Caspase-3凋亡通路参与神经病理性疼痛及其相关机制的研究
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摘要
目的及研究背景:
慢性疼痛被广泛地定义为急性组织损伤修复后疼痛超过1个月、疼痛持续或反复发作超过3个月以上或与组织损伤有关的疼痛预计持续存在或加重。慢性疼痛近来逐年增加,已经成为一个主要的社会、经济负担,全世界有超过3亿2千万的人受其影响。疼痛患者通常分为以下几类:术后疼痛、癌痛、后背痛、HIV疼痛、神经病理性疼痛、关节痛和偏头痛。
依原始刺激的时程和自然特性,疼痛分为3个不同时期,第一期:急性疼痛期,是对简单、短暂的伤害性刺激物的处理过程,是单纯、直接由外周向丘脑及皮层中枢的传递过程。外周感受器的放电与主观疼痛的感知之间有着密切的联系。第二期:炎症性状态期,由持续长时间伤害性刺激所形成,导致组织损伤和外周的炎症反应,机体出现自发性疼痛、痛觉过敏以及超敏反应(将正常的非伤害性刺激认为疼痛)。第三期:神经病理性状态期,为外周神经或CNS损害的结果,包括外周的神经病理状态和中枢的疼痛状态,此时的疼痛与原发的损伤无关。尤其要指出的是这3个时期之间并不排斥,在某些特定的神经病理状态下可以共存。
神经病理性疼痛以自发性疼痛、痛觉过敏以及超敏反应为特点,通常由外周神经、背根神经节、脊髓或脑的损伤所致,包括了疼痛的第二、第三期。神经病理性疼痛目前仍然是一个棘手的临床问题,主要是对其病理过程了解不全。近年来,有研究表明,凋亡对于细胞损伤及神经病理性疼痛具有重要意义。形态学的研究,在神经病理性疼痛的大鼠脊髓浅层及在外周神经损伤的脊髓上同时存在细胞凋亡的现象。在神经病理性疼痛的大鼠上,bcl-2家族基因表达的改变可调节细胞死亡。我们推测在细胞死亡前有较长的一段凋亡期,但是否是凋亡相关基因在脊髓的表达导致神经病理性疼痛的发生目前仍不清楚。
细胞凋亡(apoptosis)是一个细胞自我破坏的程序性生化过程,形态学特征是细胞核和细胞质发生浓缩,在多种的疾病中都起重要作用。凋亡的中心环节是蛋白裂解系统,包括半胱氨酰蛋白酶家族,称半胱氨酸天冬氨酸蛋白酶(caspase)。caspases分为两类:一类是上游启动者(initiators)caspases,如caspase8、9,可裂解并激活其它caspase;另一类是下游效应者(effectors)caspases,主要包括caspase-3、6、7,可裂解各种底物,分解细胞结构或使酶失活。而caspase-3作为“刽子手”蛋白酶,在其中起关键作用。Vitode Novellis等的研究发现大鼠坐骨神经结扎后凋亡通路激活,阻断谷氨酸mGlu5受体可防止凋亡基因在脊髓的过表达。但Erika的研究却指出在SNI模型中,脊髓背角神经元的死亡并不是机械性超敏反应所必需的。因此,凋亡通路是否参与外周神经病变后的疼痛还需进一步证实。
某些神经病理性疾病与线粒体功能障碍有关,而线粒体功能障碍与凋亡关系密切。线粒体依赖性的凋亡可被许多因素激活,如活性氧(reactive oxygen species,ROS)、神经酰胺、一氧化氮。这些因素可导致线粒体释放细胞色素C,而后形成细胞色素C、凋亡蛋白酶激活因子1和caspase-9复合体,从而裂解并激活下游的效应者caspases。氧化应激可损伤组织、蛋白、DNA和脂质。ROS是激活caspase,引起线粒体功能障碍的关键因素,ROS参与NMDA受体激活,而大鼠CCI模型上凋亡的发生又是谷氨酸依赖性,这就建立起ROS与凋亡的联系。反之,广谱的抗凋亡蛋白如BCL-2又有抗氧化作用,通过直接清除ROS或上调其它ROS清除剂如硫醇复合物或抗氧化蛋白酶,这也进一步证明ROS的产生是启动凋亡的关键之一。ROS和凋亡都同神经病理性疼痛有关表明两者之间具有某种联系。由于ROS清除剂可缓解疼痛,而且ROS是诱导凋亡的重要步骤,有理由推测ROS可快速诱导脊髓凋亡基因的过表达,并发挥其在疼痛的发展中的重要作用。
肿瘤坏死因子(tumour necrosis factor,TNF)家族受体同样可诱导凋亡。TNF-a作用于感觉神经元,在炎症性疼痛中起重要作用;神经酰胺,作为TNF-a的第二信使,可增强神经元的兴奋性。TNF-a可通过多种不同方式激活细胞内的多种信号转导通路。连接并聚合TNF-R1可导致某些adapter信号蛋白的聚集,其中之一即Fas相关的死亡区域蛋白(Fas-associated death domain protein,FADD)。FADD可聚集前caspase-8分子并激活可自我放大的caspase级联反应,该效应为细胞凋亡的必需环节。研究表明,抑制caspase可减轻TNF-a诱导的痛觉过敏;抑制TNF-a的第二信使神经酰胺,可同时抑制痛觉过敏和细胞凋亡。因此,我们有理由假设,caspase信号转导通路所引起的痛觉相关的行为学改变可能是由TNF-a所诱导的。
当然,还要一个问题需要解决,那就是神经元凋亡后为何超敏反应还持续存在,因为众所周知,痛觉信号是由神经元传递。芽生(sprouting),是神经损伤的结果,通常伴有强烈的胶质细胞反应。在此,我们假设芽生可能是神经元凋亡后神经病理性疼痛持续存在的机制。有证据表明,激活的星形胶质细胞在清除损伤突触、重新连接神经断端及芽生中起重要作用。在神经元的断端,星形胶质细胞激活,突触发生替换,同时,在与断端不相邻的远处的中枢神经系统的神经元也发生了替换。在中枢初级传入神经元的周围胶质细胞迅速激活。神经纤维在分裂的星形胶质细胞的传送下,在脊髓内生长,形成生长区带。研究表明,激活的星形胶质细胞至少可以通过两种方式促进芽生:①通过清除轴突碎片,减少髓鞘对芽生的阻碍而促进芽生;②通过释放细胞因子和生长因子调控芽生。研究表明,两种细胞因子IL-1和IL-6可调节胶质细胞的反应,并在调节神经元芽生中具有重要作用。IL-1可调节胶质细胞增殖,引导营养因子作用于新生的神经纤维。而IL-6则诱发新生纤维向外继续生长。因此,推测在凋亡时或凋亡后,大直径、厚髓、低阈值的Aa纤维或交感神经纤维可能芽生入脊髓浅层,包括Ⅰ和Ⅱ层。中小直径的、薄髓、高阈值的Ad纤维可能被低阈值Aa纤维或交感神经纤维替代,导致超敏反应的发生。
鉴于caspase-3在神经病理性疼痛中的作用,阻断该通路可能成为治疗疼痛的新方法。RNA干扰是一种抑制基因表达的新方法,一系列相关的临床前的研究都在开展。RNA干扰主要细胞浆中执行,采用非病毒载体易于达到该目的,但是要被细胞有效的摄入并实现长期、稳定的表达是比较困难的。目前更倾向于在哺乳动物细胞内表达siRNA,以维持长久的RNA干扰效应。而利用腺病毒表达干扰RNA,因其在体内的存在时间更长,而且,腺病毒的转染效率比单纯的siRNA或质粒介导的表达载体的转染效率要高得多,可大大增强体内实验的干扰效率。腺病毒的宿主范围广,可将外源基因转入静止的非分裂的细胞。有学者利用腺病毒介导小RNA在体内外实验都可抑制葡苷酸酶,说明腺病毒介导RNA的表达有一定的可行性。因此,在本研究中,我们将构建表达caspase-3小RNA的腺病毒,并观察其对CCI大鼠痛觉过敏反应的影响。
方法:
1.坐骨神经结扎大鼠脊髓背角细胞凋亡的研究
(1)大鼠分成2组:假手术组(Sham组)和手术组(CCI组)。术后1d、3d、7d、10d、14d不同的时间点测定坐骨神经结扎大鼠的热痛阈,并采用免疫荧光方法测定caspase-3的表达,TUNEL原位末端标记法检测脊髓神经细胞的凋亡。
(2)caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK阻断凋亡通路对CCI的影响。
手术当日及术后在大鼠疼痛同侧的后肢脚底皮内每日1个单位分别注射caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK,连续注射5d,观察比较大鼠热痛阈的变化,同时观察凋亡指标。
2.TNF-α、ROS参与CCI大鼠神经元凋亡的研究
(1)大鼠分成2组:假手术对照组和手术组,手术组随机分为2个亚组,N-乙酰半胱酸组(NAC组)、生理盐水(NS组)。实验前禁食12h,自由饮水。NAC组于麻醉后、结扎前腹腔注射NAC 300 mg/kg,而后每日注射NAC 300 mg/kg,生理盐水组注射等量生理盐水。
(2)术后1d、3d、7d、10d、14d不同的时间点测定坐骨神经结扎大鼠的热痛阈,采用ELISA检测脊髓TNF-α的表达、并对脊髓SOD表达进行测定。同时采用免疫荧光方法测定caspase-3的表达,TUNEL原位末端标记法检测脊髓神经细胞的凋亡。
3.CCI大鼠脊髓背角神经元凋亡、星形胶质细胞激活促进芽生形成介导神经病理性疼痛的研究。
(1)caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK阻断通路。术后在大鼠疼痛同侧的后肢脚底皮内隔日注射caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK,观察比较大鼠热痛阈的变化,同时观察凋亡指标。用westernblotting及免疫荧光方法测定GAP-43的表达,观察芽生情况。
(2)用药物丙戊茶碱抑制星形胶质细胞激活。术后在大鼠疼痛鞘内每日注射胶质细胞抑制剂丙戊茶碱,用GFAP抗体进行免疫组化,观察星形胶质细胞激活,并用ELISA方法检测IL-1β的表达,同时观察疼痛及测定GAP-43的表达。
4.腺病毒介导的小RNA干扰caspase-3对CCI大鼠的抗伤害性刺激作用
(1)体外筛选caspase-3小RNA的有效序列。
应用经典的克隆方法,首先克隆大鼠caspase-3基因,将caspase-3基因与含EGFP的质粒pEGFP-C1重组,构建报告载体pEGFP-Cas3,对3个不同表达caspase-3小RNA的质粒pShuttleH1-siCas3进行筛选,通过Lipofectamine2000将pShuttleH1-siCas3及体pEGFP-Cas3共转染HEK-293细胞株,倒置相差显微镜和流式细胞仪观察EGFP的荧光强度。
(2)采用分子克隆的方法构建表达caspase-3小RNA的腺病毒,并观察其对体外培养神经元凋亡的影响。
(3)caspase-3小RNA及重组腺病Ad-SiCas3鞘内注射对CCI大鼠热痛阈的影响。
caspase-3小RNA在术前1d开始鞘内注射,20μg/d,连续7d;重组腺病毒Ad-SiCas3单次注射,采用real time PCR及western blotting的方法检测caspase-3 mRNA及其蛋白。并观察比较大鼠热痛阈的变化。
结果:
1.坐骨神经结扎大鼠脊髓背角细胞凋亡的研究
(1)大鼠坐骨神经结扎后,右足热痛阈明显降低,与左足相比,具有明显的差异(P＜0.05),免疫荧光显示结扎侧脊髓背角caspase-3的表达明显高于未结扎侧,TUNEL法显示结扎侧脊髓背角凋亡的细胞明显高于未结扎侧。
(2)caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK注射后,caspase-3的表达明显降低,大鼠热痛阈明显增高,与NS组相比差异显著(P＜0.05),与Sham组相比无显著差异(P＞0.C5)。停止注射后,大鼠热痛阈降低,与NS组相比差异显著(P＜0.05),与CCI组相比无显著差异(P＞0.05)。
2.TNF-α、ROS参与CCI大鼠神经元凋亡的研究。
(1)坐骨神经结扎后脊髓TNF-α的表达增加,与CCI组相比差异显著(P＜0.05)。脊髓SOD表达下降,在第7天上与CCI组相比差异显著(P＜0.05),其它时间点无显著差异(P＞0.05)。
(2)NAC组脊髓caspase-3的表达及TUNEL阳性神经元数与NS组相比,明显下降(P＜0.05),在施加干预后,NAC组右足热痛阈明显升高,而NS组无明显变化,两者比较有统计学意义(P＜0.05)。
3.CCI大鼠脊髓背角神经元凋亡、星形胶质细胞激活促进芽生形成介导神经病理性疼痛的研究。
(1)caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK注射后,caspase-3的表达明显降低,大鼠热痛阈明显增高,与NS组相比差异显著(P＜0.05),与Sham组相比无显著差异(P＞0.05)。停止注射后,大鼠热痛阈降低,与NS组相比差异显著(P＜0.05),与CCI组相比无显著差异(P＞0.05)。GAP-43的表达明显降低(P＜0.05)。
(2)注射后丙戊茶碱,大鼠热痛阈明显增高,GFAP的表达明显下降,与NS组相比差异显著(P＜0.05)。IL-1及GAP-43的表达明显下降与NS组相比差异显著(P＜0.05)。
4.腺病毒介导的小RNA干扰caspase-3对CCI大鼠的抗伤害性刺激作用
(1)成功构建报告载体pEGFP-Cas3及3个质粒pShuttleH1-siCas3。共转染HEK-293细胞株,倒置相差显微镜和流式细胞仪均可见EGFP的荧光强度被明显抑制,其中,以pShuttleH1-siCas3Ⅱ的干扰效果最佳。
(2)Ad-siCas3能够较好抑制caspase-3 mRNA的表达,抑制内毒素诱导的神经元细胞凋亡。
(3)caspase-3小RNA或Ad-siCas3鞘内注射后大鼠热痛阈明显增高(P＜0.05),caspase-3 mRNA及其蛋白的表达下降(P＜0.05)。
结论:
1.大鼠坐骨神经结扎后,脊髓背角caspase-3高表达,细胞发生凋亡,阻断caspase-3通路均可抑制凋亡,减轻痛觉过敏;非特异性阻断caspase通路均亦可抑制凋亡,减轻痛觉过敏。坐骨神经结扎引起的神经病理性疼痛可能与caspase-3通路介导的细胞凋亡有关。
2.大鼠坐骨神经结扎后,脊髓背角细胞凋亡的发生可能与TNF-α表达增加,SOD下降导致ROS增多有关。抗氧化剂N-已酰半胱氨酸具有对抗大鼠神经元凋亡效应,减轻痛觉过敏。N-已酰半胱氨酸抗氧化可能将成为一种新的缓解病理性疼痛的方法。
3.阻断凋亡caspase通路,抑制星形胶质细胞激活,均可抑制GAP-43的表达,同时减轻痛觉过敏。神经元凋亡时或凋亡后,芽生可能介导神经病理性疼痛的产生,星形胶质细胞的激活,IL-1的升高有助于芽生的发展。
4.caspase-3的小RNA鞘内注射可在短期内减轻CCI大鼠热痛觉过敏;重组腺病毒Ad-SiCas3可持续抑制热痛觉过敏。
Objective and Background:
Chronic pain is defined widely as the suffering which continues for more than one month after tissue lesion,the pain continues or recurrent attacks for more than three months and the pain related to tissue lesion exists continually or aggravate in anticipation.Chronic pain is increasingly and rightfully being recognized as a major socio-economic burden,estimated to be affecting＞320 million people worldwide.The patient population is broadly divided into the following sectors:postoperative pain,cancer pain,back pain,HIV pain,neuropathies, arthritic pain and migraine.
