| Background Opium addiction is now regarded as a psychiatric disease,it places an enormous burden on society all over the world.The central problem in the treatment of opium addiction is high rates of relapse to drug use after periods of forced or self-imposed abstinence. Although the mechanisms that underlie the development of dependence on morphine remain unclear, previous studies suggested that proteome changes of brain are crucial determinants of extinction and reinstatement condition. Thus, understanding global protein changes events induced by the two phases are essential for understanding the complex mechanisms of morphine dependence.Objective To use comparative proteomics technology to find out the differential expressed protein in NAc between extinction and reinstatement to morphine addiction.There are three sections in this study.(1)To establish extinction and relapse model.(2)To optimized two-dimensional electrophoresis for proteomic analysis of NAc. (3)Screening target proteins from proteomics databases.Methods:60 male SD rats were tested randomly in group of three: saline group,morphine group and yoked-morphine group. Initially trained to self-administer drugs by active nose-poke for intravenous drug infusions in operant conditioning chambers.Subsequently, the drug-reinforced behavior is extinguished by disconnecting the infusion pumps. After extinction of the drug-reinforced behavior,each group was divided into two parts(extinction examination and reinstatement examination ).The ability of acute exposure to condition drug cues to reinstate drug seeking is determined.When all models established,rats were sacrificed by decapitation,then NAc was dissected from a 1mm section and immersed it in liquid nitrogen for five minutes ,stored in -80℃.The proteins from rat NAc were extracted,2-DE system with the first dimension using isoelectrofocusing electrophoresis and the second dimension using SDS-PAGE was employed to obtain the 2-DE gel maps.Then gels were stained with silver and differential protein spots were analyzed by software analysis,subject to in-gel digestion,and identified by matrix assistant laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS).Results:①Stable extinction and relapse behavior were successfully established②The resolution of 2-DE mappings with pH3-10 non-liner gradient strip were better than with pH3-10 liner gradient strip.(p<0.05)④Did the analysis of protein spots group with PDQuest8.0 analysis software, there were(868±27) protein spots in saline extinction group,(833±24) protein spots in saline reinstatement group,( 859±38) protein spots in morphine extinction group,( 838±33)protein spots in morphine reinstatement group, ( 846±31) protein spots in yoked extinction group, and( 863±21)protein spots in yoked reinstatement group.There were 1protein spot hyp-expression and 1 protein spot homo-expression on the NAc 2-DE mappings between saline two groups, there were 1 protein spot hyp-expression and 8 protein spots homo-expression between morphine two groups,there were 6 protein spots hyp-expression and 1protein spot homo-expression between yoked two groups.⑤We identified 11 proteins by MS and moscot: Fatty acid-binding protein( brain-FABP7),Serine/threonine-protein phosphatase 2A catalytic subunit beta isoform(PP2AB), Serine/threonine-protein phosphatase2A catalytic subunit alpha isoform(PP2AA) ,Serine/threonine-protein phosphatase 2A regulatory subunit B'' subunit gamma, Hsp90 co-chaperone Cdc37,Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial precursor,Sialidase-3,Peroxiredoxin-2,Glutathione S-transferase Mu 2, Glutathione S-transferase Mu1, Schwannomin-interacting protein 1.Conclusion : In this study,comparative proteomics technologies were systemically performed in extinction and reinstatement model,11 proteins were successfully identified with MALDI-TOF mass spectrometry. Statistical analysis revealed 5 protein spots which were significantly changed form extinction to reinstatement concerned with phosphorylation, TCA cycle and stress.These results will provide scienticfic foundation for screening the molecular biomarker used to diagnose and treat opium dependence,as well as to provide new clue for the research of drug addiction.③self-administration was related to rewarding behavior and pharmacological effects of psychoactive substance,but passive- administration was related to pharmacology only,2-DE maps were difference between the two ways,we supposed that rewarding behavior would induce protein expression changes,specific mechanisms needs further study. |
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