Analysis Of MPFC Differentially Expressed Proteins And Synaptic Plasticity In Rats Under Restraint Water-immersion Stress

Restraint water-immersion stress(RWIS)is a physiological and psychological compound stress that induces gastrointestinal disorders in a short period of time(hyperkalosis,increased gastric acid secretion,decreased gastric mucus secretion,gastric mucosal damage,and Intestinal exercise is strengthened,etc.).The medial prefrontal cortex(mPFC)is the highest level of joint cortex in mammals,with the ability to think and memory,integrate information,and direct regulation.The expression of Fos protein in mPFC increased significantly after 1 hour of stress,indicating that the mPFC nuclei participate in the RWIS process.Then,which proteins in mPFC have changed during the RWIS process? What are the possible signaling pathways and molecular mechanisms by which these differential proteins are involved in RWIS regulation? These problems have still not been resolved.In addition,synaptic plasticity plays an important role in the stability of neurons and neural circuits.So,in the RWIS process,whether the synaptic plasticity of mPFC changes?It has not been reported in detail.In view of this,we proposed the following three questions:1.what kind of proteins will be changed by RWIS in rat mPFC? What are the possible mechanisms by which these differential proteins are involved and how the signaling pathways will be changed?2.How the ultrastructure of neuronal synapses will be changed in rat mPFC by RWIS?3.How the synaptic-associated marker function proteins will be changed in rat mPFC by RWIS?To explore the above three issues,we designed the following experiments:Experiment 1: The experimental animals were randomly divided into control group(RWIS 0h)and experimental group(RWIS 4h).The differentially expressed proteins in rat mPFC were analyzed by isobaric tag for relative and absolute quantitative technique(iTRAQ).These proteins were subjected to GO analysis,KEGG analysis and IPA analysis to explore the signaling pathways and molecular mechanisms that these differential proteins may be involved in during stress?Experiment 2: Experimental animals were randomly divided into 4 groups,namely RWIS 0h(control group),RWIS 1h,RWIS 2h and RWIS 4h groups.Golgi staining was used to detect the changes of the morphology and quantity of dendritic spines in mPFC.The changes of synaptic ultrastructure such as the thickness of PSD,synaptic gap width and synaptic curvature of mPFC were observed by transmission electron microscopy.To investigate whether the synaptic plasticity of neurons in mPFC has changed and how it has changed by means of RWIS.Experiment 3: The experimental animals were grouped as simble as Experiment 2.Immunohistochemical technique,Western blotting and RT-PCR technique were used to detect the synaptic markers in different time periods of stress such as post synaptic density protein(PSD-95)and synaptophysin(SYN)protein and the relative transcript level of mRNA.It was thus determined whether RWIS caused a change in synaptic functional plasticity of rat mPFC.Experimental results show:1.iTRAQ technology identified 129 different types of proteins between RWIS 0h and RWIS 4h group,of which 88 were up-regulated and 41 were down-regulated.GO analysis showed that 22%(29)of differential proteins were directly involved in neurological function,including synaptic plasticity,axon morphology and growth,neurodevelopment and apoptosis and neural signals transmition and so on.KEGG analysis found that 129 differential proteins were enriched into 64 metabolic pathways involving synaptic vesicle cycling,toxicological metabolism and other processes.IPA analysis differential proteins are involved in 137 signaling pathways,of which only the RhoGDI signaling pathway is inhibited and the integrin signaling pathway is activated.2.Dendritic spine staining experiment showed that the number of dendritic spines per unit length(10um)was significantly reduced in each stress group compared with the coontrol group(P<0.05).The density of dendritic spines generally showed a trend of decreasing first and then recovering.Among them,the dendritic spine density of RWIS 1h group was the lowest(P<0.01),and the density of dendritic spine of RWIS 2h group was higher than that of RWIS 1h group,but there was no significant difference(P>0.05)with the extension of stress time.Clear cell structures such as nuclei,mitochondria,Golgi and synaptic structures can be observed under electron microscopy.Electron microscopic observation of the synaptic structure revealed a dense electron dense substance.At the presynaptic terminal,there are more or less vesicles that accumulate in the presynaptic membrane,and vesicles that are undergoing endocytosis or exocytosis can also be observed.The length of the synaptic active zone and the thickness and length of the postsynaptic dense zone are also different.In the control group,the cell components were clearly defined,while the stress group had a clear outline,and