【作者】 刘真；

【导师】 王怀经； 李振中； Mark S. Kindy； 邢子英；

【作者基本信息】 山东大学 ， 神经生物学， 2008， 博士

【摘要】 促生长激素神经肽（galanin,Gal）是一个由29/30个氨基酸（在大多数物种为29个氨基酸,在人为30个氨基酸）组成的神经肽,在神经系统有广泛的表达,可发挥调节伤害性刺激、神经元发育和神经营养等多种生物学功能。在周围感觉神经节神经元和交感神经节（sympathetic ganglion,SG）神经元都检测到Gal的表达。Gal在周围神经系统对损伤的适应性反应及调节痛觉传递的过程中可能发挥作用。Gal被认为是背根神经节（dorsal root ganglion,DRG）神经元损伤的标志物之一和交感神经元轴突受到严重损伤时神经元表型改变的一个标志。在正常成年动物,Gal在感觉神经元和SG神经元表达的水平很低;在周围神经损伤和炎症状态下,Gal的表达显著上调。外周去甲肾上腺素能系统与痛觉的调节有一定的关系。正常健康状态下,去甲肾上腺素（norepinephrine,NE）对痛觉的影响作用极小,在神经损伤或炎症时,去甲肾上腺素能系统发生的各种可塑性变化可影响其对抗伤害性刺激的效能。不同的α-受体的亚型在交感神经元有表达,这些受体激活后可影响交感神经系统的活性或神经递质的表达或释放,并最终导致不同的效应。有趣的是,在初级感觉神经元也有功能性的α-受体表达,α-受体在介导NE对疼痛调节作用的过程中起着关键的作用,并调节神经源性炎症和伤害性刺激的反应。但激活α-受体对Gal表达的影响作用尚有待于证实。神经生长因子（nerve growth factor,NGF）不仅是一种重要的神经营养因子,而且还是炎症和疼痛的重要调节因子。NGF不仅影响DRG感觉神经元内包括Gal在内的多种神经肽的表达,也可调节SG神经元内神经肽的表达。外源性NGF对DRG神经元和SG神经元Gal表达的影响作用目前尚不清楚。6-羟多巴胺（6-hydroxydopamine,6-OHDA）和辣椒素（capsaicin,CAP）分别可以毁损SG交感神经元和DRG感觉神经元。6-OHDA是高效的儿茶酚胺选择性的神经毒素,对SG神经元Gal的表达变化有类似于轴突切断所致的效应。然而,6-OHDA对SG神经元Gal表达的影响程度目前尚不清楚。CAP通过激活位于初级传入神经元的辣椒素受体（vanilloid receptor 1,VR1）而发挥作用,大剂量CAP可选择性毁损初级传入神经元。Gal的受体GalR2和VR1在DRG神经元共同表达。CAP是否可影响DRG神经元Gal及其受体GalR2的表达需要进一步探讨。感觉神经肽和VR1的表达反映了DRG神经元感受伤害性刺激的特性。在初级传入神经元表达的感觉神经肽P物质（substance P,SP）在介导伤害性刺激的过程中发挥作用,Gal和SP可在同一个DRG感觉神经元中共存。外源性的Gal是否能影响DRG神经元VR1以及SP的表达尚需要进一步证实。在正常情况下,Gal在感受伤害性刺激的过程中只发挥较小的作用,然而在神经病理状态下,Gal对伤害性刺激的调节可发挥重要的作用。在非炎性刺激和炎症刺激条件下,Gal是一个重要的疼痛或痛觉过敏的调节因子。然而,Gal产生这种兴奋性作用的机制并不清楚。大鼠足底注射福尔马林可诱发自发性疼痛行为反应,Gal对此是否具有影响作用及其作用机制目前尚不清楚。根据以上研究背景可知Gal与多种生理及病理生理机制相关。但是NE、CAP、NGF对DRG神经元Gal的表达的影响作用,NGF、6-OHDA、α-受体激动剂对SG神经元Gal表达的影响作用以及Gal在福尔马林诱发的炎性疼痛中的作用机制尚不清楚。因此,本研究建立了DRG神经元和颈上神经节（superiorcervical ganglion,SCG）神经元培养模型,用这两个培养模型,分别研究了NE、CAP、NGF对DRG神经元Gal的表达的影响作用,NGF、6-OHDA、α-受体激动剂对SCG神经元Gal表达的影响作用。用动物实验探讨了Gal在福尔马林诱发的伤害性刺激中的作用及其机制。此外,NGF所致的DRG和SCG神经元轴突再生与Gal表达的关系,CAP对DRG神经元GalR2表达的影响,外源性Gal对DRG神经元SP释放、VR1表达的影响作用在本实验中也进行了探讨。DRG取自胚胎15 d的Wistar大鼠,SCG神经元取自新生Wistar大鼠。DRG神经元和SCG神经元培养24 h后,再用阿糖胞苷（cytarabine,ara-C）作用24h抑制非神经元细胞生长。之后,用不同的处理因素作用4 d或正常培养4 d后用不同的处理因素作用4 h后进行检测。（1）NE及α-受体拮抗剂孵育DRG神经元:DRG神经元用NE(10^-4mol/L)孵育4 d,或在加入NE之前10 min,在培养液内预先加入α₁-受体拮抗剂哌唑嗪(10^-6mol/L)或α₂-受体拮抗剂育亨宾(10^-5mol/L)。检测对Gal及其mRNA的表达。（2）NGF孵育DRG神经元:DRG神经元用NGF（10 ng/ml）或NGF（10ng/ml）加NE(10^-4mol/L)孵育4 d,检测Gal及其mRNA的表达及单个神经元的轴突总长度。（3）CAP孵育DRG神经元:①DRG神经元用不同浓度的CAP(10^-8mol/L,10^-7mol/L,10^-6mol/L)孵育4 h,检测Gal和GalR2及其mRNA的表达;②DRG神经元用不同浓度的CAP(10^-8mol/L,10^-7mol/L,10^-6mol/L)孵育4 d,检测Gal和GalR2及其mRNA的表达。（4）外源性Gal孵育DRG神经元:DRG神经元用不同浓度的Gal(10^-9mol/L,10^-8mol/L,10^-7mol/L)孵育4 d,检测VR1及其mRNA、SPmRNA的表达及SP的释放。在加入Gal之前10 min,预先加入GalR拮抗剂M35(10^-8mol/L)或PKC抑制剂Calphostin C(10^-7mol/L),检测GalR拮抗剂及PKC抑制剂对Gal作用的影响。（5）选择性α-受体激动剂孵育SCG神经元:SCG神经元分别用α₁-受体激动剂苯肾上腺素(10^-5mol/L)和α₂-受体激动剂可乐定(10^-5mol/L)孵育4 d,检测Gal及其mRNA的表达。（6）NGF或/和6-OHDA孵育SCG神经元:SCG神经元用NGF（10 ng/ml）、6-OHDA(10^-5mol/L)、NGF（10 ng/ml）加6-OHDA(10^-5mol/L)孵育4 d,检测Gal及其mRNA的表达及单个神经元的轴突总长度。动物实验所用动物为体重为220～250g的雄性Wistar大鼠。