淫羊藿苷对缺血再灌致脑损伤保护作用的实验研究
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摘要
目的:
随着社会的发展和人口的老龄化,老年痴呆发病率呈上升趋势。血管性痴呆(vascular dementia,VD)是世界范围内困扰老年人生活质量的常见病,是老年期痴呆的主要类型之一。脑组织对缺血缺氧十分敏感。缺血性脑血管病是导致 VD 的主要原因。由于我国脑血管病的多发,VD 在老年期痴呆发病率中居于首位。目前对 VD 的防治尚无特效药物,因此寻找有效的抗脑缺血的神经保护药防治VD已成为当务之急。
淫羊藿苷(icariin,Ica)是从小檗科淫羊藿属植物中提取的有效成分,是一种黄酮醇苷类化合物。以往研究表明淫羊藿苷具有增加脑血流量、改善冠脉循环、调节免疫、影响内分泌和抗衰老等作用。我们推测淫羊藿苷有潜在的防治脑缺血再灌损伤神经细胞的作用。
本课题旨在从整体、细胞、亚细胞、分子等不同水平探讨淫羊藿苷对脑缺血再灌损伤神经元是否有保护作用及相关作用机制,以期为进一步研究脑缺血再灌损伤打下基础,并为脑缺血再灌损伤寻找一种新的有效的神经保护剂。
方法:
(1)采用大鼠乳鼠神经元进行原代培养,利用缺氧缺糖/复氧复糖损伤模型模拟脑缺血再灌作用,分别在缺氧缺糖(4h、6h、8h)和缺氧缺糖(6h)/复氧复糖(3h、12h、24h)的不同时间点进行各组神经细胞的 MTT 检测、乳酸脱氢酶(LDH)活性检测、观察其病理形态学改变、细胞凋亡率及细胞内游离 Ca2+浓度的变化来评价淫羊藿苷对脑缺血再灌损伤的神经元是否有保护作用并初步探讨相关作用机制。
Objective:
With the social development and population aging, the morbidity of aging-related dementia sharply increased. Vascular dementia is one of the main types in aging dementia, which is common disease seriously influencing life qualify of old population in the world. It is well known that brain is very susceptible to hypo-glucose and hypoxia. Ischemic cerebral disease is the primary cause of vascular dementia. Because of the high morbidity of cerebrovascular diseases in our country, the vascular dementia is main responsible for aging dementia. Unfortunately, there is rarely effective remedy that can be used to reverse or prevent the pathological course up to date. Search for novel anti-ischemia neuroprotective drugs is very important for prevention or treatment of vascular dementia.
Icariin, the major active component of traditional Chinese herb “Yinyanghuo”, has been revealed that it possesses various important pharmacological effects, such as promoting cerebral blood flow, improving circulation, regulating immunity,affecting endocrine and anti-aging effect. There is a possibility that icariin may have a beneficial role for neurons in cerebral ischemia/reperfusion caused by accident. However, it has been little known about this effect as yet.
In this study we investigate the protective effects of icariin on neurons injured by cerebral ischemia / reperfusion and further explore its possible protective mechanisms at the levels of entirety, cell, subcell, and molecular. Our object is to illustrate the mechanisms of icariin against cerebral
ischemia /reperfusion injury and hope to find novel neuro- protective drug that can decrease the neuron damages induced by ischemia/ reperfusion.
Methods:
(1) In order to observe the protective effects of icariin on neurons treated by ischemia/ reperfusion in vitro and investigate its protection mechanisms, primary culture cerebral cortical neurons of Wistar rats were studied during the different period of oxygen-glucose deprivation / reperfusion with oxygen and glucose. MTT assay was used to determine cell viability,and the activity of lactate dehydrogenase (LDH) leaked from neurons, pathological changes of neurons and cell apoptosis were measured, as well as concentration of intracellular free calcium were also evaluated,respectively.
(2) The mice model of transient cerebral ischemia/reperfusion was made by bilateral occlusion of common carotid arteries and ischemic hypotension for 20 min before reperfusion to investigate the protective effects of icariin on mice injured by transient cerebral ischemia/ reperfusion in vivo. The changes of mice behavioral (locomotive activity, passive learning and memory ability and spatial learning and memory ability) and pathological changes were detected, respectively. And to further investigate the possible mechanisms of icariin, based on the results above the total anti-oxidant capacity, the activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA), the activity of acetylcholinesterase (AchE), the activity of choline acetyltransferse (ChAT), and the changes of ultrastructure in brain tissue were measured, respectively.
