en
×

分享给微信好友或者朋友圈

使用微信“扫一扫”功能。
通讯作者:

恽惠方,E-mail:yhfdoctor@163.com

中图分类号:R364.5

文献标识码:A

文章编号:1007-4368(2022)10-1364-08

DOI:10.7655/NYDXBNS20221003

参考文献 1
TANEJA A,DELLA PASQUA O,DANHOF M.Challeng⁃ es in translational drug research in neuropathic and in⁃ flammatory pain:the prerequisites for a new paradigm[J].Eur J Clin Pharmacol,2017,73(10):1219-1236
参考文献 2
RONCHETTI S,MIGLIORATI G,DELFINO D V.Associ⁃ ation of inflammatory mediators with pain perception[J].Biomed Pharmacother,2017,96:1445-1452
参考文献 3
YE Z H,ZHANG M X,DING N,et al.Antinociceptive ef⁃ fects of dezocine on complete Freund’s adjuvant⁃induced inflammatory pain in rats[J].Exp Ther Med,2018,15(6):5469-5474
参考文献 4
DEMIR I E,SCHÄFER K H,TIEFTRUNK E,et al.Neu⁃ ral plasticity in the gastrointestinal tract:chronic inflam⁃ mation,neurotrophic signals,and hypersensitivity[J].Acta Neuropathol,2013,125(4):491-509
参考文献 5
PATNAIK S S,LAGANÀ A S,VITALE S G,et al.Etiolo⁃ gy,pathophysiology and biomarkers of interstitial cystitis/painful bladder syndrome[J].Arch Gynecol Obstet,2017,295(6):1341-1359
参考文献 6
HUANG Y Y,CHEN S R,CHEN H,et al.Endogenous transient receptor potential ankyrin 1 and vanilloid 1 ac⁃ tivity potentiates glutamatergic input to spinal lamina I neurons in inflammatory pain[J].J Neurochem,2019,149(3):381-398
参考文献 7
MULEY M M,KRUSTEV E,MCDOUGALL J J.Preclini⁃ cal assessment of inflammatory pain[J].CNS Neurosci Ther,2016,22(2):88-101
参考文献 8
MONISHA B A,KUMAR N,TIKU A B.Emodin and its role in chronic diseases[J].Anti Inflamm Nutraceuticals Chronic Dis,2016,928:47-73
参考文献 9
YI J,YANG J,HE R,et al.Emodin enhances arsenic tri⁃ oxide⁃induced apoptosis via generation of reactive oxygen species and inhibition of survival signaling[J].Cancer Res,2004,64(1):108-116
参考文献 10
LI H L,CHEN H L,LI H,et al.Regulatory effects of emo⁃ din on NF ⁃ kappaB activation and inflammatory cytokine expression in RAW 264.7 macrophages[J].Int J Mol Med,2005,16(1):41-47
参考文献 11
MA C,WEN B,ZHANG Q,et al.Emodin induces apopto⁃ sis and autophagy of fibroblasts obtained from patient with ankylosing spondylitis[J].Drug Des Dev Ther,2019,13:601-609
参考文献 12
SCHWARZ S,WANG K,YU W J,et al.Emodin inhibits current through SARS ⁃ associated coronavirus 3a protein [J].Antivir Res,2011,90(1):64-69
参考文献 13
DONG X,FU J,YIN X B,et al.Emodin:a review of its pharmacology,toxicity and pharmacokinetics[J].Phytoth⁃ er Res,2016,30(8):1207-1218
参考文献 14
XIANG H C,LIN L X,HU X F,et al.AMPK activation at⁃ tenuates inflammatory pain through inhibiting NF⁃κB acti⁃ vation and IL ⁃ 1β expression[J].J Neuroinflammation,2019,16(1):34
参考文献 15
CHOI G,YANG T J,YOO S,et al.TRPV4⁃mediated anti⁃ nociceptive effect of suberanilohydroxamic acid on me⁃ chanical pain[J].Mol Neurobiol,2019,56(1):444-453
参考文献 16
GHASEMLOU N,CHIU I M,JULIEN J P,et al.CD11b+ Ly6G ⁃ myeloid cells mediate mechanical inflammatory pain hypersensitivity[J].PNAS,2015,112(49):E6808-E6817
参考文献 17
LI F J,HUO J H,ZHUANG Y,et al.Anti⁃nociceptive and anti ⁃inflammatory effects of the ethanol extract of Arenga pinnata(Wurmb)merr.fruit[J].J Ethnopharmacol,2020,248:112349
参考文献 18
CHEN W H,TZEN J T C,HSIEH C L,et al.Attenuation of TRPV1 and TRPV4 expression and function in mouse inflammatory pain models using electroacupuncture[J].Evid Based Complementary Altern Med,2012,2012:636848
参考文献 19
NAVARRO ⁃ ALVAREZ N,GONÇALVES B M M,AN⁃ DREWS A R,et al.A CFA⁃induced model of inflammato⁃ ry skin disease in miniature swine[J].Int J Inflamm,2018,2018:6916920
参考文献 20
ZHANG C J,WANG Y,PENG Y N,et al.METTL3 regu⁃ lates inflammatory pain by modulating m6A⁃dependent pri⁃ miR⁃365⁃3p processing[J].FASEB J,2020,34(1):122-132
参考文献 21
FENG S F,YU H H,YU Y,et al.Levels of inflammatory cytokines IL⁃1β,IL⁃6,IL⁃8,IL⁃17A,and TNF⁃α in aque⁃ ous humour of patients with diabetic retinopathy[J].J Di⁃ abetes Res,2018,2018:8546423
参考文献 22
YANG Q Q,LI H N,ZHANG S T,et al.Red nucleus IL⁃6 mediates the maintenance of neuropathic pain by induc⁃ ing the productions of TNF ⁃ α and IL ⁃ 1β through the JAK2/STAT3 and ERK signaling pathways[J].Neuropa⁃ thology,2020,40(4):347-357
参考文献 23
李秋月,许海玉,杨洪军.促炎因子TNF⁃α,IL⁃1β,IL⁃6 在神经病理性疼痛中的研究进展[J].中国中药杂志,2017,42(19):3709-3712
参考文献 24
KAMEDA T,ZVICK J,VUK M,et al.Expression and ac⁃ tivity of TRPA1 and TRPV1 in the intervertebral disc:as⁃ sociation with inflammation and matrix remodeling[J].Int J Mol Sci,2019,20(7):1767
参考文献 25
VINCENT F,DUNCTON M A J.TRPV4 agonists and an⁃ tagonists[J].Curr Top Med Chem,2011,11(17):2216-2226
参考文献 26
SUI F,HUO H R,ZHANG C B,et al.Emodin down⁃regu⁃ lates expression of TRPV1 mRNA and its function in DRG neurons in vitro[J].Am J Chin Med,2010,38(4):789-800
目录contents

