神经甾体孕酮对大鼠吗啡精神依赖调节的分子机制研究
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摘要
阿片类药物成瘾是药物成瘾中最重要的一种,已经成为世界范围内严重的公共卫生和社会问题。目前,有关阿片类药物成瘾机理的研究是神经生物领域的热点课题之一。相关的研究已经从神经解剖、神经生化、受体调控、受体后信号转导、基因转录及表达调控等各个水平进行了大量的研究并取得了一定的进展,但对该类药物成瘾的确切机理仍未阐明。一般认为,机体反复与阿片类物质接触,会使中枢神经系统(central nervous system, CNS)某些神经元发生代偿适应性变化,如代谢活性、受体活性、基因表达及对环境暗示的反应性等一系列的改变,最终导致耐受、敏化、渴求、强迫用药等一系列复杂的行为。阿片类药物依赖不仅涉及阿片受体本身,还有多种神经递质及受体系统参与阿片药理作用的调节,如多巴胺(dopamine, DA)受体系统、N-甲基-D-天冬氨酸(NMDA)受体系统、5-羟色胺(5-HT)受体系统、去甲肾上腺素(NE)受体系统、乙酰胆碱(Ach)受体系统,促肾上腺皮质激素释放因子(CRF)受体系统等。随着分子生物学研究的不断深入,对阿片类物质依赖的形成机制也在不断的深入认识。阿片类药物成瘾包括对阿片类物质耐受、身体依赖性、戒断综合征和自身给药行为等特征性过程。目前,大多数学者认为,阿片依赖是一种细胞适应性反应,特别是中枢神经系统发生了细胞及分子水平上的适应,包括神经化学物质、神经电生理以及突触形态亚结构方面的变化,也包括细胞机能适应和形态适应,从而导致细胞的生理生化过程及组织结构的代偿性改变,最终达到病理状态的平衡。阿片类药物依赖时机体一系列适应性变化的发生是一个非常复杂的生物学过程,其可发生在神经组织、细胞和分子三个不同水平,受体后细胞和分子水平的适应性变化更为重要。
神经甾体是近年来发现的由中枢神经系统合成或来自外周由中枢神经系统代谢衍生而成的甾类物质的总称,包括孕烯醇酮(pregnenolone, PREG)、孕烯醇酮硫酸酯(pregnenolone sulfate, PREGS)、别孕烯醇酮(allopregnanolone, AP)、脱氢表雄酮(dehydroepiandrosterone, DHEA)、脱氢表雄酮硫酸酯(dehydroepiandrosterone sulfate, DHEAS)、孕酮(progesterone,PROG)、睾酮(testosterone, T)和脱氧皮质酮(deoxycorticosterone, DOC)等,现已经被列为继氨基酸和乙酰胆碱、儿茶酚胺和5-羟色胺、肽类之后的第四代神经递质。神经甾体可通过影响脑内多种神经递质受体的功能而产生广泛的生物学效应。有研究表明,多种神经甾体可以抑制慢性吗啡处理引起的依赖、耐受的形成;减轻纳络酮诱发的吗啡戒断症状;抑制或诱发吗啡条件性位置偏爱的形成;某些神经甾体还可以使大鼠产生辨别效应,剂量依赖性的增加伏隔核多巴胺的释放,并加强吗啡升高伏隔核细胞外多巴胺水平的作用,提示神经甾体可能影响吗啡躯体依赖和精神依赖的形成过程。本实验室前期研究发现,吗啡慢性处理可使大鼠在行为上产生躯体依赖、出现戒断反应、诱发条件性位置偏爱形成和复发等,在神经生化上表现为大鼠全脑内、伏隔核、下丘脑、杏仁核和垂体内某些甾体水平发生不同程度的变化;进一步深入研究发现,作为神经甾体之一的孕酮可以有效地抑制大鼠吗啡条件性位置偏爱效应的获得,被吗啡激活而诱发奖赏效应的中脑多巴胺系统的改变参与了孕酮对大鼠吗啡条件性位置偏爱效应的逆转。上述研究结果表明,对脑内神经甾体系统的调节可能是吗啡依赖形成的神经生物学机制之一。
阿片类药物依赖时机体可发生在神经组织、细胞和分子三个不同水平均可以发生适应性变化。本课题组前期研究发现,吗啡慢性处理导致精神依赖时,大鼠全脑内、伏隔核、下丘脑、杏仁核和垂体内某些神经甾体的水平均可发生不同程度的变化;并进一步研究证实,给与外源性神经甾体孕酮可以有效的抑制吗啡精神依赖大鼠CPP(conditioned place preference)效应的获得,同时使中枢内单胺类神经递质水平显著降低。故本实验在课题组前期工作的基础上,采用了放射免疫技术,探讨了外源性神经甾体孕酮对吗啡条件性位置偏爱大鼠下丘脑、额叶皮质、伏隔核、海马和中脑中内阿片肽水平的影响;并应用免疫组化技术研究了孕酮在逆转大鼠吗啡位置偏爱时,相关脑区中的内阿片受体和多巴胺受体表达调控的规律;最后应用免疫印迹技术观察了孕酮逆转大鼠吗啡位置偏爱时中枢相关核团内p-CREB(phosphorylated cAMP response element binding protein)水平的变化,为研究吗啡成瘾与神经甾体的关系提供了实验基础。
本研究采用的实验方法包括:
1吗啡精神依赖动物模型的建立
采用条件性位置偏爱模型,模拟吗啡精神依赖的形成过程。40只雄性SD大鼠,随机分为空白对照组(C)、吗啡组(Mor)、孕酮组(PROG)和孕酮加吗啡组(PROG+Mor) 4组,每组10只。以偏爱箱的白室为伴药侧,训练时用隔板封闭黑白两室的通道,大鼠每天训练两次,上午(8:00)注射后放入白室,下午(14:00)注射后放入黑室,两次间隔6h。C组和PROG组大鼠上午分别皮下注射10%的2-羟丙基-β-环糊精和孕酮15 mg·kg~(-1),10 min后给予腹腔注射5mg·kg~(-1)生理盐水;下午均注射等量的2-羟丙基-β-环糊精和生理盐水。Mor组和PROG+Mor组大鼠上午腹腔注射吗啡5mg·kg~(-1),给予吗啡的前10 min分别给予皮下注射10%的2-羟丙基-β-环糊精和PROG,剂量均为15 mg·kg~(-1),下午均注射等量的2-羟丙基-β-环糊精和生理盐水。大鼠每次在箱内停留45 min,连续训练10 d。最后一次训练后24 h进行CPP测试,测试时将隔板倒置,使大鼠可以在黑白箱中自由出入,记录在不给药状态下10 min内大鼠的活动情况,并软件分析大鼠在白室的累积停留时间。将动物断头处死,收集躯干血,分离下丘脑、额叶皮质、伏隔核、海马和中脑等脑区,-20℃保存备用。
2大鼠脑组织中内阿片肽水平的测定
把分离出的脑区置于玻璃匀浆管内,分别加入1mol·L~(-1) HCl 1 ml,在冰浴中充分匀浆后转入到玻璃试管中,室温放置100min,加入1 mol·L~(-1) NaOH 1 ml,以4000 rpm的速度低温离心20 min,取上清。采用放射免疫法测定内阿片肽的含量:每管加标准品100μl或待测样品100μl,稀释的抗血清100μl,缓冲液加至200μl,4℃环境下孵育24 h后再加入标记物100μl,沉淀物用计数器记数,计算并绘制竞争抑制曲线,最后得出单位重量样品中的内阿片肽含量。
3大鼠脑组织中阿片受体水平的测定
将SD大鼠腹腔注射戊巴比妥麻醉(2%,w/v, 0.3ml/100g体重),开胸,以30℃的生理盐水(50-100ml/250-350g)经左心室灌流,速度应快,然后以4%多聚甲醛200ml灌流固定,前10min速度较快,后调慢滴速,总时间大于30min。开颅,取出整个大脑,去除小脑,置4%多聚甲醛60ml于4℃后固定4h。转移至30%蔗糖溶液100ml中,4℃脱水,定时振摇,直至大脑沉底。将脱水平衡的大脑取出,石蜡包埋,进行连续冠状切片,隔二取一,每个大鼠的每个脑区做6套切片。
μ受体ABC法免疫细胞化学染色步骤如下:取出标本,用PBS漂洗一次,使标本湿润。过氧化氢室温孵育5min,以消除内源性过氧化氢酶的活性;蒸馏水漂洗2遍,PBS液浸泡3分钟;正常羊血清封闭非特异性抗原,室温孵育20分钟;倾去血清,滴加一抗工作液(1:1000稀释)室温湿盒中放置1小时后置4℃冰箱过夜;PBS液漂洗3遍,每次3分钟;滴加生物素标记的二抗工作液,室温湿盒中放置75分钟;PBS液漂洗3遍,每次3分钟;滴加辣根酶标记的链霉卵白素工作液,室温湿盒中放置30分钟;PBS液漂洗3遍,每次3分钟;DAB显色,显微镜下控制显色时间;适时终止反应,自来水冲洗后蒸馏水冲洗1遍,苏木精复染10分钟,自来水冲洗;1%盐酸酒精分色;1%氨水中和;常规梯度酒精脱水,二甲苯透明,中性树胶封片。
4大鼠脑组织中多巴胺受体水平的测定
除一抗的效价为1:250外,其余同阿片受体水平的测定。
5大鼠脑组织中p-CREB水平的测定
