Effect Of Epigallocatechin Gallate On The Activity And Expression Of Dipeptidyl Peptidase-4 In Non-alcoholic Fatty Liver Disease

Objective: Non-alcoholic fatty liver disease(NAFLD)is a major health problem worldwide.However,there is no effective treatment for NAFLD.In-depth research shows that epigallocatechin gallate(EGCG)is a major biologically active compound extracted from green tea and has shown beneficial effects in the treatment of NAFLD.However,the specific molecular mechanism of EGCG to improve the disease is not clear.This study mainly explores the possible target of EGCG to improve NAFLD.Methods: 1.Target protein acquisition: initially,the Protein Data Bank was searched for crystal structures with EGCG as a co-crystallized ligand.In order to further expand the search range,The EGCG flavonoid scaffold was taken as a substructure,and the PDB was searched for proteins co-crystallized with this structure.Proteins obtained in the above way were retrieved in the Pubmed database to find out which protein was NAFLD-related.2.Molecular docking: molecular docking between target protein and EGCG was performed.3.Molecular dynamics simulation: to further explore the combination and stability of the complex,the molecular dynamics simulation was performed.4.Enzyme activity assay: DPP4 inhibitor sitagliptin was used as a positive control,and the DPPIV-Glo? Protease kit was used to determine the effect of tea polyphenols such as EGCG on DPP4 activity.5.Cell experiment verification: culture human Hep G2,use oleic acid to induce NAFLD cell model,and then intervene with EGCG,divided into three groups: control group(NC),oleic acid group(OA),oleic acid + EGCG group(OA + EGCG).After the intervention,oil red O staining was used to observe the intracellular lipid content,the triglyceride kit was used to determine the intracellular triglyceride content,and the DPP4 expression level of each group was measured by real-time PCR.Results: 1.Target protein acquisition: five target proteins bound to EGCG were obtained by ligand search,and were not related to NAFLD.The substructure search showed that 17 compounds contained the flavonoid structure.These 17 compounds co-crystallized with 40 proteins.After screening,only dipeptidyl peptidase 4(DPP4,PDB ID: 5J3J)was NAFLD-related.2.Molecular docking: the molecular docking results showed that amino acids Glu205,Glu206,Pro550,Cys551,Arg669 and EGCG formed hydrogen bonds.In addition,?-? and ?-cations were formed between DPP4 and EGCG.3.Molecular dynamics simulation: the root-mean-square deviation(RMSD)of protein backbone atoms can be used to monitor the stability of the complex structure.The simulation results showed that the RMSD value of the main chain of DPP4 protein increased from 1.2 ? and gradually stabilized at 2.6 ?,which means that the protein did not show large fluctuations during the 100 ns simulation.EGCG bound to residue GLU206 throughout the 100 ns simulation.Other residues that had significant interactions with EGCG were VAL207,PHE357,PRO550,TYR662,ASP663,TYR666 and TYR670.When the system became stable,the stability of the complex interaction was analyzed.Hydrogen bonds and hydrophobic interactions played an important role in the binding of EGCG and DPP4.4.Enzyme activity assay: the effects of EGCG and four other tea polyphenols(C,EC,EGC,ECG)on the activity of DPP4 were measured using the DPPIV-Glo? Protease kit.Half maximal inhibitory concentration(IC50)values were determined by non-linear regression in Graph Pad Prism 8 software.The results showed that EGCG has the powerful inhibitory effect on DPP4,and the concentration of EGCG required to inhibit DPP4 activity by 50%(the IC50 value)was 28.42 ?M.5.Cell experiment verification: The results showed that the content of TG in OA group was higher than that in NC group.After EGCG intervention,the content of TG in OA+EGCG group was lower than the OA group,and the difference were all achieve statistically significant.The results were in line with the results of oil red O staining.PCR results showed that the expression of DPP4 in the OA group was higher than that in the NC group after OA induction.After EGCG intervention,compared with the OA group,DPP4 expression was reduced in the OA + EGCG group.These results indicate that EGCG reduces lipid deposition and DPP4 expression in the NAFLD cell model.Conclusion: 1.Molecular simulation results showed that EGCG bound to key amino acid residues Glu205,Glu206,Pro550,Phe357,and Tyr666 in the active site of DPP4 protein through hydrogen bonding and hydrophobic interaction.2.EGCG can inhibit the enzyme activity of DPP4 and reduce the expression of DPP4 in NAFLD cell model.