电针命门穴对FMR1基因敲除小鼠自主行为学及海马CREB蛋白表达的影响
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摘要
目的观察针刺督脉命门穴对FMR1基因敲除(KO)小鼠大脑海马区环磷腺苷效应元件结合蛋白(CREB)及其磷酸化CREB(p-CREB)蛋白表达的影响,并探讨其作用机制。方法选取适龄的FMR1基因敲除小鼠,采用聚合酶链式反应(PCR)鉴定小鼠基因表型;采用随机数表法将24只基因型为纯合子的小鼠分为空白组、非经非穴组和命门组,每组各8只。空白组在相同时间、相同条件下仅模拟抓取动作。命门组选取小鼠命门穴,非经非穴组选取小鼠尾根与肛门之间凹陷旁开约1 cm的部位,均于每日固定时间采用由0.5寸毫针自制而成的双极连体针连接电针仪进行干预,电针刺激频率2 Hz,强度2 m A,连续波,每日30 min,连续干预14 d;干预后连续两天于同一时间进行自主活动行为学测试,并采用免疫组化法及Western blot法分析海马CREB及其p-CREB蛋白表达量。结果自主行为学测试结果显示:与空白组比较,命门组活动次数和站立次数显著降低(P<0.05);免疫组化结果显示:命门组CREB和p-CREB蛋白的阳性平均光密度表达较空白组显著性升高(P<0.01,P<0.05),命门组p-CREB蛋白的阳性平均光密度表达较非经非穴组显著性升高(P<0.05);Western blot结果显示:各组CREB蛋白表达量比较无统计学意义(P>0.05),非经非穴组与命门组较空白组p-CREB蛋白表达量显著升高(P<0.05,P<0.01)。结论电针命门穴对FMR1基因敲除小鼠自主活动站立行为有影响,且能促进海马CREB、p-CREB蛋白的表达。
Objective: To observe the effects of electroacupuncture(EA) at Mingmen(GV4) on autonomous behavior and CREB and p-CREB expression in hippocampus of fragile X mental retardation 1(FMR1) gene knockout(KO) mice, and explore the mechanism of EA at GV4 on FMR1 KO mice. Methods: Fragile X-1 deficiency mice on appropriate age were chosen and the genotype were identified by polymerase chain reaction(PCR). 24 chose mice were randomly divided into blank group,non-acupoint EA group and GV4 acupoint group, with 8 mice in each group. The GV4 acupoint was selected in the GV4 acupoint EA group, and in the non-acupoints EA group, the area about 1 cm adjacent to the pit between the tail root and the anus was selected. The blank group only simulated grabbing at the same time and under the same conditions, and the rest of the groups took EA intervention by HANS electroacupuncture therapeutic apparatus and chosen the 1 inch acupuncture to make the double pole needle to puncture,the puncture were connected electroacupuncture therapeutic apparatus acupuncturing with continuewave, 2 Hz, 2 m A, 30 min. All groups of mice were treated with daily at fixed time, once a day for 14 days. The experiment mice were tested by autonomic activity during two days after the interesting treatment. And the expression of the related proteins of CREB and p-CREB was detected by immunohistochemistry and western blot. Results: The results of autonomous behavior test showed that compared with the blank group, the activities and standing times of GV4 acupoint EA group decreased significantly(P<0.05). The expression of CREB and p-CREB protein by immunohistochemistry in GV4 acupoint EA group was significantly higher than that of blank group(P<0.01, P<0.05). The expression of p-CREB protein by immunohistochemistry in GV4 acupoint EA group was significantly higher than that of the non-acupoints EA group(P<0.05). There was no statistical significance in the expression of CREB protein in each group by western blot(P>0.05).The expression of p-CREB protein by western blot in GV4 acupoint EA group and the non-acupoints EA group were significantly higher than that of the blank group(P<0.05, P<0.01). Conclusion: EA at the Mingmen(GV4) acupoint could adjust the independent activity of FMR1 gene KO mice and could improve the expression of CREB and p-CREB proteins in hippocampus region of FMR1 gene knockout mice.
