Study On The Effect And Mechanism Of Daphnetin On Human Glioblastoma Cell Line U251 Based On Network Pharmacology And Molecular Docking Technology

Part Ⅰ Network pharmacological study on antitumor mechanism of daphnetin.Objective:Prediction of common targets and possible mechanisms of daphnetin against malignant glioma,hepatecellular carcinoma,breast neoplasts,ovarian cancer and esophageal cancer based on network pharmacology and molecular docking technology.Methods:The target of daphnetin was predicted by Swiss target prediction,and the targets of "malignant glioma,hepatecellular carcinoma,breast neoplasts,ovarian cancer,esophageal carcinoma" were searched by genecards database.Cytoscape 3.7.1 was used to construct daphnetin tumor protein interaction network(PPI)and screen the core targets.GO and KEGG enrichment analysis was performed on the core targets.The molecular docking study of daphnetin with core targets were carried out using the Auto Dock Tools.Results:116 key anti-tumor targets of daphnetin were screened,which were mainly concentrated in P53 pathway and involved in biological processes such as cell cycle regulation,apoptosis,DNA biosynthesis and repair.PPI results showed that P53 was the core target,and molecular docking showed that daphnetin had a good binding effect with P53.Conclusion:The results of pharmacological analysis based on network show that:1.Daphnetin has broad-spectrum antitumor effect;2.The antitumor effect of daphnetin is related to P53 pathway;3.The antitumor mechanism of daphnetin may be related to the regulation of biological processes such as cell cycle regulation and DNA synthesis.Part Ⅱ Study on the effect and mechanism of daphnetin on human glioblastoma U251 cellsObjective:To investigate the antitumor effect and mechanism of daphnetin on glioblastoma U251 cells.Methods:U251 cells were treated with daphnetin at different concentrations for 24 and 48 hours.Cell counting kit-8 was used to detect the cell inhibition rate and scratch test was used to detect the cell migration.After U251 cells were treated with daphnetin at different concentrations for 48 hours,the protein expression levels of P53,P21,CCNB1,PUMA and RRM2 were detected by Western blotting,and the mRNA expression levels of P53,RRM2 and CCNB1 were detected by RT-PCR.Results:Daphnetin could significantly inhibit the proliferation and migration of glioblastoma cells after 24 and 48 hours(P<0.05).Daphnetin could significantly up regulate the expression of P53 protein and down regulate the expression of CCNB1,P21,PUMA and RRM2 protein in a dose-dependent manner(P<0.05).After 48 hours of daphnetin treatment of U251 cells,the expression of P53-mRNA increased significantly at 40 μg/mL(P<0.001),and decreased at 80 and 160 μg/mL(P<0.001).After 48 hours of daphnetin treatment of U251 cells,the mRNA expression of CCNB1 and RRM2 decreased significantly in a dose-dependent manner.Conclusion:The results of in vitro experiments showed that:1.Daphnetin has a significant inhibitory effect on glioblastoma U251 cells;2.The anti-tumor effect of daphnetin on U251 cells is related to P53 pathway:it can increase the expression of P53 and regulate its downstream targets(cell cycle related proteins CCNB1,P21 and DNA synthesis related protein RRM2);3.The anti-tumor effect of daphnetin on U251 cells may not be related to PUMA mediated apoptosis.Part Ⅲ Study on daphnetin induced autophagy of glioblastoma U251 cellsObjective:To investigate the autophagy of U251 cells induced by daphnetin and its related mechanism.Methods:After U251 cells were treated with daphnetin at different concentrations for 48 hours,the number and formation of autophagy bodies in U251 cells were observed by transmission electron microscope;The expression of autophagy related proteins mTOR,p62,beclin-1 and LC-3 were detected by Western blot.Results:Transmission electron microscopy showed that a large number of autophagy bodies could be observed in U251 cells in daphnetin treatment group,while autophagy bodies were rarely observed in control group;Western blot showed that daphnetin could significantly up regulate the expression of mTOR,Beclin-1 and LC-3 proteins and down regulate the expression of P62 protein in a dose-dependent manner.Conclusions:1.Daphnetin can induce autophagy in U251 cells,and its mechanism may be related to up regulating the expression of mTOR,Beclin-1 and LC-3 proteins and down regulating the expression of P62 protein;2.The anti-tumor effect of daphnetin on U251 cells may be related to its induction of autophagy.