针灸对海洛因复吸大鼠脑突触超微结构和突触骨架蛋白的影响
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摘要
目的:观察针灸对海洛因复吸大鼠脑前额叶皮层突触超微结构以及突触骨架蛋白的影响,并探讨其作用机制。方法:Wistar大鼠24只随机分为正常组、模型组和针灸组,每组8只。递增量肌肉注射海洛因8d(染毒),正常饲养5d(脱毒),染毒—脱毒3个循环形成海洛因复吸模型。针灸组在染毒期处理与模型组相同,在脱毒期给予针灸治疗,毫针斜刺"百会"穴1～2mm,留针30min,同时艾灸双侧"肾俞"穴30min。正常组染毒期注射0.9%的氯化钠溶液,脱毒期常规饲养。于实验第39天取材并用荧光定量PCR和Western blot法检测大鼠脑前额叶皮层突触骨架蛋白的表达,用透射电镜观察大鼠脑突触超微结构。结果:荧光定量PCR和Western blot检测结果显示,与正常组比较,模型组大鼠前额叶皮层脑组织中微管相关蛋白(MAP)-2、Tau较正常组表达明显降低(P<0.05),细胞骨架活性调节蛋白(Arc)的表达较正常组明显升高(P<0.05);与模型组比较,针灸组前额叶皮层脑组织中MAP-2、Tau表达明显升高(P<0.05),而Arc的表达明显降低(P<0.05)。与模型组比较,针灸组大鼠电镜下突触的超微结构有明显的修复趋势。结论:针灸可改善海洛因复吸大鼠脑前额叶皮层突触超微结构,调节突触骨架蛋白的表达,对海洛因复吸大鼠脑组织的损伤具有一定的保护作用。
Objective To observe the effect of acupuncture plus moxibustion on the synaptic ultrastructure and expression of synaptic skeleton related proteins in the prefrontal cortex(PFC)of heroin re-addicted rats,so as to reveal its mechanisms underlying improvement of heroin addiction.Methods Twenty-four Wister rats(half male and half female)were randomly divided into normal control,model and acupuncture groups(n=8 in each group).The heroin re-addicted model was established by muscular injection of heroin into the hind limbs for 8 days(incremental 0.8—3.6 mg,once daily for 6 days,and twice daily for2 days),followed by conventional breeding for 5 days(detoxification),the procedure(addition-detoxification)was repeated3 cycles.For rats of the acupuncture group,"Baihui"(GV20)was needled with filiform needles which were retained for 30 min,and moxibustion was then applied to bilateral"Shenshu"(BL23)for 30 min.The treatment was conducted once daily during the detoxification.On the 39 th day of experiment,the bilateral prefrontal cortex tissues were sampled for examining the ultrastructure by using transmission electron microscope(TEM)after fixative solution immersion and for determining the expression of genes and proteins of activity-regulated cytoskeleton-associated protein(Arc),microtubule-asso-ciated protein-2(MAP-2)and microtubuleassociated protein Tau(Tau)with quantitative real-time PCR and Western blot,respectively.Results After modeling,the expression levels of Arc mRNA and protein were significantly up-regulated,and those of MAP-2 and Tau mRNA and proteins obviously down-regulated in the model group relevant to the normal control group(P<0.05).Following the intervention,the up-regulated Arc protein and mRNA and the down-regulated MAP-2 and Tau were obviously reversed relevant to the model group(P<0.05).Outcomes of TEM showed unclear pre-and post-membranes of the synapses,narrowing of the synaptic gap and non-uniform of the density of the thickened dense plaque after modeling,which was relatively milder in the acupuncture group.Conclusion Acupuncture plus moxibustion can improve changes of synaptic ultrastructure in heroin re-addicted rats,which may be related to their effect in regulating the expression of some synaptic skeleton proteins and genes.
