Study On Network Prediction And Immune Effect Evaluation Of Cold Components Gentiopicroside And Picroside Ⅱ

The theory of four properties of traditional Chinese medicine is the core of the theory of Chinese herbal medical properties,and it is also an important basis to guide the clinical use of traditional Chinese medicine（TCM）.The material basis of the four properties of Chinese medicine originates from its material bias.The material bias of Chinese medicine originates from different sources of medicinal materials and different processing methods.The chemical composition contained in Chinese medicine is the material basis of Chinese herbal medicine’s cold and hot properties.Specific Chinese medicines often contain specific ingredients.Taking camphor and borneol,which are both composed of a single component,as an example,they have the same structural skeleton and only one valence bond is different,but camphor is hot and borneol is cold.In other words,a single chemical component can reflect the cold-heat nature,and also has the characteristics of matter.Then it raises a question,whether the components with the most content in Chinese medicine could have a more important impact on the cold-heat nature of TCM?Based on this assumption,the main active substances Gentiopicroside and Picroside Ⅱ in cold traditional Chinese medicine Gentian and Rhizoma Picrorhizae were taken as the research objects,and the network pharmacology method was introduced into the theoretical research on cold-heat nature of TCM.For the purpose of exploring whether the monomer of traditional Chinese medicine have properties and these properties through what targets and pathways expressed.Key targets were validated using molecular docking methods.Then introduce the immunoassay method to study the theory of cold-heat nature of TCM,in order to explore its material basis.Obtaining monoclonal antibodies lays the foundation for further research on cold-heat nature or for the establishment of cold-heat evaluation standards.Interpreting the scientific connotation of cold-heat nature of TCM by integrating multiple methods.Main achievements of this thesis are as follows.1.Validation and mechanism of cold properties of Gentiopicroside and Picroside Ⅱ:The potential targets of GPS and PIC were collected in TCMSP,PubChem and PharmMapper.The AURTI potential targets were screened in OMIM,DrugBank and GeneCards.The componentdisease intersection targets were obtained by Venny analysis,these targets were used to construct a PPI network and performed GO and KEGG enrichment analysis.Key targets were got by MCC calculation.The results showed that there were 18 targets for the prediction of GPS and PIC in the treatment of AURTI.The main biological process of GPS is apoptotic process and the main metabolic pathways are Apoptosis,p53 signaling pathway,Sphingolipid signaling pathway,NOD-like receptor signaling pathway and so on.The main biological process of PIC is apoptotic process and the main metabolic pathways are Prolactin signaling pathway,Estrogen signaling pathway,Fc epsilon RI signaling pathway and so on.2.Validation of the key targets of of Gentiopicroside and Picroside Ⅱ:The LibDock mode of Discovery Studio 2016 software was used to record the docking scores of GPS and PIC with their potential targets by molecular docking,and the binding energy was calculated.The AURTI model was induced by LPS（3 mg/kg）intranasally.The high and low dose GPS groups（200 mg/kg,100 mg/kg）and the high and low dose PIC groups（30 mg/kg,15 mg/kg）were administered for 5 days and the model was established 2 h after the last administration.After 24 h,the lung tissue was taken for RT-PCR to verify the mRNA expression levels of key factors in the above results.The results showed that GPS was successfully docked with 8 selected targets,including CASP8,CDK2 and CYCS,and had the best binding ability to CYCS.PIC was successfully docked with six screening targets,including HRAS,MAPK1 and MAPK3,and had the best binding ability to STK11.The AURTI mouse model was successfully constructed.RT-PCR showed that GPS could down-regulate the expression of MAPK1,MAPK3,HRAS,HSP90AA1 mRNA and up-regulate the expression of CYCS mRNA.PIC could down-regulate the mRNA expression of MAPK1,MAPK3,HRAS,HSP90AA1 and STK11.It indicated that GPS and PIC may act on multiple pathways to show coldness by regulating the expression of these genes.3.Synthesis of artificial antigens GPS-BSA and PIC-BSA:Synthesis of GPS-BSA and PC II-BSA artificial antigens by sodium periodate oxidation.After the artificial antigen was dialyzed,its protein content was measured.The successful coupling of artificial antigens was identified by UV full wavelength scanning and SDS-PAGE electrophoresis.The results showed that the two artificial antigens were successfully coupled.The concentrations of GPS-BSA and PIC-BSA were 3.12 mg/mL and 2.80 mg/mL respectively;After identification,the coupling ratio of GPS-BSA is about 13:1 and that of PIC-BSA is about 15:1.4.Preparation of polyclonal antibodies against Gentiopicroside and Picroside Ⅱ and screening of monoclonal cell lines:The immunized mice serum was obtained to screen the detection conditions of the coating solution,the blocking solution and the coating concentration,and then using the above screening conditions detect the mouse serum titer.Choosing the best mice for cell fusion by electrofusion and filter the cell lines which secreting corresponding antibodies in these hybridoma cells.The results showed that all the mice in the GPS group could undergo cell fusion except for the No.1 mouse.The fusion rate of mice No.2（1:20 000）in GPS group was about 83.06%,and the hybridoma cell line 3G9 that could secrete anti-GPS antibody was screened out.The monoclonal antibody was amplified by induced ascites induction method,and the titer of 3G9 ascites was over 24 000.In the PIC group,the titer of mice No.4 was the highest（close to 1:16 000）,and the cell fusion rate was about 80.56%.The anti-PIC antibody hybridoma cell line could not be screened.