There are three major stages or phases of pain and proposed that different neurophysiologic mechanisms are involved,depending on the nature and time course of the originating stimulus.These three phases are(1) acute pain phase,the processing of a brief noxious stimulus;(2) inflammatory pain phase,the consequences of prolonged noxious stimulation,leading to tissue damage and peripheral inflammation;and(3) neuropathies pain phase,the consequences of neurological damage,including peripheral neuropathies and central pain states.However,it is important to point out that these phases are not exclusive, and that at any given time several of the neurophysiologic mechanisms that underlie these pain states may coexist in the same individual.
Neuropathic pain,characterized by hyperalgesia,allodynia and spontaneous pain,often occurs as a result of injuries to the peripheral nerve,dorsal root ganglion(DRG),spinal cord or brain.Neuropathic pain remains a prevalent and persistent clinical problem because of our incomplete understanding of its pathogenesis.In the recent years,studies performed in all neurobiological fields have resulted in important information about apoptosis that are responsible for cell damage and neuropathic pain.Morphological studies have suggested the occurrence of apoptosis in the spinal cord following peripheral nerve,and in the spinal superficial laminae of neuropathic rats,that altered bcl-2 family gene expression may modulate cell death in a model of neuropathic pain.We hypothesized that there may be a prolonged latent phase of apoptosis before cell death.Whether or not changes in the expression of apoptosis-associated genes occur in the spinal cord during the first days leading to the establishment of neuropathic pain syndrome is unknown.
Apoptosis,a morphologically and biochemically defined form of cell death,has been demonstrated to play a role in a variety of diseases.The central component of the apoptotic machinery is a proteolytic system consisting of a family of cysteinyl proteases,termed caspases.Two groups of caspases can be distinguished:upstream initiator caspases such as caspase-8 or caspase-9,which cleave and activate other caspases,and downstream effector caspases,including caspase-3,6 and 7,which cleave a variety of cellular substrates thereby disassembling cellular structures or inactivating enzymes.Caspase-3,the 'executioner' protease,is the most intensively studied effector caspase.Study by Vito de Novellis shows that the activation of apoptotic pathways in the rat spinal cord following CCI of sciatic nerve, and that blockade of glutamate mGlu5 receptors prevents early over-expression of pro-apoptotic genes and morphological changes in dorsal horn.But results by Erika suggest that a significant loss of neurons from the dorsal horn is not necessary for the development of tactile allodynia in the SNI model.Thus,whether apoptosis signaling pathways contribute to pain associated with peripheral neuropathy remain to confirm.
Several neuropathies are associated with mitochondrial dysfunction,which is well known to contribute to apoptosis.Mitochondria-dependent apoptosis is activated by a number of factors,reactive oxygen species(ROS),ceramide and nitric oxide,which have been implicated in the pathophysiology of neuropathies.These factors cause the release of cytochrome C from mitochondria and the subsequent formation of a complex of cytochrome C,apoptotic protease activity factor-1 and caspase-9,which initiates the cleavage and activation of downstream /effector caspases.The oxidative stress can damage tissue,proteins,DNA,or lipids.ROS act as key agents in the events leading to caspase activation,mitochondrial dysfunction,which are well known to contribute to apoptosis.It has been shown that reactive oxygen species are involved in NMDA receptor activation,and that apoptosis in rats of chronic constriction injury (CCI) model is glutamate-dependent.Reciprocally,broad-spectrum of anti-apoptotic proteins such as BCL-2 have been described to have antioxidant function by maintaining cells in a more reduced state by scavenging ROS either directly or by up-regulating other ROS scavengers such as thiol compounds and antioxidant enzymes,further indicating that ROS generation is one of the crucial initiation step for apoptotic event.ROS and apoptosis involved in neuropathic pain suggest the connection between both of them.Since ROS scavengers are able to reduce pain and ROS are important steps in the induction of apoptosis,it is reasonable to hypothesize that ROS may rapidly induce over-expression of apoptotic genes in the spinal cord and play an important role in pain development.
The tumor necrosis factor(TNF) family of receptors also activates apoptosis,and TNF-a, acting on sensory neurons contributes to neuropathic pain as well as playing an important role in inflammatory diseases Ceramide,a second messenger in TNF-a signaling enhances excitability of sensory neurons.TNF-a exerts its pleiotropic and cell-dependent effects by activating multiple cellular signal transduction pathways Ligation and trimerization of TNF-R1 lead to recruitment of several adapter signaling proteins,among them Fas-associated death domain protein(FADD).FADD recruits procaspase-8 molecules and is responsible for activation of the self amplifying caspase cascade,an effect that is necessary for the process of apoptosis.The ability of caspase inhibitors to antagonize the hyperalgesia induced by TNF-a via the type-ⅠTNF death receptor on primary afferents was also studied.Inhibition of the synthesis of ceramide,a downstream second messenger in TNF-a signaling that contributes to both apoptosis and TNF-a hyperalgesia,also produces hyperalgesia that is inhibited markedly by all caspase inhibitors.Thus,we hypothesis that caspase signaling pathways contribute to pain-related behavior may induced by TNF-a.
Another question to be considered is that why allodynia remains after neurons apoptosis. Sprouting as a result of injury is accompanied by a strong glial response.We assumed that sprouting may act as a mechanism after neurons apoptosis in neuropathic pain.The evidence suggests that activated astrocytes play important and direct roles in synapse elimination and in the processes mediating collateral reinnervation and sprouting.They undergo activation close to axotomized motoneuron perikarya,where synapse displacement occurs,but not adjacent to axotomized intrinsic central nervous system neurons,where synapse displacement also occurs. Microglia are also rapidly activated around central primary sensory terminals of peripherally axotomized sensory ganglion cells.Nerve fibers growing from the spinal cord explant carry at their tips immature mitotic astrocytic cells that lead their growth cones after injury.These activated glial cells might be involved in lesion-induced plasticity in at least two ways:(1) by phagocytosis of axonal debris,because myelin sheaths act as obstacles for sprouting fibers in the central nervous system and(2) by releasing cytokines and growth factors which regulate layer-specific sprouting The two cytokines interleukin-1(IL-1) and interleukin-6(IL-6) are important modulators of the glia response.Data demonstrates their role in regulating the sprouting of neurons and the associated glia response as a means to examine the role of glia in sprouting.IL-1 plays a role in modulating glia proliferation and thereby guidance and trophic factors for new fibers,whereas IL-6 may be important in triggering the outgrowth of new fibers.During or after apoptosis,the heavily myelinated,large-diameter Aa-fibers or sympathetic fibers may sprout into the superficial dorsal horn including laminaeⅠandⅡ.The lightly myelinated,medium diameter and high-threshold Ad-fibers or sympathetic fibers replaced by low-threshold Aa-fibers may result in allodynia.
Consideration of the role of caspase-3 in neuropathic pain,the inhibition of caspase-3 may probably develop a new of pain treatment.RNAi has been used to inhibit gene expression from clinically relevant transcripts,and studies in suitable preclinical models are beginning. RNAi primarily acts within the cytoplasmic compartment,which is easier to access using nonviral methods than the nucleus,but ensuring efficient uptake and long-term stability in vivo is still likely to be difficult.In order to long-term stability and facilitate the entry of siRNA in cells,tissue and animal models that are difficult to transfect,adenovirus have recently been employed.Adenoviruses are popular gene delivery vehicles because they efficiently transduce many different cell types,including terminally differentiated cells. Adenoviruses are well suited for gene therapy because they do not integrate to the host genome,and they have a high level of RNA expression that is independent of the cell cycle, making their use for both dividing and non-dividing cells possible.Adenoviral vectors,which are able to transduce exogenous sequences into quiescent cells and sustain their long-term expression,suppressed humanβ-glucuronidase,a neurotoxic polyglutamine disease protein,in vitro and in vivo.In present study,the Ad carrying siRNA of caspase-3 is about to construct and its effect on hyperalgesia in CCI rats is about to observe.
Methods:
1.Neurons apoptosis in spinal cord of CCI rats
(1) Rats were randomly divided into two groups:the sham group,CCI group.Thermal hyperalgesia was assessed at 1,3,7,10,14 days after surgery.Caspase-3 expression was measured by immunofluorescence.Neurons apoptosis were detected with TUNEL method.
(2)Caspase 3 inhibitor Z-DEVD-FMK or broad-spectrum caspase inhibitor Z-VAD-FMK on CCI rats
Z-DEVD-FMK and broad-spectrum caspase inhibitor Z-VAD-FMK 1U per day were administered intradermally on the dorsal surface of the hind paw during surgery and 1,2,3,4 days after surgery,respectly.Thermal hyperalgesia test was performed 1,3,7,10,14 days after surgery.Caspase-3 expression was measured by immunofluorescence.Neurons apoptosis were detected with TUNEL method.
2.The role of SOD and TNF-αin apoptosis and Anti-oxidative effect of N-acetyl-L-cysteine in apoptosis and nociceptive response of CCI rats
(1) Neuropathic pain was produced by ligation of right sciatic nerve according to the technique described by Bermet and Xie.Rats were randomly divided into three groups:a sham group Intraperitoneal normal saline,i.p.NS),a CCI group(CCI+ i.p.NS),and a NAC group (CCI+ i.p.NAC).Effect of intraperitoneally administered NAC 300 mg/kg per day in rats was investigated using nociceptive behavioral tests.
(2) Caspase-3 expression by immunohistochemistry and apoptotic neurons detected by TUNEL technique were investigated.SOD and TNF-αexpression were also measured by ELISA method.Pain threshold to thermal stimulation of the right paw was measured to evaluate the effects of NAC.
3.Inhibition of caspase pathway and glial activation on sprouting in CCI rats
(1) Z-DEVD-FMK and broad-spectrum caspase inhibitor Z-VAD-FMK 1U per day were administered intradermally on the dorsal surface of the hind paw during surgery and 1,2,3,4 days after surgery,respectly.Thermal hyperalgesia test was performed at 1 day before and 1,3, 7,10,14 days after surgery.Caspase-3 expression was measured by immunofluorescence. Neurons apoptosis were detected with TUNEL method.GAP-43 expression was measured by immunofluorescence and western blotting.
(2) Propentofylline 40μg were administered intrathecally during surgery and 1,3,5 days after surgery.Thermal hyperalgesia test was performed at 1 day before and 1,3,7,10,14 days after surgery.GFAP and IL-1 expression was observed by immunofluorescence and ELISA method,respectly.caspase-3 expression was measured by immunofluorescence. Neurons apoptosis were detected with TUNEL method.GAP-43 expression was measured by immunofluorescence and western blotting.
4.RNA interference of caspase-3 on hyperalgesia in CCI rats
(1) Screening siRNA effective sequences of caspase-3 in vitro
Gene of rat caspase-3 was cloned and inserted to pEGFP-C1 to construct a reporter plasmid,pEGFP-Cas3.Then,pEGFP-Cas3 and three different plasmids pShuttleH1-siCas3 expression siRNA of caspase-3 were transfected into HEK-293 cells mediated by Lipofectamine2000.The cells were observed under inverted fluorescence microscope and flow cyometer to detect the suppression effect of all siRNAs on EGFP expression.
(3) Gene knockdown with intrathecal injection of siRNA of caspase-3 of the recombinant adenovirus Ad-SiCas3 in CCI rats
The effective siRNA of caspase-3 with 20μg per day were administered intrathecally for 7 days starting 1 day before surgery.The Ad-SiCas3 was administered intrathecally after surgery.The expression of caspase-3 mRNA and protein were assessed by real-time PCR and Western blotting.Thermal hyperalgesia test was performed at 1 day before and 1,3,7,10,14 days after surgery.
Results:
1.Neurons apoptosis in spinal cord of CCI rats
(1) PWL of right paw was decreased after Cither was a significant difference between right paw and the left paw(P＜0.05).TUNEL-positive neurons and caspase-3 expression in the dorsal horn of spinal cord were increased significantly.
(2) The reporter vector pEGFP-Cas3 and three different plasmid pShuttleH1-siCas3 were successfully constructed.After contrasfection,EGFP expression was inhibited by three plasmids observed by inverted microscope and under flow cytometry.The interference efficacy of pShuttleH1-siCas3Ⅱwas better.
(3) PWLs were increased after injection of siRNA.There was a significant difference in terms of PWL between the siRNA-Cas group and the NS group(P＜0.05).The caspase-3 expression in spinal cord was decreased detected by real time PCR and western blotting.
(4) After Z-DEVD-FMK or Z-VAD-FMK injection,caspase-3 expression was decreased and PWLs were increased.There was a significant difference compared to the NS group(P＜0.05) and no significant difference compared to the Sham group(P＞0.05).After injection was terminated,there was a significant difference compared to the Sham group (P＞0.05) and no significant difference compared to the NS group(P＜0.05).
2.Anti-oxidative effect of N-acetyl-L-cysteine decrease spinal apoptosis and nociceptive response in CCI rats
(1) CCI markedly induced up-regulation of TNF-a in spinal cord(P＜0.05 VS CCI group).SOD in spinal cord was decreased.There was a significant difference compared to the CCI group at 7d after surgery(P＜0.05) and no significant difference at other days(P＞0.05).
(2) Compared to NS group,TUNEL-positive neurons and caspase-3 expression were decreased significantly after NAC injection(P＜0.05).The right paw-withdrawal latencies (PWLs) were significantly increased compared to NS groups(P＜0.05).
3.Inhibition of caspase pathway and glial activation on sprouting in CCI rats
(1) After Z-DEVD-FMK or Z-VAD-FMK injection,caspase-3 expression was decreased and PWLs were increased.There was a significant difference compared to the NS group(P＜0.05) and no significant difference compared to the Sham group(P＞0.05).After injection was terminated,there was a significant difference compared to the Sham group (P＞0.05) and no significant difference compared to the NS group(P＜0.05).The GAP-43 expression was decreased(P＜0.05 VS NS group).
(2) Compared to NS group,PWLs were increased significantly after propentofylline injection(P＜0.05).GFAP expression were decreased significantly after propentofylline injection(P＜0.05 VS NS group).The GAP-43 and IL-1expression was decreased(P＜0.05 VS NS group).
4.RNA interference of caspase-3 on hyperalgesia in CCI rats
(1) The reporter vector pEGFP-Cas3 and three different plasmid pShuttleH1-siCas3 were successfully constructed.After cotransfection,EGFP expression was inhibited by three plasmids observed by inverted microscope and under flow cytometry.The interference efficacy of pShuttleH1-siCas3Ⅲwas better.
