针刺预处理对老年大鼠脑缺血再灌注损伤的保护作用
摘要
目的观察针刺预处理对老年大鼠短暂性缺血/再灌注损伤后海马CA1区神经元凋亡及相关基因和血清炎性因子的影响。
方法健康老年雄性Wistar大鼠120只,随机分为四组每组30只。组Ⅰ(单纯脑缺血/再灌注组)：四动脉阻断法全脑缺血4min建立老年大鼠全脑缺血模型；组Ⅱ(针刺预处理后脑缺血/再灌注组)：连续5d对百会穴实施穴位针刺预处理(疏密波,强度1mA,频率15 Hz),每天持续30 min,然后建立全脑缺血模型；组Ⅲ(单纯针刺预处理组)：仅针刺百会穴30min/d,持续5 d；组Ⅳ(假手术组),仅暴露四条动脉,不缺血。每组分别于缺血后12h、1、2、3和7d各随机处死6只大鼠,随机抽取一只动物提取海马CA1区脑组织作透射电镜观察神经元超微结构,余下的5只光镜下观察海马神经元形态和凋亡细胞,监测Caspase-3蛋白及c-fos蛋白表达；抽静脉血离心取血清,测定血清中炎性因子IL-1β和IL-6含量。
结果各组于光镜和电镜下均可见凋亡神经元及相关基因的表达,与针刺对照组和假手术组相比,脑缺血再灌注组和针刺预处理后脑缺血再灌注组细胞凋亡数和Caspase-3及c-fos表达阳性细胞数均显著增多,再灌注两天后达峰值,差异有显著统计学意义(P<0.01)；与脑缺血再灌注组相比,针刺预处理后脑缺血再灌注组细胞凋亡数和Caspase-3表达阳性细胞数显著减少,c-fos表达阳性细胞数显著增多,差异有显著统计学意义(P<0.01)。血清炎性因子IL-1β和IL-6含量针刺预处理组及脑缺血组再灌注各时间点均明显上调,与两对照组比较差异有显著统计学意义(P<0.01)；脑缺血组与针刺预处理组各时间点相比较,前者更高,两者间均有明显差异(P<0.01)。
结论针刺预处理可通过调控caspase-3蛋白及c-fos蛋白的表达,调节炎性细胞因子的水平,阻断由炎症级联反应引起的老年大鼠脑神经元缺血再灌注损伤,对老年大鼠短暂脑缺血再灌注损伤起到的保护作用。
Objective To investigate the effects of acupuncture pre-conditioning on apoptosis in hippocampal neurons and expression of apoptosis related genes & Inflammatory factor in serum following ischemia-reperfusion injury in aged rats.
Methods Totally 120 old male wistar rats aged 19-21 months(corresponding to 60 years old human being) weighting 550-710g were randomly divided into 4 groups(n=30). Cerebral ischemic group:4-vessel-blocked was conducted for 4 minutes to establish cerebral ischemic models; Acupuncture precondition group:electro acupuncture was giving on baihui with frequency of 15 Hz, amper 2mA,30 minutes once daily for 5 days, then the rats were given 4-vessel-block for 4 minutes;sham-operation group:4 vessel were exposed; sham-acupuncture group:only electroacupuncture for 5 days without operation. The rats were killed at the end of predetermined duration of reperfusion 12h、1d、2d、3d and 7d respectively. The brain were immediately removed and hippocampal CA1 region was isolated for (1) light and electron microscopic examination of hippocampal neurons, (2) detection of apoptotic neurons(TUNEL) and (3) determination of caspase-3 protein and c-fos protein expression using SABC immuno-histochemical technique. (4) determination of nflammatory factor IL-1βand IL-6 expression in serum.
