| Background:Colorectal cancer（CRC）is one of the most common malignancies in digestive system.According to the latest statistics released in 2021,more than 1.9million new cases of CRC were diagnosed,and about 935,000 patients died.The data has increased compared with the 2018 report,and onset age is becoming increasingly younger.Clinically,the combined treatment of CRC radical mastectomy and radiotherapy/chemotherapy is usually used to improve the survival rate of patients.Postoperative recurrence and metastasis are the main causes of death in CRC patients.Therefore,the key problems urgently to be solved are how to inhibit tumor recurrence,and metastasis after surgery,prolong the disease-free survival of patients,and improve the quality of life.Traditional Chinese medicine（TCM）is an important part of the comprehensive treatment of CRC.Notably,increasing clinical evidence has shown that TCM synergizes the efficacy of chemotherapy and radiotherapy,decreases the toxicity or side effects induced by chemotherapy and radiotherapy,enhances quality of life,and improves the immune function of cancer patients.Recently,the potential role of TCM in the process of postoperative recurrence or metastasis formation has attracted attention.Objective:（1）This study was designed to evaluate the clinical efficacy of combined TCM and chemotherapy in patients with CRC after radical surgery,then to analyze the TCM compounds to find the core drug pair.（2）The inhibitory effect of the core drug in combination with oxaliplatin on the invasion and metastasis ability of CRC cells,which was verified in vivo and in vitro experiments.（3）Combined with network pharmacology methods and whole transcriptome RNA sequencing（RNA-seq）,the underlying mechanism was explored.Real-time q PCR（RTq-PCR）and western blot（WB）were adopted to determine the m RNA and protein levels of the corresponding molecules.Methods:（1）This study was a multi-center retrospective cohort study.Patients after CRC surgery from January 2010 to May 2020 were divided into a TCM group and a control group.The Kaplan-Meier method was used for survival analysis,and univariable and multivariable Cox regression（MVA）were used to determine prognostic effects on progression-free survival（PFS）.At the same time,according to the TCM prescription used by the patients,the Apriori method was used for correlation analysis to find out the core drug pair.（2）The IC50（half-inhibitory concentration）values for Oldenlandia diffusa-Rhizoma Atractylodis Macrocephalae（Od-RAM）extract and oxaliplatin in CRC cancer cells were determined by the CCK-8 method after the cancer cells had been treated for 24h and 48h.Then the subsequent experimental drug concentrations of drug combination were determined.Annexin V-FITC/PI staining,clone formation experiments,wound healing assay and Transwell invasion assay were used to determine the apoptosis,proliferation,migration and invasion of CRC cells when Od-RAM,oxaliplatin and drug combination treatment.（3）Nude mice of the subcutaneous tumor models were established with HCT116 cells and were randomly divided into control group,chemotherapy group,TCM group and combination group to investigate the therapeutic effect on CRC.Furthermore,nude mice of the subcutaneous tumor models were established with HCT116 cells and were randomly divided into control group,chemotherapy group and low-dose,medium-dose,and high-dose combination groups to evaluate the effects of different doses of TCM combined with oxaliplatin on the growth of subcutaneous tumors in nude mice.A tail vein injection mouse model were established and randomly divided into control group,chemotherapy group,TCM group and combination group.The effects of different treatments on the formation of hematogenous metastasis were observed.（4）The active ingredients of Od and RAM were acquired from the Traditional Chinese Medicine System Pharmacology（TCMSP）database,and potential targets were predicted using the Swiss Target Prediction database.A Herb-Compound-Target network was established to analyze the core active ingredients.CRC metastasis-related disease targets were collected in Genecards,OMIM,Therapeutic Target Database（TTD）,Drugbank,and Dis Ge NET databases and intersected with compound targets to obtain intersection targets.A PPI network was constructed for the intersection targets,and the core targets were obtained by analysis.To gain a better insight of the potential mechanisms of Od-RAM underlying metastases of CRC,GO and KEGG enrichment analysis of intersection targets was performed.Molecular docking was used to verify the binding activity of the targets and the active ingredients.