电针对CFA大鼠感觉及情绪的二维调节及前扣带皮层-初级体感皮层p-ERK表达的影响
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摘要
目的:观察电针对慢性炎性痛大鼠痛感觉及其所诱发情绪的干预效应及对前扣带皮层(Anterior Cingulate Cortex,ACC)和初级体感皮层后肢区域(Primary Somatosensory Cortex,Hindlimb Region,S1HL)磷酸化细胞外调节蛋白激酶(Phosphorylated Extracellular Regulated Protein Kinases,p-ERK)表达的影响。方法:建立完全弗氏佐剂(Complete Freund's Adjuvant,CFA)诱导的慢性炎性痛大鼠模型。痛感觉部分:将38只成年雄性SD大鼠随机分为空白组(n=11)、模型组(n=13)、电针组(n=14),在造模前1 d、模后3 d、6 d、9 d检测大鼠患足机械缩足阈(PWTs)的变化;痛情绪部分:将62只成年雄性SD大鼠,随机分为空白组(n=21)、模型组(n=21)、电针组(n=20)进行条件性位置厌恶实验(Conditioned Place Aversion,CPA)。通过自由跑动(15 min),剔除不符合条件的大鼠,造模前1 d进行条件化前训练(45 min),模后2 h和第2天进行条件化训练(45 min),模后第3天、9天进行检测(15 min)。两部分电针组大鼠均在造模后3 d～9 d进行电针干预,选取双侧"后三里"穴,刺激参数:2/100 Hz疏密波,初始电流强度1 m A,后每10 min增加0. 5 m A,共30 min,1次/d,造模后第10天取材,采用免疫荧光法(IF)和免疫印迹法(WB)检测大鼠ACC、S1HL的p-ERK的表达情况。结果:PWTs结果显示,造模后第3天、第6天、第9天,模型组与电针组大鼠PWTs较空白组显著降低(P <0. 01),第9天电针组PWTs较模型组显著升高(P <0. 01)。CPA检测结果显示,造模后第3天,模型组和电针组CPA score值(大鼠在条件箱停留时间差,Pre-post)较空白组显著升高(P <0. 01),造模后第9天,模型组CPA score值较空白组显著升高(P <0. 01),电针组CPA score值较模型组显著降低(P <0. 01)。与造模后第3比较,造模后第9天模型组CPA score值显著增加(P <0. 01),电针组显著降低(P <0. 05)。IF检测结果显示,在左侧S1HL,模型组p-ERK免疫阳性细胞的表达较空白组和电针组有上升趋势,在右侧S1HL和双侧ACC,模型组p-ERK免疫阳性细胞的表达较空白组和电针组均显著升高(P <0. 01)。WB结果显示,在双侧S1HL,模型组p-ERK1/2蛋白表达与空白组比较,差异无统计学意义。在左侧S1HL,电针组p-ERK1/2蛋白水平与模型组比较明显降低(P <0. 05),在右侧S1HL,电针组p-ERK2蛋白水平与模型组比较明显降低(P <0. 05),在双侧ACC区,模型组p-ERK1/2蛋白水平较空白组有上升趋势,电针组p-ERK1/2蛋白水平较模型组有下降趋势。结论:电针可提高CFA模型大鼠机械痛阈并缓解CFA大鼠厌恶情绪;该效应可能与其下调右侧S1HL中p-ERK表达水平和下调双侧ACC的p-ERK表达水平有关。
Objective: To observe the intervention effects of electroacupuncture on pain sensation and induced emotion in rats with chronic inflammatory pain and on the expression of phosphorylated extracellular regulated protein kinases( p-ERK) in anterior cingulate cortex( ACC) and somatosensory cortex( S1 HL). Methods: A model of chronic inflammatory pain induced by complete Freund's adjuvant( CFA) was established. Pain sensation part: 38 adult male Sprague-Dawley rats were randomly divided into a blank group( n = 11),a model group( n = 13),and an electroacupuncture group( n = 14). The changes of mechanical paw withdrawal threshold( PWTs) of rats were measured 1 day before modeling,3 days,6 days,and 9 days after modeling. Pain affect part: 62 adult male SD rats were randomly divided into a blank group( n = 21),a model group( n = 21),an electroacupuncture group( n = 20) for conditional position aversion test( CPA). Rats that did not meet the criteria were excluded by free running( 15 min). Conditional training( 45 min) was performed one day before modeling,and conditional training( 45 min) was performed 2 hours and 2 days after modeling,and the test was performed on the 3 rd and 9 th day after modeling( 15 min). In the electroacupuncture group of the 2 parts of the experiment,electroacupuncture was performed on the 3 rd to 9 th day after modeling,one time per day,and the bilateral"Housanli"points were selected. Stimulation parameters: 2/100 Hz sparse wave,initial current intensity was 1 m A,then increase 0. 5 m A every 10 minutes for 30 min,the rats were sacrificed on the 10 th day after modeling to detect the expression of p-ERK in rat ACC and S1 HL by using immunofluorescence( IF) and Western blotting( WB). Results: PWTs showed that PWTs in the model group and the electroacupuncture group were significantly decreased than those in the blank group on the 3 rd,6 th,and 9 th day after modeling( P < 0. 01). On day 9,PWTs in the electroacupuncture group were significantly increased than those in the model group( P <0. 01). The results of CPA test showed that the CPA score of the model group and the electroacupuncture group( the difference in the residence time of the conditional box,pre-post) was significantly increased than that of the blank group on the 3 rd day after modeling( P < 0. 