Results There were apoptotic neurons in all groups. The number of apoptotic neurons and positive neurons of caspase-3 and c-fos were significantly increased and peaked at 2d after reperfusion in acupuncture precondition group and cerebral ischemic group as compared with sham-acupuncture group and sham-operation group (P<0.01). The number of apoptotic neurons and positive neurons of caspase-3 were significantly decreased and c-fos were significantly increased in acupuncture precondition groupas compared with cerebral ischemic group (P<0.01). The content of serum inflammatory factor IL-1βand IL-6 in the cerebral ischemic group and acupuncture precondition group increased obviously (P<0.01),but increased significantly in cerebral ischemic group (P<0.01)
Conclusion acupuncture pre-conditioning can decrease neuron apoptosis after ischemia-reperfusion injury by regulationg and control caspase-3 protein、c-fos protein and nflammatory factor expression in aged rats.
方法健康老年雄性Wistar大鼠120只,随机分为四组每组30只。组Ⅰ(单纯脑缺血/再灌注组)：四动脉阻断法全脑缺血4min建立老年大鼠全脑缺血模型；组Ⅱ(针刺预处理后脑缺血/再灌注组)：连续5d对百会穴实施穴位针刺预处理(疏密波,强度1mA,频率15 Hz),每天持续30 min,然后建立全脑缺血模型；组Ⅲ(单纯针刺预处理组)：仅针刺百会穴30min/d,持续5 d；组Ⅳ(假手术组),仅暴露四条动脉,不缺血。每组分别于缺血后12h、1、2、3和7d各随机处死6只大鼠,随机抽取一只动物提取海马CA1区脑组织作透射电镜观察神经元超微结构,余下的5只光镜下观察海马神经元形态和凋亡细胞,监测Caspase-3蛋白及c-fos蛋白表达；抽静脉血离心取血清,测定血清中炎性因子IL-1β和IL-6含量。
结果各组于光镜和电镜下均可见凋亡神经元及相关基因的表达,与针刺对照组和假手术组相比,脑缺血再灌注组和针刺预处理后脑缺血再灌注组细胞凋亡数和Caspase-3及c-fos表达阳性细胞数均显著增多,再灌注两天后达峰值,差异有显著统计学意义(P<0.01)；与脑缺血再灌注组相比,针刺预处理后脑缺血再灌注组细胞凋亡数和Caspase-3表达阳性细胞数显著减少,c-fos表达阳性细胞数显著增多,差异有显著统计学意义(P<0.01)。血清炎性因子IL-1β和IL-6含量针刺预处理组及脑缺血组再灌注各时间点均明显上调,与两对照组比较差异有显著统计学意义(P<0.01)；脑缺血组与针刺预处理组各时间点相比较,前者更高,两者间均有明显差异(P<0.01)。
结论针刺预处理可通过调控caspase-3蛋白及c-fos蛋白的表达,调节炎性细胞因子的水平,阻断由炎症级联反应引起的老年大鼠脑神经元缺血再灌注损伤,对老年大鼠短暂脑缺血再灌注损伤起到的保护作用。
Objective To investigate the effects of acupuncture pre-conditioning on apoptosis in hippocampal neurons and expression of apoptosis related genes & Inflammatory factor in serum following ischemia-reperfusion injury in aged rats.
Methods Totally 120 old male wistar rats aged 19-21 months(corresponding to 60 years old human being) weighting 550-710g were randomly divided into 4 groups(n=30). Cerebral ischemic group:4-vessel-blocked was conducted for 4 minutes to establish cerebral ischemic models; Acupuncture precondition group:electro acupuncture was giving on baihui with frequency of 15 Hz, amper 2mA,30 minutes once daily for 5 days, then the rats were given 4-vessel-block for 4 minutes;sham-operation group:4 vessel were exposed; sham-acupuncture group:only electroacupuncture for 5 days without operation. The rats were killed at the end of predetermined duration of reperfusion 12h、1d、2d、3d and 7d respectively. The brain were immediately removed and hippocampal CA1 region was isolated for (1) light and electron microscopic examination of hippocampal neurons, (2) detection of apoptotic neurons(TUNEL) and (3) determination of caspase-3 protein and c-fos protein expression using SABC immuno-histochemical technique. (4) determination of nflammatory factor IL-1βand IL-6 expression in serum.
