艾灸对血管性痴呆大鼠海马神经营养因子及Notch信号通路表达的影响
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摘要
目的:观察艾灸对血管性痴呆(VD)大鼠海马神经营养因子及Notch通路表达的影响,探讨艾灸治疗VD的作用机制。方法:SD大鼠随机分成假手术组、模型组、西药组、艾灸组,每组15只。采用双侧颈总动脉缺血再灌注法复制大鼠VD模型。艾灸组给予悬灸"命门""大椎""关元",每次15min,每日1次,每周治疗6d,治疗4周。西药组给予尼莫地平灌胃,每日1次,治疗4周。Morris水迷宫实验测定大鼠学习与记忆能力,氯化三苯基四氮唑(TTC)染色法检测大鼠大脑的梗死面积,HE染色法检测大鼠脑组织病理形态,实时荧光定量PCR和Western blot法检测各组大鼠海马胶质原纤维酸性蛋白(GFAP)、神经生长因子(NGF)、脑源性神经营养因子(BDNF)、Notch 1、碱性螺旋-环-螺旋转录因子3(Hes 3)mRNA和蛋白表达情况。结果:模型组水迷宫实验逃避潜伏期较假手术组延长(P<0.05);治疗后,艾灸组和西药组逃避潜伏期较模型组明显缩短(P<0.05)。TTC染色结果显示,模型组颜色较假手术组变浅,白色区域多;西药组、艾灸组白色区域较模型组减少。HE染色结果显示,模型组大鼠海马区神经元数量较假手术组明显减少,深染细胞排列紊乱,可见坏死的神经元;西药组、艾灸组细胞水肿较轻,细胞形态接近正常,细胞数量增加。模型组海马组织中BDNF、NGF、GFAP、Hes 3、Notch 1mRNA及蛋白表达均较假手术组明显升高(P<0.05,P<0.01);与模型组相比,艾灸组BDNF、NGF、GFAP、Hes 3、Notch 1 mRNA及蛋白表达明显升高(P<0.05,P<0.01),西药组NGF、GFAP mRNA及蛋白表达明显升高(P<0.05,P<0.01)。与西药组比较,艾灸组海马组织中GFAP、Hes 3、Notch 1mRNA表达明显升高(P<0.05,P<0.01)。结论:艾灸可以改善痴呆大鼠记忆和认知能力,调节神经营养因子表达,可能与调节Notch信号通路有关。
Objective To observe the effect of moxibustion on the learning ability and expression of neurotrophic factors and Notch signaling in vascular dementia(VD)rats,so as to explore its neurogenesis mechanism underlying improvement of VD.Methods Sixty SD rats were equally and randomly divided into sham operation(sham),model,medication and moxibustion groups(n=15 rats/group).The VD model was established by occlusion of the bilateral cervical common arteries and reperfusion.Moxibustion was applied to"Dazhui"(GV 14),"Guanyuan"(CV 4)and "Mingmen"(GV 4)for 15 minutes,once daily,6 times a week for 4 weeks.Rats of the medication group were treated by gavage of nimodipine(2 mg·kg-1·d-1),3 times a day,6 days a week for 4 weeks.Morris water maze tests were performed to detect the rat's learning-memory ability.The infarcted size of the brain was detected by using 2,3,5-triphenyltetrazolium chloride(TTC)staining,and H.E.staining was used to detect the histopathological changes.The expression level of glia fibrillary acidic protein(GFAP),nerve growth factor(NGF),brain derived neurotrophic factor(BDNF),Notch 1(a receptor),Hes 3(a downstream effector)mRNAs and proteins in the hippocampal tissues were detected by quantitative real-time PCR and Western blot,respectively.Results After 4 weeks' intervention,modeling-induced increase of escape latency was significantly shortened in both moxibustion and medication groups relevant to the model group(P<0.05),and the infarct size was reduced and the damage degree of nerve cells in the brain tissue alleviated.The expression levels of BDNF,NGF,GFAP,Hes 3,Notch 1 genes and proteins were significantly up-regulated in the model group relevant to the sham operation group(P<0.05,P<0.01).After the intervention,the expression levels of hippocampal BDNF,NGF,GFAP,Hes 3 and Notch 1 mRNAs and proteins in the moxibustion group,and NGF and GFAP mRNAs,and BDNF,NGF,GFAP,Hes 3 and Notch 1 proteins in the medication group were further obviously up-regulated relevant to the model group(P<0.05,P<0.01).The effect of moxibustion was significantly superior to that of medication in up-regulating GFAP,Hes 3,Notch 1 mRNAs expression(P<0.05,P<0.01).No significant differences were found in the expression of BDNF,Hes 3 and Notch 1 mRNAs in the medication group relevant to the model group(P>0.05),and between the moxibustion and medication groups in up-regulating the expression of BDNF and NGF mRNAs,and in up-regulating the expression of BDNF,NGF,GFAP,Hes3 and Notch 1 proteins(P>0.05).Conclusion Moxibustion can improve learning ability in VD rats,which may be associated with its effects in up-regulating the expression of neurotrophic factors and in potentiating Notch signaling.
