| BACKGROUND & OBJECTIVEDrug abuse is a global pervasive social health problem, which is getting muchworse in China with the heroin abusers as the main bodies. In many coutries, drugabuse has become the third leading cause of death, after cardio-cerebrovasculardisease and malignant tumor. Therefore, the central toxicity and the dependencemechanism of the drug have become a hot issue nowadays. Heroin, on the top of thehard drug list, is a kind of opioid. Study on mechanisms of the drug addiction toopioids and nerve damage mainly focus on the opioid receptor and opioid peptidessystem, which regulate many neurotransmitters, neuromodulator, endocrine, ionchannels and intracellular signal transduction system in vivo through the complexneuro-humoral mechanisms. The exact mechanism of opioid addiction has beeninvestigated.Because the structure and function of the central nervous system is complex, it isdifficult to clarify the mechanism of drug toxicity completely through traditionalmethods, such as mechanism of pathological injury and addiction. Omics opened up anovel pathway of studying life characteristic roundly and systematically. Protein isthe executant of the physiological function and the embodiment of life phenomenon.Study on protein structure and function would directly elucidate the life mechanism changed in physiological and pathological conditions.As reinforcing system to the nerve, Mesolimbic dopamine rewarding system isthe axis position of opioid dependence, and it is in the final reinforcing pathway ofopioid. Cortex - basal ganglia circuit is concerned with learning and memory-relateddrug addiction. All of Prefrontal cortex (PFC), nucleus accumbens (NAc),hippocampus and striatum corpora play a key role in two closely related pathway.Inthis study, difference protein expressing profile is analyzed by two-dimensional gelelectrophoresis (2-DE), and these different protein spots is further analyzed withMatrix-assisted laser desorption inoization-time of flight mass spectrometry(MALDI-TOF MS), by which the differential protein expression and modification isseeked to reveal the functional changes and interaction between different proteins. Itis expected that the mechanism of drug- dependence and toxicity caused by heroinwould be revealed from our research, including mechanism of pathological injury andthe latest ways to clarify the mechanism of drug addiction.METHODS1. Establishment of heroin dependent rat model and pathology inspection ofheroin-induced organ, especially brain damage.A total of 32 male wistar rats were used. The rats were random divided intothree groups. Primal heroin (purification 80%) dosage was 4mg/kg, increasedprogressively 4mg/kg from second day,three times everyday, intraperitonealinjection, continue administering 10d,as heroin addictive group. Then the rats hadbeen abstinent for 3d, as natural withdrawal group. Physiological saline was tookplace of heroin to inject, as control group. The withdrawal scores were evaluated byimproved criterion of literatures after administering naloxone.Recorded the ratsweight between control group and withdrawal group during establishing the heroinwithdrawal rat model. Organs, especially brain were observed under optical and/orelectronic microscope,and the expression of GFAP were tested byimmunohistochemical method in the brain tissues.2. Detectioin of the concentration of DA and DOPAC in correlated encephalic regionsbetween heroin addictive group and control group A total of 12 male wistar rats were used.The rats were random divided into twogroups. In accordance with stereotaxic atlas of rat brain, dissected PFC, NAc,hippocampus and striatum corpora, then prepared samples. HPLC technology wasperformed to detect the concentration of DA and DOPAC in the four brain regions,the ratio of DA and DOPAC was calculated.3. Establishment and optimization of two-dimensional polyacrylamide gelelectrophoresis (2-DE) for the proteome analysis of the different encephalic regionsof rat.In accordance with stereotaxic atlas of rat brain,dissected PFC,NAc,hippocampusand striatum corpora, Choosing suitable tissue lysis buffer, prepared samples for2-DE.The samples were precipitated with acetone in order to desalination,and theconversation were detected by improved Bradford method, pH3～10 line IPG and2-DE technology was used to separate the total proteins of the samples.The gels werestained by silver(200μg samples) or Blue silver(1mg samples),to compare thedifference about the two methods.4. Screening of differential expression protein from heroin addictive rats,withdrawalrats and control rats by 2-DE and MALDI-TOF-MS.30 male wistar rats were random divided into three groups:control goup,heroinaddctive group and withdrawal group.The narrow pH4～7 line IPG strip and 2-DEtechnology was used to separate the total proteins of the samples, and the gels werestained with Blue silver. 