海洛因成瘾戒断大鼠相关脑区差异蛋白的分离与鉴定
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摘要
研究背景和目的
吸毒是全球普遍存在的严重社会卫生问题，在我国形势也日趋严峻，以海洛因滥用者为主体。毒品滥用已成为许多国家仅次于心脑血管疾病和恶性肿瘤的第三位致死病因。因此，毒品的中枢性毒害作用以及依赖机制已成当今研究的热点问题。
海洛因作为硬性毒品之王，属于阿片类药物。对阿片类药物成瘾机制和神经损害的研究主要集中于阿片受体及其阿片肽系统，通过各种复杂的神经体液机制调节体内多种神经递质、调质、内分泌、离子通道及细胞内信号传导系统的作用。但是，阿片成瘾的确切机制尚未明了。
传统的方法很难完全阐明毒品的毒性作用，包括病理性损伤机制以及成瘾性机制等关键问题，而组学研究，开辟了全面、系统把握生命现象之特征的研究道路。蛋白质是生理功能的执行者，是生命现象的直接体现者，对蛋白质的结构和功能的研究将直接阐明生命在生理和病理条件下的变化机制。
由于中脑—边缘多巴胺(DA)奖赏或强化神经系统与皮层-基底节回路是参与药物成瘾相关的重要的通路，而且前额叶皮质(PFC)、伏隔核(NAc)、海马和纹状体在两个密切相关的通路中扮演重要角色，因此本研究通过双向电泳(2-DE)技术筛选这四个脑区的差异蛋白表达谱，结合基质辅助解析飞行时间质谱鉴定这些差异蛋白和标志性蛋白位点，寻找蛋白的差异表达以及修饰变化，揭示其功能的变化和蛋白之间的相互作用，有可能从机理上揭示海洛因导致的依赖和毒性作用，包括病理性损伤机制以及成瘾性机制等的最新途径。
方法
1．海洛因成瘾戒断大鼠模型的建立及病理学观察
成年雄性Wistar大鼠，随机分成3组，设计海洛因初始剂量为4mg／kg体重，每天递增4mg／kg体重，腹腔注射，3次／天，共10天，建立海洛因成瘾模型；停药后3天作为自然戒断大鼠模型；并以生理盐水代替海洛因注射作为对照组。利用纳洛酮催促戒断反应，观察戒断症状并根据改进的柳田知司评分标准进行评分；记录大鼠模型制作过程中不同时间体重变化。对各组的内脏器官和脑组织进行病理学观察，并应用免疫组化手段检测脑损伤标志分子GFAP的表达变化。
2．海洛因成瘾和正常大鼠相关脑区DA和代谢产物DOPAC的检测
参照大鼠脑立体定位图谱，分离海洛因成瘾组和对照组各6只大鼠PFC、NAc、海马和纹状体等脑组织，制备样品，利用高效液相色谱技术，检测对照组和海洛因成瘾组大鼠各相关脑区中的DA以及代谢产物DOPAC的含量，并观察二者比值。
3．大鼠不同脑区双向电泳条件的建立
选择合适的组织裂解液，参照大鼠脑立体定位图谱，分离PFC、NAc、海马和纹状体等脑区组织抽提蛋白，并经冷丙酮沉淀除盐，制备双向电泳蛋白样品，经改良Bradford法检测蛋白浓度；选择pH3～10线性固相pH梯度预制胶(IPG)，利用2—DE技术在pH3-10范围内分离蛋白，分别用银染法(上样量200μg)和考马斯亮蓝Blue silver染色法(上样量1mg)对凝胶进行染色，并对两种方法进行比较。
4．海洛因成瘾戒断大鼠和对照大鼠相关脑区差异蛋白的筛选和MALDI-TOF—TOF-MS鉴定
选择pH4～7线性IPG，在上述2—DE条件下对海洛因成瘾、戒断和正常对照3组大鼠相关脑区蛋白样品行2—DE分离，考马斯亮蓝Blue silver染色法对凝胶进行染色；所得凝胶图谱借助图像分析软件包ImageMaster 2D Platinum 5.0进行图像分析，同一脑区不同组之间的蛋白质表达水平上升或下降200％的点选择为目标点；随后筛选重复性和分离效果好的差异蛋白点进行MALDI-TOF—TOF-MS鉴定；所得肽质量指纹图(PMF)和串联质谱图，用GPS-MS进行数据库检索，数据库选择NCBInr，鉴定筛选的差异蛋白。