Objective To observe the effect of moxibustion on the learning ability and expression of neurotrophic factors and Notch signaling in vascular dementia(VD)rats,so as to explore its neurogenesis mechanism underlying improvement of VD.Methods Sixty SD rats were equally and randomly divided into sham operation(sham),model,medication and moxibustion groups(n=15 rats/group).The VD model was established by occlusion of the bilateral cervical common arteries and reperfusion.Moxibustion was applied to"Dazhui"(GV 14),"Guanyuan"(CV 4)and "Mingmen"(GV 4)for 15 minutes,once daily,6 times a week for 4 weeks.Rats of the medication group were treated by gavage of nimodipine(2 mg·kg-1·d-1),3 times a day,6 days a week for 4 weeks.Morris water maze tests were performed to detect the rat's learning-memory ability.The infarcted size of the brain was detected by using 2,3,5-triphenyltetrazolium chloride(TTC)staining,and H.E.staining was used to detect the histopathological changes.The expression level of glia fibrillary acidic protein(GFAP),nerve growth factor(NGF),brain derived neurotrophic factor(BDNF),Notch 1(a receptor),Hes 3(a downstream effector)mRNAs and proteins in the hippocampal tissues were detected by quantitative real-time PCR and Western blot,respectively.Results After 4 weeks' intervention,modeling-induced increase of escape latency was significantly shortened in both moxibustion and medication groups relevant to the model group(P<0.05),and the infarct size was reduced and the damage degree of nerve cells in the brain tissue alleviated.The expression levels of BDNF,NGF,GFAP,Hes 3,Notch 1 genes and proteins were significantly up-regulated in the model group relevant to the sham operation group(P<0.05,P<0.01).After the intervention,the expression levels of hippocampal BDNF,NGF,GFAP,Hes 3 and Notch 1 mRNAs and proteins in the moxibustion group,and NGF and GFAP mRNAs,and BDNF,NGF,GFAP,Hes 3 and Notch 1 proteins in the medication group were further obviously up-regulated relevant to the model group(P<0.05,P<0.01).The effect of moxibustion was significantly superior to that of medication in up-regulating GFAP,Hes 3,Notch 1 mRNAs expression(P<0.05,P<0.01).No significant differences were found in the expression of BDNF,Hes 3 and Notch 1 mRNAs in the medication group relevant to the model group(P>0.05),and between the moxibustion and medication groups in up-regulating the expression of BDNF and NGF mRNAs,and in up-regulating the expression of BDNF,NGF,GFAP,Hes3 and Notch 1 proteins(P>0.05).Conclusion Moxibustion can improve learning ability in VD rats,which may be associated with its effects in up-regulating the expression of neurotrophic factors and in potentiating Notch signaling.
引文
[1]SAKR H F,KHALIL K I,HUSSEIN A M,et al.Effect of dehydroepiandrosterone(DHEA)on memory and brain derived neurotrophic factor(BDNF)in a rat model of vascular dementia[J].J Physiol Pharmacol,2014,65(1):41-53.
[2]FENG G.Observation on efficacy of treating vascular dementia gait disorder with electro-acupuncture(EA)on Jiājǐ(夹脊EX-B 2)along lumbar vertebra combined with scalp acupuncture[J].World J Acu-moxi,2014,24(2):1-5.