随机分为5组,每组10只动物。Gal组、福尔马林组、福尔马林+Gal组、福尔马林+Gal+GalR拮抗剂组、福尔马林+Gal+PKC抑制剂组。在大鼠左侧后足底注射20μl 2%的福尔马林诱发炎症,同时注射20μl Gal（0.1 ng/μl）,之后进行缩足反射行为学检测实验。GalR拮抗剂（M35）在注射Gal前20 min、PKC抑制剂（CalphostinC）在注射Gal前60 min注射。Gal组、福尔马林组分别只注射20μl Gal（0.1ng/μl）或20μl 2%的福尔马林。行为学检测后,取左侧腰骶部DRG和腰骶部SG,检测Gal及其mRNA的表达。本研究的结果显示:（1）用NE(10^-4mol/L)卵育4 d后,培养的DRG神经元中Gal及其mRNA的表达增加;用α₁-受体阻断剂哌唑嗪(10^-6mol/L)预处理可阻断NE的这一效应;用α₂-受体阻断剂育亨宾(10^-5mol/L)预处理则不影响NE引起的Gal及其mRNA的表达增加。（2）用NGF（10 ng/ml）孵育4 d后,培养的DRG神经元中Gal及其mRNA的表达降低;NGF还可抑制NE诱发的Gal及其mRNA表达。NGF能促进DRG神经元轴突再生,而NE则不能促进DRG神经元轴突再生。（3）CAP(10^-6mol/L)急性处理（4 h）或CAP(10^-8mol/L,10^-7mol/L)长期孵育（4d）均可促进Gal及其mRNA的表达,CAP(10^-7mol/L)长期孵育（4 d）可促进GalR2及其mRNA的表达。（4）外源性Gal可增加DRG神经元CAP诱发的SP释放量,但不影响SP及其mRNA的表达以及基础SP释放量。外源性Gal可促进DRG神经元VR1及其mRNA的表达,并呈剂量依赖方式。外源性Gal所致的DRG神经元VR1表达增加可部分地被GalR拮抗剂M35或PKC抑制剂Calphostin C所抑制。（5）α₂-受体激动剂可乐定(10^-5mol/L)可抑制SCG神经元Gal及其mRNA的表达,α₁-受体激动剂苯肾上腺素(10^-5mol/L)对SCG神经元Gal及其mRNA的表达无影响。（6）NGF（10 ng/ml）可抑制SCG神经元Gal及其mRNA的表达;6-OHDA(10^-5mol/L)孵育SCG神经元,可促进Gal及其mRNA的表达;联合应用NGF和6-OHDA,NGF不能抑制6-OHDA引起的Gal及其mRNA表达的上调,但NGF能促进SCG神经元轴突再生。（7）注射福尔马林可诱发大鼠自发性疼痛行为缩足反射,注射20μl Gal（0.1 ng/μl）可增加缩足反射的次数。在注射Gal前注射30μl（20 ng/μl）Gal受体拮抗剂M35或30μl PKC抑制剂Calphostin C（0.1ng/μl）,可部分抑制Gal引起的疼痛增强效应。（8）大鼠足底内注射福尔马林可引起同侧腰骶部DRG内Gal mRNA表达增加,神经肽Gal的表达无变化。腰骶部SG内Gal及其mRNA表达均无变化。这些结果表明:（1）DRG神经元内Gal的表达可受NE、NGF、CAP等多种因素的影响。NE通过作用于α₁-受体,而不是α₂-受体,增强了DRG神经元Gal的表达,这可能是外周促进伤害性刺激的肾上腺素能调节机制之一。NGF抑制DRG神经元内Gal的表达,一定孵育时间和孵育浓度的CAP刺激DRG神经元,Gal及其受体GalR2表达上调。这反映了Gal所介导的伤害性刺激作用机制的复杂性。（2）SCG神经元内Gal的表达可受α₂-受体激动剂、NGF、6-OHDA等多种因素的影响。交感神经元α₂-受体的激活对Gal的表达有抑制作用,表明α₂-受体可能参与了Gal介导的损伤刺激或炎症反应。外源性NGF可使培养的SCG神经元Gal表达下调,而6-OHDA则可使Gal表达上调。（3）Gal可能介入了与VR1有关的伤害性刺激调节机制,GalR的活化和PKC通路被激活可能是其作用机制。Gal对福尔马林诱发的炎性疼痛的增强作用可能是由于Gal作用于GalR后激活了PKC细胞信号转导通路所致。选择性GalR拮抗剂和PKC抑制剂可能对炎性疼痛的治疗具有一定的应用价值。更多还原

【Abstract】 Galanin（Gal）,a 29-amino-acid neuropeptide in most species（30-amino-acid in human）,is widely distributed throughout the nervous system including sensory ganglion and sympathetic ganglion（SG）neurons and is involved in the regulation of manifold functions including nociception,developmental and trophic effects.Gal may play a role in the adaptive response of the peripheral nervous system to injury and modulate pain transmission.Gal is recognized as one of the dorsal root ganglion（DRG）injury markers.Increased Gal expression was used as a marker of the change of phenotype that occurs in sympathetic neuronal cell bodies when their axons are severely damaged.Gal is normally expressed at low levels in sensory neurons and SG neurons and is markedly up-regulated within these neurons following peripheral nerve injury and inflammation in the adult.Peripheral noradrenergic system is involved in intrinsic control of pain. Norepinephrine（NE）has little influence on pain in healthy tissues,whereas noradrenergic system is subject to various plastic changes that influence