【作者】 王超；

【导师】 岳寿伟； 葛洪友；

【作者基本信息】 山东大学 ， 康复医学与理疗学， 2012， 博士

【摘要】 背景背根神经节(dorsal root ganglion, DRG)的持续受压(chronic compression of DRG, CCD)和椎管狭窄或椎间盘突出压迫神经根是临床上根性神经疼痛的主要原因之一。CCD大鼠模型是一种典型的神经疼痛模型,它可以产生自发性疼痛、机械性的热痛觉过敏和异常疼痛,伴随着受压迫神经元的自发性放电增加,电流阈值和动作电位降低。外周神经损伤能够通过背根神经节和脊髓中一系列复杂的分子级联反应诱发神经病理性疼痛,包括肿瘤坏死因子(tumor necrosis factor, TNF-a)、白介素-6(interleukin-6, IL-6)和核因子κB(nuclear faetor-kappa B, NF-κ B).细胞内的第二信使环磷腺苷(cyclic adenosine monophosphate, cAMP)-依赖性的蛋白激酶A(protein kinase A, PKA)和环磷鸟苷(cyclic guanosine monophosphate, cGMP)-依赖性的蛋白激酶G (protein kinase G, PKG)介导CCD大鼠的痛觉过敏和受压神经元的高兴奋性。另外,多种离子通道被认为参与并传递持续受压后DRG神经元的伤害性刺激,如电压门控性Na+通道和K+通道、超极化激活性阳离子通道和瞬时感受器电位离子通道香草素受体亚家族4通道(transient receptor potential vanilloid, TRPV4)等。TRPV4是一种多觉型感受器,可被低渗、机械刺激、热、佛波醇酯、低pH值、柠檬酸盐、anandamide (AEA)和其代谢产物花生四烯酸(arachidonic acid, AA)、以及bisandrographolide A (BAA)等激活。前列腺素E2(PGE2)可增强低渗(17mOsm)或中度高渗溶液刺激(2%NaCl,607mOsm)引起的机械性痛觉过敏和异常疼痛,TRPV4通道参与介导上述炎症性痛觉反应。TRPV4基因敲除的小鼠表现出体液渗透压调节功能异常,对伤害性压力刺激的回避反应降低,但对非伤害性热和触觉刺激的反应正常。TRPV4基因敲除小鼠也表现出对炎症引起的热痛觉过敏降低。一氧化氮(nitric oxide, NO)是动物疼痛模型中一种重要的参与伤害活动的调质,并且介导了神经病理疼痛的发生。有文献报道,TRPV4的激动剂4a-PDD (4a-phorbol12,13-didecanoate)和低渗溶液的刺激在毛豚鼠耳蜗的细胞外层能够诱导一氧化氮的合成,而通过TRPV4的阻断剂钌红(ruthenium red, RR)能够抑制一氧化氮的生成。我们前期的研究发现,TRPV4-NO-cGMP-PKG信号通路在CCD大鼠热痛觉过敏中发挥重要的作用。然而,到目前为止,在CCD大鼠热痛觉过敏中TRPV4与NO之间的具体的机制尚不清楚。NF-κB是基因转录的调控因子,由Rel家族的多亚基蛋白组成,包括：p50,p52, p65(RelA), c-Rel and RelB。NF-κB在细胞的生长、分化、坏死和凋亡过程中起重要作用,广泛参与肿瘤、炎症等病理过程。在正常状态下或者在静息状态的细胞中,NF-κB与其抑制性蛋白I-κ B结合,以二聚体的形式存在于细胞浆中,最常见的是二聚体是p65/p50。当细胞受到刺激时,I-κB蛋白被迅速磷酸化并与NF-κB解离,游离的NF-κB转移到细胞核内,参与众多基因的诱导表达。当受到细胞因子、氧化应急、一些化学和疼痛性伤害时,NF-κ B被激活。最近,有研究者发现,在脊髓和背根神经节(DRG)中NF-κB参与伤害性感觉信息的传递。低频和高频的电针灸刺激能够降低NF-κB敲出小鼠的痛觉敏感性。Bunnell等发现NF-κB的激活发生在背根神经节和脊髓,参与伤害性信息的传递和处理,部分坐骨神经损伤后大鼠腰背根神经节NF-κB的活性增加。Chan等对大鼠的后爪皮下注射弗罗因德佐剂做痛觉过敏模型,观察大鼠腰脊髓NF-κB表达的时程差异性,证实NF-κB在弗罗因德佐剂产生的痛敏实验的炎症前状态发挥重要作用。这些证据NF-κB参与介导了神经病理疼痛的传递。因此,我们推测在CCD大鼠热痛觉过敏中NF-κB可能参与了TRPV4-NO的信号通路。目的研究NF-κB介导的TRPV4-NO信号通路在CCD大鼠热痛觉过敏中的作用。方法1.建立CCD模型将大鼠随机分为CCD组和假手术组。大鼠先用戊巴比妥钠(50mg/kg,腹腔注射)麻醉后,沿L4-S1棘突作后正中偏右切口,暴露右侧L4及L5椎间外孔,将“L”形棒的一端沿L4及L5椎间外孔的前壁上方水平插入椎管内,另一端位于椎管壁外。通过控制“L”形棒椎管壁外侧端使其椎管内端沿椎骨内壁缓慢下滑至椎管中部,使之挤压L4、L5的大鼠背根神经节。术后用生理盐水冲洗切口,依层缝合肌肉、腰背筋膜和皮肤,腹腔注射青霉素预防感染。假手术组除不插钢棒压迫神经节外,其余操作同手术组。术后大鼠没有发生白残现象,也没有表现出感觉完全缺失。2. NF-κB的阻断剂对热痛敏感性的影响为了测量NF-κ B的阻断剂对热痛敏感性的影响,大鼠被随机分为假手术组(sham组)、NF-κ B的阻断剂(PDTC或BAY)处理组、生理盐水处理组。其中通过NF-κB的阻断剂PDTC处理组又分成PDTC (10-1M)组、PDTC (10-2M)组、PDTC (10-3M)三个亚组；NF-κB的阻断剂的另外一种阻断剂BAY处理组又分成BAY (100μM)组、BAY (50μM)组、BAY (25μM)三个亚组。假手术组中未接受任何处理的大鼠作为对照,CCD组中只接受CCD手术的大鼠作为对照。NF-κ B的阻断剂(PDTC或BAY)溶于0.9%的生理盐水通过鞘内注射,40μg/d,每天一次,共7天。3神经行为学测定于手术前连续测两天,每天一次。术后7天药物处理前2小时再次测量,排除痛觉表现不明显者(<5%),药物处理后1、2、4、8、24小时分别测量。对于鞘内注射NF-κB的阻断剂(PDTC或BAY)的大鼠,痛觉测量则在最后一次注射后12小时进行。热辐射刺激缩爪反应潜伏期(thermal withdrawl latency)使用BME-410A型热痛刺激仪,将热痛刺激仪放在6mm厚的有机玻璃板下方,使光源焦距照射动物后肢足底掌心,电子秒表记录从照射开始到引起后肢回缩反应时的潜伏期作为热痛觉观测指标。取每只大鼠5次测量结果的均值为统计数据。结果1NF-κB的阻断剂PDTC对大鼠热痛觉过敏的影响与假手术组相比,CCD大鼠产生明显的热痛觉过敏(n=8, F=245.054, P<0.001)。鞘内注射不同浓度的NF-κB的阻断齐IJPDTC (10-1M、10-2M、10-3M)各40μl,分别测注射后0、1、2、4、8和24h时的热刺激缩爪时间,结果显示：与生理盐水组相比,浓度10-1M、10-2M的PDTC对CCD后大鼠的热痛觉过敏产生抑制作用(n=8,P<0.001),此抑制作用注射后1h可测得,4h作用达到最大,大约能够持续到8h,直到24h作用消失。而浓度10-3M的PDTC对CCD后大鼠的热痛觉过敏没有明显的抑制作用(n=8,P>0.05)。2NF-κ B的阻断剂BAY对大鼠热痛觉过敏的影响与假手术组相比,CCD大鼠产生明显的热痛觉过敏(n=8,F=253.816,P<0.001)。鞘内注射不同浓度的NF-κB的阻断剂BAY (100uM、50uM、25uM)各40μl,分别测注射后0、1、2、4、8和24h时的热刺激缩爪时间,结果显示：与生理盐水组相比,浓度100uM、50uM的PDTC对CCD后大鼠热痛觉过敏产生抑制作用(n=8,P<0.001),此抑制作用注射后1h可测得,4h作用达到最大,大约能够持续到8h,直到24h作用消失。而浓度25uM的PDTC对CCD后大鼠的热痛觉过敏没有明显的抑制作用(n=8,P>0.05)。3TRPV4激动齐(?)4a-PDD对NF-κB的阻断剂PDTC的作用鞘内注射NF-κB的阻断齐(?)JPDTC (10-1M),分别测注射后0、1、2、4、8和24h时的热刺激缩爪时间。与生理盐水组相比,PDTC (10-1M)对CCD后大鼠热痛觉过敏产生抑制作用(n=8,P<0.001)。鞘内注射TRPV4激动剂4α-PDD (1nmol)预处理后,能够明显逆转PDTC (10-1M)对CCD后大鼠热痛觉过敏产生抑制作用(n=8,P<0.001)。4TRPV4激动剂4a-PDD对NF-κB的阻断剂BAY的作用鞘内注射NF-κ B的阻断剂BAY (100uM),分别测注射后0、1、2、4、8和24h时的热刺激缩爪时间。与生理盐水组相比,BAY (100uM)对CCD后大鼠热痛觉过敏产生抑制作用(n=8,P<0.001)。鞘内注射TRPV4激动剂4α-PDD (1nmol)预处理后,能够明显逆转BAY (100uM)对CCD后大鼠热痛觉过敏产生抑制作用(n=8,P<0.001)。结论在CCD大鼠的热痛觉过敏中,NF-κB参与介导了TRPV4-NO信号通路。背景一氧化氮(NO, nitric oxide)是一种在中枢和外周神经内传递细胞间及细胞内信号的信号分子,能够感受病理状态下的伤害性刺激,在神经病理性疼痛及痛觉过敏的发生和持续过程中发挥重大作用。