将分离的脑区分别用0.5 ml冰冷的细胞裂解液A ( HEPES 10 mmol·L~(-1), MgCl2 1.5 mmol·L~(-1), KCl 10 mmol·L~(-1), PMSF 1 mmol·L~(-1), NaF 2 mmol·L~(-1), NaPPi 2 mmol·L~(-1)),冰浴下匀浆10s×3次,冰浴上孵育30min,期间不断将细胞弹起使细胞膜裂解,4℃离心4000×g×20 min,弃上清。用50μl细胞裂解液B (HEPES 100 mmol·L~(-1), MgCl2 1.5 mmol·L~(-1), EDTA 1 mmol·L~(-1), NaCl 0.8mmol·L~(-1), NaF 2 mmol·L~(-1), NaPPi 2 mmol·L~(-1), PMSF 1 mmol·L~(-1) ),冰浴30 min,反复吹打,使细胞核裂解,4℃离心14000×g×30 min,取上清液保存于-20℃备用。
采用紫外分光光度法测定样品中蛋白浓度。将样品与等体积的上样缓冲液(pH6.8 Tris·Cl 0.0625mmol·L~(-1),溴酚蓝0.2g·L~(-1),SDS 20g·L~(-1) ,甘油400g·L~(-1))混合,于100℃煮沸5min,冷却至室温。样品经120g·L~(-1)SDS-PAGE电泳分离(稳压,15V/cm),湿法转移至硝酸纤维素(NC)膜(稳流,2mA/cm2),50g·L~(-1)脱脂奶粉封闭,依次与一抗Ser133-p-CREB抗体、HRP-羊抗兔二抗(1:2000)反应,发光剂显色,X线胶片进一步显影。
主要实验结果及结论如下:
1孕酮对大鼠吗啡位置偏爱效应的影响
5mg·kg~(-1)吗啡连续训练10d后,吗啡处理组大鼠产生了显著的CPP,表现为在伴药侧的停留时间显著长于空白对照组(P<0.01);单独使用孕酮(15mg·kg~(-1))进行条件性训练10d后,孕酮处理组大鼠不产生CPP,表现为在伴药侧的停留时间与空白对照组比较无显著性差异(P>0.05)。在吗啡处理的同时给与大鼠15mg·kg~(-1)孕酮,进行条件性训练10d后,孕酮使大鼠的吗啡CPP效应消失,表现为在伴药侧的停留时间显著短于吗啡处理组(P<0.01)。
结果表明,15mg·kg~(-1)孕酮处理本身不产生条件性位置偏爱效应,但与吗啡同用时,可以抑制大鼠吗啡CPP效应的获得。
2孕酮抑制大鼠吗啡位置偏爱效应的内阿片肽机制
与空白对照组相比,5mg·kg~(-1)吗啡训练10d诱导CPP形成时,Mor组大鼠下丘脑、海马和额叶皮质中的β-EP水平显著降低(P<0.05,P<0.01和P<0.05 ),下丘脑、伏隔核和额叶皮质中的L-EK水平显著降低(P<0.05,P<0.05和P<0.01 ),下丘脑和额叶皮质中的DynA水平显著升高(P<0.05和P<0.05)。与空白对照组比较,单独给予15mg·kg~(-1)孕酮训练10d,上述三项指标在各个脑区中未有显著性变化(P>0.05)。与吗啡组比较,在给予吗啡的前10min给予15mg·kg~(-1)孕酮进行条件性训练10d后,大鼠下丘脑中β-EP水平的显著升高(P<0.01 ),下丘脑、伏隔核和额叶皮质中的L-EK水平显著升高(P<0.05,P<0.01和P<0.05 ),下丘脑和额叶皮质中的DynA水平显著降低(P<0.01和P<0.05)。
结果提示长期给与外源性吗啡影响了内源性阿片类物质的合成和表达;给予外源性神经甾体孕酮可以有效抑制大鼠吗啡位置偏爱效应,除中脑DA系统外,孕酮对中枢内阿片递质系统的影响可能是孕酮对吗啡精神依赖的逆转机制之一。
3孕酮抑制大鼠吗啡位置偏爱效应的阿片受体机制
与空白对照组相比,5mg·kg~(-1)吗啡训练10d诱导CPP形成时,Mor组大鼠在下丘脑、纹状体、海马和额叶皮质中μ受体的数量显著下降, (P<0.05),下丘脑、纹状体、海马和额叶皮质中分别下降27%、44%、29%和38%,表明吗啡精神依赖时可引起不同脑区μ受体不同程度的下调。与空白盐水对照组比较,单独给予15mg·kg~(-1)孕酮训练10d,μ受体的数量在各个脑区中无显著性变化(P>0.05)。与吗啡组比较,在给予吗啡的前10min给予15mg·kg~(-1)孕酮进行条件性训练10d后,下丘脑、额叶皮质和纹状体中μ受体的数量分别升高29%、40%和49%(P<0.05),但在海马中未有显著性变化(P>0.05)。
本实验中所选择的神经核团均是与神经内分泌轴尤其是阿片类药物奖赏效应密切相关的核团。结果表明,外源性慢性给予吗啡影响了内源性阿片肽受体的合成和表达,外源性神经甾体孕酮抑制大鼠吗啡位置偏爱效应时,中枢神经系统中的内阿片受体的变化可以有效改善吗啡精神依赖脑内神经递质的合成和表达,从而实现对吗啡精神依赖的逆转。
4孕酮抑制大鼠吗啡位置偏爱效应的中枢多巴胺受体机制
与空白对照组相比,5mg·kg~(-1)吗啡训练10d诱导CPP形成时,Mor组大鼠在伏隔核、纹状体和中脑腹侧被盖区中D2受体的数量显著下降, (P<0.05),伏隔核、纹状体和中脑腹侧被盖区中分别下降31%、40%和37%,表明吗啡精神依赖时可引起相关脑区D2受体不同程度的下调。与空白盐水对照组比较,单独给予15mg·kg~(-1)孕酮训练10d,D2受体的数量在各个脑区中未有显著性变化(P>0.05)。与吗啡组比较,在给予吗啡的前10min给予15mg·kg~(-1)孕酮进行条件性训练10d后,纹状体和伏隔核中D2受体的数量分别升高29%和33%(P<0.05),但在中脑腹侧被盖区无显著性变化(P>0.05)。
本实验中所选择的神经核团均是与吗啡精神依赖的神经解剖基础-奖赏回路密切相关的核团。结果表明,慢性吗啡处理影响了多巴胺受体在伏隔核、纹状体和中脑腹侧被盖区中的合成和表达,外源性神经甾体孕酮抑制大鼠吗啡位置偏爱效应时,中枢神经系统相关脑区中的多巴胺受体也发生了相应的变化,主要作用表现为逆转吗啡引起的D2受体水平的下调。
5孕酮对吗啡位置偏爱大鼠不同脑区中p-CREB水平的影响
与空白对照组相比,5mg·kg~(-1)吗啡训练10d诱导CPP形成时,Mor组大鼠在海马和纹状体中p-CREB的表达显著增加(P<0.01和P<0.01),海马和纹状体中分别增加33%和51%;但在伏隔核中p-CREB的表达显著降低(P<0.01),表明吗啡精神依赖时引起不同脑区p-CREB水平的变化具有脑区特异性。与空白对照组比较,单独给予15mg·kg~(-1)孕酮训练10d,p-CREB的表达在各个脑区中未有显著性变化(P>0.05)。与吗啡组比较,在给予吗啡的前10min给予15mg·kg~(-1)孕酮进行条件性训练10d后,纹状体中p-CREB的表达下降29% (P<0.01),在海马中无显著性变化(P>0.05),但伏隔核中的p-CREB的表达上升(P<0.05)。
结果提示,外源性慢性给予吗啡影响了中枢相关脑区内p-CREB水平的表达,外源性神经甾体孕酮在抑制大鼠吗啡位置偏爱效应的同时中枢神经系统中的p-CREB的变化参与了对吗啡精神依赖的逆转过程。
综上所述,本研究发现外源性神经甾体孕酮可以从内阿片和多巴胺两个系统的递质、受体和受体后三个水平参与对吗啡大鼠精神依赖的调节过程。下丘脑和额叶皮质中内源性阿片肽水平的变化在孕酮逆转吗啡精神依赖的形成中发挥着十分重要的作用,孕酮通过对吗啡CPP大鼠该脑区中内源性阿片肽水平的调节,从而有效的抑制大鼠吗啡CPP的形成。在孕酮逆转吗啡导致大鼠精神依赖的过程中,中枢神经系统的阿片受体和多巴胺受体均发生了适应性改变,主要表现在μ受体和D2受体在相关脑区中的表达上调。μ受体改变所涉及到的脑区较广泛,主要是和大鼠神经内分泌轴相关的下丘脑、纹状体和额叶皮质均发生显著性变化。D2受体发生显著性改变的脑区主要是与奖赏回路有关的纹状体和伏隔核两个部位。孕酮在抑制吗啡CPP形成的过程中,可以使纹状体中p-CREB水平下降,同时伴随伏隔核中p-CREB水平的升高。提示不同脑区由于在吗啡依赖形成过程中的功能不同,CREB的表达改变也不完全相同,即各脑区或核团对吗啡诱导CREB表达的改变呈现多样性。所以,孕酮作为外源性神经甾体可以通过对吗啡精神依赖大鼠相关脑区中内源性阿片肽系统的递质和受体水平、多巴胺受体水平以及核转录因子p-CREB水平均可产生不同程度的影响,从而导致行为学上表现出实现对吗啡大鼠CPP的有效逆转。这就为进一步研究神经甾体参与吗啡成瘾的机理提供的新的理论依据,为临床开发戒毒药物奠定了基础。
Long-term opiate treatment is known to induce a state of dependence, a phenomenon associated with the abstinence syndrome following cessation of opiate administration. Despite numerous studies, the neuronal mechanism of the opiate addiction has not been fully elucidated as yet.
