Danggui Buxue Decoction In The Treatment Of Metastatic Colon Cancer: Network Pharmacology Analysis And Experimental Validation

Background:Colon cancer is the third most frequently diagnosed malignant tumor and the second leading cause of cancer-related deaths worldwide.Surgery as the primary treatment for malignant tumors has significantly improved the prognosis and quality of life of cancer patients.The risk of metastatic tumor development following surgery remains the major factor associated with the limited efficacy of the operation.Evidence suggests that excision of the primary tumor can promote the development of metastases,which is the main cause of cancer-related mortality.However,conventional chemoradiotherapy cannot be used during this period,given its suppressive effects on the immune system and tissue healing.Thus,there is a need for an alternative approach.Reinforcing qi and nourishing the blood are among the approaches used in conventional traditional Chinese medicine(TCM)to correct blood loss resulting from operation.Danggui Buxue Decoction(DBD),a classic Chinese prescription,is used for treating qi and the blood deficiency syndrome in clinic practice.Pharmacological studies have shown the evidence of anti-tumor effects of DBD.However,little is known about the molecular mechanisms of DBD against Metastatic colon cancer after primary tumor removal.Purpose: This study aimed to reveal Danggui Buxue Decoction(DBD)candidate targets and mechanisms in the treatment of Metastatic colon cancer(MCC),using network pharmacology-based analyses and experimental validation.Methods: Traditional Chinese Medicine Systems Pharmacology(TCMSP)database query and text mining were used to screen active compounds in DBD,and the Swiss target prediction platform was applied to predict compound-related target proteins.Targets likely associated with MCC were determined using Gene Cards and OMIM databases.Targets common to DBD and MCC were obtained from the Venn platform;subsequently,Cytoscape was used to construct drug-compound-target-disease and protein-protein interaction networks.The hub gene was determined by R,while GO and KEGG enrichment analyses were performed on common targets to elucidate biological processes and signaling pathways involved in DBD against MCC.Finally,the subcutaneous MCC mouse model was constructed and were then randomly divided into 4 groups,primary tumor resected group(R),non-resected primary tumor group(NR),and DBD I group,and DBD II group.The primary tumor was excised and administered with DBD for 14 days.Finally,the metastatic tumor was removed and its volume and weight were measured.Morphological changes of metastatic tumors were observed by Hematoxylin-eosin(HE)staining.Immunohistochemistry assay was used to detect Caspase3(Cas3)expression of metastatic tumor.Western blot was used to detect the protein expression of Bax,Bcl2,Caspase3 and Cleaved caspase3(C-cas3).Results: A total of 28 active compounds and 61 common targets were predicted.The main compounds were quercetin,hederagenin,jaranol,methylnissolin,formononetin,calycosin,kaempferol,3.9-di-O-methylnissolin,24-propylcholesterol,and7-O-methylisomucronulatol,present in Astragalus membranaceus(Huangqi,HQ).In addition,beta-sitosterol,ferulic acid,and stigmasterol,present in Angelica sinensis(Danggui,DG),were detected.JUN,PTSG2,EGFR,ESR1 and,CASP3 genes were the top 5 hub genes in the PPI network.GO and KEGG enrichment analyses indicated that apoptosis played a major role in the biological processes and signaling pathways involved.In vivo experiments showed that NR,DBD I and DBD II inhibited the growth of subcutaneous metastases after primary tumor resection,and the inhibitory rates were 25.10%,44.53% and 74.26% respectively;IHC and Western blot analysis indicated that the expression of Bax,Cas3,and C-cas3 was up-regulated in the DBD I/DBD II groups compared to that in the R group;moreover,the expression of these proteins in the DBD II group was markedly up-regulated compared to that in the R group(P<0.0001,P<0.01,P < 0.01).Meanwhile,it was down-regulated in the R group compared to the NR group(P<0.05,P<0.01).In contrast,the expression of Bcl2 was down-regulated in the DBD I/II groups compared to that in the R group;moreover,in the DBD II group,it was significantly down-regulated compared to that in the R group(P<0.0001).Finally,the expression of Bcl2 in the R group was up-regulated compared to that in the NR group(P<0.01).In addition,we calculated the ratio of Bax/Bcl2 in four groups;in the DBD I/II groups,it increased more than it did in the R group(P<0.001),while in the R group,it was lower than in the DBD II and NR groups(P<0.01).Conclusion: Primary tumor removal can facilitate the growth of metastases.Danggui Buxue Decoction can induce the apoptosis of tumor cells by regulating the expression of apoptosis-related proteins Bax,Bcl2,Cas3 and C-cas3,thus inhibiting the growth of subcutaneous metastases after the resection of colon cancer primary tumor.And the postoperative application of DBD may achieve better overall effect on metastatic foci.