2-D gel analysis software (ImageMaster 2D Platinum 5.0)was used in this study for spot detection, gel matching and spot quantitation.Significant spots that showed at least 2-fold difference in intensity between thegroups were selected for protein MALDI-TOF—TOF-MS identification. After dataacquisition the mass spectra were searched against the GPS-MS database searchengine.5. The differences of neurogranin expression in rat brain between heroin addcitive,withdrawal and control groups.Western blot and immunohistochemistry analysis were used to further verifyneurogranin in order to ensure the reliabilty of the proteome result. RESULTS1. Successful establishment of heroin addictive, withdrawal rat modelAfter naloxone-precipitated withdrawal reactions, In heroin injection group,withdrawal symptoms scores different significantly compared with those of thecontrol group (t=19.094,P=0.000), which marks the successful establishment ofheroin dependence rat model. In the model-making process since the third day afterinjection, there was a significant difference in body weight between heroin injectiongroup and the control group (F=41.412,P=0.000); After two days of heroinwithdrawal weight losses in rat differents significantly compared with that of thecontrol group (t=9.889,P=0.000); Histology revealed livers of heroin injection grouphad hepatic lobules peripheral edema, mild steatosis lambs edema. Two addictiverats' renal tube had protein casts; and we found a small amount of PFC andhippocampus neuronal degeneration in brain tissue ultrastructure; GFAPimmunohistochemistry results showed PFC and hippocampal CA1 region, heroinaddiction,withdrawal group PU(positive units), exsisted a significant differencecompared with that of the control group (F=36.772. P=0.000;F=105.534, P=0.000),and we found no tissue-specific in GFAP positive expression of drug group.2.Changes of dopamine and its metabolite DOPAC in brain regions related to heroinaddiction ratsHPLC found except the hippocampus, the two contents of the other three brainareas changed significantly compared with the control group,where PFC and NAcincreased significantly (t=36.617,P=0.000; t=16.017,P=0.000; t=16.991,P=0.000; t=4.186, P=0.002), but in striatum corporate of addictive group rats,thetwo decreased significantly (t=32.642,P=0.000; t=50.457, P=0.000); DOPAC/Dof addiction group PFC was significantly higher than the control group (t=7.429, P=0.000); But NAc and striatum corporate was significantly decreased (t=15.535,P=0.000;t=16.673, P=0.000).3.Analysis and comparison of rat different brain regions gel maps in differentconditions of two-dimensional electrophoresisUnder premise of other electrophoresis optimal conditions, we stained the gel using silver staining and Coomassie Brilliant Blue (Blue silver), Through comparisonwe found the silver staining gel map repeated poorly, and there was less protein andso on. But through Coomassie Brilliant Blue (Blue silver),we found goodrepeatability,separation and clear points. And we found protein of different Brainsexisted mainly in the area pH4～7.4. Screening and MALDI-TOF-TOF-MS identification of variance proteins incorrelated encephalic regions of heroin addictive, Withdrawal rats and control ratsAfter 2-DE separation of PFC, NAc, hippocampus and striatum corporate proteinsof the three groups within pH4-7. By image analysis, the above four brain regionsbetween the three groups were screened 8, 10, 5, 7 variance proteins spots, andthrough MALDI-TOF-TOF MS analysis we identified successful 27 proteins spots, atotal of 18 proteins.(1) In PFC, the proteins which exists only in addictive group and withdrawal groupare glucose-regulated protein 58 (glucose regulate d protein, called GRP58;ERp57. ER60) and 26s proteasome subunit p40.5 (26S proteasome subuni tp40.5), and there are almost the same expression; Compared with the controlgroup and addiction group, ubiquitin ligase E2N (Ubiquitin-conjugating 2.1.3.3E2N) of withdrawal group had higher expression;In dependence and withdrawalgroups, the down-regulated protein is ATP synthase D chain; In 2-DEmaps,compared to control group two protein spots including Ndufa10[NADHdehydrogenase (ubiquinone) alpha subcomplex-10] and Eno1 (alpha-enolase) inthe heroin addictive and withdrawal group had been migrated; After comparisonbetween heroin addictive, withdrawal group and the control group,the MW andpI differed in Ndufa10,but the differences in MW and pI of Eno1 become moreobvious.