Study On The Material Basis And Mechanism Of Action Of Jinrong Granules Against Breast Cance

Objective: Jin Rong Granule(JRG)is an innovative traditional Chinese medicine drug for the treatment of breast hyperplasia and adjuvant treatment of breast cancer,which is composed of ten traditional Chinese medicines such as Epimedii Folium,Curcumae Radix,Cistanches Herba.It can tonifies the kidney and promotes blood circulation,eliminates phlegm and disperses stagnation,and coordinates Chong and conception channels,exerted multiple beneficial effects on the human body.Pharmacological studies have shown that JRG inhibits the growth and proliferation of tumor cells by regulating the level of estrogen and the release of inflammatory factors,but its pharmacodynamic material basis and mechanism of action for the treatment of breast cancer are still unclear.Therefore,aiming at the above problems,this study analyzed JRG and its serum components;Guided by the anti-Br Ca activity,on the basis of the analysis of blood components,network pharmacology combined with molecular docking was used to study the potential pharmacodynamic components and mechanism of JRG against Br Ca;Then the activity of the pharmacodynamic components was predicted by in vitro cell experiments,and the potential mechanism of action was preliminarily explored.Through the above research aiming to clarify the pharmacodynamic material basis of JRG in treating Br Ca,and provide a theoretical basis for expanding the clinical application and drug safety of JRG.Methods:1.Identification of JRG and its serum components: The chemical composition database of JRG was established.The sample solution of Jinrong granules was detected by UPLC-Q-TOF-MS/MS,and 14 components were selected for comparison with the control index components and key components of each medicinal herb.SD rats were selected and JRG drug serum and blank serum were prepared by administering JRG and normal saline respectively;Establish a qualitative analysis method for JRG and its serum components based on the UPLC-Q-TOF-MS/MS: Agilent ZORBAX Eclipse Plus C18(3.0× 100 mm,1.8μm);Gradient elution was carried out with acetonitrile-0.05mmol/L ammonium formate aqueous solution,and ESI ionization was used to collect mass spectrometry information of chemical components in JRG and its medicinal serum in positive and negative ion modes.Qualitative and quantitative analysis of JRG and its serum components were conducted using the analysis and processing function of the Agilent Mashhunter Qualitative Analysis workstation.2.Network Pharmacology and molecular docking study of JRG: According to the concentration of components in blood and the activity of chemical components,Stachydrine,Magnoflorine,Echinacoside,Icariin,Baohuoside I,Cryptotanshinone,Tanshinone I,Tanshinone IIA,Emodin,Salidroside,β-Elemene and Curdione were selected for network pharmacological analysis.Pub Chem and Swiss Target Prediction databases were used to predict the action targets of potential active ingredients of JRG.Malacards,Genecards,Dis Ge NET,CTD,and OMIM were searched to obtain breast cancer related targets.Venny V2.8 software and Cytoscape 3.9.0 were used for topological analysis to screen the potential targets of JRG in the treatment of Br Ca.The g:Profiler and Omicshare databases were used for GO and KEGG enrichment analysis of potential targets.The network diagram of " Traditional Chinese Medicine-Main Active Ingredients-Potential Targets-Pathways" was constructed,and the key targets,effective components and potential pathways were analyzed and obtained.Py MOL software and Auto Dock Tools1.5.7 software were used to molecularly dock the main active ingredients and key targets of JRG.The binding energy and hydrogen bond formation were used as indicators to judge the spontaneous binding ability of the target and key components.3.Validation of action of JRG serum and its mechanism of anti-breast cancer: The pharmacodynamics of potential pharmacodynamic components of JRG,such as Magnoflorine,Echinacoside,Icariin,Baohuoside I,Tanshinone IIA,Emodin,Salidroside,and Curdione were validated.With human breast cancer cell line MDA-MB-231 and mouse breast cancer cell line4T1 as the research objects,the CCK8 method was used to determine the effect of JRG drug serum and its components on the proliferation activity of breast cancer cells,and calculate the IC50 values of each component,as the intervention concentration for follow-up mechanism research.The expression of EGFR,RAS,MEK1,PI3 K,m TOR proteins after drug intervention was determined by western blotting.The above experiments were used to verify whether JRG serum and its potential active ingredients can inhibit breast cancer by regulating EGFR-PI3 K