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
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摘要
目的:观察电针对骨癌痛-吗啡耐受大鼠痛行为的影响,探讨电针抗骨癌痛大鼠吗啡耐受效应的作用机制。方法:通过胫骨内接种MRMT-1乳腺癌细胞(3×10~4个)诱发骨癌痛,联合腹腔注射盐酸吗啡注射液(10mg·kg~(-1)·d~(-1),分两次注射,连续注射11d)建立骨癌痛-吗啡耐受大鼠模型。第1部分:23只健康雌性SD大鼠随机分为假手术组(n=6)、骨癌痛组(n=9)、吗啡耐受组(n=8)。第2部分:61只健康雌性SD大鼠随机分为假手术组(n=11)、骨癌痛组(n=11)、吗啡耐受组(n=13)、电针组(n=13)和假电针组(n=13)。成功诱导吗啡耐受后1d(接种癌细胞后第22天)电针,于每日第1次吗啡腹腔注射后即刻电针,穴位选用双侧"足三里"和"昆仑",每次30min,每日1次,连续治疗7d。于癌细胞接种前和接种后10、11、21、22、24、26和28d时检测大鼠患侧机械缩足阈(PWTs);胫骨X线及HE染色观察胫骨组织形态;免疫荧光组织化学法(IHC)检测大鼠蓝斑核(LC)内μ阿片受体(MOR)、Rab5阳性细胞表达及其共表达情况。结果:癌细胞接种后10d,骨癌痛组、吗啡耐受组、电针组和假电针组大鼠PWTs均显著低于同期假手术组(P<0.01);接种后21d(即吗啡持续注射后11d),4组大鼠PWTs均显著低于同期假手术组(P<0.01);接种后22～28d(即电针1～7d),电针组大鼠患侧PWTs显著高于同期骨癌痛组、吗啡耐受组和假电针组(P<0.01)。X线结果显示骨癌痛大鼠左侧胫骨近端上1/3处骨皮质破坏,呈不连续状,且局部软组织可见明显肿胀。HE染色结果显示,假手术组大鼠胫骨结构完整,骨髓腔内未见MRMT-1癌细胞;骨癌痛组和吗啡耐受组骨髓腔内见大量MRMT-1癌细胞。IHC结果显示:骨癌痛组、吗啡耐受组大鼠LC内MOR、Rab5阳性细胞率及MOR~+/Rab5~+阳性率均低于假手术组(P<0.01);吗啡耐受组大鼠LC内Rab5阳性细胞率及MOR~+/Rab5~+阳性率均低于同期骨癌痛组(P<0.05);电针组大鼠LC内MOR、Rab5阳性细胞率及MOR~+/Rab5~+阳性率均显著高于同期骨癌痛组、吗啡耐受组和假电针组(P<0.01)。结论:电针可缓解骨癌痛-吗啡耐受大鼠的机械痛阈,部分翻转吗啡耐受现象;该效应可能与其提高大鼠LC内MOR表达、促进MOR内吞有关。
Objective To observe the effect of electroacupuncture(EA)on pain behavior and expression of!-opioid receptor(MOR)and Rab5(an important protein molecule for internalization of MOR)in the locus coeruleus(LC)region in bone cancer pain(BCP)rats with morphine tolerance(MT),so as to explore its mechanisms underlying improvement of BCP and MT.Methods The present study included two parts.In the first part,23 female SD rats were randomized into sham BCP(n=6),BCP(n=9)and BCP+MT(n=8)groups,and in the second part,61 female SD rats were randomized into 5 groups:sham BCP(n=11),BCP(n=11),BCP+MT(n=13),BCP+MT+EA(n=13)and BCP+MT+sham EA(n=13).The BCP morphine tolerance(BCP+MT)model was established by injection of 10 !L of human Walker 256 breast cancer cells(MRMT-1 breast cancer cells,1 x10~4 cells/!L)into the bone marrow cavity at the upper part of the left tibia and intraperitoneal injection of morphine hydrochloride(10 mg/kg,once per 12 h,for 11 successive days).On day 21 after inoculation,EA(2 Hz/100 Hz,0.5-1.5 mA,increasing 0.5 mA every 10 min)was began to applied to bilateral"Zusanli"(ST30)and"Kunlun"(BL60)immediately after the first intraperitoneal injection of morphine.The treatment was performed for 30 min every time,once daily for 7 successive days.The paw withdrawal threshold(PWT)was detected before and 10,11,21,22,24,26 and 28 days after inoculation.The immunoactivity of MOR and Rab5 proteins in the LC region was detected by immunofluorescence histochemistry.Results In the first part of the study,at the 10 th day after inoculation of cancer cells,the PWT of the BCP and BCP+MT groups was significantly lower than that of the sham BCP group(P<0.05),suggesting a success of BCP model.From day 11 to 19 after inoculation(during injection of morphine),the PWTs of the BCP+MT group were significantly higher than those of the BCP group(P<0.01),and on day 21,the PWT of the BCP+MT group was similar to that of the BCP group(P>0.05)but significantly lower than that of the sham BCP group(P<0.01),suggesting a success of MT.H.E.staining showed a large quantity of MRMT-1 cancer cells in the bone marrow cavity in both BCP and BCP+MT groups.In the second part of the study,the decreased PWTs from 10 th to 28 th day after inoculation were significantly increased on day 22,24,26 and 28 in the BCP+MT+EA group relevant to the BCP,BCP+MT and BCP+MT+sham EA groups(P<0.01).The ratios of MOR and Rab5 positive(+)cells and MOR+/Rab5+of the left LC region were significantly lower in the BCP and BCP+MT groups than those of the sham BCP group(P<0.01),but were considerably higher