当归芍药散对拟血管性痴呆小鼠氧化损伤治疗作用机理的实验研究
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摘要
目的：血管性痴呆（Vascular Dementia ，VD）属中医学老年呆病范畴。近年来，随着社会人口老龄化，VD的患病率越来越高，成为严重影响人们生活质量的重要因素。因此，深入探讨VD的病理机制，开发和研制防治VD的有效药物具有非常重要的意义。本研究通过观察当归芍药散对拟血管性痴呆小鼠脑组织丙二醛（MDA）、超氧化物歧化酶（SOD）、还原型谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GSH-Px）等指标的影响，从抗氧化损伤角度探讨其对VD的治疗作用机理，为临床治疗血管性痴呆提供实验依据。
方法:实验采用双侧颈总动脉结扎的方法制备血管性痴呆小鼠模型。昆明小鼠360只，随机分为假手术组(sham-operation group，S)、模型组（model group，M）、当归芍药散高剂量组（high-dose DanguiShaoyaoSan group，H）、当归芍药散低剂量组（low-dose DanguiShaoyaoSan group，L）、银杏叶提取物对照组(Extract of GinkgoBiloba control group，E)、尼莫地平对照组(Nimodipine control group，N)6组，每组60只。S组小鼠只分离颈总动脉，穿线但不结扎，尾部不放血，其余M、H、L、E、N各组结扎双侧颈总动脉阻断血流加尾部放血，制备反复脑缺血再灌注VD模型。术后第2天开始治疗。H组和L组灌服当归芍药散水煎剂（以
下简称“归芍煎”），E组灌服银杏叶提取物混悬液银杏叶液（以下简称“银杏叶液”），N组灌服尼莫地平混悬液（以下简称“尼莫地平液”），S、M两组均灌服生理盐水。分别于术后7、15、30天进行行为学实验（跳台法、水迷宫法）。行为学实验结束后断头处死小鼠，取左侧脑组织固定后切片，常规HE染色和硫堇染色，光镜下观察脑组织的形态变化；取右侧脑组织制成10%的脑组织匀浆，采用黄嘌呤氧化酶法测定脑组织SOD活力、硫代巴比妥酸比色分析法测定脑组织MDA含量、比色分析法测定脑组织GSH含量和GSH-Px活力。通过与银杏叶提取物和尼莫地平对照，观察高、低剂量当归芍药散对拟VD小鼠脑组织 MDA、GSH含量及SOD、GSH-Px活力的影响。
结果:
1当归芍药散对拟VD小鼠行为学实验的影响
1．1水迷宫成绩
术后7天、15天和30天，小鼠的学习成绩与记忆成绩改变基本一致， M组小鼠的游全程时间延长，错误次数增加，与S组比较差异具有显著性统计学意义（P<0.05或P<0.01）。H、L、E、N各组与M组比较，差异有显著性统计学意义（P<0.05或P<0.01）。H组与L、E、N各组比较，术后7天无显著性统计学意义（P>0.05），15天和30天时有显著性统计学意义（P<0.05或P<0.01）；L、E、N各组之间比较无显著性统计学意义（P>0.05）。
1．2跳台成绩
术后各时段， M组小鼠的反应时间延长，潜伏期缩短，错误次数增加，与S组比较，有显著性统计学意义（P<0.01）。H组与M组比较，反应时间缩短，潜伏期延长，错误次数减少，差异有显著性统计学意义（P<0.05）；H组与L、E、N各组比较，术后7天学习成绩无显著性统计学意义（P>0.05），术后15天和30天的学习成绩和各时段的记忆成绩均有显著性统计学意义（P<0.05或P<0.01）；而L、E、N各组之间比较无显著性统计学意义（P>0.05）。
2当归芍药散对拟VD小鼠病理形态学的影响
光镜下病理形态学显示，术后M组小鼠脑组织海马区呈缺血性病理改变。锥体细胞层次减少，排列稀疏，明显脱失，胞浆内尼氏体消失，细胞核体积变小，结构不清。随着时间的推移，模型小鼠脑组织病理改变逐步加重。各用药组小鼠病变轻于M组。
3当归芍药散对VD小鼠脑组织MDA 、GSH含量及SOD、GSH-Px活性的影响
3．1当归芍药散对VD小鼠脑组织MDA的影响
各时段M组小鼠脑组织MDA含量（7天、15天、30天分别为9.12±3.31、9.28±2.08、9.14±2.71）较S组（3.65±2.31、3.48±1.22、3.42±1.56）明显升高（P<0.01）；经用药干预后，各时段各用药组小鼠脑组织MDA含量均低于M组（P<0.05或P<0.01）；各治疗组比较，术后7天各治疗组（H、L、E、N各组分别为4.88±1.19、6.00±2.16，6.38±2.38、6.29±2.38）之间差异无显著性统计学意义（P>0.05）；术后15天和30天H组小鼠脑组织MDA含量（4.10±1.55、3.45±1.12）低于L组（5.94±
2.02、5.56±1.70）、E组（6.35±2.03、5.76±2.15）和N组（6.12±2.27、5.96±1.50）（P<0.05）；L组、E组和N组之间比较无显著性统计学意义（P>0.05）。
3．2当归芍药散对VD小鼠脑组织SOD活性的影响
各时段M组小鼠脑组织SOD活性（15.07±4.14、14.05±5.05、15.72±5.93）均较S组（32.78±7.70、32.45±10.01、35.60±9.80）明显降低（P<0.01）；经用药干预后，用药各组小鼠脑组织SOD活性（H组:28.06±6.75、32.15±10.25、37.14±6.75；E组:20.79±5.89、23.02±9.82、27.33±11.53；N组:20.33±6.00、23.56±10.19、25.84±8.88；L组:22.82±5.97、25.01±8.93、28.52±9.03）均较M组升高（P<0.05或P<0.01）；用药各组之间比较，各时段H组小鼠脑组织SOD活性均较E、N组升高（P<0.05或P<0.01），H组与L组比较，术后7天和15天，两组无显著性统计学意义（P >0.05），术后30天，H组高于L组（P <0.05）。L、E、N各组间比较，各时段均无显著性统计学意义（P