5．神经颗粒素(Ng)在海洛因成瘾戒断大鼠脑组织中的表达
采用western blot和免疫组化方法在蛋白水平上验证Ng在海洛因成瘾戒断大鼠脑组织中的表达。
结果
1．海洛因成瘾、戒断大鼠模型成功建立
纳洛酮催促戒断反应后，海洛因注射组戒断症状得分与对照组比较具有显著性差异(t=19.094，P=0.000)，标志着海洛因注射后大鼠已经产生药物依赖。海洛因注射组和对照组在模型制作过程中体重变化自注射第3天起具有显著性差异(F=41.412，P=0.000)；海洛因停药后第2天大鼠体重下降，与对照组相比差异显著(t=9.889，P=0.000)；组织学观察可见海洛因注射组肝脏部分肝小叶有水肿、轻度脂肪变，2只成瘾模型肾脏肾小管有蛋白管型；脑组织超微结构发现少量PFC和海马神经元变性；GFAP免疫组化结果显示PFC和海马CA1区，海洛因成瘾、戒断组PU值(阳性单位)与对照组比较有显著性差异(F=36.772，P=0.000；F=105.534，P=0.000)，并且发现用药组GFAP阳性表达无组织特异性。
2．海洛因成瘾大鼠相关脑区多巴胺及其代谢产物DOPAC的变化
高效液相色谱结果发现除海马外，其它3个脑区DA和DOPAC的含量发生明显变化，与对照组相比，其中PFC和NAe显著性升高(t=36.617，P=0.000；t=16.017，P=0.000；t=16.991，P=0.000；t=4.186，P=0.002)，而成瘾组大鼠的纹状体中二者含量却显著下降(t=32.642，P=0.000 t=50.457，P=0.000)；成瘾组PFC的DOPAC／DA值显著高于对照组(t=7.429，P=0.000)；而NAc和纹状体却显著降低(t=15.535，P=0.000；t=16.673，P=0.000)。
3．大鼠不同脑区双向电泳条件的凝胶图谱分析比较
其它电泳条件优化前提下，分别用银染法和马斯亮蓝Blue silver对凝胶进行染色，经比较发现各脑区银染的凝胶图谱都存在重复性差，得到的蛋白点少等等，而应用考马斯亮蓝Blue silver对凝胶进行染色，发现重复性好，分离效果二者相差不大。并发现各脑区蛋白主要集中在pH4～7范围内。
4．海洛因成瘾、戒断大鼠和对照大鼠相关脑区差异蛋白的筛选和MALDI-TOF—TOF-MS鉴定
在pH4～7范围内2—DE分离3组大鼠PFC、NAc、海马和纹状体组织的蛋白，经图像分析，以上4个脑区在3组之间分别筛选出8、10、5、7个差异蛋白点，经MALDI-TOF-TOF-MS分析，成功鉴定27个蛋白点，共计18个蛋白。
(1)在PFC中，仅在成瘾组和戒断组出现的蛋白是葡萄糖调节蛋白58(glucose regulated protein,GRP58；又称ERp57，ER60)和26s蛋白酶体亚基p40.5(26S proteasome subunit p40.5)而且表达量相当；相对于对照组和成瘾组，戒断组的泛素连接酶E2N(Ubiquitin-conjugating enzyme E2N)表达升高；成瘾组和戒断组含量下调的蛋白是ATP合酶D链；成瘾、戒断组与对照组相比，Ndufa10[NADH dehydrogenase(ubiquinone)alpha subcomplex-10]和α-烯醇化酶(Enol)发生相对分子质量和(或)等电点迁移，Enol的MW和pI差异更加明显。
