The Design?preparation And Effect Study Of Antidepressant Drugs Targeting Immune Molecule PDCD4

BackgroundsDepression is a common neurological disorder,and the main manifestations of clinical depression patients are persistent emotion stepping down,inhibition of thought and sense of meaninglessness.Moreover,patients with severe depression are at risk for suicide,which seriously restricts people's healthy life.At present,the treatment of depression is mainly based on drug remission.Selective 5-hydroxytryptamine(5-HT)reuptake inhibitors and 5-hydroxytryptamine-norepinephrine(NE)reuptake inhibitors are the main drugs for depression,which plays an antidepressant role mainly by increasing the concentration of excitatory neurotransmitters in synaptic cleft.However,long effective time,severe side effects and large differences in drug sensitivity among populations are the major obstacles.Therefore,revealing the new mechanisms of depression and finding new targets are of great value for the development of antidepressant drugs and the treatment of related disorders.Programmed cell death 4(PDCD4)is a vital negative regulator for protein translation.Previous studies have shown that PDCD4 directly or indirectly regulates the expression of about 1/3 proteins in cells,also playing crucial roles in the regulation of cytokine expression and cell growth,survival and metabolism.Our laboratory has been engaged in the study of the roles of PDCD4 in the development of diseases for a long time,such as tumors and metabolic diseases(Atherosclerosis and Obesity).More importantly,we recently discovered that PDCD4 was closely related to the occurrence of depression:1)PDCD4 was highly expressed in the hippocampus of depressive patients,and depression induced by CRS could enhance the expression of PDCD4 in hippocampus of mice;2)PDCD4 knockout mice could resist the depression-like behavior induced by CRS,while overexpression of PDCD4 in hippocampus of mice could result in spontaneous depression-like behavior;3)Mechanistically,PDCD4 inhibits the initiation of BDNF translation by binding to the protein translation initiation factor eIF4A,resulting in the development of depression.Therefore,our data indicated that PDCD4 is a potential pathogenic target of depression,and our study supply a new target for the development of antidepressant drugs and new theoretical foundation for the treatment of depression.The purpose of this project is to take PDCD4 as the target and the mechanism of PDCD4 regulating BDNF expression as the theoretical basis.The expression of PDCD4 in central nervous system was silenced by small interfering RNA,and the regulation function of PDCD4 was interfered with interference peptides and small molecule compounds.Antidepressants were designed,screened and prepared,and the effects of these candidate drugs on the expression of BDNF and the role in the treatment of depression were analyzed by cell model and CRS-induced mice depression model.Which laid the foundation for the development of new antidepressant drugs.Therefore,the main research contents of this project are as follows:1.The preparation,modification and antidepressant effect of PDCD4 specific small interfering RNA;2.The screening,preparation and antidepressant effect of PDCD4 interfering peptide;3.The screening and antidepressant effect of PDCD4 ligand small molecule.MethodsI.The preparation,modification and antidepressant effect of PDCD4 specific small interfering RNA1.The PDCD4 small interfering RNA and its effect on BDNF expression in nerve cells was detected(1)Screening PDCD4 small interfering RNA(siPdcd4),and the silencing efficiency of siPdcd4 was detected by RT-PCR and Western blot;(2)siPdcd4 was transfected into nerve cells by liposome method,and the changes of BDNF expression was detected by Western blot after 24 hours.2.The effect of siPdcd4 on the expression of inflammatory cytokines in microglia were explored(1)siPdcd4 was transfected into microglia by liposome method,after 24 hours,LPS and ATP were added.The cell culture supernatant and total RNA with different time gradients were collected;(2)RT-PCR