(2) Neurons apoptosis and caspase-3 mRNA were greatly reduced after virus infection.
(3) PWLs were increased after injection of siRNA or Ad-SiCas.The anti-hyperalgesia effect of Ad-SiCas last for 28 days.The caspase-3 mRNA and its protein expression in spinal cord were decreased detected by real time PCR and western blotting.
Conclusions:
1.The expression of caspase-3 and apoptosis increase in the dorsal horn of spinal cord after constriction of sciative nerve.Apoptosis and hyperalgesia decrease through the inhibition of caspase-3 pharmaceutic blocker.Apoptosis and hyperalgesia are also inhibited by non-selected caspase blocker.Cell apoptosis mediated by caspase-3 are probably responsible for neuropathic pain in CCI rats.
2.Up-regulation of TNF-αand increasing of ROS resulted from down-regulation of SOD may involve in apoptosis in spinal cord of CCI rats.This study identifies that NAC inhibits neural apoptosis in the spinal cord dorsal horn and alleviates thermal hyperalgesia induced by CCI.NAC may be a potential candidate for alleviation of neuropathic pain.
3.Sprouting marked by GAP-43 and hyperalgesia measured by PWL are inhibited by caspase inhibitor or glial modulating agent.During or after apoptosis in the models of CCI rats, sprouting may probably play a role in the development of persistent neuropathic pain states. Astrocytes activation or IL-1 up-regulation may contribute to the development of sprouting.
4.Thermal hyperalgesia were inhibited by caspase-3 siRNA and Ad-SiCas3,respectly. Ad-SiCas3 may probably be used in the treatment of chronic pain.
慢性疼痛被广泛地定义为急性组织损伤修复后疼痛超过1个月、疼痛持续或反复发作超过3个月以上或与组织损伤有关的疼痛预计持续存在或加重。慢性疼痛近来逐年增加,已经成为一个主要的社会、经济负担,全世界有超过3亿2千万的人受其影响。疼痛患者通常分为以下几类:术后疼痛、癌痛、后背痛、HIV疼痛、神经病理性疼痛、关节痛和偏头痛。
依原始刺激的时程和自然特性,疼痛分为3个不同时期,第一期:急性疼痛期,是对简单、短暂的伤害性刺激物的处理过程,是单纯、直接由外周向丘脑及皮层中枢的传递过程。外周感受器的放电与主观疼痛的感知之间有着密切的联系。第二期:炎症性状态期,由持续长时间伤害性刺激所形成,导致组织损伤和外周的炎症反应,机体出现自发性疼痛、痛觉过敏以及超敏反应(将正常的非伤害性刺激认为疼痛)。第三期:神经病理性状态期,为外周神经或CNS损害的结果,包括外周的神经病理状态和中枢的疼痛状态,此时的疼痛与原发的损伤无关。尤其要指出的是这3个时期之间并不排斥,在某些特定的神经病理状态下可以共存。
神经病理性疼痛以自发性疼痛、痛觉过敏以及超敏反应为特点,通常由外周神经、背根神经节、脊髓或脑的损伤所致,包括了疼痛的第二、第三期。神经病理性疼痛目前仍然是一个棘手的临床问题,主要是对其病理过程了解不全。近年来,有研究表明,凋亡对于细胞损伤及神经病理性疼痛具有重要意义。形态学的研究,在神经病理性疼痛的大鼠脊髓浅层及在外周神经损伤的脊髓上同时存在细胞凋亡的现象。在神经病理性疼痛的大鼠上,bcl-2家族基因表达的改变可调节细胞死亡。我们推测在细胞死亡前有较长的一段凋亡期,但是否是凋亡相关基因在脊髓的表达导致神经病理性疼痛的发生目前仍不清楚。
细胞凋亡(apoptosis)是一个细胞自我破坏的程序性生化过程,形态学特征是细胞核和细胞质发生浓缩,在多种的疾病中都起重要作用。凋亡的中心环节是蛋白裂解系统,包括半胱氨酰蛋白酶家族,称半胱氨酸天冬氨酸蛋白酶(caspase)。caspases分为两类:一类是上游启动者(initiators)caspases,如caspase8、9,可裂解并激活其它caspase;另一类是下游效应者(effectors)caspases,主要包括caspase-3、6、7,可裂解各种底物,分解细胞结构或使酶失活。而caspase-3作为“刽子手”蛋白酶,在其中起关键作用。Vitode Novellis等的研究发现大鼠坐骨神经结扎后凋亡通路激活,阻断谷氨酸mGlu5受体可防止凋亡基因在脊髓的过表达。但Erika的研究却指出在SNI模型中,脊髓背角神经元的死亡并不是机械性超敏反应所必需的。因此,凋亡通路是否参与外周神经病变后的疼痛还需进一步证实。
某些神经病理性疾病与线粒体功能障碍有关,而线粒体功能障碍与凋亡关系密切。线粒体依赖性的凋亡可被许多因素激活,如活性氧(reactive oxygen species,ROS)、神经酰胺、一氧化氮。这些因素可导致线粒体释放细胞色素C,而后形成细胞色素C、凋亡蛋白酶激活因子1和caspase-9复合体,从而裂解并激活下游的效应者caspases。氧化应激可损伤组织、蛋白、DNA和脂质。ROS是激活caspase,引起线粒体功能障碍的关键因素,ROS参与NMDA受体激活,而大鼠CCI模型上凋亡的发生又是谷氨酸依赖性,这就建立起ROS与凋亡的联系。反之,广谱的抗凋亡蛋白如BCL-2又有抗氧化作用,通过直接清除ROS或上调其它ROS清除剂如硫醇复合物或抗氧化蛋白酶,这也进一步证明ROS的产生是启动凋亡的关键之一。ROS和凋亡都同神经病理性疼痛有关表明两者之间具有某种联系。由于ROS清除剂可缓解疼痛,而且ROS是诱导凋亡的重要步骤,有理由推测ROS可快速诱导脊髓凋亡基因的过表达,并发挥其在疼痛的发展中的重要作用。
肿瘤坏死因子(tumour necrosis factor,TNF)家族受体同样可诱导凋亡。TNF-a作用于感觉神经元,在炎症性疼痛中起重要作用;神经酰胺,作为TNF-a的第二信使,可增强神经元的兴奋性。TNF-a可通过多种不同方式激活细胞内的多种信号转导通路。连接并聚合TNF-R1可导致某些adapter信号蛋白的聚集,其中之一即Fas相关的死亡区域蛋白(Fas-associated death domain protein,FADD)。FADD可聚集前caspase-8分子并激活可自我放大的caspase级联反应,该效应为细胞凋亡的必需环节。研究表明,抑制caspase可减轻TNF-a诱导的痛觉过敏;抑制TNF-a的第二信使神经酰胺,可同时抑制痛觉过敏和细胞凋亡。因此,我们有理由假设,caspase信号转导通路所引起的痛觉相关的行为学改变可能是由TNF-a所诱导的。
当然,还要一个问题需要解决,那就是神经元凋亡后为何超敏反应还持续存在,因为众所周知,痛觉信号是由神经元传递。芽生(sprouting),是神经损伤的结果,通常伴有强烈的胶质细胞反应。在此,我们假设芽生可能是神经元凋亡后神经病理性疼痛持续存在的机制。有证据表明,激活的星形胶质细胞在清除损伤突触、重新连接神经断端及芽生中起重要作用。在神经元的断端,星形胶质细胞激活,突触发生替换,同时,在与断端不相邻的远处的中枢神经系统的神经元也发生了替换。在中枢初级传入神经元的周围胶质细胞迅速激活。神经纤维在分裂的星形胶质细胞的传送下,在脊髓内生长,形成生长区带。研究表明,激活的星形胶质细胞至少可以通过两种方式促进芽生:①通过清除轴突碎片,减少髓鞘对芽生的阻碍而促进芽生;②通过释放细胞因子和生长因子调控芽生。研究表明,两种细胞因子IL-1和IL-6可调节胶质细胞的反应,并在调节神经元芽生中具有重要作用。IL-1可调节胶质细胞增殖,引导营养因子作用于新生的神经纤维。而IL-6则诱发新生纤维向外继续生长。因此,推测在凋亡时或凋亡后,大直径、厚髓、低阈值的Aa纤维或交感神经纤维可能芽生入脊髓浅层,包括Ⅰ和Ⅱ层。中小直径的、薄髓、高阈值的Ad纤维可能被低阈值Aa纤维或交感神经纤维替代,导致超敏反应的发生。
鉴于caspase-3在神经病理性疼痛中的作用,阻断该通路可能成为治疗疼痛的新方法。RNA干扰是一种抑制基因表达的新方法,一系列相关的临床前的研究都在开展。RNA干扰主要细胞浆中执行,采用非病毒载体易于达到该目的,但是要被细胞有效的摄入并实现长期、稳定的表达是比较困难的。目前更倾向于在哺乳动物细胞内表达siRNA,以维持长久的RNA干扰效应。而利用腺病毒表达干扰RNA,因其在体内的存在时间更长,而且,腺病毒的转染效率比单纯的siRNA或质粒介导的表达载体的转染效率要高得多,可大大增强体内实验的干扰效率。腺病毒的宿主范围广,可将外源基因转入静止的非分裂的细胞。有学者利用腺病毒介导小RNA在体内外实验都可抑制葡苷酸酶,说明腺病毒介导RNA的表达有一定的可行性。因此,在本研究中,我们将构建表达caspase-3小RNA的腺病毒,并观察其对CCI大鼠痛觉过敏反应的影响。
方法:
1.坐骨神经结扎大鼠脊髓背角细胞凋亡的研究
(1)大鼠分成2组:假手术组(Sham组)和手术组(CCI组)。术后1d、3d、7d、10d、14d不同的时间点测定坐骨神经结扎大鼠的热痛阈,并采用免疫荧光方法测定caspase-3的表达,TUNEL原位末端标记法检测脊髓神经细胞的凋亡。
(2)caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK阻断凋亡通路对CCI的影响。
手术当日及术后在大鼠疼痛同侧的后肢脚底皮内每日1个单位分别注射caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK,连续注射5d,观察比较大鼠热痛阈的变化,同时观察凋亡指标。
2.TNF-α、ROS参与CCI大鼠神经元凋亡的研究
(1)大鼠分成2组:假手术对照组和手术组,手术组随机分为2个亚组,N-乙酰半胱酸组(NAC组)、生理盐水(NS组)。实验前禁食12h,自由饮水。NAC组于麻醉后、结扎前腹腔注射NAC 300 mg/kg,而后每日注射NAC 300 mg/kg,生理盐水组注射等量生理盐水。
(2)术后1d、3d、7d、10d、14d不同的时间点测定坐骨神经结扎大鼠的热痛阈,采用ELISA检测脊髓TNF-α的表达、并对脊髓SOD表达进行测定。同时采用免疫荧光方法测定caspase-3的表达,TUNEL原位末端标记法检测脊髓神经细胞的凋亡。
3.CCI大鼠脊髓背角神经元凋亡、星形胶质细胞激活促进芽生形成介导神经病理性疼痛的研究。
(1)caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK阻断通路。术后在大鼠疼痛同侧的后肢脚底皮内隔日注射caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK,观察比较大鼠热痛阈的变化,同时观察凋亡指标。用westernblotting及免疫荧光方法测定GAP-43的表达,观察芽生情况。
(2)用药物丙戊茶碱抑制星形胶质细胞激活。术后在大鼠疼痛鞘内每日注射胶质细胞抑制剂丙戊茶碱,用GFAP抗体进行免疫组化,观察星形胶质细胞激活,并用ELISA方法检测IL-1β的表达,同时观察疼痛及测定GAP-43的表达。
4.腺病毒介导的小RNA干扰caspase-3对CCI大鼠的抗伤害性刺激作用
(1)体外筛选caspase-3小RNA的有效序列。
应用经典的克隆方法,首先克隆大鼠caspase-3基因,将caspase-3基因与含EGFP的质粒pEGFP-C1重组,构建报告载体pEGFP-Cas3,对3个不同表达caspase-3小RNA的质粒pShuttleH1-siCas3进行筛选,通过Lipofectamine2000将pShuttleH1-siCas3及体pEGFP-Cas3共转染HEK-293细胞株,倒置相差显微镜和流式细胞仪观察EGFP的荧光强度。
(2)采用分子克隆的方法构建表达caspase-3小RNA的腺病毒,并观察其对体外培养神经元凋亡的影响。
(3)caspase-3小RNA及重组腺病Ad-SiCas3鞘内注射对CCI大鼠热痛阈的影响。
caspase-3小RNA在术前1d开始鞘内注射,20μg/d,连续7d;重组腺病毒Ad-SiCas3单次注射,采用real time PCR及western blotting的方法检测caspase-3 mRNA及其蛋白。并观察比较大鼠热痛阈的变化。
结果:
1.坐骨神经结扎大鼠脊髓背角细胞凋亡的研究
(1)大鼠坐骨神经结扎后,右足热痛阈明显降低,与左足相比,具有明显的差异(P＜0.05),免疫荧光显示结扎侧脊髓背角caspase-3的表达明显高于未结扎侧,TUNEL法显示结扎侧脊髓背角凋亡的细胞明显高于未结扎侧。
(2)caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK注射后,caspase-3的表达明显降低,大鼠热痛阈明显增高,与NS组相比差异显著(P＜0.05),与Sham组相比无显著差异(P＞0.C5)。停止注射后,大鼠热痛阈降低,与NS组相比差异显著(P＜0.05),与CCI组相比无显著差异(P＞0.05)。
2.TNF-α、ROS参与CCI大鼠神经元凋亡的研究。
(1)坐骨神经结扎后脊髓TNF-α的表达增加,与CCI组相比差异显著(P＜0.05)。脊髓SOD表达下降,在第7天上与CCI组相比差异显著(P＜0.05),其它时间点无显著差异(P＞0.05)。
(2)NAC组脊髓caspase-3的表达及TUNEL阳性神经元数与NS组相比,明显下降(P＜0.05),在施加干预后,NAC组右足热痛阈明显升高,而NS组无明显变化,两者比较有统计学意义(P＜0.05)。
3.CCI大鼠脊髓背角神经元凋亡、星形胶质细胞激活促进芽生形成介导神经病理性疼痛的研究。
(1)caspase-3抑制剂Z-DEVD-FMK及caspase家族抑制剂Z-VAD-FMK注射后,caspase-3的表达明显降低,大鼠热痛阈明显增高,与NS组相比差异显著(P＜0.05),与Sham组相比无显著差异(P＞0.05)。停止注射后,大鼠热痛阈降低,与NS组相比差异显著(P＜0.05),与CCI组相比无显著差异(P＞0.05)。GAP-43的表达明显降低(P＜0.05)。
(2)注射后丙戊茶碱,大鼠热痛阈明显增高,GFAP的表达明显下降,与NS组相比差异显著(P＜0.05)。IL-1及GAP-43的表达明显下降与NS组相比差异显著(P＜0.05)。
4.腺病毒介导的小RNA干扰caspase-3对CCI大鼠的抗伤害性刺激作用
(1)成功构建报告载体pEGFP-Cas3及3个质粒pShuttleH1-siCas3。共转染HEK-293细胞株,倒置相差显微镜和流式细胞仪均可见EGFP的荧光强度被明显抑制,其中,以pShuttleH1-siCas3Ⅱ的干扰效果最佳。
(2)Ad-siCas3能够较好抑制caspase-3 mRNA的表达,抑制内毒素诱导的神经元细胞凋亡。
(3)caspase-3小RNA或Ad-siCas3鞘内注射后大鼠热痛阈明显增高(P＜0.05),caspase-3 mRNA及其蛋白的表达下降(P＜0.05)。
结论:
1.大鼠坐骨神经结扎后,脊髓背角caspase-3高表达,细胞发生凋亡,阻断caspase-3通路均可抑制凋亡,减轻痛觉过敏;非特异性阻断caspase通路均亦可抑制凋亡,减轻痛觉过敏。坐骨神经结扎引起的神经病理性疼痛可能与caspase-3通路介导的细胞凋亡有关。
2.大鼠坐骨神经结扎后,脊髓背角细胞凋亡的发生可能与TNF-α表达增加,SOD下降导致ROS增多有关。抗氧化剂N-已酰半胱氨酸具有对抗大鼠神经元凋亡效应,减轻痛觉过敏。N-已酰半胱氨酸抗氧化可能将成为一种新的缓解病理性疼痛的方法。
3.阻断凋亡caspase通路,抑制星形胶质细胞激活,均可抑制GAP-43的表达,同时减轻痛觉过敏。神经元凋亡时或凋亡后,芽生可能介导神经病理性疼痛的产生,星形胶质细胞的激活,IL-1的升高有助于芽生的发展。
4.caspase-3的小RNA鞘内注射可在短期内减轻CCI大鼠热痛觉过敏;重组腺病毒Ad-SiCas3可持续抑制热痛觉过敏。
Objective and Background:
Chronic pain is defined widely as the suffering which continues for more than one month after tissue lesion,the pain continues or recurrent attacks for more than three months and the pain related to tissue lesion exists continually or aggravate in anticipation.Chronic pain is increasingly and rightfully being recognized as a major socio-economic burden,estimated to be affecting＞320 million people worldwide.The patient population is broadly divided into the following sectors:postoperative pain,cancer pain,back pain,HIV pain,neuropathies, arthritic pain and migraine.