in the BCP+MT+EA group than those in the BCP,BCP+MT and BCP+MT+sham EA groups(P<0.01).The ratios of Rab5+and MOR+/Rab5~+cells of the BCP+MT group were significantly lower than those of the BCP group(P<0.05).No significant changes were found in the ratios of MOR+and Rab5~+cells and MOR+/Rab5~+cells after BCP+MT+sham EA in comparison with the BCP+MT group(P>0.05).Conclusion EA intervention can relieve pain and MT in bone cancer pain rats with MT,which may be related to its effects in increasing MOR expression and promoting endocytosis of MOR in LC region.
Objective To observe the effect of electroacupuncture(EA)on pain behavior and expression of!-opioid receptor(MOR)and Rab5(an important protein molecule for internalization of MOR)in the locus coeruleus(LC)region in bone cancer pain(BCP)rats with morphine tolerance(MT),so as to explore its mechanisms underlying improvement of BCP and MT.Methods The present study included two parts.In the first part,23 female SD rats were randomized into sham BCP(n=6),BCP(n=9)and BCP+MT(n=8)groups,and in the second part,61 female SD rats were randomized into 5 groups:sham BCP(n=11),BCP(n=11),BCP+MT(n=13),BCP+MT+EA(n=13)and BCP+MT+sham EA(n=13).The BCP morphine tolerance(BCP+MT)model was established by injection of 10 !L of human Walker 256 breast cancer cells(MRMT-1 breast cancer cells,1 x10~4 cells/!L)into the bone marrow cavity at the upper part of the left tibia and intraperitoneal injection of morphine hydrochloride(10 mg/kg,once per 12 h,for 11 successive days).On day 21 after inoculation,EA(2 Hz/100 Hz,0.5-1.5 mA,increasing 0.5 mA every 10 min)was began to applied to bilateral"Zusanli"(ST30)and"Kunlun"(BL60)immediately after the first intraperitoneal injection of morphine.The treatment was performed for 30 min every time,once daily for 7 successive days.The paw withdrawal threshold(PWT)was detected before and 10,11,21,22,24,26 and 28 days after inoculation.The immunoactivity of MOR and Rab5 proteins in the LC region was detected by immunofluorescence histochemistry.Results In the first part of the study,at the 10 th day after inoculation of cancer cells,the PWT of the BCP and BCP+MT groups was significantly lower than that of the sham BCP group(P<0.05),suggesting a success of BCP model.From day 11 to 19 after inoculation(during injection of morphine),the PWTs of the BCP+MT group were significantly higher than those of the BCP group(P<0.01),and on day 21,the PWT of the BCP+MT group was similar to that of the BCP group(P>0.05)but significantly lower than that of the sham BCP group(P<0.01),suggesting a success of MT.H.E.staining showed a large quantity of MRMT-1 cancer cells in the bone marrow cavity in both BCP and BCP+MT groups.In the second part of the study,the decreased PWTs from 10 th to 28 th day after inoculation were significantly increased on day 22,24,26 and 28 in the BCP+MT+EA group relevant to the BCP,BCP+MT and BCP+MT+sham EA groups(P<0.01).The ratios of MOR and Rab5 positive(+)cells and MOR+/Rab5+of the left LC region were significantly lower in the BCP and BCP+MT groups than those of the sham BCP group(P<0.01),but were considerably higher in the BCP+MT+EA group than those in the BCP,BCP+MT and BCP+MT+sham EA groups(P<0.01).The ratios of Rab5+and MOR+/Rab5~+cells of the BCP+MT group were significantly lower than those of the BCP group(P<0.05).No significant changes were found in the ratios of MOR+and Rab5~+cells and MOR+/Rab5~+cells after BCP+MT+sham EA in comparison with the BCP+MT group(P>0.05).Conclusion EA intervention can relieve pain and MT in bone cancer pain rats with MT,which may be related to its effects in increasing MOR expression and promoting endocytosis of MOR in LC region.
