To Explore The Mechanism Of Huoluo Xiaoling Pill In The Treatment Of Colorectal Cancer Based On Network Pharmacology

Objectiv:This paper analyzed the mechanism of action of Huoluo Xiaoling Pill in the treatment of colorectal cancer by network pharmacological method,and applied bioinformatics technology to obtain CRC related differential genes,so as to further explore the key genes associated with CRC prognosis.At the same time,molecular docking was applied to simulate and validate the core compounds with prognosis related hub genes,provide theoretical support for further clinical or experimental research.Methods: The chemical constituents and their targets of Salvia miltiorrhiza,Angelica sinensis,frankincense and Myrrh were obtained by searching TCMSP database.Gene Cards,Drug Bank,OMIM and TTD databases were searched to obtain CRC related targets.Construct“drug-active ingredient-target ”network using Cytoscape3.7.2.PPI network was constructed using STRING database.GO and KEGG enrichment analysis was performed using DAVID database.GSE35279 and GSE22598 gene chips were screened based on GEO database,and CRC related differential genes(DEGs)were obtained by GEO2 R tool,and intersection genes were obtained by Winn diagram.The TCGA online tool GEPIA was used to map the relative expression levels of the above intersection genes in normal and tumor tissues.at the same time,the HPA database was applied to acquire the immunohistochemical outcome of the intersection genes for expression verification.The GEPIA online tool was used for survival analysis of intersection genes,and prognostic related genes were screened out.At the same time,R language was used to draw ROC curve for the genes with prognostic value,and the diagnostic accuracy of prognostic related genes was evaluated.R language was used to analyze the correlation between the expression level of prognostic genes and clinicopathologic stage,lymph node metastasis status,and age of CRC patients.The gene modules in TIMER database were applied to estimate the correlation between CRC prognosis related gene expression and the levels of 6 kinds of immune cell infiltration.Auto Dock software was applied to verification of the screened core compounds and prognostic related hub genes were validated by molecular docking simulation,and the results were visualized.Results: A total of 101 chemical components of Huoluo Xiaoling Pill for the treatment of CRC were filtered,including 59 kinds of salvia miltiorrhiza,2 kinds of angelica,6 kinds of frankincense and 34 kinds of myrrh,and 229 corresponding targets were obtained.A total of 1844 CRC related targets were obtained,and 124 targets were obtained after intersection.Among them,the core compounds of Huoluo Xiaoling Pill are quercetin,luteolin,β-sitosterol,tanshinone IIA,ellagic acid,etc.These components act on the core targets of TP53,AKT1,VEGFA,EGFR,MYC,TNF,ESR1,CASP3,HIF1 A,IL6,etc.Go functions were enriched to 806 entries,and 152 signaling pathways were screened by KEGG pathway analysis.A total of 168 CRC differential genes were obtained by comprehensive analysis of GSE35279 and GSE22598 microarray expression data,and the intersection of differential genes and potential drug-disease targets was identified,namely,8 intersection genes such as ADH1 C,CDK1,TOP2 A,PLAU,MET,SPP1,MYC and CXCL8 were obtained.GEPIA analysis showed that the expression levels of the screened hub genes CDK1,TOP2 A,PLAU,MET,SPP1,MYC and CXCL8 were significantly up-regulated in CRC tissues compared with their normal tissues,and seven genes except ADH1 C were statistically significant(p < 0.01).Immunohistochemical staining images of proteins obtained from HPA database confirmed that CDK1,TOP2 A,PLAU,MET,MYC and CXCL8 proteins were more highly expressed in CRC tissues than in normal tissues.The results of Kaplan-Meier map made by GEPIA online tool showed that CXCL8,MET and SPP1 genes were identified as prognostic related hub genes.ROC diagnosis results explained that the area under ROC curve of CXCL8,MET and SPP1 were 0.898,0.971 and 0.849,respectively,indicating that MET could be used as a prognostic indicator of CRC with high sensitivity and specificity.Correlation analysis of clinical data showed that the expression of SPP1 in CRC was significantly correlated with pathological stage,lymph node metastasis and age,but not with the expression of CXCL8 or MET.Correlation analysis of immune cell infiltration level showed that CXCL8(IL8),MET and SPP1 prognostic related genes played a key role in regulating the immune cell infiltration level of CRC.Molecular docking results showed that CXCL8,MET and SPP1 prognostic related hub genes had stable binding activity with quercetin,luteolin,β-sitosterol,tanshinone IIA,ellagic acid and other core compounds.Conclusion:Through the network pharmacological analysis on the treatment of CRC by Huoluo Xiaoling Pill,It was found that 124 potential targets of TP53,AKT1,VEGFA,EGFR,MYC,TNF,ESR1,CASP3,HIF1 A,IL6 were affected by 101 active compounds such as quercetin,luteolin,β-sitosterol,tanshinone IIA,ellagic acid,etc.Involved Pathways in cancer,PI3K-Akt,lipid and atherosclerosis,hepatitis B,AGE-RAGE,proteoglycan in cancer,Cellular senescence,TNF,endocrine resistance,HIF-1,p53,IL-17 and other signaling pathways in the treatment of CRC Function.Therefore,Huoluo Xiaoling Pill was mainly used to treatment of CRC through multi-component,multi-target and multi-pathway methods.After screening by bioinformatics methods,CXCL8,MET and SPP1 were shown to be hub genes related to CRC prognosis.By binding to these hub genes,core compounds could participate in the regulation of PI3K-Akt signaling pathway and inhibit epithelium-mesenchymal transformation,thus effectively inhibiting the proliferation,migration and anti-apoptosis of CRC cells.