NOS有三种类型：神经元型NOS(neuronal NOS, nNOS),内皮型NOS(endothelial NOS, eNOS),诱导型NOS (inducible NOS, iNOS)。通常在有氧的环境下,NOS介导L-精氨酸生成NO。在病理状态或缺氧情况下,神经组织的NOS表达升高,NO是由NOS在一定的条件下合成,因此神经系统NO的病理作用可能受控于NOS表达的变化和活性的变化。在神经组织,nNOS主要表达于神经元内,促进NO的合成。多种神经病理疼痛模型实验已经证实nNOS诱导产生的NO参与介导伤害性疼痛发展过程。近年来,iNOS也逐渐成为研究神经痛病理机制的一个重要介质。有学者指出,iNOS在脊髓损伤后的痛觉高敏过程发挥特殊作用,因此认为,iNOS在神经组织的高表达可能参与介导痛觉过敏过程。另外,神经组织内nNOS和iNOS在坐骨神经持续缩窄性损伤和脊神经结扎模型中表达升高,也证实了两者在神经痛中的作用。虽然已经有大量的证据表明nNOS和iNOS在上述两种神经痛模型的痛觉过敏中发挥作用,但是两者是否参与其它神经痛动物模型的痛觉高敏过程还不甚清楚。NF-κB是基因转录的调控因子,由Rel家族的多亚基蛋白组成,包括：p50,p52, p65, c-Rel and RelB。NF-κB在细胞的生长、分化、坏死和凋亡过程中起重要作用,广泛参与肿瘤、炎症等病理过程。在正常状态下或者在静息状态的细胞中,NF-κB与其抑制性蛋白I-κB结合,以二聚体的形式存在于细胞浆中,最常见的是二聚体是p65/p50。当细胞受到刺激时,I-κB蛋白被迅速磷酸化并与NF-κB解离,游离的NF-κ B转移到细胞核内,参与众多基因的诱导表达。当受到细胞因子、氧化应急、一些化学和疼痛性伤害时,NF-κB被激活。体内NF-κB的活化过程调控包括两条途径。一是经细胞外的正反馈途径：这是炎症反应放大的重要机制,前炎症介质TNF-α、IL-1及活性氧介质(ROI)是NF-κB的强诱导剂。前炎症介质对NF-κB的活化就属于正反馈调节,在其诱导下,NF-κB活化后,可增强TNF-α和IL-1β的基因转录,使其产生和释放增多,进而再次激活NF-κB;还可使IL-6和IL-8产生和释放增多,导致最初的炎症信号进一步放大。二是经细胞内、外的负反馈途径：在细胞内,NF-κB活化后,在启动炎症介质基因转录的同时,抑制蛋白如Iκ B-α和p105的基因转录亦被上调,IκB基因上的启动子区域含有κB序列,κB序列被降解后,激活的NF-κB可使其生成增加,从而又可与NF-κB结合,甚至可进入核内与活化的NF-κB结合,竞争性抑制NF-κB与DNA上的结合位点结合,从而下调细胞核中NF-κ B的活性,终止炎症介质的生成；在细胞外,刺激NF-κB由活化的因素,如LPS、TNF-α和IL-1等也可导致抗炎因子如IL-10、IL-13等的产生,后者可抑制NF-κB的激活,反馈抑制促炎因子的转录和表达。但IκB-β则不能被NF-κB诱导再生,故在Iκ B-β占优势的细胞内NF-κB的活化有持续存在的可能。NF-κB活性有两种类型,即NF-κ B组成活性和NF-κB诱导活性,NF-κB组成活性是指细胞处于静息状态时,其细胞内即有一定水平的NF-κB活性,往往与机体某些重要的发挥生理作用的大分子表达有关,在体内表现出持续性存在的特点。NF-κB诱导性活性指某些因素刺激使细胞内NF-κB活性水平迅速增加,多与机体应急有关,刺激因素解除后,诱导活性在一定的时间内大都可以平息下来,否则将扰乱体内神经内分泌一细胞因子网络,带来严重的病理性危害。最近,有研究者发现,在脊髓和背根神经节(DRG)中NF-κB参与伤害性感觉信息的传递。低频和高频的电针灸刺激能够降低NF-κB敲出小鼠的痛觉敏感性。Bunnell等发现NF-κB的激活发生在背根神经节和脊髓,参与伤害性信息的传递和处理,部分坐骨神经损伤后大鼠腰背根神经节NF-κB的活性增加。Chan等对大鼠的后爪皮下注射弗罗因德佐剂做痛觉过敏模型,观察大鼠腰脊髓NF-κB表达的时程差异性,证实NF-κB在弗罗因德佐剂产生的痛敏实验的炎症前状态发挥重要作用。这些证据NF-κB参与介导了神经病理疼痛的传递。因此,我们推测在CCD大鼠背根神经节持续机械刺激后NF-κB可能参与了其中的分子机制及这些类型的NOS是否与TRPV4有相关性。目的1.观察CCD对DRG中NO的含量。2.观察NF-κB在CCD大鼠背根神经节持续机械刺激后的表达及在组织化学中的定位。3.明确NF-κB参与介导TRPV4-NO信号通路中nNOS的作用。方法1.建立CCD模型将大鼠随机分为CCD组和假手术组。大鼠先用戊巴比妥钠(50mg/kg,腹腔注射)麻醉后,沿L4-S1棘突作后正中偏右切口,暴露右侧L4及L5椎间外孔,将“L”形棒的一端沿L4及L5椎间外孔的前壁上方水平插入椎管内,另一端位于椎管壁外。通过控制“L”形棒椎管壁外侧端使其椎管内端沿椎骨内壁缓慢下滑至椎管中部,使之挤压L4、L5的大鼠背根神经节。术后用生理盐水冲洗切口,依层缝合肌肉、腰背筋膜和皮肤,腹腔注射青霉素预防感染。假手术组除不插钢棒压迫神经节外,其余操作同手术组。术后大鼠没有发生自残现象,也没有表现出感觉完全缺失。2.亚硝酸盐产物(NO)测量亚硝酸盐水平间接反应DRG内NO产物的含量,按照亚硝酸盐检测试剂盒进行操作。3.细胞核蛋白/浆蛋白的提取从各组大鼠DRG中提取蛋白质,按照组织细胞核蛋白/浆蛋白抽提试剂盒进行操作。4. Western blotting检测从各组大鼠DRG中提取蛋白质,Western blotting检测胞核中NF-κB、胞浆中I-K B蛋白表达量的变化以及大鼠DRG中nNOS的表达变化。5.实时定量PCR检测从各组大鼠DRG中提取RNA,实时定量PCR检测胞核中NF-κB、胞浆中I-κB mRNA的改变以及大鼠DRG中nNOS mRNA的改变。6.免疫组织化学大鼠麻醉后经颈动脉灌注4%多聚甲醛固定,然后迅速取出DRG,4%多聚甲醛固定7-8小时,经过脱水浸蜡过程将组织石蜡包埋,切成厚约5微米的石蜡切片,行nNOS、NF-κB的免疫组化染色。结果1.NF-κB的阻断齐(?)PDTC和BAY对大鼠DRG中NO衍生产物亚硝酸盐浓度的影响鞘内注射NF-κB的阻断剂PDTC(10-1M)40μl,分别测注射后0、1、2、4、8和24h时DRG中的亚硝酸盐浓度,结果显示：与鞘内注射生理盐水组相比,10-1M的PDTC组明显降低DRG亚硝酸盐浓度,1h后有所降低(n=8,P<0.05),4h作用最大(n=8,P<0.05),大约能够持续到8h,但是,24h后没有明显的作用(n=8,P>0.05)。其浓度的变化与热痛觉过敏反应呈负相关(r=-0.966,P<0.05)。鞘内注射NF-κB的阻断齐(?)BAY(100uM)40μl,分别测注射后0、1、2、4、8和24h时DRG中的亚硝酸盐浓度,结果显示：与鞘内注射生理盐水组相比,100uM的BAY组明显降低DRG亚硝酸盐浓度,1h后有所降低(n=8,P<0.05),4h作用最大(n=8,P<0.05),大约能够持续到8h,但是,24h后没有明显的作用(n=8,P>0.05)。其浓度的变化与热痛觉过敏反应呈负相关(r==-0.952,P<0.05)。2. TRPV4激动齐(?)4α-PDD对大鼠DRG中NO衍生产物亚硝酸盐浓度的影响鞘内注射NF-κB的阻断齐(?)PDTC(10-1M)后,大鼠DRG中NO衍生产物亚硝酸盐浓度明显降低(P<0.05)。鞘内注射TRPV4激动剂4α-PDD(1nmol)预处理后,能够明显逆转PDTC(10-1M)对CCD后大鼠DRG中NO衍生产物亚硝酸盐的浓度(n=8,P<0.05)。鞘内注射NF-κB的阻断剂BAY(100uM)后,大鼠DRG中NO衍生产物亚硝酸盐浓度明显降低(P<0.05)。鞘内注射TRPV4激动齐(?)4α-PDD(1nmol)预处理后,能够明显逆转BAY(100uM)对CCD后大鼠DRG中NO衍生产物亚硝酸盐的浓度(n=8,P<0.05)。3.Western blotting示胞浆中I-κ B、胞核中NF-κB以及不同处理后大鼠DRG中nNOS蛋白表达量的变化Western blotting结果显示；与大鼠CCD组相比,鞘内注射NF-κB的阻断剂PDTC后,Westem blotting显示胞浆中I-κB的表达量明显升高(P<0.05)。鞘内注射TRPV4激动齐(?)4a-PDD (1nmol)预处理后,能够明显逆转PDTC对CCD后大鼠DRG中NF-κB的表达量(n=4,P<0.05)。与大鼠CCD组相比,鞘内注射NF-κB的阻断齐(?)PDTC后,胞核中NF-κB的表达量明显降低(n=4,P<0.05)。鞘内注射TRPV4激动剂4a-PDD (1nmol)预处理后,能够明显逆转PDTC对CCD后大鼠DRG中NF-κB的表达量(n=4,P<0.05)。另外,与大鼠假手术组相比,CCD组大鼠DRG中nNOS的表达量明显升高(n=4,P<0.05)。鞘内注射NF-κB的阻断剂PDTC后预处理后,能够明显逆转CCD后大鼠DRG中nNOS的表达量(n=4,P<0.05)。