Exploration Of The Molecular Mechanism Of Euphorbia Fischeriana Steud Extract For The Treatment Of HCC Based On Network Pharmacology And Experimental Verification

Background:Primary liver cancer(PLC)is the second most common malignant tumor in China and its incidence and mortality rate are growing year by year,which extremely threatens the life and health of the population.Hepatocellular carcinoma(HCC),as the main pathological type of primary liver cancer,accounts for about 75%to 85%of the primary liver.Euphorbia fischeriana steud.(E.fischeriana)has been employed as a traditional Chinese medicine for tuberculosis and tumors.Previous studies have confirmed that E.fischeriana plays an important role in the treatment of cancers such as breast cancer,melanoma,gastric cancer,laryngeal cancer and other cancer types.Nevertheless,the potential mechanism of action of E.fischeriana for the treatment of HCC has not been reported.Objective:This study aimed to identify the antitumor activity of E.fischeriana extract in HCC and to explore its potential targets and molecular mechanisms for improving sorafenib resistance.Methods:Liquid chromatography-mass spectrometry(LC-MS)was used to evaluate all the compounds of E.fischeriana extract.Public databases(TCMSP,Swiss ADME and Swiss Target Prediction)and LC-MS results were employed to identify the major compounds and the major targets of E.fischeriana extract.The causative genes of HCC were obtained using the GEO,DisGeNET and GeneCards databases.The online analysis platform,STRING database and Cytoscape software were used to construct protein-protein interaction network maps.Molecular docking of major compounds to core targets was performed using AutoDockTools software.The DAVID database was used to perform GO and KEGG pathway enrichment analysis.The effect of E.fischeriana extract on the proliferation of HCC cells as well as sorafenib-resistant HCC cells were assessed by CCK-8 assay,EdU assay and plate cloning assay.The effect of E.fischeriana extract on the apoptosis of HCC cells as well as sorafenib-resistant HCC cells were measured by Annexin V/7-AAD assay and Tunel assay.The effect of E.fischeriana extract on the invasion and migration of HCC cells as well as sorafenib-resistant HCC cells were examined by transwell assay and scratch migration assay.The effect of E.fischeriana extract on the cycle progression of HCC cells were observed by PI staining.The effect of E.fischeriana extract on signaling pathways of HCC cells as well as sorafenib-resistant HCC cells were analyzed by Western Blot.Finally,the anti-cancer effect of E.fischeriana extract in vivo was explored by xenograft nude mouse model of human liver cancer.Results:A total of 37 major compounds in E.fischeriana extract were identified by LC-MS coupled with ADME screening conditions.Based on the public databases,678 targets corresponding to 37 compounds and 921 HCC-associated genes were obtained.Through the network topology model and algorithm,132 E.fischeriana extract-HCC common targets were obtained from the online analysis website,and 14 core targets were screened out from the 132 common targets.Through GO and KEGG enrichment analysis of 132 common targets,it was hypothesized that E.fischeriana extract exerted anti-cancer effects on HCC by regulating multiple signaling pathways,particularly PI3K/AKT pathway.Additionally,the molecular docking results disclosed that the main compounds of E.fischeriana extract had significant binding effects on the core targets.The results from in vitro experiments showed that E.fischeriana extract could significantly inhibit the proliferation of HCC cells,alter the cycle process of HCC cells,induce apoptosis of HCC cells,and inhibit the migration and invasion of HCC cells.Moreover,in vitro experiments also confirmed that E.fischeriana extract could improve the sensitivity of sorafenib-resistant cells to sorafenib by altering the above phenotype of sorafenib-resistant HCC cells.Western Blot analysis showed that E.fischeriana extract significantly reduced the phosphorylation levels of p-PI3K(p85a-Y607)and p-AKT in a dose-dependent manner.Furthermore,in vivo experiments have shown that E.fischeriana extract,alone or in combination with sorafenib,significantly inhibited tumor growth and improved the efficacy of sorafenib in tumor-bearing nude mice.Conclusion:E.fischeriana extract has potent anticancer activity in HCC by regulating multi-target and multi-pathway,especially PI3K/AKT signaling pathway,suggesting that E.fischeriana is a promising agent for the treatment of HCC.