(3) The injury model of mitochondria induced by oxygen free radical was made by ferrous sulfate/ ascorbic acid (Fe2+/VitC) in vitro. To investigate the protective effects of icariin, the swelling and the activities of complexⅠ-Ⅳ, as well as the content of MDA of mitochondria injured by
different concentration Fe2+/VitC were measured,respectively.
(4) In order to investigate the effects of cerebral ischemia/ reperfusion on the expression of cytochrome C oxidase subunit Ⅱ (COⅡ) mRNA and the effects of icariin, the mice model of transient cerebral ischemia/ reperfusion was made by similar method above. The changes of the expression of COⅡ mRNA were measured at different reperfusion time points by RT – PCR . Results:
(1) During the different period of oxygen-glucose deprivation (4, 6, 8h) or oxygen-glucose deprivation (6h)/ reperfusion with oxygen and glucose (3, 12, 24h) incubation in
随着社会的发展和人口的老龄化,老年痴呆发病率呈上升趋势。血管性痴呆(vascular dementia,VD)是世界范围内困扰老年人生活质量的常见病,是老年期痴呆的主要类型之一。脑组织对缺血缺氧十分敏感。缺血性脑血管病是导致 VD 的主要原因。由于我国脑血管病的多发,VD 在老年期痴呆发病率中居于首位。目前对 VD 的防治尚无特效药物,因此寻找有效的抗脑缺血的神经保护药防治VD已成为当务之急。
淫羊藿苷(icariin,Ica)是从小檗科淫羊藿属植物中提取的有效成分,是一种黄酮醇苷类化合物。以往研究表明淫羊藿苷具有增加脑血流量、改善冠脉循环、调节免疫、影响内分泌和抗衰老等作用。我们推测淫羊藿苷有潜在的防治脑缺血再灌损伤神经细胞的作用。
本课题旨在从整体、细胞、亚细胞、分子等不同水平探讨淫羊藿苷对脑缺血再灌损伤神经元是否有保护作用及相关作用机制,以期为进一步研究脑缺血再灌损伤打下基础,并为脑缺血再灌损伤寻找一种新的有效的神经保护剂。
方法:
(1)采用大鼠乳鼠神经元进行原代培养,利用缺氧缺糖/复氧复糖损伤模型模拟脑缺血再灌作用,分别在缺氧缺糖(4h、6h、8h)和缺氧缺糖(6h)/复氧复糖(3h、12h、24h)的不同时间点进行各组神经细胞的 MTT 检测、乳酸脱氢酶(LDH)活性检测、观察其病理形态学改变、细胞凋亡率及细胞内游离 Ca2+浓度的变化来评价淫羊藿苷对脑缺血再灌损伤的神经元是否有保护作用并初步探讨相关作用机制。
Objective:
With the social development and population aging, the morbidity of aging-related dementia sharply increased. Vascular dementia is one of the main types in aging dementia, which is common disease seriously influencing life qualify of old population in the world. It is well known that brain is very susceptible to hypo-glucose and hypoxia. Ischemic cerebral disease is the primary cause of vascular dementia. Because of the high morbidity of cerebrovascular diseases in our country, the vascular dementia is main responsible for aging dementia. Unfortunately, there is rarely effective remedy that can be used to reverse or prevent the pathological course up to date. Search for novel anti-ischemia neuroprotective drugs is very important for prevention or treatment of vascular dementia.
Icariin, the major active component of traditional Chinese herb “Yinyanghuo”, has been revealed that it possesses various important pharmacological effects, such as promoting cerebral blood flow, improving circulation, regulating immunity,affecting endocrine and anti-aging effect. There is a possibility that icariin may have a beneficial role for neurons in cerebral ischemia/reperfusion caused by accident. However, it has been little known about this effect as yet.