01). On the 9 th days,the CPA score of the model group was significantly increased than that of the blank group( P < 0. 01),and the CPA score of the electroacupuncture group was significantly decreased than that of the model group( P <0. 01). Compared with the 3 rd day after modeling,the CPA score of the model group increased significantly on the 9 th day after modeling( P < 0. 01),and the electroacupuncture group decreased significantly( P < 0. 05). The results of IF showed that the expression of p-ERK immunoreactive cells in the model group was increased than that in the blank group and the electroacupuncture group on the left S1 HL. In the right S1 HL and bilateral ACC regions,the expression of p-ERK immunoreactive cells in the model group was significantly increased than that in the blank group and the electroacupuncture group( P < 0. 01). Western Blotting results showed that the expression of p-ERK1/2 protein in model group was not significantly different from that in blank group in bilateral S1 HL. In left S1 HL,the level of p-ERK1/2 protein in electroacupuncture group was significantly decreased than that in model group( P < 0. 05). On the right side of S1 HL,the level of p-ERK2 protein in electroacupuncture group was significantly decreased than that in model group( P < 0. 05). In bilateral ACC,the level of p-ERK1/2 protein in model group was increased than that in blank group,while that in electro-acupuncture group was decreased than that in model group. Conclusion: Electroacupuncture can increase the mechanical pain threshold of CFA model rats and alleviate the aversion of CFA rats; this effect may be related to down-regulation of p-ERK expression in the right S1 HL and down-regulation of p-ERK expression in bilateral ACC regions.
Objective: To observe the intervention effects of electroacupuncture on pain sensation and induced emotion in rats with chronic inflammatory pain and on the expression of phosphorylated extracellular regulated protein kinases( p-ERK) in anterior cingulate cortex( ACC) and somatosensory cortex( S1 HL). Methods: A model of chronic inflammatory pain induced by complete Freund's adjuvant( CFA) was established. Pain sensation part: 38 adult male Sprague-Dawley rats were randomly divided into a blank group( n = 11),a model group( n = 13),and an electroacupuncture group( n = 14). The changes of mechanical paw withdrawal threshold( PWTs) of rats were measured 1 day before modeling,3 days,6 days,and 9 days after modeling. Pain affect part: 62 adult male SD rats were randomly divided into a blank group( n = 21),a model group( n = 21),an electroacupuncture group( n = 20) for conditional position aversion test( CPA). Rats that did not meet the criteria were excluded by free running( 15 min). Conditional training( 45 min) was performed one day before modeling,and conditional training( 45 min) was performed 2 hours and 2 days after modeling,and the test was performed on the 3 rd and 9 th day after modeling( 15 min). In the electroacupuncture group of the 2 parts of the experiment,electroacupuncture was performed on the 3 rd to 9 th day after modeling,one time per day,and the bilateral"Housanli"points were selected. Stimulation parameters: 2/100 Hz sparse wave,initial current intensity was 1 m A,then increase 0. 5 m A every 10 minutes for 30 min,the rats were sacrificed on the 10 th day after modeling to detect the expression of p-ERK in rat ACC and S1 HL by using immunofluorescence( IF) and Western blotting( WB). Results: PWTs showed that PWTs in the model group and the electroacupuncture group were significantly decreased than those in the blank group on the 3 rd,6 th,and 9 th day after modeling( P < 0. 