Studies On The Effect Of 14-3-3ζ Protein In The Anti-depressive Behavior Of Mice By Protopanaxadiol

It is recorded in "Shen Nong’s Materia Medica" that ginseng can "mainly nourish the five internal organs,calm the spirit,calm the soul,and stop panic." In recent years,multiple studies at home and abroad have confirmed that ginsenoside,the main active ingredient of ginseng,can antagonize the plastic damage of hippocampal structure caused by long-term stress and improve the depression-like behavior of model animals.However,compared with the research on the antidepressant mechanism of ginsenosides,the search for the target of antidepressant proteins in the brain of ginsenosides is relatively scarce,which needs to be discovered and clarified.In the research of this paper,we discovered a potential target 14-3-3^protein in the brain tissue through the direct affinity "fishing" technology of total ginsenosides.Biolayer interferometry(BLI)further discovered that protopanaxadiol,a metabolite of ginsenosides in vivo,has a direct interaction with 14-3-3ζ protein,suggesting that 14-3-3ζ protein may be a potential protein in the brain for protopanaxadiol directly act on the target.Therefore,the target verification of 14-3-3ζ protein through a variety of technical methods is the primary research goal of this thesis.On the basis of determining it as a target,further explaining the antidepressant mechanism of protopanaxadiol based on this target is the secondary research goal of this thesis.Aiming at the above two research goals,the designed research content and the experimental results obtained are as follows:Research contents:1.Primary screening of ginsenoside protein targets in the brain(1)The total saponins of ginseng are used as the "ligand" to connect to the solid-phase carrier through its functional group(glycosyl),and the protein in the brain tissue is fished out using the direct affinity "fishing" method.Thus,the protein target of ginsenoside in the brain was discovered.The experimental steps mainly include:preparation of affinity resin,affinity "fishing",protein electrophoresis analysis and subsequent protein identification by mass spectrometry.(2)The 14-3-3ζ His fusion protein was obtained by the construction of prokaryotic expression vector and the induction and purification of the fusion protein.Then use BLI to detect the direct interaction between ginsenoside monomer and sapogenin and 14-3-3ζ protein.Thus it is clear whether there is a direct interaction between the suspected target protein 14-3-3ζ and ginsenoside,and which ginsenoside monomer or sapogenin has the strongest direct interaction with 14-3-3C protein.2.Determination of affinity constant of protopanaxadiol and 14-3-3ζ protein and analysis of binding mode(1)The BLI kinetic method was used to determine the intermolecular interaction between the strongest direct acting protopanaxadiol and His-14-3-3ζ fusion protein.Determine the kinetic parameters such as the affinity constant,binding constant and dissociation constant of the protopanaxadiol and His-14-3-3ζ fusion protein.(2)Isothermal titration calorimetry(ITC)was used to detect the interaction between protopanaxadiol and 14-3-3ζ protein to obtain the thermodynamic parameters of the combination of the two.(3)X-ray crystallography is one of the main methods for studying the three-dimensional structure of biological macromolecules or biological macromolecule-compound small molecule complexes.Using protein structure information can explain the interaction between protein molecules and small substrate molecules,and clearly reveal the binding mode and biologically active conformation.In this research,the method of co-crystallization of traditional Chinese medicine monomer-protein was used to study the binding mode between protopanaxadiol and 14-3-3ζprotein.(4)The three amino acid residues Arg56,Arg127 and Tyr128 on the 14-3-3ζ protein found in the co-crystal structure analysis process combined with protopanaxadiol were subjected to site-directed mutations using overlap extension PCR technology.Then the BLI technology was used to detect the affinity between the mutant and protopanaxadiol,so as to confirm the binding site of protopanaxadiol and the 14-3-3ζ protein.3.Target pathway based on 14-3-3ζ protein glycogen synthase kinase 3β(GSK3β)-cAMP response element binding protein(CREB)-brain-derived neurotrophic factor(BDNF)to explain the antidepressant mechanism of protopanaxadiol(1)Using molecular docking and BLI technology to study the affinity between 14-3-3ζ protein and GSK3β.The competition method was used to detect the effect of protopanaxadiol on the affinity of GSK3P and 14-3-3ζ protein.BLI combined with Co-IP technology to analyze the effect of protopanaxadiol on 14-3-3ζ protein binding to GSK3β Ser9 phosphorylation site.Finally,in vitro analysis of the effect of protopanaxadiol and 14-3-3ζ protein binding on GSK3P enzyme activity.In order to explain in vitro whether the binding of protopanaxadiol and 14-3-3ζ protein affects the binding of 14-3-3ζ protein to GSK3β and the enzyme activity of GSK3β.