4. RT-PCR示胞浆中I-κ B mRNA、胞核中NF-κ B mRNA以及大鼠DRG中nNOS mRNA表达量的变化RT-PCR结果显示：与大鼠CCD组相比,鞘内注射NF-κB的阻断剂PDTC后,RT-PCR显示胞浆中I-κ B mRNA的表达量明显升高(n=3,P<0.05)。鞘内注射TRPV4激动剂4a-PDD (1nmol)预处理后,能够明显逆转PDTC对CCD后大鼠DRG中I-κ B mRNA的表达量(n=3,P<0.05)。与大鼠CCD组相比,鞘内注射NF-κ B的阻断剂PDTC后,胞核中NF-κ B mRNA的表达量明显降低(P<0.05)。鞘内注射TRPV4激动剂4a-PDD (1nmol)预处理后,能够明显逆转PDTC对CCD后大鼠DRG中NF-κ B mRNA的表达量(n=3,P<0.05)。另外,与大鼠假手术组相比,CCD组大鼠DRG中nNOS mRNA的表达量明显升高(n=3,P<0.05)。鞘内注射NF-κ B的阻断剂PDTC后预处理后,RT-PCR显示能够明显逆转CCD后大鼠DRG中nNOS mRNA的表达量(n=3,P<0.05)。5.免疫组化检测不同处理后大鼠DRG中NF-κB, nNOS表达的改变免疫组化结果显示：与大鼠假手术组相比,CCD组大鼠DRG中NF-κB的阳性细胞数明显升高(n=3,P<0.05)。鞘内注射NF-κB的阻断剂IPDTC后预处理后,大鼠DRG中NF-κB的阳性细胞数明显减少(n=3,P<0.05)。另外,与大鼠假手术组相比,CCD组大鼠DRG中nNOS的阳性细胞数明显升高(P<0.05)。鞘内注射NF-κ B的阻断剂PDTC后预处理后,大鼠DRG中nNOS的阳性细胞数明显减少(n=3,P<0.05)。结论NF-κB可能诱导产生了nNOS,生成NO,并且参与介导大鼠背根神经节持续机械刺激后TRPV4-NO痛觉过敏的分子机制信号通路。更多还原

【Abstract】 BackgroundMechanical compression of the dorsal root ganglion (DRG) and the nerve root by spinal canal stenosis or disc herniation is one of the primary causes of radicular pain syndrome in humans. Chronic compression of DRG (CCD) in animals, a typical model of neuropathic pain, results in spontaneous pain, mechanical allodynia and thermal hyperalgesia. Peripheral nerve injury induces neuropathic pain by upregulating a complicated network of molecules in DRG and spinal cord, including the proinflammatory cytokine tumor necrosis factor (TNF)-a, interleukin (IL)-6and nuclear factor-kappa B (NF-κB). Neurotransmitter and second messenger signals, including nitric oxide (NO), cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA), cyclic guanosine monophosphate (cGMP)-dependent protein kinase G (PKG), have been shown to participate in maintaining neuronal hyperexcitability and behavioral hyperalgesia in CCD treatment. Moreover, several types of ion channels such as voltage-gated Na+and K+, hyperpolarization-activated cation current, and transient receptor potential vanilloid4(TRPV4) may contribute to the transmission of nociceptive stimuli in CCD neurons. NO has been shown to be a key mediator of nociceptive activity in animal pain models and involved in the maintenance of pathological pain. TRPV4synthetic activator,4a-PDD, and hypoosmotic stimulation could induce NO production in outer hair cells of the guinea pig cochlea, while NO production is inhibited by ruthenium red, an inhibitor of TRPV4. Our recent study indicated that TRPV4enhances pain-related behavior through a NO-cGMP-PKG cascade that may occur within the DRG in CCD rat. However, up to date, the detailed mechanisms between ion channels (TRPV4) and neurotransmitter (NO) in behavioral hyperalgesia remain unclear in the CCD model of neuropathic pain in rats.NF-κB consists of multi-submit proteins of Rel family (p50, p52, p65, c-Rel, and RelB). Under normal conditions or in resting cells, NF-κB is located in the cytoplasm and bound with inhibitory proteins, generically called inhibitory factor B (I-κB). When the cell is activated, proteins are degraded via the ubiquitin-proteasome pathway, leading to the release of the active form of NF-κB which translocates to the nuclear, where it regulates the expression of multiple genes involved neuropathic pain. Recently, activation of NF-κB has been shown to be involved in the transmission and processing of nociceptive information in the DRG and spinal cord. The analgesic effects of both low and high frequency electroacupuncture significantly decreased in NF-κB knockout mice. Further, intrathecal administration of NF-κB inhibitors, PDTC (pyrrolidine dithiocarbamate) and SN50, prior to glycoprotein partially attenuated glycoprotein-induced allodynia and pretreatment with NF-κB antisense oligodeoxynucleotides (ODNs) attenuated mechanical allodynia and thermal hyperalgesia in a chronic constriction injury (CCI) model of rats. These evidence suggests that NF-κB may be involved in the neuropathic pain. Therefore, we hypothesized that NF-κB might be involved in TRPV4-NO in thermal hyperalgesia in the CCD model. In order to test