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
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摘要
目的:观察针刺长强穴对脆性X精神发育迟缓1(FMR1)基因敲除(KO)小鼠小脑CypA-ERK1/2通路相关蛋白表达的影响,并探讨其作用机理。方法:选取FMR1基因缺失且日龄为28日的KO小鼠24只,通过聚合酶链式反应(PCR)鉴定小鼠基因表型,采用数字随机表法分为空白组、长强穴组、阻断剂+长强穴组和非经非穴组,每组6只。空白组仅给予抓取动作,其余组别采取针刺干预,Western blot手段检测小脑CypA、P-ERK1/2、ERK1/2相关蛋白的表达情况。结果:长强穴组CypA的表达明显高于非经非穴组和空白组(P<0.05);长强穴组和非经非穴组P-ERK1/2的表达明显高于空白组(P<0.05);长强穴组ERK1/2的表达明显高于非经非穴组和空白组(P<0.05);而上述效应能被ERK通路阻断剂PD98059所逆转,但阻断剂+长强穴组与长强穴组比较差异无统计学意义(P>0.05)。结论:针刺长强穴能促进FMR1基因敲除小鼠小脑区域CypA-ERK1/2通路相关蛋白的表达,其作用能被ERK通路阻断剂所阻断。
Objective: To observe the effect of DU1 acupoint on the expression of CypA-ERK1/2 pathway related proteins from the cerebellum of FMR1 gene knockout(KO) mice, so as to explore its mechanism. Methods: Identification of murine gene phenotype by polymerase chain reaction(PCR), and choosing a Fragile X-1(FMR1) deficiency in the KO mice with 28 days after birth, total 24 KO mice were randomly divided into blank group, DU1 acupoint group, DU1 acupoint group with blocking and non-acupoint group,with 6 mice in each group. The blank group was given grab action, the other groups were taken acupuncture intervention. The expression of the related proteins of cerebellar CypA, P-ERK1/2 and ERK1/2 was detected by Western blot method. Results: The expression of Cyp A in the DU1 acupoint group was significantly higher than that of the non-acupoint group and the blank group(P<0.05). The expression of P-ERK1/2 in the DU1 acupoint group and the non-acupoint group was significantly higher than that of the blank group(P<0.05); the expression of ERK1/2 in the DU1 acupoint group was higher than that of the non-acupoint group and the blank group(P<0.05). The relative protein expression of the DU1 acupoint group with blocking can be converted by PD98059. Conclusion:Acupuncture on DU1 acupoint can promote the expression of CypA-ERK1/2 pathway related proteins in the cerebellar region of FMR1 gene KO mice, and this effection can be broken off by the blocker PD98059.
Objective: To observe the effect of DU1 acupoint on the expression of CypA-ERK1/2 pathway related proteins from the cerebellum of FMR1 gene knockout(KO) mice, so as to explore its mechanism. Methods: Identification of murine gene phenotype by polymerase chain reaction(PCR), and choosing a Fragile X-1(FMR1) deficiency in the KO mice with 28 days after birth, total 24 KO mice were randomly divided into blank group, DU1 acupoint group, DU1 acupoint group with blocking and non-acupoint group,with 6 mice in each group. The blank group was given grab action, the other groups were taken acupuncture intervention. The expression of the related proteins of cerebellar CypA, P-ERK1/2 and ERK1/2 was detected by Western blot method. Results: The expression of Cyp A in the DU1 acupoint group was significantly higher than that of the non-acupoint group and the blank group(P<0.05). The expression of P-ERK1/2 in the DU1 acupoint group and the non-acupoint group was significantly higher than that of the blank group(P<0.05); the expression of ERK1/2 in the DU1 acupoint group was higher than that of the non-acupoint group and the blank group(P<0.05). The relative protein expression of the DU1 acupoint group with blocking can be converted by PD98059. Conclusion:Acupuncture on DU1 acupoint can promote the expression of CypA-ERK1/2 pathway related proteins in the cerebellar region of FMR1 gene KO mice, and this effection can be broken off by the blocker PD98059.
引文
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[22]董玉霞,孙晓红,宋卫科,等.脆性X智力低下蛋白致病机制研究进展[J].中国现代神经疾病杂志,2010,10(3):387-389.
[23]CHEN L,YUN S W,SETO J,et al.The fragile X mental retardation protein binds and regulates a novel class of m RNAs containing U rich target sequences[J].Neuroscience,2003,120(4):1005-1017.
[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]OBCHOEI S,SAWANYAWISUTH K,WONGKHAM C,et al.Secreted cyclophilin A mediates G1/S phase transition of cholangiocarcinoma cells via CD147/ERK1/2 pathway[J].Tumour Biol,2015,36(2):849-859.
[26]CHEN Y,DUAN Y,YANG X,et al.Inhibition of ERK1/2 and activation of LXR synergistically reduce atherosclerotic lesions in Apo E-deficient mice[J].Arterioscler Thromb Vasc Biol,2015,35(4):948-959.