引文
[1]CONNOR M,OSBORNE P B,CHRISTIE M J.Mu-opioid receptor desensitization:is morphine different[J].Br JPharmacol,2004,143(6):685-696.
[2]ANAND K J,CLARK A E,WILLSON D F,et al.Opioid analgesia in mechanically ventilated children:results from the multicenter measuring opioid tolerance induced by fentanyl study[J].Pediatr Crit Care Med,2013,14(1):27-36.
[3]张怀奇.阿片样镇痛药过量的研究进展[J].医学新知杂志,2014,24(1):51-53.
[4]BAILEY C P,OLDFIELD S,LLORENTE J,et al.Involvement of PKCαand G-protein-coupled receptor kinase 2in agonist-selective desensitization of!-opioid receptors in mature brain neurons[J].Br J Pharmacol,2009,158(1):157-164.
[5]WANG F,CHEN X,ZHANG X,et al.Phosphorylation state of mu-opioid receptor determines the alternative recycling of receptor via Rab4or Rab11pathway[J].Mol Endocrinol,2008,22(8):1881-1892.
[6]ZHU H,LIANG Z,LI A G.Rabex-5is a Rab22effector and mediates a Rab22-Rab5signaling cascade in endocytosis[J].Mol Biol Cell,2009,20(22):4720-4729.
[7]KAEHLER S T,SINNER C,PHILIPPU A.Release of catecholamines in the locus coeruleus of freely moving and anaesthetized normotensive and spontaneously hypertensive rats:effects of cardiovascular changes and tail pinch[J].Naunyn Schmiedebergs Arch Pharmacol,2000,361(4):433-439.
[8]KHAKPAY R,SEMNANIAN S,JAVAN M,et al.The effect of intra-locuscoeruleus injection of 17beta-estradiol on inflammatory pain modulation in male rat[J].Behav Brain Res,2010,214(2):409-416.
[9]BAILEY C P,COUCH D,JOHNSON E,et al.Mu-opioid receptor desensitization in mature rat neurons:lack of interaction between DAMGO and morphine[J].J Neurosci,2003,23(33):10515-10520.
[10]CONNOR M,BORGLAND S L,CHRISTIE M J.Continued morphine modulation of calcium channel currents in acutely isolated locuscoeruleus neurons from morphine-dependent rats[J].Br J Pharmacol,1999,128(7):1561-1569.
[11]司马蕾,刘波涛,厉建春,等.电针对骨癌痛-吗啡耐受大鼠痛行为和降钙素基因相关肽的影响[J].中华行为医学与脑科学杂志,2013,22(5):388-390.
[12]MEDHURST S J,WALKER K,BOWES M,et al.A rat model of bone cancer pain[J].Pain,2002,96(1):129-140.
[13]QIN Z,DONG F,JIE C,et al.Enhanced excitability of small dorsal root ganglion neurons in rats with bone cancer pain[J].Mol Pain,2012,8(1):24-42.
[14]DUCOURNEAU V R,DOLIQUE T,HACHEMDELAU-NAY S,et al.Cancer pain is not necessarily correlated with spinal overexpression of reactive glia markers[J].Pain,2014,155(2):275-291.
[15]HASSANZADEH K,KHODADADI B,MOLOUDI M R,et al.A new pharmacological role for thalidomide:attenuation of morphine-induced tolerance in rats[J].Acta Anaesthesiol Taiwan,2016,54(2):65-69.
[16]华兴邦,周浩良.大鼠穴位图谱的研制[J].实验动物与动物实验,1991,1(1):1-5.
[17]聂鋆,刘淑俊,邸立军,等.癌痛及其对癌症患者生活质量影响的调查[J].中华肿瘤杂志,2000,23(5):432-434.
[18]CARACENI A,HANKS G,KAASA S,et al.Use of opioid analgesics in the treatment of cancer pain:evidence-based recommendations from the EAPC[J].Lancet Oncol,2012,13(2):58-68.
[19]司马蕾,厉建春,蔡淑呈,等.P物质和降钙素基因相关肽在骨癌痛-吗啡耐受模型中的表达[J].中国癌症杂志,2012,22(8):561-565.
[20]LEFKOWITZ R J.Historical review:a brief history and personal retrospective of seven-transmembrane receptors[J].Trends Pharmacol Sci,2004,25(8):413-422.