In this study we investigate the protective effects of icariin on neurons injured by cerebral ischemia / reperfusion and further explore its possible protective mechanisms at the levels of entirety, cell, subcell, and molecular. Our object is to illustrate the mechanisms of icariin against cerebral
ischemia /reperfusion injury and hope to find novel neuro- protective drug that can decrease the neuron damages induced by ischemia/ reperfusion.
Methods:
(1) In order to observe the protective effects of icariin on neurons treated by ischemia/ reperfusion in vitro and investigate its protection mechanisms, primary culture cerebral cortical neurons of Wistar rats were studied during the different period of oxygen-glucose deprivation / reperfusion with oxygen and glucose. MTT assay was used to determine cell viability,and the activity of lactate dehydrogenase (LDH) leaked from neurons, pathological changes of neurons and cell apoptosis were measured, as well as concentration of intracellular free calcium were also evaluated,respectively.
(2) The mice model of transient cerebral ischemia/reperfusion was made by bilateral occlusion of common carotid arteries and ischemic hypotension for 20 min before reperfusion to investigate the protective effects of icariin on mice injured by transient cerebral ischemia/ reperfusion in vivo. The changes of mice behavioral (locomotive activity, passive learning and memory ability and spatial learning and memory ability) and pathological changes were detected, respectively. And to further investigate the possible mechanisms of icariin, based on the results above the total anti-oxidant capacity, the activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA), the activity of acetylcholinesterase (AchE), the activity of choline acetyltransferse (ChAT), and the changes of ultrastructure in brain tissue were measured, respectively.
(3) The injury model of mitochondria induced by oxygen free radical was made by ferrous sulfate/ ascorbic acid (Fe2+/VitC) in vitro. To investigate the protective effects of icariin, the swelling and the activities of complexⅠ-Ⅳ, as well as the content of MDA of mitochondria injured by
different concentration Fe2+/VitC were measured,respectively.
(4) In order to investigate the effects of cerebral ischemia/ reperfusion on the expression of cytochrome C oxidase subunit Ⅱ (COⅡ) mRNA and the effects of icariin, the mice model of transient cerebral ischemia/ reperfusion was made by similar method above. The changes of the expression of COⅡ mRNA were measured at different reperfusion time points by RT – PCR . Results:
(1) During the different period of oxygen-glucose deprivation (4, 6, 8h) or oxygen-glucose deprivation (6h)/ reperfusion with oxygen and glucose (3, 12, 24h) incubation in
引文
[1]Strub R. Vascular Dementia[J]. South Medic J, 2003,96(4): 363-366
[2]Roman GC. Vascular dementia may be the most common form of dementia in the elderly[J]. J Neurol Sci, 2002,203-204: 7-10
[3]Decarli C. Vascular factors in dementia: an overview[J]. J Neurol Sci, 2004, 226(1-2): 19-23
[4]Boyle PA, Paul RH, Moser DJ, et al. Executive impairments predict functional declines in vascular dementia[J]. Clin Neuropsychol, 2004, 18(1): 75-82
