Study On The Anti-tumor Active Ingredients Of Cortex Fraxini And Its Molecular Mechanism Of Inhibiting Warburg Effect

Objective: Based on glycolysis pathway,the potential interaction between target protein and target protein of th e active components of Cortex Fraxini was explored,and the anti-tumor mechanism and target protein interaction were revealed.Methods: In this paper,the ethanol extract of Cortex Fraxini was divided into petroleum ether extract,chloroform extract,ethyl acetate extract and n-butanol extract by systematic solvent method.Then MTT method was used to screen the anti-tumor activity of each extract with the survival rate of Hep G2 cells as the research index.The chemical constituents of the anti-tumor extract of Cortex Fraxini were isolated and identified by LC-MS.Then,ultrafiltration centrifugation combined with LC-MS analysis was used to screen the active components that interact with the total protein of Hep G2 cells.High-throughput sequencing technique was used to determine the transcriptional group of the Hep G2 cell intervened b y the active extract of Cortex Fraxini,to analyze the effect of the active extract on tumor cell transcriptional group,and to determine the target protein and molecular pathway of the anti-tumor effect of Cortex Fraxini.By using the method of network ph armacological analysis,the biological network of "component-target-pathway" of the anti-tumor effect of Cortex Fraxini was constructed to find the potential target proteins.The correlation between proteins was speculated by STRING analysis and literature review,combined with computer virtual docking technology to predict the binding and key active components of target proteins and compounds.The binding constants between active components and target proteins,target proteins and target proteins were dete rmined by MST technique.Combined with the results of MST and Co-IP,the effects of the active compound between target proteins and target proteins and the binding between target proteins and antibodies were studied,so as to speculate the binding sites and active groups between active compound and target proteins.The effects of active components of Cortex Fraxini on the growth of tumor cells and transplanted tumor in mice were investigated by MTT method and H&E,and the anti-tumor effect was determined.The changes of glucose utilization and lactic acid production of Hep G2 cells and transplanted tumor before and after administration were detected by kit,and the effect of drugs on glycolysis rate was calculated.The effect of scopolin on the regulation of glycolysis-related proteins was determined by immunohistochemistry and Elisa test.Results: Taking Hep G2 cells as subjects,the ethyl acetate extract of Cortex Fraxini was determined by MTT method,and 14 compounds such as esculetin were identified by LC-MS.The results of transcriptome study on Hep G2 cells interfered with ethyl acetate showed that the main biological functions and signal pathways of differentially expressed genes were related to glycolysis.The results of ultrafiltration centrifugation combined with LC-MS analysis showed that the potential anti-tumor active components of Cortex Fraxini included 8 compounds such as esculin,esculetin,scopolin and so on.Through network pharmacology,it was found that GPI,GPD2,PGK2,FASN and heat shock protein 90 alpha subunit(Heatshockprotein90 ?,HSP90 ?)may be the target proteins of esculin and scopolin.The binding constants and binding sites of active compounds t o target proteins were determined by computer virtual docking technique,and the binding was verified by MST technique.At the same time,it was found that there was a strong binding between GPI and GPD2,FASN and GPI,and the MST curve between FASN and GPI was positive s-shaped,indicating that the conformation of FASN or GPI changed greatly due to the binding between FASN and GPI.Co-IP test confirmed that the active component scopolin only affected the interaction between GPI and FASN,but had no effect on the binding of GPI and GPD2.The results of MST assay also showed that scopolin interfered with the binding of GPI and FASN mainly by changing the spatial structure of FASN.According to the phenomenon that scopolin interferes with the binding of FASN p rotein and its antibody,combined with the results of computer virtual molecular docking,it is inferred that the binding site of scopolin and target protein FASN is located near the glycine(Glycine,GLY46)residue of the thioesterase domain on the three-dimensional structure of FASN protein.The results of cell test in vitro and tumor-bearing mice showed that scopolin could significantly reduce the glucose consumption and lactic acid production of tumor cells and tumor tissue,and reduce the rate of glycol ysis of tumo r.The results of Elisa and immunohistochemistry showed that high and middle doses of scopolin could significantly increase the expression level of glycolysis pathway and related proteins,which may be related to the inhibition of the activity of related proteins and the inhibition of glycolysis metabolism and the threat of tumor growth,which may be related to the compensatory increase of glycolysis key proteins expression after scopolin binding to target proteins.Conclusion: 1.Scopolin and esculetin are the main effective components of the anti-tumor effect of Cortex Fraxini.2.GPI,GPD2,PGK2,FASN and HSP90 ? proteins are the main targets of esculetin and scopolin,which are the anti-tumor active components of Cortex Fraxini.3.Esculetin a nd scopolin exert their anti-tumor effect mainly by inhibiting the activity of glycolysis key proteins GPI,GPD2,PGK2,FASN and HSP90 ?.The effect of scopolin on the interaction between GPI and FASN proteins provides a basis for exploring a new anti-tumor mechanism.