[3]陈浩,王频,杨骏,等.艾灸对血管性痴呆患者症状及脑脊液中神经肽类物质水平的影响[J].中国针灸,2011,31(1):22-24.
[4]朱才丰,杨骏,杨坤,等.通督化瘀灸法治疗血管性认知障碍临床观察[J].上海针灸杂志,2013,32(4):242-244.
[5]余静,楚佳梅,高灵爱,等.麦粒灸对阿尔茨海默病小鼠学习记忆能力及脑内淀粉样蛋白沉积的影响[J].针刺研究,2014,39(1):58-62.
[6]朱才丰,孙健健,韩为,等.通督调神灸法抗轻度认知功能障碍大鼠海马β-淀粉样蛋白过度表达的机制研究[J].针刺研究,2016,41(2):131-137.
[7]CHOI D H,LEE K H,LEE J.Effect of exercise-induced neurogenesis on cognitive function deficit in a rat model of vascular dementia[J].Mol Med Rep,2016,13(4):2981-2990.
[8]侯志涛,孙忠人,刘松涛,等.电针对缺血性学习记忆障碍大鼠氧自由基及凋亡相关蛋白表达的影响[J].针刺研究,2015,40(6):431-438.
[9]张会珍,梁玉磊,张雪静,等.不同时间电针对血管性痴呆小鼠行为学及海马单胺类神经递质的影响[J].针刺研究,2014,39(2):142-147.
[10]林文注.实验针灸学[M].上海:上海科学技术出版社,1999:276-299.
[11]包伍叶,焦爽,卢峻,等.电针对慢性应激抑郁大鼠学习记忆与海马神经元损伤的影响[J].针刺研究,2014,39(2):136-141.
[12]田文静,黄琳娜,王瑞辉,等.方氏头针对血管性痴呆大鼠海马CA 1区星形胶质细胞凋亡的影响[J].针刺研究,2015,40(2):6-12.
[13]何国永,黄海威,邓哲治,等.鼠神经生长因子对全脑照射大鼠空间认知功能的修复作用[J].中华医学杂志,2016,96(19):1530-1534.
[14]DENG R,PI M,YANG Z X,et al.Effect of electroacupuncture at conception vessel and governor vessel on correlative signals of stress-injury-repair in cerebral ischemia reperfusion rats[J].World J Acu-moxi,2015,25(3):22-29.
[15]XING M,SUN Q,WANG Y,et al.Hydroxysafflor yellow A increases BDNF and NMDARs in the hippocampus in a vascular dementia rat model[J].Brain Res,2016,1642:419-425.
[16]唐勇,卢圣锋,乔秀兰,等.艾灸对快速老化模型小鼠海马神经干细胞分化的影响[J].中国康复医学杂志,2010,25(4):301-304.
[17]李怀斌,吴锋,熊克仁,等.针药结合对局灶性脑缺血大鼠海马CA 1和CA 3区神经干细胞增殖与分化相关蛋白表达的影响[J].针刺研究,2015,40(6):455-460.
[18]刘敏,缪化春,李怀斌,等.针药结合对脑缺血大鼠下丘脑室旁核脑源性神经营养因子和血管内皮生长因子表达的影响[J].针刺研究,2016,41(2):119-123.
[19]黄锐,赵健,吴锋,等.电针联合天麻多糖对脑缺血大鼠基底外侧杏仁核巢蛋白、脑源性神经营养因子表达的影响[J].针刺研究,2016,41(3):230-234.
[20]王晓兰,孙国杰,杜艳军,等.艾灸预处理对阿尔茨海默病模型大鼠海马CA 1区NGF和BDNF表达的影响[J].中国康复,2011,26(5):323-326.
[21]李熙,孙国杰,王述菊,等.艾灸预处理对阿尔茨海默病模型大鼠海马CA 1区NGF及其受体Trk A表达的影响[J].湖北中医杂志,2012,34(3):20-22.
[22]YAGI H,SAITO T,YANAGISAWA M,et al.Lewis Xcarrying N-glycans regulate the proliferation of mouse embryonic neural stem cells via the Notch signaling pathway[J].J Biol Chem,2012,287(29):24356-24364.