Results There were apoptotic neurons in all groups. The number of apoptotic neurons and positive neurons of caspase-3 and c-fos were significantly increased and peaked at 2d after reperfusion in acupuncture precondition group and cerebral ischemic group as compared with sham-acupuncture group and sham-operation group (P<0.01). The number of apoptotic neurons and positive neurons of caspase-3 were significantly decreased and c-fos were significantly increased in acupuncture precondition groupas compared with cerebral ischemic group (P<0.01). The content of serum inflammatory factor IL-1βand IL-6 in the cerebral ischemic group and acupuncture precondition group increased obviously (P<0.01),but increased significantly in cerebral ischemic group (P<0.01)
Conclusion acupuncture pre-conditioning can decrease neuron apoptosis after ischemia-reperfusion injury by regulationg and control caspase-3 protein、c-fos protein and nflammatory factor expression in aged rats.
引文
1 Kitagawa K. Matsumoto M, Tagaya M, et al. "Ischemic tolerance" phenomenon found in the brain. Brain Res,1990,528(1):21-4
2 王明山,周海鹏,马世龙.针刺预处理对脑缺血及再灌注损伤的保护作用.国外医学麻醉与复苏分册,2009,30(2)：189-192.
3 Wang MS, Ma FG, Chen HL. Protective effect of acupuncture on brain tissue following ischemia-reperfusion injury. NRR,2008,3:309-312.
4 Canese R, Fortuna S, Lorenzini P, et al. Transient global brain ischemia in young and aged rats:differences in severity and progression, but not localization of lesions evaluated by magnetic resonance imaging. MA-GMA,1998,7(1):28-34.
5 王明山,冀翔宇,张丽娜,等.短暂性脑缺血对老年大鼠海马神经元凋亡的影响.中华麻醉学,2007,27(2)：140-143
6王明山,冀翔宇,张丽娜,等.短暂性脑缺血对老年大鼠海马组织AQP4表达及神元凋亡的影响.中华医学杂志,2008,16：2504-2507.
7华兴帮.大白鼠穴位谱的研制实验动物与动物实验,1991,(1)：1
8 Pulsinelli WA, Brierly JB. A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke,1979,10:267-272.
9 Akins PT, Liu PK, Hsu CY. Immediate early gene expression in response to cerebral ischemia. Friend or foe? Stroke,1996,27(9):1682-1687.
10 amnura S, Zhu J, FinkK, et al. Activation and cleavage of Caspase-3 in atpoptos is induced by experimental cerebral ischemia. Neurosci,1998,18:3659
11 Marcia B. Stroke-damaged neurons may commit cellular suicid Science,1998,281:1302
12 Davis M, Mendelow AD, Perry RH, et al. Experimental stroke and neuro-protection in the aging rat brain. Stroke,2003,26(6):1072-1078.
13施新猷.现代医学实验动物学.北京：人民军医出版社,2000：39.12 14赵卫东,王子灿,李麟仙,等.大鼠脑缺血再灌流综合征模型初探.中国神经精神疾病杂志,1997,13(4)：193.
15王明山,冀翔宇,张丽娜,等.短暂性脑缺血-再灌注损伤对老年大鼠海马组织AQP4表达及Caspase-3蛋白表达的影响.临床麻醉学杂志.2007,23(3)：223-225
16贾健民,贾健平,饶明俐.啮齿动物全脑缺血模型和海马选择易损伤性研究进展.国外医学脑血管疾病分册,1994,2(1)：34-38.