and ELISA were used to test the effect of siPdcd4 on the expression of IL-6 and IL-1 ? in microglia.3.Nerve targeting modification and interference efficiency detection of siPdcd4(1)The rabies virus glycoprotein transport polypeptides(RVG-9dR)were synthesized;(2)The Cy5 fluorescein-labeled siPdcd4(Cy5-siPdcd4)was modified for neuro-targeted transport(RVG/Cy5-siPdcd4);(3)Nerve cells were treated with RVG/Cy5-siPdcd4,and the distribution of red fluorescence in the cells was detected by high-content imaging;(4)The expression of PDCD4 were detected by RT-PCR and Western blot,after nerve cells were treated with RVG/Cy5-siPdcd4 for 48h.4.The effect of RVG/siPdcd4 on BDNF expression under mTORC1 inactivation(1)The specific mTORC1 inhibitor rapamycin was used to block the neuronal mTORC1 activity,and the changes of BDNF and PDCD4 expression in different time gradients were detected by Western blot;(2)RVG/siPdcd4 silenced the endogenous PDCD4 in nerve cells.After 24 hours,rapamycin was added to block the mTORC1 signaling pathway.The effect of RVG/siPdcd4 on BDNF expression was detected by Western blot.5.Whether RVG/siPdcd4 could cross the blood-brain barrier and its effect on expression of BDNF and PDCD4(1)RVG/Cy5 siPdcd4 was injected into mice via tail vein,the distribution of red fluorescence in mice was detected by three-dimensional imaging system after 6 hours,24 hours,48 hours and 72 hours;(2)RT-PCR and Western blot were used to detected the silencing efficiency of PDCD4 and the expression changes of BDNF in hippocampus and prefrontal cortex;(3)The optimal concentration of RVG/siPdcd4 was determined by the silencing efficiency of PDCD4 and the expression of BDNF.6.The effect of RVG/siPdcd4 on CRS induced depression was evaluated by behavioral test(1)The depressed mice were induced by chronic restraint stress.RVG/siPdcd4 was injected into the mice through tail vein,the silencing efficiency of PDCD4 in hippocampus and prefrontal cortex was detected by RT-PCR,and the expression of BDNF in hippocampus were detected by Western blot and Quantitative ELISA;(2)To clarifies the effect of RVG/siPdcd4 on the inflammatory of depression.The changes of inflammatory cytokines expression in the hippocampus and peripheral blood were detected by Quantitative ELISA;(3)The brain tissue was stained by Golgi,and the changes of synaptic plasticity were evaluated by the density of dendritic spines;(4)The effect of RVG/siPdcd4 on depression-like behavior were evaluated by tail suspension test,forced swimming test and sucrose preference test.?.The screening,preparation and antidepressant effect of PDCD4 interfering peptide1.The amino acid sequences that specifically interfere with the binding of PDCD4 to eIF4A was screened(1)The eIF4A-HA,PDCD4-FLAG and BDNF-GFP overexpression vectors were constructed;(2)The key amino acid sites and main domains of interaction between PDCD4 and eIF4A were found by literature and protein structure database;(3)The fusion protein expression vector of key amino acid sequence with GFP was constructed by molecular cloning;(4)The key amino acid sequence could up-regulates the expression of BDNF and interfere with the binding of PDCD4 and eIF4A were determined by co-immunoprecipitation and Western blot;(5)The screened amino acid sequence was coupled with the transmembrane sequence 9R(N terminal)to synthesise the corresponding interference peptides.2.The effect of interfering peptides on the binding of PDCD4 and eIF4A(1)The transmembrane efficiency,intracellular stability and co-localization of interfering peptides with PDCD4 was detected by Immunofluorescence assay;(2)The interference efficiency of interfering peptides on the binding of PDCD4 and elF4A were detected by co-immunoprecipitation and double fluorescence complementary assay.3.The influence of interfering peptides on the expression of BDNF(1)The PDCD4-FLAG and BDNF-GFP were overexpressed in HEK-293 cells,and the influence of interfering peptides on the expression of BDNF was detected by Western blot;(2)The effect of interfering peptides on the expression of BDNF in neurons were detected by Western blot