通督调神固本法对血管性痴呆大鼠学习记忆能力及海马区mTOR、PTEN表达的影响
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摘要
目的:观察通督调神固本法对血管性痴呆(VD)大鼠学习记忆能力及海马区mTOR、PTEN表达的影响,探讨通督调神固本法治疗VD的作用机制。方法:SD大鼠随机分为假手术组、模型组、电针组、LY模型组、LY电针组、DMSO电针组,每组6只。采用四血管阻断法(4-VO)复制VD模型。对电针组、LY电针组、DMSO电针组选取"百会""大椎""脾俞""肾俞"进行电针干预,每日1次,25 min/次,治疗两周。采用Morris水迷宫测试大鼠空间学习记忆能力,TUNEL法检测大鼠海马中神经元细胞凋亡数量,Western Blot法检测大鼠海马中mTOR、PTEN蛋白表达情况。结果:与假手术组相比,模型组大鼠的平均逃避潜伏期及首次穿越平台时间明显延长(P<0.05),模型组海马区凋亡细胞数目明显增多(P<0.01)及mTOR表达减少(P<0.05),PTEN表达增加(P<0.05);与模型组相比,电针组平均逃避潜伏期及首次穿越平台时间明显缩短(P<0.05),LY模型组、LY电针组平均逃避潜伏期及首次穿越平台时间均无差异(P>0.05),电针组凋亡细胞减少(P<0.01),LY电针组、LY模型组凋亡细胞均无统计学差异(P>0.05),电针组mTOR表达增加(P<0.05),PTEN表达减少(P<0.05);LY模型组与LY电针组比较,平均逃避潜伏期、首次穿越平台时间、凋亡细胞数、海马区mTOR及PTEN表达均无差异(P>0.05);与LY电针组相比,DMSO电针组平均逃避潜伏期及首次穿越平台时间均有所缩短(P<0.05),海马区凋亡细胞数减少(P<0.01)及mTOR表达增加(P<0.05),PTEN表达减少(P<0.05)。结论:通督调神固本法改善了VD模型大鼠的学习记忆能力,海马中mTOR蛋白的表达增加,而PTEN蛋白的表达减少,减少了细胞凋亡数,对受损的神经元细胞起到保护作用,这可能是通督调神固本法治疗VD的重要作用机制之一。
Objective:To observe the effect of electroacupuncture(EA) on learning-memory ability and expressions of hippocampal mTOR and PTEN in vascular dementia rats so as to reveal its mechanism underlying improving VD. Methods:SD rats were randomly divided into sham operation,model,EA group,LY model group,LY electroacupuncture group and DMSO electroacupuncture group,6 rats in each. The rats in the EA group were treated 25 minutes per day ato Baihui(DU20), Dazhui(DU14) and Pishu(BL20),Shenshu(BL23). The rats learning-memory ability was detected by Morris water maze tests and the state of hippocampal apoptosis cells was observed by light microscope after TUNEL staining, and the expressions of hippocampal mTOR,PTEN proteins were detected by western blot. Results:In comparion with sham group, the average escape latency of place navigation task and the duration for crossing the target-platform for the first time were evidently prolonged(P<0.05). The number of apoptotic cells in hippocampal was significantly increased(P<0.01)and the expression levels of mTOR proteins was decreased(P<0.05) and PTEN proteins was increased(P<0.05). Compared with model group,the average escape latency of place navigation task and the duration for crossing the target-platform for the first time were evidently shortened(P<0.05). LY model group and LY electroacupuncture group had no significant difference(P>0.05). The number of apoptotic cells in EA group was decreased(P<0.01). LY model group and LY electroacupuncture group had no significant difference. The expression levels of mTOR proteins were increased(P<0.05)and PTEN decreased(P<0.05). There was no significant difference between the LY model group and the LY group(P>0.05). Compared with LY electroacupuncture group,the average escape latency of place navigation task and the duration for crossing the target-platform for the first time were shortened(P<0.05). The number of apoptotic cells in EA group was decreased(P<0.01). The expression levels of mTOR proteins was increased(P<0.05)and PTEN decreased(P<0.05). Conclusion:Electroacupunture can improve the learning-memory ability of VD model rats and the expression of hippocampal mTOR was increased and PTEN decreased. It reduced the number of apoptotic cells and protected damaged nerve cells, which may be contribute to the mechanisms of treatment of vascular dementia.