Neurosteroids is a new group of neurotransmitters with wide physical and pharmalogical functions. The term neurosteroids applie to those steroids that are synthesized within the central and peripheral nervous system, either de novo from cholesterol or from steroid hormone precursors. Neurosteroids including dehydroepiandresterone (DHEA), dihydroepiandresterone sulfate (DHEAS), pregnenolone (PREG), pregnenolone sulfate (PREGS), allopregnanolone (AP), deoxycorticosterone (DOC), testosterone (T), and progesterone (PROG) were named as the forth generation of neurotransmitter. It is demonstrated that neurosteroids can widely influence the function of traditional neurotransmitter receptors. Neuroactive steroids may modulate neuronal function through their concurrent influence on neuronal excitability and gene expression. This intracellular cross-talk between genomic and non-genomic steroid effects provides the molecular basis for such compounds in neuropsychopharmacology, both with regard to putative clinical effects and side effects. Neurosteroids, particularly PROG and AP can act as positive modulators at the GABAA receptor. In vivo, effects of these neurosteroids are similar to those produced by other positive modulator of GABAA receptors such as benzodiazepines and barbiturates. Many of these neurosteroids showed anticonvulsant, Myorelaxant, anesthetic and anxiolytic effects when they were administered to animals. Neurosteroid sulfate esters, such as PREGS and DHEAS, have an excitatory cellular action, since they antagonize the action of GABAA and potentiate the activation of NMDA receptor. Moreover, they can improve memory and learning, increase dopamine release in the rat nucleus accumbens, and enhance the dopaminergic response to morphine.
Endogenous opioids is the term used to describe a family of peptides, which include endorphines, enkephalins, dynorphins, and endomorphine,that act like opiates in biological systems. The discovery of these endogenous substances has spearheaded the recent interest in the biological functions of numerous peptide hormonse and neurotransmitters. Indeed, over the last two decades, at least 50 peptides found in the skin, brain, and the gut have been added to the list of neurotransmitter candidates. However, the endogenous opioids have attracted more attention than some of the other bioactive peptides. As a general rule, like morphine, endogenous opioids, which are inhibitory substances, reduce neuronal firing rates and neurotransmitters release. Endogenous opioids may inhibit nerve cells by depressing presynaptic neurotransmitter release, by altering the ability of other neurotransmitters to produce postsynaptic ion conductance, or by directly interfering with the postsynaptic nerve impulse.
Based on the previous results of our lab, this study was carried out to further test the effect neurosteroids on morphine-induced psychological dependence from the levels of neurotransmitters, receptors and postreceptors respectively in related brain areas such as nucleus accumbens(NA), frontal cortex (Fc), hippocampus (Hc), hypothalamus (Ht), striatum (Str) in rats. We first investigate the effect of PROG administration on the morphine-induced rewarding effect. Then, a simplified radioimmunoassay method has been established to detect the Endogenous opioids transmitters in rat Ht, NAc, Hc, Fc, and Str. By using the method of immunohistochemistry and western- blotting, we will further investigated the machenism of how PROG reverse the effects of morphine induced psychological dependence from receptors and nuclear factors. Therefore, this study will take a basis for the further study of the relationship between morphine addiction and neurosteroids.
