蛇床子素对髓核致炎神经根痛大鼠脊髓背角p38 MAPK表达的影响
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摘要
目的研究蛇床子素(osthole,Ost)对髓核致炎神经根痛大鼠脊髓背角p38 MAPK信号相关通路表达的影响。方法♂成年SD大鼠156只随机分为5组:Blank组(n=36)、Sham组(n=36)、手术模型组(NP,n=36)、DMSO组(n=24)、蛇床子素组(Ost组,n=24),检测所有大鼠术前1 d,术后1、2、6、7、12、14、21、28 d的50%机械痛阈值(mechanical withdrawal threshold,MWT)。DMSO组和Ost组于术后d2经硬膜外腔分别注射100 m L·L~(-1)DMSO和20 g·L~(-1)蛇床子素,体积均为50μL。分别于术后7、14 d取出术侧腰段脊髓背角,Western blot检测p-p38、IL~(-1)8、IL~(-1)8R蛋白的表达,q PCR检测IL~(-1)8 mRNA表达。结果与Blank组相比,Sham组在术后1 d 50%MWT降低(P<0.05),而NP组、DMSO组及Ost组术后各时点的50%MWT降低(P<0.05);术后d 7 p-p38、IL~(-1)8蛋白及IL~(-1)8 mRNA表达上调(P<0.05),而IL~(-1)8R蛋白在术后d 14表达上调(P<0.05);硬膜外给予蛇床子素后,与DMSO组相比,Ost组各个时间点的50%MWT升高,持续至术后28 d(P<0.05),同时,术后d 7伴有p-p38、IL~(-1)8蛋白及IL~(-1)8 mRNA表达下降;术后d 14,IL~(-1)8R蛋白表达降低(P<0.05);p-p38蛋白相对浓度和IL~(-1)8 mRNA含量进行相关性分析,Spearman秩相关系数R=0.9(P<0.05)。结论髓核致炎大鼠脊髓背角p-p38蛋白及IL~(-1)8可能参与了早期炎性痛形成,而IL~(-1)8R在疼痛的维持中发挥作用,蛇床子素长期有效缓解髓核致炎大鼠神经根痛,可能与抑制p38 MAPK信号相关通路有关。
Aim To observe the effect of epidurally application of osthole on the model of nucleus pulposusinduced inflammatory radicular pain and the expression of p38 MAPK signaling related pathway in the spinal dorsal horn of rats. Methods The model of radicular pain was generated by putting nucleus pulposus to the L5 dorsal root ganglion( DRG). 50% MWT was measured using Von Frey filaments to calculate mechanical pain threshold before and after operation. 50 μL of 20 g · L~(-1) osthole was administered epidurally in group Ost and 50 μL of 100 m L·L~(-1) DMSO in group DMSO at postoperative day( POD). The expression of phosphorylated p38( p-p38),IL~(-1)8 and IL~(-1)8 R in the lumbar spinal dorsal horn was detected by Western blot. IL~(-1)8 mRNA was assessed by real-time PCR.Results The mechanical pain threshold significantly decreased after operation( P < 0. 05),while the expression of protein p-p38 MAPK,IL~(-1)8,IL~(-1)8 R and IL~(-1)8 mRNA was significantly different. Compared with DMSO group,50% MWT was significantly increased and accompanied with the decrease of protein p-p38,IL~(-1)8,IL~(-1)8 R and IL~(-1)8 mRNA in Ost group after drug administration( P < 0. 05). The correlation analysis between protein concentration of p38 MAPK and IL~(-1)8 mRNA showed that the Spearman correlation coefficient was 0. 9( P < 0. 05). Conclusion p-p38 and IL~(-1)8 of spinal dorsal horn participate in the rat model with inflammatory radicular pain induced by nucleus pulposus,and IL~(-1)8 R plays a role in maintenance of the pain. Osthole administered epidurally in the early stage of pain could alleviate the pain for a long time,which may be related with inhibiting p38 MAPK signaling related pathways.