its antinociceptive efficacy after injury or inflammation.Distinct alpha-adrenoreceptors are expressed in sympathetic neurons.Activation of distinct alpha-adrenoreceptors influences tone of sympathetic nervous system or neurotransmitter synthesis or release and finally results in different effects.Interestingly,functional alpha-adrenoreceptors are expressed in primary sensory neurons and regulate neurogenic inflammation and nociceptive responses.Alpha-adrenoreceptors have a key role in mediating pain regulatory effects of NE.These adrenoreceptors are functionally active may vary with the presence of nerve injury,inflammation or other physiological and pathophysiological conditions.It is not known whether activation of alpha adrenoreceptors could affect Gal expression in DRG and SG neurons.Nerve growth factor（NGF）initially interested neurobiologists because of its effects in the developing nervous system of the survival,differentiation and maturation.In the course of the last years,several lines of evidence converged to indicate that NGF is a major regulator of inflammatory and homeostatic pain states, influencing both sensory neuron phenotype and physiologic responses.NGF could influence the expression of several neuropeptides including Gal in both DRG and SG neurons.It is not clear whether exogenous NGF could affect Gal expression in DRG or SG neurons.6-Hydroxydopamine（6-OHDA）and capsaicin（CAP）could cause neurotoxicity on SG neurons and DRG neurons,respectively.The highly potent and catecholamine selective neurotoxin 6-OHDA could causes changes in the expression of Gal mRNA in the SCG similar to those seen after axotomy.It is not known to what extent Gal expression is affected by 6-OHDA in cultured SCG neurons.CAP,the pungent component of hot peppers,elicits a sensation of burning pain,via activation of vanilloid receptor 1（VRI,CAP receptor）expressed in primary sensory neurons that convey information about noxious stimuli to the central nervous system.Large dosage of CAP could selectively destroy primary sensory neurons.Interestingly,co-expression of GalR2 and VR1 in DRG neurons suggested that Gal-induced effects are mediated by GalR2 on CAP-sensitive primary sensory neurons.Whether expression of Gal and GalR2 was affected by CAP should be further studied. Neuropeptide expression and VR1 expression may reflect nociceptive properties of DRG neurons.Substance P（SP）,an 11-amino acid-long neuropeptide, is expressed in primary sensory neurons and plays an important role in nociception. Gal immunoreactive cells are colocalized with SP immunoreactive cells in single sensory neurons of the rat DRG suggesting the functional significance of these neurotransmitters in the modulation of sensory action and neuropathic pain transmission.Whether exogenous Gal could affect expression of VR1 and SP should