| Objective:In the comprehensive treatment of prostate cancer,the status of Chinese medicine has become increasingly prominent.In this study,we explored the therapeutic mechanism of Danggui Beimu Kushen pill on prostate cancer,analyzed the components contained in the compound and its target by network pharmacology,and targeted the drug itself with prostate cancer.The disease-related targets are matched to find the main target and main active ingredients of Danggui Beimu Kushen pill in the treatment of prostate cancer.Subsequently,the main component of the compound,quercetin(Que),was selected for in vitro experiments to verify the validity and accuracy of the previous analysis results.Methods:1.The compounds and targets contained in the Danggui Beimu Kushen pill were searched by the Chinese Medicine System Pharmacology Analysis Platform(TCMSP),and the gene names of each target protein were obtained through the Uni Prot KB web platform and the Genecards database;the compounds-targets network of Danggui Beimu Kushen pill was constructed using Cytoscape software.Prostate cancer disease-related targets were obtained by consulting literatures and querying CTD database and TTD database.Molecular docking was conducted between prostate cancer disease-related targets and the action targets of Danggui Beimu Kushen pill.A protein-protein interaction network(PPI)was constructed in a STRING database,and Cytoscape was used for network visualization analysis.Gene ontology(GO)analysis and enrichment pathway analysis of Kyoto gene and genome encyclopedia(KEGG)of key target proteins were conducted through DAVID data platform,and the results of the analysis were summarized and classified.2.According to the results of network pharmacology analysis,the main component Que in the network was selected for in vitro cytology experiments.Firstly,the prostate cancer PC3 cells were cultured,the effects of different concentrations of Que on the morphology of prostate cancer cells were observed,and the effect of Que on the proliferation of prostate cancer cells was observed by trypan blue staining.The effect of Que on the migration ability of prostate cancer cells was observed by Transwell experiment.The effects of different concentrations of Que on the apoptosis of prostate cancer cells were detected by flow cytometry.Finally,the effects of different concentrations of Que on the proliferation and apoptosis-related proteins in prostate cancer cells were detected by Western-blot assay.Results:1.Results of network pharmacological analysis(1)A total of 255 active ingredients were screened out from the TCMSP Chinese medicine database.After evaluation of the OB value and the DL value,26 active ingredients were obtained,and the 26 active ingredients had a total of 246 targets.(2)Cytoscape software was used to construct the compounds-targets network map of Danggui Beimu Kushen pill.The network shows that the representative compounds of Danggui Beimu Kushen pill are quercetin(MOL000098),luteolin(MOL000006),etc.;and prostaglandin G/H synthase 2(PTGS2),heat shock protein90(HSP 90),etc.are the main targets of this formula.(3)There are 135 targets of Danggui Beimu Kushen pill acting on prostate cancer.The protein-protein interaction network map analysis found that there were as many as 3,066 interactions before these targets.Through the plug-ins and algorithms in Cytoscape,34 key genes among the 135 targets were analyzed.(4)Through gene ontology(GO)analysis and KEGG enrichment pathway analysis,it was concluded that Danggui Beimu Kushen pill could exert its anti-tumor effect by promoting cell apoptosis,inhibiting cell proliferation,inhibiting cell migration and invasion,and inhibiting tumor angiogenesis.2.In vitro experimental verification results:(1)Effect of Quercetin on the Proliferation of Prostate Cancer Cells: After 24 h and48 h,the number of cells in the dosing group was significantly lower than that in the control group,and it was dose-dependent(P<0.0001).(2)Effect of Quercetin on Apoptosis of Prostate Cancer Cells: The results showed that the number of apoptosis of prostate cancer PC3 cells increased with the increase of drug concentration.When the concentration was 150 um,the apoptosis-promoting effect of Que was the most obvious,and the apoptosis rate was 26.13±3.36%.(3)Effect of Quercetin on the migration of prostate cancer cells: It was found that Que could inhibit the migration ability of PC3 cells in a dose-dependent manner(P <0.005).(4)Effect of Quercetin on the expression of proliferation and apoptosis-related proteins in prostate cancer cells: The results showed that Que can significantly reduce the expression of p-AKT,a protein related to proliferation in PC3 cells.In addition,it also reduced the expression of Bcl-2,which inhibits apoptosis,and increased the expression of proapoptotic protein BAX.In addition,Que also has a certain inhibitory effect on the chaperone protein HSP90.Conclusions:1.According to network pharmacological analysis,the traditional Chinese medicine formula Danggui Beimu Kushen pill contains a variety of effective anti-cancer compounds,which can exert its anti-prostate cancer effect by the following four methods: inhibiting cell proliferation,promoting apoptosis,inhibiting cell migration and invasion,and inhibiting tumor angiogenesis.2.Experimental results show that quercetin,one of the main compounds of Danggui Beimu Kushen pill,can exert its anti-cancer effect by inhibiting the proliferation of prostate cancer PC3 cells,promoting apoptosis of PC3 cells,and inhibiting the migration of PC3 cells.This experimental verification is consistent with the previous analysis of network pharmacology partly. |
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