There are three major stages or phases of pain and proposed that different neurophysiologic mechanisms are involved,depending on the nature and time course of the originating stimulus.These three phases are(1) acute pain phase,the processing of a brief noxious stimulus;(2) inflammatory pain phase,the consequences of prolonged noxious stimulation,leading to tissue damage and peripheral inflammation;and(3) neuropathies pain phase,the consequences of neurological damage,including peripheral neuropathies and central pain states.However,it is important to point out that these phases are not exclusive, and that at any given time several of the neurophysiologic mechanisms that underlie these pain states may coexist in the same individual.
Neuropathic pain,characterized by hyperalgesia,allodynia and spontaneous pain,often occurs as a result of injuries to the peripheral nerve,dorsal root ganglion(DRG),spinal cord or brain.Neuropathic pain remains a prevalent and persistent clinical problem because of our incomplete understanding of its pathogenesis.In the recent years,studies performed in all neurobiological fields have resulted in important information about apoptosis that are responsible for cell damage and neuropathic pain.Morphological studies have suggested the occurrence of apoptosis in the spinal cord following peripheral nerve,and in the spinal superficial laminae of neuropathic rats,that altered bcl-2 family gene expression may modulate cell death in a model of neuropathic pain.We hypothesized that there may be a prolonged latent phase of apoptosis before cell death.Whether or not changes in the expression of apoptosis-associated genes occur in the spinal cord during the first days leading to the establishment of neuropathic pain syndrome is unknown.
Apoptosis,a morphologically and biochemically defined form of cell death,has been demonstrated to play a role in a variety of diseases.The central component of the apoptotic machinery is a proteolytic system consisting of a family of cysteinyl proteases,termed caspases.Two groups of caspases can be distinguished:upstream initiator caspases such as caspase-8 or caspase-9,which cleave and activate other caspases,and downstream effector caspases,including caspase-3,6 and 7,which cleave a variety of cellular substrates thereby disassembling cellular structures or inactivating enzymes.Caspase-3,the 'executioner' protease,is the most intensively studied effector caspase.Study by Vito de Novellis shows that the activation of apoptotic pathways in the rat spinal cord following CCI of sciatic nerve, and that blockade of glutamate mGlu5 receptors prevents early over-expression of pro-apoptotic genes and morphological changes in dorsal horn.But results by Erika suggest that a significant loss of neurons from the dorsal horn is not necessary for the development of tactile allodynia in the SNI model.Thus,whether apoptosis signaling pathways contribute to pain associated with peripheral neuropathy remain to confirm.
Several neuropathies are associated with mitochondrial dysfunction,which is well known to contribute to apoptosis.Mitochondria-dependent apoptosis is activated by a number of factors,reactive oxygen species(ROS),ceramide and nitric oxide,which have been implicated in the pathophysiology of neuropathies.These factors cause the release of cytochrome C from mitochondria and the subsequent formation of a complex of cytochrome C,apoptotic protease activity factor-1 and caspase-9,which initiates the cleavage and activation of downstream /effector caspases.The oxidative stress can damage tissue,proteins,DNA,or lipids.ROS act as key agents in the events leading to caspase activation,mitochondrial dysfunction,which are well known to contribute to apoptosis.It has been shown that reactive oxygen species are involved in NMDA receptor activation,and that apoptosis in rats of chronic constriction injury (CCI) model is glutamate-dependent.Reciprocally,broad-spectrum of anti-apoptotic proteins such as BCL-2 have been described to have antioxidant function by maintaining cells in a more reduced state by scavenging ROS either directly or by up-regulating other ROS scavengers such as thiol compounds and antioxidant enzymes,further indicating that ROS generation is one of the crucial initiation step for apoptotic event.ROS and apoptosis involved in neuropathic pain suggest the connection between both of them.Since ROS scavengers are able to reduce pain and ROS are important steps in the induction of apoptosis,it is reasonable to hypothesize that ROS may rapidly induce over-expression of apoptotic genes in the spinal cord and play an important role in pain development.
The tumor necrosis factor(TNF) family of receptors also activates apoptosis,and TNF-a, acting on sensory neurons contributes to neuropathic pain as well as playing an important role in inflammatory diseases Ceramide,a second messenger in TNF-a signaling enhances excitability of sensory neurons.TNF-a exerts its pleiotropic and cell-dependent effects by activating multiple cellular signal transduction pathways Ligation and trimerization of TNF-R1 lead to recruitment of several adapter signaling proteins,among them Fas-associated death domain protein(FADD).FADD recruits procaspase-8 molecules and is responsible for activation of the self amplifying caspase cascade,an effect that is necessary for the process of apoptosis.The ability of caspase inhibitors to antagonize the hyperalgesia induced by TNF-a via the type-ⅠTNF death receptor on primary afferents was also studied.Inhibition of the synthesis of ceramide,a downstream second messenger in TNF-a signaling that contributes to both apoptosis and TNF-a hyperalgesia,also produces hyperalgesia that is inhibited markedly by all caspase inhibitors.Thus,we hypothesis that caspase signaling pathways contribute to pain-related behavior may induced by TNF-a.
Another question to be considered is that why allodynia remains after neurons apoptosis. Sprouting as a result of injury is accompanied by a strong glial response.We assumed that sprouting may act as a mechanism after neurons apoptosis in neuropathic pain.The evidence suggests that activated astrocytes play important and direct roles in synapse elimination and in the processes mediating collateral reinnervation and sprouting.They undergo activation close to axotomized motoneuron perikarya,where synapse displacement occurs,but not adjacent to axotomized intrinsic central nervous system neurons,where synapse displacement also occurs. Microglia are also rapidly activated around central primary sensory terminals of peripherally axotomized sensory ganglion cells.Nerve fibers growing from the spinal cord explant carry at their tips immature mitotic astrocytic cells that lead their growth cones after injury.These activated glial cells might be involved in lesion-induced plasticity in at least two ways:(1) by phagocytosis of axonal debris,because myelin sheaths act as obstacles for sprouting fibers in the central nervous system and(2) by releasing cytokines and growth factors which regulate layer-specific sprouting The two cytokines interleukin-1(IL-1) and interleukin-6(IL-6) are important modulators of the glia response.Data demonstrates their role in regulating the sprouting of neurons and the associated glia response as a means to examine the role of glia in sprouting.IL-1 plays a role in modulating glia proliferation and thereby guidance and trophic factors for new fibers,whereas IL-6 may be important in triggering the outgrowth of new fibers.During or after apoptosis,the heavily myelinated,large-diameter Aa-fibers or sympathetic fibers may sprout into the superficial dorsal horn including laminaeⅠandⅡ.The lightly myelinated,medium diameter and high-threshold Ad-fibers or sympathetic fibers replaced by low-threshold Aa-fibers may result in allodynia.
Consideration of the role of caspase-3 in neuropathic pain,the inhibition of caspase-3 may probably develop a new of pain treatment.RNAi has been used to inhibit gene expression from clinically relevant transcripts,and studies in suitable preclinical models are beginning. RNAi primarily acts within the cytoplasmic compartment,which is easier to access using nonviral methods than the nucleus,but ensuring efficient uptake and long-term stability in vivo is still likely to be difficult.In order to long-term stability and facilitate the entry of siRNA in cells,tissue and animal models that are difficult to transfect,adenovirus have recently been employed.Adenoviruses are popular gene delivery vehicles because they efficiently transduce many different cell types,including terminally differentiated cells. Adenoviruses are well suited for gene therapy because they do not integrate to the host genome,and they have a high level of RNA expression that is independent of the cell cycle, making their use for both dividing and non-dividing cells possible.Adenoviral vectors,which are able to transduce exogenous sequences into quiescent cells and sustain their long-term expression,suppressed humanβ-glucuronidase,a neurotoxic polyglutamine disease protein,in vitro and in vivo.In present study,the Ad carrying siRNA of caspase-3 is about to construct and its effect on hyperalgesia in CCI rats is about to observe.
Methods:
1.Neurons apoptosis in spinal cord of CCI rats
(1) Rats were randomly divided into two groups:the sham group,CCI group.Thermal hyperalgesia was assessed at 1,3,7,10,14 days after surgery.Caspase-3 expression was measured by immunofluorescence.Neurons apoptosis were detected with TUNEL method.
(2)Caspase 3 inhibitor Z-DEVD-FMK or broad-spectrum caspase inhibitor Z-VAD-FMK on CCI rats
Z-DEVD-FMK and broad-spectrum caspase inhibitor Z-VAD-FMK 1U per day were administered intradermally on the dorsal surface of the hind paw during surgery and 1,2,3,4 days after surgery,respectly.Thermal hyperalgesia test was performed 1,3,7,10,14 days after surgery.Caspase-3 expression was measured by immunofluorescence.Neurons apoptosis were detected with TUNEL method.
2.The role of SOD and TNF-αin apoptosis and Anti-oxidative effect of N-acetyl-L-cysteine in apoptosis and nociceptive response of CCI rats
(1) Neuropathic pain was produced by ligation of right sciatic nerve according to the technique described by Bermet and Xie.Rats were randomly divided into three groups:a sham group Intraperitoneal normal saline,i.p.NS),a CCI group(CCI+ i.p.NS),and a NAC group (CCI+ i.p.NAC).Effect of intraperitoneally administered NAC 300 mg/kg per day in rats was investigated using nociceptive behavioral tests.
(2) Caspase-3 expression by immunohistochemistry and apoptotic neurons detected by TUNEL technique were investigated.SOD and TNF-αexpression were also measured by ELISA method.Pain threshold to thermal stimulation of the right paw was measured to evaluate the effects of NAC.
3.Inhibition of caspase pathway and glial activation on sprouting in CCI rats
(1) Z-DEVD-FMK and broad-spectrum caspase inhibitor Z-VAD-FMK 1U per day were administered intradermally on the dorsal surface of the hind paw during surgery and 1,2,3,4 days after surgery,respectly.Thermal hyperalgesia test was performed at 1 day before and 1,3, 7,10,14 days after surgery.Caspase-3 expression was measured by immunofluorescence. Neurons apoptosis were detected with TUNEL method.GAP-43 expression was measured by immunofluorescence and western blotting.
(2) Propentofylline 40μg were administered intrathecally during surgery and 1,3,5 days after surgery.Thermal hyperalgesia test was performed at 1 day before and 1,3,7,10,14 days after surgery.GFAP and IL-1 expression was observed by immunofluorescence and ELISA method,respectly.caspase-3 expression was measured by immunofluorescence. Neurons apoptosis were detected with TUNEL method.GAP-43 expression was measured by immunofluorescence and western blotting.
4.RNA interference of caspase-3 on hyperalgesia in CCI rats
(1) Screening siRNA effective sequences of caspase-3 in vitro
Gene of rat caspase-3 was cloned and inserted to pEGFP-C1 to construct a reporter plasmid,pEGFP-Cas3.Then,pEGFP-Cas3 and three different plasmids pShuttleH1-siCas3 expression siRNA of caspase-3 were transfected into HEK-293 cells mediated by Lipofectamine2000.The cells were observed under inverted fluorescence microscope and flow cyometer to detect the suppression effect of all siRNAs on EGFP expression.
(3) Gene knockdown with intrathecal injection of siRNA of caspase-3 of the recombinant adenovirus Ad-SiCas3 in CCI rats
The effective siRNA of caspase-3 with 20μg per day were administered intrathecally for 7 days starting 1 day before surgery.The Ad-SiCas3 was administered intrathecally after surgery.The expression of caspase-3 mRNA and protein were assessed by real-time PCR and Western blotting.Thermal hyperalgesia test was performed at 1 day before and 1,3,7,10,14 days after surgery.
Results:
1.Neurons apoptosis in spinal cord of CCI rats
(1) PWL of right paw was decreased after Cither was a significant difference between right paw and the left paw(P＜0.05).TUNEL-positive neurons and caspase-3 expression in the dorsal horn of spinal cord were increased significantly.
(2) The reporter vector pEGFP-Cas3 and three different plasmid pShuttleH1-siCas3 were successfully constructed.After contrasfection,EGFP expression was inhibited by three plasmids observed by inverted microscope and under flow cytometry.The interference efficacy of pShuttleH1-siCas3Ⅱwas better.
(3) PWLs were increased after injection of siRNA.There was a significant difference in terms of PWL between the siRNA-Cas group and the NS group(P＜0.05).The caspase-3 expression in spinal cord was decreased detected by real time PCR and western blotting.
(4) After Z-DEVD-FMK or Z-VAD-FMK injection,caspase-3 expression was decreased and PWLs were increased.There was a significant difference compared to the NS group(P＜0.05) and no significant difference compared to the Sham group(P＞0.05).After injection was terminated,there was a significant difference compared to the Sham group (P＞0.05) and no significant difference compared to the NS group(P＜0.05).
