乌头汤缓解神经病理性疼痛的炎症网络调控机制研究
|
摘要
痛痹要方乌头汤临床治疗神经病理性疼痛(neuropathic pain, NP)疗效确切,但作用机制尚不完全清楚,本研究旨在探究乌头汤缓解NP的炎症网络调控机制。前期基于脊神经结扎(spinal nerve ligation, SNL)小鼠模型,通过全基因表达谱分析发现SNL发病相关基因集及乌头汤干预SNL药效相关基因集。本研究中"疾病基因-药物效应基因"互作网络及通路富集分析结果表明,乌头汤的镇痛网络靶标参与丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号通路的富集显著性最高(P=4.04E-12),其中AKT激酶、MAP激酶的激酶4 (MAP kinase kinase 4, MKK4)、终端激酶(c-Jun N-terminal kinase, JNK)和转录因子AP-1 (JUN)与NP发生、发展中具有重要意义的神经炎症密切相关。基于SNL大鼠模型的药效学实验结果表明,乌头汤可显著升高SNL大鼠的机械痛阈值(P<0.01)和冷痛阈值(P<0.05);机制层面的验证结果表明, SNL大鼠术后脊髓组织中AKT、MKK4、JNK和JUN磷酸化蛋白的表达量均显著升高(P<0.001);乌头汤可上调具有神经保护作用的AKT磷酸化蛋白的表达量(P<0.001),并降低促进神经炎症的MKK4、JNK和JUN磷酸化蛋白的表达量(P<0.01)。上述结果表明,乌头汤可有效缓解SNL大鼠的疼痛程度,其作用机制可能是通过调节AKT-MKK4-JNK-JUN信号轴而缓解神经炎症。本研究丰富了经方乌头汤治疗痛痹的科学内涵,也为基于NP发病机制的多靶点镇痛药物的研发提供了方法学示范。本研究中的动物实验获得中国中医科学院实验动物伦理委员会批准。
Wu-tou decoction(WTD) was originally recorded in the synopsis of the golden chamber and it had been widely used for the treatment of neuropathic pain(NP) with exact therapeutic efficacy. However, the underlying molecular mechanisms still remain unclarified. Thus, in this research, we aimed at clarifying the underlying molecular mechanisms of WTD against NP by combining network analysis and experimental validation based on the spinal nerve ligation(SNL) model. Firstly, the network analysis indicated that key targets of WTD were significantly involved in the MAPK signaling pathway(P = 4.04 E-12) and four important components of the above pathway, AKT kinase(AKT), MAP kinase kinase 4(MKK4), c-Jun N-terminal kinase(JNK) and transcription factor AP-1(JUN) had been reported to play a vital role in neuroinflammation during the disease process of NP. Then, experimental validation results proved that WTD markedly reduce the severity of mechanical allodynia(P<0.01) and cold hypersensitivity(P<0.05) of SNL rats. In addition, Western blot results provided evidence that the phosphorylated protein expression levels of AKT, MKK4, JNK and JUN in the superficial lamina of spinal cord of SNL rats were markedly increased(P<0.001), and WTD could improve the phosphorylated protein expression level of AKT(P<0.001) which was reported to be nerve protective and attenuate the phosphorylated protein expression levels of MKK4, JNK and JUN(P<0.01) which were closely involved into neuroinflammation.In conclusion, this study indicated that WTD might exert anti-hyperalgesia action through the inhibition of neuroin‐flammation mediated by AKT-MKK4-JNK-JUN which belong to the MAPK signaling pathway. These findings also provided scientific evidences that WTD might be a promising candidate for NP. Animal experiments in this study were approved by the Ethics Committee of Experimental Animals of the China Academy of Chinese Medical Sciences.
