三七总皂苷对D-半乳糖联合Aβ(1-40)致老年痴呆大鼠模型治疗作用的研究
摘要
[目的]通过构建D-半乳糖(D-gal)腹腔注射加Aβ(1-40)侧脑室注射致复合型SD大鼠老年痴呆(AD)模型,从分子、细胞、组织及整体层面探讨三七总皂苷对老年痴呆的治疗作用和对神经细胞凋亡的影响机制。
[材料和方法]成年雄性SD大鼠60只随机分为假手术组、AD模型组(AD组)、PNS治疗组(PNS组),每组20只。AD模型组和PNS治疗组SD大鼠腹腔注射D-半乳糖60mg/kg.d,假手术组SD大鼠腹腔注射等体积生理盐水;PNS治疗组灌胃给药PNS 200mg/kg,每日一次,连续8周;AD组和假手术组以等体积的生理盐水灌胃,连续8周。第42天,SD大鼠在3.6%水合氯醛(10ml/Kg)麻醉,用脑立体定位仪,前囟后1.3mm,右侧旁开2.0mm,DV4.0mm即为侧脑室注射点,在颅骨上用牙钻打孔,微量进样器垂直进入右侧脑室,AD组和PNS组缓慢注射5μl(10μg)Aβ(1-40),假手术组注射5μl生理盐水。第49天开始对各组大鼠进行Morris水迷宫(MWM)实验,评估动物的学习记忆能力。用TBA比色法测定丙二醛(MDA)的含量;分光光度法测定一氧化氮(NO)、过氧化氢(H_2O_2)、还原型谷胱甘肽(GSH)、谷胱甘肽还原酶(GR)、谷胱甘肽过氧化物酶(GSH-Px)、总抗氧化能力(T-AOC)、谷胱甘肽S-转移酶(GSH-ST)、总超氧化物歧化酶(T-SOD)的含量。常规免疫组织化学法检测大脑皮质凋亡相关因子Bcl-2、Caspase-3蛋白的表达变化。Western Blot检测分析Bcl-2蛋白的表达变化和Procaspase-3蛋白的变化。
[结果]PNS治疗组在Morris水迷宫(MWM)测试中,逃避潜伏期明显缩短,寻求次数,原平台象限比、跨越原平台次数明显增加,与AD模型组比较有显著意义(P＜0.05)。PNS能减少模型大鼠大脑皮质NO、H_2O_2、MDA的含量(P＜0.05)、增加大脑皮质GR、GSH-ST、GSH-Px、GSH、T-AOC、T-SOD水平(P＜0.05)。PNS治疗组大鼠脑皮质Bcl-2表达的阳性细胞数量无明显变化(P＞0.05),Caspase-3表达的阳性细胞数量明显下降(P＜0.05)。Bcl-2的Western Blot检测表达结果与免疫组化基本一致;AD模型组较假手术组大鼠脑皮质procaspase-3分解增加(P＜0.05),PNS治疗组较AD模型组Procaspase-3分解减少(P＜0.05)。
[结论](1)PNS对D-半乳糖腹腔注射-Aβ(1-40)AD大鼠空间学习记忆损害具有明显改善作用;(2)活性氧(reactive oxygen species,ROS)水平在AD模型大脑皮质有明显增高,ROS可能是诱导AD大鼠模型记忆力减退的重要原因,PNS可以降低大鼠脑皮质ROS水平从而改善模型大鼠的记忆能力;(3)PNS可以提高SD大鼠脑皮质巯基抗氧化物的含量,从而改善模型大鼠的学习记忆能力,这可能是通过增加大脑的抗氧化能力而抑制了神经细胞凋亡的信号通路;(4)PNS可通过调控巯基抗氧化物,减少凋亡因子Caspase-3蛋白表达,抑制神经元细胞凋亡从而改善模型大鼠的学习记忆能力。
Objective:To investigate the therapeutic action of panax notoginsenoside(PNS) in rat model of Alzheimer's disease(AD) and the mechansm of PNS' influence in the nerve cell apoptosis and necrosis in molecular,cellular,systemic and integrate aspects. The Alzheimer's composite rat models were established by D-galactose(D-gal) intraperitoneal injection and Aβ(1-40) intracerebroventricular injection.