(2)在NAc中，仅在成瘾组和戒断组出现的蛋白是Grp58，在两个实验组中都表达升高的蛋白是参与ATP合成的线粒体F0复合体F6亚基(ATP synthase，H+transporting，mitochondrial F0 complex，subunit F6)，而载脂蛋白酶E(Apolipoprotein E，apoE)、低分子量磷酸酪氨酸蛋白磷酸酶同工酶AcP2(low M(r)phosphotyrosine protein phosphatase isoenzyme AcP2)和ADP—精氨酸核糖基化水解酶(ADP-ribosylarginine hydrolase，AAH)在成瘾组中表达下降，仅在对照组出现的蛋白是表皮型脂肪酸结合蛋白(fatty acid binding protein 5，epidermal)；Ndufa10的表达情况与PFC脑区基本相同。
(3)在海马组织中，神经颗粒素(neurogranin，Ng)曲在海洛因成瘾、戒断组表达明显升高；戒断组微管蛋白α1表达升高；但mVps29p表达量在正常组、成瘾组和戒断组中依次降低；Ndufa10的表达情况与PFC脑区基本相同。
(4)在纹状体中，海洛因成瘾组和戒断组表达升高的蛋白是Zn结合乙醇脱氢酶(Zinc binding alcohol dehydrogenase)和G蛋白解离抑制因子2[Guanosine diphosphate(GDP)dissociation inhibitor 2,GDI2]，而两组的NAD~+依赖的异柠檬酸脱氢酶[isocitrate dehydrogenase 3(NAD+)alpha]表达降低；原肌球调节蛋白2(tropomodulin,TMOD2)在戒断组表达下降；Ndufa10的表达情况与PFC脑区基本相同。
5．神经颗粒素(Ng)在海洛因成瘾戒断大鼠脑组织中的表达验证
Western blot结果表明各组在6.5～14.4Kda之间均显示一条较粗的Ng免疫印迹反应条带，海洛因成瘾、戒断组含量明显高于对照组；免疫组化结果显示Ng阳性表达主要在海洛因成瘾、戒断脑组织的皮质和海马组织，其它脑区未见明显表达。PFC和海马CA1区Ng的阳性产物的PU值各组总体比较差异显著，有统计学意义(F=53.552，P=0.000；F=48.861，P=0.000)；PFC脑区，成瘾组、戒断组的PU值与对照组比较均有显著性差异(P=0.000，P=0.000)，而成瘾组和戒断组比较没有统计学差异(P=0.535)；海马CA1区，成瘾组、戒断组的PU值与对照组比较均有显著性差异(P=0.000，P=0.000)，成瘾组和戒断组比较差异有统计学意义(P=0.022)。
结论
1．成功建立了海洛因成瘾和自然戒断大鼠模型，并且发现海洛因对中枢神经系统造成轻度损害。
2．各组DA和DOPAC结果进一步证明了以NAc为核心的中脑多巴胺奖赏系统参与阿片类药物成瘾的形成过程，DA是参与海洛因成瘾形成的重要的神经递质。
3．建立了良好的2—DE条件，在此基础上成功鉴定了27个蛋白位点即18个蛋白，根据功能可以划分为分子伴侣蛋白，蛋白质降解相关蛋白，功能蛋白修饰相关蛋白，信号转导蛋白，细胞骨架蛋白，能量及物质代谢相关的酶，功能涉及神经保护和毒害两个方面。由结果可知，海洛因成瘾、戒断必然引起神经细胞的适应性病理改变，这个过程必然涉及神经细胞结构、突触可塑性、细胞运输与突触运输、信号转导、功能蛋白的降解或修饰以及能量代谢等方面的改变。
4．海洛因成瘾、戒断后在四个脑区中被特异修饰的Ndufa10广泛分布；在PFC和NAc脑区，海洛因成瘾、戒断大鼠表达新蛋白Grp58。两个蛋白可能是海洛因依赖的标志性蛋白，但需要进一步研究证实。
5．Ng在成瘾戒断大鼠的海马和PFC表达上调，提示学习记忆通路中的关键蛋白Ng在海洛因依赖相关学习记忆形成中扮演重要角色。
BACKGROUND & OBJECTIVE
Drug 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.
METHODS
1. 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.