Objective: To observe the effects of electroacupuncture(EA) at Mingmen(GV4) on autonomous behavior and CREB and p-CREB expression in hippocampus of fragile X mental retardation 1(FMR1) gene knockout(KO) mice, and explore the mechanism of EA at GV4 on FMR1 KO mice. Methods: Fragile X-1 deficiency mice on appropriate age were chosen and the genotype were identified by polymerase chain reaction(PCR). 24 chose mice were randomly divided into blank group,non-acupoint EA group and GV4 acupoint group, with 8 mice in each group. The GV4 acupoint was selected in the GV4 acupoint EA group, and in the non-acupoints EA group, the area about 1 cm adjacent to the pit between the tail root and the anus was selected. The blank group only simulated grabbing at the same time and under the same conditions, and the rest of the groups took EA intervention by HANS electroacupuncture therapeutic apparatus and chosen the 1 inch acupuncture to make the double pole needle to puncture,the puncture were connected electroacupuncture therapeutic apparatus acupuncturing with continuewave, 2 Hz, 2 m A, 30 min. All groups of mice were treated with daily at fixed time, once a day for 14 days. The experiment mice were tested by autonomic activity during two days after the interesting treatment. And the expression of the related proteins of CREB and p-CREB was detected by immunohistochemistry and western blot. Results: The results of autonomous behavior test showed that compared with the blank group, the activities and standing times of GV4 acupoint EA group decreased significantly(P<0.05). The expression of CREB and p-CREB protein by immunohistochemistry in GV4 acupoint EA group was significantly higher than that of blank group(P<0.01, P<0.05). The expression of p-CREB protein by immunohistochemistry in GV4 acupoint EA group was significantly higher than that of the non-acupoints EA group(P<0.05). There was no statistical significance in the expression of CREB protein in each group by western blot(P>0.05).The expression of p-CREB protein by western blot in GV4 acupoint EA group and the non-acupoints EA group were significantly higher than that of the blank group(P<0.05, P<0.01). Conclusion: EA at the Mingmen(GV4) acupoint could adjust the independent activity of FMR1 gene KO mice and could improve the expression of CREB and p-CREB proteins in hippocampus region of FMR1 gene knockout mice.
引文
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[14]范郁山,叶子维,汤昌华,等.命门穴之助阳作用刍议[J].河北中医,2015,37(3):469-472.
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[16]林羽,徐伟,张玉琴,等.栝楼桂枝颗粒抗缺血性脑卒中大鼠神经元及原代海马神经元凋亡研究[J].康复学报,2015,25(1):38-43.
[17]BARCO A,MARIE H.Genetic approaches to investigate the role of CREB in neuronal plasticity and memory[J].Mol Neurobiol,2011,44(3):330-349.
[18]YAMAMOTO K K,GONZALEZ G A,BIGGS W H,et al.Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB[J].Nature,1988,334(6182):494-498.
[19]MONTMINY M R,BILEZIKJIAN L M.Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene[J].Nature,1987,328(6126):175-178.
[20]马莉,王诗陶,刘征.电针额区对血管性痴呆大鼠行为学及脑内cAMP-PKA-CREB信号通路的影响[J].针灸临床杂志,2017,33(1):50-52.
[21]LIN D,ZHANG J,ZHUANG W Y,et al.The effect of electroacupuncture versus manual acupuncture through the expression of TrkB/NF-κB in the subgranular zone of the dentate gyrus of telomerase-deficient mice[J].Evid Based Complement Alternat Med,2018,2018:1013978.
[22]SHENG S,HUANG J,REN Y,et al.Neuroprotection against hypoxic/ischemic injury:δ-opioid receptors and BDNF-TrkB pathway[J].Cell Physiol Biochem,2018,47(1):302-315.
[23]YUN Y C,JANG D,YOON S B,et al.Laser acupuncture exerts neuroprotective effects via regulation of Creb,Bdnf,Bcl-2,and Bax gene expressions in the hippocampus[J].Evid Based Complement Alternat Med,2017,2017:7181637.
[24]LIN D,WU Q,LIN X,et al.Brain-derived neurotrophic factor signaling pathway:modulation by acupuncture in telomerase knockout mice[J].Altern Ther Health Med,2015,21(6):36-46.
[25]LIN D,DE LA PENA I,LIN L,et al.The neuroprotective role of acupuncture and activation of the bdnf signaling pathway[J].Int J Mol Sci,2014,15(2):3234-3252.
[26]FANG J Q,FANG J F,LIANG Y,et al.Electroacupuncture mediates extracellular signal-regulated kinase 1/2 pathways in the spinal cord of rats with inflammatory pain[J].BMC Complement Altern Med,2014,14:285.
[27]SHAO X M,SUN J,JIANG B Y,et al.Inhibition of the cAMP/PKA/CREB pathway contributes to the analgesic effects of electroacupuncture in the anterior cingulate cortex in a rat pain memory model[J].Neural Plast,2016,2016:5320641.
[28]张占伟,薛丽君.针刺对急性脑挫裂伤大鼠脑组织内p-CREB表达的影响[J].中国微侵袭神经外科杂志,2012,17(10):469-471.
[2]LAVAL S H,BLAIR H J,HIRST M C,et al.Mapping of FMR1,the gene implicated in fragile X-linked mental retardation,on the mouse X chromosome[J].Genomics,1992,12(4):818-821.
[3]徐琴,竺智伟,赵正言.脆性X综合征树突棘形态发育研究进展[J].神经解剖学杂志,2009,25(5):587-590.