and Quantitative ELISA;(3)The expression of inflammatory cytokines in the supernatant was detected by Quantitative ELISA.4.The effect of Interfering peptides on BDNF expression under mTORC1 inactivationUnder the condition that the mTORC1 signaling pathway was completely blocked,the effect of interfering peptides on the expression of endogenous BDNF in primary hippocampal neurons was detected by Western blot.5.The effect of interfering peptides on anxiety and depression-like behaviors induced by CRS(1)Wild type C57BL/6J male mice(aged 4-6weeks)were induced depression by chronic restraint stress;(2)According to the coronal brain atlas of mice,the micro drug delivery tubes were embedded in the ventral side of the hippocampus.And the diffusion of interfering peptides in hippocampus was detected by immunofluorescence assay;(3)The interfering peptides was injected into hippocampus of depression mice,and the changes of BDNF expression were detected by Western blot and Quantitative ELISA;(4)The brain tissues of mice were stained with Golgi.and the density of axon dendritic spines in hippocampal neurons was statistically analyzed to evaluate the influence of interfering peptides on synaptic plasticity;(5)Behavioral tests were conducted to evaluate the effects of interfering peptides on anxiety and depression-like behaviors of mice.?.The screening,and antidepressant effect of PDCD4 ligand small molecule1.The small ligand molecules of PDCD4 were screenedThe quantitative structure-activity relationship regression prediction(QSAR)based on Gaussian process was used to high-throughput virtual screening in drug-like biological compound libraries.And the small molecular compounds with high or medium affinity with PDCD4 protein at the micromolar level were screened.2.The functional verification of PDCD4 ligand small molecule(1)The effect of PDCD4 ligand small molecules on the binding of PDCD4 and eIF4A was detected by co-immunoprecipitation;(2)The effect of PDCD4 ligand small molecule on the expression of BDNF in nerve cells was detected by Western blot(3)The PDCD4-FALG and BDNF-GFP overexpression vectors were transfected into HEK-293 cells,and the effect of PDCD4 ligand small molecules on BDNF expression was detected by Western blot;(4)The effect of PDCD4 ligand small molecule on BDNF expression during mTORC1 inactivation was detected by Western blot.3.The effect of PDCD4 ligand small molecule on depression-like behavior induced by CRS(1)The depressed mice were induced by chronic restraint stress,and different concentrations of PDCD4 ligand small molecules were intraperitoneally injected into mice,the expression of BDNF in hippocampus and prefrontal cortex were detected by Western blot and Quantitative ELISA;(2)The brain tissue was stained with Golgi,and the changes of synaptic plasticity were evaluated by the density of axon dendritic spines;(3)The effect of PDCD4 ligand small molecules on depression-like behavior was evaluated by behavioral testResults?.The preparation,modification and antidepressant effect of PDCD4 specific small interfering RNAPrevious studies have shown that the expression of PDCD4 in the hippocampus is elevated in patients with depression,and overexpression PDCD4 in the hippocampus of mice showed spontaneous depression-like behavior.However,PDCD4 knockout mice were completely resistant to depressive behavior caused by chronic stress,suggesting that PDCD4 is an important target for the treatment of depression.Therefore,in this part,we used small interfering RNA to interfere with the expression of PDCD4 in the central nervous system to study its therapeutic effect on depression.1.The interference efficiency of siPdcd4 and its effect on PDCD4 downstream target gene expression(1)First,the PDCD4 small interfering RNA(siPdcd4)was screened and synthesized from the nucleic acid database by analyzed the nucleic acid sequence of PDCD4 mRNA.And the silence efficiency of siPdcd4 were detected by RT-PCR and Western blot.