this hypothesis, we investigated the effects of PDTC and BAY, two specific NF-κB inhibitors, on paw withdrawal latency in the DRG in CCD model.ObjectiveTo investigate the effects of the two specific NF-κB inhibitors, PDTC and BAY, on paw withdrawal latency in the DRG in CCD model.Methods1. Establish CCD modelMale Wistar rats (n=80), weighing150-180g, were purchased from the Animal Center of Shandong University and were housed in a controlled environment (50-60%relative humidity,12h light/dark cycle, room temperature23±2℃), with free access to food and water for at least7days before surgery. Rats were randomly divided into CCD groups and sham groups. In CCD rats, under pentobarbital sodium anesthesia (Nembutal,50mg/kg ip), the transverse process and intervertebral foramina of L4and L5were exposed unilaterally as previously described. A stainless steel L-shaped rod (0.63-mm diameter and4-mm length) was inserted into each foramen, one at L4and the other at the L5level, to compress the DRG. The incision site was sutured in layers and penicillin was injected to prevent infection. The sham group underwent the same surgical procedure as described, but without the insertion of the rods. The animals did not show any autotomy and any loss of sensation following the surgery. All efforts were made to minimize animal suffering and the number of animals used. The study was approved by the Chinese Institutional Care Committee for the use of animals and was performed in accordance with the Helsinki declaration.2. Chemicals and treatment4a-phorbol12,13-didecanoate (4α-PDD, TRPV4synthetic activator) and pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor), were purchased from Sigma Company (St Louis, MO), BAY11-7082(BAY, an NF-κB inhibitor) was purchased from Beyotime Institute Biotechnology of (Shanghai, China). All chemicals were dissolved in saline. PDTC and BAY were respectively reconstituted in nuclease-free0.9%NaCl (10μg/μl) and administered into the spinal intrathecal space once a day for7days until the animals were sacrificed.3. Behavioral testingThermal hyperalgesia was assessed using the paw withdrawal latency (PWL) to radiant heat according to the protocol of Villetti and colleagues. Rats were placed in clean cages with a6-mm thick glass floor and acclimate to their surroundings for15-20min before testing. A high intensity light beam beneath the glass floor was focused on the plantar surface of the ipsilateral hind paw through the glass plate. The nociceptive endpoints in the radiant heat test were the characteristic lifting or withdrawal of the hind paw, while the time to the endpoint was defined as the paw withdrawal latency. To avoid tissue damaged, the cut-off time was set at20s. There were three trials per rat with5min intervals between trials. The rats were tested on each of2successive days before surgery. Postoperative tests were performed2h before chemicals treatment on the7th day after surgery. Additional tests were conducted1,2,4,8, and24h after injection of chemicals or saline into the subarachnoid space on the midline between the L4and L5vertebrae on the7th day after surgery.4. Data analysisA two-way repeated measures analysis of variance (RM ANOVA) was used to compare the differences in PWL over time between several treatment and control groups.Results1. Effects of PDTC (a specific inhibitor of NF-κB) on CCD-induced thermal hyperalgesia in ratsThe PWL was significantly shorter in CCD saline group (n=8) than in sham group (n=8, F=245.054, P<0.001). The intrathecal administration of the inhibitor of NF-κB PDTC in concentration range of10-1-10-2M produced a significant dose-dependent reduction of the PWL when compared with saline group, but PDTC in a concentration of the10-3M had no effects on thermal hyperalgesia (n=8, P>0.05). The significant reduction of the thermal hyperalgesia was observed at1h (P<0.05), peaked at4h (P<0.05), and lasted for about8h post-injection (P<0.05). There were no significant differences between groups in CCD rats at24h after administration of PDTC (n=8, all P>0.05).2. Effects of BAY (a specific inhibitor of NF-κB) on CCD-induced thermal hyperalgesia in ratsThe PWL was significantly shorter in CCD saline group (n=8) than in sham group (n=8, F=253.816, P<0.001). Intrathecal pretreatment with BAY (100-50μM) significantly and dose-dependently inhibited CCD-induced thermal hyperalgesia when compared with saline group. The significant decrease of the thermal hyperalgesia was observed from1to8h (P<0.05), peaked at4h (P<0.05). The intrathecal administration of BAY in concentration of (25μM) had no effects on thermal hyperalgesia when compared to saline group (n=8, P>0.05).3. Effects of4a-PDD on the PDTC-induced suppression of thermal hyperalgesia in rats4α-PDD (the TRPV4agonist,1nm) attenuated the suppressive effects of PDTC (10-1M) on CCD-induced thermal hyperalgesia when compared to PDTC (10-1M) alone (n=8, P<0.05).4. Effects of4α-PDD on the BAY-induced suppression of thermal hyperalgesia in rats4α-PDD (the TRPV4agonist,1nm) attenuated the suppressive effects of BAY (100μM) on CCD-induced thermal hyperalgesia when compared to BAY (100μM) alone (n=8, P<0.05).ConclusionsThis study demonstrates that NF-κB could be involved in TRPV4-NO pathway in CCD-induced thermal hyperalgesia following chronic compression of the dorsal root ganglion in rat. BackgroundNitric oxide (NO) as a mediator of pathological nociception, acting as an inter-and intracellular messenger molecule in the peripheral and central nervous system, plays a pivotal role in the development and maintenance of hyperalgesia and neuropathic pain. Three isoforms of NO synthase (NOS), including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), mediate NO synthesis from L-arginine in the presence of oxygen. These NOS isoforms’expression can be upregulated in nervous tissues under pathological conditions, suggesting that the pathophysiologic functions of NO in the nervous system may be regulated by altering expression and activity of NOS isoforms. Neuronal NOS is expressed in the neurons and produces predominantly NO in neuronal tissues. The contribution of nNOS-synthesized NO to nociceptive processing has been characterized in several neuropathic pain models. In recent years iNOS has become of significant interest in the pathophysiology of neuropathic pain. iNOS expressed in neuronal tissues is thought to be involved in mechanisms of hyperalgesia with some authors showing a specific role in the pain hypersensitivity associated with models of spinal cord injury. Increased nNOS and iNOS expression in neuronal tissues have also been observed in the chronic constriction injury (CCI) model and spinal nerve ligation, suggesting their role in the development of neuropathic pain, lines of evidence have demonstrated that nNOS and iNOS contribute to NO production involved in the maintenance of behavioural hyperalgesia developing after spinal nerve transection and sciatic nerve chronic constriction injury. However, its contribution to behavioural hypersensitivity in other animal models of neuropathic pain still remains unclear.NF-κB consists of multi-submit proteins of Rel family (p50, p52, p65, c-Rel, and RelB). Under normal conditions or in resting cells, NF-κB is located in the cytoplasm and bound with inhibitory proteins, generically called inhibitory factor B (I-κB). When the cell is activated, proteins are degraded via the ubiquitin-proteasome pathway, leading to the release of the active form of NF-κB which translocates to the nuclear, where it regulates the expression of multiple genes involved neuropathic pain. The NF-κB activation process has two pathways. First, it is the positive feedback pathway in the extracellular, this is an important mechanism for amplification of inflammatory reactions pro-inflammatory mediators TNF-alpha, IL-1and reactive oxygen