Objective To observe the effect of acupuncture plus moxibustion on the synaptic ultrastructure and expression of synaptic skeleton related proteins in the prefrontal cortex(PFC)of heroin re-addicted rats,so as to reveal its mechanisms underlying improvement of heroin addiction.Methods Twenty-four Wister rats(half male and half female)were randomly divided into normal control,model and acupuncture groups(n=8 in each group).The heroin re-addicted model was established by muscular injection of heroin into the hind limbs for 8 days(incremental 0.8—3.6 mg,once daily for 6 days,and twice daily for2 days),followed by conventional breeding for 5 days(detoxification),the procedure(addition-detoxification)was repeated3 cycles.For rats of the acupuncture group,"Baihui"(GV20)was needled with filiform needles which were retained for 30 min,and moxibustion was then applied to bilateral"Shenshu"(BL23)for 30 min.The treatment was conducted once daily during the detoxification.On the 39 th day of experiment,the bilateral prefrontal cortex tissues were sampled for examining the ultrastructure by using transmission electron microscope(TEM)after fixative solution immersion and for determining the expression of genes and proteins of activity-regulated cytoskeleton-associated protein(Arc),microtubule-asso-ciated protein-2(MAP-2)and microtubuleassociated protein Tau(Tau)with quantitative real-time PCR and Western blot,respectively.Results After modeling,the expression levels of Arc mRNA and protein were significantly up-regulated,and those of MAP-2 and Tau mRNA and proteins obviously down-regulated in the model group relevant to the normal control group(P<0.05).Following the intervention,the up-regulated Arc protein and mRNA and the down-regulated MAP-2 and Tau were obviously reversed relevant to the model group(P<0.05).Outcomes of TEM showed unclear pre-and post-membranes of the synapses,narrowing of the synaptic gap and non-uniform of the density of the thickened dense plaque after modeling,which was relatively milder in the acupuncture group.Conclusion Acupuncture plus moxibustion can improve changes of synaptic ultrastructure in heroin re-addicted rats,which may be related to their effect in regulating the expression of some synaptic skeleton proteins and genes.
引文
[1]尹恝,潘速跃,周亮,等.海洛因海绵状白质脑病病理分析[J].南方医科大学学报,2007,27(6):881-883.
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[6] GAO Y L,ZHANG Y,CAO J P,et al.Regulation of the endoplasmic reticulum stress response and neuroprotective effects of acupuncture on brain injury caused by heroin addiction[J].Acupunct Med,2017,35(5):366-373.
[7] ZHANG Y,CAI X H,ZHANG R J,et al.Acupuncture regulates the unfolded protein response and inhibits apoptosis in a rat model of heroin relapse[J].Acupunct Med,2016,34(6):441-448.
[8] HOU X R,ZHANG R J,LV H,et al.Acupuncture at Baihui and Dazhui reduces brain cell apoptosis in heroin readdicts[J].Neural Regene Res,2014,9(2):164-170.
[9] ZHANG R J,CAI X H,SONG X G,et al.Normalization of wentral tegmental area structure following acupuncture in a rat model of heroin relapse[J].Neural Regene Res,2014,9(3):301-307.
[10]王念,张荣军,侯晓蓉,等.针刺对海洛因复吸大鼠脑神经细胞凋亡的影响[J].安徽中医学院学报.2013,32(5):44-48.
[11]韦献良,叶峻,郑毅.海洛因成瘾复吸大鼠模型的建立[J].广西医学,2004,26(6):783-785.
[12]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):1-5.
[13]包新民,舒斯云.大鼠脑立体定位图谱[M].北京:人民卫生出版社,1991:95-104.
[14] CAO J P,ZHANG Y,FU G J,et al.Effect of acupuncture on expression of△FosB and CREB in hippocampal heroin-relapse rats[J].World J Acupunct-moxibustion,2015,25(2):34-37.
[15] HWANG A W,TRZECIAKIEWICZ H,FRIEDMANN D,et al.Conserved lysine acetylation within the microtubule-binding domain regulates MAP-2/Tau family members[J].PLoS One,2016,11(12):e0168913.
[16]赵子艾,周元国.Tau蛋白过度磷酸化的致病机制及其在创伤性脑损伤中的作用[J].生理科学进展,2014,45(3):213-216.
[17]刘志娟.脑源性神经营养因子在突触可塑性中的作用[J].国际神经病学神经外科学杂志,2015,42(2):185-188.
[18] MESSAOUDI E,KANHEMA T,SOULEJ,et al.Sustained Arc/Arg3.1synthesis controls long-term potentiation consolidation through regulation of local actin polymerization in the dentate gyrus in vivo[J].J Neurosci,2007,27(39):10445-10455.
[19]徐振华,许能贵,易玮,等.不同针刺量对大鼠脑缺血后突触可塑性的影响[J].广州中医药大学学报,2009,26(1):32-37.
[20]姜美驰,梁静,张玉杰,等.针刺“四关”穴对阿尔茨海默病大鼠学习记忆及海马区β淀粉样蛋白42、白介素-1β和白介素-2的影响[J].针刺研究,2016,41(2):113-118.
[21]朱晶,国海东,邵水金.针灸治疗阿尔茨海默病机制的研究进展[J].针刺研究,2012,37(5):422-427.
