督脉电针对急性脊髓损伤大鼠脊髓损伤区NR2B表达的影响
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摘要
目的:观察督脉电针"大椎""命门"对急性脊髓损伤(SCI)大鼠脊髓损伤区N-甲基-D-天门冬氨酸受体(NMDAR)亚基NR2B表达的影响,探讨督脉电针促进急性SCI脊髓前角神经修复的分子机制。方法:SD大鼠随机分为假手术组、模型组、电针组及药物组,每组24只。采用脊髓打击器打击复制急性SCI模型。电针组于造模成功后电针大鼠"大椎""命门"穴,每次治疗30min,共治疗3次。药物组先予尾静脉注射甲基强的松龙(MP)30mg/kg冲击治疗,然后予5.4mg·kg~(-1)·h-1 MP维持,共24h。采用BBB行为学评分观察大鼠造模前及造模后6、24、48h运动功能的变化。采用实时荧光定量PCR法检测脊髓损伤区NR2B mRNA表达水平,采用蛋白免疫印迹法和免疫荧光技术检测脊髓损伤区NR2B蛋白表达水平。结果:与同时段假手术组相比,模型组BBB行为学评分于造模后6、24、48h均显著降低(P<0.001)。与同时段模型组相比,电针组和药物组的BBB评分于造模后各时点均未见明显改变(P>0.05)。与假手术组相比,模型组脊髓损伤区病理学改变明显,NR2B阳性神经元数量显著增加(P<0.001),NR2BmRNA和蛋白表达水平均显著升高(P<0.05)。与模型组相比,电针组和药物组脊髓损伤区病理学改变明显减轻,NR2B阳性神经元数量显著减少(P<0.001),NR2BmRNA和蛋白表达水平显著降低(P<0.05)。电针组与药物组比较,各指标变化的差异均无统计学意义(P>0.05)。结论:督脉电针"大椎""命门"穴可能通过抑制脊髓损伤区NR2B的表达,从而促进脊髓前角损伤神经的修复。
Objective To observe the influence of eletroacupuncture(EA)at"Dazhui"(EX-B2)and"Mingmen"(GV4)on expression of NR2 Bsubunit of N-methyl-D-aspartate receptor(NMDA)in the injured anterior horn(AH)area of rats with acute spinal cord injury(SCI),so as to explore its mechanisms underlying improvement of neural repair.Methods A total of 96 male Sprague-Dawley(SD)rats were randomly and equally divided into four groups:sham operation(sham),model,medication(Methyl-prednisone,MP)and EA(n=24 in each group).The acute SCI model was established by using a MASCIS spinal cord impactor.EA(2 Hz,0.5 mA)was applied to EX-B2 and GV4 for 30 min,once at 0.5 h,12 and 24 hafter SCI.Rats of the medication group were treated by tail intravenous injection of MP 30 mg/kg within 15 min(impact therapy)and 5.4 mg·kg~(-1)·h-1(maintaining treatment)45 min thereafter for 23 h.The Basso,Beattie and Bresnahan(BBB)rating scale(0 to 21 points)was used to assess changes of locomotor function 6,24 and 48 hafter SCI.Histopathological changes of the injured spinal cord AH region were observed after sectioning and hematoxylin-eosin(H.E.)staining,and the expression levels of NR2 BmRNA and protein of AH were measured by quantitative real-time PCR,Western blot and immunofluorescence,respectively.Results After SCI,the BBB scores at 6,24 and 48 hwere significantly decreased in the model group compared with those of the sham group(P<0.001),but had no remarkable changes in both medication and EA groups after the intervention in comparison with those of the model group(P>0.05).After modeling,the histopathological changes(blurred border of the grey-white matter,cellular karyopyknosis,deepening of the cytoplasmic red stain,and rupture,dissolution and disordered arrangement of myelinated nerve fibers)in the injury area of the spinal cord in the model group were apparent,the number of NR2 Bpositive neurons and the relative expression levels of NR2 BmRNA and protein were significantly increased in the model group relevant to the sham group(P<0.05).In contrast to the model group,the injured severity of the spinal cord AH region was relatively milder,and the expression levels of NR2 BmRNA and protein were considerably down-regulated in both EA and MP groups(P<0.05).However,there were no significant differences between the EA and MP groups in the expression levels of NR2 BmRNA and protein(P>0.05).Conclusion EA at EX-B2 and GV4 may inhibit the expression of NR2 BmRNA and protein in acute SCI rats,which may contribute to its action in promoting nerve repair of the injured ventricolumna area of the thoracic spinal cord.