Following methods were employed in this study:
1 The establishment of morphine psychological dependence rat models
The rat model of conditioned place preference (CPP) was used to mimic morphine psychological dependence. 40 male SD rats were designated to 4 groups randomly with 10 in each, including blank control group, morphine addiction group, progesterone group and progesterone plus morphine group. The white room was used as the drug-pairing room. In the morning, the groups of black control and progesterone had hypodermic injection of cyclodextrin and progesterone of 15mg·kg~(-1), 10 min later, had intraperitoneal injection of Saline 5mg·kg~(-1). The morphine and progesterone plus mrophine groups had hypodermic injection of cyclodextrin and progesterone of 15mg·kg~(-1), 10 min later, had intraperitoneal injection of morphine of 5mg·kg~(-1). All the rats were put into the white room for a 45-min training. And in the afternoon, all rats had hypodermic injection of cyclodextrin and intraperitoneal injection of Saline of the same dose, and were put into the black room for the same time training. After trained for 10 days, CPP test was scored during a 10-min session 24 h after the last training. Then all the rats were rapidly sacrificed by decapitation. The brain regions including NA, Fc, Hc, ST and Ht were separated and frozen until being taken other procedures.
2. Determination of the endogenoud opoiod peptides in rat brain
The procedure of CPP was all the same in this study. Then the rats were rapidly sacrificed by decapitation. The brain regions including VTA, NAc, Ht, and Str were separated and frozen until the further studying.
Neuropeptide radioimmunoassay established by the Department of Neorobiology of the Second Militery Medical University of Chinese PLA was applied to measure brain endogenoud opioid peptide levels. Sequential saturation sample addition was adopted. The radioactivity count per minute (cpm) of the whole reaction was 7000 per minute. The activated carbon was separated, centrifuged, and the cpm of the sedent was measured. The standard curve was made accroding to the study. Then, according to the curve, the content of brain endogenoud opioid peptide in each tube was figured out, and it was then translated into the content of endogenoud opioid peptide per miligrame of brain.
3. Determination of the mu opoiod receptor in rat brain
The procedure of CPP was as the same as before. Then the rats were rapidly sacrificed by decapitation. The brain regions including VTA, NAc, Ht, and Str were separated and frozen until the further studying.
Paraffin sections were used for immunohistochemical staining according to the protocol recently described by the researchers. Briefly, after treatment with 3% H2O2 and 10% normal rabbit serum for 10 and 30 minutes at 37℃, respectively, the deparaffinized and rehydrated sections were incubated with polyclonal rabbit-anti-mu opoiod receptor (1:1000) over night. Biotinylated goat anti-rabbit IgG secondary antibody and ABC complex solution were applied to the sections for 2 hours and 30 minutes at room temperature. The horseradish peroxidase reaction was detected with diaminobenzidine-H2O2 at room temperature for 5-10 minutes. Among each step, 0.01 M PBS (pH 7.4) was used to wash the sections three times with five minutes each time by swaying. Finally, sections were mounted, dehydrated, cleared, and sealed. Slides were observed under a Zeiss Axioskop2 Plus microscope linked to a digital camera from Diagnostics Instruments. Images were captured using the Spot software (Diagnostics) and analyzed with the Image Pro Plus 5.0 image analysis software, resulting in quantification of the protein levels as the mean of integrated optical density (IOD).
4. Determination of the dopamine 2 receptor in rat brain
Beside the deparaffinized and rehydrated sections were incubated with polyclonal rabbit-anti-D2R (1:250) over night, the other procedures were the same as section 3.
5. Determination of the p-CREB levels in rat brain
CPP test was used to investigate the morphine rewarding effect. The neuclear lysates were extracted and screened by using Western blot to observe the changes of p-CREB levels in NA, Fc, Hc, Ht, Str and VTA in rat brain. All frozen specimens were grinded in liquid nitrogen and homogenized in lysis buffer on ice. The lystate was centrifuged and the supernatant was prepared. Total protein concentration of the supernatant was measured by BCA Ptotein Assay Bit. 50μg of each protein sample was separated by SDS-PAGE and transferred to nitrocellulose membrane. After blocking in skimmed milk for 1 hour, the membranes were incubated with antibody over night. The membrane were then developed with peroxidase-conjugated secondary antibody. The immunoreactive proteins were visulized by enhanced chemiluminescence system. The immunoblots were quantitated using analystic software.
The principal results and conclusions were as follows:
1 Effects of progesterone administration on morphine-induced conditioned place preference(CPP) and its relationship to endogenous opioids in the related rat brain regions
Compared with NS control group, 5 mg·kg~(-1) morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg~(-1) progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect.
Compared with NS control group, the levels ofβ-EP following morphine administration were decreased in hypothalamus, hippocampus and frontal cortex(P<0.05、P<0.01 and P<0.05). Compared with morphine group, the levels ofβ-EP were increased by co-administration with 15 mg·kg~(-1) dose of progesterone in hypothalamus (P<0.01). Compared with NS control group, the levels of L-EK following morphine administration were decreased in hypothalamus, nucleus accumbens and frontal cortex(P<0.05, P<0.05 and P<0.01). Compared with morphine group, the levels of L-EK were increased by co-administration with 15mg·kg~(-1) dose of progesterone in hypothalamus, nucleus accumbens and frontal cortex (P<0.05,P<0.01 and P<0.05). Compared with NS control group, the levels of DynA following morphine administration were increased in hypothalamus and frontal cortex (P<0.05 and P<0.05). Compared with morphine group, the levels of DynA were decreased by co-administration with 15mg·kg~(-1) dose of progesterone in hypothalamus and frontal cortex (P<0.01 and P<0.05).
2 Effects of progesterone administration on morphine-induced conditioned place preference and its relation toμ-receptors in rat brain regions
Compared with NS control group, 5 mg·kg~(-1) morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg~(-1) progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect.
Compared with NS control group, the levels of mu opioid receptor following morphine administration were decreased in hypothalamus, hippocampus , striatum and frontal cortex(P<0.05、P<0.01、P<0.01 and P<0.01). Compared with morphine group, the levels of mu opioid receptor were increased by co-administration with 15 mg·kg~(-1) dose of progesterone in hypothalamus, striatum and frontal cortex (P<0.05、P<0.01 and P<0.01).
3 Effects of progesterone administration on morphine-induced conditioned place preference and its relation to D2-receptors in rat brain regions
Compared with NS control group, 5 mg·kg~(-1) morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg~(-1) progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect.
Compared with NS control group, the levels of D2 receptor following morphine administration were decreased in nucleus accumbens ,striatum and ventral tegmental area (P<0.01、P<0.01 and P<0.01). Compared with morphine group, the levels of D2 receptor were increased by co-administration with 15 mg·kg~(-1) dose of progesterone in nucleus accumbens and striatum (P<0.05 and P<0.01).
4 Effects of progesterone administration on morphine-induced conditioned place preference and its relation to p-CREB in rat brain regions
Compared with NS control group, 5 mg·kg~(-1) morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg~(-1) progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect(P<0.01).
Compared with NS control group, the levels of p-CREB following morphine administration were increased in hippocampus and striatum(P<0.01 and P<0.01)but decreased in nucleus accumbens (P<0.01). Compared with morphine group, the levels of p-CREB were decreased by co-administration with 15 mg·kg~(-1) dose of progesterone in striatum (P<0.01) but increased in nucleus accumbens (P<0.05).
