| Part Ⅰ.Pinpoint the target protein and signaling pathway of perampanel induced aggression behavior in mice by TMT combined with LC-MS/MS.Objective To explore the key protein and the related signaling pathways of perampanel induced aggressive behavior in C57BL/6J mice.Methods C57BL/6J mice were administered intragastrically with perampanel for 60 days to establish the aggressive behavior model.TMT-lab el combined with liquid chromat-ography-tandem mass spectrometry were used to identify the differentially expressed proteins in the hippocampal tissues of the aggressive behavior mice and the control mice.Gene ontology functional annotation and enrichment of the differentially expressed protein were performed by bioinformatics software,and the most differentially expressed proteins were selected.The obtained protein information was input into the Kyoto Encyclopedia of Genes and Genomes pathway database for pathway interpretation and enrichment analysis of target proteins and STRING interaction network analysis of differentially expressed proteins to find out the target proteins and key signaling pathways related to perampanel induced aggressive behavior.Results Long-term exposure to perampanel increased the aggressive behavior of C57BL/J mice,showed more attacks,significantly increased the attack duration and decreased incubation period of first attack.A total of 93 differentially expressed proteins were identified in hippocampal tissues(n=6849,95%confidence interval:1.07-1.011,p<0.05),which 59 proteins were up-regulated and 34 proteins were down-regulated.These proteins are mainly associated with synaptic function,Glutamate APMA receptor transport,Ras/R-ap GTPase,synaptogenesis,synaptic plasticity,postsynaptic density protein,vesicle transport and neurite growth.The enrichment analysis of GO showed that among the top 20 en-richment items,biological processes included regulation of synaptic plasticity,regulation of AMPA receptor activity,regulation of postsynaptic dense and synaptic structure,regulation of p-ERK mediated folding protein response,vesicle-mediated transport regulation and synaptic structural homeostasis.Molecular functions include neuroprotein family,SH3 do-main,structural components of postsynaptic dense,nerve growth factor,neurolinker family protein and brain-derived neurotrophic factor.The cellular components involved in the postsynaptic membrane,presynaptic membrane,dendritic spinous membrane,dendritic membrane and neuron projection membrane.KEGG pathway enrichment analysis showed that Ras Signaling Pathway and Axon guidance were the most significant changes in pathways with protein up-regulation and down-regulation.The STRING interaction network diagram showed that the differential proteins contained four main network clusters,namely neural development and dendritic growth,Ras pathway,postsynaptic density protein and Glutamate AMPA receptor.Conclusion Perampanel altered the protein profile expression in the hippocampus of mice.Glutamate AMPA receptor and Ras pathway are potential targets of PER-induced aggression in mice.Part Ⅱ.Molecular mechanism of perampanel induced aggressive behavior in miceObjective To study the correlation between aggressive behavior and AMP A receptor subunit induced by perampanel in mice and the mechanism of TNF-α mediated aggressive behavior.Methods The model of aggressive behavior induced by perampanel was established.The resident intruder test was used to evaluate the aggressive behavior,and the damage of perampanel on hippocampal neuron morphology was observed by Nissl staining.The expression of GluAl subunit and GluA2 subunit in hippocampus was detected by immun-ofluorescence.Ultrastructural changes of synapses in hippocampal CA3 region were observed by transmission electron microscopy.The morphological changes of dendrites and dendritic spines in hippocampal CA3 region were analyzed by Golgi staining.The correlation between inflammatory factors TNF-α,IL-6,IL-1β and aggressive behavior was determined by ELISA.Western blot was used to verify the changes of GluA1 subunit and related proteins in Ras signaling pathway.Results The nissl bodies of the hippocampus neurons in the aggressive mice were clearly visible and structurally intact.Immunofluorescence showed that the number of Glu-A1 subunit in hippocampal CA3 region increased.TEM showed that the length of synaptic vesicles in the hippocampus of the aggressive mice was shorter,the thickness of the postsynaptic dense became thicker,and the synaptic interface curvature became smaller,with statistically significant differences,but there was no difference in synaptic cleft.Golgi staining showed that dendritic spines of hippocampal neurons increased,but the length and number of dendrites had no effect.The expression of inflammatory cytokines TNF-α,IL-6 and IL-1β in the hippocampus of mice was detected by ELISA.The expression of Glual receptor subunit protein in mouse hippocampus was detected by Western blot.GluA2 subunit protein expression decreased;Synaptophysin protein expression decreased;Postsynaptic dense protein protein expression decreased;The protein expression of p-ERK1/2 increased.ERK protein expression decreased;The expression of BDNF protein decreased,which was statistically significant.Conclusion Perampanel induced TNF-α to activation the ERK1/2/BDNF signaling pathway that mediates GluA1 involvement in aggression behavior.Part Ⅲ.Ameliorative effect of glycyrrhetinic acid on aggressive behavior induced by perampanelObjective To investigate the ameliorative effect of glycyrrhetinic acid on aggressive behavior in mice induced by perampanel.Methods The 330 male C57BL/6J mice were randomly divided into 5 groups:Control group(CON group,n=15);Glycyrrhetinic acid group(GA Group,n=15);aggressive behavior model group(PER group,n=13/100);Aggressive behavior model&glycyrrhetinic acid group(PER&GA group,n=14/100)and Perampanel&glycyrrhetinic ac-id&epilepsy group(PER&GA&EP group,n=14/100).The model of aggressive behavior was established by continuous intragastric administration of perampanel,and the model of epilepsy was established by pentylenetrazol lighting.The expression of GluAl and GluA2 subunit in hippocampus of mice in each group was detected by immunofluorescence,and the expression of TNF-α in hippocampus of mice in each group was detected byElisa.The expressions of GluAl and GluA2,SYN,PSD-95,ERK1/2,P-ERK/1/2 and BDNF proteins in hippocampus of each group were detected by Western blot.Results After 3 days of treatment with glycyrrhetinic acid,the total number and duration of attack in PER&GA group were significantly decreased compared with PER group,the incubation period of attack was significantly increased.However,compared with CON group,the incubation period of attack was still shortened,and the difference was statistically significant(**p<0.01).After 3 days of treatment with glycyrrhetinic acid,the total number of attacks in PER&GA&EP group was significantly lower than that in PER group,attack duration increased,prolonged incubation period of attack,the difference was statistically significant;Co-mpared with PER&GA group,the total number of attacks increased,the attack duration was significantly prolonged,shorten the incubation period of attack,the difference was statistically significant.Glycyrrhetinic acid reduced the expression of TNF-α in hippocampus of mice in PER group.Glycyrrhetinic acid intervention reduced the expression of GluA1 in hippocampus of PER group,and there was no significant change in the expression of GluA2.Glycyrrhetinic acid increased SYN and decreased PSD-95 protein expression in hippocampus of PER group,respectively.Glycyrrhetinic acid increased the expression levels of ERK1/2 and BDNF protein in hippocampus of PER group,and decreased the expression level of p-ERK/1/2.Conclusion Glycyrrhetinic acid can improve the aggressive behavior induced by perampanel,the possible mechanism is to regulate the elevation of GluA1 subunit by inhibiting the TNF-α pathway. |
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