    摘要

    目的:研究大黄素(emodin)对弗氏完全佐剂(complete Freund’s adjuvant,CFA)诱发的小鼠炎性疼痛的治疗作用及其可能的分子机制。方法:CFA注射于C57BL/6小鼠右后爪足背侧皮下诱导炎性疼痛模型,并用大黄素溶液腹腔注射进行干预。疼痛阈值通过 von Frey 试验和热板试验进行评估。RT-qPCR 和 ELISA 测定炎症细胞因子肿瘤坏死因子(tumor necrosis factor, TNF)-α、白细胞介素(interleukin,IL)-1β和IL-6的表达。Western blot检测背根神经节(dorsal root ganglion,DRG)中瞬时受体电位香草酸1(transient receptor potential vanillic acid 1,TRPV1)和瞬时受体电位香草酸4(transient receptor potential vanillic acid 4, TRPV4)蛋白表达。结果:CFA组小鼠的机械疼痛和热痛阈值显著低于对照组(P < 0.05)。大黄素治疗可显著提高 CFA 诱导的炎性疼痛小鼠的机械疼痛和热痛阈值(P < 0.05)。与对照组相比,大黄素干预能够显著降低CFA炎性疼痛小鼠DRG和血清中炎症细胞因子TNF-α、IL-1β、IL-6以及DRG中TRPV1和TRPV4的表达水平(P < 0.05)。结论:大黄素可能通过下调DRG 和血清中炎症细胞因子TNF-α、IL-1β、IL-6以及DRG中疼痛相关离子通道TRPV1和TRPV4的表达来缓解CFA诱导的炎性疼痛。

    Abstract

    Objective:To study the therapeutic effect of emodin on inflammatory pain induced by complete Freund’s adjuvant (CFA)in mice and its possible molecular mechanism. Methods:CFA was injected subcutaneously on the dorsal side of the right hind paw of C57BL/6 to induce an inflammatory pain model,and emodin solution was injected intraperitoneally for intervention. The pain threshold was evaluated by the von Frey test and the hot plate test. qRT - PCR and ELISA measure the expression of inflammatory cytokines tumor necrosis factor α(TNF -α),interleukin -1β(IL -1β)and interleukin -6(IL -6). Western blot was used to detect the expression of transient receptor potential vanillic acid 1(TRPV1)and transient receptor potential vanillic acid 4(TRPV4)in dorsal root ganglion(DRG). Results:The mechanical pain and thermal pain thresholds of mice in the CFA group were significantly lower than those of the control group(P < 0.05). Emodin treatment significantly increased mechanical pain and thermal pain threshold in mice with induced inflammatory pain(P < 0.05). Compared with the control group,emodin intervention can not only significantly reduce the inflammatory cytokines TNF - α,IL - 1β and IL - 6 in DRG and serum of mice with CFA inflammatory pain,but also reduce the expression levels of TRPV1 and TRPV4 in DRG(P < 0.05). Conclusion:Emodin relieves CFA - induced inflammatory pain by reducing the content of inflammatory cytokines TNF-α,IL-1β and IL-6 in DRG and serum,and down-regulating the expression of pain- related ion channels TRPV1 and TRPV4 in DRG.

  • 疼痛是日常生活和临床中最常见的疾病之一,其中炎性疼痛最为常见[1]。炎性疼痛与炎症发生的区域紧密相关,这些炎症发生部位中的伤害感受器 [位于三叉神经和背根神经节(dorsal root ganglion, DRG)中]通常在检测到外界有害的化学、热和机械刺激后,将相关信息传递至大脑并产生痛觉[2]。在炎症发生部位,机体通常对有害刺激的反应增强 (痛觉过敏)或在正常无害刺激的条件下(痛觉异常)触发疼痛[3]。炎症如不及时治愈,炎性疼痛就会逐渐发展成为慢性疼痛,即使后期导致炎症的伤害得到干预,疼痛依然长时间持续存在[4]。众所周知,炎性疼痛将严重影响患者的生活质量和劳动能力,由炎性疼痛发展成为慢性疼痛无疑给患者的家庭和社会带来巨大的经济负担[5]。据报道,机械损伤和高温等物理因素以及神经系统中的化学物质或炎性介质可引起炎性疼痛,但引起疼痛的确切机制目前尚未明确[6]。同时,限于目前的研究基础和治疗水平,炎性疼痛的临床治疗仍是一个重要问题[7]