(2)The effect of siPdcd4 on the expression of BDNF:siPdcd4 was transfected into nerve cells by liposome method,and the effect of siPdcd4 on the expression of BDNF was detected by Western blot.The results showed that siPdcd4 could significantly upregulate the BDNF in nerve cells after silencing PDCD4 expression.(3)The effect of siPdcd4 on the expression of cytokines:It is known that PDCD4 not only inhibit BDNF expression,but also negatively regulate the expression of pro-inflammatory cytokines.Therefore,we further investigated the effect of siPdcd4 on the expression of cytokines in microglia,and the results showed that siPdcd4 could significantly inhibit the expression of IL-6 and IL-1 ? induced by LPS and ATP stimulation.2.The nerve cell targeted modification and specific detection of siPdcd4(1)The nerve cell targeted modification of siPdcd4:In order to solve the technical bottleneck that siPdcd4 could not enter the brain,we synthesized the neurotropic rabies virus glycoprotein polypeptide(RVG-9dR).It has been proved that the 9 D-arginines of the RVG-9dR polypeptide binding to siPdcd4 through charge absorption,which can be used for neuro-targeting modification of siPdcd4(RVG/siPdcd4).In order to facilitate in vivo tracing,Cy5 fluorescein(red)was used to label siPdcd4(Cy5-siPdcd4),and the RVG/Cy5-siPdcd4 complex with both nerve specificity and traceability was prepared.(2)The specific detection of RVG/siPdcd4 targeting of nerve cells:In order to detect the neuro-targeting specificity of siPdcd4 modified by RVG-9dR(RVG/siPdcd4).RVG/Cy5-siPdcd4 was used to infected nerve cells SH-SY5Y,HT-22,BV2 and non-neural cells HeLa respectively,and the distribution of siPdcd4 was detected by high content imaging system.The results showed that RVG/Cy5-siPdcd4 could specifically enter the cytoplasm of SH-SY5Y,HT-22 and BV2,but there was no obvious red fluorescence in HeLa.(3)The silencing efficiency of RVG/siPdcd4 was detected:RT-PCR and Western blot results showed that RVG/siPdcd4 could silence the endogenous PDCD4 expression in SH-SY5Y,HT-22 and BV2 at the mRNA and protein levels,but had no significant effect on HeLa cells.These results indicate that RVG-9dR peptide-modified siPdcd4(RVG/siPdcd4)specifically enters nerve cells and silences the expression of PDCD4.3.RVG/siPdcd4 reversed the inhibitory effect of neuronal mTORC1 inactivation on BDNFOur previous research found that the up-regulation of PDCD4 in the hippocampus during depression was due to the decrease of mTORC1 activity,which results the obstruction of the PDCD4 phosphorylation-ubiquitination degradation pathway.So,we used rapamycin the specific inhibitor of mTORC1 to block the mTORC1 signaling pathway of HT-22 and BV2 to establish a cell model that mimics changes in the brain of depression mice.We found that PDCD4 accumulates after mTORC1 inhibition,and BDNF expression is significantly inhibited,while RVG/siPdcd4 can reverse the inhibitory effect of mTORC1 inactivation on BDNF.4.RVG/siPdcd4 could cross the BBB and target the brainTo test whether RVG-9dR could carry siRNA across the blood-brain barrier.We injected RVG/Cy5-siPdcd4 into mice via tail vein,and the distribution of red fluorescence was detected by in vivo imaging.The results showed that at 6 hours Cy5-siPdcd4 mainly present in the abdomen of mice only a small amount of siRNA enters the brain.while,at 24 hours Cy5-siPdcd4 mainly concentrated in brain,with no obvious fluorescence in the abdomen.At 48 hours,red fluorescence was still present in the brain,but significantly decreased compared with 24hours.No fluorescent was observed in the brain at 72 hours.These results suggest that RVG/siPdcd4 could efficiently cross the blood-brain barrier and target into the brain with good stability.We also detected the changes of PDCD4 and BDNF by RT-PCR and Western blot.The results showed that RVG/siPdcd4 could significantly silenced PDCD4 in hippocampus and prefrontal cortex while up-regulating the expression of BDNF.5.Intravenous injection of RVG/siPdcd4 could resist depression-like behavior induced by CRSIn order to further explore the therapeutic effect of RVG/siPdcd4 on depression like behavior.We induced mice depression model by chronic restraint stress,and then RVG/siPdcd4 was injected through tail vein for treatment.