01). On day 9,PWTs in the electroacupuncture group were significantly increased than those in the model group( P <0. 01). The results of CPA test showed that the CPA score of the model group and the electroacupuncture group( the difference in the residence time of the conditional box,pre-post) was significantly increased than that of the blank group on the 3 rd day after modeling( P < 0. 01). On the 9 th days,the CPA score of the model group was significantly increased than that of the blank group( P < 0. 01),and the CPA score of the electroacupuncture group was significantly decreased than that of the model group( P <0. 01). Compared with the 3 rd day after modeling,the CPA score of the model group increased significantly on the 9 th day after modeling( P < 0. 01),and the electroacupuncture group decreased significantly( P < 0. 05). The results of IF showed that the expression of p-ERK immunoreactive cells in the model group was increased than that in the blank group and the electroacupuncture group on the left S1 HL. In the right S1 HL and bilateral ACC regions,the expression of p-ERK immunoreactive cells in the model group was significantly increased than that in the blank group and the electroacupuncture group( P < 0. 01). Western Blotting results showed that the expression of p-ERK1/2 protein in model group was not significantly different from that in blank group in bilateral S1 HL. In left S1 HL,the level of p-ERK1/2 protein in electroacupuncture group was significantly decreased than that in model group( P < 0. 05). On the right side of S1 HL,the level of p-ERK2 protein in electroacupuncture group was significantly decreased than that in model group( P < 0. 05). In bilateral ACC,the level of p-ERK1/2 protein in model group was increased than that in blank group,while that in electro-acupuncture group was decreased than that in model group. Conclusion: Electroacupuncture can increase the mechanical pain threshold of CFA model rats and alleviate the aversion of CFA rats; this effect may be related to down-regulation of p-ERK expression in the right S1 HL and down-regulation of p-ERK expression in bilateral ACC regions.
引文
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[11]Buitrago M M,Luft A R,Thakor N V,et al. Effects of somatosensory electrical stimulation on neuronal injury after global hypoxia-ischemia[J]. Experimental Brain Research,2004,158(3):336-344.
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[13]Jones A K P,Brown W D,Friston K J,et al. Cortical and subcortical localization of response to pain in man using positron emission tomography[J]. Proc Biol Sci,1991,244(1309):39-44.
[14]Zussy C,Gómez-Santacana,X,Rovira X,et al. Dynamic modulation of inflammatory pain-related affective and sensory symptoms by optical control of amygdala metabotropic glutamate receptor 4[J]. Molecular Psychiatry,2016,223:509-520.
[15]Apkarian A V,Bushnell M C,Treede R D,et al. Human brain mechanisms of pain perception and regulation in health and disease[J]. European Journal of Pain,2005,9(4):463-463.
[16]Grant JA,Courtemanche J,Rainville P. A non-elaborative mental stance and decoupling of executive and pain-related cortices predicts low pain sensitivity in Zen meditators[J]. Pain,2011,152(1):150-156.
[17]Wei F,Qiu C S,Kim S J,et al. Genetic elimination of behavioral sensitization in mice lacking calmodulin-stimulated adenylyl cyclases[J]. Neuron,2002,36(4):713-726.
[18]Ji R R,Befort K,Brenner G J,et al. ERK MAP Kinase Activation in Superficial Spinal Cord Neurons Induces Prodynorphin and NK1 Upregulation and Contributes to Persistent Inflammatory Pain Hypersensitivity[J]. Journal of Neuroscience the Official Journal of the Society for Neuroscience,2002,22(2):478.