  • 大黄素(1,3,8⁃三羟基⁃6⁃甲基蒽醌,emodin)是天然的蒽醌衍生物,是大黄和茜草科、鼠李科和豆科等多种药用中草药的主要成分[8]。研究表明,大黄素具有多种生物学特性,如抗病毒、抗菌、抗炎、免疫抑制和诱导凋亡等[9-12]。更重要的是,在大量的中医临床实践中,大黄常被用于治疗便秘或疼痛相关疾病[13]。为明确大黄素在炎性疼痛中的作用,本研究利用弗氏完全佐剂(complete Freund’s adjuvant, CFA)诱发的炎性疼痛小鼠模型,评估大黄素对CFA诱发的炎性疼痛小鼠模型的镇痛作用,并进一步阐明其镇痛的可能分子机制。

  • 1 材料和方法

  • 1.1 材料

  • C57BL/6小鼠购自赛业模式生物研究中心(太仓)有限公司[许可证号:SCXK(苏)2018⁃0003]。 CFA(Sigma公司,美国);大黄素(Sigma ⁃Aldrich公司,美国);TRIzol试剂(Invitrogen公司,美国);Prim⁃ erScriptMT RT Master Mix试剂盒、SYBR® Premix Ex TaqTM 试剂盒(TaKaRa公司,日本);肿瘤坏死因子α(tumor necrosis factor α,TNF⁃α)、白细胞介素 (interleukin,IL)⁃1β和IL⁃6免疫测定试剂盒(R&D Systems公司,美国);兔抗小鼠瞬时受体电位香草酸1 (transient receptor potential vanillic acid 1,TRPV1)、瞬时受体电位香草酸4(transient receptor potential vanillic acid 4,TRPV4)、GAPDH一抗和羊抗兔二抗 (Abcam公司,美国);Nanodrop 2000分光光度仪 (Thermo Fisher Scientific公司,美国)。

  • 1.2 方法

  • 1.2.1 动物处理

  • 所有动物实验经南京医科大学附属常州第二人民医院动物保护福利委员会批准。选取8周龄,体重(25±1)g健康雄性C57BL/6小鼠进行疼痛行为学试验以及炎性疼痛小鼠模型制作。将小鼠饲养在温度(25±1)℃可控的微型小鼠笼中,湿度保持在 (55±5)%,光照时间维持12h光照/12h黑暗周期,并给予充足的食物和无菌蒸馏水供其自由采食。

  • 在炎性疼痛小鼠模型制作前1天,将12只小鼠分为Sham组和炎性疼痛模型组两组,每组6只,并测定小鼠基础伤害感受性阈值。炎性疼痛模型制作如Xiang等[14] 报道,将25 μL CFA通过皮下注射于小鼠右后爪足背侧表面诱发炎性疼痛模型,Sham组则向小鼠右后爪足背侧表面注射等体积无菌生理盐水。在注射CFA后的7d内,每隔1d评估1次小鼠的机械痛敏和热痛敏行为。

  • 大黄素用DMSO溶解后,腹腔注射时用生理盐水稀释到工作浓度。将36只小鼠随机分为6组,每组6只,前5组在小鼠足背侧注射CFA 4d后分别将不同剂量的大黄素液(0、5、25、50、100mg/kg)通过腹腔注射给药。空白组则于小鼠足背侧表面和腹腔注射等体积无菌生理盐水。

  • 在确定最适镇痛效果的大黄素溶液剂量后,将24只小鼠分为4组,每组6只,CFA组和CFA+Emodin组注射CFA制作炎性疼痛模型,另外Sham组和Sham+Emodin组则注射等体积生理盐水。在CFA注射后第4天,分别给CFA+Emodin组和Sham+Emodin组小鼠腹腔注射最适镇痛效果的大黄素,同时以等体积的生理盐水分别对CFA组和Sham组小鼠进行腹腔注射。在大黄素腹腔注射0.5、2、4、8、12、24、 48h后分别评估小鼠的机械疼痛阈值和热痛阈值。

  • 1.2.2 疼痛行为学试验

  • 1.2.2.1机械性异常性疼痛测试

  • 使用冯·弗雷(von Frey)尼龙丝测量小鼠机械疼痛敏感性,以评估小鼠对连续增加的尼龙丝张力作出的缩爪反应。具体操作如下,将小鼠放置在底部带有金属丝网的塑料笼中,让每只小鼠在测试前适应15~30min。在每只小鼠右后脚掌底部的中间区域使用von Frey尼龙丝,刺激力起始量为0.008g,范围为0.008~2.000g,细丝接触后爪直至弯曲,而细丝应保持垂直于爪的作用侧约6~8s [15-16]。撤出阈值数据以g为单位确定为爪撤出阈值(paw withdrawal thresholds,PWT)。

