枫叶黄酮对老年性痴呆动物模型的抗氧化作用及分子机制
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摘要
目的：采用侧脑室注射β淀粉样蛋白1-42(Aβ1-42)联合腹腔注射D-半乳糖(D-gal)构建SD大鼠老年性痴呆动物模型,从行为学和分子水平探讨枫叶黄酮对衰老的防治作用及分子机制。
材料和方法：36只成年雄性Sprague Dawley (SD)大鼠随机分为3组,假手术组(sham group)、模型组(model group)和治疗组(treatment group),每组12只。采用侧脑室注射p淀粉样蛋白1-42(Aβ1-42)联合腹腔注射D-半乳糖(D-gal)构建SD大鼠衰老模型,并同时给予枫叶黄酮预防性治疗。采用Morris水迷宫实验(MWM)进行空间学习记忆能力检测,用化学比色法检测大脑皮质组织中丙二醛(MDA)、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)、超氧化物歧化酶(SOD)的含量。用免疫印迹法(Western blotting)检测大脑皮质组织中丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)家族相关蛋白：细胞外信号调节蛋白激酶(extracellular signal-regulated protein kinase, ERK)、P38、c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)的磷酸化水平。
结果：模型组与假手术组进行比较发现：模型组逃避潜伏期明显延长(P<0.05),在第Ⅲ象限逗留的时间明显减少(P<0.05),跨越平台次数明显减少(P<0.05),大脑皮质组织中MDA的含量明显增加(P<0.05),GR和GSH-PX的含量明显降低(P<0.05),JNK、P38的磷酸化明显增强,ERK的磷酸化明显受抑制(P<0.05)。治疗组与模型组进行比较发现：治疗组逃避潜伏期明显缩短(P<0.05),在第Ⅲ象限逗留的时间明显增加(P<0.05),跨越平台次数明显增加(P<0.05),大脑皮质组织中MDA的含量明显降低(P<0.05),GR、GSH-PX的含量明显升高(P<0.05), JNK、p38的磷酸化明显受抑制,ERK的磷酸化明显增强(P<0.05)。SOD、GSH在三组间没有统计学差异(P>0.05)。
结论：(1)侧脑室注射β淀粉样蛋白1-42(Aβ1-42)联合腹腔注射D-半乳糖(D-gal)能诱发脑内氧化应激,同时活性氧(reactive oxygen species, ROS)作为第二信使影响MAPK信号通路激活(P38、JNK激活明显增强,ERK激活明显受抑制),导致大鼠空间探索学习记忆能力减退,出现老年性痴呆的表现。
(2)枫叶黄酮能通过上调衰老模型大鼠大脑皮质组织中抗氧化物(酶)GR、GSH-PX的含量,抗氧化应激损伤,抑制P38、JNK的激活,增强ERK激活,改善空间探索学习记忆能力,抑制或延缓老年性痴呆的发生。
Objective:To explore the anti-oxidation molecule mechanism of Acer truncatum Bunge Flavonoid from the behavioral and molecular level and provide the experimental foundations for clinical treatment of AD, the Aβ1-42 and D-galactose AD rat model was established.
Methods:Thirty-six rats were divided into three groups at random:sham group, model group and treatment group. Aging rats model were established by lateral ventricle injection of Aβ1-42 and abdominal cavity injection of D-Galactose to the rats. Meantime, rats were treated by intragastric administration Acer truncatum Bunge Flavonoid. Then experimental rats were examined spatial memory with the Morris water maze (MWM). Malondialdehyde (MDA),and antioxidants including glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activities were examined by colorimetric method in cerebral cortex. The concentration of the extracellular signal-regulated kinase(ERK), p38 and c-Jun N-terminal kinases(JNK), P-ERK, P-P38, P-JNK were examined by western blotting methods.
Results:The AD model rats when compared with control group exhibited a significant increase in escape latencies (P<0.05), while a decrease in the time of staying at the first quadrants of platform and the degree of crossing over a platform. The cerebral cortex concentration of the MDA was increased, and the concentration of the GR, GSH-PX were decreased (P<0.05). The expression of P-p38, P-JNK were increased while P-ERK was decreased (P<0.05). After the treatment of the he Flavonoid from the Leaves of Acer truncatum Bunge, the AD model rats exhibited a significant decrease in escape latencies (P<0.05), an obvious increase in the time of staying the first quadrants of platform (P<0.05)and the increase of crossing over a platform(P<0.05) when compared with the AD group(P<0.05). The concentration of the MDA was decreased, the concentration of GR, GSH-PX were increased (P<0.05). The expression of the P-p38, P-JNK were less than the AD model while P-ERK was more than the AD model (P<0.05). But there were no significant differences between the three groups in the expression of theP38, JNK, ERK (P>0.05).
Conclusions:(1) Aβ1-42 combinated with D-galactose could impair the oriented learning and memory capacity and induce the neurodegeneration of central nervous systems in model rats by oxidative stress wich results in P38, JNK phosphorylation increased and ERK phosphorylation inhibition. (2) Acer truncatum Bunge Flavonoid could improve the oriented learning and memory capacity and prevent the neurodegeneration of central nervous systems in aging model rats by antioxidants increased wich induce P38, JNK phosphorylation inhibition and ERK phosphorylation increased.