In conclusion, our study indicated that chronic morphine treatment could induce the development of morphine psycological dependence and progesterone as an ectogenic neurosteroid could reverse morphine-induced rewarding effects. Durig the procedure of progesterone abolishing morpnine- induced dependence, the levels ofβ-EP, L-EK and DynA were increased or decreased in rat brain. Opioid receptors and dopamine receptors were involved in the regulation of morphine dependence by progesterone. We also found that with the changing in neurotransmitters and receptors, the levels of p-CREB were diversified in rat brain rewarding circuit. Thus, our data provide a new insight for the research of the neurobiochemical mechanism of the relationship between morphine dependence and neurosteroid.
神经甾体是近年来发现的由中枢神经系统合成或来自外周由中枢神经系统代谢衍生而成的甾类物质的总称,包括孕烯醇酮(pregnenolone, PREG)、孕烯醇酮硫酸酯(pregnenolone sulfate, PREGS)、别孕烯醇酮(allopregnanolone, AP)、脱氢表雄酮(dehydroepiandrosterone, DHEA)、脱氢表雄酮硫酸酯(dehydroepiandrosterone sulfate, DHEAS)、孕酮(progesterone,PROG)、睾酮(testosterone, T)和脱氧皮质酮(deoxycorticosterone, DOC)等,现已经被列为继氨基酸和乙酰胆碱、儿茶酚胺和5-羟色胺、肽类之后的第四代神经递质。神经甾体可通过影响脑内多种神经递质受体的功能而产生广泛的生物学效应。有研究表明,多种神经甾体可以抑制慢性吗啡处理引起的依赖、耐受的形成;减轻纳络酮诱发的吗啡戒断症状;抑制或诱发吗啡条件性位置偏爱的形成;某些神经甾体还可以使大鼠产生辨别效应,剂量依赖性的增加伏隔核多巴胺的释放,并加强吗啡升高伏隔核细胞外多巴胺水平的作用,提示神经甾体可能影响吗啡躯体依赖和精神依赖的形成过程。本实验室前期研究发现,吗啡慢性处理可使大鼠在行为上产生躯体依赖、出现戒断反应、诱发条件性位置偏爱形成和复发等,在神经生化上表现为大鼠全脑内、伏隔核、下丘脑、杏仁核和垂体内某些甾体水平发生不同程度的变化;进一步深入研究发现,作为神经甾体之一的孕酮可以有效地抑制大鼠吗啡条件性位置偏爱效应的获得,被吗啡激活而诱发奖赏效应的中脑多巴胺系统的改变参与了孕酮对大鼠吗啡条件性位置偏爱效应的逆转。上述研究结果表明,对脑内神经甾体系统的调节可能是吗啡依赖形成的神经生物学机制之一。
阿片类药物依赖时机体可发生在神经组织、细胞和分子三个不同水平均可以发生适应性变化。本课题组前期研究发现,吗啡慢性处理导致精神依赖时,大鼠全脑内、伏隔核、下丘脑、杏仁核和垂体内某些神经甾体的水平均可发生不同程度的变化;并进一步研究证实,给与外源性神经甾体孕酮可以有效的抑制吗啡精神依赖大鼠CPP(conditioned place preference)效应的获得,同时使中枢内单胺类神经递质水平显著降低。故本实验在课题组前期工作的基础上,采用了放射免疫技术,探讨了外源性神经甾体孕酮对吗啡条件性位置偏爱大鼠下丘脑、额叶皮质、伏隔核、海马和中脑中内阿片肽水平的影响;并应用免疫组化技术研究了孕酮在逆转大鼠吗啡位置偏爱时,相关脑区中的内阿片受体和多巴胺受体表达调控的规律;最后应用免疫印迹技术观察了孕酮逆转大鼠吗啡位置偏爱时中枢相关核团内p-CREB(phosphorylated cAMP response element binding protein)水平的变化,为研究吗啡成瘾与神经甾体的关系提供了实验基础。
本研究采用的实验方法包括:
1吗啡精神依赖动物模型的建立
采用条件性位置偏爱模型,模拟吗啡精神依赖的形成过程。40只雄性SD大鼠,随机分为空白对照组(C)、吗啡组(Mor)、孕酮组(PROG)和孕酮加吗啡组(PROG+Mor) 4组,每组10只。以偏爱箱的白室为伴药侧,训练时用隔板封闭黑白两室的通道,大鼠每天训练两次,上午(8:00)注射后放入白室,下午(14:00)注射后放入黑室,两次间隔6h。C组和PROG组大鼠上午分别皮下注射10%的2-羟丙基-β-环糊精和孕酮15 mg·kg~(-1),10 min后给予腹腔注射5mg·kg~(-1)生理盐水;下午均注射等量的2-羟丙基-β-环糊精和生理盐水。Mor组和PROG+Mor组大鼠上午腹腔注射吗啡5mg·kg~(-1),给予吗啡的前10 min分别给予皮下注射10%的2-羟丙基-β-环糊精和PROG,剂量均为15 mg·kg~(-1),下午均注射等量的2-羟丙基-β-环糊精和生理盐水。大鼠每次在箱内停留45 min,连续训练10 d。最后一次训练后24 h进行CPP测试,测试时将隔板倒置,使大鼠可以在黑白箱中自由出入,记录在不给药状态下10 min内大鼠的活动情况,并软件分析大鼠在白室的累积停留时间。将动物断头处死,收集躯干血,分离下丘脑、额叶皮质、伏隔核、海马和中脑等脑区,-20℃保存备用。
2大鼠脑组织中内阿片肽水平的测定
把分离出的脑区置于玻璃匀浆管内,分别加入1mol·L~(-1) HCl 1 ml,在冰浴中充分匀浆后转入到玻璃试管中,室温放置100min,加入1 mol·L~(-1) NaOH 1 ml,以4000 rpm的速度低温离心20 min,取上清。采用放射免疫法测定内阿片肽的含量:每管加标准品100μl或待测样品100μl,稀释的抗血清100μl,缓冲液加至200μl,4℃环境下孵育24 h后再加入标记物100μl,沉淀物用计数器记数,计算并绘制竞争抑制曲线,最后得出单位重量样品中的内阿片肽含量。
3大鼠脑组织中阿片受体水平的测定
将SD大鼠腹腔注射戊巴比妥麻醉(2%,w/v, 0.3ml/100g体重),开胸,以30℃的生理盐水(50-100ml/250-350g)经左心室灌流,速度应快,然后以4%多聚甲醛200ml灌流固定,前10min速度较快,后调慢滴速,总时间大于30min。开颅,取出整个大脑,去除小脑,置4%多聚甲醛60ml于4℃后固定4h。转移至30%蔗糖溶液100ml中,4℃脱水,定时振摇,直至大脑沉底。将脱水平衡的大脑取出,石蜡包埋,进行连续冠状切片,隔二取一,每个大鼠的每个脑区做6套切片。
μ受体ABC法免疫细胞化学染色步骤如下:取出标本,用PBS漂洗一次,使标本湿润。过氧化氢室温孵育5min,以消除内源性过氧化氢酶的活性;蒸馏水漂洗2遍,PBS液浸泡3分钟;正常羊血清封闭非特异性抗原,室温孵育20分钟;倾去血清,滴加一抗工作液(1:1000稀释)室温湿盒中放置1小时后置4℃冰箱过夜;PBS液漂洗3遍,每次3分钟;滴加生物素标记的二抗工作液,室温湿盒中放置75分钟;PBS液漂洗3遍,每次3分钟;滴加辣根酶标记的链霉卵白素工作液,室温湿盒中放置30分钟;PBS液漂洗3遍,每次3分钟;DAB显色,显微镜下控制显色时间;适时终止反应,自来水冲洗后蒸馏水冲洗1遍,苏木精复染10分钟,自来水冲洗;1%盐酸酒精分色;1%氨水中和;常规梯度酒精脱水,二甲苯透明,中性树胶封片。
4大鼠脑组织中多巴胺受体水平的测定
除一抗的效价为1:250外,其余同阿片受体水平的测定。
5大鼠脑组织中p-CREB水平的测定
将分离的脑区分别用0.5 ml冰冷的细胞裂解液A ( HEPES 10 mmol·L~(-1), MgCl2 1.5 mmol·L~(-1), KCl 10 mmol·L~(-1), PMSF 1 mmol·L~(-1), NaF 2 mmol·L~(-1), NaPPi 2 mmol·L~(-1)),冰浴下匀浆10s×3次,冰浴上孵育30min,期间不断将细胞弹起使细胞膜裂解,4℃离心4000×g×20 min,弃上清。用50μl细胞裂解液B (HEPES 100 mmol·L~(-1), MgCl2 1.5 mmol·L~(-1), EDTA 1 mmol·L~(-1), NaCl 0.8mmol·L~(-1), NaF 2 mmol·L~(-1), NaPPi 2 mmol·L~(-1), PMSF 1 mmol·L~(-1) ),冰浴30 min,反复吹打,使细胞核裂解,4℃离心14000×g×30 min,取上清液保存于-20℃备用。
采用紫外分光光度法测定样品中蛋白浓度。将样品与等体积的上样缓冲液(pH6.8 Tris·Cl 0.0625mmol·L~(-1),溴酚蓝0.2g·L~(-1),SDS 20g·L~(-1) ,甘油400g·L~(-1))混合,于100℃煮沸5min,冷却至室温。样品经120g·L~(-1)SDS-PAGE电泳分离(稳压,15V/cm),湿法转移至硝酸纤维素(NC)膜(稳流,2mA/cm2),50g·L~(-1)脱脂奶粉封闭,依次与一抗Ser133-p-CREB抗体、HRP-羊抗兔二抗(1:2000)反应,发光剂显色,X线胶片进一步显影。
主要实验结果及结论如下:
1孕酮对大鼠吗啡位置偏爱效应的影响