RESULTS
1. Successful establishment of heroin addictive, withdrawal rat model
After 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 rats
HPLC 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 electrophoresis
Under 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 rats
Western 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
吸毒是全球普遍存在的严重社会卫生问题，在我国形势也日趋严峻，以海洛因滥用者为主体。毒品滥用已成为许多国家仅次于心脑血管疾病和恶性肿瘤的第三位致死病因。因此，毒品的中枢性毒害作用以及依赖机制已成当今研究的热点问题。
海洛因作为硬性毒品之王，属于阿片类药物。对阿片类药物成瘾机制和神经损害的研究主要集中于阿片受体及其阿片肽系统，通过各种复杂的神经体液机制调节体内多种神经递质、调质、内分泌、离子通道及细胞内信号传导系统的作用。但是，阿片成瘾的确切机制尚未明了。
传统的方法很难完全阐明毒品的毒性作用，包括病理性损伤机制以及成瘾性机制等关键问题，而组学研究，开辟了全面、系统把握生命现象之特征的研究道路。蛋白质是生理功能的执行者，是生命现象的直接体现者，对蛋白质的结构和功能的研究将直接阐明生命在生理和病理条件下的变化机制。
由于中脑—边缘多巴胺(DA)奖赏或强化神经系统与皮层-基底节回路是参与药物成瘾相关的重要的通路，而且前额叶皮质(PFC)、伏隔核(NAc)、海马和纹状体在两个密切相关的通路中扮演重要角色，因此本研究通过双向电泳(2-DE)技术筛选这四个脑区的差异蛋白表达谱，结合基质辅助解析飞行时间质谱鉴定这些差异蛋白和标志性蛋白位点，寻找蛋白的差异表达以及修饰变化，揭示其功能的变化和蛋白之间的相互作用，有可能从机理上揭示海洛因导致的依赖和毒性作用，包括病理性损伤机制以及成瘾性机制等的最新途径。
方法
1．海洛因成瘾戒断大鼠模型的建立及病理学观察
成年雄性Wistar大鼠，随机分成3组，设计海洛因初始剂量为4mg／kg体重，每天递增4mg／kg体重，腹腔注射，3次／天，共10天，建立海洛因成瘾模型；停药后3天作为自然戒断大鼠模型；并以生理盐水代替海洛因注射作为对照组。利用纳洛酮催促戒断反应，观察戒断症状并根据改进的柳田知司评分标准进行评分；记录大鼠模型制作过程中不同时间体重变化。对各组的内脏器官和脑组织进行病理学观察，并应用免疫组化手段检测脑损伤标志分子GFAP的表达变化。
2．海洛因成瘾和正常大鼠相关脑区DA和代谢产物DOPAC的检测
参照大鼠脑立体定位图谱，分离海洛因成瘾组和对照组各6只大鼠PFC、NAc、海马和纹状体等脑组织，制备样品，利用高效液相色谱技术，检测对照组和海洛因成瘾组大鼠各相关脑区中的DA以及代谢产物DOPAC的含量，并观察二者比值。
3．大鼠不同脑区双向电泳条件的建立
选择合适的组织裂解液，参照大鼠脑立体定位图谱，分离PFC、NAc、海马和纹状体等脑区组织抽提蛋白，并经冷丙酮沉淀除盐，制备双向电泳蛋白样品，经改良Bradford法检测蛋白浓度；选择pH3～10线性固相pH梯度预制胶(IPG)，利用2—DE技术在pH3-10范围内分离蛋白，分别用银染法(上样量200μg)和考马斯亮蓝Blue silver染色法(上样量1mg)对凝胶进行染色，并对两种方法进行比较。
4．海洛因成瘾戒断大鼠和对照大鼠相关脑区差异蛋白的筛选和MALDI-TOF—TOF-MS鉴定
选择pH4～7线性IPG，在上述2—DE条件下对海洛因成瘾、戒断和正常对照3组大鼠相关脑区蛋白样品行2—DE分离，考马斯亮蓝Blue silver染色法对凝胶进行染色；所得凝胶图谱借助图像分析软件包ImageMaster 2D Platinum 5.0进行图像分析，同一脑区不同组之间的蛋白质表达水平上升或下降200％的点选择为目标点；随后筛选重复性和分离效果好的差异蛋白点进行MALDI-TOF—TOF-MS鉴定；所得肽质量指纹图(PMF)和串联质谱图，用GPS-MS进行数据库检索，数据库选择NCBInr，鉴定筛选的差异蛋白。
