To Explore The Mechanism Of Inhibition Of Esophageal Squamous Cell Carcinma By Ranunculus Ternatus Thunb Based On Network Pharmacology And Experimental Verification

China is one of the regions with high incidence of esophageal cancer in the world.According to the latest statistics from the International Agency for Research on Cancer(IARC)of the World Health Organization in 2020,the incidence and death of esophageal cancer account for more than half of the world.In China,more than 90%of Esophageal cancer is pathologically manifested as squamous cell carcinoma(ESCC),with an overall five-year survival rate of 15-25%.Esophageal cancer is called"tumor with Chinese characteristics" due to its high incidence and unique pathological classification.Although the treatment of esophageal cancer has made progress in surgical resection,chemotherapy,radiotherapy and chemoradiotherapy over the years,it is still a malignant tumor with high mortality.Traditional Chinese medicine is widely used in the treatment of various forms of cancer,most of which are used together with antineoplastic drugs to assist anti-tumor therapy,improve the quality of life of patients,and reduce various adverse drug reaction,it is considered to have great potential.Ranunculus ternatus Thunb is a kind of traditional Chinese medicine produced in Henan province of China.It is widely used in the treatment of lymph node tuberculosis,tuberculosis,pharyngitis and so on.In recent years,it has been gradually found to have a good anticancer effect.However,the pharmacological mechanism of traditional Chinese medicine is not yet clear because of its complex composition,and it acts on the human body with multi-component and multi-target characteristics Network pharmacology,explores the relationship between traditional Chinese medicine and human body and disease from the perspective of system and molecular level,making the treatment of traditional Chinese medicine more systematic and objective.Therefore,this study mainly explored the antineoplastic mechanism of Ranunculus ternatus Thunb in esophageal squamous cell carcinoma through network pharmacology program.ObjectiveRanunculus ternatus Thunb has the functions of Dissipate phlegm,dispelling knots,detoxification and detumescence.In recent years,many studies have shown that Ranunculus ternatus Thunb has a broad-spectrum of antitumor effects.It is commonly used to compose "Jia Shu Ercao Decoction" with Ranunculus ternatus Thunb,Carapax Trionycis,Zedoray and Rubeidus rubeidus to treat liver cancer,ovarian cancer and breast cancer.It has the effect of nourishing Yin and softening firmness,reducing phlegm and dispersing stagnation.At present,most of the researches on the anti-tumor mechanism of Ranunculus ternatus Thunb are limited to the level of in vitro cell research,and the single target and single component of western medicine is used to study Traditional Chinese medicine,which is difficult to reflect the systematic nature of traditional Chinese medicine and cannot scientifically explain its mechanism of action.If the network pharmacology analysis can be combined and verified from the perspective of molecular mechanism,the level of empirical evidence will be greatly improved and the foundation will be laid for clinical promotion and application.Therefore,this study uses network pharmacology and in vivo experiments to investigate the effective components of Ranunculus ternatus Thunb and its antitumor treatment mechanism for esophageal squamous cell carcinoma.Methods1.Detection of the inhibitory effect of different Ranunculus ternatus Thunb extracts on the proliferation of human esophageal squamous cell carcinoma cells.Select the appropriate ethanol concentration through pre-experiment,and then the extracts of petroleum ether,ethyl acetate and n-butanol were extracted and isolated from Ranunculus ternatus Thunb by reflux extraction method and systematic solvent method.The proliferation inhibition of petroleum ether,ethyl acetate and n-butanol extracts on ESCC cells include TE-1,TE-13 and EC A-109 cell line was determined by MTT assay.Calculate the IC50 value of the inhibitory concentration of the drug,and the results were compared and statistically analyzed to obtain the extract with the strongest inhibitory effect.2.To search the active ingredients of Ranunculus ternatus Thunb and to predict and explore the molecular mechanism of treatment of esophageal cancer through network pharmacologyCollect Ranunculus ternatus Thunb medicinal components through literature search,conduct component verification and transformation in PubChem and CAS,and collect chemical components-related targets in PubChem,TCMSP,SEA,TargetNet,PharmMapper,and Swiss Target prediction databases.The related targets of ESCC were searched in Genecards and OMIM databases using the keywords " esophageal squamous cell carcinoma ",and the intersection targets of Ranunculus ternatus Thunb and ESCC were screened out as research targets.The intersection targets were imported into String database to construct PPI network,and produce an active ingredient-target network diagram by using the software of Cytoscape 3.2.7 version.Finally,combine the above two network diagrams to obtain the core targets of Ranunculus ternatus Thunb acting on ESCC,and then use Metascape and Bioinformatics database for GO analysis and KEGG signal pathway enrichment analysis.3.To verify the molecular mechanism of inhibition of proliferation of esophageal squamous cell carcinoma by ethyl acetate fractions of Ranunculus ternatus Thunb.Western Blot was performed to verify the core targets,related pathways and biological processes of the network pharmacological analysis results,to detect the signal pathways and related proteins that may be regulated by Ranunculus ternatus Thunb in the inhibition of the proliferation of esophageal squamous cell carcinoma.Result1.Reflux extraction was carried out with 60%and 95%ethanol solutions,respectively,and a preliminary experiment was conducted to obtain 60%ethanol extract and 95%ethanol extract of Ranunculus ternatus Thunb.The two extracts were dissolved with DMSO and then MTT assay was performed to compare the inhibitory effects of the two extracts on esophageal squamous cell carcinoma cells.The results showed that the 95%ethanol extract had better inhibitory effect,but there was no statistical difference between the two extracts.Combined with the preliminary experimental results and previous research data,95%ethanol was selected for the extraction of Ranunculus ternatus Thunb.The Ranunculus ternatus Thunb is extracted with 95%ethanol,and the n-butanol extract,ethyl acetate extract and petroleum ether extract of Ranunculus ternatus Thunb are obtained after extraction and drying with organic solvent.MTT experiments were performed on TE-1,TE-13,and Eca-109 cells line after the extraction with different solvents was dissolved and quantified.The RTB had no obvious inhibitory effect on the proliferation of ESCC cells,while the RTE and RTP had remarkable proliferation inhibition on the proliferation of TE-1,TE-13,and Eca-109 cell lines,especially on Eca-109.2.Combined with the MTT results of various extracted parts and previous literature studies,a total of 32 chemical compounds in the ethyl acetate extract of Ranunculus ternatus Thunb were collected,and 472 targets related to the chemical compounds were retrieved from the database.And There are 4838 ESCC related targets,and 281overlapping targets of ESCC and RTE.After screening,14 main targets of RTE were obtained for worked on ESCC,are HSP90AA1,MAPK1,RELA,ESR1,CASP3,PPARG,AR,PTGS,CDK2,F2,MAPK10,ESR2,PGR and MMP3.GO analysis showed 52 related biological processes,9 molecular functions and 30 cellular components.It mainly involves biological processes such as signal transduction,cytokine-mediated signaling pathway and response to drug.There were 36 KEGG signaling pathways,mainly involved in cancer pathway,TNF signaling pathway,MAPK signaling pathway,PI3K/Akt signaling pathway,and NOD-like receptor signaling pathway etc.3.Two cell lines,TE-13 and Eca-109,were selected for Western Blot experiments.TE-13 and Eca-109 cell lines were treated with RTE at IC50 concentration for 48h,and compared with the control group.The results showed that the RTE could inhibit the proliferation of ESCC cell by regulating the MEK/ERK and PI3K/Akt pathways,and reducing the expression levels of downstream related carcinogens factors HSP90 and cMyc.