17熊利泽,路志红.重复电针预处理可减轻大鼠急性短暂性脑缺血损伤.第四军医大学学报,2001：22(21)封2
18 LAO-Ning, XIONG Li-Ze, LU Zhi-Hong, et al Baihui electroacupuncture in the induction of brain ischemic tolerance by pre-conditioning. Journal of Fourth Military Medical University,2003,24(5):418-420
19 Lu Zhihong, Xiong Lize, Zhu Zhenghua, et al. Acupoint Specificity of Electroacupuncture Preconditioning Effect on Cerebral Ischemia Injury in Rats Chinese Acupuncture and Moxibustion,2002,22(10):671-673
20 LAO Nrng; XIONG Li-ze:LU Zhi-hong, et al. Difference of electroacupuncture with various frequencies on the degree of induced brain ischemia tolerance. Chinese Journal of Clinical Rehabilitation,2005,9(9):116-117
21 Kamme F, Campbell K, Wieloch T. Biphasic expression of the fos and jun families of transcription factors following transient forebrain ischaemia in the rat, Effect of hypothermia. Eur J Neurosci,1995,7(10):2007-16
22 Huang J, Upadhyay UM, Tamargo RJ. Inflammation in stroke and focal cerebral ischemia. SurgNeurol,2006,66(3):232-245.
23 Danton GH, DietrichWD. Inflammatory mechanisms after ischemia and stroke. Neuropathol Exp Neuro,2003,62(2):127-136.
1 TANG Wei, ZOU Safeng. Effect of acupuncture precondition on cerebral edema and adenosine in rats with cerebral ischemia-reperfusion. Chin J TCM WM Crit Care2007,14(3):166-168
2唐伟,王威,白鹰等.针刺预处理对全脑缺血大鼠微血管损伤及脑水肿的保护作用大连大学学报2007,28(3)：65-68
3 ZHANG Min, LIU Jianbo, ZHANG Hong-liang, et al. Rapid and delayed tolerance to focal cerebral ischemia induced by pretreatment with single electroacupuncture in rats. Chinese Journal of Anesthesiology 2003,23(5):355-357.
4 DING Jiong, GU Zhen, WU Wen-zhong, et al. Electroacupuncture preconditioning in treatment of focal cerebral ischemia in rats. Chinese Journal of Clinical Rehabilitation.2004,8(1):106-108
5 ZHANG Min, LIU Jian-bo, ZHANG Hong-liang, et al. Preventive effects of electroacupuncture pre-management and Shenfu injection on focal cerebral ischemia in rats. Shanghai Journal of Acupuncture and Moxibustion2003,22(7)10-13.
6 WANG Qiang, LIU Yan hong, XIONG Li ze, et al. Effect of adenosine Al receptor in the rapid ischemic tolerance induced by pretreatment with single electroacupuncture in rats. Chin J TCM WM Crit Care2004,11(1):13-16
7 TIAN Dai-Shi, DENG Yi-Yu, WANG Guang-Hai; et al. Role of electroacupuncture preconditioning against neuronal apoptosis in rats with focal cerebral ischemia. Journal of Fourth Military Medical University2005,25(1):27-29.
8 熊利泽,路志红.重复电针预处理可减轻大鼠急性短暂性脑缺血损伤.第四军医大学学报2001：22(21)封2
9 唐伟,孙忠人,王卓尔.针刺预处理对脑缺血再灌注损伤的保护作用。辽宁中医杂志2006,33(5)：627-628
10 LEI Yi, XIONGLi-ze, ZHANG Long-fang, et al. Effect of preconditioning with electroacupuncture on changes of intracellular Ca2 in spinal ischemia-reperfusion injury in rabbits. Chinese Acupuncture and Moxibustion2004,24(4):279-282.
11 JIANG Ke-wen, ZHANG Ying, SHUI Quan-xian. Possible mechanism of electroacupuncture preconditioning for hypoxia/ischemic brain injury protection effect in neonatal rats. Chinese journal of ITWM 2003,23(12):914-917.
12郭佳,王磊,张莉,等. 电针预处理对缺血再灌注损伤大鼠脑自由基含量的影响.安徽中医学院学报2003,22(1)：29-31.
13 ZENG Yi, XIONG Li ze, CHEN Shao yang, et al. Effects of pretreatment with repeated electroacupuncture on biochemistry variables after spinal cord ischemia-reperfusion injury in rabbits. Chin J TCM WM Crit Care2004,11(2):80-82.
14陈泽斌,段妍君,袁芳,等.穴位针刺预处理脑组织提取液对小鼠缺氧存活时间的影响 湖北中医学院学报2002,4(4)：15-17.