（5）RNA-seq for subcutaneous tumor tissue in different group was performed,and gene set enrichment analysis was performed to identify the gene sets/pathways that were enriched in the differential expression profiles.The RNA and protein expression levels of related molecules were determined by RTq-PCR,WB and immunohistostaining.Results:（1）A total of 436 cases were collected,including 216 in the TCM group and 220 in the control group.After 1:1 propensity score matching,the m PFS of the TCM group and the control group were 36.000 months and 32.000 months,respectively（HR:0.553,95%CI:0.401-0.761,P<0.05）,multivariate analysis showed that the systematic use of TCM（HR:0.486,95%CI:0.348-0.678,P<0.05）was a protective factor affecting the prognosis of patients.By analyzing the TCM prescriptions used by 179 patients,a total of 72 high-frequency TCM herbs（frequency>10%）were obtained.Further correlation analysis showed that Od-RAM was the core drug pair,and it had certain advantages in the TCM group.（2）The IC50values of Od-RAM in HCT116 cells at 24h and 48h were 7.01±0.82mg/ml and5.86±0.61mg/ml,respectively;the IC50 values of Od-RAM in SW620 cells at 24h and48h were 11.01±0.21mg/ml and 10.35±0.43mg/ml,respectively.The IC50 values of oxaliplatin in HCT116 cells at 24h and 48h were 26.39±0.03μM and 10.28±0.92μM,respectively;the IC50 values of oxaliplatin in SW620 cells at 24h and 48h were39.42±1.98μM and 14.28±1.33μM,respectively.The IC50 values of drug combination in HCT116 cells at 24h and 48h were 20μM+5mg/ml and 7.5μM+3mg/ml,respectively;the IC50 values of drug combination in SW620 cells at 24h and 48h were30μM+7.5mg/ml and 12.5μM+5mg/ml,respectively.Apoptosis experiments showed that all drug-treated groups could effectively induce apoptosis（P<0.05）.The cell clone formation experiment showed that the cell clone formation in each drug-treated group was inhibited,and the combination group was the most significant（P<0.05）.The wound healing assay showed that the cell migration ability of each drug-treated group was inhibited,and the combination group was the most significant（P<0.05）.Transwell invasion assay showed that the cell invasion ability of each drug-treated group was inhibited,and the combined drug group was the most significant（P<0.05）.（3）Subcutaneous tumor bearing CRC cells in a nude mice model was established,and the results showed that drug combination group inhibited tumor growth than TCM group and chemotherapy group.Additionally,it showed a dose-dependent inhibitory effect on the growth of subcutaneous tumor in nude mice.Nude mice hematogenous metastasis model showed that the hematogenous metastasis of each drug-treated group was inhibited to varying degrees,and the combination group had the most obvious effect,and the TCM treatment had a certain improvement on the weight loss of nude mice.（4）A total of 14 bioactive components and 104 related targets were obtained from Od-RAM via network pharmacology analysis.A total of 71 potential targets were obtained by the intersection of active ingredients-related targets and disease-related targets.AKT1,EGFR,SRC,MMP9 and AR were included in the top five targets of the PPI network with highest degree.Further GO and KEGG analysis results showed that the intersection targets were involved in a variety of biological processes,including regulation of kinase activity,response to external stimuli,etc.and participated in the regulation of various cancer-related signaling pathways,such as PI3K-Akt pathway,Adherens junction and Focal adherin.Molecular docking results showed that the main active compounds had a good binding activity with the hub targets.（5）The analysis results of RNA-seq showed that the regulation of multiple genes and pathways changed after the combined treatment.The results of RTq-PCR and WB showed that the expression of fibronectin 1（FN1）was significantly inhibited after the combined drug treatment;the measurement results of EMT-related protein molecular expression showed that the combined drug treatment up-regulated the expression of epithelial cell markers and down-regulated the expression of mesenchymal cell markers.WB results showed that the activation of PI3K-Akt signaling pathway was inhibited after combined treatment.Conclusion:Traditional Chinese medicine is an effective adjuvant therapy for postoperative CRC cancer patients,and has the potential advantage of reducing the risk of postoperative recurrence and metastasis.The core drug pair represented by Od-RAM combined with oxaliplatin can effectively inhibit the invasion and metastasis of CRC cells.Inhibition of FN1 by the core drug pair combined with oxaliplatin could reduce the activation of PI3K-Akt signaling pathways,thereby suppressing EMT and metastasis of CRC.This study provides clinical medication ideas for the application of TCM in postoperative CRC patients,and provides a new perspective for preventing recurrence and metastasis after CRC surgery. |
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