5．神经颗粒素(Ng)在海洛因成瘾戒断大鼠脑组织中的表达
采用western blot和免疫组化方法在蛋白水平上验证Ng在海洛因成瘾戒断大鼠脑组织中的表达。
结果
1．海洛因成瘾、戒断大鼠模型成功建立
纳洛酮催促戒断反应后，海洛因注射组戒断症状得分与对照组比较具有显著性差异(t=19.094，P=0.000)，标志着海洛因注射后大鼠已经产生药物依赖。海洛因注射组和对照组在模型制作过程中体重变化自注射第3天起具有显著性差异(F=41.412，P=0.000)；海洛因停药后第2天大鼠体重下降，与对照组相比差异显著(t=9.889，P=0.000)；组织学观察可见海洛因注射组肝脏部分肝小叶有水肿、轻度脂肪变，2只成瘾模型肾脏肾小管有蛋白管型；脑组织超微结构发现少量PFC和海马神经元变性；GFAP免疫组化结果显示PFC和海马CA1区，海洛因成瘾、戒断组PU值(阳性单位)与对照组比较有显著性差异(F=36.772，P=0.000；F=105.534，P=0.000)，并且发现用药组GFAP阳性表达无组织特异性。
2．海洛因成瘾大鼠相关脑区多巴胺及其代谢产物DOPAC的变化
高效液相色谱结果发现除海马外，其它3个脑区DA和DOPAC的含量发生明显变化，与对照组相比，其中PFC和NAe显著性升高(t=36.617，P=0.000；t=16.017，P=0.000；t=16.991，P=0.000；t=4.186，P=0.002)，而成瘾组大鼠的纹状体中二者含量却显著下降(t=32.642，P=0.000 t=50.457，P=0.000)；成瘾组PFC的DOPAC／DA值显著高于对照组(t=7.429，P=0.000)；而NAc和纹状体却显著降低(t=15.535，P=0.000；t=16.673，P=0.000)。
3．大鼠不同脑区双向电泳条件的凝胶图谱分析比较
其它电泳条件优化前提下，分别用银染法和马斯亮蓝Blue silver对凝胶进行染色，经比较发现各脑区银染的凝胶图谱都存在重复性差，得到的蛋白点少等等，而应用考马斯亮蓝Blue silver对凝胶进行染色，发现重复性好，分离效果二者相差不大。并发现各脑区蛋白主要集中在pH4～7范围内。
4．海洛因成瘾、戒断大鼠和对照大鼠相关脑区差异蛋白的筛选和MALDI-TOF—TOF-MS鉴定
在pH4～7范围内2—DE分离3组大鼠PFC、NAc、海马和纹状体组织的蛋白，经图像分析，以上4个脑区在3组之间分别筛选出8、10、5、7个差异蛋白点，经MALDI-TOF-TOF-MS分析，成功鉴定27个蛋白点，共计18个蛋白。
(1)在PFC中，仅在成瘾组和戒断组出现的蛋白是葡萄糖调节蛋白58(glucose regulated protein,GRP58；又称ERp57，ER60)和26s蛋白酶体亚基p40.5(26S proteasome subunit p40.5)而且表达量相当；相对于对照组和成瘾组，戒断组的泛素连接酶E2N(Ubiquitin-conjugating enzyme E2N)表达升高；成瘾组和戒断组含量下调的蛋白是ATP合酶D链；成瘾、戒断组与对照组相比，Ndufa10[NADH dehydrogenase(ubiquinone)alpha subcomplex-10]和α-烯醇化酶(Enol)发生相对分子质量和(或)等电点迁移，Enol的MW和pI差异更加明显。
(2)在NAc中，仅在成瘾组和戒断组出现的蛋白是Grp58，在两个实验组中都表达升高的蛋白是参与ATP合成的线粒体F0复合体F6亚基(ATP synthase，H+transporting，mitochondrial F0 complex，subunit F6)，而载脂蛋白酶E(Apolipoprotein E，apoE)、低分子量磷酸酪氨酸蛋白磷酸酶同工酶AcP2(low M(r)phosphotyrosine protein phosphatase isoenzyme AcP2)和ADP—精氨酸核糖基化水解酶(ADP-ribosylarginine hydrolase，AAH)在成瘾组中表达下降，仅在对照组出现的蛋白是表皮型脂肪酸结合蛋白(fatty acid binding protein 5，epidermal)；Ndufa10的表达情况与PFC脑区基本相同。