2.Anti-oxidative effect of N-acetyl-L-cysteine decrease spinal apoptosis and nociceptive response in CCI rats
(1) CCI markedly induced up-regulation of TNF-a in spinal cord(P＜0.05 VS CCI group).SOD in spinal cord was decreased.There was a significant difference compared to the CCI group at 7d after surgery(P＜0.05) and no significant difference at other days(P＞0.05).
(2) Compared to NS group,TUNEL-positive neurons and caspase-3 expression were decreased significantly after NAC injection(P＜0.05).The right paw-withdrawal latencies (PWLs) were significantly increased compared to NS groups(P＜0.05).
3.Inhibition of caspase pathway and glial activation on sprouting in CCI rats
(1) After Z-DEVD-FMK or Z-VAD-FMK injection,caspase-3 expression was decreased and PWLs were increased.There was a significant difference compared to the NS group(P＜0.05) and no significant difference compared to the Sham group(P＞0.05).After injection was terminated,there was a significant difference compared to the Sham group (P＞0.05) and no significant difference compared to the NS group(P＜0.05).The GAP-43 expression was decreased(P＜0.05 VS NS group).
(2) Compared to NS group,PWLs were increased significantly after propentofylline injection(P＜0.05).GFAP expression were decreased significantly after propentofylline injection(P＜0.05 VS NS group).The GAP-43 and IL-1expression was decreased(P＜0.05 VS NS group).
4.RNA interference of caspase-3 on hyperalgesia in CCI rats
(1) The reporter vector pEGFP-Cas3 and three different plasmid pShuttleH1-siCas3 were successfully constructed.After cotransfection,EGFP expression was inhibited by three plasmids observed by inverted microscope and under flow cytometry.The interference efficacy of pShuttleH1-siCas3Ⅲwas better.
(2) Neurons apoptosis and caspase-3 mRNA were greatly reduced after virus infection.
(3) PWLs were increased after injection of siRNA or Ad-SiCas.The anti-hyperalgesia effect of Ad-SiCas last for 28 days.The caspase-3 mRNA and its protein expression in spinal cord were decreased detected by real time PCR and western blotting.
Conclusions:
1.The expression of caspase-3 and apoptosis increase in the dorsal horn of spinal cord after constriction of sciative nerve.Apoptosis and hyperalgesia decrease through the inhibition of caspase-3 pharmaceutic blocker.Apoptosis and hyperalgesia are also inhibited by non-selected caspase blocker.Cell apoptosis mediated by caspase-3 are probably responsible for neuropathic pain in CCI rats.
2.Up-regulation of TNF-αand increasing of ROS resulted from down-regulation of SOD may involve in apoptosis in spinal cord of CCI rats.This study identifies that NAC inhibits neural apoptosis in the spinal cord dorsal horn and alleviates thermal hyperalgesia induced by CCI.NAC may be a potential candidate for alleviation of neuropathic pain.
3.Sprouting marked by GAP-43 and hyperalgesia measured by PWL are inhibited by caspase inhibitor or glial modulating agent.During or after apoptosis in the models of CCI rats, sprouting may probably play a role in the development of persistent neuropathic pain states. Astrocytes activation or IL-1 up-regulation may contribute to the development of sprouting.
4.Thermal hyperalgesia were inhibited by caspase-3 siRNA and Ad-SiCas3,respectly. Ad-SiCas3 may probably be used in the treatment of chronic pain.
引文
[1]吴飞翔,俞卫锋.疼痛治疗靶点:N型及T型钙通道.麻醉与监护论坛,2006,13(6):332-334.
[2]De Novellis V,Siniscalo D,Galderisi U,Fuccio R,Nolano M,Santoro L,Cascino A,Roth KA,Rossi N,Maione S.Blockade of glutamate mGlu5 receptors in a rat model of neuropathic pain prevents early overexpression of pro-apoptotic genes and morphological changes in dorsal horn lamina Ⅱ.Neuropharmacology,2004,46:468-479.
[3]Kimberly A,Moore,Tatsuro Kohno,Laurie A,Karchewski,Joachim Scholz.Partial peripheral nerve injury promotes a selective loss of GABAergic inhibition in the superficial dorsal horn of the spinal cord.The J Neurosci,2002,22(15):6724-6731.
[4]Scholz J,Broom DC,Youn DH,Mills CD,Kohno T,Suter MR,Moore KA,Decosterd I,Coggeshall RE,Woolf CJ.Blocking caspase activity prevents transsynaptic neuronal apoptosis and the loss of inhibition in lamina Ⅱ of the dorsal horn after peripheral nerve injury.J Neurosci,2005,25(32):7317-23.
[5]Hassanzadeh P,Ahmadiani A.Nitric oxide and c-Jun N-terminal kinase are involved in the development of dark neurons induced by inflammatory pain.Synapse,2006,59(2):101-6.
[6]Polgar E, Hughes DI, Arham AZ, Todd AJ. Loss of neurons from laminas Ⅰ-Ⅲ of the spinal dorsal horn is not required for development of tactile allodynia in the spared nerve injury model of neuropathic pain. J Neurosci, 2005, 13, 25(28): 6658-66.
[7]Craig A. Pain mechanisms: labelled lines versus convergence in central processing.Ann Rev Neurosci,2003, 26: 1-30.
[8]Woof CJ, Salter MW. Neuronal plasticity: increasing the gain in pain. Seience, 2000, 288: 1765-1768.
[9]Sindrup SH, Jensen TS. Pharmacologic treatment of pain in polyneuropathy. Neurology, 2000, 55, 915-920.
[10]Bomerc. Apoptosis without caspases: an inefficient molecular guillotine[J]. Cell Death Differ, 1999, 6(6): 497-502.
[11]Miljanich GP. Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. Curr Med Chem, 2004, 11: 3029-40.
[12]MicheauO, TschoppJ. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell, 2003, 114: 181-190.
[13]Schafers, M, Lee DH, Brors D, Yaksh TL, Sorkin LS. Increased sensitivity of injured and adjacent uninjured rat primary sensory neurons to exogenous tumor necrosis factor-alpha after spinal nerve ligation. J Neurosci, 2003, 23: 3028-3038.
[14]Zhang YH, Vasko MR, Nicol GD. Ceramide, a putative second messenger for nerve growth factor, modulates the TTX-resistant Na+ current and delayed rectifier K+ current in rat sensory neurons. J Physiol(Lond), 2002, 544: 385-402.
[15]Cossarizza A, Moyle G. Antiretroviral nucleoside and nucleotide analogues and mitochondria. AIDS, 2004, 18: 137-151.
[16]Mazumder S, Plesca D, Almasan A. caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis.Methods Mol Biol, 2008, 414:13-21.
[17]D'Costa AM, Denning MF.A caspase-resistant mutant of PKC-delta protects keratinocytes from UV-induced apoptosis. Cell Death Differ, 2005, 12(3):224-32.
[18]Logette E, Solary E, Corcos L.Identification of a functional DNA binding site for the SREBP-1c transcription factor in the first intron of the human caspase-2 gene. Biochim Biophys Acta, 2005, 1738(1-3):1-5.
[19]Lee SC, Chan J, Clement MV, Pervaiz S. Functional proteomics of resveratrol-induced colon cancer cell apoptosis: caspase-6-mediated cleavage of lamin A is a major signaling loop.Proteomics, 2006, 6(8):2386-94.
[20]Sgorbissa A,Benetti R,Marzinotto S,Schneider C,Brancolini C.caspase-3 and caspase-7 but not caspase-6 cleave Gas2 in vitro:implications for microfilament reorganization during apoptosis.J Cell Sci,1999,112(Pt23):4475-82.
[21]Shakibaei M,John T,Seifarth C,Mobasheri A.Resveratrol inhibits IL-1 beta-induced stimulation of caspase-3 and cleavage of PARP in human articular chondrocytes in vitro.Ann N Y Acad Sci,2007,1095:554-63.
[22]Related Articles,LinksSlee EA,Adrain C,Martin SJ.Executioner caspase-3,-6,and-7 perform distinct,non-redundant roles during the demolition phase of apoptosis.J Biol Chem,2001,276(10):7320-6.
[23]Joseph EK,Levine JD.caspase signalling in neuropathic and inflammatory pain in the rat.Eur J Neurosci,2004,20(11):2896-902.
[24]傅开元,张非煜,侯波等.福尔马林致炎症疼痛动物脊髓后角神经元凋亡.中国疼痛医学杂志,2005,11(1):25-28.
[25]解小丽,周青山,刘先义等.坐骨神经结扎大鼠脊髓神经元凋亡与痛敏的关系.中国疼痛医学杂志,2006,12(2):98-102.
[26]Campana WM,Myers RR.Exogenous erythropoietin protects against dorsal root ganglion apoptosis and pain following peripheral nerve injury.Eur J Neurosci,2003,18(6):1497-506.
[1]Walczak H,Haas.TLBiochemical analysis of the native TRAIL death-inducing signaling complex.Methods Mol Biol,2008,414:221-39.
[2]Czeschik JC,Hagenacker T,Schafers M,Busselberg D.TNF-alpha differentially modulates ion channels of nociceptive neurons.Neurosci Lett,2008,434(3):293-8.
[3]Yuan B,Ohyama K,Bessho T,Uchide N,Toyoda H.Imbalance between ROS production and elimination results in apoptosis induction in primary smooth chorion trophoblast cells prepared from human fetal membrane tissues.Life Sci,2008,82(111-12):623-30.
[4]Gao X,Kim HK,Chung JM,Chung K.Reactive oxygen species(ROS) are involved in enhancement of NMDA-receptor phosphorylation in animal models of pain.Pain,2007,131(3):262-71.
[5]Wagner R,Myers RR.Endoneurial injection of TNF-alpha produces neuropathic pain behaviors.Neuroreport.1996,7(18):2897-901.
[6]Lindenlaub T,Teuteberg P,Hartung T,Sommer C.Effects of neutralizing antibodies to TNF-alpha on pain-related behavior and nerve regeneration in mice with chronic constriction injury.Brain Res,2000,866(1-2):15-22.
[7]Sommer C,Lindenlaub T,Teuteberg P,Schafers M,Hartung T,Toyka KV.Anti-TNF-neutralizing antibodies reduce pain-related behavior in two different mouse models of painful mononeuropathy.Brain Res, 2001,913(1):86-9.
[8] Yeh WC,Pompa,McCurrach ME, et al.FADD:essential for embryo development an d signaling from some, but not all, inducers of apoptosis.Science, 1998, 279(5358): 1954-1957.
[9]MacFarlane M. TRAIL-induced signalling and apoptosis [J]. Toxicol Lett, 2003, 139(2-3): 89-97.
[10] Philchenkov A.caspases: potential targets for regulating cell death.J Cell Mol Med, 2004, 8(4):432-444.
[11] Chen G, Goeddel DV. TNF-R1 signaling: A beautiful pathway [J]. Science, 2002, 296(5573): 1634-1635.
[12]Wullaert A, Heyninck K, Beyaert R.Mechanisms of crosstalk between TNF-induced NF-kappaB and JNK activation in hepatocytes.Biochem Pharmacol, 2006, 72(9):1090-101.
[13]Niedbala B, Sanchez A, Feria M.Nitric oxide mediatesneuropathie pain behavior in peripherally denervared rats.Neurosci Lett, 1995, 188:57-60.
[14]Rokyta R, Holecek V, Pekarkova I, Krejcova J, Racek J, Trefil L, et al. Free radicals after painful stimulation are influenced by antioxidants and analgesics. Neuro Endocrinol Lett, 2003, 24(5):304-309.
[15]Chung JM. The role of reactive oxygen species (ROS) in persistent pain. Mol Interv 2004, 4(5):248-250.
[16]Hacimuftuoglu A, Handy CR, Goettl VM, Lin CG, Dane S, Stephens RL Jr.Antioxidants attenuate multiple phases of formalin-induced nociceptive response in mice. Behav Brain Res, 2006, 173(2):211-6.
[17]Adraln C, Martin SJ.The mitochondrial apeptosome: A killer unleashed by the cytochrome seas. Trends Biochem Sci, 2001, 26(6):390-397.
[18]Loh KP, Huang SH, De Silva R, Tan BK, Zhu YZ.Oxidative stress: apoptosis in neuronal injury. Curr Alzheimer Res, 2006, 3(4):327-37.
[19]Yoon- DY, Cho YS, Park JW, Kim SH, Kim JW.Up-regulation of reactive oxygen species (ROS) and resistance to Fas-mediated apoptosis in the C33A cervical cancer cell line transfected with IL-18 receptor.Clin Chem Lab Med, 2004, 42(5):499-506.
[20]Salazar M, Pariente JA, Salido GM, Gonzalez A.Ethanol induces glutamate secretion by Ca(2+) mobilization and ROS generation in rat hippocampal astrocytes.Neurochem Int, 2008, 52(6):1061-7.
[21]Sharikabad MN, Ostbye KM, Lyberg T, Br0rs O.Effect of extracellular Mg(2+) on ROS and Ca(2+) accumulation during reoxygenation of rat cardiomyocytes.Am J Physiol Heart Circ Physiol, 2001, 280(1):H344-53.
[22]Li J, Li Q, Xie C, Zhou H, Wang Y, Zhang N, Shao H, Chan SC, Peng X, Lin SC, Han J.Beta-actin is required for mitochondria clustering and ROS generation in TNF-induced, caspase-independent cell death. J Cell Sci,2004,117(Pt 20):4673-80.
[23]Yamada M,Kojima N,Paranjpe A,Att W,Aita H,Jewett A,Ogawa T.N-acetyl Cysteine(NAC)-assisted Detoxification of PMMA Resin.J Dent Res,2008,87(4):372-377.
[24]Spagnuolo G,D'Anto V,Cosentino C,Schmalz G,Schweikl H,Rengo S.Effect of N-acetyl-L-cysteine on ROS production and cell death caused by HEMA in human primary gingival fibroblasts.Biomaterials,2006,27(9):1803-9.
[25]Naik AK,Tandan SK,Dudhgaonkar SP,et al.Role of oxidative stress in pathophysiology of peripheral neuropathy and modulation by N-acetyl-L-cysteine in rats.Eur J Pain,2006,10(7):573-539.
[26]Chae HJ,Chae SW,Kim HR.N-acetyl cysteine regulates TNF-alpha-inhibited differentiation in ROS 17/2.8 osteoblasts.Immunopharmacol Immunotoxicol,2004,26(2):203-13.
[1]Ginsberg SD,Martin LJ.Axonal transection in adult rat brain induced transynaptic apoptosis and persistent atrophy of target neurons.Neurotrauma,2002,19(1):99-109.
[2]Csuka E,Hans VH,Ammann E,et al.Cell activation and inflammatory response following traumatic axonal injury in the rat.Neuroreport,2000,11(11):2587-90
[3]Camand E,Morel MP,Faissner A,et al.Long-term changes in the molecular composition of the glial scar and progressive increase of serotoninergic fibre sprouting after hemisection of the mouse spinal cord.Eur J Neurosci,2004,20(5):1161-76.