  • 1.2.2.2热痛敏感性测试

  • 使用热板测试所有小鼠的爪缩回潜伏期(paw retraction latency,PWL),以评估小鼠的热痛敏感性。如Li等[17] 报道,热板由25cm×25cm的金属板和带玻璃的笼子组成。在测试之前,将每只小鼠放置在热板装置中10min,以使其在不加热的情况下适应。应确保小鼠的两个后爪在不施加任何力的情况下轻轻接触板的表面,并且将热板设置在55℃,截止时间为30s。疼痛反应通过小鼠抖动、缩回或舔脚掌的反应时间来界定,每隔15min进行重复测量,小鼠热痛敏感性程度的量化根据3次重复测量试验的平均值来确定。

  • 1.2.3 逆转录实时荧光定量聚合酶链反应(reverse transcription quantitative PCR,RT⁃qPCR)

  • 在CFA注射后第4天,以50mg/kg的大黄素溶液对小鼠进行腹腔注射,并在大黄素腹腔注射4h后收集各组小鼠L4~L6DRG。DRG收集方法如下:剪开小鼠背部皮肤,分离脊柱两旁的肌肉,用咬骨钳轻轻剥去棘突及两侧横突,暴露脊髓后,用镊子挑起脊髓可看到脊神经后根及其椎管处膨大的DRG,收集CFA组和Sham组小鼠L4~L6DRG。使用TRIzol提取每个样品的总RNA,并通过分光光度法对每个样品的RNA浓度进行定量。使用Primer⁃ ScriptMT RT Master Mix试剂盒将每个样品1 μg总RNA反转录为cDNA,然后进行qPCR实验。根据SYBR® Premix Ex TaqTM试剂盒操作步骤准备qPCR反应体系。将qPCR反应体系在95℃下孵育3min,然后将目标基因片段在95℃下10s和58℃下10s扩增40个循环。qPCR实验中所使用的引物序列来自Primer Bank在线网站,并使用NCBI Primer⁃BLAST进行引物特异性验证,具体序列如下:TNF⁃α正向引物5′⁃CAGGCGGTGCCTATGTCTC⁃3′,反向引物5′⁃ CGATCACCCCGAAGTTCAGTAG⁃3′。IL⁃1β正向引物5′⁃CAACCAACAAGTGATATTCTCCATG⁃3′,反向引物5′⁃GATCCACACTCTCCAGCTGCA⁃3′。IL⁃6正向引物5′⁃CCAAGAGGTGAGTGCTTCCC⁃3′,反向引物5′ ⁃ CTGTTGTTCAGACTCTCTCCCT ⁃ 3′。GAPDH正向引物5′⁃AGGTCGGTGTGAACGGATTTG⁃3′,反向引物5′⁃GGGGTCGTTGATGGCAACA⁃3′。根据公式2-ΔΔCt计算基因的表达水平,并将GAPDH基因的表达水平用作内部对照。

  • 1.2.4 酶联免疫吸附实验(enzyme⁃linked immuno⁃ sorbent assay,ELISA)

  • 为了检测小鼠血清炎症因子的表达水平,采用血清分离管分离血清,使血样室温凝集30min,然后1 000 g 离心15min。吸取血清样本之后即刻用于检测。使用酶联免疫试剂盒测定各组小鼠血清炎症细胞因子TNF⁃α、IL⁃1β和IL⁃6的含量。

  • 1.2.5 蛋白免疫印迹

  • 向各组小鼠DRG加入组织裂解液并于匀浆器中低温匀浆,将组织裂解液置于12 000r/min,4℃离心5min。采用BCA法测定各组样本蛋白浓度,向蛋白裂解液中加入SDS上样缓冲液并煮沸变性。蛋白样品经10%SDS聚丙烯酰胺凝胶电泳,转膜至PVDF膜,使用5%脱脂奶粉室温封闭1h后,加入兔抗小鼠TRPV1(ab6166)、TRPV4(ab39260)和GAPDH (ab9485)一抗(1∶1 000),于4℃过夜孵育,羊抗兔二抗(ab205718)(1∶3 000)室温孵育2h。滴加发光液,使用天能ECL发光仪捕获蛋白条带,Image J软件测定蛋白条带灰度值,与内参GAPDH比较,计算目的蛋白的相对表达水平。

  • 1.3 统计学方法

  • 所有数据统计分析和绘图均在GraphPad Prism 8中处理。实验数据表示为均数±标准差(x-±s), Student’s t 检验用于比较两组间差异,单因素或重复测量的方差分析用于多组间或不同时间点间比较,P< 0.05为差异有统计学意义。

  • 2 结果

  • 2.1 建立CFA诱导的小鼠炎性疼痛模型

  • 为了建立小鼠炎性疼痛模型,将25 μL CFA注射到8周龄C57BL/6小鼠后爪的足背侧。注射后数小时,可在注射部位观察到明显的局部炎症迹象 (如红紫色肿胀)。与Sham组相比,CFA组在机械敏感性(图1A)和热敏感性(图1B)测试中的疼痛阈值在CFA注射后的第1天显著降低(P< 0.001),并持续到第7天。结果表明,成功建立了CFA诱导的小鼠炎性疼痛模型。

  • 2.2 大黄素以剂量依赖方式缓解CFA引起的炎性疼痛

  • 为了评估大黄素对炎性疼痛的镇痛作用,在CFA注射后第4天,分别以5、25、50、100mg/kg的大黄素溶液对小鼠进行腹腔注射,并在大黄素腹腔注射2h后评估小鼠的机械疼痛阈值和热痛敏阈值。机械疼痛敏感性测试结果表明,腹腔注射大黄素可以剂量依赖方式缓解由CFA诱导的机械痛觉过敏 (图2A)。当注射大黄素的剂量为50mg/kg时,小鼠的机械疼痛阈值从CFA组(Emodin=0mg/kg)的 (0.02 ± 0.01)g显著升高到(0.27 ± 0.15)g(图2A, P< 0.001)。