Objective:To observe the effect of electroacupuncture(EA) on learning-memory ability and expressions of hippocampal mTOR and PTEN in vascular dementia rats so as to reveal its mechanism underlying improving VD. Methods:SD rats were randomly divided into sham operation,model,EA group,LY model group,LY electroacupuncture group and DMSO electroacupuncture group,6 rats in each. The rats in the EA group were treated 25 minutes per day ato Baihui(DU20), Dazhui(DU14) and Pishu(BL20),Shenshu(BL23). The rats learning-memory ability was detected by Morris water maze tests and the state of hippocampal apoptosis cells was observed by light microscope after TUNEL staining, and the expressions of hippocampal mTOR,PTEN proteins were detected by western blot. Results:In comparion with sham group, the average escape latency of place navigation task and the duration for crossing the target-platform for the first time were evidently prolonged(P<0.05). The number of apoptotic cells in hippocampal was significantly increased(P<0.01)and the expression levels of mTOR proteins was decreased(P<0.05) and PTEN proteins was increased(P<0.05). Compared with model group,the average escape latency of place navigation task and the duration for crossing the target-platform for the first time were evidently shortened(P<0.05). LY model group and LY electroacupuncture group had no significant difference(P>0.05). The number of apoptotic cells in EA group was decreased(P<0.01). LY model group and LY electroacupuncture group had no significant difference. The expression levels of mTOR proteins were increased(P<0.05)and PTEN decreased(P<0.05). There was no significant difference between the LY model group and the LY group(P>0.05). Compared with LY electroacupuncture group,the average escape latency of place navigation task and the duration for crossing the target-platform for the first time were shortened(P<0.05). The number of apoptotic cells in EA group was decreased(P<0.01). The expression levels of mTOR proteins was increased(P<0.05)and PTEN decreased(P<0.05). Conclusion:Electroacupunture can improve the learning-memory ability of VD model rats and the expression of hippocampal mTOR was increased and PTEN decreased. It reduced the number of apoptotic cells and protected damaged nerve cells, which may be contribute to the mechanisms of treatment of vascular dementia.
引文
[1] 赖新生,黄泳.血管性痴呆的针灸治疗和实验研究近况[J].广州中医药大学学报,2006,23(1):81-84.
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[6] Ciuffreda L,Falcome I,Incani UC,et al.PTEN expression and function in adult cancer stem cells and prospects for therapeutic targeting[J].Adv Biol Regul,2014(56):66-80.
[7] Christie KJ,Webber CA,Martinez JA,et al.PTEN inhibition to facilitate intrinsic regenerative outgrowth of adult peripheral axons[J].JNeurosci,2010,30(27):9306-9315.
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[11] 王维刚,周嘉斌,朱明莉,等.小鼠动物实验方法系列专题(一)——Morris水迷宫实验在小鼠表型分析中的应用[J].中国细胞生物学学报,2011,33(1):8-14.
[12] 谢洲,甘君学,刘慧慧.针灸治疗血管性痴呆临床研究进展[J].辽宁中医药大学学报,2014,16(3):231-234.
[13] 赵奕.通督调神针刺法治疗血管性痴呆的临床研究[D].广州:广州中医药大学,2013.
[14] 吕晓红,王岩,张淑琴,等.局灶性脑缺血再灌流过程中凋亡细胞相关基因的研究[J].中风与神经疾病杂志,2001,18(2):70.
[15] 赖新生,王黎,唐纯志.电针对实验性血管性痴呆大鼠学习记忆能力和脑组织细胞凋亡的影响[J].中国康复医学杂志,2003,18(3):140-143.
[16] 刘振寰,潘佩光,祁岩超,等.通督醒神针刺法对脑性瘫痪幼鼠脑组织神经细胞凋亡及神经生长因子蛋白表达的影响[J].中医药临床杂志,2010,22(1):36-40.
[17] Kang S,Dong SM,Kim BR,et al.Thioridazine induces apoptosis by targeting the PI3K/Akt/mTOR pathway in cervical and endometrial cancer cells.Apoptosis,2012,17(9):989-997.
[18] 彭沛,宋志明,刘勇,等. 人参皂苷Rb1抗小鼠脑自然衰老及其对m TOR/P70S6K 通路的影响[J]. 中山大学学报: 医学科学版,2015,36(2): 176-180.
[19] Feng MJ, Ding XS. Akt and cell surviving[J]. J Med Molec Biol,2002,24(5):283-285.