Objective:Vascular Dementia (called VD in short ) belongs to the disease scope of dementia of old age in the light of tradditional Chinese medincine(TCM).In the recent years,with the aging of population the incidence of vascular dementia is on the rise and affects seriously the people's working ability as well as living quality.Therefore,it's of great significance to make deeper research on it's pathological mechanism and develop the effective medicine with the integration of both Chinese and western medical theory.In order to find the relationship between disequilibrium of oxidation--anti-oxidation and Vascular Dementia ,then further to analyse the therapeutical pathogenicity with DangguiShaoyaoSan，we detected malonaldehyde（MDA）,superoxide dismutase（SOD）,glutathione（GSH） and glutathione peroxidase（GSH-Px） of cerebral tissue on mice with Vascular Dementia .
Methods: 360 Kunming mice were divided into six groups at random，which were sham-operation group（which were abbreviated to “S group”in the following）,model group（which were abbreviated to “M group” in the following,
high dose of DangguiShaoyaoSan group（which were abbreviated to “H group” in the following）, low dose of DangguiShaoyaoSan group（which were abbreviated to “ L group” in the following）, extract of Ginkgobiloba control group（which were abbreviated to “E group”）and Nimodipine control group（which were abbreviated to “N group” in the following）. There were 60 mice in each group. We duplicated the model of vascular dementia mice with the operation of cerebral ischemic reperfusion except S group. And then 6 group were continuously filled medicine or saline through the stomach from the second day after the operation of cerebral ischemic reperfusion. S group and M group were filled saline and H, L,E and N group were given medicine. On the 7th,15th and 30th day after praxiology experiment,the mice were cut head for obtaining cerebral tissue， the left part of which was fixed by 10% for maldehyde solution. The tissue was stained by HE and Thionine. We observed constitutional morphologic changes of cerebral tissue under light microscope and the right part of which were made into tissue homogenation for check up.