Objective To observe the influence of eletroacupuncture(EA)at"Dazhui"(EX-B2)and"Mingmen"(GV4)on expression of NR2 Bsubunit of N-methyl-D-aspartate receptor(NMDA)in the injured anterior horn(AH)area of rats with acute spinal cord injury(SCI),so as to explore its mechanisms underlying improvement of neural repair.Methods A total of 96 male Sprague-Dawley(SD)rats were randomly and equally divided into four groups:sham operation(sham),model,medication(Methyl-prednisone,MP)and EA(n=24 in each group).The acute SCI model was established by using a MASCIS spinal cord impactor.EA(2 Hz,0.5 mA)was applied to EX-B2 and GV4 for 30 min,once at 0.5 h,12 and 24 hafter SCI.Rats of the medication group were treated by tail intravenous injection of MP 30 mg/kg within 15 min(impact therapy)and 5.4 mg·kg~(-1)·h-1(maintaining treatment)45 min thereafter for 23 h.The Basso,Beattie and Bresnahan(BBB)rating scale(0 to 21 points)was used to assess changes of locomotor function 6,24 and 48 hafter SCI.Histopathological changes of the injured spinal cord AH region were observed after sectioning and hematoxylin-eosin(H.E.)staining,and the expression levels of NR2 BmRNA and protein of AH were measured by quantitative real-time PCR,Western blot and immunofluorescence,respectively.Results After SCI,the BBB scores at 6,24 and 48 hwere significantly decreased in the model group compared with those of the sham group(P<0.001),but had no remarkable changes in both medication and EA groups after the intervention in comparison with those of the model group(P>0.05).After modeling,the histopathological changes(blurred border of the grey-white matter,cellular karyopyknosis,deepening of the cytoplasmic red stain,and rupture,dissolution and disordered arrangement of myelinated nerve fibers)in the injury area of the spinal cord in the model group were apparent,the number of NR2 Bpositive neurons and the relative expression levels of NR2 BmRNA and protein were significantly increased in the model group relevant to the sham group(P<0.05).In contrast to the model group,the injured severity of the spinal cord AH region was relatively milder,and the expression levels of NR2 BmRNA and protein were considerably down-regulated in both EA and MP groups(P<0.05).However,there were no significant differences between the EA and MP groups in the expression levels of NR2 BmRNA and protein(P>0.05).Conclusion EA at EX-B2 and GV4 may inhibit the expression of NR2 BmRNA and protein in acute SCI rats,which may contribute to its action in promoting nerve repair of the injured ventricolumna area of the thoracic spinal cord.
引文
[1] MEKHAIL M,ALMAZAN G,TABRIZIAN M.Oligodendrocyte-protection and remyelination post-spinal cord injuries:a review[J].Prog Neurobiol,2012,96(3):322-339.
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[3] CHOI D C,LEE J Y,MOON Y J,et al.Acupuncture-mediated inhibition of inflammation facilitates significant functional recovery after spinal cord injury[J].Neurobiol Dis,2010,39(3):272-282.
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[6]邹恩苗,王赛,李江筎,等.针刺水沟、风府穴对急性大鼠脊髓损伤后前炎性因子表达的影响[J].中国中医急症,2013,22(6):883-886.
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[12] COLON J M,GONZLEZ P A,CAJIGAS,et al.Continuous tamoxifen delivery improves locomotor recovery 6hafter spinal cord injury by neuronal and glial mechanisms in male rats[J].Exp Neurol,2018,299(Pt A):109-121.
[13] BRACKEN M B,SHEPARD M J,HOLFORD T R,et al.Administration of methylprednisolone for 24or 48hours or tirilazad mesylate for 48hours in the treatment of acute spinal cord injury.results of the third national acute spinal cord injury randomized controlled trial.national acute spinal cord injury study[J].JAMA,1997,277(20):1597-1604.
[14] BRACKEN M B,SHEPARD M J,COLLINS W F,et al.Methylprednisolone or naloxone treatment after acute spinal cord injury:1-year follow-up data.results of the second national acute spinal cord injury study[J].J Neurosurg,1992,76(1):23-31.