[4]Ling Yan and Xie Yi-kuan,Dynamic changes of mechanically activated channels and K+chanells at injury site of peripheral nerve in rat.Science inChina,1996,2:71-79.
[5]Kajander KC,Wakisaka S,Bennett GJ.Spontaneous discharge originates in the dorsal root ganglion at the onset of a painful peripheral neuropathy in the rat.NeurosciLett,1992,138:225-228.
[6]Lehner B,Koeck FX,Capellino S,Schubert TE,Hofbauer R,Straub RH.Preponderance of sensory versus sympathetic nerve fibers and increased cellularity in the infrapatellar fat pad in anterior knee pain patients after primary arthroplasty.J Orthop Res,2008,26(3):342-50.
[7]Jortner BS.The returns of the dark neuron.A histological artifact complicating contemporary neurotoxicologic evaluation.Neurotoxicology,2006,27(4):628-34.
[8]Murata R,Ohtori S,Ochiai N,et al.Extracorporeal shockwaves induce the expression of ATF3 and GAP-43 in rat dorsal root ganglion neurons.Auton Neurosci,2006,128(1-2):96-100.
[9]Macias MY,Syring MB,Pizzi MA,et al.Pain with no gain:allodynia following neural stem cell transplantation in spinal cord injury.Exp Neurol,2006,201(2):335-48.
[10]Kotulska K,LePecheur M,Marcol W,et al.Overexpression of copper/zinc-superoxide dismutase in transgenic mice markedly impairs regeneration and increases development of neuropathic pain after sciatic nerve injury.J Neurosci Res,2006,84(5):1091-7.
[11]Kyungsoon Chunga,Jin Mo Chung.Sympathetic sprouting in the dorsal root ganglion after spinal nerve ligation:evidence of regenerative collateral sprouting.Brain Research,2001,895:204-212.
[12]Znaor L,Lovric S,Hogan Q,et al.Association of neural inflammation with hyperalgesia following spinal nerve ligation.Croat Med J,2007,48(1):35-42.
[13]Obata H,Eisenach JC,Hussain H,et al.Spinal glial activation contributes to postoperative mechanical hypersensitivity in the rat.J Pain,2006,7(11):816-22.
[14]Kuzumaki N,Narita M,Narita M,et al.Chronic pain-induced astrocyte activation in the cingulate cortex with no change in neural or glial differentiation from neural stem cells in mice.Neurosci Lett,2007,415:22-27.
[15]Parish CL,Finkelstein DI,et al.The role of interleukin-1,interleukin-6,and glia in inducing growth of neuronal terminal arbors in mice.J Neurosci,2002,22(18):8034-41.
[16]Sobkowicz HM,Waclawik AJ,August BK.The astroglial cell that guides nerve fibers from growth cone to synapse in organotypic cultures of the fetal mouse spinal cord.Synapse,2006,59(4):183-200.
[17]Bechmann I,Nitsch R.Involvement of non-neuronal cells in entorhinal-hippocampal reorganization following lesions.Ann N Y Acad Sci,2000,911:192-206.
[18]Xiao WH,Bennett GJ.Persistent low-frequency spontaneous discharge in A-fiber and C-fiber primary afferent neurons during an inflammatory pain condition.Anesthesiology,2007,107(5):813-21.
[19]董承海,谢作磊,樊继云,谢益宽.异位电活动促使背根神经节交感节后纤维的芽生.中国科学,2001,31:531-536.
[1]Hassanzadeh P,Ahmadiani A.Nitric oxide and c-Jun N-terminal kinase are involved in the development of dark neurons induced by inflammatory pain.Synapse,2006,59(2):101-106.
[2]Gabriele Dorn,Sadhana Patel1,Glen Wotherspoon.siRNA relieves chronic neuropathic pain.Nucleic Acids Res(2004) 32(5):e49.
[3]Fire A,Xu AQ,Montgomery MK,et al.Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans.Nature,1998,391:806-811.
[4]Lipardi C,Wei Q,Paterson BM.RNAi as random degradative PCR siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs.Cell,2001,107:297-307.
[5]Knight SW,Bass BL.A role for the RNaseⅢ enzyme DICR-1 in RNA interference and germ line development in Caenorhabditis elegans.Science,2001,293(5538):2269-2271.
[6]Sijen T,Fleenor J,Simmer F,et al.On the role of RNA amplification in dsRNA triggered gene silencing.Cell,2001,107:465-476.
[7]Mantyh P,Clohisy D,Koltzenburg M,et al.Molecular mechanisms of cancer pain.Nat Rev,2002,20:201-209.
[8]Hains B,Klein J,Saab C et al.Upregulation of sodium channel Nav1.3 and functional involvement in neuronal hyperexcitability associated with central neuropathic pain after spinal cord injury.J Neurosci,2003,23(26):8881-8892.
[9]Christoph T,Grunweller A,Mika J,Schafer MK,Wade EJ,Weihe E,Erdmann VA,Frank R,Gillen C,Kurreck J.Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo.Biochem Biophys Res Commun,2006,350(1):238-43.
[10]Gabriele Dorn,Sadhana Patel1,Glen Wotherspoon.SiRNA relieves chronic neuropathic pain.Nucleic Acids Res,2004,32(5):e49.
[11]Achenbach T,Brunner B and Heermeier K.Oligonucleotide-based knockdown technologies: antisense versus RNA interference.Chembiochem,2003,4(10):928-935.
[12]Ganju P,Hall J.Potential applications of siRNA for pain therapy.ExpertOpin Biol Ther,2004,4(4):531-542.
[13]吴飞翔,吴军珍,俞卫锋.RNA干扰在慢性疼痛研究分析及治疗中的应用.麻醉与监护论坛,2006,13(5):285-287.
[14]Chiou SH,Chen SJ,Peng CH,et al.Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibites LPS-induced apoptosis in hippocampus-derived neural stem cell.Biochem Biophys Res Commun,2006,343(2):391-400.
[1]Fire A,Xu AQ,Montgomery MK,et al.Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans.Nature,1998,391:806-811.
[2]Lipardi C,Wei Q.Paterson BM.RNAi as random degradative PCR.siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs.Cell,2001,107:297-307.
[3]Moffat J,Sabatini DM.Building mammalian signalling pathways with RNAi screens.Nat Rev Mol Cell Biol,2006,7:177-87.
[4]Sijen T,Fleenor J,Simmer F,et al.On the role of RNA amplification in dsRNA triggered gene silencing.Cell,2001,107:465-476.
[5]Nishikura K.A short primer on RNAi:RNA directed RNA polymerase acts as a key catalyst.Cell,2001,107:415-418.
[6]Craig A.Pain mechanisms:labelled lines versus convergence in central processing.Ann Rev Neurosci,2003,26:1-30.
[7]Woof CJ,Salter MW.Neuronal plasticity:increasing the gain in pain.Seience,2000,288:1765-1768.
[8]Rondinone CM.RNAi:for functional analysis and target validation.Expert Opin Ther Targets,2006,10:337-40.
[9]QinghHa Liu,m A R,Savitha K,et al.a Bridge Between the Initiation and Efector Steps of the Drosophila RNAi Pathway.Science,2003,301(26):1921-192.
[10]Fraser AG,Kamath RS,Zipperlen P,et al.Functional genomic analysis of C.elegans chromosome I by systematic RNA interference.Nature,2000,408:325-330.
[11]Berns K,Hijmans EM,Mullenders J,et al.A large-scale RNAi screen in human cells identifies new components of the p53 pathway.Nature,2004,428:431-437.
[12]何承伟,刘芳等.RNAi技术在大规模基因功能研究中的应用.国外医学,临床生物化学与检验学分册,2005,26:539-541.
[13]Hemmings-Mieszczak M,Dorn G,Natt FJ,et al.Independent combinatorial effect of antisense oligonucleotides and RNAi-mediated specific inhibition of the recombinant rat P2X3 receptor.Nucleic Acids Res,2003,31:2117-2126.
[14]Gabriele Dorn,Sadhana Patel1,Glen Wotherspoon.SiRNA relieves chronic neuropathic pain.Nucleic Acids Res,2004,32:e49.
[15]Grunweller A,Wyszko E,Bieber B.Comparison of different antisense strategies in mammalian cells using locked nucleic acids,2'-O-methyl RNA,phosphorothioates and small interfering RNA.Nucleic Acids Res,2003,31:3185-3193.
[16]Lai J,Porreca F,Hunter J,et al.Voltage-gated sodium channels and hyperalgesia.Ann Rev Pharmacol Toxico,2004,44:371-397.
[17]Mikami M,Yang J.Short hairpin RNA-mediated selective knockdown of NaV1.8tetrodotoxin-resistant voltage-gated sodium channel in dorsal root ganglion neurons.Anesthesiology,2005,103(4):828-36.
[18]Miljanich GP.Ziconotide:neuronal calcium channel blocker for treating severe chronic pain.Curr Med Chem, 2004, 11: 3029-40.
[19]Tan PH, Yang LC, Shih HC, et al.Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat.Gene Ther, 2005, 12: 59-66.
[20] Hains B, Klein J, Saab C, et al.Upregulation of sodium channel Nav1.3 and functional involvement in neuronal hyperexcitability associated with central neuropathic pain after spinal cord injury.J Neurosci, 2003, 23: 8881-8892.
[21] Zhang WB, Zhang Z, Ni YX, et al. A novel function of Goa: Mediation of extracellular signal-regulated kinase activation by opioid receptors in neural cells.J Neurochem.2003, 86: 1213-1222.
[22] Zhang Y, Boado RJ, Pardridge WM.In vivo knockdown of gene expression in brain cancer with intravenous RNAi in adult rats. J Gene Med, 2003, 5: 1039-1045.
1 Coutaux A,Adam F,Willer JC,et al.Hyperalgesia and allodynia:peripheral mechanisms.Joint Bone Spine,2005,72(5):359-371.
2 Attal N,Bouhassira D.Translating basic research on sodium channels in human neuropathic pain.J Pain,2006,7(S1):S31-S37.
3 Casula MA,Facer P,Powell AJ,et al.Expression of the sodium channel β3 subunit in injured human sensory neurons.Neuroreport,2004,15(10):1629-1632.
4 Hains BC,Klein JP,Saab CY,et al.Upregulation of sodium channel Nav1.3 and functional involvement in neuronal hyperexcitability associated with central neuropathic pain after spinal cord injury.J Neurosci,2003,23(26):8881-8892.
5 Black JA,Liu S,Tanaka M,et al.Changes in the expression of tetrodotoxin-sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain.Pain,2004,108(3):237-247.
6 Nassar MA,Stirling LC,Forlani G,et al.Nociceptor-specific gene deletion reveals a major role for Nav1.7(PN1) in acute and inflammatory pain.Proc Natl Acad Sci U S A,2004,101(34):12706-12711.
7 Porreca F,Lai J,Bian D,et al.A comparison of the potential role of the tetrodotoxin-insensitive sodium channels,PN3/SNS and NaN/SNS2,in rat models of chronic pain.Proc Natl Acad Sci U S A,1999,96(14):7640-7644.
8 Lai J,Gold MS,Kim CS,et al.Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel,Nav1.8.Pain,2002,95(1-2):143-152.
9 Baker MD,Chandra SY,Ding Y,et al.GTP-induced tetrodotoxin-resistant Na~+ current regulates excitability in mouse and rat small diameter sensory neurones.J Physiol,2003,548(2):373-382.
10 Bourinet E,Zamponi GW.Voltage gated calcium channels as targets for analgesics.Curr Top Med Chem,2005,5(6):539-546.
11 Bourinet E,Alloui A,Monteil A,et al.Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception.EMBO J,2005,24(2):315-324.
12 Pareek TK,Keller J,Kesavapany S,et al.Cyclin-dependent kinase 5 activity regulates pain signaling.Proc Natl Acad Sci,2006,103(3):791-796.
13 Song XS,Cao JL,Xu YB,et al.Activation of ERK/CREB pathway in spinal cord contributes to chronic constrictive injury-induced neuropathic pain in rats.Acta Pharmacol Sin,2005,26:789-98.
14 Sweitzera SM,Petersa MC,Mab JY,et al.Peripheral and central p38MAPK mediates capsaicin-induced hyperalgesia.Pain,2004,111:278-285.
15 Jin SX. Zhuang ZY, Woolf CB, et al. P38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J Neurosci, 2003, 23: 4017-4022.
16 Doya H, Ohtori S, Fujitani M, et al. C-Jun N-terminal kinase activation in dorsal root ganglion contributes to pain hypersensitivity. Biochem Biophys Res Commun, 2005, 335: 132-138.
17 Hassanzadeh P, Ahmadiani A. Nitric oxide and c-Jun N-terminal kinase are involved in the development of dark neurons induced by inflammatory pain. Synapse, 2006, 59: 101-106.
18 Ji RR, Rupp F. Phosphorylation of transcription factor CREB in rat spinal cord after formalin-induced hyperalgesia: relationship to c-fos induction. J Neurosci, 1997, 17:1776-1785.
19 Yasuhiko Kawasaki, Tatsuro Kohno, Zhi-Ye Zhuang. Ionotropic and metabotropic receptors, protein kinase A, protein kinase C, and Src contribute to C-fiber-induced ERK activation and cAMP response element-binding protein phosphorylation in dorsal horn neurons, leading to central sensitization. J Neurosci, 2004, 24:8310-8321.
20 Ru-Rong Ji, Clifford J. Neuronal Plasticity and Signal Transduction in Nociceptive Neurons: Implications for the Initiation and Maintenance of Pathological Pain. Neurobiology of Disease, 2001, 8:1-10.
21 Bomerc. Apoptosis without caspases: an inefficient molecular guillotine. Cell Death Differ, 1999, 6(6): 497-502.
22 De Novellis V, Siniscalo D, Galderisi U, et al. Blockade of glutamate mGlu5 receptors in a rat model of neuropathic pain prevents early overexpression of pro-apoptotic genes and morphological changes in dorsal horn lamina II. Neuropharmacology, 2004, 46: 468-479.
23 Joseph EK, Levine JD. caspase signalling in neuropathic and inflammatory pain in the rat. Eur J Neurosci, 2004, 20(11): 2896-2902.
24 Kimberly A, Moore, Tatsuro Kohno, et al. Partial peripheral nerve injury promotes a selective loss of GABAergic inhibition in the superficial dorsal horn of the spinal cord. The J Neurosci, 2002, 22(15): 6724-6731.
25 Scholz J, Broom DC, Youn DH, et al. Blocking caspase activity prevents transsynaptic neuronal apoptosis and the loss of inhibition in lamina II of the dorsal horn after peripheral nerve injury. J Neurosci, 2005, 25(32): 7317-7323.
26 Polgar E, Hughes DI, Arham AZ, Todd AJ. Loss of neurons from laminas Ⅰ-Ⅲ of the spinal dorsal horn is not required for development of tactile allodynia in the spared nerve injury model of neuropathic pain. J Neurosci, 2005, 13, 25(28): 6658-6666.