the perforated synapses were more.The vacuolated mitochondria could be observed,indicating that the oxidative stress response caused some damage to the mitochondria.Compared with the control group,the number of synapses per unit area in the RWIS 4h group was significantly decreased(P<0.05),and the number of synapses in the RWIS 1h and RWIS 2h groups was decreased compared with the control group,but the difference was not significant(P>0.05).In the curved synapse,it can be divided into U-shaped and inverted Ushaped synapses.Compared with the control group,the proportion of flat synapses and U-type synapses in the RWIS 4h group was significantly decreased(P<0.05),while the proportion of inverted U-type synapses was significantly increased(P<0.05).There was no significant difference between the RWIS 1h/2h group with the control group(P>0.05).The number of presynaptic vesicles in the RWIS 4h group was significantly lower than control group(P<0.05),and there was no significant difference between the RWIS 1h and RWIS 2h groups compared with the control group(P>0.05).The width of synaptic gap decreased first and then increased with the increase of stress time.The width of synaptic gap in RWIS 1h and RWIS 2h group was significantly lower than that in RWIS 0h group(P<0.05),but There was no significant difference between the RWIS 4h group and the control group(P>0.05).The thickness of PSD in each group was significantly lower than that in the control group at different time points of stress(P<0.05).With the prolongation of stress time,the thickness of PSD decreased first and then recovered.Among them,the PSD thickness was lowest in the RWIS 2h group(P<0.01),and the PSD thickness in the RWIS 4h group was higher than that in the RWIS 1h and RWIS 2h groups,but there was no significant difference(P>0.05).3.Immunohistochemistry results showed that PSD-95 protein was mainly expressed on the cytoplasm and membrane surface,which was brown or brownish yellow.With the prolongation of stress time,the number of PSD-95 positive cells decreased first and then recovered,and the number of PSD-95 positive cells in each stress group was significantly lower than that of the control group(P<0.05).Among them,the number of PSD-5 positive expression in RWIS 1h group was the lowest(P<0.01),and the expression of PSD-95 was increased in RWIS 2h and RWIS 4h group compared with RWIS 1h group,but there was no significant difference(P>0.05).SYN is expressed throughout the cytoplasm and is brownish yellow.Compared with the control group,the expression of SYN in the RWIS 4h group was significantly decreased(P<0.05),while the RWIS 1h and RWIS 2h groups were slightly increased compared with the control group,but there was no significant difference(P>0.05).The result of immunoblotting were consistent with the results of immunohistochemistry.Compared with the control group,the relative expression of PSD-95 stress group decreased,and there was significant difference(P<0.05).Among them,the relative expression of PSD-95 was the lowest in RWIS 1h group(P<0.01),and the expression of PSD-95 in RWIS 2h and RWIS 4h group was higher than that in RWIS 1h group,but there was no significant difference.The expression of PSD-95 was generally decreased first and then increased.The relative expression of SYN in the RWIS 4h group was the lowest,compared with the stress group(P<0.05).The expression of SYN in the RWIS 1h group and the RWIS 4h group was slightly higher than that in the control group,but there was no significant difference.The expression of SYN generally showed a trend of increasing first and then decreasing.The result of PCR showed that the relative expression of SYN protein mRNA was consistent with the results of immunohistochemistry and immunoblotting,the expression of mRNA in RWIS 4h group was significantly lower than that of RWIS 0h group(P<0.05).Compared with control group,the RWIS 1h and RWIS 2h groups were increased,but there was no no significant difference.Compared with control group,the expression of PSD-95 protein mRNA was significantly up-regulated in RWIS 1h and RWIS 2h groups(P<0.05),and there was no significant change in mRNA expression in RWIS 4h group.The expression of RWIS 4h group was lower than that of RWIS 2h group(P<0.05),but there was no significant difference compared with RWIS 1h group(P>0.05).Based on the above experiments,it was found that RWIS had a certain degree of damage to neurons in rat mPFC at different time points,and synaptic plasticity showed a trend of reduction.Differential proteins are involved in biological processes such as oxidative stress,toxicological metabolism,transcriptional translation and apoptosis.In particular,NDRG1 and NYAP2 proteins may be involved in the protective mechanism of the body during stress.With the prolongation of RWIS time,the body initiates the mechanism of stress damage.The inhibition of Rho-GDI signaling and activation of integrin signaling pathway in mPFC may play an important role in neuronal damage repair and synaptic remodeling.