and Ras-MAPK signaling pathways.Results:1.The composition database of JRG was established,and a total of 1367 chemical components were obtained.A total of 99 compounds were identified from the sample solution of JRG,36 from Epimedium brevicomu Maxim,including flavonoids;12 from Cistanche deserticola Y.C.Ma,including phenylethanol glycosides,iridoid glycosides and lignans;23 from Salvia miltiorrhiza Bge,including diterpenoids and phenolic acids;17 from Ligustrum lucidum Ait,including iridoids,phenolic acids,and flavonoids.On this basis,56 prototype components in JRG serum were identified,among which are mainly flavonoids,diterpenes and phenolic acids,and the blood concentration of flavonoids is higher.2.22794 gene targets related to Br Ca were retrieved from Malacards,Gene Cards and other databases.A total of 309 related targets were obtained by using Pub Chem and Swiss Target Prediction databases to predict the action targets of potential active ingredients in JRG.308 targets intersected with each other.After analysis and screening,21 proteins were identified as key target proteins.Go functional enrichment analysis yielded 35 items,suggesting that treatment of Br Ca with JRG is related to the regulation of multiple pathways,as well as the regulation of cell proliferation,survival,migration and metabolism,platelet activation and leukocyte migration.A total of 44 pathways were obtained through KEGG pathway analysis,including 23 pathways closely related to breast cancer,such as VEGF pathway,Erb B pathway,Estrogen pathway,m TOR pathway and PI3K/Akt pathway.Combined with PPI network protein screening and KEGG screening,a total of six target proteins closely related to the occurrence and development of breast cancer were obtained: EGFR,PI3 K,MEK1,RAS,Akt,m TOR protein,which were respectively docked with potential components such as Stachydrine,Magnoflorine,Echinacoside,Icariin,Baohuoside I,Cryptotanshinone,Tanshinone I,Tanshinone IIA,Emodin,Salidroside,and Curdione,and all of them could spontaneously bind.It is suggested that these components may have anti-Br Ca activity,and they are potential effective components of JRG in the treatment of Br Ca,and JRG mechanism of action is closely related to the regulation of PI3K/Akt signaling pathway and RAS/MAPK signaling pathway.3.Validation of action of JRG serum:(1)MDA-MB-231 cells: after intervention with different percentage of JRG serum,the cell proliferation rate slows down.When the percentage of drug serum reaches 10%,the inhibition rate of cells exceeds 50%.Icariin,Baohuoside I,Magnoflorine,Echinacoside,Tanshinone IIA,Emodin,Curdione,Salidroside intervened cells respectively,and the IC50 of each component was 6.627,17.510,4.710,157.900,6.564,14.230,6.163,and 93.784μM.(2)4T1 cells: JRG serum intervenes cells.When the serum content is 2%,it has inhibitory effect on cells,and when the content reaches 10%,the inhibitory rate on cells exceeds 50%;Icariin,Baohuoside I,Magnoflorine,Echinacoside,Tanshinone IIA,Emodin,Curdione,Salidroside intervened the cells respectively,and the IC50 of each component was 2.136,3.263,12.900,4.132,2.209,15.250,0.113,and 79.785 μM.JRG mechanism of anti-breast cancer: The protein expressions of RAS,MEK1,PI3 K,EGFR and m TOR were significantly down regulated in MDAMB-231 cells and 4T1 cells treated with JRG serum.Icariin,Baohuoside I,Magnoflorine,Echinacoside,Tanshinone IIA,Emodin,Curdione,Salidroside intervened MDA-MB-231 cells and4T1 cells,respectively.Magnoflorine,Echinacoside,Tanshinone IIA significantly downregulated PI3 K,EGFR,and m TOR proteins.After Icariin intervention,MEK1,PI3 K,EGFR,and m TOR expressions were downregulated,but had no significant effect on Ras.Baohuoside I,Tanshinone IIA,Emodin,Curdione,and Salidroside all have a downregulation effect on RAS,MEK1,PI3 K,EGFR,and m TOR,which is significantly different from the blank group.Conclusion: In this experiment,a UPLC-Q-TOF-MS method for the detection of JRG and its serum components was established,and the chemical composition of JRG was preliminarily clarified;The potential pharmacodynamic components and signal pathways of JRG against Br Ca were predicted;According to the in vitro cell experiment,the inhibitory activity of JRG on Br Ca may be related to the inhibition of RAS,MEK1,PI3 K,EGFR,m TOR protein expression,and may participate in the regulation of PI3K/Akt/m TOR signaling pathway and RAS/MAPK signaling pathway.Among them,Icariin,Baohuoside I,Magnoflorine,Echinacoside,Tanshinone IIA,Emodin,Curdione,and Salidroside may be the main pharmacodynamic components of JRG.The experimental results provide a scientific foundation for the formulation of quality standards that reflect the precise treatment and efficacy of JRG in clinical practice.