27 Campana WM, Myers RR. Exogenous erythropoietin protects against dorsal root ganglion apoptosis and pain following peripheral nerve injury.Eur J Neurosci,2003,18(6):1497-1506.
28 Tanga FY,Nutile-McMenemy N,DeLeo.The CNS role of toll-like receptor 4 in innate neuroimmunity and painful neuropathy.Proc Natl Acad Sci USA,2005,102:5856-5861.
29 Tanga FY,Raghavendra V,DeLeo JA.Quantitative realtime RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain.Neurochem Int,2004,45:397-407.
30 Sakaue G,Shimaoka M,Fukuoka T,et al.NF-kappa B decoy suppresses cytokine expression and thermal hyperalgesia in rat neuropathic pain model.Neuroreport,2001,12(10):2079-2084.
31 Leslie M.Inflaming chronic pain.Sci Aging Knowledge Environ,2005,13(15):nf28.
32 Wieseler-Frank J,Maier SF,Watkins LR.Immune-to-brain communication dynamically modulates pain:physiological and pathological consequences.Brain Behav Immun,2005,19(2):104-11.
33 李卉丽,秦绿叶,万有等.疼痛研究的新亮点:星形胶质细胞.生理科学进展,2003,34(1):45-46.
34 Milligan ED,Connor KAO,Nguyen KT,et al.Intrathecal H1V-1 envelope glycoprotein gp120induces enhan ced pain states mediated by spinal cord proinflammatory cytokines.J Neurosci,2001,21:2808-2819.
35 Inoue K,Tsuda M.The role of microglia and ATP receptors in a mechanism of neuropathic pain.Nippon Yakurigaku Zasshi.2006,127:14-17.
36 Tsuda M,Shigemoto-Mogami Y,Koizumi S,et al.P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury.Nature,2003,424:778-783.
[2]De Novellis V,Siniscalo D,Galderisi U,Fuccio R,Nolano M,Santoro L,Cascino A,Roth KA,Rossi N,Maione S.Blockade of glutamate mGlu5 receptors in a rat model of neuropathic pain prevents early overexpression of pro-apoptotic genes and morphological changes in dorsal horn lamina Ⅱ.Neuropharmacology,2004,46:468-479.
[3]Kimberly A,Moore,Tatsuro Kohno,Laurie A,Karchewski,Joachim Scholz.Partial peripheral nerve injury promotes a selective loss of GABAergic inhibition in the superficial dorsal horn of the spinal cord.The J Neurosci,2002,22(15):6724-6731.
[4]Scholz J,Broom DC,Youn DH,Mills CD,Kohno T,Suter MR,Moore KA,Decosterd I,Coggeshall RE,Woolf CJ.Blocking caspase activity prevents transsynaptic neuronal apoptosis and the loss of inhibition in lamina Ⅱ of the dorsal horn after peripheral nerve injury.J Neurosci,2005,25(32):7317-23.
[5]Hassanzadeh P,Ahmadiani A.Nitric oxide and c-Jun N-terminal kinase are involved in the development of dark neurons induced by inflammatory pain.Synapse,2006,59(2):101-6.
[6]Polgar E, Hughes DI, Arham AZ, Todd AJ. Loss of neurons from laminas Ⅰ-Ⅲ of the spinal dorsal horn is not required for development of tactile allodynia in the spared nerve injury model of neuropathic pain. J Neurosci, 2005, 13, 25(28): 6658-66.
[7]Craig A. Pain mechanisms: labelled lines versus convergence in central processing.Ann Rev Neurosci,2003, 26: 1-30.
[8]Woof CJ, Salter MW. Neuronal plasticity: increasing the gain in pain. Seience, 2000, 288: 1765-1768.
[9]Sindrup SH, Jensen TS. Pharmacologic treatment of pain in polyneuropathy. Neurology, 2000, 55, 915-920.
[10]Bomerc. Apoptosis without caspases: an inefficient molecular guillotine[J]. Cell Death Differ, 1999, 6(6): 497-502.
[11]Miljanich GP. Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. Curr Med Chem, 2004, 11: 3029-40.
[12]MicheauO, TschoppJ. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell, 2003, 114: 181-190.
[13]Schafers, M, Lee DH, Brors D, Yaksh TL, Sorkin LS. Increased sensitivity of injured and adjacent uninjured rat primary sensory neurons to exogenous tumor necrosis factor-alpha after spinal nerve ligation. J Neurosci, 2003, 23: 3028-3038.
[14]Zhang YH, Vasko MR, Nicol GD. Ceramide, a putative second messenger for nerve growth factor, modulates the TTX-resistant Na+ current and delayed rectifier K+ current in rat sensory neurons. J Physiol(Lond), 2002, 544: 385-402.
[15]Cossarizza A, Moyle G. Antiretroviral nucleoside and nucleotide analogues and mitochondria. AIDS, 2004, 18: 137-151.
[16]Mazumder S, Plesca D, Almasan A. caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis.Methods Mol Biol, 2008, 414:13-21.
[17]D'Costa AM, Denning MF.A caspase-resistant mutant of PKC-delta protects keratinocytes from UV-induced apoptosis. Cell Death Differ, 2005, 12(3):224-32.
[18]Logette E, Solary E, Corcos L.Identification of a functional DNA binding site for the SREBP-1c transcription factor in the first intron of the human caspase-2 gene. Biochim Biophys Acta, 2005, 1738(1-3):1-5.
[19]Lee SC, Chan J, Clement MV, Pervaiz S. Functional proteomics of resveratrol-induced colon cancer cell apoptosis: caspase-6-mediated cleavage of lamin A is a major signaling loop.Proteomics, 2006, 6(8):2386-94.
[20]Sgorbissa A,Benetti R,Marzinotto S,Schneider C,Brancolini C.caspase-3 and caspase-7 but not caspase-6 cleave Gas2 in vitro:implications for microfilament reorganization during apoptosis.J Cell Sci,1999,112(Pt23):4475-82.
[21]Shakibaei M,John T,Seifarth C,Mobasheri A.Resveratrol inhibits IL-1 beta-induced stimulation of caspase-3 and cleavage of PARP in human articular chondrocytes in vitro.Ann N Y Acad Sci,2007,1095:554-63.
[22]Related Articles,LinksSlee EA,Adrain C,Martin SJ.Executioner caspase-3,-6,and-7 perform distinct,non-redundant roles during the demolition phase of apoptosis.J Biol Chem,2001,276(10):7320-6.
[23]Joseph EK,Levine JD.caspase signalling in neuropathic and inflammatory pain in the rat.Eur J Neurosci,2004,20(11):2896-902.
[24]傅开元,张非煜,侯波等.福尔马林致炎症疼痛动物脊髓后角神经元凋亡.中国疼痛医学杂志,2005,11(1):25-28.
[25]解小丽,周青山,刘先义等.坐骨神经结扎大鼠脊髓神经元凋亡与痛敏的关系.中国疼痛医学杂志,2006,12(2):98-102.
[26]Campana WM,Myers RR.Exogenous erythropoietin protects against dorsal root ganglion apoptosis and pain following peripheral nerve injury.Eur J Neurosci,2003,18(6):1497-506.
[1]Walczak H,Haas.TLBiochemical analysis of the native TRAIL death-inducing signaling complex.Methods Mol Biol,2008,414:221-39.
[2]Czeschik JC,Hagenacker T,Schafers M,Busselberg D.TNF-alpha differentially modulates ion channels of nociceptive neurons.Neurosci Lett,2008,434(3):293-8.
[3]Yuan B,Ohyama K,Bessho T,Uchide N,Toyoda H.Imbalance between ROS production and elimination results in apoptosis induction in primary smooth chorion trophoblast cells prepared from human fetal membrane tissues.Life Sci,2008,82(111-12):623-30.
[4]Gao X,Kim HK,Chung JM,Chung K.Reactive oxygen species(ROS) are involved in enhancement of NMDA-receptor phosphorylation in animal models of pain.Pain,2007,131(3):262-71.
[5]Wagner R,Myers RR.Endoneurial injection of TNF-alpha produces neuropathic pain behaviors.Neuroreport.1996,7(18):2897-901.
[6]Lindenlaub T,Teuteberg P,Hartung T,Sommer C.Effects of neutralizing antibodies to TNF-alpha on pain-related behavior and nerve regeneration in mice with chronic constriction injury.Brain Res,2000,866(1-2):15-22.
[7]Sommer C,Lindenlaub T,Teuteberg P,Schafers M,Hartung T,Toyka KV.Anti-TNF-neutralizing antibodies reduce pain-related behavior in two different mouse models of painful mononeuropathy.Brain Res, 2001,913(1):86-9.
[8] Yeh WC,Pompa,McCurrach ME, et al.FADD:essential for embryo development an d signaling from some, but not all, inducers of apoptosis.Science, 1998, 279(5358): 1954-1957.
[9]MacFarlane M. TRAIL-induced signalling and apoptosis [J]. Toxicol Lett, 2003, 139(2-3): 89-97.
[10] Philchenkov A.caspases: potential targets for regulating cell death.J Cell Mol Med, 2004, 8(4):432-444.
[11] Chen G, Goeddel DV. TNF-R1 signaling: A beautiful pathway [J]. Science, 2002, 296(5573): 1634-1635.
[12]Wullaert A, Heyninck K, Beyaert R.Mechanisms of crosstalk between TNF-induced NF-kappaB and JNK activation in hepatocytes.Biochem Pharmacol, 2006, 72(9):1090-101.
[13]Niedbala B, Sanchez A, Feria M.Nitric oxide mediatesneuropathie pain behavior in peripherally denervared rats.Neurosci Lett, 1995, 188:57-60.
[14]Rokyta R, Holecek V, Pekarkova I, Krejcova J, Racek J, Trefil L, et al. Free radicals after painful stimulation are influenced by antioxidants and analgesics. Neuro Endocrinol Lett, 2003, 24(5):304-309.
[15]Chung JM. The role of reactive oxygen species (ROS) in persistent pain. Mol Interv 2004, 4(5):248-250.
[16]Hacimuftuoglu A, Handy CR, Goettl VM, Lin CG, Dane S, Stephens RL Jr.Antioxidants attenuate multiple phases of formalin-induced nociceptive response in mice. Behav Brain Res, 2006, 173(2):211-6.
[17]Adraln C, Martin SJ.The mitochondrial apeptosome: A killer unleashed by the cytochrome seas. Trends Biochem Sci, 2001, 26(6):390-397.
[18]Loh KP, Huang SH, De Silva R, Tan BK, Zhu YZ.Oxidative stress: apoptosis in neuronal injury. Curr Alzheimer Res, 2006, 3(4):327-37.
[19]Yoon- DY, Cho YS, Park JW, Kim SH, Kim JW.Up-regulation of reactive oxygen species (ROS) and resistance to Fas-mediated apoptosis in the C33A cervical cancer cell line transfected with IL-18 receptor.Clin Chem Lab Med, 2004, 42(5):499-506.
[20]Salazar M, Pariente JA, Salido GM, Gonzalez A.Ethanol induces glutamate secretion by Ca(2+) mobilization and ROS generation in rat hippocampal astrocytes.Neurochem Int, 2008, 52(6):1061-7.
[21]Sharikabad MN, Ostbye KM, Lyberg T, Br0rs O.Effect of extracellular Mg(2+) on ROS and Ca(2+) accumulation during reoxygenation of rat cardiomyocytes.Am J Physiol Heart Circ Physiol, 2001, 280(1):H344-53.
[22]Li J, Li Q, Xie C, Zhou H, Wang Y, Zhang N, Shao H, Chan SC, Peng X, Lin SC, Han J.Beta-actin is required for mitochondria clustering and ROS generation in TNF-induced, caspase-independent cell death. J Cell Sci,2004,117(Pt 20):4673-80.
[23]Yamada M,Kojima N,Paranjpe A,Att W,Aita H,Jewett A,Ogawa T.N-acetyl Cysteine(NAC)-assisted Detoxification of PMMA Resin.J Dent Res,2008,87(4):372-377.
[24]Spagnuolo G,D'Anto V,Cosentino C,Schmalz G,Schweikl H,Rengo S.Effect of N-acetyl-L-cysteine on ROS production and cell death caused by HEMA in human primary gingival fibroblasts.Biomaterials,2006,27(9):1803-9.
[25]Naik AK,Tandan SK,Dudhgaonkar SP,et al.Role of oxidative stress in pathophysiology of peripheral neuropathy and modulation by N-acetyl-L-cysteine in rats.Eur J Pain,2006,10(7):573-539.
[26]Chae HJ,Chae SW,Kim HR.N-acetyl cysteine regulates TNF-alpha-inhibited differentiation in ROS 17/2.8 osteoblasts.Immunopharmacol Immunotoxicol,2004,26(2):203-13.
[1]Ginsberg SD,Martin LJ.Axonal transection in adult rat brain induced transynaptic apoptosis and persistent atrophy of target neurons.Neurotrauma,2002,19(1):99-109.
[2]Csuka E,Hans VH,Ammann E,et al.Cell activation and inflammatory response following traumatic axonal injury in the rat.Neuroreport,2000,11(11):2587-90
[3]Camand E,Morel MP,Faissner A,et al.Long-term changes in the molecular composition of the glial scar and progressive increase of serotoninergic fibre sprouting after hemisection of the mouse spinal cord.Eur J Neurosci,2004,20(5):1161-76.
[4]Ling Yan and Xie Yi-kuan,Dynamic changes of mechanically activated channels and K+chanells at injury site of peripheral nerve in rat.Science inChina,1996,2:71-79.
[5]Kajander KC,Wakisaka S,Bennett GJ.Spontaneous discharge originates in the dorsal root ganglion at the onset of a painful peripheral neuropathy in the rat.NeurosciLett,1992,138:225-228.
[6]Lehner B,Koeck FX,Capellino S,Schubert TE,Hofbauer R,Straub RH.Preponderance of sensory versus sympathetic nerve fibers and increased cellularity in the infrapatellar fat pad in anterior knee pain patients after primary arthroplasty.J Orthop Res,2008,26(3):342-50.
[7]Jortner BS.The returns of the dark neuron.A histological artifact complicating contemporary neurotoxicologic evaluation.Neurotoxicology,2006,27(4):628-34.
[8]Murata R,Ohtori S,Ochiai N,et al.Extracorporeal shockwaves induce the expression of ATF3 and GAP-43 in rat dorsal root ganglion neurons.Auton Neurosci,2006,128(1-2):96-100.
[9]Macias MY,Syring MB,Pizzi MA,et al.Pain with no gain:allodynia following neural stem cell transplantation in spinal cord injury.Exp Neurol,2006,201(2):335-48.
[10]Kotulska K,LePecheur M,Marcol W,et al.Overexpression of copper/zinc-superoxide dismutase in transgenic mice markedly impairs regeneration and increases development of neuropathic pain after sciatic nerve injury.J Neurosci Res,2006,84(5):1091-7.
[11]Kyungsoon Chunga,Jin Mo Chung.Sympathetic sprouting in the dorsal root ganglion after spinal nerve ligation:evidence of regenerative collateral sprouting.Brain Research,2001,895:204-212.