The experiment used TBA compared color analytical method to detect the content changes of MDA in cerebral tissue of Kunming mice, and used xanthine oxidase method to detect the activity changes of SOD, and used compared color analytical method to detect the activity changes of GSH and GSH-Px in cerebal tissue of Kunming mice. With
Ginkgobiloba extract and Nimodipine as the control medicine，we observed the effect of high and low dose of DangguiShaoyaoSan on MDA and GSH content and the activity of SOD and GSH-Px in cerebral ischemic reperfusion. Then we detected the mechanism on vascular dementia with DangguiShaoyaoSan from the direction of anti-oxidation injury.
Results:
1 Results of praxiology experiment
1.1 The mice's performance record of water-labyrinth
There was no significant difference between the learning and memorizing performance. The wrong times and the time of swimming along the whole line of M group was more than that of S group (P<0.05or P<0.01). At the same time, the swimming time and the wrong times of H ,L,E and N group was less than that of M group(P<0.05or P<0.01). Comparing with those of L,E and N group ,The swimming time and the wrong times of H group was less on the 15th and 30th day(P<0.05or P<0.01)，but not on the 7th day after the operation of cerebral ischemic reperfusion(P>0.05).There was no statistical difference among L, E and N group according to the learning and memorizing performance record ( P>0.05).
1.2 The mice's performance record of water-labyrinth Experiment of jumping stand
M group has shorter reaction time(the time of jumping up the stand), longer latency time(the time of jumping down the
stand) and more wrong times than S group (P<0.05or P<0.01).At the same time， H group has longer reaction t
方法:实验采用双侧颈总动脉结扎的方法制备血管性痴呆小鼠模型。昆明小鼠360只，随机分为假手术组(sham-operation group，S)、模型组（model group，M）、当归芍药散高剂量组（high-dose DanguiShaoyaoSan group，H）、当归芍药散低剂量组（low-dose DanguiShaoyaoSan group，L）、银杏叶提取物对照组(Extract of GinkgoBiloba control group，E)、尼莫地平对照组(Nimodipine control group，N)6组，每组60只。S组小鼠只分离颈总动脉，穿线但不结扎，尾部不放血，其余M、H、L、E、N各组结扎双侧颈总动脉阻断血流加尾部放血，制备反复脑缺血再灌注VD模型。术后第2天开始治疗。H组和L组灌服当归芍药散水煎剂（以
下简称“归芍煎”），E组灌服银杏叶提取物混悬液银杏叶液（以下简称“银杏叶液”），N组灌服尼莫地平混悬液（以下简称“尼莫地平液”），S、M两组均灌服生理盐水。分别于术后7、15、30天进行行为学实验（跳台法、水迷宫法）。行为学实验结束后断头处死小鼠，取左侧脑组织固定后切片，常规HE染色和硫堇染色，光镜下观察脑组织的形态变化；取右侧脑组织制成10%的脑组织匀浆，采用黄嘌呤氧化酶法测定脑组织SOD活力、硫代巴比妥酸比色分析法测定脑组织MDA含量、比色分析法测定脑组织GSH含量和GSH-Px活力。通过与银杏叶提取物和尼莫地平对照，观察高、低剂量当归芍药散对拟VD小鼠脑组织 MDA、GSH含量及SOD、GSH-Px活力的影响。