(2)Prepare a mouse depression model to explore how protopanaxadiol can regulate the GSK3β-CREB-BDNF signaling pathway to play an antidepressant effect by binding to 14-3-3ζ protein.A mouse depression model was prepared by repeatedly injecting corticosterone.After giving fluoxetine and high,medium and low protopanaxadiol treatments,forced swimming test(FST)and tail suspension test(TST)to detect depression-like behavior in mice.Real-time PCR detects the mRNA expression level of BDNF and neurofilament light chain(NF-L).Western Blot technology detects the protein expression of GSK3β,p-GSK3β,CREB,p-CREB and BDNF.And the enzyme activity of GSK3β was determined by spectrophotometry.Research results:1.In the brain tissue homogenate,there are 10 protein strips that have affinity with the high-purity ginsenoside affinity resin.After removing the same bands as the blank control group resin,there are 8 bands in the total ginsenoside group.And after identifying these bands by protein mass spectrometry,five highly suspicious proteins were obtained:14-3-3ζ protein(gi | 1433Z-MOUSE,mass:27925),14-3-3ε protein(gi | 1433Z-MOUSE,Mass:29326),actin(gi | ACTG,mass:42108),type B creatine kinase(gi | KCRB,mass:42971)and ATP synthase subunit beta(gi | ATPB,mass:56265).BLI detects the direct interaction between ginsenoside monomer and sapogenin and 14-3-3ζ protein.Only the protopanaxadiol and 14-3-3ζ protein have a strong direct interaction.2.The BLI kinetic method detected that the affinity between protopanaxadiol and 14-3-3ζ protein was 4.68×10-5 M.The ITC technology determines that the affinity between the two was(3.10±0.20)×10-6 M.Crystallographic studies have shown that protopanaxadiol directly binds to the active site of 14-3-3ζ protein,and the main interaction between them occurs at residues R56,R127 and Y128 of 14-3-3ζ protein.Furthermore,the overlap extension PCR technology was used to obtain three mutants of the active site in the structure analysis.The affinity between protopanaxadiol and wild 14-3-3ζ protein and 14-3-3ζ single amino acid mutants were detected by BLI technology.The results showed that:mutation of any one of the three amino acids will result in a significant decrease in the affinity between protopanaxadiol and 14-3-3ζ protein.It is implied that 14-3-3ζ is the target protein of protopanaxadiol,and R56,R127 and Y128 are the three important amino acid residues at the binding site.3.BLI technology and molecular docking confirmed that there is an interaction between GSK3βand 14-3-3ζ protein,but only with Ser9 phosphorylated GSK3β polypeptide.Competition experiment,Co-IP combined with BLI technology and Western Blot experiment found that the combination of protopanaxadiol and 14-3-3ζ protein can enhance its binding to GSK3β Ser9 phosphorylation site and promote 14-3-3ζ protein pair Inhibition of GSK3β enzyme activity.4.In the corticosterone-induced depression model in mice,the behavioral results of FST and TST showed that long-term administration of protopanaxadiol can shorten the immobility time of model mice and have obvious antidepressant effects.Protopanaxadiol(50,25,12.5 mg/kg/d)can significantly reversed the decrease of BDNF and NF-L mRNA expression in hippocampus of model mice.Propanaxadiol(50,25,12.5 mg/kg/d)can significantly reversed the decrease of the p-GSK3β/GSK3β ratio,p-CREB/CREB ratio and BDNF protein expression level in hippocampus.Enzyme activity test results showed that protopanaxadiol(50,25,12.5 mg/kg/d)can significantly antagonize the increase of GSK3β enzyme activity in the hippocampus of model mice.Conclusion:The combination of protopanaxadiol and 14-3-3ζ protein in the brain can promote the binding of 14-3-3ζ protein and GSK3β Ser9 phosphorylation site,thereby enhancing the inhibitory effect of Ser9 phosphorylation on GSK3β enzyme activity.Furthermore,it antagonizes the inhibitory effect of GSK3β on the CREB-BDNF signaling pathway in the state of depression,improves the plasticity of nerve structure,and exerts antidepressant effects.