Study On The Molecular Mechanism Of Seizures And Ginseng's Preventive And Therapeutic Effects Based On Synaptosomal Proteomics

Febrile seizures caused by infection and fever are very common in clinic.If the seizures happen repeatedly or prolong for a long-time,it will lead to acute hippocampal injury and hippocampal atrophy,and inevitably affect the patient's cognitive,learning and memory abilities.It was also reported that febrile seizures may be one of causes for the occurrence of epilepsy in future.Nowadays,for febrile seizures,supportive care is mainly adopted for the patients,and for persistent seizures some antiepileptic drugs will be administrated to enhance the inhibitory effects of ?-aminobutyric acid.However,it is not clearly how the hyperthermia-and infection-factor synergistically induce seizures,and what will be changed for the protein networks in synapses of the central nerves system(especially for hippocampi).In this study,we aimed to establish a synaptosomal proteomic method for rat brain tissues.Then using the proteomic method we investigate the effects of hyperthermia and infections on the expressions of synaptosome proteins in hippocampi to reveal the key molecules or molecular mechanism for the occurrence of seizures.Moreover,we investigate the effects of ginseng which has been widely used for nervous diseases on the expressions of synaptosome proteins to reveal the therapy targets of ginseng in nervous system,and we also try to find its active ingredients that inhibit the occurrence of seizures.Part one Study on the synaptosomal proteomics in different brain regions of rats Objective: To establish a proteomics method for cerebral synaptosomes and to study the protein expressions in the synaptosomes of rat frontal cortex,parietal cortex,hippocampus,striatum,olfactory bulb and cerebellar cortex. Methods: The synaptosomes were prepared by centrifugation using Percoll as the density gradient medium,and the obtained synaptosomes were confirmed by electron microscopy observation and Western Blot.Then we investigated the extraction buffers for synaptosomal proteins,and optimized the conditions for reductive alkylation reactions and enzymatic cleavages.The enzymatic peptides were analyzed by Nano-liquid chromatography coupled with mass spectrometry(Nano LC-MS).Finally,the proteins in the synaptosomes were inferred according to the detected peptides and the protein data base.Results: The synaptosomes were obtained after the density gradient centrifugation.Many synaptosomes with complete structure and different size are observed in the synaptosome-enriched fraction using the transmission electron microscope.The postsynaptic dense layer,vesicles and mitochondria were also clearly seen in the amplified synaptosomes.Synaptophysin(a specific marker for the presynaptic membrane)and PSD 95(a specific marker for the postsynaptic membrane)in the synaptosome-enriched fraction were also validated by Western Blot.The optimized sample preparation method was as followed: the synaptosomal proteins were extracted by the 8 mol/L urea lysis buffer,and subjected to reductive alkylation of dithiothreitol and iodoacetamide followed by sequential enzymatic digestion with trypsin and Lys-C.The synaptosomal proteins were identified and quantified according to the peptides analyzed by Nano LC-MS.The method was used to study the synaptosomal proteomics in different brain regions of rats.For two replicated experiments,there are 2920 identified proteins including synaptic proteins,postsynaptic dense layer proteins,mitochondrial proteins,vesicle proteins and presynaptic membrane proteins,etc.Based on the prtotein expression data,the synaptosomal proteins with relatively high-level expression in specific brain regions were clustered by several clustering methods,and then analyzed by the bioinformatic methods.The bioinformatic analysis revealed that the proteins with high-level expression can represent the function of the brain region where they are located.The results approved that the established synaptosomal proteomic method was feasible.Brief summary: We established the synaptosomal proteomic method for rat brain tissues.And the synaptosomal protein expression data in different brain regions are useful references for other studies.Part two Study on the molecular mechanism of seizures based on the hippocampal synaptosomal proteomics Objective: To reveal the molecular mechanism of seizures by investigating the effects of