TMT-based Quantitative Proteomic Analysis Of Hippocampus In A Rat Model Of Posttraumatic Stress Disorder

Background:Posttraumatic stress disorder(PTSD)is a chronic mental disorder that occurred after people experienced severe trauma events.lt is almost impossible for people to avoid experiencing a severe trauma event at some time in their lives,but it’s not everyone who will develop into PTSD.Only a few people who experienced severe trauma events will develop into PTSD.That is to say,People appear to be susceptible and resilient to traumatic events,The mechanisms associated to the susceptibility and resilience are still unclear.PTSD is a debilitating disease,The core symptoms of the disease are:flashback,avoided reactions to traumatic cues,hypervigilance,negative alterations of mood and cognition,insomnia,etc.These symptoms are intense and persistent,PTSD affects work and life of patients to a large extent and imposes a huge burden on society.The rate of suicide and self-injury in patients with PTSD is high,so is the incidence of alcohol and drug abuse.The treatment of PTSD is not so effective,and the recurrence rate is high after stopping treatment.Post-traumatic stress disorder is currently a hot topic in medical research.Objective:The hippocampus is one of the key brain regions involved in PTSD.We used a PTSD rat model and divided the rats into PTSD-susceptible and PTSD-resilient subgroup based on behavioral results.Rat hippocampus was selected as the research object,and TMT quantitative proteomics technology was used to identify proteins with significant changes in expression levels.By analyzing differentially expressed proteins,we hope to find the proteins that mediate the susceptibility and resilience of PTSD,and provide a theoretical basis for the prevention and treatment of PTSD.Method:1.A PTSD rat model was prepared by a protocol which is 10 paired conditional stimulation(sound stimulation and context)and unconditional stimulation(foot shock)randomly assigned at 15 minutes.Rats developed a conditioned fear after the shock procedure,and after 28 days of feeding in a standard environment,the rats were exposed to the initial shock environment again(no foot shock presentation)to test behavioral indicators(freezing time),the shocked rats were divided into PTSD susceptible and PTSD resilient rats based on their freezing time.Rats in the control group Rats do not receive the shock treatment.2.On the second day after the end of the rat behavior test,the hippocampus of the susceptible group,the resilient group,and the control group were extracted.Samples were randomly mixed into three biological replicates per group.after protein extraction,and peptide digestion.The sample was subjected to TMT labeling,high pH reverse peptide fractionation,combined with LC-MS/MS data acquisition technology for protein quantification and analysis,differentially expressed proteins were screened according to the standard(expression fold change greater than 1.2 times or down-regulation less than 0.83 times and P value<0.05).Bioinformatics analysis of differentially expressed proteins followed.Results:1.Rat models of conditioned fear PTSD were constructed,the shocked rats were divided into PTSD-susceptible and PTSD-resilient subgroups based on freezing time.Compared with the control group and the resilient group,the freezing time of the susceptible group was significantly longer(P<0.001).2.Using the TMT quantitative proteomics technique,6151 proteins were identified in the Rattus norvegicus(Rat)database.The S_Vs_C comparison group,the R_Vs_C comparison group,and the R_Vs_S comparison group were screened for 51,46,and 44 differentially expressed proteins that met the screening criteria.Conclusin:1.The rat models of PTSD were constructed successfully,and we provided a method to screen susceptibility and resilience of PTSD.2.The differentially expressed proteins selected from the hippocampus of the susceptible and resilient groups were mainly related to oxidative stress,antioxidant protection,synaptic function,and signal transduction.The results may provide a reference for the pathogenesis of PTSD and provide the theoretical basis for developing PTSD prevention and treatment strategies.