15 CHEN Ze-bin, YUAN Fang, LIANG Feng-xia, et al. Effect of brain tissue extract of rats after acupuncture preconditioning on cerebral ischemia-reperfusion injury. Chinese Acupuncture and Moxibustion2004,24(5):347-350
16 YANG Jing, XIONG Lize, WANG Qiang, et al. Role of opioid receptors in cerebral ischemic tolerance induced by electroacupuncture preconditioning in rats. Chinese journal of Mult-Organ Disease Elderiy2006,5(3):218-291.
17 WANG Jun, YU Zhen, JIA Shi-qi, et al. Effect of electroacupuncture on inflammatory factors in blood of rat with cerebral Ischemia reperfusion. China Prac Med 2008,3 (6):4-5.
18 TANG Wei, SUN Zhong-ren, ZHANG Li, et al. Effect of acupuncture precondition on apoptosis and heat shock protein 70 mRNA expression in hippocamal CA1 of rats with global cerebral ischemia. Chinese Journal of Clinical Rehabilitation 2005,9(37): 90-92.
19 LU Zhi-hong, XIONG Li-ze, TIAN Lei, et al. Effect of pretreatment with repeated electroacupuncture on heat shock protein 70 after cerebral ischemia-reperfusion injury in rats. Chinese Journal of Anesthesiology.2004,24(6):453-456
20 陈泽斌,柯晖,邹锋.针刺预处理对脑缺血再灌注大鼠中枢杏仁核CRF样蛋白表达的影响.中医药学刊2006,34(3)：14-16.
21 CHEN Ze-bin, ZOU Feng, YUAN Fang, et al. Effect of acupuncture preconditioning on protein expression of N methyl D aspartate receptor subunits NR2A and NR2B in parietal cortex during ischemia-reperfusion in rats. Chin J TCM WM Crit Care.2005, 12(2):79-83
22 CHEN Ze-bin, Wang Hua. Effect of acupuncture on expression of neurology-related genes in the brain tissue of the rat. Chinese Acupuncture and Moxibustion 2005, 25(8):537-6
23 孙忠人,张力,郑美华等.针刺预处理对全脑缺血大鼠脑组织Bcl-2蛋白表达影响的实验研究. 中医药学刊2006,24(11)：1976-1977.
24 唐伟,王威,孙忠人等.针刺预处理全脑缺血大鼠海马CA1区Bcl-2 mRNA的表达.中国中医基础医学杂志2007,13(2)：156-158
25 SUN Zhong-ren, TANG Wei, WANG Wei, et al. Effect of acupuncture precondition on c-fosmRNA and its protein expression in hippocampal CA1 of rats with global cerebral ischemia. Chinese Journal of Clinical Rehabilitation.2006,10(19):125-127.
26 LAO Ning, XIONG Li-Ze, LU Zhi-Hong, et al. Baihui electroacupuncture in the induction of brain ischemic tolerance by pre-conditioning. Journal of Fourth Military Medical University.2003,24(5):418-420
27 Lu Zhihong, Xiong Lize, Zhu Zhenghua, et al. Acupoint Specificity of Electroacupuncture Preconditioning Effect on Cerebral Ischemia Injury in Rats Chinese Acupuncture and Moxibustion,2002,22(10):671-673
28 LAO Ning, XIONG Li-ze, LU Zhi-hong, et al. Difference of electroacupuncture with various frequencies on the degree of induced brain ischemia tolerance. Chinese Journal of Clinical Rehabilitation.2005,9(9):116-117.
29 CHEN Ze-bin, WANG Hua. Influence of Electroacupuncture Preconditioning on Surviving Neuron Density of Cerebral Cortex in Rats with Temporary Cerebral Ischemia-reperfusion. Acupuncture Research2003,28(3):165-169.
2 王明山,周海鹏,马世龙.针刺预处理对脑缺血及再灌注损伤的保护作用.国外医学麻醉与复苏分册,2009,30(2)：189-192.
3 Wang MS, Ma FG, Chen HL. Protective effect of acupuncture on brain tissue following ischemia-reperfusion injury. NRR,2008,3:309-312.