Aim To observe the effect of epidurally application of osthole on the model of nucleus pulposusinduced inflammatory radicular pain and the expression of p38 MAPK signaling related pathway in the spinal dorsal horn of rats. Methods The model of radicular pain was generated by putting nucleus pulposus to the L5 dorsal root ganglion( DRG). 50% MWT was measured using Von Frey filaments to calculate mechanical pain threshold before and after operation. 50 μL of 20 g · L~(-1) osthole was administered epidurally in group Ost and 50 μL of 100 m L·L~(-1) DMSO in group DMSO at postoperative day( POD). The expression of phosphorylated p38( p-p38),IL~(-1)8 and IL~(-1)8 R in the lumbar spinal dorsal horn was detected by Western blot. IL~(-1)8 mRNA was assessed by real-time PCR.Results The mechanical pain threshold significantly decreased after operation( P < 0. 05),while the expression of protein p-p38 MAPK,IL~(-1)8,IL~(-1)8 R and IL~(-1)8 mRNA was significantly different. Compared with DMSO group,50% MWT was significantly increased and accompanied with the decrease of protein p-p38,IL~(-1)8,IL~(-1)8 R and IL~(-1)8 mRNA in Ost group after drug administration( P < 0. 05). The correlation analysis between protein concentration of p38 MAPK and IL~(-1)8 mRNA showed that the Spearman correlation coefficient was 0. 9( P < 0. 05). Conclusion p-p38 and IL~(-1)8 of spinal dorsal horn participate in the rat model with inflammatory radicular pain induced by nucleus pulposus,and IL~(-1)8 R plays a role in maintenance of the pain. Osthole administered epidurally in the early stage of pain could alleviate the pain for a long time,which may be related with inhibiting p38 MAPK signaling related pathways.
引文
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[3]吴海璇,冯璐璐,徐辉,等.蛇床子素对髓核致神经根炎性痛大鼠ERK/MAPK信号通路及COX-2 mRNA表达的影响[J].中国药理学通报,2014,30(8):1096-101.[3]Wu H X,Feng L L,Xu H,et al.Effect of osthole on ERK/MAPKsignaling pathway and the expression of COX-2 mRNA in the spinal dorsal horn of rats with nucleus pulposus-induced inflammatory radicular pain[J].Chin Pharmacol Bull,2014,30(8):1096-101.
[4]吴海璇,冯璐璐,贺秋兰,等.蛇床子素对髓核致坐骨神经痛大鼠DRG中CGRPR1表达的影响[J].中国药理学通报,2014,30(1):49-54.[4]Wu H X,Feng L L,He Q L,et al.Effect of osthole on expression of CGRPR1 in DRG of rats with nucleus pulposus-induced sciatica[J].Chin Pharmacol Bull,2014,30(1):49-54.
[5]贺秋兰,魏明,李梅娜,等.蛇床子素对酸诱发髓核致炎大鼠神经节神经元的膜电位改变抑制的作用[J].中国药理学通报,2010,26(11):1459-62.[5]He Q L,Wei M,Li M N,et al.Osthole inhibited the acid-evoked change of membrane potential of rats’dorsal root ganglion neuron following application of nucleus pulposus[J].Chin Pharmacol Bull,2010,26(11):1459-62.
[6]魏明,张劲军,贺秋兰,等.新型腰椎间盘突出致坐骨神经痛大鼠模型及硬膜外腔置管方法的建立[J].中国疼痛医学杂志,2011,17(12):744-8.[6]Wei M,Zhang J J,He Q L.Establishment of new rat model for sciatica induced by lumbar disc herniation and new method for epidural catheterization[J].Chin J Pain Med,2011,17(12):744-8.
[7]Obata K,Noguchi K.MAPK activation in nociceptive neurons and pain hypersensitivity[J].Life Sci,2004,74(21):2643-53.
[8]Kumar S,Boehm J,Lee J C.p38 MAP kinases:key signalling molecules as therapeutic targets for inflammatory diseases[J].Nat Rev Drug Discov,2003,2:717-26.