[19]Guo SW,Liu MG,Long YL,et al. Region-or state-related differences in expression and activation of extracellular signal-regulated kinases(ERKs)in nave and pain-experiencing rats[J]. BMC Neurosci,2007,8:53-65.
[20]Cao H,Ren W H,Zhu M Y,et al. Activation of glycine site and Glu N2B subunit of NMDA receptors is necessary for ERK/CREB signaling cascade in rostral anterior cingulate cortex in rats:Implications for affective pain[J]. Neuroscience Bulletin,2012,28(1):77-87.
[21]Maoying Q L,Ren D H,Mi W L,et al. Analgesic effects of electroacupuncture combined with celebrex on rats with tibial cancer pain[J]. Journal of Chinese Integrative Medicine,2008,6(8):830-835.
[22]Zhao Z L,Zhao G W,Li H Z,et al. Acupuncture Attenuates Anxiety-Like Behavior by Normalizing Amygdaloid Catecholamines during Ethanol Withdrawal in Rats[J]. Evid Based Complement Alternat Med,2010,2011(2):429843.
[23]Xiao-Mei S,Zui S,Jing S,et al. Strong Manual Acupuncture Stimulation of“Huantiao”(GB 30)Reduces Pain-Induced Anxiety and pERK in the Anterior Cingulate Cortex in a Rat Model of Neuropathic Pain[J]. Evidence-Based Complementary and Alternative Medicine,2015,2015:1-11.
[24]Johansen J P,Fields H L,Manning B H. The affective component of pain in rodents:Direct evidence for a contribution of the anterior cingulate cortex[J]. Proceedings of the National Academy of Sciences,2001,98(14):8077-8082.
[25]Du J Y,Fang J Q,Liang Y,et al. Electroacupuncture attenuates mechanical allodynia by suppressing the spinal JNK1/2 pathway in a rat model of inflammatory pain[J]. Brain Research Bulletin,2014,108:27-36.
[26]Zhang R,Lao L,Ren K,et al. Mechanisms of Acupuncture-Electroacupuncture on Persistent Pain[J]. Anesthesiology,2014,120(2):482-503.
[27]Li Q,Yue N,Liu S B,et al. Effects of Chronic Electroacupuncture on Depression-and Anxiety-Like Behaviors in Rats with Chronic Neuropathic Pain[J]. Evidence-Based Complementary and Alternative Medicine,2014,2014:1-10.
[2]CADTH. Gabapentin for Adults with Neuropathic Pain:A Review of the Clinical Evidence and Guidelines[J]. Canadian Agency for Drugs and Technologies in Health,2014,Sep 26:1569.
[3]Du J Y,Fang J Q,Liang Y,et al. Electroacupuncture attenuates mechanical allodynia by suppressing the spinal JNK1/2 pathway in a rat model of inflammatory pain[J]. Brain Research Bulletin,2014,108:27-36.
[4]Zhang R,Lao L,Ren K,et al. Mechanisms of Acupuncture-Electroacupuncture on Persistent Pain[J]. Anesthesiology,2014,120(2):482-503.
[5]施任怡,付桃芳,蔡杨乾,等.电针抗骨癌痛大鼠吗啡耐受效应及其对蓝斑核μ阿片受体内吞的调控[J].针刺研究,2019,44(3):161-169.
[6]Smith C A,Hay P P,Macpherson H. Acupuncture for depression[J].Cochrane database of systematic reviews(Online),2010,20(1):CD004046.
[7]Chen Y F,Liu J H,Xu N G,et al. Effects of acupuncture treatment on depression insomnia:a study protocol of a multicenter randomized controlled trial[J]. Trials,2013,14(1):2.
[8]吴敏范,滕国玺.扣带回的形态结构[J].解剖科学进展,2000,6(4):329-331+343.
[9]Sikes R W,Vogt B A. Nociceptive neurons in area 24 of rabbit cingulate cortex[J]. Journal of Neurophysiology,1992,68(5):1720-1732.
[10]Dupont E,Stevens L,Cochon L,et al. ERK Is Involved in the Reorganization of Somatosensory Cortical Maps in Adult Rats Submitted to Hindlimb Unloading[J]. PLOS ONE,2011,6(3):e17564.