be verified.Gal is thought to play only a minor role in nociception under normal conditions. However,it may have a critical role in modulation of nociception in neuropathic states.Several lines of evidence demonstrated that Gal was involved in peripheral pain processing.In both non-inflammatory stimulation and inflammatory conditions, Gal is one of the important mediators in the processing of pain sensation or hypersensitivity.However,the possible mechanisms by which Gal exerts an excitatory action are still unknown.Based on the above research backgrounds,we know that Gal was involved in so many physiological and pathophysiological conditions.Whereas whether NE, CAP and NGF influence Gal expression in DRG neurons and NGF,6-OHDA and adrenoreceptor agonists affect Gal expression in SG neurons in vitro needs to be clarified.The effects and mechanisms of Gal in formalin-induced nociception remain unknown.In the present study,both DRG and superior cervical ganglion （SCG）neuronal culture models were established.Gal expression in DRG neurons induced by NE,CAP and NGF and Gal expression in SCG neurons induced by NGF, 6-OHDA and alpha-adrenoreceptor agonists were investigated using these two culture models,respectively.The effects and mechanisms of Gal in formalin-induced inflammatory pain were also investigated.In addition,the relationship between NGF-induced axonal regeneration and Gal expression,the effect of CAP on GalR2 expression in DRG neurons and the effect of exogenous Gal on SP release and VR1 expression in DRG neurons were investigated in the present experiment. DRG and SCG culture experiments.DRG and SCG were dissected out from embryonic 15-day-old or newborn Wistar rats,respectively.DRG and SCG cells were cultured in Dulbecco’s Modified Eagle Medium with F-12 supplement （DMEM/F-12）media at 37℃with 5%CO₂ for 24 hours and then maintained in culture media containing cytarabine（ara-C）（5μg/ml）for another 24 hours to inhibit growth of non-neuronal cells.After that,DRG and SCG cells were maintained in different culture conditions for additional 4 days with media change every 2 days. Neurons were cultured continuously in culture media for 6 days as control.（1） Exposure of NE on DRG neurons:DRG neurons were exposed to NE(10^-4mol/L) for 4 days.When requested,DRG neurons were pretreated with alpha 1-adrenoreceptor antagonist prazosin(10^-6mol/L)or alpha 2-adrenoreceptor antagonist yohimbine(10^-5mol/L),10 minutes prior to the NE challenge.（2） Exposure of NGF on DRG neurons:DRG neurons were exposed to NGF（10 ng/ml）for 4 days.When requested,DRG neurons we,re exposed to NE(10^-4mol/L) during the 4 days NGF treatment.（3）Exposure of CAP on DRG neurons:DRG neurons were exposed to CAP(10^-8,10^-7,10^-6mol/L)for 4 hours as the acute treatment and exposed to CAP(10^-8,10^-7,10^-6mol/L)for 4 days as the chronic treatment.