[15] HAN J S,CHEN X H,SUN S L,et al.Effect of low-and high-frequency TENS on Met-enkephalin-Arg-Phe and dynorphin A immunoreactivity in human lumbar CSF[J].Pain,1991,47(3):295-298.
[16] LI W J,LI S M,DING Y,et al.Electro-acupuncture upregulates CGRP expression after rat spinal cord transection[J].Neurochem Int,2012,61(8):1397-1403.
[17] GENG X,SUN T,LI J H,et al.Electroacupuncture in the repair of spinal cord injury:inhibiting the Notch signaling pathway and promoting neural stem cell proliferation[J].Neural Regen Res,2015,10(3):394-403.
[18] MO Y P,YAO H J,LV W,et al.Effects of electroacupuncture at governor vessel acupoints on neurotrophin-3in rats with experimental spinal cord injury[J].Neural Plast,2016,2016:2371875.
[19] DING Y,YAN Q,RUAN J W,et al.Bone marrow mesenchymal stem cells and electroacupuncture downregulate the inhibitor molecules and promote the axonal regeneration in the transected spinal cord of rats[J].Cell Transplant,2011,20(4):475-491.
[20] WANG Y,QIN Z H.Molecular and cellular mechanisms of excitotoxic neuronal death[J].Apoptosis,2010,15(11):1382-1402.
[21] HILDEBRAND M E,PITCHER G M,HARDING E K,et al.GluN2Band GluN2D NMDARs dominate synaptic responses in the adult spinal cord[J].Sci Rep,2014,4:4094.
[22]翁启芳,龙儒桃,张策.NMDA受体NR2B亚单位结构和功能的研究进展[J].现代预防医学,2013,40(16):3132-3134.
[23] LI Y,SUN W,HAN S,et al.IGF-1-involved negative feedback of NR2BNMDA subunits protects cultured hippocampal neurons against NMDA-induced excitotoxicity[J].Mol Neurobiol,2017,54(1):684-696.
[24] GROSSMAN S D,WOLFE B B,YASUDA R P,et al.Changes in NMDA receptor subunit expression in response to contusive spinal cord injury[J].J Neurochem,2000,75(1):174-184.
[25] KIM Y,CHO H Y,AHN Y J,et al.Effect of NMDA NR2Bantagonist on neuropathic pain in two spinal cord injury models[J].Pain,2012,153(5):1022-1029.
[26]李潇潇,卢圣锋,朱冰梅,等.兴奋性氨基酸毒性与缺血性脑中风及针刺的调整作用[J].针刺研究,2016,41(2):180-185.
[27] KIM H N,KIM Y R,JANG J Y,et al.Effects of electroacupuncture on N-Methyl-D-aspartate receptor-related signaling pathway in the spinal cord of normal rats[J].Evid Based Complement Alternat Med,2012,2012:492471.
[2] KIM Y,PARK Y K,CHO H Y,et al.Long-term changes in expressions of spinal glutamate transporters after spinal cord injury[J].Brain Res,2011,1389:194-199.
[3] CHOI D C,LEE J Y,MOON Y J,et al.Acupuncture-mediated inhibition of inflammation facilitates significant functional recovery after spinal cord injury[J].Neurobiol Dis,2010,39(3):272-282.
[4] NORRBRINK C,LUNDEBERG T.Acupuncture and massage therapy for neuropathic pain following spinal cord injury:an exploratory study[J].Acupunct Med,2011,29(2):108-115.
[5] TU W Z,CHEN W C,XIA W,et al.The regulatory effect of electro-acupuncture on the expression of NMDA receptors in a SCI rat model[J].Life Sci,2017,177:8-14.
[6]邹恩苗,王赛,李江筎,等.针刺水沟、风府穴对急性大鼠脊髓损伤后前炎性因子表达的影响[J].中国中医急症,2013,22(6):883-886.
[7] PREGNO G,ZAMBURLIN P,GAMBAROTTA G,et al.Neuregulin1/ErbB4-induced migration in ST14Astriatal progenitors:calcium-dependent mechanisms and modulation by NMDA receptor activation[J]. BMC Neurosci,2011,12:103.
[8] KARTHICK C,PERIYASAMY S,JAYACHANDRAN K S,et al.Intrahippocampal administration of ibotenic acid induced cholinergic dysfunction via NR2A/NR2Bexpression:implications of resveratrol against Alzheimer disease pathophysiology[J].Front Mol Neurosci,2016,9:28.
