sham operation group were 2:1,1:1,and 3:2 group.Among them,the center of the 2:1 group was the shortest from the sham operation group,indicating that the ratio 2:1 of AC had the best intervention effect on colon cancer in mice.2.1.2 Determination of the content of Astragali Radix-Curcumae Rhizoma in different proportionsA total of 14 main active components in Astragali Radix and Curcumae Rhizoma(Astragaloside Ⅰ,Astragaloside Ⅱ,Astragaloside Ⅳ,Formononetin,Ononin,Calycosin,calycosin7-O-beta-D-glucoside,β-elemene,Germacron,Curzerene,curedione,curcumin,demethoxycurcumin,Bisdemethoxycurcumin)were determined by UPLC-Qtrap-MS.The results showed that the content of each component of the two-flavor compatibility group was higher than that of the single-flavor drug group,indicating that AC decoction can increase the dissolution of the components.Among them,the contents of Calycosin,astragaloside Ⅳ,curcumin and βelemene,which have the clear anti-tumor activity in the 2:1 group,were higher than those in the other ratio groups.Combined with the above research results,the compatibility ratio of AC at 2:1 was selected for follow-up experimental research.2.2 The effect of AC optimal ratio on tumor growth and intestinal tract of colon cancer orthotopic transplantation tumor model miceOrthotopic transplantation mice model of colon cancer was used to study the effect of AC on intestinal tight junction protein,gut microbiota and flora metabolites(SCFAs).2.2.1 The effect of AC on tumor growth and intestinal mechanical barrier of colon cancer model miceThe results showed that different dose groups of AC had a certain inhibitory effect on the growth of colon cancer tumors in mice,and the middle dose group had a more obvious effect on reducing tumor volume.AC improved tumor and colonic histopathological changes to varying degrees.After colon cancer modelling,the expression of TJP(ZO-1,Occludin)protein in the intestine of mice was significantly reduced,indicating that the mechanical barrier of the intestine of mice with colon cancer was destroyed.After the intervention of AC,the expression of TJP in each group was equal significantly up-regulated,indicating that the AC can significantly improve the intestinal mechanical barrier tight junction protein of colon cancer model mice.2.2.2 The effect of AC on the gut microbiota of mice with colon cancer orthotopic transplantation tumor model16S rRNA technology was used to detect the structure of gut microbiota in the feces of each group of mice.At the phylum level,9 main phyla were detected.The number of Proteobacteria and Cyanobacteria in the gut microbiota of mice in the model group increased significantly,while Firmicutes and Bacteroidetss abundance decreased significantly.AC can adjust the relative abundance of the gut microbiota.The ratio of Firmicutes to Bacteroides in the low and mediumdose administration groups were significantly reduced,indicating that AC can improve the imbalanced gut microbiota structure.At the genus level,compared with the sham operation group,the relative abundance of Escherichia-Shigella,Streptococcus,and Enterococcus in the model group increased significantly,while the relative abundance of Lactobacillus,Prevotellaceae_UCG-001,Roseburia and Mucispirillum were significantly reduced.After the intervention of AC,the number of these bacterial genera was restored.It showed that the AC drug pair can balance the gut microbiota.2.2.3 The effect of AC on the SCFAs of the gut microbiota metabolites in mice with colon cancer orthotopic transplantation tumor modelGC-MS was used to measure the 7 SCFAs contents in the intestinal of each group of mice.The SCFAs content of the model group were significantly lower than that of the sham operation group.Except for caproic acid,the content of other short-chain fatty acids changed significantly.After administration of AC,the content of other SCFAs increased to varying degrees.Only in the middle dose group,the content of propionic acid and butyric acid increase significantly.It indicates that AC may improve the intestinal injury by improving the content of short-chain fatty acids in the stool of colon cancer model mice.2.3 The mechanism of AC anti-colon cancer via gut microbiota mediated SDF-1/CXCR4/NFκB pathway2.3.1 Prediction of intermolecular interaction based on molecular dockingMolecular docking was used to verify the geometric and energetic matching degree of the 14 active components with the three protein targets.Taking the level of binding free energy as the evaluation standard for the degree of combination of the two,the docking results showed that the flavonoids and astragaloside Ⅱ in Astragali Radix have a strong binding ability with CXCR4,and the volatile oil components of Curcumae Rhizoma have a strong binding ability with CXCR4.The flavonoids in Astragali Radix and the volatile oil in Curcumae Rhizoma can also form a strong and stable combination with NF-κB p65.The flavonoids in Astragali Radix can have better docking activity with SDF-1,while the saponins in Astragali Radix and the volatile oil in Curcumae Rhizoma have the weaker binding activity.It shows that the main active components in Astragali Radix and Curcumae Rhizoma can interact with SDF-1/CXCR4/NF-κB signaling pathway proteins.2.3.2 The tumor-bearing mice of colon cancer in vivo verifies the mechanism of AC in regulating the SDF-1/CXCR4/NF-κB pathwayCompared with the sham operation group,the protein expression of SDF-1,CXCR4,p-NFκB p65 in the colon tissue of the model group increased significantly.And the SDF-1,CXCR4,NF-κB p65 and Cyclin D1,C-myc protein and mRNA expression in the tumor tissue increased significantly.After the administration of AC,the related protein and mRNA expression levels were significantly reduced.The results verified the results of molecular docking,indicating that AC may act on the protein and mRNA expression in this signaling pathway to play an anti-growth effect on colon cancer.The research results of the paper indicated that the compatibility of AC at the ratio of 2:1 exerted a better anti-colon cancer effect,and AC may act on the gut microbiota mediated SDF1/CXCR4/NF-κB pathway to inhibit the growth of colon cancer.It provides a basis for the more effective clinical application of AC in the treatment of colon cancer,and provides more ideas for the application and development of classic drug pairs. |
PDF Full Text Request