(1)The RT-PCR and Western blot results showed that RVG/siPdcd4 effectively silenced PDCD4 in the hippocampus and prefrontal cortex of depressed mice,decreased the expression of pro-inflammatory cytokines IL-6 and IL-1?,and significantly increased the expression of BDNF;(2)The result of Golgi staining of brain tissue showed that RVG/siPdcd4 could increase the density of axon dendritic spines of hippocampal neurons and improve synaptic plasticity;(3)The behavioral test results showed that RVG/siPdcd4 could reduce the immobility time of tail suspension test and forced swimming test,and enhance the sucrose water preference index of the depressed mice.These results suggest that RVG/siPdcd4 could resist CRS-induced depression-like behavior by silencing PDCD4,inhibiting the expression of pro-inflammatory cytokines,up-regulating expression of BDNF and repairing synaptic plasticity.?The screening,preparation and antidepressant effect of PDCD4 interfering polypeptideOur previous research found that PDCD4 inhibits the translation of BDNF and promotes the development of depression by binding eIF4A,suggesting that interference with the binding of PDCD4 and eIF4A will relieve the inhibitory effect of PDCD4 on BDNF and treat depression.Therefore,in this part interfering peptides that could interfere with the combination between PDCD4 and eIF4A were selected according to the binding characteristics of PDCD4 and eIF4A,and their antidepressant effect were studied.1.The key sequence of eIF4A binding to PDCD4 was screenedWe found eIF4A P56,K83,G137,T159 and R360 were the main amino sites of eIF4A binding with PDCD4 by consulting literature and protein structure database.According to the position of the amino acid site,four possible PDCD4 binding sequences were designed according to the location of amino acid sites,which were named eIF4A-Q,eIF4A-I,eIF4A-Ia and eIF4A-VI.In order to verify the function of each amino acid sequence,we constructed the fusion protein expression vectors of eIF4A-Q,eIF4A-I,eIF4A-la and eIF4A-VI with GFP,and the amino acid sequence was linked to the GFP protein through Linker.The results of co-immunoprecipitation and Western blot show that eIF4A-VI interfere with the combination of PDCD4 and eIF4A,at the same time up-regulate the expression of BDNF.Therefore,eIF4A-VI becomes the candidate sequence for subsequent studies.2.The transmembrane modification of eIF4A-VI and its effect on binding of PDCD4 to eIF4AThe binding of PDCD4 to eIF4A occurs in the cytoplasm.In order to further explore the function of eIF4A-VI,we synthesized 9R-eIF4A-VI interference peptide based on the amino acid sequence of eIF4A-VI,to ensure the transmembrane efficiency TAT-like transmembrane sequence 9R was conjugated at the N terminal of eIF4A-VI.At the same time to confirm the specificity of 9R-eIF4A-VI,we synthesized Mu-9R-eIF4A-VI peptide(R363 mutation to Q363)as negative control.The results of immunofluorescence showed that 9R-eIF4A-VI interfering peptide could cross the cell membrane within 1 hour and stably exist in the cytoplasm for 48 hours.Furthermore,9R-eIF4A-VI interference peptides was found have significant co-location with PDCD4 protein.The results of co-immunocoprecipitation and double fluorescence molecular complementary experiments showed that 9R-eIF4A-VI interfering peptides could interfere with the binding of PDCD4 to eIF4A,but the mutant peptide has no such effect.3.The interference peptides specifically up-regulated the expression of BDNFPDCD4-FLAG and BDNF-GFP overexpression vectors were transfected into HEK-293 cells at a mass ratio of 3:1,and then treated with 9R-eIF4A-VI interference peptide.The changes of BDNF expression were detected by Western blot.The results showed that 9R-eIF4A-VI interfering peptide could reverse the inhibitory effect of PDCD4 overexpression on BDNF,but the up-regulation effeect of mu-9R-eIF4A-VI peptide on BDNF expression disappeared at the same concentration.In order to verify the effect of interfering peptides on endogenous BDNF expression in nerve cells,we stimulated nerve cells(primary hippocampal neurons,microglial