[9] HERRING W J,ASSAID C,BUDD K,et al.A phase Ib randomized controlled study to evaluate the effectiveness of a single-dose of the NR2Bselective N-Methyl-D-Aspartate antagonist MK-0657on levodopa-induced dyskinesias and motor symptoms in patients with parkinson disease[J].Clin Neuropharmacol,2017,40(6):255-260.
[10] BRACKEN M B,SHEPARD M J,COLLINS W F,et al.A randomized,controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury.results of the second national acute spinal cord injury study[J].N Engl J Med,1990,322(20):1405-1411.
[11] BASSO D M,BEATTIE M S,BRESNAHAN J C.Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection[J].Exp Neurol,1996,139(2):244-256.
[12] COLON J M,GONZLEZ P A,CAJIGAS,et al.Continuous tamoxifen delivery improves locomotor recovery 6hafter spinal cord injury by neuronal and glial mechanisms in male rats[J].Exp Neurol,2018,299(Pt A):109-121.
[13] BRACKEN M B,SHEPARD M J,HOLFORD T R,et al.Administration of methylprednisolone for 24or 48hours or tirilazad mesylate for 48hours in the treatment of acute spinal cord injury.results of the third national acute spinal cord injury randomized controlled trial.national acute spinal cord injury study[J].JAMA,1997,277(20):1597-1604.
[14] BRACKEN M B,SHEPARD M J,COLLINS W F,et al.Methylprednisolone or naloxone treatment after acute spinal cord injury:1-year follow-up data.results of the second national acute spinal cord injury study[J].J Neurosurg,1992,76(1):23-31.
[15] HAN J S,CHEN X H,SUN S L,et al.Effect of low-and high-frequency TENS on Met-enkephalin-Arg-Phe and dynorphin A immunoreactivity in human lumbar CSF[J].Pain,1991,47(3):295-298.
[16] LI W J,LI S M,DING Y,et al.Electro-acupuncture upregulates CGRP expression after rat spinal cord transection[J].Neurochem Int,2012,61(8):1397-1403.
[17] GENG X,SUN T,LI J H,et al.Electroacupuncture in the repair of spinal cord injury:inhibiting the Notch signaling pathway and promoting neural stem cell proliferation[J].Neural Regen Res,2015,10(3):394-403.
[18] MO Y P,YAO H J,LV W,et al.Effects of electroacupuncture at governor vessel acupoints on neurotrophin-3in rats with experimental spinal cord injury[J].Neural Plast,2016,2016:2371875.
[19] DING Y,YAN Q,RUAN J W,et al.Bone marrow mesenchymal stem cells and electroacupuncture downregulate the inhibitor molecules and promote the axonal regeneration in the transected spinal cord of rats[J].Cell Transplant,2011,20(4):475-491.
[20] WANG Y,QIN Z H.Molecular and cellular mechanisms of excitotoxic neuronal death[J].Apoptosis,2010,15(11):1382-1402.
[21] HILDEBRAND M E,PITCHER G M,HARDING E K,et al.GluN2Band GluN2D NMDARs dominate synaptic responses in the adult spinal cord[J].Sci Rep,2014,4:4094.
[22]翁启芳,龙儒桃,张策.NMDA受体NR2B亚单位结构和功能的研究进展[J].现代预防医学,2013,40(16):3132-3134.
[23] LI Y,SUN W,HAN S,et al.IGF-1-involved negative feedback of NR2BNMDA subunits protects cultured hippocampal neurons against NMDA-induced excitotoxicity[J].Mol Neurobiol,2017,54(1):684-696.
[24] GROSSMAN S D,WOLFE B B,YASUDA R P,et al.Changes in NMDA receptor subunit expression in response to contusive spinal cord injury[J].J Neurochem,2000,75(1):174-184.
[25] KIM Y,CHO H Y,AHN Y J,et al.Effect of NMDA NR2Bantagonist on neuropathic pain in two spinal cord injury models[J].Pain,2012,153(5):1022-1029.
[26]李潇潇,卢圣锋,朱冰梅,等.兴奋性氨基酸毒性与缺血性脑中风及针刺的调整作用[J].针刺研究,2016,41(2):180-185.
[27] KIM H N,KIM Y R,JANG J Y,et al.Effects of electroacupuncture on N-Methyl-D-aspartate receptor-related signaling pathway in the spinal cord of normal rats[J].Evid Based Complement Alternat Med,2012,2012:492471.