眼针对脑缺血再灌注损伤大鼠神经血管单元细胞凋亡的影响
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摘要
目的:观察眼针对脑缺血再灌注损伤大鼠脑组织微血管内皮细胞、星形胶质细胞、神经元凋亡及大脑皮质Bad、Bcl-xL表达的影响,探讨眼针对脑缺血再灌注损伤大鼠神经血管单元保护作用的机制。方法:SD大鼠随机分为空白组、假手术组、模型组(3、24、72h组),眼针组(3、24、72h组),每组12只。采用改良线栓法制备脑缺血再灌注损伤大鼠模型。眼针组针刺"肝区""肾区""上焦""下焦",每次20min,每12h治疗1次。造模后3、24、72h采用Western blot法检测缺血侧大脑皮质Bad、Bcl-xL蛋白的表达;造模后72h采用凋亡免疫荧光双标记法检测神经元、微血管内皮细胞、星形胶质细胞凋亡情况。结果:与空白组比较,脑缺血再灌注72h后模型大鼠缺血侧大脑皮质的神经元、微血管内皮细胞、星形胶质细胞均可见不同程度的凋亡(P<0.01)。眼针72h组与模型72h组比较,神经元、微血管内皮细胞、星形胶质细胞凋亡数减少(P<0.01)。与空白组比较,模型组3、24、72h大脑皮质Bad蛋白表达上调(P<0.01);与同时间点模型组比较,眼针24h组、眼针72h组Bad蛋白表达减少(P<0.05)。与空白组比较,模型3h组、24h组、72h组大脑皮质Bcl-xL蛋白表达上调(P<0.01);与同时间点模型组比较,眼针3h组、24h组、72h组Bcl-xL蛋白表达上调(P<0.01)。结论:眼针治疗可以通过减少Bad蛋白的表达,促进Bcl-xL的表达,减少脑缺血后神经血管单元的主要成分神经元、微血管内皮细胞、星形胶质细胞的凋亡,从而对脑缺血后的神经血管单元起到保护作用。
Objective To observe the effect of eye-acupuncture intervention on cerebro-cortical apoptosis of microvascular endothelial cells,neurons and astrocytes(main components of neurovascular unit)and the expression of Bad(an apoptosis promoter)and B-cell lymphoma-extra large(Bcl-xL)proteins in focal cerebral ischemia-reperfusion injury(CIRI)rats,so as to explore its mechanisms underlying improvement of CIRI.Methods SD rats were randomly divided into control,sham-operation,model 3 h,model 24 h,model 72 h,eye-acupuncture 3 h,eye-acupuncture 24 hand eye-acupuncture 72 hgroups(n=12 in each group).The CIRI model was established by middle cerebral artery occlusion/reperfusion(MCAO/R).Eye-acupuncture was applied to bilateral"Gan"(Liver)regions,"Shen"(Kidney)regions,"Shangjiao"(Upper-energizer)and"Xiajiao"(Lower-energizer)for 20 min,once 3 hand every 12 hafter modeling.The expression levels of Bad and Bcl-xL in the ischemic cerebral cortex tissue were detected by Western blot.The apoptotic neurons,microvascular endothelial cells and astrocytes were assayed by immunofluorescence double labeling(Nestin/TUNEL,CD34/TUNEL and glial fibrillary acidic protein[GFAP]/TUNEL)separately.Results After modeling,the numbers of apoptotic neurons,microvascular endothelial cells and astrocytes in the ischemic cerebral cortex tissue were significantly increased in the model 72 hgroup than in the sham-operation group(P<0.01).Following the treatment,the numbers of the 3 types of apoptotic cells were markedly lower in the eye-acupuncture 72 hgroup than in the model72 hgroup(P<0.01).The expression levels of Bad and Bcl-xL proteins were notably up-regulated in the model 3 h,model 24 h and model 72 hgroups than in the sham operation group(P<0.01).Following eye-acupuncture intervention,modeling induced increase of the Bad expression were obviously reversed in eye-acupuncture 24 hand eye-acupuncture 72 hgroups than those in the2 model groups(P<0.05).And the increase of Bcl-xL expression levels were further increased in the eye-acupuncture groups in comparison with those in the 3 model groups(P<0.01).Conclusion Eye-acupuncture can down-regulate the expression of Bad protein,and up-regulate the expression of Bcl-xL protein in the ischemic cerebral cortex in CIRI rats,which may contribute to its function in reducing apoptotic neurons,microvascular endothelial cells and astrocytes,suggesting aprotective effect of eye-acupuncture intervention on neurovascular unit.