4 Canese R, Fortuna S, Lorenzini P, et al. Transient global brain ischemia in young and aged rats:differences in severity and progression, but not localization of lesions evaluated by magnetic resonance imaging. MA-GMA,1998,7(1):28-34.
5 王明山,冀翔宇,张丽娜,等.短暂性脑缺血对老年大鼠海马神经元凋亡的影响.中华麻醉学,2007,27(2)：140-143
6王明山,冀翔宇,张丽娜,等.短暂性脑缺血对老年大鼠海马组织AQP4表达及神元凋亡的影响.中华医学杂志,2008,16：2504-2507.
7华兴帮.大白鼠穴位谱的研制实验动物与动物实验,1991,(1)：1
8 Pulsinelli WA, Brierly JB. A new model of bilateral hemispheric ischemia in the unanesthetized rat. Stroke,1979,10:267-272.
9 Akins PT, Liu PK, Hsu CY. Immediate early gene expression in response to cerebral ischemia. Friend or foe? Stroke,1996,27(9):1682-1687.
10 amnura S, Zhu J, FinkK, et al. Activation and cleavage of Caspase-3 in atpoptos is induced by experimental cerebral ischemia. Neurosci,1998,18:3659
11 Marcia B. Stroke-damaged neurons may commit cellular suicid Science,1998,281:1302
12 Davis M, Mendelow AD, Perry RH, et al. Experimental stroke and neuro-protection in the aging rat brain. Stroke,2003,26(6):1072-1078.
13施新猷.现代医学实验动物学.北京：人民军医出版社,2000：39.12 14赵卫东,王子灿,李麟仙,等.大鼠脑缺血再灌流综合征模型初探.中国神经精神疾病杂志,1997,13(4)：193.
15王明山,冀翔宇,张丽娜,等.短暂性脑缺血-再灌注损伤对老年大鼠海马组织AQP4表达及Caspase-3蛋白表达的影响.临床麻醉学杂志.2007,23(3)：223-225
16贾健民,贾健平,饶明俐.啮齿动物全脑缺血模型和海马选择易损伤性研究进展.国外医学脑血管疾病分册,1994,2(1)：34-38.
17熊利泽,路志红.重复电针预处理可减轻大鼠急性短暂性脑缺血损伤.第四军医大学学报,2001：22(21)封2
18 LAO-Ning, XIONG Li-Ze, LU Zhi-Hong, et al Baihui electroacupuncture in the induction of brain ischemic tolerance by pre-conditioning. Journal of Fourth Military Medical University,2003,24(5):418-420
19 Lu Zhihong, Xiong Lize, Zhu Zhenghua, et al. Acupoint Specificity of Electroacupuncture Preconditioning Effect on Cerebral Ischemia Injury in Rats Chinese Acupuncture and Moxibustion,2002,22(10):671-673
20 LAO Nrng; XIONG Li-ze:LU Zhi-hong, et al. Difference of electroacupuncture with various frequencies on the degree of induced brain ischemia tolerance. Chinese Journal of Clinical Rehabilitation,2005,9(9):116-117
21 Kamme F, Campbell K, Wieloch T. Biphasic expression of the fos and jun families of transcription factors following transient forebrain ischaemia in the rat, Effect of hypothermia. Eur J Neurosci,1995,7(10):2007-16
22 Huang J, Upadhyay UM, Tamargo RJ. Inflammation in stroke and focal cerebral ischemia. SurgNeurol,2006,66(3):232-245.
23 Danton GH, DietrichWD. Inflammatory mechanisms after ischemia and stroke. Neuropathol Exp Neuro,2003,62(2):127-136.
1 TANG Wei, ZOU Safeng. Effect of acupuncture precondition on cerebral edema and adenosine in rats with cerebral ischemia-reperfusion. Chin J TCM WM Crit Care2007,14(3):166-168
2唐伟,王威,白鹰等.针刺预处理对全脑缺血大鼠微血管损伤及脑水肿的保护作用大连大学学报2007,28(3)：65-68
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