medium (ROI) is a strong inducer of NF-kappa B. Pro-inflammatory mediators in the activation of NF-κB belongs to the positive feedback regulation, the induction of NF-κB activation can enhance TNF-a and IL-1β gene transcription, increased production and release, and then re-activation of NF-kappa B; also allows the increase in IL-6and IL-8production and release, resulting in further amplification of the initial inflammatory signal. Secondly, NF-kappa B activation start at the gene transcription of inflammatory mediators such as IκB-α and p105gene transcription were also upregulated. IκB gene the promoter region containing the κB sequence and kappa B sequence degradation, and thus can be combined with NF-κB, or even to enter the nucleus and activation of NF-κB binding, inhibition of NF-κ B and DNA binding site.Recently, activation of NF-κB has been shown to be involved in the transmission and processing of nociceptive information in the DRG and spinal cord. The analgesic effects of both low and high frequency electroacupuncture significantly decreased in NF-κB knockout mice. Further, intrathecal administration of NF-κB inhibitors, PDTC (pyrrolidine dithiocarbamate) and SN50, prior to glycoprotein partially attenuated glycoprotein-induced allodynia and pretreatment with NF-κB antisense oligodeoxynucleotides (ODNs) attenuated mechanical allodynia and thermal hyperalgesia in a chronic constriction injury (CCI) model of rats. These evidence suggests that NF-κB may be involved in the neuropathic pain. Therefore, we hypothesized that NF-κB might be involved in TRPV4-NO in thermal hyperalgesia in the CCD model. In order to test this hypothesis, we investigated the effects of PDTC and BAY, two specific NF-κB inhibitors, on paw withdrawal latency in the DRG in CCD model.Objective1. To detect the NO (nitric oxide) content in rats in the CCD model. 2. To investigate the effects of CCD on the expression levels of mRNA and protein of NF-κB and I-κB.3. To determine the role of NF-κB in CCD-induced TRPV4-NO pathway.Methods1. Establish CCD modelMale Wistar rats (n=80), weighing150-180g, were purchased from the Animal Center of Shandong University and were housed in a controlled environment (50-60%relative humidity,12h light/dark cycle, room temperature23±2℃), with free access to food and water for at least7days before surgery. Rats were randomly divided into CCD groups and sham groups. In CCD rats, under pentobarbital sodium anesthesia (Nembutal,50mg/kg ip), the transverse process and intervertebral foramina of L4and L5were exposed unilaterally as previously described. A stainless steel L-shaped rod (0.63-mm diameter and4-mm length) was inserted into each foramen, one at L4and the other at the L5level, to compress the DRG. The incision site was sutured in layers and penicillin was injected to prevent infection. The sham group underwent the same surgical procedure as described, but without the insertion of the rods. The animals did not show any autotomy and any loss of sensation following the surgery. All efforts were made to minimize animal suffering and the number of animals used.2. Measurement of NO content in DRGThe level of nitrite as a measure of NO production in DRG was determined with modified Griess reagent. A nitrite detection kit (Beyotime Biotech Inc., Jiangsu, People’s Republic of China) was used according to instructions provided by the manufacturer.3. Nuclear and cytoplasmic protein extraction Frozen tissues were homogenized in cytoplasmic extraction reagent A CERA (0.2ml)(BioTeke Corporation, Beijing, China). After vortexing for10seconds, the homogenates were centrifuged at15,000rpm for10minutes at4℃, the supernatant containing cytoplasmic proteins were collected and stored at-80℃. After centrifugation at15,000rpm for10min at4℃, the supernatant was collected as nuclear extracts and stored at-80℃.4. Western blotSix DRGs from3rats were pooled to extract protein as one sample