hyperthermia and infections on the expressions of synaptosome proteins in hippocampus.Methods: The changes of protein expressions profiles in hippocampal synaptosomes of animals with hyperthermia-or LPS-treatment were analyzed by the proteomic methods.Then we analyzed the differential proteins by bioinformatic methods to reveal the molecular mechanism of seizures.And we also proved the hypothetical mechanism by the electroencephalographic(EEG)recording.Results: The 1313 differential proteins in hippocampal synaptosomes were induced by hyperthermia-or LPS-treatment.Among the 1313 proteins,the expressions of 1312 proteins altered with the same trend,which indicated that hyperthermia-and LPS-treatment have common action to synergistically induce seizures.Pathway analysis and protein-protein interaction analysis revealed that the glutamatergic synaptic pathway was the major target for the onset of seizures.Especially,the protein network in which AMPA receptors were regards as the core changed greatly(i.e.the protein expressions were up-regulated).It was demonstrated that NBQX,a specific inhibitor of AMPA receptor,inhibited hippocampal epileptiform EEG discharges induced by seizures,suggesting that AMPA receptors are closely related to the seizures caused by hyperthermia and infection.Brief summary: The expressions of some synaptic proteins altered with the same trend when the animals treated with LPS or heat.The protein network with AMPA receptor as the core is closely related to the seizures caused by infection and fever.Part three Study on the preventive and therapeutic effects of ginseng on seizures based on synaptosomal proteomics Objective: To reveal what were altered on the expressions of synaptosomal proteins after ginseng administration,and to find and verify the effective chemical substances of ginseng for preventing the occurrence of seizures.Methods: By the synaptosomal proteomics,we studied the expression changes of hippocampal and cortical synaptosomal proteins in rats after a long-term administration of ginseng(14 days).After the bioinformatic analysis,the effect of ginseng on the expressions of mitochondrial oxidative phosphorylation pathway-related proteins was confirmed.Then we tested the effects of ginsenosides on mitochondrial oxidative phosphorylation in vitro.Finally,we observed the preventive effects of representative ginsenoside on the occurrence of seizures by EEG recording.Results: The expressions of mitochondria-associated proteins in rat hippocampal and cortical synaptosomes ware down-regulated.The bioinformatic analysis revealed the altered proteins were closely associated with mitochondrial oxidative phosphorylation.Mitochondrial respiration tests confirmed that ginseng extract and ginsenosides Rb1,Rg1,Rc,Rb2,Rd could inhibit the process of mitochondrial oxidative phosphorylation and the activity of ATP synthase in vitro.Among test ginsenosides,ginsenoside Rd is the most powerful inhibitor for mitochondrial oxidative phosphorylation.It is proved that ginsenoside Rd could inhibit hippocampal epileptiform EEG discharges induced by seizures,suggesting that ginsenoside Rd is a neuroprotector for seizures.Brief summary: Ginseng mainly affects the oxidative phosphorylation of mitochondria in the central nervous system.Ginsenoside,especially ginsenoside Rd,is the main active component of Panax ginseng to inhibit the process of oxidative phosphorylation.Ginsenoside Rd can inhibit epileptic epileptiform discharge caused by seizures and can be used to prevent the occurrence of febrile seizures.Conclusions:1.We established a synaptosomal proteomic method for brain tissues.After investigating the expressions of synaptosomal proteins,it is approved that the established proteomic method was feasible.And the expression data of the synaptosomal proteins in different brain regions are useful references for other studies.2.The expressions of some synaptosamal proteins was altered with the same trend after the rats treated by hyperthermia-or LPS-factor,which may be the main reason that hyperthermia-and LPS-treatment have the ability to synergistically induce seizures.Proteomics analysis reveals that AMPA receptors are closely related to the seizures caused by hyperthermia and infection.3.Ginseng extract and some ginsenosides could inhibit the process of mitochondrial oxidative phosphorylation and the activity of ATP synthase.Ginsenoside Rd could inhibit hippocampal epileptiform discharges induced by seizures.