材料和方法：36只成年雄性Sprague Dawley (SD)大鼠随机分为3组,假手术组(sham group)、模型组(model group)和治疗组(treatment group),每组12只。采用侧脑室注射p淀粉样蛋白1-42(Aβ1-42)联合腹腔注射D-半乳糖(D-gal)构建SD大鼠衰老模型,并同时给予枫叶黄酮预防性治疗。采用Morris水迷宫实验(MWM)进行空间学习记忆能力检测,用化学比色法检测大脑皮质组织中丙二醛(MDA)、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)、超氧化物歧化酶(SOD)的含量。用免疫印迹法(Western blotting)检测大脑皮质组织中丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)家族相关蛋白：细胞外信号调节蛋白激酶(extracellular signal-regulated protein kinase, ERK)、P38、c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)的磷酸化水平。
结果：模型组与假手术组进行比较发现：模型组逃避潜伏期明显延长(P<0.05),在第Ⅲ象限逗留的时间明显减少(P<0.05),跨越平台次数明显减少(P<0.05),大脑皮质组织中MDA的含量明显增加(P<0.05),GR和GSH-PX的含量明显降低(P<0.05),JNK、P38的磷酸化明显增强,ERK的磷酸化明显受抑制(P<0.05)。治疗组与模型组进行比较发现：治疗组逃避潜伏期明显缩短(P<0.05),在第Ⅲ象限逗留的时间明显增加(P<0.05),跨越平台次数明显增加(P<0.05),大脑皮质组织中MDA的含量明显降低(P<0.05),GR、GSH-PX的含量明显升高(P<0.05), JNK、p38的磷酸化明显受抑制,ERK的磷酸化明显增强(P<0.05)。SOD、GSH在三组间没有统计学差异(P>0.05)。
结论：(1)侧脑室注射β淀粉样蛋白1-42(Aβ1-42)联合腹腔注射D-半乳糖(D-gal)能诱发脑内氧化应激,同时活性氧(reactive oxygen species, ROS)作为第二信使影响MAPK信号通路激活(P38、JNK激活明显增强,ERK激活明显受抑制),导致大鼠空间探索学习记忆能力减退,出现老年性痴呆的表现。
(2)枫叶黄酮能通过上调衰老模型大鼠大脑皮质组织中抗氧化物(酶)GR、GSH-PX的含量,抗氧化应激损伤,抑制P38、JNK的激活,增强ERK激活,改善空间探索学习记忆能力,抑制或延缓老年性痴呆的发生。
Objective:To explore the anti-oxidation molecule mechanism of Acer truncatum Bunge Flavonoid from the behavioral and molecular level and provide the experimental foundations for clinical treatment of AD, the Aβ1-42 and D-galactose AD rat model was established.
Methods:Thirty-six rats were divided into three groups at random:sham group, model group and treatment group. Aging rats model were established by lateral ventricle injection of Aβ1-42 and abdominal cavity injection of D-Galactose to the rats. Meantime, rats were treated by intragastric administration Acer truncatum Bunge Flavonoid. Then experimental rats were examined spatial memory with the Morris water maze (MWM). Malondialdehyde (MDA),and antioxidants including glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activities were examined by colorimetric method in cerebral cortex. The concentration of the extracellular signal-regulated kinase(ERK), p38 and c-Jun N-terminal kinases(JNK), P-ERK, P-P38, P-JNK were examined by western blotting methods.
Results:The AD model rats when compared with control group exhibited a significant increase in escape latencies (P<0.05), while a decrease in the time of staying at the first quadrants of platform and the degree of crossing over a platform. The cerebral cortex concentration of the MDA was increased, and the concentration of the GR, GSH-PX were decreased (P<0.05). The expression of P-p38, P-JNK were increased while P-ERK was decreased (P<0.05). After the treatment of the he Flavonoid from the Leaves of Acer truncatum Bunge, the AD model rats exhibited a significant decrease in escape latencies (P<0.05), an obvious increase in the time of staying the first quadrants of platform (P<0.05)and the increase of crossing over a platform(P<0.05) when compared with the AD group(P<0.05). The concentration of the MDA was decreased, the concentration of GR, GSH-PX were increased (P<0.05). The expression of the P-p38, P-JNK were less than the AD model while P-ERK was more than the AD model (P<0.05). But there were no significant differences between the three groups in the expression of theP38, JNK, ERK (P>0.05).
Conclusions:(1) Aβ1-42 combinated with D-galactose could impair the oriented learning and memory capacity and induce the neurodegeneration of central nervous systems in model rats by oxidative stress wich results in P38, JNK phosphorylation increased and ERK phosphorylation inhibition. (2) Acer truncatum Bunge Flavonoid could improve the oriented learning and memory capacity and prevent the neurodegeneration of central nervous systems in aging model rats by antioxidants increased wich induce P38, JNK phosphorylation inhibition and ERK phosphorylation increased.
引文
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