  • 与此类似,在热板试验中,腹腔注射大黄素可以剂量依赖方式显著缓解由CFA诱导的热痛觉过敏 (图2B)。当注射大黄素的剂量为50mg/kg时,小鼠的PWL从CFA组(Emodin=0mg/kg)的(5.71±1.04)s升高到(8.11±1.11)s(图2B,P< 0.001)。以上结果表明,大黄素以剂量依赖的方式缓解CFA引起的机械疼痛过敏和热痛觉过敏。

  • 图1 CFA诱导的小鼠炎性疼痛模型的行为评估

  • Fig.1 Behavioral evaluation of CFA⁃induced inflammatory pain model in mice

  • 图2 大黄素以剂量依赖性方式缓解CFA引起的炎性疼痛

  • Fig.2 Emodin relieves CFA⁃induced inflammatory pain in a dose⁃dependent manner

  • 2.3 大黄素缓解CFA引起的炎性疼痛具有明显的时效性

  • 如图3A所示,在大黄素注射2、4、8h后,与对照组(CFA组)相比,腹腔注射大黄素可以显著缓解由CFA诱导的机械痛觉过敏(P< 0.05)。而在大黄素作用早期(注射0.5h后)和较长时间(注射12、24和48h)后,CFA组和CFA+Emodin组小鼠的机械疼痛敏感程度无显著差异。

  • 热板试验结果显示,在大黄素注射2、4h后,与CFA组相比,腹腔注射大黄素可以显著缓解由CFA诱导的热痛觉敏感(P< 0.01)。而在大黄素作用早期(注射0.5h后)和较长时间(注射8、12、24、48h) 后,CFA组和CFA+Emodin组小鼠的热疼痛敏感程度无显著差异。以上结果表明,大黄素缓解CFA引起的炎性疼痛具有明显时效性,大黄素在腹腔注射2~4h内,具有显著缓解CFA诱导的炎性疼痛的作用。

  • 图3 大黄素缓解CFA引起的炎性疼痛明显的时效性

  • Fig.3 Emodin relieves CFA⁃induced inflammatory pain with obvious timeliness

  • 2.4 大黄素下调CFA诱导的炎性疼痛小鼠的TNF⁃α、 IL⁃1β和IL⁃6水平

  • 为了揭示大黄素缓解CFA引起的炎性疼痛的可能机制,在CFA注射后第4天,以50mg/kg的大黄素溶液对小鼠进行腹腔注射(对照组腹腔注射等体积生理盐水),并在大黄素腹腔注射4h后分别获取各组小鼠L4~L5DRG和血清,通过RT⁃qPCR测定炎性细胞因子TNF⁃α、IL⁃1β和IL⁃6mRNA表达水平, ELISA测定各组小鼠血清中炎症细胞因子TNF⁃α、 IL⁃1β和IL⁃6的蛋白水平。如图4所示,相较于Sham组CFA能够显著促进小鼠DRG和血清中炎性细胞因子TNF⁃α、IL⁃1β和IL⁃6的表达;而当以50mg/kg的大黄素溶液对小鼠进行腹腔注射,4h后可显著降低由CFA诱导的炎性细胞因子TNF⁃α、IL⁃1β和IL⁃6的水平(P< 0.01)。结果表明,将大黄素注射到CFA引起的炎症性疼痛小鼠中,可显著降低炎性细胞因子TNF⁃α、IL⁃1β和IL⁃6的水平。

  • 2.5 大黄素抑制CFA诱导的TRPV1和TRPV4的表达

  • TRPV1和TRPV4被广泛报道参与疼痛进程,可以被诸如炎症等生理过程激活[1518]。结果如图5所示,相较于Sham组(1.00±0.13和1.00±0.12),CFA能显著上调DRG中TRPV1(1.95±0.21)和TRPV4 (1.72±0.18)的表达。当以50mg/kg的大黄素溶液腹腔注射后,可显著降低DRG中由CFA诱导的TRPV1 (1.28±0.10)和TRPV4(1.27±0.10)的水平。

  • 3 讨论

  • CFA由矿物油组成,含有完整或粉碎的热灭活分枝杆菌的悬浮液,其佐剂活性来自油性沉积物中抗原的持续释放和局部先天免疫反应的刺激,导致注射部位迟发的超敏反应,伴有强烈的炎症反应和痛觉过敏,因而被广泛应用于炎症和炎症性疼痛动物模型的研究[19]。与Zhang等[20] 的结果一致,将CFA注射到C57BL/6小鼠后爪的足背侧,能够在注射数小时后观察到后爪背侧出现明显的局部炎症迹象(红紫色肿胀)。于此同时,相较于Sham组, CFA组能够在长时间内出现异常的机械疼痛敏感性和热敏感性,在进一步的试验中也显示了CFA能够显著诱导小鼠DRG和血清中炎症细胞因子TNF⁃α、IL⁃1β和IL⁃6的表达。