[23]陶静,柳维林,黄佳,等.基于Notch 1信号通路观察电针对脑缺血再灌注损伤大鼠缺血周边皮质与SVZ区神经干细胞增殖的影响[J].康复学报,2015,25(3):23-33.
[24]郭娜,何金婷,胥桂华,等.Notch 1受体在脑缺血损伤中表达变化及作用的体外研究[J].中风与神经疾病杂志,2013,30(4):345-347.
[25]王珏,邓宇斌,万勇.Notch信号通路对神经干细胞增殖分化的作用[J].中国细胞生物学学报,2014,36(5):708-712.
[26]娄远蕾,匡助才,鲁友明,等.大鼠脑缺血后Notch分子上调调控血管新生[J].基础医学与临床,2015,35(1):97-98.
[27]陈娉婷,周小青,曹泽标,等.丹龙醒脑方对脑缺血再灌注大鼠海马区Notch信号通路相关蛋白表达的影响[J].湖南中医药大学学报,2016,36(1):10-14.
[28]GROTH C,FORTINI M E.Therapeutic approaches to modulating Notch signaling:current challenges and future prospects[J].Semin Cell Dev Biol,2012,23(4):465-672.
[29]莽靖,王姣琦,刘洪雨,等.脑缺血损伤中Notch信号通路活性的非配体依赖性改变及其作用[J].中国老年学杂志,2014,35(10):2781-2783.
[30]方继良,王小玲,王寅,等.电针正常人足三里和关元穴中枢效应的fMRI脑功能成像比较[J].针刺研究,2012,37(1):46-52.
[31]罗燕,廖金玲,冯枫,等.电针对大鼠脑缺血灶周围区少突胶质细胞超微结构及蛋白表达的影响[J].针刺研究,2015,40(3):192-198.
[32]朱才丰,潘洪萍,贺成功.蔡圣朝主任医师针灸治疗血管性痴呆学术思想探析[J].辽宁中医药大学学报,2015,17(8):110-112.
[2]FENG G.Observation on efficacy of treating vascular dementia gait disorder with electro-acupuncture(EA)on Jiājǐ(夹脊EX-B 2)along lumbar vertebra combined with scalp acupuncture[J].World J Acu-moxi,2014,24(2):1-5.
[3]陈浩,王频,杨骏,等.艾灸对血管性痴呆患者症状及脑脊液中神经肽类物质水平的影响[J].中国针灸,2011,31(1):22-24.
[4]朱才丰,杨骏,杨坤,等.通督化瘀灸法治疗血管性认知障碍临床观察[J].上海针灸杂志,2013,32(4):242-244.
[5]余静,楚佳梅,高灵爱,等.麦粒灸对阿尔茨海默病小鼠学习记忆能力及脑内淀粉样蛋白沉积的影响[J].针刺研究,2014,39(1):58-62.
[6]朱才丰,孙健健,韩为,等.通督调神灸法抗轻度认知功能障碍大鼠海马β-淀粉样蛋白过度表达的机制研究[J].针刺研究,2016,41(2):131-137.
[7]CHOI D H,LEE K H,LEE J.Effect of exercise-induced neurogenesis on cognitive function deficit in a rat model of vascular dementia[J].Mol Med Rep,2016,13(4):2981-2990.
[8]侯志涛,孙忠人,刘松涛,等.电针对缺血性学习记忆障碍大鼠氧自由基及凋亡相关蛋白表达的影响[J].针刺研究,2015,40(6):431-438.
[9]张会珍,梁玉磊,张雪静,等.不同时间电针对血管性痴呆小鼠行为学及海马单胺类神经递质的影响[J].针刺研究,2014,39(2):142-147.
[10]林文注.实验针灸学[M].上海:上海科学技术出版社,1999:276-299.
[11]包伍叶,焦爽,卢峻,等.电针对慢性应激抑郁大鼠学习记忆与海马神经元损伤的影响[J].针刺研究,2014,39(2):136-141.
[12]田文静,黄琳娜,王瑞辉,等.方氏头针对血管性痴呆大鼠海马CA 1区星形胶质细胞凋亡的影响[J].针刺研究,2015,40(2):6-12.