BV2,mouse hippocampal neurons HT-22)with different concentrations of 9R-eIF4A-VI peptides.Western blot results showed that 9R-eIF4A-VI interfering peptides could significantly up-regulate the expression of endogenous BDNF in nerve cells.At the same time,we evaluated the side effects of 9R-eIF4A-VI interfering peptides on nerve cells.ELISA results showed that 9R-eIF4A-VI interfering peptides did not induce the up-regulation of cytokines IL-6 and IL-10.4.The interfering peptides reverse the inhibitory effect of mTORC1 inactivation on BDNF in hippocampal neuronsIn previous work,we successfully constructed a cell model to simulate the changes in the brain of depressed mice using mTORC1 inhibitors.In order to investigate the effect of 9R-eIF4A-VI interference peptides on the expression of BDNF in neurons,rapamycin was used to inhibit the activity of mTORC1 in primary hippocampal neurons.We found that,consistent with the previous results,PDCD4 accumulated after mTORC1 inactivation,and BDNF expression was significantly inhibited.While,9R-eIF4A-VI interfering peptides could reverse the inhibitory effect of primary mTORC1 inactivation on BDNF in primary hippocampal neurons.5.Injecting interfering peptides into the hippocampus alleviates anxiety and depression-like behavior induced by CRSTo test the effects of 9R-eIF4A-VI interfering peptides on anxiety-like and depression-like behavior.The mice depression model was induced by chronic restraint stress,and the microinjection tube were buried into the ventral side of the hippocampus.(1)The results of immunofluorescence staining of brain tissue showed that 9R-eIF4A-VI interfering peptides could diffuse to the entire hippocampus through targeted injection;(2)Western blot and Quantitative ELISA results showed that 9R-eIF4A-VI interference peptides could significantly up-regulate the BDNF expression in the hippocampus of depressed mice;(3)Golgi staining of brain tissue results showed that 9R-eIF4A-VI interfering peptides repair the synaptic plasticity by increasing the density of dendritic spines.(4)The behavioral results showed that the immobility time of mice in 9R-eIF4A-VI group was significantly reduced compared with the CRS group in the tail suspension test and forced swimming test.In sucrose preference test the sucrose water preference index of the mice in the 9R-eIF4A-VI group was significantly higher than in the CRS group.The residence time of mice in open field test and elevated plus maze interfering peptides group was significantly lower than that of the CRS group.The above results indicate that the 9R-eIF4A-VI interfering peptides injected into the hippocampus could up-regulate the expression of BDNF,improve synaptic plasticity,and alleviate CRS-induced anxiety and depression-like behaviors.?.The screening and antidepressant effect of PDCD4 ligand small molecularAlthough interfering peptides and siRNA drugs have the advantages of high specificity and low immunogenicity,their preparation cost is high and they cannot be administered orally,so the future clinical application will be limited.In order to overcome this deficiency,in this part,we mainly explore the feasibility of PDCD4 ligand small molecule compounds in the treatment of depression1.The screening of PDCD4 ligand small moleculesPrevious studies have reported that six small molecule compounds may bind to PDCD4 were screened from the small molecule library of drugs through QSAR.Further study found that one small molecule compound had a high affinity with PDCD4,and the other small molecule had a medium affinity with PDCD4.The simulation results showed that the two compounds were mainly linked to amino acids in the C-terminal MA-3 domain of PDCD4 by hydrogen bonds and ?-? bonds,but the function of small molecule still unclear.Therefore,this topic selected these two small molecules as candidate compounds(named ZHJ-204 and ZHJ-690,respectively)to test their effects on BDNF expression.2.The effect of PDCD4 ligand small molecule on the expression of BDNF(1)First,we examined the effects of small molecules ZHJ-204 and ZHJ-690 on endogenous BDNF expression in nerve cells.Results showed that ZHJ-204 and ZHJ-690 at 10pM to 1nM could significantly up-regulate the expression of HT-22 and BV2 BDNF in a concentration-dependent manner.