Methods:60 male adult SD rats were randomly divided into three groups 20 each, including the sham-operation group,the AD model group and the AD model treated with PNS group.The rats of the AD model group and the PNS treatement group were injected intraperitoneal with D-galactose 60mg/kg.d.The sham group were given intraperitoneal injection with the same Volume of normal saline.The PNS treatement grotip were given PNS(200mg/kg) by intragastric administration once daily for 56days.The sham group and AD model group were given the same Volume of normal saline by intragastric administ ration.At the 42th day,the rats were given intraperitoneal injection with 3.6%chloral hydrate(10ml/kg) for anesthetization and were fixed in brain three-dimensional positioning.Amyloidβ-peptides(1-40)(5μL = 10μg) were injected into the right lateral ventricle of the AD model group and PNS treatement group,with stereotaxic coordinates from the bregma being,in mm,A -1.3, L/R-2.0,and V 4.0.The rats of the sham group were injected 5μL NS into the right lateral ventricle.All experimental animals were tested by Morris water maze (MWM)test from the 49th day to estimate the ability of leaning and memory.The Nitric Oxide(NO),Hydrogen peroxide(H_2O_2),glutathione(GSH),Glutathione Reductase(GR),glutathione peroxidase(GSH-Px),anti-oxidative capabilities(T-AOC), Glutathione-s-transferase(GSH-ST) and total superoxide dismutase(T-SOD) levels were measured with the spectrophotometry,the Malonyldialdehyed(MDA) concentration was determined with thiobarbituric acid reaction(TBA).The expression of Bcl-2 and Caspase-3 in the cortex were measured with immunohistochemistry. The expression of Bcl-2 and the change of Procaspase-3 protein were measured with Western Blot.
Results:All of the test indexes in MWM showed that the abilities of space search, learning and memory in PNS group remarkably improved compared with that of the model group(P＜0.05).PNS could decrease ROS in cortex,including a decrease in NO,H_2O_2 and MDA(P＜0.05),while an increase in GR,GSH-ST,GSH-Px,GSH, T-AOC and T-SOD activities(P＜0.05).The cortex's immunohistochemistry expression of Bcl-2 was no obvious change(P＞0.05).The expression of Caspase-3 decreased obviously(P＜0.05).The Western Blot expression of Bcl-2 was according with immunohistochemistry.The AD model group cerebral cortex increased procaspase-3 decomposition than the sham group(P＜0.05).The Procaspase-3 decomposition of PNS group was reduced than the AD model group(P＜0.05).
Conclusions:(1) PNS has a significant relieving effect on the lesion of the abilities of space search,learning and memory in D-galactose-amyloidβ-peptide(1-40) AD model rats.(2) The level of reactive oxygen species(ROS) was increased obviously in the cortex of AD model rats.ROS maybe an important factor which inducing a spatial memory deficit.PNS may improve the abilities of learning and memory through decreasing ROS level of rats'cortex.(3) PNS may increased the content of sulfhydryl compound in the cortex to improve the abilities of learning and memory,by enhancing the organism antioxygen and restraining the signal transduction pathway of the neuroglial cell apoptosis.(4) PNS may relieved the lesion of the abilities of space search,learning and memory in AD rats model,by restraining the neuroglial cell apoptosis induced by the apoptosis factor Caspase-3,which were controled by sulfhydryl compound.
[材料和方法]成年雄性SD大鼠60只随机分为假手术组、AD模型组(AD组)、PNS治疗组(PNS组),每组20只。AD模型组和PNS治疗组SD大鼠腹腔注射D-半乳糖60mg/kg.d,假手术组SD大鼠腹腔注射等体积生理盐水;PNS治疗组灌胃给药PNS 200mg/kg,每日一次,连续8周;AD组和假手术组以等体积的生理盐水灌胃,连续8周。第42天,SD大鼠在3.6%水合氯醛(10ml/Kg)麻醉,用脑立体定位仪,前囟后1.3mm,右侧旁开2.0mm,DV4.0mm即为侧脑室注射点,在颅骨上用牙钻打孔,微量进样器垂直进入右侧脑室,AD组和PNS组缓慢注射5μl(10μg)Aβ(1-40),假手术组注射5μl生理盐水。第49天开始对各组大鼠进行Morris水迷宫(MWM)实验,评估动物的学习记忆能力。用TBA比色法测定丙二醛(MDA)的含量;分光光度法测定一氧化氮(NO)、过氧化氢(H_2O_2)、还原型谷胱甘肽(GSH)、谷胱甘肽还原酶(GR)、谷胱甘肽过氧化物酶(GSH-Px)、总抗氧化能力(T-AOC)、谷胱甘肽S-转移酶(GSH-ST)、总超氧化物歧化酶(T-SOD)的含量。常规免疫组织化学法检测大脑皮质凋亡相关因子Bcl-2、Caspase-3蛋白的表达变化。Western Blot检测分析Bcl-2蛋白的表达变化和Procaspase-3蛋白的变化。
[结果]PNS治疗组在Morris水迷宫(MWM)测试中,逃避潜伏期明显缩短,寻求次数,原平台象限比、跨越原平台次数明显增加,与AD模型组比较有显著意义(P＜0.05)。PNS能减少模型大鼠大脑皮质NO、H_2O_2、MDA的含量(P＜0.05)、增加大脑皮质GR、GSH-ST、GSH-Px、GSH、T-AOC、T-SOD水平(P＜0.05)。PNS治疗组大鼠脑皮质Bcl-2表达的阳性细胞数量无明显变化(P＞0.05),Caspase-3表达的阳性细胞数量明显下降(P＜0.05)。Bcl-2的Western Blot检测表达结果与免疫组化基本一致;AD模型组较假手术组大鼠脑皮质procaspase-3分解增加(P＜0.05),PNS治疗组较AD模型组Procaspase-3分解减少(P＜0.05)。
[结论](1)PNS对D-半乳糖腹腔注射-Aβ(1-40)AD大鼠空间学习记忆损害具有明显改善作用;(2)活性氧(reactive oxygen species,ROS)水平在AD模型大脑皮质有明显增高,ROS可能是诱导AD大鼠模型记忆力减退的重要原因,PNS可以降低大鼠脑皮质ROS水平从而改善模型大鼠的记忆能力;(3)PNS可以提高SD大鼠脑皮质巯基抗氧化物的含量,从而改善模型大鼠的学习记忆能力,这可能是通过增加大脑的抗氧化能力而抑制了神经细胞凋亡的信号通路;(4)PNS可通过调控巯基抗氧化物,减少凋亡因子Caspase-3蛋白表达,抑制神经元细胞凋亡从而改善模型大鼠的学习记忆能力。
Objective:To investigate the therapeutic action of panax notoginsenoside(PNS) in rat model of Alzheimer's disease(AD) and the mechansm of PNS' influence in the nerve cell apoptosis and necrosis in molecular,cellular,systemic and integrate aspects. The Alzheimer's composite rat models were established by D-galactose(D-gal) intraperitoneal injection and Aβ(1-40) intracerebroventricular injection.