[27]黄靓,李国庆,毛振江,等.ERK1/2信号通路阻断剂PD98059对胃癌细胞MGC-803中RECK、MMP-9基因表达的影响[J].世界华人消化杂志,2011,19(20):2097-2103.
[2]宋道辉,朱幼玲,唐南,等.小脑与认知关系的研究进展[J].山东医药,2011,51(44):108-109.
[3]DEMIRTAS-TATLIDEDE A,FREITAS C,CROMER J R,et al.Safety and proof of principle study of cerebellar vermal theta burst stimulation in refractory schizophrenia[J].Schizophr Res,2010,124(1/3):91-100.
[4]KOSSOROTOFF M,GONIN-FLAMBOIS C,GITIAUX C,et al.A cognitive and affective pattern in posterior fossa strokes in children:a case series[J].Dev Med Child Neurol,2010,52(7):626-631.
[5]李忠仁.实验针灸学[M].北京:中国中医药出版社,2003:327-329.
[6]MOORE C J,DALY E M,TASSONE F,et al.The effect of premutation of X chromosome CGG trinucleotide repeats on brain anatomy[J].Brain,2004,127(Pt 12):2672-2681.
[7]颜华,张惠佳,喻佩君,等.小脑电刺激治疗对脑性瘫痪患儿运动功能和智力发育的影响[J].中国临床康复,2004,8(33):7486-7487.
[8]周继宏,张志强,韦建芳.电刺激小脑顶核对缺血性脑血管病的影响[J].中国临床康复,2003,7(28):3860-3861.
[9]王茂林,汪恩焕,周钟阳,等.血清亲环素A与急性动脉粥样硬化性脑梗死关系研究[J].淮海医药,2014,32(3):221-222.
[10]张田田,张俊峰.亲环素A及其受体CD147对动脉粥样硬化的影响[J].上海交通大学学报:医学版,2011,31(12):1778-1781.
[11]CHIU R,REY O,ZHENG J Q,et al.Effects of altered expression and localization of cyclophilin A on differentiation of p19embryonic carcinoma cells[J].Cell Mol Neurobiol,2003,23(6):929-943.
[12]GOLDNER F M,PATRICK J W.Neuronal localization of the cyclophilin.A protein in the adult rat brain[J].J Comp Neurol,1996,372(2):283-293.
[13]BOULOS S,MELONI B P,ARTHUR P G,et al.Evidence that intracellular cyclophilin A and cyclophilin A/CD147 receptormediated ERK1/2 signalling can protect neurons against in vitro oxidative and ischemic injury[J].Neurobiol Dis,2007,25(1):54-64.
[14]韩学哲,赵刚,刘兴吉,等.亲环素A蛋白水平与脑胶质瘤预后相关性研究[J].中国实验诊断学,2009,13(1):54-56.
[15]王佳,周文胜,洪秀琴.高尔基体应激与抗氧化系统在缺血脑损伤中的作用[J].中国老年学杂志,2014,34(1):278-281.
[16]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.
[17]廖娟,兰风华.脆性X智力低下蛋白结构及功能的研究进展[J].福建医科大学学报,2010,44(4):311-314.
[18]邢洲.脆性X智力低下蛋白通过MEK/ERK信号通路促进星形胶质细胞瘤增殖[D].广州:暨南大学,2015:71-72.
[19]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.
[20]BROWN V,JIN P,CEMAN S,et al.Microarray identification of FMRP-associated brain m RNAs and altered m RNA translational profiles in fragile X syndrome[J].Cell,2001,107(4):477-487.
[21]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.
[22]董玉霞,孙晓红,宋卫科,等.脆性X智力低下蛋白致病机制研究进展[J].中国现代神经疾病杂志,2010,10(3):387-389.
[23]CHEN L,YUN S W,SETO J,et al.The fragile X mental retardation protein binds and regulates a novel class of m RNAs containing U rich target sequences[J].Neuroscience,2003,120(4):1005-1017.
[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]OBCHOEI S,SAWANYAWISUTH K,WONGKHAM C,et al.Secreted cyclophilin A mediates G1/S phase transition of cholangiocarcinoma cells via CD147/ERK1/2 pathway[J].Tumour Biol,2015,36(2):849-859.
[26]CHEN Y,DUAN Y,YANG X,et al.Inhibition of ERK1/2 and activation of LXR synergistically reduce atherosclerotic lesions in Apo E-deficient mice[J].Arterioscler Thromb Vasc Biol,2015,35(4):948-959.
[27]黄靓,李国庆,毛振江,等.ERK1/2信号通路阻断剂PD98059对胃癌细胞MGC-803中RECK、MMP-9基因表达的影响[J].世界华人消化杂志,2011,19(20):2097-2103.