[11]Buitrago M M,Luft A R,Thakor N V,et al. Effects of somatosensory electrical stimulation on neuronal injury after global hypoxia-ischemia[J]. Experimental Brain Research,2004,158(3):336-344.
[12]G. Watson C. Paxinos. The Rat Brain in Stereotaxic Coordinates[M]. New York:Academic Press,2013.
[13]Jones A K P,Brown W D,Friston K J,et al. Cortical and subcortical localization of response to pain in man using positron emission tomography[J]. Proc Biol Sci,1991,244(1309):39-44.
[14]Zussy C,Gómez-Santacana,X,Rovira X,et al. Dynamic modulation of inflammatory pain-related affective and sensory symptoms by optical control of amygdala metabotropic glutamate receptor 4[J]. Molecular Psychiatry,2016,223:509-520.
[15]Apkarian A V,Bushnell M C,Treede R D,et al. Human brain mechanisms of pain perception and regulation in health and disease[J]. European Journal of Pain,2005,9(4):463-463.
[16]Grant JA,Courtemanche J,Rainville P. A non-elaborative mental stance and decoupling of executive and pain-related cortices predicts low pain sensitivity in Zen meditators[J]. Pain,2011,152(1):150-156.
[17]Wei F,Qiu C S,Kim S J,et al. Genetic elimination of behavioral sensitization in mice lacking calmodulin-stimulated adenylyl cyclases[J]. Neuron,2002,36(4):713-726.
[18]Ji R R,Befort K,Brenner G J,et al. ERK MAP Kinase Activation in Superficial Spinal Cord Neurons Induces Prodynorphin and NK1 Upregulation and Contributes to Persistent Inflammatory Pain Hypersensitivity[J]. Journal of Neuroscience the Official Journal of the Society for Neuroscience,2002,22(2):478.
[19]Guo SW,Liu MG,Long YL,et al. Region-or state-related differences in expression and activation of extracellular signal-regulated kinases(ERKs)in nave and pain-experiencing rats[J]. BMC Neurosci,2007,8:53-65.
[20]Cao H,Ren W H,Zhu M Y,et al. Activation of glycine site and Glu N2B subunit of NMDA receptors is necessary for ERK/CREB signaling cascade in rostral anterior cingulate cortex in rats:Implications for affective pain[J]. Neuroscience Bulletin,2012,28(1):77-87.
[21]Maoying Q L,Ren D H,Mi W L,et al. Analgesic effects of electroacupuncture combined with celebrex on rats with tibial cancer pain[J]. Journal of Chinese Integrative Medicine,2008,6(8):830-835.
[22]Zhao Z L,Zhao G W,Li H Z,et al. Acupuncture Attenuates Anxiety-Like Behavior by Normalizing Amygdaloid Catecholamines during Ethanol Withdrawal in Rats[J]. Evid Based Complement Alternat Med,2010,2011(2):429843.
[23]Xiao-Mei S,Zui S,Jing S,et al. Strong Manual Acupuncture Stimulation of“Huantiao”(GB 30)Reduces Pain-Induced Anxiety and pERK in the Anterior Cingulate Cortex in a Rat Model of Neuropathic Pain[J]. Evidence-Based Complementary and Alternative Medicine,2015,2015:1-11.
[24]Johansen J P,Fields H L,Manning B H. The affective component of pain in rodents:Direct evidence for a contribution of the anterior cingulate cortex[J]. Proceedings of the National Academy of Sciences,2001,98(14):8077-8082.
[25]Du J Y,Fang J Q,Liang Y,et al. Electroacupuncture attenuates mechanical allodynia by suppressing the spinal JNK1/2 pathway in a rat model of inflammatory pain[J]. Brain Research Bulletin,2014,108:27-36.
[26]Zhang R,Lao L,Ren K,et al. Mechanisms of Acupuncture-Electroacupuncture on Persistent Pain[J]. Anesthesiology,2014,120(2):482-503.
[27]Li Q,Yue N,Liu S B,et al. Effects of Chronic Electroacupuncture on Depression-and Anxiety-Like Behaviors in Rats with Chronic Neuropathic Pain[J]. Evidence-Based Complementary and Alternative Medicine,2014,2014:1-10.