[21]YAMAMOTO J,KAWAMATA T,NIIYAMA Y,et al.Down-regulation of mu opioid receptor expression within distinct subpopulations of dorsal root ganglion neurons in a murine model of bone cancer pain[J].Neuroscience,2008,151(3):843-853.
[22]SIM-SELLEY L J,SELLEY D E,VOGT L J,et al.Chronic heroin self-administration desensitizes mu opioid receptor-activated G-proteins in specific regions of rat brain[J].J Neurosci,2000,20(12):4555-4562.
[23]吕庆琴,陈霆隽,洪炎国.!阿片受体的内吞抑制吗啡耐受的形成[J].中国临床药理学与治疗学,2012,17(10):1185-1190.
[24]STAHL B,MOLLARD G F V,WALCHSOLIMENA C,et al.GTP cleavage by the small GTP-binding protein Rab3Ais associated with exocytosis of synaptic vesicles induced by alpha-latrotoxin[J].J Bio Chem,1994,269(40):24770-24776.
[25]QUELCH D R,KATSOURI L,NUTT D J,et al.Imaging endogenous opioid peptide release with[11C]carfentanil and[3H]diprenorphine:influence of agonist-induced internalization[J].J Cereb Blood Flow Metab,2014,34(10):1604-1612.
[26]CHIA K L.Electroacupuncture treatment of acute low back pain:unlikely to be a placebo response[J].Acupunct Med,2014,32(4):354-355.
[27]DU J Y,FANG J Q,LIANG Y,et al.Electroacupuncture attenuates mechanical allodynia by suppressing the spinal JNK1/2pathway in a rat model of inflammatory pain[J].Brain Res Bull,2014,108:27-36.
[28]黄美娜,郑宇欣,于泳浩,等.电针对吗啡耐受大鼠辣椒素受体磷酸化水平变化的影响[J].天津医药,2011,39(6):542-545.
[29]杜俊英,房军帆,陈宜恬,等.电针抗大鼠骨癌痛的参数优选及其对阿片受体和前体mRNA表达的干预[J].中国针灸,2015,35(2):161-168.
[30]HAMZA M A,WHITE P F,AHMED H E,et al.Effect of the frequency of transcutaneous electrical nerve stimulation on the postoperative opioid analgesic requirement and recovery profile[J].Anesthesiology,1999,91(5):1232-1238.
[31]吴鎏桢,崔彩莲,韩济生.韩氏穴位神经刺激仪治疗阿片戒断综合征的临床研究[J].中国疼痛医学杂志,1995,1(1):30-38.
[2]ANAND K J,CLARK A E,WILLSON D F,et al.Opioid analgesia in mechanically ventilated children:results from the multicenter measuring opioid tolerance induced by fentanyl study[J].Pediatr Crit Care Med,2013,14(1):27-36.
[3]张怀奇.阿片样镇痛药过量的研究进展[J].医学新知杂志,2014,24(1):51-53.
[4]BAILEY C P,OLDFIELD S,LLORENTE J,et al.Involvement of PKCαand G-protein-coupled receptor kinase 2in agonist-selective desensitization of!-opioid receptors in mature brain neurons[J].Br J Pharmacol,2009,158(1):157-164.
[5]WANG F,CHEN X,ZHANG X,et al.Phosphorylation state of mu-opioid receptor determines the alternative recycling of receptor via Rab4or Rab11pathway[J].Mol Endocrinol,2008,22(8):1881-1892.
[6]ZHU H,LIANG Z,LI A G.Rabex-5is a Rab22effector and mediates a Rab22-Rab5signaling cascade in endocytosis[J].Mol Biol Cell,2009,20(22):4720-4729.
[7]KAEHLER S T,SINNER C,PHILIPPU A.Release of catecholamines in the locus coeruleus of freely moving and anaesthetized normotensive and spontaneously hypertensive rats:effects of cardiovascular changes and tail pinch[J].Naunyn Schmiedebergs Arch Pharmacol,2000,361(4):433-439.
[8]KHAKPAY R,SEMNANIAN S,JAVAN M,et al.The effect of intra-locuscoeruleus injection of 17beta-estradiol on inflammatory pain modulation in male rat[J].Behav Brain Res,2010,214(2):409-416.
[9]BAILEY C P,COUCH D,JOHNSON E,et al.Mu-opioid receptor desensitization in mature rat neurons:lack of interaction between DAMGO and morphine[J].J Neurosci,2003,23(33):10515-10520.