(2) In NAc, the proteins which exists only in addiction group and abstinent group areGrp58, In the two experimental groups the same increased expression of proteinwas mitochondria F0 complex F6subunit involved in ATP synthase (ATPsynthase H+transporting, mitochondrial F0 complex, subunit F6), Expression ofapoE,low M(r) phosphotyrosine protein phosphatase isoenzyme AcP2 and ADP-ribosylarginine hydrolase in addictive group decreased, the proteins whichexists only in control group was epidermal fatty acid binding protein 5; theexpression of Ndufa10 in NAc and PFC brain regions was the same basically.(3) In the hippocampus,Ng expressed highly in heroin addictive group andwithdrawal group; Phase tubulin protein.α1 expressed highly; mVps29pExpression in the normal group, dependence and withdrawal group went downgradually; the expression of Ndufa10 in hippocampus and PFC brain regions wasthe same basically.(4) In the striatum corpora, the increased expression protein of Heroin Addictive andthe withdrawal Group were Zinc binding alcohol dehydrogenase and Guanosinediphosphate (GDP) dissociation inhibitor 2(GDI2), But isocitrate dehydrogenase3 (NAD+) alpha had less expression in the two groups; expression of TMOD2decreased in the withdrawal Group; the expression of Ndufa10 in striatum corporaand PFC brain regions was the same basically.5. Certification of Ng expression in the brain tissues of heroin addictive andwithdrawal ratsWestern blot results showed that among 6.5～14.4Kda of all groups there was acoarse Ng Western blot strip.The intensity of strip in heroin addictive, withdrawalgroup were significantly higher than the control group. Immunohistochemistry resultsshowed Ng expression was mainly in the cortex and hippocampus of heroin injectedrats,but not obvious expression in other brain areas. The poisitive Ng product PU ofPFC and the hippocampal CA1 region in three groups were statistically significant(F=53.552, P=0.000; F=48.861,P=0.000); In PFC brain regions, the PU ofdependence, withdrawal group and the control group were significantly different (P=0.000, P=0.000), but there was no difference between the addiction Group andwithdrawal group (P=0.535); In hippocampus CA1 region, the PU of dependence,withdrawal group and the control group were significantly different (P=0.000. P=0.000), and there was difference between the addiction Group and withdrawal group(P=0.022).CONCLUSIONS 1. The rat model of Heroin Addiction and spontaneous abstinence from drugaddiction is established successfully, and it was found to cause mild damage to thecentral nervous system.2. Results from all of DA and DOPAC group further showed that mesolimbicdopamine system, which core is the NAc, had participated in the process of becomingopioid addiction, and the mechanism of DA neurotransmitter involved in the heroinaddiction.3. A good condition for 2-DE is established, and 27 sites, that is 18 proteins, had beensuccessfully identified based on this technique. According to its functions, thoseproteins can be classified as molecular chaperone protein, proteindegradation-associated protein, protein modification-associated protein, signaltransduction proteins, cytoskeletal proteins and metabolism and energy-relatedenzyme. The results suggest that heroin addiction and withdrawal inevitably lead toadaptability of pathological changes in nerve cells. The structure of nerve cells,synaptic plasticity, cell transport, synaptic transport, signal transduction, modificationor degradation of functional function and the changes of energy metabolism musthave been involved in this process.4. The modified Ndufa10 in four specific brain regions after both Heroin addictionand withdrawal is possible the molecular marker associated with heroin-dependentand widely distributed. A novel protein, named as Grp58 and expressed in rat PFCand NAc brain regions after both Heroin addiction and withdrawal, is also a probableprotein marker associated with heroin-dependent.5. Ng expression in the hippocampus and PFC is up-regulated in rat of heroinaddiction and withdrawal, which suggest that the key protein- Ng play an importantrole in learning and memory process involved in heroin dependence... |
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