Wu-tou decoction(WTD) was originally recorded in the synopsis of the golden chamber and it had been widely used for the treatment of neuropathic pain(NP) with exact therapeutic efficacy. However, the underlying molecular mechanisms still remain unclarified. Thus, in this research, we aimed at clarifying the underlying molecular mechanisms of WTD against NP by combining network analysis and experimental validation based on the spinal nerve ligation(SNL) model. Firstly, the network analysis indicated that key targets of WTD were significantly involved in the MAPK signaling pathway(P = 4.04 E-12) and four important components of the above pathway, AKT kinase(AKT), MAP kinase kinase 4(MKK4), c-Jun N-terminal kinase(JNK) and transcription factor AP-1(JUN) had been reported to play a vital role in neuroinflammation during the disease process of NP. Then, experimental validation results proved that WTD markedly reduce the severity of mechanical allodynia(P<0.01) and cold hypersensitivity(P<0.05) of SNL rats. In addition, Western blot results provided evidence that the phosphorylated protein expression levels of AKT, MKK4, JNK and JUN in the superficial lamina of spinal cord of SNL rats were markedly increased(P<0.001), and WTD could improve the phosphorylated protein expression level of AKT(P<0.001) which was reported to be nerve protective and attenuate the phosphorylated protein expression levels of MKK4, JNK and JUN(P<0.01) which were closely involved into neuroinflammation.In conclusion, this study indicated that WTD might exert anti-hyperalgesia action through the inhibition of neuroin‐flammation mediated by AKT-MKK4-JNK-JUN which belong to the MAPK signaling pathway. These findings also provided scientific evidences that WTD might be a promising candidate for NP. Animal experiments in this study were approved by the Ethics Committee of Experimental Animals of the China Academy of Chinese Medical Sciences.
引文
[1] Van HO, Austin SK, Khan RA, et al. Neuropathic pain in the general population:a systematic review of epidemiological studies[J]. Pain, 2014, 155:654-662.
[2] Colloca L, Ludman T, Bouhassira D, et al. Neuropathic pain[J].Nat Rev Dis Primers,2017, 3:1-45.
[3] Gao YJ,Ji RR. Chemokines,neuronal-glial interactions,and central processing of neuropathic pain[J]. Pharmacol Ther,2010, 126:56-68.
[4] Skaper SD,Facci L,Zusso M,et al. An inflammation-centric view of neurological disease:beyond the neuron[J]. Front Cell Neurosci, 2018, 12:1-26.
[5] Qiu AW, Liu Z, Guo J, et al. Relationship between neuroinflammation and neurodegenerative diseases[J]. Prog Physiol Sci(生理科学进展),2011,42:353-358.
[6] Wu HG, Gao Z, Luo FW, et al. Signaling pathways associated with neuroinflammation in microglia[J]. Curr Immunol(现代免疫学),2014, 34:501-505, 496.
[7] Skaper SD, Facci L. Mast cell-glia axis in neuroinflammation and therapeutic potential of the anandamide congener palmitoylethanolamide[J]. Philos Trans R Soc Lond B Biol Sci, 2012, 367:3312-3325.
[8] Cao RL, Yuan B, Gu YL, et al. Research progress on molecular mechanism of astrocyte-mediated neuroinflammation regulating neuropathic pain and the intervention of traditional Chinese medicine[J]. E J Transl Med(转化医学电子杂志),2018, 9:20-25.
[9] Gao YJ, Zhang ZJ, Cao DL. Chemokine mediated neuroinflammatory response and neuropathic pain[J]. Chin J Cell Biol(中国细胞生物学学报),2014, 36:297-307.
[10] Hurley RW, Adams MC, Benzon HT. Neuropathic pain:treatment guidelines and updates[J]. Curr Opin Anaesthesiol, 2013,26:580-587.
[11] Guo QY, Zhang YQ, Lin N. Research progress on mechanism of Chinese medicine acting on neuropathic pain[J]. E J Transl Med(转化医学电子杂志),2017, 4:1-6.
[12] Bai GH, Zhang JH. Clinical observation of 28 cases of trigeminal neuralgia treated with modified Wu tou Decoction[J]. Mod J Integr Tradit Chin West Med(现代中西医结合杂志),2003,4:385.
[13] Zhou H, He XY, Zou QF. Clinical observation of 48 cases of metastatic bone cancer pain treated by Wu tou Decoction[J]. J Sichuan Tradit Chin Med(四川中医),2013, 31:92-93.