[22]曹香玲,吴新贵,张俊川,等.电针治疗对MCAO大鼠运动功能及梗死对侧大脑皮层GAP-43、MAP-2表达的影响[J].华中科技大学学报(医学版),2018,47(3):274-279.
[23]刘瑶,周启心,徐林,等.突触骨架重塑调控吗啡戒断负性记忆的分子机制进展[J].科学通报,2014,59(1):15-20.
[2]张瑛,苗玲,钱可久,等.海洛因海绵状白质脑病(附2例报道)[J].卒中与神经疾病,2004,11(1):47-49.
[3]张荣军,蔡兴慧,宋小鸽,等.海洛因复吸对大鼠脑神经元的超微结构的影响[J].中国药物依赖性杂志,2008,17(6):424-426.
[4]张荣军,宋小鸽,蔡兴慧,等.针灸对海洛因复吸大鼠条件性位置偏爱及脑前额叶皮质超微结构的影响[J].针刺研究,2009,34(2):97-100.
[5]张荣军,宋小鸽,唐照亮,等,电针抗吗啡戒断大鼠胸腺细胞凋亡的实验观察[J].针刺研究,2005,30(2):94-96.
[6] GAO Y L,ZHANG Y,CAO J P,et al.Regulation of the endoplasmic reticulum stress response and neuroprotective effects of acupuncture on brain injury caused by heroin addiction[J].Acupunct Med,2017,35(5):366-373.
[7] ZHANG Y,CAI X H,ZHANG R J,et al.Acupuncture regulates the unfolded protein response and inhibits apoptosis in a rat model of heroin relapse[J].Acupunct Med,2016,34(6):441-448.
[8] HOU X R,ZHANG R J,LV H,et al.Acupuncture at Baihui and Dazhui reduces brain cell apoptosis in heroin readdicts[J].Neural Regene Res,2014,9(2):164-170.
[9] ZHANG R J,CAI X H,SONG X G,et al.Normalization of wentral tegmental area structure following acupuncture in a rat model of heroin relapse[J].Neural Regene Res,2014,9(3):301-307.
[10]王念,张荣军,侯晓蓉,等.针刺对海洛因复吸大鼠脑神经细胞凋亡的影响[J].安徽中医学院学报.2013,32(5):44-48.
[11]韦献良,叶峻,郑毅.海洛因成瘾复吸大鼠模型的建立[J].广西医学,2004,26(6):783-785.
[12]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):1-5.
[13]包新民,舒斯云.大鼠脑立体定位图谱[M].北京:人民卫生出版社,1991:95-104.
[14] CAO J P,ZHANG Y,FU G J,et al.Effect of acupuncture on expression of△FosB and CREB in hippocampal heroin-relapse rats[J].World J Acupunct-moxibustion,2015,25(2):34-37.
[15] HWANG A W,TRZECIAKIEWICZ H,FRIEDMANN D,et al.Conserved lysine acetylation within the microtubule-binding domain regulates MAP-2/Tau family members[J].PLoS One,2016,11(12):e0168913.
[16]赵子艾,周元国.Tau蛋白过度磷酸化的致病机制及其在创伤性脑损伤中的作用[J].生理科学进展,2014,45(3):213-216.
[17]刘志娟.脑源性神经营养因子在突触可塑性中的作用[J].国际神经病学神经外科学杂志,2015,42(2):185-188.
[18] MESSAOUDI E,KANHEMA T,SOULEJ,et al.Sustained Arc/Arg3.1synthesis controls long-term potentiation consolidation through regulation of local actin polymerization in the dentate gyrus in vivo[J].J Neurosci,2007,27(39):10445-10455.
[19]徐振华,许能贵,易玮,等.不同针刺量对大鼠脑缺血后突触可塑性的影响[J].广州中医药大学学报,2009,26(1):32-37.
[20]姜美驰,梁静,张玉杰,等.针刺“四关”穴对阿尔茨海默病大鼠学习记忆及海马区β淀粉样蛋白42、白介素-1β和白介素-2的影响[J].针刺研究,2016,41(2):113-118.
[21]朱晶,国海东,邵水金.针灸治疗阿尔茨海默病机制的研究进展[J].针刺研究,2012,37(5):422-427.
[22]曹香玲,吴新贵,张俊川,等.电针治疗对MCAO大鼠运动功能及梗死对侧大脑皮层GAP-43、MAP-2表达的影响[J].华中科技大学学报(医学版),2018,47(3):274-279.
[23]刘瑶,周启心,徐林,等.突触骨架重塑调控吗啡戒断负性记忆的分子机制进展[J].科学通报,2014,59(1):15-20.