结果:
1当归芍药散对拟VD小鼠行为学实验的影响
1．1水迷宫成绩
术后7天、15天和30天，小鼠的学习成绩与记忆成绩改变基本一致， M组小鼠的游全程时间延长，错误次数增加，与S组比较差异具有显著性统计学意义（P<0.05或P<0.01）。H、L、E、N各组与M组比较，差异有显著性统计学意义（P<0.05或P<0.01）。H组与L、E、N各组比较，术后7天无显著性统计学意义（P>0.05），15天和30天时有显著性统计学意义（P<0.05或P<0.01）；L、E、N各组之间比较无显著性统计学意义（P>0.05）。
1．2跳台成绩
术后各时段， M组小鼠的反应时间延长，潜伏期缩短，错误次数增加，与S组比较，有显著性统计学意义（P<0.01）。H组与M组比较，反应时间缩短，潜伏期延长，错误次数减少，差异有显著性统计学意义（P<0.05）；H组与L、E、N各组比较，术后7天学习成绩无显著性统计学意义（P>0.05），术后15天和30天的学习成绩和各时段的记忆成绩均有显著性统计学意义（P<0.05或P<0.01）；而L、E、N各组之间比较无显著性统计学意义（P>0.05）。
2当归芍药散对拟VD小鼠病理形态学的影响
光镜下病理形态学显示，术后M组小鼠脑组织海马区呈缺血性病理改变。锥体细胞层次减少，排列稀疏，明显脱失，胞浆内尼氏体消失，细胞核体积变小，结构不清。随着时间的推移，模型小鼠脑组织病理改变逐步加重。各用药组小鼠病变轻于M组。
3当归芍药散对VD小鼠脑组织MDA 、GSH含量及SOD、GSH-Px活性的影响
3．1当归芍药散对VD小鼠脑组织MDA的影响
各时段M组小鼠脑组织MDA含量（7天、15天、30天分别为9.12±3.31、9.28±2.08、9.14±2.71）较S组（3.65±2.31、3.48±1.22、3.42±1.56）明显升高（P<0.01）；经用药干预后，各时段各用药组小鼠脑组织MDA含量均低于M组（P<0.05或P<0.01）；各治疗组比较，术后7天各治疗组（H、L、E、N各组分别为4.88±1.19、6.00±2.16，6.38±2.38、6.29±2.38）之间差异无显著性统计学意义（P>0.05）；术后15天和30天H组小鼠脑组织MDA含量（4.10±1.55、3.45±1.12）低于L组（5.94±
2.02、5.56±1.70）、E组（6.35±2.03、5.76±2.15）和N组（6.12±2.27、5.96±1.50）（P<0.05）；L组、E组和N组之间比较无显著性统计学意义（P>0.05）。
3．2当归芍药散对VD小鼠脑组织SOD活性的影响
各时段M组小鼠脑组织SOD活性（15.07±4.14、14.05±5.05、15.72±5.93）均较S组（32.78±7.70、32.45±10.01、35.60±9.80）明显降低（P<0.01）；经用药干预后，用药各组小鼠脑组织SOD活性（H组:28.06±6.75、32.15±10.25、37.14±6.75；E组:20.79±5.89、23.02±9.82、27.33±11.53；N组:20.33±6.00、23.56±10.19、25.84±8.88；L组:22.82±5.97、25.01±8.93、28.52±9.03）均较M组升高（P<0.05或P<0.01）；用药各组之间比较，各时段H组小鼠脑组织SOD活性均较E、N组升高（P<0.05或P<0.01），H组与L组比较，术后7天和15天，两组无显著性统计学意义（P >0.05），术后30天，H组高于L组（P <0.05）。L、E、N各组间比较，各时段均无显著性统计学意义（P
Objective:Vascular Dementia (called VD in short ) belongs to the disease scope of dementia of old age in the light of tradditional Chinese medincine(TCM).In the recent years,with the aging of population the incidence of vascular dementia is on the rise and affects seriously the people's working ability as well as living quality.Therefore,it's of great significance to make deeper research on it's pathological mechanism and develop the effective medicine with the integration of both Chinese and western medical theory.In order to find the relationship between disequilibrium of oxidation--anti-oxidation and Vascular Dementia ,then further to analyse the therapeutical pathogenicity with DangguiShaoyaoSan，we detected malonaldehyde（MDA）,superoxide dismutase（SOD）,glutathione（GSH） and glutathione peroxidase（GSH-Px） of cerebral tissue on mice with Vascular Dementia .