（4）Exposure of Gal on DRG neurons:DRG neurons were exposed to Gal(10^-9,10^-8,10^-7mol/L)for 4 days.When requested,DRG neurons were pretreated with GalR antagonist M35(10^-8mol/L)or PKC inhibitor(10^-7mol/L),10 minutes prior to the Gal treatment.（5）Exposure of selective alpha-adrenoreceptor agonists on SCG neurons:SCG neurons were exposed to alpha 1-adrenoreceptor agonist phenylephrine(10^-5mol/L)or alpha 2-adrenoreceptor agonist clonidine(10^-5mol/L).（6）Exposure of NGF or/and 6-OHDA on SCG neurons:SCG neurons were exposed to NGF（10 ng/ml）, 6-OHDA(10^-5mol/L)and NGF（10 ng/ml）plus 6-OHDA(10^-5mol/L)for 4 days.Animal behavioral experiments.Male Wistar rats weighing 220-250 g were used in this experiment.Animals were randomly divided into 5 groups（n=10 per group）:Gal group,formalin group,formalin+Gal group,formalin+Gal+GalR antagonist group,and formalin+Gal+PKC inhibitor group.Inflammation in the formalin-induced arthritis of the left tibiotarsal joint of male Wistar rats was induced by subcutaneous injection of 20μl 2%formalin into the plantar surface of the left hind paw.20μl Gal（0.1 ng/μl）was injected into the left hind paw simultaneously and flinching animal behavior would be examined at this time.GalR antagonist M35 was injected 20 min prior to Gal injection.PKC inhibitor Calphostin C was injected 60 min prior to Gal injection.Animals in Gal or formalin group,only 20μl Gal（0.1 ng/μl）or 20μl 2%formalin was injected into the left hind paw, respectively.After animal behavior examination,the expression of Gal mRNA and peptide in lumbosacral DRG and SG were examined.The results are as follows:（1）NE(10^-4mol/L)promoted Gal mRNA and Gal peptide expression in cultured DRG neurons after 4 days incubation.Pretreatment with alpha 1-adrenoreceptor antagonist prazosin(10^-6mol/L)could block the effects caused by NE,whereas alpha 2-adrenoreceptor antagonist yohimbine(10^-5mol/L) did not have the effects on NE induced elevation of Gal mRNA and Gal peptide levels.（2）NGF（10 ng/ml）inhibited Gal mRNA and Gal peptide expression as compared with control at the same time point.NGF also inhibited NE-induced the elevation Gal mRNA and Gal peptide expression in DRG cultures.NGF but not NE could promote axonal regeneration of DRG neurons.（3）Acute exposure（4 hours） of CAP(10^-6mol/L)and chronic exposure（4 days）of CAP(10^-8mol/L,10^-7mol/L) promoted Gal mRNA and Gal peptide expression in DRG cultures.Chronic exposure（4 days）of 10^-7mol/L CAP promoted GalR2 mRNA and GalR2 protein expression in DRG cultures.（4）Exogenous Gal sensitized CAP-evoked SP release, but did not have effects on SP mRNA,SP peptide expression and basal SP release in DRG cultures.Exogenous Gal promoted VR1 mRNA and VR1 protein expression in a dose-dependent manner in cultured DRG neurons after 4 days incubation.The elevation of VR1 expression could partially be inhibited by GalR antagonist M35 or PKC inhibitor Calphostin C.