Study On The Molecular Mechanism Of Oridonin Against The Proliferation Of Cervical Cancer Cells Based On Metabonomics

Research Background:Cervical cancer is one of the most common malignant tumors in women.Currently,chemotherapy and surgical treatment are common strategies for clinical treatment of cervical cancer.However,due to the serious side effects and postoperative complications of chemotherapy drugs,its clinical application has certain limitations.Therefore,the development of new and effective anti-tumor drugs with low toxicity and side effects has become a hot spot in the current tumor treatment research field.Oridonin is a tetracyclic diterpenoid natural organic compound with strong biological activity.It has strong antioxidant properties and can induce apoptosis of a variety of tumor cells.At present,the molecular mechanism of oridonin inhibiting the proliferation of cervical cancer Hela cells is still unclear.Based on this,this study uses systems biology technology to study the molecular mechanism of oridonin inhibiting the proliferation of Hela cells,in order to provide basic theoretical data support for oridonin in the treatment of cervical cancer.Research purposes:Based on the technical methods of network pharmacology and metabolomics,analyzed the small molecular differential metabolites and related metabolic pathways in Hela cells after the intervention of oridonin,and explained the molecular mechanism of oridonin inhibiting Hela cell proliferation at the molecular level,with a view to provide basic theory for oridonin to treat cervical cancer.Research methods:1.The MTT experiment was used to investigate the toxic effect of oridonin on Hela cells,and the Graph Pad Prism software was used to fit the IC₅₀ value of oridonin for inhibiting Hela cells.2.Use flow cytometry to detect the effects of oridonin on Hela cell cycle arrest,mitochondrial membrane potential and apoptosis.3.Use the method of network pharmacology to screen out the targets of oridonin through the Pharm mapper database,screen out the therapeutic targets for cervical cancer through the Gene Cards database,use Cytoscape software to construct a network for the common target of oridonin and cervical cancer,and use R software and DAVID database to perform GO function analysis and KEGG pathway analysis on the common target.4.UPLC-MS/MS-based metabolomics technology was used to analyze the effect of oridonin on small molecular metabolites in Hela cells.The compound was qualitatively performed by the small molecule qualitative software OSI/SMMS,the VIP value was calculated by SIMCA software,the differential metabolites were screened by VIP>1,P<0.05,and the differential metabolites were imported into the Metabo Analyst website to analyze the relevant differential metabolic pathways.5.Use derivatization reagent N-（1-pyridine）maleimide to derivatize cysteine,a small molecule compound that synthesizes glutathione in cells,and use UPLC-MS/MS to determine the intracellular cysteine content of the control group and the oridonin group.6.Enzyme-linked immunosorbent assay was used to detect the effect of oridonin on the activity of glutamate-cysteine ligase catalytic subunit（GCLC）and glutathione peroxidase 4（GPX4）in Hela cells.7.Use flow cytometry to detect the effect of oridonin on the content of ROS and ATP in Hela cells.Research results:1.MTT results show that with the increase of the concentration,the inhibitory rate of oridonin on Hela cells gradually increases,which is positively correlated.The IC₅₀ values of oridonin on Hela cells for 24 h,48 h and 72 h were 20.61μM,15.06μM and 11.88μM,respectively.As the concentration of oridonin increases,the ratio of Hela cells stagnating in G₂/M phase gradually increases,the mitochondrial membrane potential presents a significant downward trend,and the cell apoptosis rate gradually increases.2.The results of network pharmacology show that there are 280 oridonin targets and 838 targets for cervical cancer treatment,and a total of 75 targets were obtained after the intersection.Network visualization and protein-protein interaction network analysis of common targets were carried out,and AKT1,MAPK,EGFR and other core targets were obtained.In the results of GO enrichment analysis,biological functions include nuclear receptor activity,ligand-activated transcription factor activity,membrane receptor protein kinase activity,etc.KEGG pathway analysis results include glutathione metabolism pathway,m TOR signaling pathway,AMPK signaling pathway,cysteine and methionine metabolism.Non-targeted metabolomics model discriminant analysis results show that in the OPLS-DA model,the control group and the oridonin group are significantly separated,and there is no over-fitting phenomenon between the two groups.A total of 21 small molecular metabolites with significant differences were screened.The 15 metabolites with elevated levels include glutamine,tryptophan,phenylalanine,methionine,isoleucine,valine and citric acid.The 6 metabolites with reduced levels include glutathione,5-oxoproline and glutamic acid.The results of enrichment analysis and metabolic pathway analysis showed that the intracellular glutathione metabolism,the metabolism of glutamine and glutamate,the metabolism of valine,leucine and isoleucine,tricarboxylic acid cycle,cysteine and methionine were significantly changed after oridonin treatment.3.Combined with the results of network pharmacology and metabolomics,oridonin may inhibit the proliferation of Hela cells by affecting the anabolism of glutathione.The content of cysteine,the upstream compound of synthetic glutathione,showed that the content of cysteine in the cell was significantly reduced after the action of oridonin;the results of enzyme-linked immunoassay showed that compared with the control group,the activity of GCLC,a key enzyme for glutathione synthesis in the oridonin group,was significantly reduced,indicating that oridonin inhibited the synthesis of glutathione in Hela cells.And oridonin can significantly reduce the activity of glutathione peroxidase-4（GPX4）that maintains the redox balance in the body,and increase the ROS content in Hela cells,it shows that oridonin can increase the level of oxidative stress in Hela cells and induces Hela cell apoptosis.ATP content measurement results show that after 2 h of oridonin treatment,the intracellular ATP content has been significantly reduced,within 2-12 hours,the intracellular ATP content was significantly lower than that of the control group,indicating that oridonin can inhibit Hela cellular energy production.Research conclusions:In this study,network pharmacology combined with metabonomics technology was used to analyze the mechanism of oridonin inhibiting the proliferation of cervical cancer Hela cells at the molecular level.Oridonin mainly reduces the cysteine content in Hela cells,reduces the synthesis of glutathione,increases the level of intracellular ROS,increases the level of intracellular oxidative stress,and induces Hela cell apoptosis.