[5]Gunstad J, Brickman AM, Paul RH,et al. Progressive morphometric and cognitive changes in vascular dementia[J]. Arch Clin Neuropsychol, 2005, 20(2): 229-241
[6]Schreinzer D, Ballaban T, Brannath W, et al. Components of behavioral pathology in dementia[J]. Int J Geriatr Psychiatry, 2005, 20(2): 137-145
[7]Kalaria RN, Kenny RA, Ballard CG, et al. Towards defining the neuropathological substrates of vascular dementia[J]. J Neurol Sci, 2004, 226(1-2): 75-80
[8]Jellinger KA. The pathology of ischemic-vascular dementia: an update [J]. J Neurol Sci, 2002,203-204: 153-157
[9]Ishunina TA, Kamphorst W, Swaab DF. Metabolic alterations in the hypothalamus and basal forebrain in vascular dementia[J]. J Neuropathol Exp Neurol, 2004, 63(12): 1243-1254
[10]Reed BR, Mungas DM, Kramer JH, et al. Clinical and neuropsychological features in autopsy-defined vascular dementia[J]. Clin Neuropsychol, 2004, 18(1): 63-74
[11]Roman GC. Facts, myths, and controversies in vascular dementia[J]. J Neurol Sci, 2004, 226(1-2): 49-52
[12]Lopez OL, Kuller LH, Becker JT. Diagnosis, risk factors, and treatment of vascular dementia[J]. Curr Neurol Neurosci Rep, 2004,4(5): 358-367
[13]Gold G. Diagnosis and treatment of vascular dementia[J]. Schweiz Rundsch Med Prax, 2004, 93(33): 1311-1316
[14]Wolf H, Gertz HJ. Vascular dementia-diagnosis, prevention and treatment[J]. Psychiatr Prax, 2004, 31(7): 330-338
[15]Raja PV, Blumenthal JA, Doraiswamy PM. Cognitive deficits following coronary artery bypass grafting: prevalence, prognosis, and therapeutic strategies[J]. CNS Spectr, 2004, 9(10): 763-772
[16]Erkinjuntti T, Roman G, Gauthier S. Treatment of vascular dementia -evidence from clinical trials with cholinesterase inhibitors[J]. J Neurol Sci, 2004, 226(1-2): 63-66
[17]Pantoni L. Treatment of vascular dementia: evidence from trials with non-cholinergic drugs[J]. J Neurol Sci, 2004, 226(1-2): 67-70
[18]叶丽卡,陈济民. 淫羊藿的药理研究进展[J]. 中国中药杂志,2001,26(5):293-295
[19]侯集瑞,盛吉明,王秀全,等. 淫羊藿研究进展[J]. 吉林农业大学学报,2004,26(1):59-65
[20]关利新,衣欣,杨履艳,等. 淫羊藿苷扩血管作用机制的研究[J]. 中国药理学通报, 1996, 12(4):320-322
[21]王敏,刘崇铭. 淫羊藿甙对血管平滑肌的作用[J]. 沈阳药学院学报,1993,10(3):185-190
[22]Zhang YW, Morita I, Shao G, et al. Screening of anti-hypoxia/ reoxygenation agents by an in vitro model. Part 1:Natural inhibitors for protein tyrosine kinase activated by hypoxia/reoxygenation in cultured human umbilical vein endothelial cells[J]. Planta Med, 2000, 66(2): 114-118
[23]李芳芳,李思,吕占军,等. 淫羊藿苷对大鼠卵泡颗粒细胞和肾上腺皮质细胞分泌功能的影响[J]. 中国中药杂志, 1997, 22(8): 499-500
[24]Lee MK, Choi YJ, Sung SH, et al. Antihepatotoxic activity of icariin, a major constituent of Epimedium koreanum[J]. Planta Med, 1995, 61(6):
523-536
[25]赵勇,张玲,王芸,等. 淫羊藿甙的体外免疫调节作用研究[J].中草药,1996,27(11): 669-672
[26]秦路平,石汉平,郑水庆,等. Osthol 和 Icariin 对甲减小鼠血清甲状腺激素的影响[J]. 第二军医大学学报, 1998, 19(1):48-50
[27]李晓冰,金东庆,郭良君,等. 淫羊藿甙在辐射小鼠免疫学功能恢复中作用的研究[J]. 中国辐射卫生, 2002, 11(3):171
[28]Kuroda M, Mimaki Y, Sashida Y, et al. Flavonol glycosides from Epimedium sagittatum and their neurite outgrowth activity on PC12 cells[J]. Planta Med, 2000, 66(6): 575-577
[2]Roman GC. Vascular dementia may be the most common form of dementia in the elderly[J]. J Neurol Sci, 2002,203-204: 7-10
[3]Decarli C. Vascular factors in dementia: an overview[J]. J Neurol Sci, 2004, 226(1-2): 19-23