(2)The results of cell depression model showed that ZHJ-204 and ZHJ-690 could relieve the inhibitory effect of rapamycin on BDNF,and the small molecule ZHJ-204 had a more significant up-regulation effect on BDNF than ZHJ-690.(3)In the PDCD4 overexpression model,we found that small molecules ZHJ-204 and ZHJ-690 could reverse the inhibition effect of PDCD4 overexpression on BDNF.3.The antidepressant effect of PDCD4 ligand small moleculeThe mice depression model was induced by chronic restraint stress.And then we injected different concentrations of small molecules into mice by intraperitoneal,the control group was injected with the same amount of saline,once a day,a total of 10 times.Behavioral and brain biochemical markers were analyzed.(1)First,we examined the BDNF expression in hippocampus and prefrontal cortex.Western blot results showed that CRS reduced BDNF leves in the hippocampus and prefrontal cortex,while ZHJ-204(5mg/kg)significantly up-regulated the expression of BDNF in hippocampus and prefrontal cortex of depressed mice.(2)The behavioral results showed that the immobility time of mice in ZHJ-204(5mg/kg)group was significantly lower than that in the CRS group in the tail suspension test and forced swimming test.The sucrose preference test result showed that the sucrose water preference index of mice in the ZHJ-204(5mg/kg)group was significantly higher than that in the CRS groupThese results suggest that intraperitonal injection of PDCD4 ligand ZHJ-204 could alleviate CRS-induced depression-like behavior by up-regulating BDNF expression.Conclusions1.The neuro-targeted PDCD4 small interfering RNA(RVG/siPdcd4)treated CRS-induced depression by specifically silence PDCD4,inhibiting the expression of pro-inflammatory cytokines,up-regulating the expression of BDNF and improve synaptic plasticity;2.9R-eIF4A-VI interference peptide alleviates CRS-induced anxiety and depression-like behavior by up-regulating BDNF expression;3.Intraperitoneal injection of PDCD4 ligand molecule ZHJ-204 alleviates depression-like behavior induced by CRS by upregulating BDNF expression in the hippocampus and prefrontal cortex..Innovation and significance1.We used small nucleic acid drugs for the treatment of depression for the first time,and found that the neuro-targeted modification of siPdcd4(RVG/siPdcd4)alleviates CRS-induced depression-like behavior by intravenous administration.RVG/siPdcd4 may become a new antidepressant drug.2.In the study of peptide antidepressants,we screened and designed 9R-eIF4A-VI interference peptides that specifically interfere with the binding of PDCD4 and eIF4A.In vivo studies have demonstrated that 9R-eIF4A-VI injected into hippocampus could alleviate anxiety and depression-like behaviors induced by CRS.This study further demonstrated the molecular mechanism of PDCD4 promoting depression and provides a new strategy for the study of antidepressant drugs,and laid a foundation for the research and development of antidepressant drugs of interfere peptides.3.In the study of small molecule antidepressants,PDCD4 ligand small molecules were screened from the drug-like small molecule library.We also demonstrated that intraperitoneal injection of PDCD4 ligand small molecule ZHJ-204 could alleviate CRS induced depression-like behavior by up-regulating BDNF expression.Our work provides a preliminary basis for the study of novel small molecule antidepressant drugs.Limitations1.The pharmacokinetics and drug safety evaluation of RVG/siPdcd4 have not been completed.2.Although intracerebral injection of 9R-eIF4A-VI interfering peptides showed significant antidepressant effects,the modification and effects of 9R-eIF4A-VI peripheral administration need to be further studied.3.Although PDCD4 ligand small molecules have been proved to have antidepressant effects,further transformation and modification of small molecule compounds are still needed to improve their activity and medicinal properties.