[10]CONNOR M,BORGLAND S L,CHRISTIE M J.Continued morphine modulation of calcium channel currents in acutely isolated locuscoeruleus neurons from morphine-dependent rats[J].Br J Pharmacol,1999,128(7):1561-1569.
[11]司马蕾,刘波涛,厉建春,等.电针对骨癌痛-吗啡耐受大鼠痛行为和降钙素基因相关肽的影响[J].中华行为医学与脑科学杂志,2013,22(5):388-390.
[12]MEDHURST S J,WALKER K,BOWES M,et al.A rat model of bone cancer pain[J].Pain,2002,96(1):129-140.
[13]QIN Z,DONG F,JIE C,et al.Enhanced excitability of small dorsal root ganglion neurons in rats with bone cancer pain[J].Mol Pain,2012,8(1):24-42.
[14]DUCOURNEAU V R,DOLIQUE T,HACHEMDELAU-NAY S,et al.Cancer pain is not necessarily correlated with spinal overexpression of reactive glia markers[J].Pain,2014,155(2):275-291.
[15]HASSANZADEH K,KHODADADI B,MOLOUDI M R,et al.A new pharmacological role for thalidomide:attenuation of morphine-induced tolerance in rats[J].Acta Anaesthesiol Taiwan,2016,54(2):65-69.
[16]华兴邦,周浩良.大鼠穴位图谱的研制[J].实验动物与动物实验,1991,1(1):1-5.
[17]聂鋆,刘淑俊,邸立军,等.癌痛及其对癌症患者生活质量影响的调查[J].中华肿瘤杂志,2000,23(5):432-434.
[18]CARACENI A,HANKS G,KAASA S,et al.Use of opioid analgesics in the treatment of cancer pain:evidence-based recommendations from the EAPC[J].Lancet Oncol,2012,13(2):58-68.
[19]司马蕾,厉建春,蔡淑呈,等.P物质和降钙素基因相关肽在骨癌痛-吗啡耐受模型中的表达[J].中国癌症杂志,2012,22(8):561-565.
[20]LEFKOWITZ R J.Historical review:a brief history and personal retrospective of seven-transmembrane receptors[J].Trends Pharmacol Sci,2004,25(8):413-422.
[21]YAMAMOTO J,KAWAMATA T,NIIYAMA Y,et al.Down-regulation of mu opioid receptor expression within distinct subpopulations of dorsal root ganglion neurons in a murine model of bone cancer pain[J].Neuroscience,2008,151(3):843-853.
[22]SIM-SELLEY L J,SELLEY D E,VOGT L J,et al.Chronic heroin self-administration desensitizes mu opioid receptor-activated G-proteins in specific regions of rat brain[J].J Neurosci,2000,20(12):4555-4562.
[23]吕庆琴,陈霆隽,洪炎国.!阿片受体的内吞抑制吗啡耐受的形成[J].中国临床药理学与治疗学,2012,17(10):1185-1190.
[24]STAHL B,MOLLARD G F V,WALCHSOLIMENA C,et al.GTP cleavage by the small GTP-binding protein Rab3Ais associated with exocytosis of synaptic vesicles induced by alpha-latrotoxin[J].J Bio Chem,1994,269(40):24770-24776.
[25]QUELCH D R,KATSOURI L,NUTT D J,et al.Imaging endogenous opioid peptide release with[11C]carfentanil and[3H]diprenorphine:influence of agonist-induced internalization[J].J Cereb Blood Flow Metab,2014,34(10):1604-1612.
[26]CHIA K L.Electroacupuncture treatment of acute low back pain:unlikely to be a placebo response[J].Acupunct Med,2014,32(4):354-355.
[27]DU J Y,FANG J Q,LIANG Y,et al.Electroacupuncture attenuates mechanical allodynia by suppressing the spinal JNK1/2pathway in a rat model of inflammatory pain[J].Brain Res Bull,2014,108:27-36.
[28]黄美娜,郑宇欣,于泳浩,等.电针对吗啡耐受大鼠辣椒素受体磷酸化水平变化的影响[J].天津医药,2011,39(6):542-545.
[29]杜俊英,房军帆,陈宜恬,等.电针抗大鼠骨癌痛的参数优选及其对阿片受体和前体mRNA表达的干预[J].中国针灸,2015,35(2):161-168.
[30]HAMZA M A,WHITE P F,AHMED H E,et al.Effect of the frequency of transcutaneous electrical nerve stimulation on the postoperative opioid analgesic requirement and recovery profile[J].Anesthesiology,1999,91(5):1232-1238.
[31]吴鎏桢,崔彩莲,韩济生.韩氏穴位神经刺激仪治疗阿片戒断综合征的临床研究[J].中国疼痛医学杂志,1995,1(1):30-38.