  • 异常性疼痛是周围神经损伤具有的普遍症状,由CFA诱导的炎性疼痛模型的异常性疼痛有两种表现形式,即机械刺激诱发痛与热刺激诱发痛[7]。本研究通过在C57BL/6小鼠后爪的足背侧注射CFA诱导显著的机械疼痛敏感性和热敏感性,在该模型鼠上腹腔注射不同浓度的大黄素溶液发现,当注射较高浓度(25、50、100mg/kg)的大黄素溶液时,大黄素能够以剂量依赖的方式提高CFA引起的机械疼痛和热痛觉阈值。这也说明了大黄素具有提高CFA小鼠机械疼痛和热痛觉阈值,改善炎性疼痛的作用。

  • 炎性疼痛是一种常见的病理生理过程,是机体对外部物理或化学因素的应激反应,该过程通常涉及炎细胞因子(如TNF⁃α、IL⁃1β和IL⁃6)的积累[21]。大量研究表明,机体脊髓、前扣带回皮层和各种神经组织中炎性细胞因子TNF⁃α、IL⁃1β和IL⁃6的高表达通常伴有疼痛等症状的产生[22]。诸如TNF⁃α、IL⁃1β 和IL⁃6等炎症细胞因子被广泛认为是与疼痛发生和发展有关的重要生物分子[23]。于此同时,先前的研究表明大黄素具有抑菌和抗炎作用[8]。这也提示大黄素对CFA诱导的炎性疼痛的缓解作用可能与缓解炎症的进程有关。为揭示大黄素镇痛作用的可能机制,本研究在CFA诱导的炎性疼痛小鼠中,以50mg/kg的大黄素溶液给予腹腔注射干预,在大黄素处理4h后收集各组小鼠的L4~L6DRG组织和血清并检测TNF⁃α、IL⁃1β和IL⁃6的表达水平。与无大黄素干预的CFA组相比,以50mg/kg的大黄素溶液腹腔注射4h后能够显著降低CFA小鼠DRG和血清中炎症细胞因子TNF⁃α、IL⁃1β和IL⁃6的水平。

  • 图4 大黄素下调CFA诱导的炎性疼痛小鼠的TNF⁃α、IL⁃1β和IL⁃6水平

  • Fig.4 Emodin down⁃regulates the levels of TNF⁃α,IL⁃1β and IL⁃6in mice with CFA⁃induced inflammatory pain

  • 图5 大黄素抑制CFA诱导的TRPV1和TRPV4的表达

  • Fig.5 Emodin inhibits the expression of TRPV1and TRPV4induced by CFA

  • 研究表明,TRPV1和TRPV4可以作为伤害性机械刺激的感觉传感器,或在机械性痛觉过敏中起重要作用[24-25]。同时也有报道表明,注射CFA可以刺激痛觉过敏相关离子通道中TRPV1和TRPV4的表达[18]。与此一致,在注射CFA后,小鼠L4~L6背神经节中TRPV1和TRPV4的表达水平显著上调。当以50mg/kg的大黄素溶液腹腔注射后,可显著降低DRG中由CFA诱导的TRPV1和TRPV4表达水平。据报道,大黄素均能够在体外显著下调DRG神经元Trpv1的mRNA表达和辣椒素诱发的细胞钙离子信号[26]。大黄素调控TRPV1和TRPV4表达的分子机制目前仍未明确,仍需要进一步深入研究。

  • 综上所述,本研究表明大黄素对CFA诱导的炎性疼痛具有良好的镇痛作用,机制研究揭示大黄素可能通过下调DRG和血清中的炎症细胞因子TNF⁃α、IL⁃1β和IL⁃6以及DRG中疼痛相关离子通道TRPV1和TRPV4的表达来缓解炎性疼痛。

  • 参考文献

    • [1] TANEJA A,DELLA PASQUA O,DANHOF M.Challeng⁃ es in translational drug research in neuropathic and in⁃ flammatory pain:the prerequisites for a new paradigm[J].Eur J Clin Pharmacol,2017,73(10):1219-1236

    • [2] RONCHETTI S,MIGLIORATI G,DELFINO D V.Associ⁃ ation of inflammatory mediators with pain perception[J].Biomed Pharmacother,2017,96:1445-1452

    • [3] YE Z H,ZHANG M X,DING N,et al.Antinociceptive ef⁃ fects of dezocine on complete Freund’s adjuvant⁃induced inflammatory pain in rats[J].Exp Ther Med,2018,15(6):5469-5474

    • [4] DEMIR I E,SCHÄFER K H,TIEFTRUNK E,et al.Neu⁃ ral plasticity in the gastrointestinal tract:chronic inflam⁃ mation,neurotrophic signals,and hypersensitivity[J].Acta Neuropathol,2013,125(4):491-509

    • [5] PATNAIK S S,LAGANÀ A S,VITALE S G,et al.Etiolo⁃ gy,pathophysiology and biomarkers of interstitial cystitis/painful bladder syndrome[J].Arch Gynecol Obstet,2017,295(6):1341-1359

    • [6] HUANG Y Y,CHEN S R,CHEN H,et al.Endogenous transient receptor potential ankyrin 1 and vanilloid 1 ac⁃ tivity potentiates glutamatergic input to spinal lamina I neurons in inflammatory pain[J].J Neurochem,2019,149(3):381-398

    • [7] MULEY M M,KRUSTEV E,MCDOUGALL J J.Preclini⁃ cal assessment of inflammatory pain[J].CNS Neurosci Ther,2016,22(2):88-101

    • [8] MONISHA B A,KUMAR N,TIKU A B.Emodin and its role in chronic diseases[J].Anti Inflamm Nutraceuticals Chronic Dis,2016,928:47-73