Methods: 360 Kunming mice were divided into six groups at random，which were sham-operation group（which were abbreviated to “S group”in the following）,model group（which were abbreviated to “M group” in the following,
high dose of DangguiShaoyaoSan group（which were abbreviated to “H group” in the following）, low dose of DangguiShaoyaoSan group（which were abbreviated to “ L group” in the following）, extract of Ginkgobiloba control group（which were abbreviated to “E group”）and Nimodipine control group（which were abbreviated to “N group” in the following）. There were 60 mice in each group. We duplicated the model of vascular dementia mice with the operation of cerebral ischemic reperfusion except S group. And then 6 group were continuously filled medicine or saline through the stomach from the second day after the operation of cerebral ischemic reperfusion. S group and M group were filled saline and H, L,E and N group were given medicine. On the 7th,15th and 30th day after praxiology experiment,the mice were cut head for obtaining cerebral tissue， the left part of which was fixed by 10% for maldehyde solution. The tissue was stained by HE and Thionine. We observed constitutional morphologic changes of cerebral tissue under light microscope and the right part of which were made into tissue homogenation for check up.
The experiment used TBA compared color analytical method to detect the content changes of MDA in cerebral tissue of Kunming mice, and used xanthine oxidase method to detect the activity changes of SOD, and used compared color analytical method to detect the activity changes of GSH and GSH-Px in cerebal tissue of Kunming mice. With
Ginkgobiloba extract and Nimodipine as the control medicine，we observed the effect of high and low dose of DangguiShaoyaoSan on MDA and GSH content and the activity of SOD and GSH-Px in cerebral ischemic reperfusion. Then we detected the mechanism on vascular dementia with DangguiShaoyaoSan from the direction of anti-oxidation injury.
Results:
1 Results of praxiology experiment
1.1 The mice's performance record of water-labyrinth
There was no significant difference between the learning and memorizing performance. The wrong times and the time of swimming along the whole line of M group was more than that of S group (P<0.05or P<0.01). At the same time, the swimming time and the wrong times of H ,L,E and N group was less than that of M group(P<0.05or P<0.01). Comparing with those of L,E and N group ,The swimming time and the wrong times of H group was less on the 15th and 30th day(P<0.05or P<0.01)，but not on the 7th day after the operation of cerebral ischemic reperfusion(P>0.05).There was no statistical difference among L, E and N group according to the learning and memorizing performance record ( P>0.05).
