| The World Health Organization has named obesity as a disease.Obesity poses a growing threat to humanity,leading to chronic diseases such as diabetes,high blood pressure,and impaired heart function,which are a serious threat to the health of obese people of all ages.Therefore,the treatment and mechanism research of obesity have become an urgent problem in research.Drug targets,an important part of drug development,can provide a breakthrough for drug screening.In recent years,some emerging search techniques for drug targets have been extended.This brings the gospel to obese patients.The experiment mainly carried out preliminary identification and verification of the weight loss target of phillyrin.Firstly,the weight loss effect of phillyrin was reconfirmed at the cellular level.Secondly,the phillyrin target of weight loss was predicted by network pharmacology.Then,the phillyrin target was found using the DARTS combined with electrophoresis and the interaction between phillyrin and PDE3 B was studied by the molecular docking.Finally,based on the analysis of the above research and literature research,it was speculated that the weight loss target of phillyrin might be PDE3 B,and the verification experiment was carried out from the cellular level and molecular level respectively.The main research contents and results are summarized as follows:1.Reconfirm the weight loss effect of phillyrin.The effect of phillyrin on weight loss was studied by the method of MTT and fluorescent staining.The results showed that phillyrin had no effect on the survival rate of Hep G2 cells at a concentration of 1-100 μM.Then Hep G2 cells were incubated with DMEM high glucose medium(30 m M D-glucose)to construct a cell model of lipid accumulation,and the cells were incubated with different concentrations of phillyrin.Hep G2 hepatocytes were stained with Nile Red,and the fluorescence intensity was decreased with the increase of phillyrinIV concentration(1.25,2.5,5 μM).When the concentration of phillyrin was ≥ 5μM,the fluorescence of Hep G2 cells was remained basically unchanged.The above studies indicated that phillyrin can inhibit the lipid accumulation induced by high glucose in Hep G2 hepatocytes.That was phillyrin does have a weight-loss effect at the cellular level.2.Virtual screening of phillyrin potential weight loss targets.Screening was carried out using the network pharmacology.First,the phillyrin targets(component target)was predicted by the Pharm Mapper method,and then compared with the obese targets(disease target)screened by Gene Cards and Coo LGe N database,with targets interaction network constructed by Cytoscape software.The GO and KEGG pathways involved in the targets were analyzed by Clue GO software.The results showed that 27 targets of phillyrin were predicted.The biological process analysis indicated that they mainly involved in the regulation of fibroblast proliferation,pancreatic juice secretion,nitric oxide-mediated signal transduction,superoxide metabolism,I-κB kinase/NF-κB signaling,retinoic acid receptor signaling,fatty acid metabolism,biosynthesis of unsaturated fatty acids and secretion and transportation of chitosan.The molecular function analysis indicated that the relevant targets were mainly participated in nuclear receptor activity,affected oxidoreductase activity,acted as a receptor in peroxidation,and binded to retinoid X receptors.And the KEGG pathway analysis indicated that the predicted targets involved the small cell lung cancer pathway and the regulation of lipolysis in adipocytes pathway.In addition,PDE3 B is one of27 targets.3.Experimental screening of phillyrin target in losing weight.The DARTS combined with SDS-PAGE electrophoresis was used to find the phillyrin targets.The experiments were explored from the concentration of pronase E,hydrolysis duration and hydrolysis temperature.The results showed that no significant unhydrolyzed protein bands were observed in the DARTS/SDS-PAGE bands.After analyzing the results,the technique of DARTS was more difficult to find low-abundance target proteins.And afterresearching literatures and combining network pharmacology,it was speculated that PDE3 B might be a potential weight loss target for phillyrin.According to the comparison of Total-Score displayed by molecular docking,it was concluded that the interaction ability of phillyrin with PDE3 B was stronger than that of milrinone of PDE3 inhibitor.Meanwhile,there were hydrogen bonds formation between phillyrin and PDE3 B.Therefore,phillyrin were most likely a potential inhibitor of PDE3 B.4.Verification of phillyrin target in losing weight.Verification was performed at the cellular level and molecular level,respectively.At the cellular level,it was first verified whether phillyrin and milrinone competed for the same target PDE3 B.Secondly,it was verified whether phillyrin could inhibit the target PDE3 B by ELISA kit,thereby improving the c AMP content.At the molecular level,the PDE3 B was synthesized.And p ET-28a-PDE3 B was constructed and the expression,refolding,purification and validation of PDE3 B protein were explored.The results showed that milrinone inhibited the accumulation of lipids in Hep G2 hepatocytes,and phillyrin competed with milrinone for the same active site.Phillyrin increased the c AMP content in hepatocytes.Therefore,phillyrin might a potential inhibitor of PDE3 B.That was PDE3 B might be a weight loss target for phillyrin.PDE3 B contained more hydrophobic amino acids,so it was mainly in the form of inclusion bodies after induction at different IPTG concentration,temperature and Ca2+ concentration in E.coli prokaryotic expression system.Refolding and molecular level validation experiments for PDE3 B proteins are still in progress. |