    • [9] YI J,YANG J,HE R,et al.Emodin enhances arsenic tri⁃ oxide⁃induced apoptosis via generation of reactive oxygen species and inhibition of survival signaling[J].Cancer Res,2004,64(1):108-116

    • [10] LI H L,CHEN H L,LI H,et al.Regulatory effects of emo⁃ din on NF ⁃ kappaB activation and inflammatory cytokine expression in RAW 264.7 macrophages[J].Int J Mol Med,2005,16(1):41-47

    • [11] MA C,WEN B,ZHANG Q,et al.Emodin induces apopto⁃ sis and autophagy of fibroblasts obtained from patient with ankylosing spondylitis[J].Drug Des Dev Ther,2019,13:601-609

    • [12] SCHWARZ S,WANG K,YU W J,et al.Emodin inhibits current through SARS ⁃ associated coronavirus 3a protein [J].Antivir Res,2011,90(1):64-69

    • [13] DONG X,FU J,YIN X B,et al.Emodin:a review of its pharmacology,toxicity and pharmacokinetics[J].Phytoth⁃ er Res,2016,30(8):1207-1218

    • [14] XIANG H C,LIN L X,HU X F,et al.AMPK activation at⁃ tenuates inflammatory pain through inhibiting NF⁃κB acti⁃ vation and IL ⁃ 1β expression[J].J Neuroinflammation,2019,16(1):34

    • [15] CHOI G,YANG T J,YOO S,et al.TRPV4⁃mediated anti⁃ nociceptive effect of suberanilohydroxamic acid on me⁃ chanical pain[J].Mol Neurobiol,2019,56(1):444-453

    • [16] GHASEMLOU N,CHIU I M,JULIEN J P,et al.CD11b+ Ly6G ⁃ myeloid cells mediate mechanical inflammatory pain hypersensitivity[J].PNAS,2015,112(49):E6808-E6817

    • [17] LI F J,HUO J H,ZHUANG Y,et al.Anti⁃nociceptive and anti ⁃inflammatory effects of the ethanol extract of Arenga pinnata(Wurmb)merr.fruit[J].J Ethnopharmacol,2020,248:112349

    • [18] CHEN W H,TZEN J T C,HSIEH C L,et al.Attenuation of TRPV1 and TRPV4 expression and function in mouse inflammatory pain models using electroacupuncture[J].Evid Based Complementary Altern Med,2012,2012:636848

    • [19] NAVARRO ⁃ ALVAREZ N,GONÇALVES B M M,AN⁃ DREWS A R,et al.A CFA⁃induced model of inflammato⁃ ry skin disease in miniature swine[J].Int J Inflamm,2018,2018:6916920

    • [20] ZHANG C J,WANG Y,PENG Y N,et al.METTL3 regu⁃ lates inflammatory pain by modulating m6A⁃dependent pri⁃ miR⁃365⁃3p processing[J].FASEB J,2020,34(1):122-132

    • [21] FENG S F,YU H H,YU Y,et al.Levels of inflammatory cytokines IL⁃1β,IL⁃6,IL⁃8,IL⁃17A,and TNF⁃α in aque⁃ ous humour of patients with diabetic retinopathy[J].J Di⁃ abetes Res,2018,2018:8546423

    • [22] YANG Q Q,LI H N,ZHANG S T,et al.Red nucleus IL⁃6 mediates the maintenance of neuropathic pain by induc⁃ ing the productions of TNF ⁃ α and IL ⁃ 1β through the JAK2/STAT3 and ERK signaling pathways[J].Neuropa⁃ thology,2020,40(4):347-357

    • [23] 李秋月,许海玉,杨洪军.促炎因子TNF⁃α,IL⁃1β,IL⁃6 在神经病理性疼痛中的研究进展[J].中国中药杂志,2017,42(19):3709-3712

    • [24] KAMEDA T,ZVICK J,VUK M,et al.Expression and ac⁃ tivity of TRPA1 and TRPV1 in the intervertebral disc:as⁃ sociation with inflammation and matrix remodeling[J].Int J Mol Sci,2019,20(7):1767

    • [25] VINCENT F,DUNCTON M A J.TRPV4 agonists and an⁃ tagonists[J].Curr Top Med Chem,2011,11(17):2216-2226

    • [26] SUI F,HUO H R,ZHANG C B,et al.Emodin down⁃regu⁃ lates expression of TRPV1 mRNA and its function in DRG neurons in vitro[J].Am J Chin Med,2010,38(4):789-800

  • 参考文献

    • [1] TANEJA A,DELLA PASQUA O,DANHOF M.Challeng⁃ es in translational drug research in neuropathic and in⁃ flammatory pain:the prerequisites for a new paradigm[J].Eur J Clin Pharmacol,2017,73(10):1219-1236

    • [2] RONCHETTI S,MIGLIORATI G,DELFINO D V.Associ⁃ ation of inflammatory mediators with pain perception[J].Biomed Pharmacother,2017,96:1445-1452

    • [3] YE Z H,ZHANG M X,DING N,et al.Antinociceptive ef⁃ fects of dezocine on complete Freund’s adjuvant⁃induced inflammatory pain in rats[J].Exp Ther Med,2018,15(6):5469-5474

    • [4] DEMIR I E,SCHÄFER K H,TIEFTRUNK E,et al.Neu⁃ ral plasticity in the gastrointestinal tract:chronic inflam⁃ mation,neurotrophic signals,and hypersensitivity[J].Acta Neuropathol,2013,125(4):491-509