and were quickly frozen in liquid nitrogen, then stored at-80℃for further examination. The amount of protein in the cytoplasmic or nuclear extracts was quantified by Protein Quantitative Analysis kit (k3001-BCA; Shenergy Biocolor, Shanghai, China). The body homogenates were electrophoresed and transferred to nitrocellulose membranes. Membranes were blocked for1h at room temperature in blocking buffer, washed in TTBS (0.1%Tween20,50mM Tris, and150mM NaCl), and incubated overnight with rabbit anti-NF-KB antibody (1:500, sc8008; Santa Cruz) or rabbit anti-IκB antibody (1:800, sc371; Santa Cruz). After washing three times, membranes were incubated for1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000).5. Real-time PCRThe mRNA levels of NF-κB,I-Kb and nNOS in the DRG in each group were quantified by real-time reverse-transcriptase polymerase chain reaction (RT PCR) using SYBR Green technology. 7. ImmunohistochemistryAt7days post-surgery rats were anesthetized with isoflurane and then transcardially perfused with4%paraformaldehyde in phosphate-buffered saline (PBS, pH7.4);the DRGs was immediately removed and post-fixed in4%paraformaldehyde (PFA) for7-8h, then the tissues were treated with dehydration, embedded in paraffin and cut into serial paraffin sections (5μm). The paraffin-embedded sections were heated for2h at120℃, deparaffinized in xylene, and rehydrated through graded ethanol at room temperature. After three rinses in PBS, microwave accentuation was used for10minutes; then the sections were washed in PBS,3%H2O2to eliminate endogenous peroxydase, and blocked with10%normal goat serum; then sections were incubated overnight at4℃with primary antibodies, anti-SP polyclonal antibody (1:100, Santa Cruz) or anti-CGRP polyclonal antibody (1:100, Santa Cruz). After the sections were washed, they were incubated with anti-rabbit IgG peroxidase conjugate (1:300, Zhongshan Gold Bridge, Beijing, China) for1h at room temperature. After several rinses, peroxidase was revealed by a3,3’-diaminobenzidine tetrahydrochloride substrate kit (Zhongshan Gold Bridge, Beijing, China). Finally, the sections were weakly counterstained with hematoxylin. In negative controls, the sections were incubated with PBS instead of primary antibody. All images were captured and analyzed by use of a color image analysis system composed of a video camera (Olympus DP71, Olympus Co., Japan), a light microscope (Olympus BX51, Olympus Co.), and Image-Pro Plus5.0software (Media Cybernetics Inc., USA).Results 1. Effects of PDTC and BAY (two specific inhibitors of NF-κB) on CCD-induced thermal hyperalgesia and the NO content in the DRGThe PWL was significantly shorter in CCD saline group (n=8) than in sham group (n=8, F=245.054, P<0.001). The intrathecal administration of the inhibitor of NF-κB PDTC in concentration range of10-1-10-2M produced a significant dose-dependent reduction of the PWL when compared with saline group, but PDTC in a concentration of the10-3M had no effects on thermal hyperalgesia (n=8, P>0.05). The significant reduction of the thermal hyperalgesia was observed at1h (P<0.05), peaked at4h (P<0.05),and lasted for about8h post-injection (P<0.05). There were no significant differences between groups in CCD rats at24h after administration of PDTC (n=8, all P>0.05). Intrathecal injection of PDTC (10-1M) significantly decreased the content of NO metabolites nitrite when compared to saline (n=8, P<0.05) in DRG in CCD rats. The level of NO in DRG showed the decrease1h post-injection significantly (P<0.05) and reached the minimum4h post-injection (P <0.05), followed by a gradual recovery (P<0.05), and the NO content was not significantly different from the saline group at24h post-injection (n=8, P>0.05). The reduction of NO content in DRG was negatively associated with the increase in PWL (r=-0.966, P<0.05) post-operation.The PWL was significantly shorter in CCD saline group (n=8) than in sham group (n=8, F=253.816, P<0.001). Intrathecal pretreatment with BAY (100-50μM) significantly and dose-dependently inhibited CCD-induced thermal hyperalgesia when compared with saline group. The significant decrease of the thermal hyperalgesia was observed from1to8h (P<0.05), peaked at4h (P<0.05). The intrathecal administration of BAY in concentration of (25μM) had no effects on thermal hyperalgesia when compared to saline group (n=8, P>0.05). Moreover, the level of NO in the DRG of CCD-treated rats was significantly suppressed by intrathecal pretreatment with BAY (100μM). The level of NO began to decrease1h post-injection (P<0.05) and reached the minimum4h post-injection (P<0.05), followed by a gradual recovery (P<0.05), and the NO content was not significantly different from the saline group at24h post-injection (n=8,P>0.05). The reduction of NO content in DRG was negatively associated with the increase in PWL (r=-0.952, P<0.05) post-operation.2. Effects of4a-PDD on the two specific inhibitors of NF-κB (PDTC and BAY) induced suppression of thermal hyperalgesia and NO content in the DRG4a-PDD (the TRPV4agonist, lnm) attenuated the suppressive effects of PDTC (10-1M) on CCD-induced thermal hyperalgesia when compared to PDTC (10-1M) alone (n=8, P<0.05). Moreover, the decrease in the content of NO induced by intrathecal injection of PDTC (10-1M) was also attenuated by pretreatment of4α-PDD (1nm)1h post-injection (n=8, P<0.05).4a-PDD (the TRPV4agonist, lnm) attenuated the suppressive effects of BAY (100μM) on CCD-induced thermal hyperalgesia when compared to BAY (100μM) alone (n=8, P<0.05). Moreover, the reduced content of NO induced by intrathecal injection of BAY (100μM) was also attenuated by pretreatment with4a-PDD (1nm)1h post-injection (n=8, P<0.05).3. Western blot of nuclear NF-κB、cytoplasmic I-κB and nNOS expression in the DRGThe effects of PDTC on the inhibition of NF-κB expression were confirmed at the protein level by western blot. Compared with the sham group, CCD rats exhibited significantly higher levels of NF-κB expression (P<0.05). The expression of NF-κB was markedly attenuated after intrathecal injection of PDTC (P<0.05). CCD rats showed significantly lower levels of cytoplasmic I-κB expression when compared with the sham group (P<0.05). The reduction of I-κB expression was reversed after intrathecal injection of PDTC (P<0.05). In addition, compared with the sham group, CCD rats showed significantly higher nNOS (P<0.05). The expression of nNOS was markedly attenuated after intrathecal injection of PDTC (P<0.05).4. Effects of CCD on gene expression of nuclear NF-κB、cytoplasmic I-κB and nNOS in the DRGThe effects of PDTC on the inhibition of NF-κB mRNA expression were confirmed at the protein level by RT-PCR. Compared with the sham group, CCD rats exhibited significantly higher levels of NF-κB mRNA expression (P<0.05). The expression of NF-κB mRNA was markedly attenuated after intrathecal injection of PDTC (P<0.05). CCD rats showed significantly lower levels of cytoplasmic I-κB mRNA expression when compared with the sham group (P<0.05). The reduction of I-κB mRNA expression was reversed after intrathecal injection of PDTC (P<0.05). In addition, compared with the sham group, CCD rats showed significantly higher nNOS mRNA (P<0.05). The expression of nNOS mRNA was markedly attenuated after intrathecal injection of PDTC (P<0.05).5. Effects of CCD on positive cell of NF-κB and nNOS in the DRGThe positive cell of NF-κB and nNOS in DRG were determined by immunohistochemistry. Our data showed that there was a significant increase in positive cell of NF-κB and nNOS in DRG compared with that in sham group rats (all P<0.05). The positive cell of NF-κB and nNOS in DRG were decreased intrathecal injection of PDTC as compared to CCD groups (all P<0.05).ConclusionsNF-κB might induce nitric oxide synthase, which mediated in TRPV4-NO pathway in CCD-induced molecular mechanisms.更多还原