Study On Molecular Mechanism Of Oridonin Against Esophageal Cancer Based On Metabolomics

Background:Esophageal cancer is a common malignant tumor of the digestive tract and the sixth leading cause of cancer-related death worldwide.China is one of the countries with a high incidence of esophageal cancer,especially in Henan,Sichuan,and Fujian provinces.Rabdosia rubescens(Hemsl.)Hara was first discovered in Wangwu Mountain,Jiyuan City,Henan Province,and it has a unique therapeutic effect on esophageal cancer.Studies have shown that the tetracyclic diterpenoids in Rabdosia rubescens,oridonin,is the main anti-cancer active ingredient of Rabdosia rubescens,which has a good inhibitory effect on the growth of various cancer cells.At present,the anti-esophageal cancer effect of oridonin has been extensively studied at the level of genes and proteins and other biological macromolecules,while few people have systematically studied the molecular mechanism of oridonin's anti-esophageal cancer effects from the perspective of small molecule metabolism.Objective:From the perspective of systems biology,the molecular mechanism of oridonin's anti-esophageal cancer effect was studied using UPLC-MS/MS metabolomics technology,with a view to providing a theoretical basis for the clinical application of oridonin and the development of anticancer drugs.Methods:1.The effect of oridonin on TE1 cells activity was studied by MTT experiment;2.The effect of oridonin on TE1 cells apoptosis,cell cycle and intracellular reactive oxygen(ROS)levels were studied using flow cytometry;3.Intracellular Fe2+ content and malondialdehyde(MDA)content determination,as well as the effect of ferroptosis inhibitor deferoxamine on cell viability,were used to study the effect of oridonin on ferroptosis in TE1 cells.4.UPLC-MS/MS metabolomics technology was used to detect endogenous small molecule metabolites in TE1 cells samples of oridonin group and control group;Small molecule compound rapid identification and analysis software OSI-SMMS was used to identify and analyze metabolites.The pattern discriminant analysis was performed by SIMCA software;The differential metabolites between the control group and the oridonin group were screened according to VIP>1 and P<0.05,and Metaboanalyst website was used to analyze the metabolic pathways.5.The activities of y-glutamyltransferase 1(GGT1),glutamylcysteine ligase catalytic subunit(GCLC),and glutathione peroxidase 4(GPX4)were analyzed by enzyme-linked immunosorbent assay(ELISA).6.High-resolution mass spectrometry was used to confirm the structure of the conjugates of oridonin with cysteine,glutathione,and y-glutamylcysteine,respectively.7.UPLC-MS/MS was used to analyze the contents of oridonin conjugates with cysteine,glutathione and ?-glutamylcysteine,respectively.Meanwhile,the intracellular GSH and GSSG were measured and the ratio of GSH/GSSG was calculated.8.Fluorescence microscopy was used to take fluorescent pictures to analyze the effect of oridonin on mitochondrial membrane potential and intracellular ROS level in TE1 cells.Results:1.Inhibitory effect of oridonin on the proliferation of esophageal cancer cell TE1:MTT experiments showed that the IC50 values of oridonin on TE1 cells after 24 h and 48 h were 26.93 ?M and 18.95 ?M.Flow cytometry analysis revealed that oridonin significantly induced apoptosis and induced cell arrest at G2/M phase of TE1 cells.Experimental results related to ferroptosis showed that ferroptosis inhibitor deferoxamine can significantly reduce the inhibitory effect of oridonin on TE1 cells.At the same time,oridonin increased intracellular Fe2+,MDA and ROS levels in a dose-dependent manner.This indicated that oridonin can induce ferroptosis in TE1 cells.2.Inhibitory effect of oridonin on y-glutamyl cycle in TE1 cells:The results of model discriminant analysis showed that there was no overfitting in the model established in this experiment.OPLS-DA analysis showed a clear separation between the control group and the oridonin group.According to the criteria of VIP>1 and P<0.05,a total of 23 endogenous metabolites with significant differences were screened between the control group and the oridonin group,including GSH,GSSG,glutamate,5-oxyproline,?-glutamylleucine,y-glutamylvaline,glutamine,methionine,and the like.Pathway analysis and enrichment analysis showed that these differential metabolites were mainly involved in glutathione metabolism,glutamate metabolism,phenylalanine and tyrosine metabolism and arginine metabolism,among which glutathione metabolism was the most significant.The results of enzyme-linked immunosorbent assay showed that the activities of key enzymes GGT1 and GCLC in the y-glutamyl cycle(glutathione salvage pathway)were significantly reduced in the oridonin group,indicating that oridonin can significantly inhibit the y-glutamyl cycle in TE1 cells.Meanwhile,the activity of GPX4 was significantly reduced after the action of oridonin,indicating that the glutathione redox balance in TE1 cells was imbalanced.3.Oridonin inhibits the y-glutamyl cycle by covalently binding cysteine thiol:By comparing the retention time and secondary spectra with the chemically synthesized oridonin conjugates,it was confirmed that oridonin can covalently bind to cysteine,glutathione,and y-glutamylcysteine in TE1 cells to form conjugates Ori-Cys,Ori-GSH and Ori-Glu-Cys,respectively.And the results showed that this binding effect was significant after the treatment with oridonin for 2 h.UPLC-MS/MS analysis showed that the content of GSH and GSSG and the ratio of GSH/GSSG in the cells were significantly reduced after 2 h of oridonin treatment.At the same time,the results of ROS fluorescence photographs showed that with the prolonged action time of oridonin,the level of ROS gradually increased.This indicated that oridonin covalently binds to the cysteine thiol to inhibit the synthesis and activity of GSH,thereby inhibiting the ?-glutamyl cycle.Conclusions:1.Oridonin can inhibit the proliferation of TE1 cells by inducing apoptosis,promoting cell cycle arrest in the G2/M phase,and inducing ferroptosis.2.Oridonin inhibits the y-glutamyl cycle by reducing the activity of GGT1 and covalently binding to cysteine thiol,which leads to an imbalance of intracellular glutathione redox balance and eventually inhibits the proliferation of TE1 cells.