（5）The levels of Gal mRNA and Gal peptide expression in cultured SCG neurons decreased significantly after stimulation with alpha 2-adrenoreceptor agonist clonidine(10^-5mol/L).Alpha 1-adrenoreceptor agonist phenylephrine(10^-5mol/L)stimulation did not have effects on Gal mRNA and Gal peptide expression.（6）NGF（10 ng/ml）inhibited Gal mRNA and Gal peptide expression in cultured SCG neurons.Exposure of 6-OHDA(10^-5mol/L) promoted Gal mRNA and Gal peptide expression in SCG cultures,whereas NGF had no effect on the increase of Gal mRNA and Gal peptide expression induced by 6-OHDA treatment.NGF could promote axonal regeneration of SCG neurons.（7） Intraplantar injection of 20μl Gal（0.1 ng/μl）to formalin-induced inflammation male Wistar rats produced spontaneous flinches of the injected hindpaw. Intraplantar administration of M35 or calphostin C partially reversed the Gal potentiation of formalin-induced nociception.（8）The levels of Gal mRNA in lumbosacral DRG were increased after intraplantar injection of formalin.The expression of Gal peptide was not increased.Both Gal mRNA and Gal peptide expression in lumbosacral SG were not affected by intraplantar injection of formalin.The results in the present study indicate that:（1）Gal expression in DRG neurons might be affected by different stimulators such as NE,NGF,or CAP.NE,due to action on alpha 1-adrenoreceptors but not alpha 2-adrenoreceptors,increases Gal expression in DRG neurons indicating that it is one of the pronociceptive noradrenergic mechanisms in the periphery.NGF inhibits Gal expression in the absence or presence of NE on cultured DRG neurons. Certain concentrations or exposure time of CAP stimulation may be relevant to up-regulation of Gal and its receptor GalR2 expression in DRG cultures.These results implicated the complexity of the mechanisms of Gal-related nociception.（2）Gal expression in SCG neurons might be modulated by different factors such as alpha-adrenoreceptor,NGF,or 6-OHDA.Gal may be regulated by activation alpha 2-adrenoreceptors,but not alpha 1-adrenoreceptors in sympathetic neurons suggested alpha 2-adrenoreceptors may be involved in the Gal related injury or inflammatory responses.Gal expression was attenuated by administration of exogenous NGF and enhanced by administration of exogenous 6-OHDA in SCG cultures.（3）Exogenous Gal could induce CAP-evoked SP release and increase VR1 expression suggested that Gal may be,at least in part,correlated with VR1-related nociception.Activation of GalR and PKC pathway may be involved in the enhancement of formalin-induced inflammatory pain caused by exogenous Gal.The use of GalR antagonists and PKC inhibitor in the periphery may have therapeutic value in the treatment of inflammatory pain.更多还原