Research On The Effect And Mechanism Of Tanshinone I On Cervical Cancer Based On Network Pharmacology

Objective:Salvia miltiorrhiza has the function of tonifying qi and removing blood stasis.Salvia miltiorrhiza related products combined with radiotherapy and chemotherapy have a good clinical efficacy in cancer treatment.But the specific mechanism is still elusive.Tanshinone I is an an effective component of Salvia miltiorrhiza.This thesis aims to elucidate the function and molecular effective mechanism of tanshinone I on cervical cancer by network pharmacology and experimental methods.Contents and Methods:The full text is divided into two parts.The first part mainly predicts the target and molecular mechanism of tanshinone I,an effective component of Salvia miltiorrhiza,on cervical cancer based on network pharmacology.The main methods are as follows:multiple databases and literature-mining method were utilized for searching the active ingredients of Salvia miltiorrhiza through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).Major active ingredients of Salvia miltiorrhiza was screened based on ADME calculation combined with five principles of analogous drugs.Tanshinone I target and cervical cancer target were obtained by CTD and STITCH databases,Moreover,the intersection target of compound and disease was screened.Gene annotation was carried out by Biological information annotation databases(DAVID)to analyze the gene function and signal pathway of the potentialtargets.The protein-protein interactionnetworkwas analyzed and screened by STRING platform.Moreover,we constructed the PPInetwork by Cytoscape software.The key targets were screened by MCC algorithm of cytoHubba.The network model of key gene targets was established,predicting the effective mechanism of tanshinone I on cervical cancer in molecular level.The second part is to investigate the effect oftanshinone I on the proliferation and migration of cervical cancerthrough experimental verification O methods.Methods:We used CCK-8 assay and cell counting to examine the cell proliferation and quantity after the treatment by using tanshinone I.Effect of Tanshinone I on Colony formation of Hela cellswas detected by colony formation assay.Cell migration was measured by wound healing mobility assay,adhesion assay,transwell cell migration assay.Transmission electron microscope was used to explore the effect on ultrastructure of Hela cells influenced by tanshinone I.ResuIts:The results of network pharmacologysuggest that salvia miltiorrhiza has 202 active ingredients in total.There are 42 major active ingredients that had been screened out by ADME calculation method and there are 28281 targets associated with cervical cancer;Tanshinone I has 127 targets.There are 123 targets of tanshinone I for cervical cancer.According to combined score>0.7,172 results were obtained by protein-protein interaction of 123 targets and 20 key targets were obtained by using MCC algorithm of cytoHubba.According to Inference Score>=60 and literature search,43 potential targets were selected from 123 intersecting targets.The potential targets involved 115 biological processes,25 molecular functions,20 cell components and 52 pathways through the GO and KEGG signaling pathway analysis by using the DAVID database.Biological processes involved the regulation of apoptosis,cell proliferation and cell cycle,as well as the metabolism of some functional substances such as protein,lipid,reactive oxygen species,glucose.Cytosol occupies the first place of enrichment in cellular components,and spherical high-density lipoprotein particle is arranged behind.Molecular functions mainly involved heme binding;transcriptional activator activity,RNA polymerase II core promoter proximal region sequence-specific binding;aromatase activity and so on.Molecular mechanisms mainly involved cancer pathways,apoptotic pathways,transcriptional misregulation in cancer,p53 signaling pathway,HIF-1 signaling path way,PI3K-Akt signaling pathway,NF-kappa B signaling pathway,as well as some amino acid metabolism such as arginine,proline;glycometabolism and cytochrome P450 exogenous substances metabolic pathways.Through the perimental verification found thatWith the25-100μMconcentrations of tanshinone I significantly inhibited the proliferation andnumber of Hela cellsin a concentration-and time-dependent manner.Colony formation assay demonstrated that compared with the control group,the25-100μMconcentrations of tanshinone I significantly inhibited the colony formation of Hela cells.Adhesion assay demonstrated that tanshinone I significantly inhibited the adhesion ability of Hela cells and was positively correlated with drug concentration.Wound healing mobility assay and transwell cell migration assays indicated that25-100μMconcentrations of tanshinone I inhibited the migration ability of Hela cells in a concentration-dependent manner.The results of transmission electron microscope suggested thattanshinone I could induce ultrastructural changes of Hela cells by altering the microvilli structure on the surface of cervical cancer cells,inducing nuclear concentration of cervical cancer cells,inducing mitochondrial damage,showing swelling of mitochondria,autophagy and apoptosis.Conclusion:(1)tanshinone I acts on the key targets of cervical cancer including TP53,MYC,CASP3,MMP9,CCND1,VEGFA and other genes and the main mechanismis related to regulating cancer pathway,apoptosis pathway,cell cycle related pathway and other signaling pathways.(2)tanshinone I significantlyinhibits Hela cell proliferation,adhesion and migration in a concentration-and time-dependent manner.(3)Tanshinone I induces autophagy and apoptosis by inducing mitochondrial damageof Hela cells.