| Objectives:To observe the clinical efficacy of Huachansu combined with chemotherapy in preventing and treating postoperative recurrence and metastasis of colorectal cancer through clinical study.To explore the mechanism of Huachansu against colorectal cancer through metabolomics and network pharmacology.Methods:1.Meta-analysis:we searched the Chinese and English databases to collect study of clinical randomized controlled trials of oral Huachansu combined with chemotherapy in the treatment of colorectal cancer.Two researchers independently conducted literature screening and data extraction,and evaluated the risk of bias in the included studies.Then Revman 5.3 software was used for data analysis.2.Clinical study:250 patients with TNM stageⅢandⅡwith high risks colorectal cancer were randomly divided into treatment group(124 cases,Huachansu combined with chemotherapy)and control group(126 cases,only chemotherapy)by random number table method.They were treated for 6 months and followed up for 3 years.The main outcome measures were 3-year disease-free survival(DFS),overall survival(OS),recurrence and metastasis rate and Karnofsky(KPS)score.The secondary outcome measures were TCM symptom score,tumor marker level,immune function index and adverse reactions.3.Metabonomics study:24 BALB/C tumor bearing mice were randomly divided into 4groups:control group,Huachansu group,FOLFOX group and combined treatment group.Another 6 healthy mice in the same batch were set as normal group.After 2 weeks of treatment,the general condition and tumor condition of mice in each group were observed. Immunohistochemistry,ELISA,and flow cytometry were used to verify the efficacy of Huachansu combined with chemotherapy in the treatment of colorectal cancer.The metabolites in the plasma between each group were detected liquid chromatography mass spectrometry(LC-MS)to find the different metabolites between each group.4.Network pharmacology research:The predicted target of Huachansu component were obtained through the target prediction databases,and the disease genes of colorectal cancer were obtained through the disease databases.Then carry out gene ontology(GO)function enrichment analysis and genome encyclopedia(KEGG)pathway analysis on the common target of Huachansu predicted targets and colorectal cancer disease genes.Protein interaction(PPI)networks and drug-target-disease interaction network were constructed by Cytoscape software to screen out key compounds and targets.Correlations between key targets and the prognosis of g colorectal cancer were analyzed through the Onco Lnc online website,and the binding status between Huachasnu components and key targets were analyzed through molecular docking.Finally,preliminary verification was carried out through animal experiments.Results:1.Meta-analysis:14 eligible articles were included.Jadad scores were all low-quality articles.Meta-analysis showed that compared with chemotherapy alone,oral Huachansu combined with chemotherapy can improve the objective remission rate,quality of life improvement rate,pain relief rate,reduce the level of CEA,reduce the incidence of leucopenia,nausea and vomiting in patients with colorectal cancer.2.Clinical study:Compared with the control group,the treatment group can prolong the DFS of 3 years(P<0.05).There was no difference between the two groups in the OS of 3 years(P>0.05).The recurrence and metastasis rates of the treatment group and the control group in 3years were 25.8%and 38.1%respectively,and the recurrence and metastasis time were 32.02±9.60 months and 28.39±11.15 months respectively(P<0.05).Subgroup analysis of DFS showed that patients with≥60 years old,FOLFOX chemotherapy regimen and KPS score of70 could benefit from DFS more significantly by oral Huachansu combined with chemotherapy(P<0.05).The improvement rate of KPS score and the effective rate of TCM syndrome score in the treatment group were significantly higher than those in the control group(P<0.05).After treatment,the level of CA125 in the treatment group was significantly lower than that in the control group(P<0.05).After treatment,the levels of CD3~+and NK cells in the treatment group were higher than those in the control group,and the levels of CD8~+in the treatment group were lower than those in the control group(P<0.05).During the treatment,the incidence of leucopenia,neutropenia and diarrhea in the treatment group were all lower than those in the control group(P<0.05).3.Metabonomics study:Animal experiments show that,after administration,the tumor inhibition rates of Huachansu group,FOLFOX group and combined treatment group were34.58%,80.02%and 87.76%respectively,and the tumor volume,weight and imaging value of the three groups were significantly lower than those of the control group(P<0.05).The relative expression levels of Ki-67 and PCNA protein in Huachansu group,FOLFOX group and combined treatment group were lower than those of the control group(P<0.05).Immunohistochemistry and flow cytometry showed that compared with the control group,the proportion of CD8+cells in the tumor tissues of Huachansu group and combination treatment group was significantly increased,and the proportion of Treg cells was significantly reduced(P<0.05);compared with the FOLFOX group,the proportion of CD8+cells in the tumor tissues of combination treatment group was significantly increased,and the proportion of Treg cells was significantly reduced(P<0.05).ELISA showed that compared with the control group and the FOLFOX group,IL-2 and IFN-γin the tumor tissues of Huachansu group and combination treatment group were significantly increased,IL-10 and TGF-βwere significantly decreased(P<0.01).Metabonomics showed that Huachansu can improve the disordered material metabolism of tumor-bearing mice,and there were 7 metabolites had been changed,and Huachansu can reverse the disordered material metabolism caused by FOLFOX chemotherapy,and there were 11 metabolites had been changed.Metabolic pathways involved in the different metabolites both Huachansu group and control group,combined treatment group and FOLFOX group were 11.4.Network pharmacology research:GO analysis showed that the effect of Huachansu and colorectal cancer affected 30 cell components,605 biological processes,and 37 molecular functions.KEGG pathway analysis included 127 signaling pathways.Through drug target disease pathway analysis,5 key compounds and 10 key targets were screened from the disease interaction network,among which two were related to the prognosis of colorectal cancer.Molecular docking showed that the main components of Huachansu had good binding activity with the key targets of colorectal cancer.Immunohistochemistry showed that Huachansu can reduce the expression of p-EGFR and p-STAT3 in mouse tumor.Conclusions:1.Meta-analysis showed that oral Huachansu combined with chemotherapy could improve the short-term efficacy,improve the quality of life,relieve pain,reduce the level of CEA,and reduce the adverse reactions of chemotherapy in patients with colorectal cancer.However,due to the low quality of the included literature,large sample,multi center,scientific and standardized,long-term clinical trials were still needed for further investigation.2.Huachansu combined with chemotherapy could reduce the recurrence and metastasis after colorectal surgery,improve the 3-year DFS of patients,improve the quality of life,TCM syndrome,immune function,decrease the level of tumor markers,and relieve the adverse reactions of chemotherapy.3.Huachansu enhanced the efficacy of chemotherapy by improving tumor immunity and metabolic disorder of tumor bearing mice,and the different metabolites maybe used as the metabolic markers of its efficacy evaluation.4.Network pharmacology showed that the treatment of colorectal cancer by Huachansu work through multiple components,multiple targets,and multiple pathways,and had a certain regulatory effect on the core targets of EGFR and STAT3. |
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