(3)在海马组织中，神经颗粒素(neurogranin，Ng)曲在海洛因成瘾、戒断组表达明显升高；戒断组微管蛋白α1表达升高；但mVps29p表达量在正常组、成瘾组和戒断组中依次降低；Ndufa10的表达情况与PFC脑区基本相同。
(4)在纹状体中，海洛因成瘾组和戒断组表达升高的蛋白是Zn结合乙醇脱氢酶(Zinc binding alcohol dehydrogenase)和G蛋白解离抑制因子2[Guanosine diphosphate(GDP)dissociation inhibitor 2,GDI2]，而两组的NAD~+依赖的异柠檬酸脱氢酶[isocitrate dehydrogenase 3(NAD+)alpha]表达降低；原肌球调节蛋白2(tropomodulin,TMOD2)在戒断组表达下降；Ndufa10的表达情况与PFC脑区基本相同。
5．神经颗粒素(Ng)在海洛因成瘾戒断大鼠脑组织中的表达验证
Western blot结果表明各组在6.5～14.4Kda之间均显示一条较粗的Ng免疫印迹反应条带，海洛因成瘾、戒断组含量明显高于对照组；免疫组化结果显示Ng阳性表达主要在海洛因成瘾、戒断脑组织的皮质和海马组织，其它脑区未见明显表达。PFC和海马CA1区Ng的阳性产物的PU值各组总体比较差异显著，有统计学意义(F=53.552，P=0.000；F=48.861，P=0.000)；PFC脑区，成瘾组、戒断组的PU值与对照组比较均有显著性差异(P=0.000，P=0.000)，而成瘾组和戒断组比较没有统计学差异(P=0.535)；海马CA1区，成瘾组、戒断组的PU值与对照组比较均有显著性差异(P=0.000，P=0.000)，成瘾组和戒断组比较差异有统计学意义(P=0.022)。
结论
1．成功建立了海洛因成瘾和自然戒断大鼠模型，并且发现海洛因对中枢神经系统造成轻度损害。
2．各组DA和DOPAC结果进一步证明了以NAc为核心的中脑多巴胺奖赏系统参与阿片类药物成瘾的形成过程，DA是参与海洛因成瘾形成的重要的神经递质。
3．建立了良好的2—DE条件，在此基础上成功鉴定了27个蛋白位点即18个蛋白，根据功能可以划分为分子伴侣蛋白，蛋白质降解相关蛋白，功能蛋白修饰相关蛋白，信号转导蛋白，细胞骨架蛋白，能量及物质代谢相关的酶，功能涉及神经保护和毒害两个方面。由结果可知，海洛因成瘾、戒断必然引起神经细胞的适应性病理改变，这个过程必然涉及神经细胞结构、突触可塑性、细胞运输与突触运输、信号转导、功能蛋白的降解或修饰以及能量代谢等方面的改变。
4．海洛因成瘾、戒断后在四个脑区中被特异修饰的Ndufa10广泛分布；在PFC和NAc脑区，海洛因成瘾、戒断大鼠表达新蛋白Grp58。两个蛋白可能是海洛因依赖的标志性蛋白，但需要进一步研究证实。
5．Ng在成瘾戒断大鼠的海马和PFC表达上调，提示学习记忆通路中的关键蛋白Ng在海洛因依赖相关学习记忆形成中扮演重要角色。
BACKGROUND & OBJECTIVE
Drug 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.
METHODS
1. 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.
RESULTS
1. Successful establishment of heroin addictive, withdrawal rat model
After 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 rats
HPLC 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 electrophoresis
Under 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 rats
Western 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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