[13]何国永,黄海威,邓哲治,等.鼠神经生长因子对全脑照射大鼠空间认知功能的修复作用[J].中华医学杂志,2016,96(19):1530-1534.
[14]DENG R,PI M,YANG Z X,et al.Effect of electroacupuncture at conception vessel and governor vessel on correlative signals of stress-injury-repair in cerebral ischemia reperfusion rats[J].World J Acu-moxi,2015,25(3):22-29.
[15]XING M,SUN Q,WANG Y,et al.Hydroxysafflor yellow A increases BDNF and NMDARs in the hippocampus in a vascular dementia rat model[J].Brain Res,2016,1642:419-425.
[16]唐勇,卢圣锋,乔秀兰,等.艾灸对快速老化模型小鼠海马神经干细胞分化的影响[J].中国康复医学杂志,2010,25(4):301-304.
[17]李怀斌,吴锋,熊克仁,等.针药结合对局灶性脑缺血大鼠海马CA 1和CA 3区神经干细胞增殖与分化相关蛋白表达的影响[J].针刺研究,2015,40(6):455-460.
[18]刘敏,缪化春,李怀斌,等.针药结合对脑缺血大鼠下丘脑室旁核脑源性神经营养因子和血管内皮生长因子表达的影响[J].针刺研究,2016,41(2):119-123.
[19]黄锐,赵健,吴锋,等.电针联合天麻多糖对脑缺血大鼠基底外侧杏仁核巢蛋白、脑源性神经营养因子表达的影响[J].针刺研究,2016,41(3):230-234.
[20]王晓兰,孙国杰,杜艳军,等.艾灸预处理对阿尔茨海默病模型大鼠海马CA 1区NGF和BDNF表达的影响[J].中国康复,2011,26(5):323-326.
[21]李熙,孙国杰,王述菊,等.艾灸预处理对阿尔茨海默病模型大鼠海马CA 1区NGF及其受体Trk A表达的影响[J].湖北中医杂志,2012,34(3):20-22.
[22]YAGI H,SAITO T,YANAGISAWA M,et al.Lewis Xcarrying N-glycans regulate the proliferation of mouse embryonic neural stem cells via the Notch signaling pathway[J].J Biol Chem,2012,287(29):24356-24364.
[23]陶静,柳维林,黄佳,等.基于Notch 1信号通路观察电针对脑缺血再灌注损伤大鼠缺血周边皮质与SVZ区神经干细胞增殖的影响[J].康复学报,2015,25(3):23-33.
[24]郭娜,何金婷,胥桂华,等.Notch 1受体在脑缺血损伤中表达变化及作用的体外研究[J].中风与神经疾病杂志,2013,30(4):345-347.
[25]王珏,邓宇斌,万勇.Notch信号通路对神经干细胞增殖分化的作用[J].中国细胞生物学学报,2014,36(5):708-712.
[26]娄远蕾,匡助才,鲁友明,等.大鼠脑缺血后Notch分子上调调控血管新生[J].基础医学与临床,2015,35(1):97-98.
[27]陈娉婷,周小青,曹泽标,等.丹龙醒脑方对脑缺血再灌注大鼠海马区Notch信号通路相关蛋白表达的影响[J].湖南中医药大学学报,2016,36(1):10-14.
[28]GROTH C,FORTINI M E.Therapeutic approaches to modulating Notch signaling:current challenges and future prospects[J].Semin Cell Dev Biol,2012,23(4):465-672.
[29]莽靖,王姣琦,刘洪雨,等.脑缺血损伤中Notch信号通路活性的非配体依赖性改变及其作用[J].中国老年学杂志,2014,35(10):2781-2783.
[30]方继良,王小玲,王寅,等.电针正常人足三里和关元穴中枢效应的fMRI脑功能成像比较[J].针刺研究,2012,37(1):46-52.
[31]罗燕,廖金玲,冯枫,等.电针对大鼠脑缺血灶周围区少突胶质细胞超微结构及蛋白表达的影响[J].针刺研究,2015,40(3):192-198.
[32]朱才丰,潘洪萍,贺成功.蔡圣朝主任医师针灸治疗血管性痴呆学术思想探析[J].辽宁中医药大学学报,2015,17(8):110-112.
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