5mg·kg~(-1)吗啡连续训练10d后,吗啡处理组大鼠产生了显著的CPP,表现为在伴药侧的停留时间显著长于空白对照组(P<0.01);单独使用孕酮(15mg·kg~(-1))进行条件性训练10d后,孕酮处理组大鼠不产生CPP,表现为在伴药侧的停留时间与空白对照组比较无显著性差异(P>0.05)。在吗啡处理的同时给与大鼠15mg·kg~(-1)孕酮,进行条件性训练10d后,孕酮使大鼠的吗啡CPP效应消失,表现为在伴药侧的停留时间显著短于吗啡处理组(P<0.01)。
结果表明,15mg·kg~(-1)孕酮处理本身不产生条件性位置偏爱效应,但与吗啡同用时,可以抑制大鼠吗啡CPP效应的获得。
2孕酮抑制大鼠吗啡位置偏爱效应的内阿片肽机制
与空白对照组相比,5mg·kg~(-1)吗啡训练10d诱导CPP形成时,Mor组大鼠下丘脑、海马和额叶皮质中的β-EP水平显著降低(P<0.05,P<0.01和P<0.05 ),下丘脑、伏隔核和额叶皮质中的L-EK水平显著降低(P<0.05,P<0.05和P<0.01 ),下丘脑和额叶皮质中的DynA水平显著升高(P<0.05和P<0.05)。与空白对照组比较,单独给予15mg·kg~(-1)孕酮训练10d,上述三项指标在各个脑区中未有显著性变化(P>0.05)。与吗啡组比较,在给予吗啡的前10min给予15mg·kg~(-1)孕酮进行条件性训练10d后,大鼠下丘脑中β-EP水平的显著升高(P<0.01 ),下丘脑、伏隔核和额叶皮质中的L-EK水平显著升高(P<0.05,P<0.01和P<0.05 ),下丘脑和额叶皮质中的DynA水平显著降低(P<0.01和P<0.05)。
结果提示长期给与外源性吗啡影响了内源性阿片类物质的合成和表达;给予外源性神经甾体孕酮可以有效抑制大鼠吗啡位置偏爱效应,除中脑DA系统外,孕酮对中枢内阿片递质系统的影响可能是孕酮对吗啡精神依赖的逆转机制之一。
3孕酮抑制大鼠吗啡位置偏爱效应的阿片受体机制
与空白对照组相比,5mg·kg~(-1)吗啡训练10d诱导CPP形成时,Mor组大鼠在下丘脑、纹状体、海马和额叶皮质中μ受体的数量显著下降, (P<0.05),下丘脑、纹状体、海马和额叶皮质中分别下降27%、44%、29%和38%,表明吗啡精神依赖时可引起不同脑区μ受体不同程度的下调。与空白盐水对照组比较,单独给予15mg·kg~(-1)孕酮训练10d,μ受体的数量在各个脑区中无显著性变化(P>0.05)。与吗啡组比较,在给予吗啡的前10min给予15mg·kg~(-1)孕酮进行条件性训练10d后,下丘脑、额叶皮质和纹状体中μ受体的数量分别升高29%、40%和49%(P<0.05),但在海马中未有显著性变化(P>0.05)。
本实验中所选择的神经核团均是与神经内分泌轴尤其是阿片类药物奖赏效应密切相关的核团。结果表明,外源性慢性给予吗啡影响了内源性阿片肽受体的合成和表达,外源性神经甾体孕酮抑制大鼠吗啡位置偏爱效应时,中枢神经系统中的内阿片受体的变化可以有效改善吗啡精神依赖脑内神经递质的合成和表达,从而实现对吗啡精神依赖的逆转。
4孕酮抑制大鼠吗啡位置偏爱效应的中枢多巴胺受体机制
与空白对照组相比,5mg·kg~(-1)吗啡训练10d诱导CPP形成时,Mor组大鼠在伏隔核、纹状体和中脑腹侧被盖区中D2受体的数量显著下降, (P<0.05),伏隔核、纹状体和中脑腹侧被盖区中分别下降31%、40%和37%,表明吗啡精神依赖时可引起相关脑区D2受体不同程度的下调。与空白盐水对照组比较,单独给予15mg·kg~(-1)孕酮训练10d,D2受体的数量在各个脑区中未有显著性变化(P>0.05)。与吗啡组比较,在给予吗啡的前10min给予15mg·kg~(-1)孕酮进行条件性训练10d后,纹状体和伏隔核中D2受体的数量分别升高29%和33%(P<0.05),但在中脑腹侧被盖区无显著性变化(P>0.05)。
本实验中所选择的神经核团均是与吗啡精神依赖的神经解剖基础-奖赏回路密切相关的核团。结果表明,慢性吗啡处理影响了多巴胺受体在伏隔核、纹状体和中脑腹侧被盖区中的合成和表达,外源性神经甾体孕酮抑制大鼠吗啡位置偏爱效应时,中枢神经系统相关脑区中的多巴胺受体也发生了相应的变化,主要作用表现为逆转吗啡引起的D2受体水平的下调。
5孕酮对吗啡位置偏爱大鼠不同脑区中p-CREB水平的影响
与空白对照组相比,5mg·kg~(-1)吗啡训练10d诱导CPP形成时,Mor组大鼠在海马和纹状体中p-CREB的表达显著增加(P<0.01和P<0.01),海马和纹状体中分别增加33%和51%;但在伏隔核中p-CREB的表达显著降低(P<0.01),表明吗啡精神依赖时引起不同脑区p-CREB水平的变化具有脑区特异性。与空白对照组比较,单独给予15mg·kg~(-1)孕酮训练10d,p-CREB的表达在各个脑区中未有显著性变化(P>0.05)。与吗啡组比较,在给予吗啡的前10min给予15mg·kg~(-1)孕酮进行条件性训练10d后,纹状体中p-CREB的表达下降29% (P<0.01),在海马中无显著性变化(P>0.05),但伏隔核中的p-CREB的表达上升(P<0.05)。
结果提示,外源性慢性给予吗啡影响了中枢相关脑区内p-CREB水平的表达,外源性神经甾体孕酮在抑制大鼠吗啡位置偏爱效应的同时中枢神经系统中的p-CREB的变化参与了对吗啡精神依赖的逆转过程。
综上所述,本研究发现外源性神经甾体孕酮可以从内阿片和多巴胺两个系统的递质、受体和受体后三个水平参与对吗啡大鼠精神依赖的调节过程。下丘脑和额叶皮质中内源性阿片肽水平的变化在孕酮逆转吗啡精神依赖的形成中发挥着十分重要的作用,孕酮通过对吗啡CPP大鼠该脑区中内源性阿片肽水平的调节,从而有效的抑制大鼠吗啡CPP的形成。在孕酮逆转吗啡导致大鼠精神依赖的过程中,中枢神经系统的阿片受体和多巴胺受体均发生了适应性改变,主要表现在μ受体和D2受体在相关脑区中的表达上调。μ受体改变所涉及到的脑区较广泛,主要是和大鼠神经内分泌轴相关的下丘脑、纹状体和额叶皮质均发生显著性变化。D2受体发生显著性改变的脑区主要是与奖赏回路有关的纹状体和伏隔核两个部位。孕酮在抑制吗啡CPP形成的过程中,可以使纹状体中p-CREB水平下降,同时伴随伏隔核中p-CREB水平的升高。提示不同脑区由于在吗啡依赖形成过程中的功能不同,CREB的表达改变也不完全相同,即各脑区或核团对吗啡诱导CREB表达的改变呈现多样性。所以,孕酮作为外源性神经甾体可以通过对吗啡精神依赖大鼠相关脑区中内源性阿片肽系统的递质和受体水平、多巴胺受体水平以及核转录因子p-CREB水平均可产生不同程度的影响,从而导致行为学上表现出实现对吗啡大鼠CPP的有效逆转。这就为进一步研究神经甾体参与吗啡成瘾的机理提供的新的理论依据,为临床开发戒毒药物奠定了基础。
Long-term opiate treatment is known to induce a state of dependence, a phenomenon associated with the abstinence syndrome following cessation of opiate administration. Despite numerous studies, the neuronal mechanism of the opiate addiction has not been fully elucidated as yet.
Neurosteroids is a new group of neurotransmitters with wide physical and pharmalogical functions. The term neurosteroids applie to those steroids that are synthesized within the central and peripheral nervous system, either de novo from cholesterol or from steroid hormone precursors. Neurosteroids including dehydroepiandresterone (DHEA), dihydroepiandresterone sulfate (DHEAS), pregnenolone (PREG), pregnenolone sulfate (PREGS), allopregnanolone (AP), deoxycorticosterone (DOC), testosterone (T), and progesterone (PROG) were named as the forth generation of neurotransmitter. It is demonstrated that neurosteroids can widely influence the function of traditional neurotransmitter receptors. Neuroactive steroids may modulate neuronal function through their concurrent influence on neuronal excitability and gene expression. This intracellular cross-talk between genomic and non-genomic steroid effects provides the molecular basis for such compounds in neuropsychopharmacology, both with regard to putative clinical effects and side effects. Neurosteroids, particularly PROG and AP can act as positive modulators at the GABAA