[12]Znaor L,Lovric S,Hogan Q,et al.Association of neural inflammation with hyperalgesia following spinal nerve ligation.Croat Med J,2007,48(1):35-42.
[13]Obata H,Eisenach JC,Hussain H,et al.Spinal glial activation contributes to postoperative mechanical hypersensitivity in the rat.J Pain,2006,7(11):816-22.
[14]Kuzumaki N,Narita M,Narita M,et al.Chronic pain-induced astrocyte activation in the cingulate cortex with no change in neural or glial differentiation from neural stem cells in mice.Neurosci Lett,2007,415:22-27.
[15]Parish CL,Finkelstein DI,et al.The role of interleukin-1,interleukin-6,and glia in inducing growth of neuronal terminal arbors in mice.J Neurosci,2002,22(18):8034-41.
[16]Sobkowicz HM,Waclawik AJ,August BK.The astroglial cell that guides nerve fibers from growth cone to synapse in organotypic cultures of the fetal mouse spinal cord.Synapse,2006,59(4):183-200.
[17]Bechmann I,Nitsch R.Involvement of non-neuronal cells in entorhinal-hippocampal reorganization following lesions.Ann N Y Acad Sci,2000,911:192-206.
[18]Xiao WH,Bennett GJ.Persistent low-frequency spontaneous discharge in A-fiber and C-fiber primary afferent neurons during an inflammatory pain condition.Anesthesiology,2007,107(5):813-21.
[19]董承海,谢作磊,樊继云,谢益宽.异位电活动促使背根神经节交感节后纤维的芽生.中国科学,2001,31:531-536.
[1]Hassanzadeh P,Ahmadiani A.Nitric oxide and c-Jun N-terminal kinase are involved in the development of dark neurons induced by inflammatory pain.Synapse,2006,59(2):101-106.
[2]Gabriele Dorn,Sadhana Patel1,Glen Wotherspoon.siRNA relieves chronic neuropathic pain.Nucleic Acids Res(2004) 32(5):e49.
[3]Fire A,Xu AQ,Montgomery MK,et al.Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans.Nature,1998,391:806-811.
[4]Lipardi C,Wei Q,Paterson BM.RNAi as random degradative PCR siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs.Cell,2001,107:297-307.
[5]Knight SW,Bass BL.A role for the RNaseⅢ enzyme DICR-1 in RNA interference and germ line development in Caenorhabditis elegans.Science,2001,293(5538):2269-2271.
[6]Sijen T,Fleenor J,Simmer F,et al.On the role of RNA amplification in dsRNA triggered gene silencing.Cell,2001,107:465-476.
[7]Mantyh P,Clohisy D,Koltzenburg M,et al.Molecular mechanisms of cancer pain.Nat Rev,2002,20:201-209.
[8]Hains B,Klein J,Saab C et al.Upregulation of sodium channel Nav1.3 and functional involvement in neuronal hyperexcitability associated with central neuropathic pain after spinal cord injury.J Neurosci,2003,23(26):8881-8892.
[9]Christoph T,Grunweller A,Mika J,Schafer MK,Wade EJ,Weihe E,Erdmann VA,Frank R,Gillen C,Kurreck J.Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo.Biochem Biophys Res Commun,2006,350(1):238-43.
[10]Gabriele Dorn,Sadhana Patel1,Glen Wotherspoon.SiRNA relieves chronic neuropathic pain.Nucleic Acids Res,2004,32(5):e49.
[11]Achenbach T,Brunner B and Heermeier K.Oligonucleotide-based knockdown technologies: antisense versus RNA interference.Chembiochem,2003,4(10):928-935.
[12]Ganju P,Hall J.Potential applications of siRNA for pain therapy.ExpertOpin Biol Ther,2004,4(4):531-542.
[13]吴飞翔,吴军珍,俞卫锋.RNA干扰在慢性疼痛研究分析及治疗中的应用.麻醉与监护论坛,2006,13(5):285-287.
[14]Chiou SH,Chen SJ,Peng CH,et al.Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibites LPS-induced apoptosis in hippocampus-derived neural stem cell.Biochem Biophys Res Commun,2006,343(2):391-400.
[1]Fire A,Xu AQ,Montgomery MK,et al.Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans.Nature,1998,391:806-811.
[2]Lipardi C,Wei Q.Paterson BM.RNAi as random degradative PCR.siRNA primers convert mRNA into dsRNA that are degraded to generate new siRNAs.Cell,2001,107:297-307.
[3]Moffat J,Sabatini DM.Building mammalian signalling pathways with RNAi screens.Nat Rev Mol Cell Biol,2006,7:177-87.
[4]Sijen T,Fleenor J,Simmer F,et al.On the role of RNA amplification in dsRNA triggered gene silencing.Cell,2001,107:465-476.
[5]Nishikura K.A short primer on RNAi:RNA directed RNA polymerase acts as a key catalyst.Cell,2001,107:415-418.
[6]Craig A.Pain mechanisms:labelled lines versus convergence in central processing.Ann Rev Neurosci,2003,26:1-30.
[7]Woof CJ,Salter MW.Neuronal plasticity:increasing the gain in pain.Seience,2000,288:1765-1768.
[8]Rondinone CM.RNAi:for functional analysis and target validation.Expert Opin Ther Targets,2006,10:337-40.
[9]QinghHa Liu,m A R,Savitha K,et al.a Bridge Between the Initiation and Efector Steps of the Drosophila RNAi Pathway.Science,2003,301(26):1921-192.
[10]Fraser AG,Kamath RS,Zipperlen P,et al.Functional genomic analysis of C.elegans chromosome I by systematic RNA interference.Nature,2000,408:325-330.
[11]Berns K,Hijmans EM,Mullenders J,et al.A large-scale RNAi screen in human cells identifies new components of the p53 pathway.Nature,2004,428:431-437.
[12]何承伟,刘芳等.RNAi技术在大规模基因功能研究中的应用.国外医学,临床生物化学与检验学分册,2005,26:539-541.
[13]Hemmings-Mieszczak M,Dorn G,Natt FJ,et al.Independent combinatorial effect of antisense oligonucleotides and RNAi-mediated specific inhibition of the recombinant rat P2X3 receptor.Nucleic Acids Res,2003,31:2117-2126.
[14]Gabriele Dorn,Sadhana Patel1,Glen Wotherspoon.SiRNA relieves chronic neuropathic pain.Nucleic Acids Res,2004,32:e49.
[15]Grunweller A,Wyszko E,Bieber B.Comparison of different antisense strategies in mammalian cells using locked nucleic acids,2'-O-methyl RNA,phosphorothioates and small interfering RNA.Nucleic Acids Res,2003,31:3185-3193.
[16]Lai J,Porreca F,Hunter J,et al.Voltage-gated sodium channels and hyperalgesia.Ann Rev Pharmacol Toxico,2004,44:371-397.
[17]Mikami M,Yang J.Short hairpin RNA-mediated selective knockdown of NaV1.8tetrodotoxin-resistant voltage-gated sodium channel in dorsal root ganglion neurons.Anesthesiology,2005,103(4):828-36.
[18]Miljanich GP.Ziconotide:neuronal calcium channel blocker for treating severe chronic pain.Curr Med Chem, 2004, 11: 3029-40.
[19]Tan PH, Yang LC, Shih HC, et al.Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat.Gene Ther, 2005, 12: 59-66.
[20] Hains B, Klein J, Saab C, et al.Upregulation of sodium channel Nav1.3 and functional involvement in neuronal hyperexcitability associated with central neuropathic pain after spinal cord injury.J Neurosci, 2003, 23: 8881-8892.
[21] Zhang WB, Zhang Z, Ni YX, et al. A novel function of Goa: Mediation of extracellular signal-regulated kinase activation by opioid receptors in neural cells.J Neurochem.2003, 86: 1213-1222.
[22] Zhang Y, Boado RJ, Pardridge WM.In vivo knockdown of gene expression in brain cancer with intravenous RNAi in adult rats. J Gene Med, 2003, 5: 1039-1045.
1 Coutaux A,Adam F,Willer JC,et al.Hyperalgesia and allodynia:peripheral mechanisms.Joint Bone Spine,2005,72(5):359-371.
2 Attal N,Bouhassira D.Translating basic research on sodium channels in human neuropathic pain.J Pain,2006,7(S1):S31-S37.
3 Casula MA,Facer P,Powell AJ,et al.Expression of the sodium channel β3 subunit in injured human sensory neurons.Neuroreport,2004,15(10):1629-1632.
4 Hains BC,Klein JP,Saab CY,et al.Upregulation of sodium channel Nav1.3 and functional involvement in neuronal hyperexcitability associated with central neuropathic pain after spinal cord injury.J Neurosci,2003,23(26):8881-8892.
5 Black JA,Liu S,Tanaka M,et al.Changes in the expression of tetrodotoxin-sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain.Pain,2004,108(3):237-247.
6 Nassar MA,Stirling LC,Forlani G,et al.Nociceptor-specific gene deletion reveals a major role for Nav1.7(PN1) in acute and inflammatory pain.Proc Natl Acad Sci U S A,2004,101(34):12706-12711.
7 Porreca F,Lai J,Bian D,et al.A comparison of the potential role of the tetrodotoxin-insensitive sodium channels,PN3/SNS and NaN/SNS2,in rat models of chronic pain.Proc Natl Acad Sci U S A,1999,96(14):7640-7644.
8 Lai J,Gold MS,Kim CS,et al.Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel,Nav1.8.Pain,2002,95(1-2):143-152.
9 Baker MD,Chandra SY,Ding Y,et al.GTP-induced tetrodotoxin-resistant Na~+ current regulates excitability in mouse and rat small diameter sensory neurones.J Physiol,2003,548(2):373-382.
10 Bourinet E,Zamponi GW.Voltage gated calcium channels as targets for analgesics.Curr Top Med Chem,2005,5(6):539-546.
11 Bourinet E,Alloui A,Monteil A,et al.Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception.EMBO J,2005,24(2):315-324.
12 Pareek TK,Keller J,Kesavapany S,et al.Cyclin-dependent kinase 5 activity regulates pain signaling.Proc Natl Acad Sci,2006,103(3):791-796.
13 Song XS,Cao JL,Xu YB,et al.Activation of ERK/CREB pathway in spinal cord contributes to chronic constrictive injury-induced neuropathic pain in rats.Acta Pharmacol Sin,2005,26:789-98.
14 Sweitzera SM,Petersa MC,Mab JY,et al.Peripheral and central p38MAPK mediates capsaicin-induced hyperalgesia.Pain,2004,111:278-285.
15 Jin SX. Zhuang ZY, Woolf CB, et al. P38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J Neurosci, 2003, 23: 4017-4022.
16 Doya H, Ohtori S, Fujitani M, et al. C-Jun N-terminal kinase activation in dorsal root ganglion contributes to pain hypersensitivity. Biochem Biophys Res Commun, 2005, 335: 132-138.
17 Hassanzadeh P, Ahmadiani A. Nitric oxide and c-Jun N-terminal kinase are involved in the development of dark neurons induced by inflammatory pain. Synapse, 2006, 59: 101-106.
18 Ji RR, Rupp F. Phosphorylation of transcription factor CREB in rat spinal cord after formalin-induced hyperalgesia: relationship to c-fos induction. J Neurosci, 1997, 17:1776-1785.
19 Yasuhiko Kawasaki, Tatsuro Kohno, Zhi-Ye Zhuang. Ionotropic and metabotropic receptors, protein kinase A, protein kinase C, and Src contribute to C-fiber-induced ERK activation and cAMP response element-binding protein phosphorylation in dorsal horn neurons, leading to central sensitization. J Neurosci, 2004, 24:8310-8321.
20 Ru-Rong Ji, Clifford J. Neuronal Plasticity and Signal Transduction in Nociceptive Neurons: Implications for the Initiation and Maintenance of Pathological Pain. Neurobiology of Disease, 2001, 8:1-10.
21 Bomerc. Apoptosis without caspases: an inefficient molecular guillotine. Cell Death Differ, 1999, 6(6): 497-502.
22 De Novellis V, Siniscalo D, Galderisi U, et al. Blockade of glutamate mGlu5 receptors in a rat model of neuropathic pain prevents early overexpression of pro-apoptotic genes and morphological changes in dorsal horn lamina II. Neuropharmacology, 2004, 46: 468-479.
23 Joseph EK, Levine JD. caspase signalling in neuropathic and inflammatory pain in the rat. Eur J Neurosci, 2004, 20(11): 2896-2902.
24 Kimberly A, Moore, Tatsuro Kohno, et al. Partial peripheral nerve injury promotes a selective loss of GABAergic inhibition in the superficial dorsal horn of the spinal cord. The J Neurosci, 2002, 22(15): 6724-6731.
25 Scholz J, Broom DC, Youn DH, et al. Blocking caspase activity prevents transsynaptic neuronal apoptosis and the loss of inhibition in lamina II of the dorsal horn after peripheral nerve injury. J Neurosci, 2005, 25(32): 7317-7323.
26 Polgar E, Hughes DI, Arham AZ, Todd AJ. Loss of neurons from laminas Ⅰ-Ⅲ of the spinal dorsal horn is not required for development of tactile allodynia in the spared nerve injury model of neuropathic pain. J Neurosci, 2005, 13, 25(28): 6658-6666.
27 Campana WM, Myers RR. Exogenous erythropoietin protects against dorsal root ganglion apoptosis and pain following peripheral nerve injury.Eur J Neurosci,2003,18(6):1497-1506.
28 Tanga FY,Nutile-McMenemy N,DeLeo.The CNS role of toll-like receptor 4 in innate neuroimmunity and painful neuropathy.Proc Natl Acad Sci USA,2005,102:5856-5861.
29 Tanga FY,Raghavendra V,DeLeo JA.Quantitative realtime RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain.Neurochem Int,2004,45:397-407.
30 Sakaue G,Shimaoka M,Fukuoka T,et al.NF-kappa B decoy suppresses cytokine expression and thermal hyperalgesia in rat neuropathic pain model.Neuroreport,2001,12(10):2079-2084.
31 Leslie M.Inflaming chronic pain.Sci Aging Knowledge Environ,2005,13(15):nf28.
32 Wieseler-Frank J,Maier SF,Watkins LR.Immune-to-brain communication dynamically modulates pain:physiological and pathological consequences.Brain Behav Immun,2005,19(2):104-11.
33 李卉丽,秦绿叶,万有等.疼痛研究的新亮点:星形胶质细胞.生理科学进展,2003,34(1):45-46.
34 Milligan ED,Connor KAO,Nguyen KT,et al.Intrathecal H1V-1 envelope glycoprotein gp120induces enhan ced pain states mediated by spinal cord proinflammatory cytokines.J Neurosci,2001,21:2808-2819.
35 Inoue K,Tsuda M.The role of microglia and ATP receptors in a mechanism of neuropathic pain.Nippon Yakurigaku Zasshi.2006,127:14-17.
36 Tsuda M,Shigemoto-Mogami Y,Koizumi S,et al.P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury.Nature,2003,424:778-783.