[4]Boyle PA, Paul RH, Moser DJ, et al. Executive impairments predict functional declines in vascular dementia[J]. Clin Neuropsychol, 2004, 18(1): 75-82
[5]Gunstad J, Brickman AM, Paul RH,et al. Progressive morphometric and cognitive changes in vascular dementia[J]. Arch Clin Neuropsychol, 2005, 20(2): 229-241
[6]Schreinzer D, Ballaban T, Brannath W, et al. Components of behavioral pathology in dementia[J]. Int J Geriatr Psychiatry, 2005, 20(2): 137-145
[7]Kalaria RN, Kenny RA, Ballard CG, et al. Towards defining the neuropathological substrates of vascular dementia[J]. J Neurol Sci, 2004, 226(1-2): 75-80
[8]Jellinger KA. The pathology of ischemic-vascular dementia: an update [J]. J Neurol Sci, 2002,203-204: 153-157
[9]Ishunina TA, Kamphorst W, Swaab DF. Metabolic alterations in the hypothalamus and basal forebrain in vascular dementia[J]. J Neuropathol Exp Neurol, 2004, 63(12): 1243-1254
[10]Reed BR, Mungas DM, Kramer JH, et al. Clinical and neuropsychological features in autopsy-defined vascular dementia[J]. Clin Neuropsychol, 2004, 18(1): 63-74
[11]Roman GC. Facts, myths, and controversies in vascular dementia[J]. J Neurol Sci, 2004, 226(1-2): 49-52
[12]Lopez OL, Kuller LH, Becker JT. Diagnosis, risk factors, and treatment of vascular dementia[J]. Curr Neurol Neurosci Rep, 2004,4(5): 358-367
[13]Gold G. Diagnosis and treatment of vascular dementia[J]. Schweiz Rundsch Med Prax, 2004, 93(33): 1311-1316
[14]Wolf H, Gertz HJ. Vascular dementia-diagnosis, prevention and treatment[J]. Psychiatr Prax, 2004, 31(7): 330-338
[15]Raja PV, Blumenthal JA, Doraiswamy PM. Cognitive deficits following coronary artery bypass grafting: prevalence, prognosis, and therapeutic strategies[J]. CNS Spectr, 2004, 9(10): 763-772
[16]Erkinjuntti T, Roman G, Gauthier S. Treatment of vascular dementia -evidence from clinical trials with cholinesterase inhibitors[J]. J Neurol Sci, 2004, 226(1-2): 63-66
[17]Pantoni L. Treatment of vascular dementia: evidence from trials with non-cholinergic drugs[J]. J Neurol Sci, 2004, 226(1-2): 67-70
[18]叶丽卡,陈济民. 淫羊藿的药理研究进展[J]. 中国中药杂志,2001,26(5):293-295
[19]侯集瑞,盛吉明,王秀全,等. 淫羊藿研究进展[J]. 吉林农业大学学报,2004,26(1):59-65
[20]关利新,衣欣,杨履艳,等. 淫羊藿苷扩血管作用机制的研究[J]. 中国药理学通报, 1996, 12(4):320-322
[21]王敏,刘崇铭. 淫羊藿甙对血管平滑肌的作用[J]. 沈阳药学院学报,1993,10(3):185-190
[22]Zhang YW, Morita I, Shao G, et al. Screening of anti-hypoxia/ reoxygenation agents by an in vitro model. Part 1:Natural inhibitors for protein tyrosine kinase activated by hypoxia/reoxygenation in cultured human umbilical vein endothelial cells[J]. Planta Med, 2000, 66(2): 114-118
[23]李芳芳,李思,吕占军,等. 淫羊藿苷对大鼠卵泡颗粒细胞和肾上腺皮质细胞分泌功能的影响[J]. 中国中药杂志, 1997, 22(8): 499-500
[24]Lee MK, Choi YJ, Sung SH, et al. Antihepatotoxic activity of icariin, a major constituent of Epimedium koreanum[J]. Planta Med, 1995, 61(6):
523-536
[25]赵勇,张玲,王芸,等. 淫羊藿甙的体外免疫调节作用研究[J].中草药,1996,27(11): 669-672
[26]秦路平,石汉平,郑水庆,等. Osthol 和 Icariin 对甲减小鼠血清甲状腺激素的影响[J]. 第二军医大学学报, 1998, 19(1):48-50
[27]李晓冰,金东庆,郭良君,等. 淫羊藿甙在辐射小鼠免疫学功能恢复中作用的研究[J]. 中国辐射卫生, 2002, 11(3):171
[28]Kuroda M, Mimaki Y, Sashida Y, et al. Flavonol glycosides from Epimedium sagittatum and their neurite outgrowth activity on PC12 cells[J]. Planta Med, 2000, 66(6): 575-577