[14] Zhang YQ, Wang Chao, Guo QY, et al. Molecular mechanisms of the analgesic action of Wu-tou Decoction on neuropathic pain in mice revealed using microarray and network analysis[J]. Acta Pharmacol Sin, 2018, 39:988-997.
[15] Le CC, Castagne V. Face-to-face comparison of the predictive validity of two models of neuropathic pain in the rat:analgesic activity of pregabalin, tramadol and duloxetine[J]. Eur J Pharmacol, 2014, 735:17-25.
[16] Kim S,Chung J. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat[J]. Pain,1992, 50:355-363.
[17] Chaplan SR, Bach FW, Pogrel JW, et al. Quantitative assessment of tactile allodynia in the rat paw[J]. J Neurosci Meth, 1994, 53:55-63.
[18] Wang R, Zhang QG, Han D. Inhibition of MLK3-MKK4/7-JNK1/2 pathway by Aktl in exogenous estrogen-induced neuroprotection against transient global cerebral ischemia by a nongenomic mechanism in male rats[J]. J Neurochem, 2006, 99:1543-1554.
[19] Ji RR, Suter MR. p38 MAPK, microglial signaling, and neuropathic pain[J]. Mol Pain, 2007, 3:33.
[20] Ellis A, Bennett DL. Neuroinflammation and the generation of neuropathic pain[J]. Br J Anaesth, 2013, 111:26-37.
[21] Yu HM, Wang Q, Sun WB. Silencing of FKBP51 alleviates the mechanical pain threshold, inhibits DRG inflammatory factors and pain mediators through the NF-κB signaling pathway[J].Gene, 2017, 627:169-175.
[22] Leung L, Cahill CM. TNF-a and neuropathic pain-a review[J].J Neuroinflammation, 2010, 7:27.
[23] Nakanishi M, Nakae A, Kishida Y. Go-sha-jinki-Gan(GJG)ameliorates allodynia in chronic constriction injury-model mice via suppression of TNF-αexpression in the spinal cord[J]. Mol Pain, 2016. DOI:10.1177/1744806916656382.
[24] Calvo M, Zhu N, Grist J, et al. Following nerve injury neuregulin-1 drives microglial proliferation and neuropathic pain via the MEK/ERK pathway[J]. Glia, 2011, 59:554-568.
[25] Chen CY, Weng YH, Chien KY, et al.(G2019S)LRRK2 activates MKK4-JNK pathway and causes degeneration of SN dopaminergic neurons in a transgenic mouse model of PD[J]. Cell Death Differ, 2012, 19:1623-1633.
[26] Xu HY, Peng XJ, Chen YB, et al. Exploration of"principlerecipe-composition-target-activity"association of Bupleuri Radixand Scutellariae Radix drug pair for diabetes treatment based on network pharmacology[J]. Acta Pharm Sin(药学学报),2018,53:1414-1421.
[27] Li JL, Liang Hui, Cai SZ, et al. Mechanism of detoxification of Chebulae Fructus against Aconiti kusnezoffii radix toxicity based on network pharmacology[J]. Acta Pharm Sin(药学学报),2018, 53:1670-1679.
[28] Chai X, Meng YK, Bai ZF, et al. Mechanism of anti-hepatitis B virus activity of tonkinensis based on biological targets network[J]. Acta Pharm Sin(药学学报),2018, 53:396-402.
[29] Li WJ, Lu JQ, Mao X, et al. A comparative study on the mechanisms of two classical herbal formulae for rheumatoid arthritis applying cold and heat patterns based on target network[J]. Acta Pharm Sin(药学学报),2018, 53:1387-1397.
[30] Wang DH, Liu CF, Tan SF, et al. Analgesic effect of Wutou Decoction on rats and its preliminary mechanism[J]. Chin J Exp Tradit Med Form(中国实验方剂学杂志),2014, 20:109-112.
[31] He LH, Liu CF, Sun CC, et al. Wu-Tou Decoction inhibits angiogenesis in experimental arthritis by targeting VEGFR2 signaling pathway[J]. Rejuv Res, 2018, 21:442-455.
[32] Mao ZY, Xu QH, Zhu CY, et al. Mechanism of Wutoutang in alleviating neuropathic pain by improving damages of descending inhibitory system[J]. Chin J Exp Tradit Med Form(中国实验方剂学杂志),2018, 24:82-87.