Objective To observe the effect of eye-acupuncture intervention on cerebro-cortical apoptosis of microvascular endothelial cells,neurons and astrocytes(main components of neurovascular unit)and the expression of Bad(an apoptosis promoter)and B-cell lymphoma-extra large(Bcl-xL)proteins in focal cerebral ischemia-reperfusion injury(CIRI)rats,so as to explore its mechanisms underlying improvement of CIRI.Methods SD rats were randomly divided into control,sham-operation,model 3 h,model 24 h,model 72 h,eye-acupuncture 3 h,eye-acupuncture 24 hand eye-acupuncture 72 hgroups(n=12 in each group).The CIRI model was established by middle cerebral artery occlusion/reperfusion(MCAO/R).Eye-acupuncture was applied to bilateral"Gan"(Liver)regions,"Shen"(Kidney)regions,"Shangjiao"(Upper-energizer)and"Xiajiao"(Lower-energizer)for 20 min,once 3 hand every 12 hafter modeling.The expression levels of Bad and Bcl-xL in the ischemic cerebral cortex tissue were detected by Western blot.The apoptotic neurons,microvascular endothelial cells and astrocytes were assayed by immunofluorescence double labeling(Nestin/TUNEL,CD34/TUNEL and glial fibrillary acidic protein[GFAP]/TUNEL)separately.Results After modeling,the numbers of apoptotic neurons,microvascular endothelial cells and astrocytes in the ischemic cerebral cortex tissue were significantly increased in the model 72 hgroup than in the sham-operation group(P<0.01).Following the treatment,the numbers of the 3 types of apoptotic cells were markedly lower in the eye-acupuncture 72 hgroup than in the model72 hgroup(P<0.01).The expression levels of Bad and Bcl-xL proteins were notably up-regulated in the model 3 h,model 24 h and model 72 hgroups than in the sham operation group(P<0.01).Following eye-acupuncture intervention,modeling induced increase of the Bad expression were obviously reversed in eye-acupuncture 24 hand eye-acupuncture 72 hgroups than those in the2 model groups(P<0.05).And the increase of Bcl-xL expression levels were further increased in the eye-acupuncture groups in comparison with those in the 3 model groups(P<0.01).Conclusion Eye-acupuncture can down-regulate the expression of Bad protein,and up-regulate the expression of Bcl-xL protein in the ischemic cerebral cortex in CIRI rats,which may contribute to its function in reducing apoptotic neurons,microvascular endothelial cells and astrocytes,suggesting aprotective effect of eye-acupuncture intervention on neurovascular unit.
引文
[1]中华医学会神经病学分会,急性缺血性脑卒中介入诊疗指南撰写组.中国急性缺血性脑卒中早期血管内介入诊疗指南[J].中华神经科杂志,2015,48(5):356-361.
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[3]韩永升,徐银,韩永竹,等.电针对局灶性脑缺血再灌注大鼠神经血管单元的保护[J].针刺研究,2013,38(3):173-180.
[4]LOVE S.Apoptosis and brain ischaemia[J].Prog Neuropsychopharmacol Biol Psychiatry,2003,27(2):267-282.
[5]PERFETTINI J L,REED J C,ISRAE¨L N,et al.Role of Bcl-2family members in caspase-independent apoptosis during chlamydia infection[J].Infect Immun,2002,70(1):55-61.
[6]候志涛,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.
[7]张立德,于丹,曲怡,等.眼针对脑缺血再灌注损伤大鼠脑组织细胞间黏附因子-1表达的影响[J].针刺研究,2011,36(6):409-413.
[8]赵丹玉,苗兰英,曹阳,等.眼针对脑缺血再灌注损伤模型大鼠脑皮层组织TLR4、MyD88表达的影响[J].中国老年病学杂志,2016,10(4):1547-1549.
[9]马贤德,孙宏伟.线栓法制备大鼠脑缺血再灌注模型的方法研究[J].中华中医药学刊,2009,27(6):1200-1201.
[10]LONGA E Z,WEINSTEIN P R,CARLSON S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[11]彭静山.眼针疗法[M].沈阳:辽宁科学技术出版社,1990:11.
[12]虞有智,祝亚猛,王颖,等.TUNEL与免疫组织化学双染色技术的应用[J].中华病理学杂志,2003,32(6):286-288.
[13]宓丹,王德成,陈雪.眼针对脑缺血再灌注损伤大鼠PI3K/AKt通路影响的实验研究[J].中国中医急症杂志,2017,26(2):212-215.
[14]PARK S E,KIM S H,HOSSAIN M A,et al.Korean red ginseng extract induces apoptosis and decreases telomerase activity in human leukemia cells[J].J Ethnopharmacol,2009,121(2):304-312.
[15]韩雪,胡楚文,李玉娟,等.右旋美托咪啶通过Akt/Bad通路抑制异氟醚引起的新生大鼠海马细胞凋亡[J].中国药理学通报,2013,29(12):1702-1706.
[16]何敏韩,江全倪,白云,等.永久性大脑中动脉闭塞大鼠梗死周围组织p-Akt(Sear3)、p-Bad(Serl36)表达与细胞凋亡[J].国际脑血管病杂志,2013,21(8):624-627.
[17]王晓天,刘晓梅,尤红娟,等.PI3K/Akt通路在脑缺血Bad线粒体转位中的神经保护作用[J].徐州医学院学报,2014,34(11):715-719.
[18]杨帆,昂文平,沈德凯,等.磷脂酰肌醇-3-激酶/蛋白激酶B信号转导通路与针刺保护癫痫继发海马神经元损伤的关系[J].针刺研究,2013,38(1):20-26.