[9]Ito T,Ohtori S,Inoue G,et al.Glial phosphorylated p38 MAPkinase mediates pain in a rat model of lumbar disc herniation and induces motor dysfunction in a rat model of lumbar spinal canal stenosis[J].Spine,2007,32(2):159-67.
[10]Seki S,Sekiguchi M,Konno S I.Association between neurotrophic factor expression and pain-related behavior induced by nucleus pulposus applied to rat nerve root[J].Spine,2017 Apr 12.[Epub ahead of print]
[11]颜玮茹,廖春梅.白细胞介素-18与神经系统疾病关系的研究进展[J].中西医结合心脑血管病杂志,2016,14(8):855-9.[11]Yan W R,Liao C M,Advances in the study of the relationship between interleukin-18 and nervous system diseases[J].Chin J Integr Med Cardio-Cerebrovasc Dis,2016,14(8):855-9.
[12]Cao W,Zou Y,Su S,et al.Chlamydial plasmid-encoded protein p ORF5 induces production of IL-1βand IL-18 via NALP3 inflammasome activation and p38 MAPK pathway[J].Int J Clin Exp Med,2015,8(11):20368-79.
[13]Porreca F,Ossipov M H,Gebhart G F.Chronic pain and medullary descending facilitation[J].Trends Neurosci,2002,25(6):319-25.
[14]龚晓丽,郭秀萍,周涤,等.独活寄生汤口服及离子导入治疗腰椎间盘突出症临床研究[J].中医学报,2016,220(9):1394-7.[14]Gong X L,Guo X P,Zhou D,et al.Clinical research of Duhuojisheng decoction combined with iontophoresis in treatment of lumbar disc herniation[J].China J Chin Med,2016,220(9):1394-7.
[15]孔令雷,胡金凤,陈乃宏.香豆素类化合物药理和毒理作用的研究进展[J].中国药理学通报,2012,28(2):165-9.[15]Kong L L,Hu J F,Chen N H.Advances in pharmacology and toxicology of coumarins[J].Chin Pharmacol Bull,2012,28(2):165-9.
[16]孔亮,姚璎珈,教亚男,等.蛇床子素对机械性脑损伤小鼠的抗炎抗凋亡作用研究[J].中国药理学通报,2015,31(7):999-1004.[16]Kong L,Yao Y J,Jiao Y N,et al.Anti-apoptosis and anti-inflammatory effect of osthole in mice following stab wound injury[J].Chin Pharmacol Bull,2015,31(7):999-1004.
[17]Wu H X,Wang Y M,Xu H,et al.Osthole,a coumadin analog from Cnidium monnieri(L.)Cusson,ameliorates nucleus pulposus-induced radicular inflammatory pain by inhibiting the activation of extracellular signal-regulated kinase in rats[J].Pharmacology,2017,100:74-82.
[2]Wei M,Mo S L,Nabar N R,et al.Modification of rat model of sciatica induced by lumber disc herniation and anti-inflammatory effect of osthole given by epidural catheterization[J].Pharmacology,2012,90(5-6):251-63.
[3]吴海璇,冯璐璐,徐辉,等.蛇床子素对髓核致神经根炎性痛大鼠ERK/MAPK信号通路及COX-2 mRNA表达的影响[J].中国药理学通报,2014,30(8):1096-101.[3]Wu H X,Feng L L,Xu H,et al.Effect of osthole on ERK/MAPKsignaling pathway and the expression of COX-2 mRNA in the spinal dorsal horn of rats with nucleus pulposus-induced inflammatory radicular pain[J].Chin Pharmacol Bull,2014,30(8):1096-101.
[4]吴海璇,冯璐璐,贺秋兰,等.蛇床子素对髓核致坐骨神经痛大鼠DRG中CGRPR1表达的影响[J].中国药理学通报,2014,30(1):49-54.[4]Wu H X,Feng L L,He Q L,et al.Effect of osthole on expression of CGRPR1 in DRG of rats with nucleus pulposus-induced sciatica[J].Chin Pharmacol Bull,2014,30(1):49-54.