    • [5] PATNAIK S S,LAGANÀ A S,VITALE S G,et al.Etiolo⁃ gy,pathophysiology and biomarkers of interstitial cystitis/painful bladder syndrome[J].Arch Gynecol Obstet,2017,295(6):1341-1359

    • [6] HUANG Y Y,CHEN S R,CHEN H,et al.Endogenous transient receptor potential ankyrin 1 and vanilloid 1 ac⁃ tivity potentiates glutamatergic input to spinal lamina I neurons in inflammatory pain[J].J Neurochem,2019,149(3):381-398

    • [7] MULEY M M,KRUSTEV E,MCDOUGALL J J.Preclini⁃ cal assessment of inflammatory pain[J].CNS Neurosci Ther,2016,22(2):88-101

    • [8] MONISHA B A,KUMAR N,TIKU A B.Emodin and its role in chronic diseases[J].Anti Inflamm Nutraceuticals Chronic Dis,2016,928:47-73

    • [9] YI J,YANG J,HE R,et al.Emodin enhances arsenic tri⁃ oxide⁃induced apoptosis via generation of reactive oxygen species and inhibition of survival signaling[J].Cancer Res,2004,64(1):108-116

    • [10] LI H L,CHEN H L,LI H,et al.Regulatory effects of emo⁃ din on NF ⁃ kappaB activation and inflammatory cytokine expression in RAW 264.7 macrophages[J].Int J Mol Med,2005,16(1):41-47

    • [11] MA C,WEN B,ZHANG Q,et al.Emodin induces apopto⁃ sis and autophagy of fibroblasts obtained from patient with ankylosing spondylitis[J].Drug Des Dev Ther,2019,13:601-609

    • [12] SCHWARZ S,WANG K,YU W J,et al.Emodin inhibits current through SARS ⁃ associated coronavirus 3a protein [J].Antivir Res,2011,90(1):64-69

    • [13] DONG X,FU J,YIN X B,et al.Emodin:a review of its pharmacology,toxicity and pharmacokinetics[J].Phytoth⁃ er Res,2016,30(8):1207-1218

    • [14] XIANG H C,LIN L X,HU X F,et al.AMPK activation at⁃ tenuates inflammatory pain through inhibiting NF⁃κB acti⁃ vation and IL ⁃ 1β expression[J].J Neuroinflammation,2019,16(1):34

    • [15] CHOI G,YANG T J,YOO S,et al.TRPV4⁃mediated anti⁃ nociceptive effect of suberanilohydroxamic acid on me⁃ chanical pain[J].Mol Neurobiol,2019,56(1):444-453

    • [16] GHASEMLOU N,CHIU I M,JULIEN J P,et al.CD11b+ Ly6G ⁃ myeloid cells mediate mechanical inflammatory pain hypersensitivity[J].PNAS,2015,112(49):E6808-E6817

    • [17] LI F J,HUO J H,ZHUANG Y,et al.Anti⁃nociceptive and anti ⁃inflammatory effects of the ethanol extract of Arenga pinnata(Wurmb)merr.fruit[J].J Ethnopharmacol,2020,248:112349

    • [18] CHEN W H,TZEN J T C,HSIEH C L,et al.Attenuation of TRPV1 and TRPV4 expression and function in mouse inflammatory pain models using electroacupuncture[J].Evid Based Complementary Altern Med,2012,2012:636848

    • [19] NAVARRO ⁃ ALVAREZ N,GONÇALVES B M M,AN⁃ DREWS A R,et al.A CFA⁃induced model of inflammato⁃ ry skin disease in miniature swine[J].Int J Inflamm,2018,2018:6916920

    • [20] ZHANG C J,WANG Y,PENG Y N,et al.METTL3 regu⁃ lates inflammatory pain by modulating m6A⁃dependent pri⁃ miR⁃365⁃3p processing[J].FASEB J,2020,34(1):122-132

    • [21] FENG S F,YU H H,YU Y,et al.Levels of inflammatory cytokines IL⁃1β,IL⁃6,IL⁃8,IL⁃17A,and TNF⁃α in aque⁃ ous humour of patients with diabetic retinopathy[J].J Di⁃ abetes Res,2018,2018:8546423

    • [22] YANG Q Q,LI H N,ZHANG S T,et al.Red nucleus IL⁃6 mediates the maintenance of neuropathic pain by induc⁃ ing the productions of TNF ⁃ α and IL ⁃ 1β through the JAK2/STAT3 and ERK signaling pathways[J].Neuropa⁃ thology,2020,40(4):347-357

    • [23] 李秋月,许海玉,杨洪军.促炎因子TNF⁃α,IL⁃1β,IL⁃6 在神经病理性疼痛中的研究进展[J].中国中药杂志,2017,42(19):3709-3712

    • [24] KAMEDA T,ZVICK J,VUK M,et al.Expression and ac⁃ tivity of TRPA1 and TRPV1 in the intervertebral disc:as⁃ sociation with inflammation and matrix remodeling[J].Int J Mol Sci,2019,20(7):1767

    • [25] VINCENT F,DUNCTON M A J.TRPV4 agonists and an⁃ tagonists[J].Curr Top Med Chem,2011,11(17):2216-2226

    • [26] SUI F,HUO H R,ZHANG C B,et al.Emodin down⁃regu⁃ lates expression of TRPV1 mRNA and its function in DRG neurons in vitro[J].Am J Chin Med,2010,38(4):789-800

  • 通知关闭
    郑重声明