Methods:60 male adult SD rats were randomly divided into three groups 20 each, including the sham-operation group,the AD model group and the AD model treated with PNS group.The rats of the AD model group and the PNS treatement group were injected intraperitoneal with D-galactose 60mg/kg.d.The sham group were given intraperitoneal injection with the same Volume of normal saline.The PNS treatement grotip were given PNS(200mg/kg) by intragastric administration once daily for 56days.The sham group and AD model group were given the same Volume of normal saline by intragastric administ ration.At the 42th day,the rats were given intraperitoneal injection with 3.6%chloral hydrate(10ml/kg) for anesthetization and were fixed in brain three-dimensional positioning.Amyloidβ-peptides(1-40)(5μL = 10μg) were injected into the right lateral ventricle of the AD model group and PNS treatement group,with stereotaxic coordinates from the bregma being,in mm,A -1.3, L/R-2.0,and V 4.0.The rats of the sham group were injected 5μL NS into the right lateral ventricle.All experimental animals were tested by Morris water maze (MWM)test from the 49th day to estimate the ability of leaning and memory.The Nitric Oxide(NO),Hydrogen peroxide(H_2O_2),glutathione(GSH),Glutathione Reductase(GR),glutathione peroxidase(GSH-Px),anti-oxidative capabilities(T-AOC), Glutathione-s-transferase(GSH-ST) and total superoxide dismutase(T-SOD) levels were measured with the spectrophotometry,the Malonyldialdehyed(MDA) concentration was determined with thiobarbituric acid reaction(TBA).The expression of Bcl-2 and Caspase-3 in the cortex were measured with immunohistochemistry. The expression of Bcl-2 and the change of Procaspase-3 protein were measured with Western Blot.
Results:All of the test indexes in MWM showed that the abilities of space search, learning and memory in PNS group remarkably improved compared with that of the model group(P＜0.05).PNS could decrease ROS in cortex,including a decrease in NO,H_2O_2 and MDA(P＜0.05),while an increase in GR,GSH-ST,GSH-Px,GSH, T-AOC and T-SOD activities(P＜0.05).The cortex's immunohistochemistry expression of Bcl-2 was no obvious change(P＞0.05).The expression of Caspase-3 decreased obviously(P＜0.05).The Western Blot expression of Bcl-2 was according with immunohistochemistry.The AD model group cerebral cortex increased procaspase-3 decomposition than the sham group(P＜0.05).The Procaspase-3 decomposition of PNS group was reduced than the AD model group(P＜0.05).
Conclusions:(1) PNS has a significant relieving effect on the lesion of the abilities of space search,learning and memory in D-galactose-amyloidβ-peptide(1-40) AD model rats.(2) The level of reactive oxygen species(ROS) was increased obviously in the cortex of AD model rats.ROS maybe an important factor which inducing a spatial memory deficit.PNS may improve the abilities of learning and memory through decreasing ROS level of rats'cortex.(3) PNS may increased the content of sulfhydryl compound in the cortex to improve the abilities of learning and memory,by enhancing the organism antioxygen and restraining the signal transduction pathway of the neuroglial cell apoptosis.(4) PNS may relieved the lesion of the abilities of space search,learning and memory in AD rats model,by restraining the neuroglial cell apoptosis induced by the apoptosis factor Caspase-3,which were controled by sulfhydryl compound.
引文
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[1]Rukhsana S,Debra BK,Fai HP,et al.Oxidative modification and down-regulation of Pinl in Aizheimer's disease hippocampus:A redox proteomics analysis[J].Neurobiology of Aging,2006,27(7):918-925
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