[5]贺秋兰,魏明,李梅娜,等.蛇床子素对酸诱发髓核致炎大鼠神经节神经元的膜电位改变抑制的作用[J].中国药理学通报,2010,26(11):1459-62.[5]He Q L,Wei M,Li M N,et al.Osthole inhibited the acid-evoked change of membrane potential of rats’dorsal root ganglion neuron following application of nucleus pulposus[J].Chin Pharmacol Bull,2010,26(11):1459-62.
[6]魏明,张劲军,贺秋兰,等.新型腰椎间盘突出致坐骨神经痛大鼠模型及硬膜外腔置管方法的建立[J].中国疼痛医学杂志,2011,17(12):744-8.[6]Wei M,Zhang J J,He Q L.Establishment of new rat model for sciatica induced by lumbar disc herniation and new method for epidural catheterization[J].Chin J Pain Med,2011,17(12):744-8.
[7]Obata K,Noguchi K.MAPK activation in nociceptive neurons and pain hypersensitivity[J].Life Sci,2004,74(21):2643-53.
[8]Kumar S,Boehm J,Lee J C.p38 MAP kinases:key signalling molecules as therapeutic targets for inflammatory diseases[J].Nat Rev Drug Discov,2003,2:717-26.
[9]Ito T,Ohtori S,Inoue G,et al.Glial phosphorylated p38 MAPkinase mediates pain in a rat model of lumbar disc herniation and induces motor dysfunction in a rat model of lumbar spinal canal stenosis[J].Spine,2007,32(2):159-67.
[10]Seki S,Sekiguchi M,Konno S I.Association between neurotrophic factor expression and pain-related behavior induced by nucleus pulposus applied to rat nerve root[J].Spine,2017 Apr 12.[Epub ahead of print]
[11]颜玮茹,廖春梅.白细胞介素-18与神经系统疾病关系的研究进展[J].中西医结合心脑血管病杂志,2016,14(8):855-9.[11]Yan W R,Liao C M,Advances in the study of the relationship between interleukin-18 and nervous system diseases[J].Chin J Integr Med Cardio-Cerebrovasc Dis,2016,14(8):855-9.
[12]Cao W,Zou Y,Su S,et al.Chlamydial plasmid-encoded protein p ORF5 induces production of IL-1βand IL-18 via NALP3 inflammasome activation and p38 MAPK pathway[J].Int J Clin Exp Med,2015,8(11):20368-79.
[13]Porreca F,Ossipov M H,Gebhart G F.Chronic pain and medullary descending facilitation[J].Trends Neurosci,2002,25(6):319-25.
[14]龚晓丽,郭秀萍,周涤,等.独活寄生汤口服及离子导入治疗腰椎间盘突出症临床研究[J].中医学报,2016,220(9):1394-7.[14]Gong X L,Guo X P,Zhou D,et al.Clinical research of Duhuojisheng decoction combined with iontophoresis in treatment of lumbar disc herniation[J].China J Chin Med,2016,220(9):1394-7.
[15]孔令雷,胡金凤,陈乃宏.香豆素类化合物药理和毒理作用的研究进展[J].中国药理学通报,2012,28(2):165-9.[15]Kong L L,Hu J F,Chen N H.Advances in pharmacology and toxicology of coumarins[J].Chin Pharmacol Bull,2012,28(2):165-9.
[16]孔亮,姚璎珈,教亚男,等.蛇床子素对机械性脑损伤小鼠的抗炎抗凋亡作用研究[J].中国药理学通报,2015,31(7):999-1004.[16]Kong L,Yao Y J,Jiao Y N,et al.Anti-apoptosis and anti-inflammatory effect of osthole in mice following stab wound injury[J].Chin Pharmacol Bull,2015,31(7):999-1004.
[17]Wu H X,Wang Y M,Xu H,et al.Osthole,a coumadin analog from Cnidium monnieri(L.)Cusson,ameliorates nucleus pulposus-induced radicular inflammatory pain by inhibiting the activation of extracellular signal-regulated kinase in rats[J].Pharmacology,2017,100:74-82.