[19]陈乾,冯飞,陈文明,等.红花注射液对脑缺血再灌注损伤大鼠大脑皮质Bax及Bcl-xl/Bcl-2的影响[J].神经药理学报,2015,5(1):15-19.
[20]贾丽娜,宋友.缺血后处理对局灶性脑缺血再灌注大鼠BclxL、Bax的影响[J].黑龙江医药科学,2012,35(6):11-14.
[21]WIESSNER C,ALLEGRINI P R,RUPALLA K,et al.Neuron-specific transgene expression of Bcl-xl But not Bcl-2genes reduced lesion size after permanent middle cerebral artery occulusion in mice[J].Neurosci Lett,1999,268(3):119-122.
[22]del ZOPPO G D.The neurovascular unit in the setting of stroke[J].Intern Med,2010,267(2):156-171.
[23]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.
[24]王世军,崔可密,卢岩,等,针刺对MCAO大鼠海马CA3区微血管数目及神经元死亡率的影响[J].山东中医药大学学报,2005,29(1):59-60.
[2]JAUCH E C,SAVER J L,ADAMS H P Jr,et al.Guidelines for the early management of patients with acute ischemic stroke:aguideline for healthcare professionals from the A-merican Heart Association/American Stroke Association[J].Stroke,2013,44(3):870-947.
[3]韩永升,徐银,韩永竹,等.电针对局灶性脑缺血再灌注大鼠神经血管单元的保护[J].针刺研究,2013,38(3):173-180.
[4]LOVE S.Apoptosis and brain ischaemia[J].Prog Neuropsychopharmacol Biol Psychiatry,2003,27(2):267-282.
[5]PERFETTINI J L,REED J C,ISRAE¨L N,et al.Role of Bcl-2family members in caspase-independent apoptosis during chlamydia infection[J].Infect Immun,2002,70(1):55-61.
[6]候志涛,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.
[7]张立德,于丹,曲怡,等.眼针对脑缺血再灌注损伤大鼠脑组织细胞间黏附因子-1表达的影响[J].针刺研究,2011,36(6):409-413.
[8]赵丹玉,苗兰英,曹阳,等.眼针对脑缺血再灌注损伤模型大鼠脑皮层组织TLR4、MyD88表达的影响[J].中国老年病学杂志,2016,10(4):1547-1549.
[9]马贤德,孙宏伟.线栓法制备大鼠脑缺血再灌注模型的方法研究[J].中华中医药学刊,2009,27(6):1200-1201.
[10]LONGA E Z,WEINSTEIN P R,CARLSON S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[11]彭静山.眼针疗法[M].沈阳:辽宁科学技术出版社,1990:11.
[12]虞有智,祝亚猛,王颖,等.TUNEL与免疫组织化学双染色技术的应用[J].中华病理学杂志,2003,32(6):286-288.
[13]宓丹,王德成,陈雪.眼针对脑缺血再灌注损伤大鼠PI3K/AKt通路影响的实验研究[J].中国中医急症杂志,2017,26(2):212-215.
[14]PARK S E,KIM S H,HOSSAIN M A,et al.Korean red ginseng extract induces apoptosis and decreases telomerase activity in human leukemia cells[J].J Ethnopharmacol,2009,121(2):304-312.
[15]韩雪,胡楚文,李玉娟,等.右旋美托咪啶通过Akt/Bad通路抑制异氟醚引起的新生大鼠海马细胞凋亡[J].中国药理学通报,2013,29(12):1702-1706.
[16]何敏韩,江全倪,白云,等.永久性大脑中动脉闭塞大鼠梗死周围组织p-Akt(Sear3)、p-Bad(Serl36)表达与细胞凋亡[J].国际脑血管病杂志,2013,21(8):624-627.
[17]王晓天,刘晓梅,尤红娟,等.PI3K/Akt通路在脑缺血Bad线粒体转位中的神经保护作用[J].徐州医学院学报,2014,34(11):715-719.
[18]杨帆,昂文平,沈德凯,等.磷脂酰肌醇-3-激酶/蛋白激酶B信号转导通路与针刺保护癫痫继发海马神经元损伤的关系[J].针刺研究,2013,38(1):20-26.
[19]陈乾,冯飞,陈文明,等.红花注射液对脑缺血再灌注损伤大鼠大脑皮质Bax及Bcl-xl/Bcl-2的影响[J].神经药理学报,2015,5(1):15-19.
[20]贾丽娜,宋友.缺血后处理对局灶性脑缺血再灌注大鼠BclxL、Bax的影响[J].黑龙江医药科学,2012,35(6):11-14.
[21]WIESSNER C,ALLEGRINI P R,RUPALLA K,et al.Neuron-specific transgene expression of Bcl-xl But not Bcl-2genes reduced lesion size after permanent middle cerebral artery occulusion in mice[J].Neurosci Lett,1999,268(3):119-122.
[22]del ZOPPO G D.The neurovascular unit in the setting of stroke[J].Intern Med,2010,267(2):156-171.
[23]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.
[24]王世军,崔可密,卢岩,等,针刺对MCAO大鼠海马CA3区微血管数目及神经元死亡率的影响[J].山东中医药大学学报,2005,29(1):59-60.