[33] Wang C, Liu C, Wan H, et al. Wu-tou decoction inhibits chronic inflammatory pain in mice:participation of TRPV1 and TRPA1ion channels[J]. Biomed Res Int, 2015, 2015:328707.
[34] Chen BQ,Li CW,Shi YP,et al. Pharmacological effects and clinical evaluation of pregabalin[J]. Chin J New Drugs Clin Rem(中国新药与临床杂志),2010, 29:11-14.
[35] Toth C. Pregabalin:latest safety evidence and clinical implications for the management of neuropathic pain[J]. Ther Adv Drug Saf,2014, 5:38-56.
[36] Qu Y, Jia L, Zhang X, et al. MAPK pathways are involved in neuropathic pain in rats with chronic compression of the dorsal root ganglion[J]. Evid Based Complement Alternat Med, 2016,2016:6153215.
[37] Liu BN, Han BX, Liu F. Neuroprotective effect of pAkt and HIF-1 on ischemia rats[J]. Asian Pac J Trop Med,2014,7:221-225.
[38] Hietakangas V, Elo I, Rosenstrom H. Activation of the MKK4-JNK pathway during erythroid differentiation of K562 cells is inhibited by the heat shock factor 2-βisoform[J]. FEBS Lett,2001, 505:168-172.
[39] Yamasaki T, Kawasaki H, Nishina H. Diverse roles of JNK and MKK pathways in the brain[J]. J Signal Transduct, 2012, 2012:459265.
[40] Sundarrajan M, Boyle DL, Chabaud-Riou M, et al. Expression of the MAPK kinases MKK-4 and MKK-7 in rheumatoid arthritis and their role as key regulators of JNK[J]. Arthritis Rheum,2003,48:2450-2460.
[41] Sanna MD, Stark H, Lucarini L, et al. Histamine H4 receptor activation alleviates neuropathic pain through differential regulation of ERK, JNK, and P38 MAPK phosphorylation[J]. Pain,2015, 156:2492-2504.
[42] Cao J, Wang JS, Ren XH, et al. Spinal sample showing p-JNK and P38 associated with the pain signaling transduction of glial cell in neuropathic pain[J]. Spinal Cord, 2015, 53:92-97.
[43] Mei XP, Zhang H, Wang W, et al. Inhibition of spinal astrocyticc-Jun N-terminal kinase(JNK)activation correlates with the analgesic effects of ketamine in neuropathic pain[J]. J Neuroinflammation, 2011, 8:6.
[44] Zhuang ZY, Wen YR, Zhang DR, et al. A peptide c-Jun N-terminal kinase(JNK)inhibitor blocks mechanical allodynia after spinal nerve ligation:respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain development and maintenance[J]. J Neurosci, 2006, 26:3551-3560.
[2] Colloca L, Ludman T, Bouhassira D, et al. Neuropathic pain[J].Nat Rev Dis Primers,2017, 3:1-45.
[3] Gao YJ,Ji RR. Chemokines,neuronal-glial interactions,and central processing of neuropathic pain[J]. Pharmacol Ther,2010, 126:56-68.
[4] Skaper SD,Facci L,Zusso M,et al. An inflammation-centric view of neurological disease:beyond the neuron[J]. Front Cell Neurosci, 2018, 12:1-26.
[5] Qiu AW, Liu Z, Guo J, et al. Relationship between neuroinflammation and neurodegenerative diseases[J]. Prog Physiol Sci(生理科学进展),2011,42:353-358.
[6] Wu HG, Gao Z, Luo FW, et al. Signaling pathways associated with neuroinflammation in microglia[J]. Curr Immunol(现代免疫学),2014, 34:501-505, 496.
[7] Skaper SD, Facci L. Mast cell-glia axis in neuroinflammation and therapeutic potential of the anandamide congener palmitoylethanolamide[J]. Philos Trans R Soc Lond B Biol Sci, 2012, 367:3312-3325.
[8] Cao RL, Yuan B, Gu YL, et al. Research progress on molecular mechanism of astrocyte-mediated neuroinflammation regulating neuropathic pain and the intervention of traditional Chinese medicine[J]. E J Transl Med(转化医学电子杂志),2018, 9:20-25.