receptor. In vivo, effects of these neurosteroids are similar to those produced by other positive modulator of GABAA receptors such as benzodiazepines and barbiturates. Many of these neurosteroids showed anticonvulsant, Myorelaxant, anesthetic and anxiolytic effects when they were administered to animals. Neurosteroid sulfate esters, such as PREGS and DHEAS, have an excitatory cellular action, since they antagonize the action of GABAA and potentiate the activation of NMDA receptor. Moreover, they can improve memory and learning, increase dopamine release in the rat nucleus accumbens, and enhance the dopaminergic response to morphine.
Endogenous opioids is the term used to describe a family of peptides, which include endorphines, enkephalins, dynorphins, and endomorphine,that act like opiates in biological systems. The discovery of these endogenous substances has spearheaded the recent interest in the biological functions of numerous peptide hormonse and neurotransmitters. Indeed, over the last two decades, at least 50 peptides found in the skin, brain, and the gut have been added to the list of neurotransmitter candidates. However, the endogenous opioids have attracted more attention than some of the other bioactive peptides. As a general rule, like morphine, endogenous opioids, which are inhibitory substances, reduce neuronal firing rates and neurotransmitters release. Endogenous opioids may inhibit nerve cells by depressing presynaptic neurotransmitter release, by altering the ability of other neurotransmitters to produce postsynaptic ion conductance, or by directly interfering with the postsynaptic nerve impulse.
Based on the previous results of our lab, this study was carried out to further test the effect neurosteroids on morphine-induced psychological dependence from the levels of neurotransmitters, receptors and postreceptors respectively in related brain areas such as nucleus accumbens(NA), frontal cortex (Fc), hippocampus (Hc), hypothalamus (Ht), striatum (Str) in rats. We first investigate the effect of PROG administration on the morphine-induced rewarding effect. Then, a simplified radioimmunoassay method has been established to detect the Endogenous opioids transmitters in rat Ht, NAc, Hc, Fc, and Str. By using the method of immunohistochemistry and western- blotting, we will further investigated the machenism of how PROG reverse the effects of morphine induced psychological dependence from receptors and nuclear factors. Therefore, this study will take a basis for the further study of the relationship between morphine addiction and neurosteroids.
Following methods were employed in this study:
1 The establishment of morphine psychological dependence rat models
The rat model of conditioned place preference (CPP) was used to mimic morphine psychological dependence. 40 male SD rats were designated to 4 groups randomly with 10 in each, including blank control group, morphine addiction group, progesterone group and progesterone plus morphine group. The white room was used as the drug-pairing room. In the morning, the groups of black control and progesterone had hypodermic injection of cyclodextrin and progesterone of 15mg·kg~(-1), 10 min later, had intraperitoneal injection of Saline 5mg·kg~(-1). The morphine and progesterone plus mrophine groups had hypodermic injection of cyclodextrin and progesterone of 15mg·kg~(-1), 10 min later, had intraperitoneal injection of morphine of 5mg·kg~(-1). All the rats were put into the white room for a 45-min training. And in the afternoon, all rats had hypodermic injection of cyclodextrin and intraperitoneal injection of Saline of the same dose, and were put into the black room for the same time training. After trained for 10 days, CPP test was scored during a 10-min session 24 h after the last training. Then all the rats were rapidly sacrificed by decapitation. The brain regions including NA, Fc, Hc, ST and Ht were separated and frozen until being taken other procedures.
2. Determination of the endogenoud opoiod peptides in rat brain
The procedure of CPP was all the same in this study. Then the rats were rapidly sacrificed by decapitation. The brain regions including VTA, NAc, Ht, and Str were separated and frozen until the further studying.