1.2 The mice's performance record of water-labyrinth Experiment of jumping stand
M group has shorter reaction time(the time of jumping up the stand), longer latency time(the time of jumping down the
stand) and more wrong times than S group (P<0.05or P<0.01).At the same time， H group has longer reaction t
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13 王永炎，刘金民，谢颖桢.中风病智能障碍的中医康复.见:北京中医药大学校庆40周年论文汇编.北京:学苑出版社，1996.561～566
14谢颖桢，梅建勋，王同胜.近十年老年期痴呆的中医研究概况.北京中医药大学学报，1997，20(5):42～44
15韩祖成，史凡凡，王凌.当归芍药散治疗脑血管性痴呆36例.陕西中医，1996，19（1）：16
16冀宏. 当归芍药散治疗血管性痴呆的临床研究.山西中医，2000，16(2): 10～11
17张志耘，胡志洁.当归芍药散研究的新进展.中草药，2000，31(7):87～91
18 陈主初.病理生理学.北京：人民卫生出版社 .2001:83～94
19 Bunn H.G，Poyon R.O:Oxygen sensing and molecular adaptation to hypoxia .Physiological Reviews.1996，76:839～885
20雷载权，张廷模主编.中华临床中药学.下册.人民卫生出版社，1998:1615～1619
21胡永新，陈红，陈士明等.几种天然药物清除超氧阴离子自由基的作用 .第二军医大学学报，1993，14(1):48～51
22李怀荆.白术水煎剂对老年小鼠抗衰老作用影响.佳木斯医学院学报，1996，19(1):9～10
23樊景坡.苍耳子、细辛、枸杞子、白术对小鼠组织自由基代谢的影响 .中医药信息，1994，（2）：48～50
24林晓明，冯建英，龙珠等.银耳、茯苓、绞股蓝对小鼠免疫功能和清除自由基的作用.北京医科大学学报，1995，27（6）：455～473
25黄建邦，刘雪芬，陈树元. 川芎嗪对人血多形核白细胞呼吸暴发与氧自由基的抑制作用.中国中西医结合杂志，1994，14(10):607～609
26王洁，汪钟.冠心病患者血液脂质过氧化物及有关酶变化的临床意义.中华心血管病杂志，1992，（2）:104～107
27阴健,郭力弓.中药现代研究与临床应用. 北京:学苑出版社,1994:287
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8 赵建新，田元祥，孙彦辉，等.电针肾俞、膈俞、百会穴对拟血管性痴呆学习记忆障碍小鼠学习记忆的影响.中国行为医学科学，2000，9(2):101～103
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10秦震.神经病学. 上海：上海医科大学出版社，2000：193～196
11 赵建新，田元祥，李国明，等.拟血管性痴呆小鼠模型皮层及海马细胞病理组织学动态观察. 中国病理生理杂志，2000，16(11):1214～1216
12刘汇波，叶翠飞，李斌，等.双侧颈总动脉结扎对大鼠学习记忆功能和海马组织形态学影响.基础医学与临床，1998，4:48-52
13 王永炎，刘金民，谢颖桢.中风病智能障碍的中医康复.见:北京中医药大学校庆40周年论文汇编.北京:学苑出版社，1996.561～566
14谢颖桢，梅建勋，王同胜.近十年老年期痴呆的中医研究概况.北京中医药大学学报，1997，20(5):42～44
15韩祖成，史凡凡，王凌.当归芍药散治疗脑血管性痴呆36例.陕西中医，1996，19（1）：16
16冀宏. 当归芍药散治疗血管性痴呆的临床研究.山西中医，2000，16(2): 10～11
17张志耘，胡志洁.当归芍药散研究的新进展.中草药，2000，31(7):87～91
18 陈主初.病理生理学.北京：人民卫生出版社 .2001:83～94
19 Bunn H.G，Poyon R.O:Oxygen sensing and molecular adaptation to hypoxia .Physiological Reviews.1996，76:839～885
20雷载权，张廷模主编.中华临床中药学.下册.人民卫生出版社，1998:1615～1619
21胡永新，陈红，陈士明等.几种天然药物清除超氧阴离子自由基的作用 .第二军医大学学报，1993，14(1):48～51
22李怀荆.白术水煎剂对老年小鼠抗衰老作用影响.佳木斯医学院学报，1996，19(1):9～10
23樊景坡.苍耳子、细辛、枸杞子、白术对小鼠组织自由基代谢的影响 .中医药信息，1994，（2）：48～50
24林晓明，冯建英，龙珠等.银耳、茯苓、绞股蓝对小鼠免疫功能和清除自由基的作用.北京医科大学学报，1995，27（6）：455～473
25黄建邦，刘雪芬，陈树元. 川芎嗪对人血多形核白细胞呼吸暴发与氧自由基的抑制作用.中国中西医结合杂志，1994，14(10):607～609
26王洁，汪钟.冠心病患者血液脂质过氧化物及有关酶变化的临床意义.中华心血管病杂志，1992，（2）:104～107
27阴健,郭力弓.中药现代研究与临床应用. 北京:学苑出版社,1994:287