[9] Gao YJ, Zhang ZJ, Cao DL. Chemokine mediated neuroinflammatory response and neuropathic pain[J]. Chin J Cell Biol(中国细胞生物学学报),2014, 36:297-307.
[10] Hurley RW, Adams MC, Benzon HT. Neuropathic pain:treatment guidelines and updates[J]. Curr Opin Anaesthesiol, 2013,26:580-587.
[11] Guo QY, Zhang YQ, Lin N. Research progress on mechanism of Chinese medicine acting on neuropathic pain[J]. E J Transl Med(转化医学电子杂志),2017, 4:1-6.
[12] Bai GH, Zhang JH. Clinical observation of 28 cases of trigeminal neuralgia treated with modified Wu tou Decoction[J]. Mod J Integr Tradit Chin West Med(现代中西医结合杂志),2003,4:385.
[13] Zhou H, He XY, Zou QF. Clinical observation of 48 cases of metastatic bone cancer pain treated by Wu tou Decoction[J]. J Sichuan Tradit Chin Med(四川中医),2013, 31:92-93.
[14] Zhang YQ, Wang Chao, Guo QY, et al. Molecular mechanisms of the analgesic action of Wu-tou Decoction on neuropathic pain in mice revealed using microarray and network analysis[J]. Acta Pharmacol Sin, 2018, 39:988-997.
[15] Le CC, Castagne V. Face-to-face comparison of the predictive validity of two models of neuropathic pain in the rat:analgesic activity of pregabalin, tramadol and duloxetine[J]. Eur J Pharmacol, 2014, 735:17-25.
[16] Kim S,Chung J. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat[J]. Pain,1992, 50:355-363.
[17] Chaplan SR, Bach FW, Pogrel JW, et al. Quantitative assessment of tactile allodynia in the rat paw[J]. J Neurosci Meth, 1994, 53:55-63.
[18] Wang R, Zhang QG, Han D. Inhibition of MLK3-MKK4/7-JNK1/2 pathway by Aktl in exogenous estrogen-induced neuroprotection against transient global cerebral ischemia by a nongenomic mechanism in male rats[J]. J Neurochem, 2006, 99:1543-1554.
[19] Ji RR, Suter MR. p38 MAPK, microglial signaling, and neuropathic pain[J]. Mol Pain, 2007, 3:33.
[20] Ellis A, Bennett DL. Neuroinflammation and the generation of neuropathic pain[J]. Br J Anaesth, 2013, 111:26-37.
[21] Yu HM, Wang Q, Sun WB. Silencing of FKBP51 alleviates the mechanical pain threshold, inhibits DRG inflammatory factors and pain mediators through the NF-κB signaling pathway[J].Gene, 2017, 627:169-175.
[22] Leung L, Cahill CM. TNF-a and neuropathic pain-a review[J].J Neuroinflammation, 2010, 7:27.
[23] Nakanishi M, Nakae A, Kishida Y. Go-sha-jinki-Gan(GJG)ameliorates allodynia in chronic constriction injury-model mice via suppression of TNF-αexpression in the spinal cord[J]. Mol Pain, 2016. DOI:10.1177/1744806916656382.
[24] Calvo M, Zhu N, Grist J, et al. Following nerve injury neuregulin-1 drives microglial proliferation and neuropathic pain via the MEK/ERK pathway[J]. Glia, 2011, 59:554-568.
[25] Chen CY, Weng YH, Chien KY, et al.(G2019S)LRRK2 activates MKK4-JNK pathway and causes degeneration of SN dopaminergic neurons in a transgenic mouse model of PD[J]. Cell Death Differ, 2012, 19:1623-1633.
[26] Xu HY, Peng XJ, Chen YB, et al. Exploration of"principlerecipe-composition-target-activity"association of Bupleuri Radixand Scutellariae Radix drug pair for diabetes treatment based on network pharmacology[J]. Acta Pharm Sin(药学学报),2018,53:1414-1421.
