Study On Network Pharmacology And Effect On Cell Proliferation In Treatment Of Condyloma Acuminatum With FFGL

Condyloma acuminatum(CA)is a sexually transmitted disease caused by human papillomavirus(HPV)infection.CA infection with high-risk HPV is more likely to have a longer course of disease and persistent infection.It is also associated with a high recurrence rate and a risk of cancer.An important link in the pathogenesis of CA is the excessive proliferation of epithelial keratinocytes to produce warts.The treatment of CA with Fu Fang Gang Liu liquid(FFGL)of Chinese herbal medicine has been used for decades and achieved good clinical effect.It has high efficiency and safety,and can significantly reduce recurrence.But its mechanism of action remains unclear and needs further exploration.This research will be studied from the following two aspects:(1)Study on the mechanism of FFGL on HeLa cell proliferation;(2)Network pharmacology study of FFGL in treatment of CA.Part 1 Study on the mechanism of FFGL on HeLa cell proliferationObjective:To observe the effect of FFGL on the proliferation of HPV 18+human cervical cancer cell line HeLa and explore the related molecular mechanism.Methods:(1)After treating HeLa for 24h,48h and 72h with different concentrations of FFGL,the proliferation of HeLa was detected by CCK-8 method,and the IC50 value of FFGL in the intervention of HeLa cell proliferation was calculated to provide reference concentration for subsequent experiments.(2)HeLa cells were treated with 160μg/mL FFGL for 0h,24h and 48h,respectively.After staining HeLa cells with propidium iodide,DNA content was determined by flow cytometry to analyze the distribution of cell cycle in HeLa cells.(3)The blank group and 160μg/mL of FFGL group were set,and the cell division rate was detected by CFSE fluorescence labeling.The fluorescence intensity at 0h,24h and 48h after intervention of HeLa cells for 0h,24h and 48h was detected respectively,and the ratio of fluorescence intensity at 24h and 48h to 0h was calculated.(4)Short time(5min,15min,30min,60min,180min)and long time(12h)intervention of HeLa were performed at the concentration of 80μg/mL and 160μg/mL of FFGL,respectively.Western Blot was used to detect the expression of HPV oncoprotein E7,cyclin dependent kinase 1(CDK1/cdc2)and cyclin B1.Results:(1)FFGL inhibit the proliferation of HeLa cells in a time and dose dependent manner.The IC50 value of 24h and 48h was 160μg/mL.80μg/mL and 160μg/mL were selected as the concentration of subsequent experiments.(2)The HeLa cells in S and G2/M phase increased in a time dependent manner.(3)FFGL could reduce the division rate of HeLa cells.The fluorescence intensity of 160μg/mL FFGL group was significantly higher than that of the control group at 24h,and the increase was more obvious at 48h.(4)FFGL inhibited E7 protein expression and CDK1 activity in a time and dose dependent manner.Conclusion:FFGL could inhibited the proliferation of HeLa cells,resulting in S and G2/M cycle arrest.The mechanism might be related to the decreased expression of E7 oncoprotein and the inhibition of CDK1 activity.Part1 Network pharmacology study on treatment of CA with FFGLObjective:To predict and screen the therapeutic CA targets of FFGL by network pharmacology and explore the potential mechanism of action.Methods:(1)TCMSP,ETCM,PharmMapper and SwisstargetPrediction databases were used to retrieve the active components and predict the related targets of FFGL.The related targets of CA were searched and predicted in DisGeNET and GeneCards databases.FFGL targets and CA targets were sorted out and duplicated items were removed,and then the intersection of FFGL for CA targets were obtained in the biological information database.Use UniProt database for human species identification and standardize gene names.Cytoscape software was used to construct the Herb-Compound-Target Network diagram.(2)The therapeutic CA targets of FFGL and drug active ingredients fluid were imported into Cytoscape software.Then Construct Herb-Compound-Target-Disease Network diagram.Key active ingredients were screened according to degree value.(3)The therapeutic CA targets of FFGL were imported into String database for protein-protein interaction network analysis.The results were imported into Cytoscape software,and topological analysis was performed on the therapeutic CA targets of FFGL using Analyzer tool.The key targets in the protein interaction network were screened according to the degree value.(4)The therapeutic CA targets of FFGL were imported into Metascape database for GO:Cellular Component(CC),Molecular function(MF),biological process(BP)and KEGG(Kyoto Encyclopedia)Analysis of Genes and Genomes.Results:(1)A total of 163,114,105 and 539 targets were predicted from the four drug-related databases,and 610 targets were obtained after removing duplicates.340 and 16 CA targets were predicted in the two disease-related databases,respectively,and the number was 346 after removing duplicates.A total of 59 therapeutic CA targets of FFGL were obtained by combining compound FFGL with CA targets.(2)Based on the network parameters of 78 drug active ingredients and therapeutic CA targets of FFGL,the key active ingredients of FFGL for the treatment of CA were screened,and the top two were ursolic acid and beta-sitosterol,respectively.(3)According to the topological parameters of 59 target networks,the key targets for the treatment of CA by FFGL were screened.The first 8 positions were rac-alpha serine/threonine-protein kinase(AKT1)、Cellular tumor antigen P53(TP53)、Proto-oncogene tyrosine protein kinase Src(SRC)、Tumor necrosis factor Factor(TNF)、Epidermal growth factor receptor Receptor(EGFR)、Albumin(ABL)、Interleukin-6(IL6)、Vascular endothelial growth factor A(AVEGFA).(4)Enrichment analysis of CA targets treated by FFGL showed that The higher-ranked pathways involved in positive regulation of protein phosphorylation,transferase complexes containing phosphoric acid groups,Transferring-containing groups,Pathways in cancer,EGFR Tyrosine kinase inhibitor resistance pathway,Cell cycle pathway.Conclusion:Ursolic acid and β-sitosterol may play an important role in the treatment of CA by FFGL.AKT1 is a potential therapeutic target,which is closely related to the regulatory mechanism of protein kinase phosphorylation.This may depend on EGFR lysine kinase inhibitor resistance and cell cycle pathways,which need further experimental verification.