[20] Granville CA,Memmott RM,Gill JJ,et al.Handicapping the race to develop inhibitors of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway[J].Clin Cancer Res,2006:12(3 Pt 1):679-689.
[21] 杨光,李美子,崔春爱.肉豆蔻提取物对慢性血管性痴呆大鼠脑组织NGF和mTOR表达的影响[J].广东医学,2017,38(2):194-196.
[22] 危国军,董大明,姚猛,等.PTEN和mTOR信号转导通路在实验性脊髓损伤中的表达[J].现代生物医学进展,2009,9(10):1850-1851.
[2] 邵瑛,赖新生,关楚威,等.“通督调神固本”电针法对高血压-高血脂复合血管性痴呆模型大鼠学习记忆干预的初步研究[J].针刺研究,2009,34(6):368-375.
[3] Saiki S,Sasazawa Y,Imamichi Y,et al.Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition[J].Autophagy,2011,7(2):176-187.
[4] Benjamin D, Colombi M, Moroni C, et al. Rapamycin passes the torch:A new generation of mTOR inhibitors[J]. Nat. Rev. Drug Discov, 2011, 10: 868-880.
[5] Wang HY,Wang GL,Yu YH, et al. The role of hosphoinositide-3-kinase/Akt pathway in propofol-induced postconditioning against focal cerebral ischemia-reperfusion injury in rats[J]. Brain Res, 2009,1297(10):177-184.
[6] Ciuffreda L,Falcome I,Incani UC,et al.PTEN expression and function in adult cancer stem cells and prospects for therapeutic targeting[J].Adv Biol Regul,2014(56):66-80.
[7] Christie KJ,Webber CA,Martinez JA,et al.PTEN inhibition to facilitate intrinsic regenerative outgrowth of adult peripheral axons[J].JNeurosci,2010,30(27):9306-9315.
[8] Park KK,Liu K,Hu Y,et al.Promoting axon regeneration in the adult CNS by modulation of the PTEN /mTOR athway[J].Science,2008,322(5903):963-966.
[9] 罗祖明,董佑忠,彭国之.脑血管疾病治疗学[M].北京:人民卫生出版社,1999:25.
[10] 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.
[11] 王维刚,周嘉斌,朱明莉,等.小鼠动物实验方法系列专题(一)——Morris水迷宫实验在小鼠表型分析中的应用[J].中国细胞生物学学报,2011,33(1):8-14.
[12] 谢洲,甘君学,刘慧慧.针灸治疗血管性痴呆临床研究进展[J].辽宁中医药大学学报,2014,16(3):231-234.
[13] 赵奕.通督调神针刺法治疗血管性痴呆的临床研究[D].广州:广州中医药大学,2013.
[14] 吕晓红,王岩,张淑琴,等.局灶性脑缺血再灌流过程中凋亡细胞相关基因的研究[J].中风与神经疾病杂志,2001,18(2):70.
[15] 赖新生,王黎,唐纯志.电针对实验性血管性痴呆大鼠学习记忆能力和脑组织细胞凋亡的影响[J].中国康复医学杂志,2003,18(3):140-143.
[16] 刘振寰,潘佩光,祁岩超,等.通督醒神针刺法对脑性瘫痪幼鼠脑组织神经细胞凋亡及神经生长因子蛋白表达的影响[J].中医药临床杂志,2010,22(1):36-40.
[17] Kang S,Dong SM,Kim BR,et al.Thioridazine induces apoptosis by targeting the PI3K/Akt/mTOR pathway in cervical and endometrial cancer cells.Apoptosis,2012,17(9):989-997.
[18] 彭沛,宋志明,刘勇,等. 人参皂苷Rb1抗小鼠脑自然衰老及其对m TOR/P70S6K 通路的影响[J]. 中山大学学报: 医学科学版,2015,36(2): 176-180.
[19] Feng MJ, Ding XS. Akt and cell surviving[J]. J Med Molec Biol,2002,24(5):283-285.
[20] Granville CA,Memmott RM,Gill JJ,et al.Handicapping the race to develop inhibitors of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway[J].Clin Cancer Res,2006:12(3 Pt 1):679-689.
[21] 杨光,李美子,崔春爱.肉豆蔻提取物对慢性血管性痴呆大鼠脑组织NGF和mTOR表达的影响[J].广东医学,2017,38(2):194-196.
[22] 危国军,董大明,姚猛,等.PTEN和mTOR信号转导通路在实验性脊髓损伤中的表达[J].现代生物医学进展,2009,9(10):1850-1851.