[27] Li JL, Liang Hui, Cai SZ, et al. Mechanism of detoxification of Chebulae Fructus against Aconiti kusnezoffii radix toxicity based on network pharmacology[J]. Acta Pharm Sin(药学学报),2018, 53:1670-1679.
[28] Chai X, Meng YK, Bai ZF, et al. Mechanism of anti-hepatitis B virus activity of tonkinensis based on biological targets network[J]. Acta Pharm Sin(药学学报),2018, 53:396-402.
[29] Li WJ, Lu JQ, Mao X, et al. A comparative study on the mechanisms of two classical herbal formulae for rheumatoid arthritis applying cold and heat patterns based on target network[J]. Acta Pharm Sin(药学学报),2018, 53:1387-1397.
[30] Wang DH, Liu CF, Tan SF, et al. Analgesic effect of Wutou Decoction on rats and its preliminary mechanism[J]. Chin J Exp Tradit Med Form(中国实验方剂学杂志),2014, 20:109-112.
[31] He LH, Liu CF, Sun CC, et al. Wu-Tou Decoction inhibits angiogenesis in experimental arthritis by targeting VEGFR2 signaling pathway[J]. Rejuv Res, 2018, 21:442-455.
[32] Mao ZY, Xu QH, Zhu CY, et al. Mechanism of Wutoutang in alleviating neuropathic pain by improving damages of descending inhibitory system[J]. Chin J Exp Tradit Med Form(中国实验方剂学杂志),2018, 24:82-87.
[33] Wang C, Liu C, Wan H, et al. Wu-tou decoction inhibits chronic inflammatory pain in mice:participation of TRPV1 and TRPA1ion channels[J]. Biomed Res Int, 2015, 2015:328707.
[34] Chen BQ,Li CW,Shi YP,et al. Pharmacological effects and clinical evaluation of pregabalin[J]. Chin J New Drugs Clin Rem(中国新药与临床杂志),2010, 29:11-14.
[35] Toth C. Pregabalin:latest safety evidence and clinical implications for the management of neuropathic pain[J]. Ther Adv Drug Saf,2014, 5:38-56.
[36] Qu Y, Jia L, Zhang X, et al. MAPK pathways are involved in neuropathic pain in rats with chronic compression of the dorsal root ganglion[J]. Evid Based Complement Alternat Med, 2016,2016:6153215.
[37] Liu BN, Han BX, Liu F. Neuroprotective effect of pAkt and HIF-1 on ischemia rats[J]. Asian Pac J Trop Med,2014,7:221-225.
[38] Hietakangas V, Elo I, Rosenstrom H. Activation of the MKK4-JNK pathway during erythroid differentiation of K562 cells is inhibited by the heat shock factor 2-βisoform[J]. FEBS Lett,2001, 505:168-172.
[39] Yamasaki T, Kawasaki H, Nishina H. Diverse roles of JNK and MKK pathways in the brain[J]. J Signal Transduct, 2012, 2012:459265.
[40] Sundarrajan M, Boyle DL, Chabaud-Riou M, et al. Expression of the MAPK kinases MKK-4 and MKK-7 in rheumatoid arthritis and their role as key regulators of JNK[J]. Arthritis Rheum,2003,48:2450-2460.
[41] Sanna MD, Stark H, Lucarini L, et al. Histamine H4 receptor activation alleviates neuropathic pain through differential regulation of ERK, JNK, and P38 MAPK phosphorylation[J]. Pain,2015, 156:2492-2504.
[42] Cao J, Wang JS, Ren XH, et al. Spinal sample showing p-JNK and P38 associated with the pain signaling transduction of glial cell in neuropathic pain[J]. Spinal Cord, 2015, 53:92-97.
[43] Mei XP, Zhang H, Wang W, et al. Inhibition of spinal astrocyticc-Jun N-terminal kinase(JNK)activation correlates with the analgesic effects of ketamine in neuropathic pain[J]. J Neuroinflammation, 2011, 8:6.
[44] Zhuang ZY, Wen YR, Zhang DR, et al. A peptide c-Jun N-terminal kinase(JNK)inhibitor blocks mechanical allodynia after spinal nerve ligation:respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain development and maintenance[J]. J Neurosci, 2006, 26:3551-3560.