[4]张伟雯,黄越玲,刘国彬,等.30日龄Fmr1基因敲除小鼠的自主活动观察[J].国际医药卫生导报,2011,17(1):1-4.
[5]张志灿,杨晓婷,林栋.电针长强穴对FMR1基因敲除小鼠皮质区CREB蛋白表达的影响[J].福建中医药,2018,49(1):56-57.
[6]林栋,卜婉萍,杨晓婷.电针长强穴对FMR1基因敲除小鼠小脑CypA相关蛋白表达的影响及其阻断效应研究[J].康复学报,2017,27(5):29-33.
[7]张晨骥,张学君,陈兰芳,等.电针“长强”穴对FMR1基因敲除小鼠海马CA1区NLGN-3蛋白表达的影响[J].辽宁中医杂志,2017,44(4):858-861.
[8]陈兰芳,张学君,林栋,等.电针长强穴对FMR1基因敲除小鼠海马CA1区PSD-95、CREB蛋白表达的影响[J].康复学报,2015,25(4):18-21,26.
[9]韩平,俞萍,陈可爱,等.针刺长强穴对FMR1基因敲除小鼠海马CA1区BDNF和SYN表达的影响[J].福建中医药大学学报,2012,22(5):14-18.
[10]韩平,王振宇,林栋,等.浅谈针刺长强穴改善儿童智力残疾[J].中华中医药杂志,2011,26(5):1238-1240.
[11]林栋,吴强.针灸对精神发育迟滞的作用机制研究进展[J].福建中医药大学学报,2011,21(2):60-61,69.
[12]林栋,吴强.针灸治疗精神发育迟滞的现代研究及古籍记载的相关性探讨[J].辽宁中医杂志,2011,38(9):1915-1917.
[13]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:326.
[14]范郁山,叶子维,汤昌华,等.命门穴之助阳作用刍议[J].河北中医,2015,37(3):469-472.
[15]翟伟,陈俊军,熊健,等.聪脑通络法针刺治疗小儿脑瘫疗效观察[J].中国针灸,2009,29(11):868-872.
[16]林羽,徐伟,张玉琴,等.栝楼桂枝颗粒抗缺血性脑卒中大鼠神经元及原代海马神经元凋亡研究[J].康复学报,2015,25(1):38-43.
[17]BARCO A,MARIE H.Genetic approaches to investigate the role of CREB in neuronal plasticity and memory[J].Mol Neurobiol,2011,44(3):330-349.
[18]YAMAMOTO K K,GONZALEZ G A,BIGGS W H,et al.Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB[J].Nature,1988,334(6182):494-498.
[19]MONTMINY M R,BILEZIKJIAN L M.Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene[J].Nature,1987,328(6126):175-178.
[20]马莉,王诗陶,刘征.电针额区对血管性痴呆大鼠行为学及脑内cAMP-PKA-CREB信号通路的影响[J].针灸临床杂志,2017,33(1):50-52.
[21]LIN D,ZHANG J,ZHUANG W Y,et al.The effect of electroacupuncture versus manual acupuncture through the expression of TrkB/NF-κB in the subgranular zone of the dentate gyrus of telomerase-deficient mice[J].Evid Based Complement Alternat Med,2018,2018:1013978.
[22]SHENG S,HUANG J,REN Y,et al.Neuroprotection against hypoxic/ischemic injury:δ-opioid receptors and BDNF-TrkB pathway[J].Cell Physiol Biochem,2018,47(1):302-315.
[23]YUN Y C,JANG D,YOON S B,et al.Laser acupuncture exerts neuroprotective effects via regulation of Creb,Bdnf,Bcl-2,and Bax gene expressions in the hippocampus[J].Evid Based Complement Alternat Med,2017,2017:7181637.
[24]LIN D,WU Q,LIN X,et al.Brain-derived neurotrophic factor signaling pathway:modulation by acupuncture in telomerase knockout mice[J].Altern Ther Health Med,2015,21(6):36-46.
[25]LIN D,DE LA PENA I,LIN L,et al.The neuroprotective role of acupuncture and activation of the bdnf signaling pathway[J].Int J Mol Sci,2014,15(2):3234-3252.
[26]FANG J Q,FANG J F,LIANG Y,et al.Electroacupuncture mediates extracellular signal-regulated kinase 1/2 pathways in the spinal cord of rats with inflammatory pain[J].BMC Complement Altern Med,2014,14:285.
[27]SHAO X M,SUN J,JIANG B Y,et al.Inhibition of the cAMP/PKA/CREB pathway contributes to the analgesic effects of electroacupuncture in the anterior cingulate cortex in a rat pain memory model[J].Neural Plast,2016,2016:5320641.
[28]张占伟,薛丽君.针刺对急性脑挫裂伤大鼠脑组织内p-CREB表达的影响[J].中国微侵袭神经外科杂志,2012,17(10):469-471.