Neuropeptide radioimmunoassay established by the Department of Neorobiology of the Second Militery Medical University of Chinese PLA was applied to measure brain endogenoud opioid peptide levels. Sequential saturation sample addition was adopted. The radioactivity count per minute (cpm) of the whole reaction was 7000 per minute. The activated carbon was separated, centrifuged, and the cpm of the sedent was measured. The standard curve was made accroding to the study. Then, according to the curve, the content of brain endogenoud opioid peptide in each tube was figured out, and it was then translated into the content of endogenoud opioid peptide per miligrame of brain.
3. Determination of the mu opoiod receptor in rat brain
The procedure of CPP was as the same as before. Then the rats were rapidly sacrificed by decapitation. The brain regions including VTA, NAc, Ht, and Str were separated and frozen until the further studying.
Paraffin sections were used for immunohistochemical staining according to the protocol recently described by the researchers. Briefly, after treatment with 3% H2O2 and 10% normal rabbit serum for 10 and 30 minutes at 37℃, respectively, the deparaffinized and rehydrated sections were incubated with polyclonal rabbit-anti-mu opoiod receptor (1:1000) over night. Biotinylated goat anti-rabbit IgG secondary antibody and ABC complex solution were applied to the sections for 2 hours and 30 minutes at room temperature. The horseradish peroxidase reaction was detected with diaminobenzidine-H2O2 at room temperature for 5-10 minutes. Among each step, 0.01 M PBS (pH 7.4) was used to wash the sections three times with five minutes each time by swaying. Finally, sections were mounted, dehydrated, cleared, and sealed. Slides were observed under a Zeiss Axioskop2 Plus microscope linked to a digital camera from Diagnostics Instruments. Images were captured using the Spot software (Diagnostics) and analyzed with the Image Pro Plus 5.0 image analysis software, resulting in quantification of the protein levels as the mean of integrated optical density (IOD).
4. Determination of the dopamine 2 receptor in rat brain
Beside the deparaffinized and rehydrated sections were incubated with polyclonal rabbit-anti-D2R (1:250) over night, the other procedures were the same as section 3.
5. Determination of the p-CREB levels in rat brain
CPP test was used to investigate the morphine rewarding effect. The neuclear lysates were extracted and screened by using Western blot to observe the changes of p-CREB levels in NA, Fc, Hc, Ht, Str and VTA in rat brain. All frozen specimens were grinded in liquid nitrogen and homogenized in lysis buffer on ice. The lystate was centrifuged and the supernatant was prepared. Total protein concentration of the supernatant was measured by BCA Ptotein Assay Bit. 50μg of each protein sample was separated by SDS-PAGE and transferred to nitrocellulose membrane. After blocking in skimmed milk for 1 hour, the membranes were incubated with antibody over night. The membrane were then developed with peroxidase-conjugated secondary antibody. The immunoreactive proteins were visulized by enhanced chemiluminescence system. The immunoblots were quantitated using analystic software.
The principal results and conclusions were as follows:
1 Effects of progesterone administration on morphine-induced conditioned place preference(CPP) and its relationship to endogenous opioids in the related rat brain regions
Compared with NS control group, 5 mg·kg~(-1) morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg~(-1) progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect.
Compared with NS control group, the levels ofβ-EP following morphine administration were decreased in hypothalamus, hippocampus and frontal cortex(P<0.05、P<0.01 and P<0.05). Compared with morphine group, the levels ofβ-EP were increased by co-administration with 15 mg·kg~(-1) dose of progesterone in hypothalamus (P<0.01). Compared with NS control group, the levels of L-EK following morphine administration were decreased in hypothalamus, nucleus accumbens and frontal cortex(P<0.05, P<0.05 and P<0.01). Compared with morphine group, the levels of L-EK were increased by co-administration with 15mg·kg~(-1) dose of progesterone in hypothalamus, nucleus accumbens and frontal cortex (P<0.05,P<0.01 and P<0.05). Compared with NS control group, the levels of DynA following morphine administration were increased in hypothalamus and frontal cortex (P<0.05 and P<0.05). Compared with morphine group, the levels of DynA were decreased by co-administration with 15mg·kg~(-1) dose of progesterone in hypothalamus and frontal cortex (P<0.01 and P<0.05).
2 Effects of progesterone administration on morphine-induced conditioned place preference and its relation toμ-receptors in rat brain regions
Compared with NS control group, 5 mg·kg~(-1) morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg~(-1) progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect.
Compared with NS control group, the levels of mu opioid receptor following morphine administration were decreased in hypothalamus, hippocampus , striatum and frontal cortex(P<0.05、P<0.01、P<0.01 and P<0.01). Compared with morphine group, the levels of mu opioid receptor were increased by co-administration with 15 mg·kg~(-1) dose of progesterone in hypothalamus, striatum and frontal cortex (P<0.05、P<0.01 and P<0.01).
3 Effects of progesterone administration on morphine-induced conditioned place preference and its relation to D2-receptors in rat brain regions
Compared with NS control group, 5 mg·kg~(-1) morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg~(-1) progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect.
Compared with NS control group, the levels of D2 receptor following morphine administration were decreased in nucleus accumbens ,striatum and ventral tegmental area (P<0.01、P<0.01 and P<0.01). Compared with morphine group, the levels of D2 receptor were increased by co-administration with 15 mg·kg~(-1) dose of progesterone in nucleus accumbens and striatum (P<0.05 and P<0.01).
4 Effects of progesterone administration on morphine-induced conditioned place preference and its relation to p-CREB in rat brain regions
Compared with NS control group, 5 mg·kg~(-1) morphine successfully induced the formation of CPP(P<0.01). 15 mg·kg~(-1) progesterone was not able to induce CPP effect itself, but abolished the morphine CPP effect(P<0.01).
Compared with NS control group, the levels of p-CREB following morphine administration were increased in hippocampus and striatum(P<0.01 and P<0.01)but decreased in nucleus accumbens (P<0.01). Compared with morphine group, the levels of p-CREB were decreased by co-administration with 15 mg·kg~(-1) dose of progesterone in striatum (P<0.01) but increased in nucleus accumbens (P<0.05).
In conclusion, our study indicated that chronic morphine treatment could induce the development of morphine psycological dependence and progesterone as an ectogenic neurosteroid could reverse morphine-induced rewarding effects. Durig the procedure of progesterone abolishing morpnine- induced dependence, the levels ofβ-EP, L-EK and DynA were increased or decreased in rat brain. Opioid receptors and dopamine receptors were involved in the regulation of morphine dependence by progesterone. We also found that with the changing in neurotransmitters and receptors, the levels of p-CREB were diversified in rat brain rewarding circuit. Thus, our data provide a new insight for the research of the neurobiochemical mechanism of the relationship between morphine dependence and neurosteroid.
引文
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45 侯艳宁, 王娜, 吴红海. 孕酮对大鼠吗啡位置偏爱效应及中枢单胺递质水平的影响. 中国药理学通报, 2006, 22(8): 980-983.
46 王娜, 吴红海, 任进民. 孕酮对大鼠吗啡奖赏效应及下丘脑和纹状体内单胺递质水平的影响. 中国药物依赖性杂志, 2006, 15(2): 16-19.
47 于动震,吴红海,侯艳宁.孕酮对吗啡条件性位置偏爱大鼠下丘脑中内阿片肽水平的影响.中国药理学通报,2007, 2007, 23(10):1309-1312
48 于动震,吴红海,侯艳宁.孕酮对吗啡条件性位置偏爱大鼠相关脑区中内啡肽水平的影响.华北国防医药, 2008, 20(2):5-7
49 于动震,吴红海,侯艳宁.孕酮对吗啡精神依赖大鼠相关脑区中强啡肽 A水平的影响.中国药物依赖性杂志, 2008,17(2):67-70
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