【摘要】 目的:探讨地黄饮子对阿尔茨海默病动物模型凋亡基因及超微结构的影响。方法:实验于2003-08/2004-08在黑龙江中医药大学基础理论实验室完成。选择120只Wistar大鼠,随机分成6组:假损伤组,模型组,抗脑衰对照组,石杉碱甲对照组,地黄饮子5.4g/(kg·d)和地黄饮子2.7g/(kg·d)组,每组20只。模型组以D-半乳糖腹腔注射合并淀粉样β蛋白注射海马制备阿尔茨海默病动物模型,假损伤组腹腔注射生理盐水,其余各组分别在造模的同时,地黄饮子治疗组分别给予地黄饮子灌胃[相当含生药5.4g/(kg·d)和2.7g/(kg·d)],抗脑衰对照组、石杉碱甲对照组分别给予抗脑衰胶囊、石杉碱甲溶液灌胃[含生药分别为2.403g/(kg·d),0.018mg/(kg·d)],1次/d,共5周。运用反转录-聚合酶链反应测定各组海马凋亡基因的表达,及采用透射电镜观察各组海马神经元超微结构的变化。结果:120只动物中,因造模死亡共20只,模型组5只、石杉碱甲对照组6只,抗脑衰对照组3只,地黄饮子5.4g/(kg·d)组3只,地黄饮子2.7g/(kg·d)组3只,进入结果分析100只。①大鼠海马半胱氨酸天冬氨酸蛋白酶3、核转录因子κB、hsp70基因mRNA的表达:反转录-聚合酶链反应结果示,淀粉样β蛋白可诱发大鼠海马半胱氨酸天冬氨酸蛋白酶3mRNA表达增加,与模型组比较,抗脑衰对照组,石杉碱甲对照组,地黄饮子5.4g/(kg·d),2.7g/(kg·d)组均可不同程度降低大鼠海马区域的表达(P<0.05)。地黄饮子5.4g/(kg·d)组下调幅度最大。与假损伤组比较,模型组鼠脑海马核转录因子κBmRNA、hsp70mRNA水平显著降低;与模型组比较,抗脑衰对照组,石杉碱甲对照组,地黄饮子5.4g/(kg·d),2.7g/(kg·d)组均有明显的上调作用(P<0.05)。地黄饮子5.4g/(kg·d)组对hsp70mRNA水平的上调作用优于抗脑衰对照组,石杉碱甲对照组。hsp70mRNA电泳强度比较显示:假损伤组>地黄饮子5.4g/(kg·d)组>地黄饮子2.7g/(kg·d)组>抗脑衰对照组>石杉碱甲对照组>模型组。②海马神经元超微结构的观察:模型组海马神经元显现凋亡早期的形态特点,各治疗组和对照组对凋亡皆有抑制作用。结论:地黄饮子对痴呆鼠海马神经元细胞凋亡有抑制作用,且呈剂量依赖性,其作用机制可能与地黄饮子上调核转录因子κB、hsp70的表达,抑制线粒体释放细胞色素C,控制半胱氨酸天冬氨酸蛋白酶3的激活有关。更多还原

【Abstract】 AIM: To investigate effects of rehmanniae decoction on the apoptosis gene and ultrastrusture in the rat models of Alzheimer disease. METHODS: The experiment was carried out in the Basic Theory Laboratory, Heilongjiang University of Traditional Chinese Medicine between August 2003 and August 2004. Totally 120 Wistar rats were randomly divided into 6 groups with 20 rats in each group: sham injury group, model group, Kangnaoshuai control group, huperzine A control group, rehmanniae decoction 5.4 and 2.7 g/kg per day groups. In the model group, the rats were treated with intraperitoneal injection of D-galactose and hippocampal injection of amyloid β to induce models of Alzheimer disease. Rats in the sham injury group were treated with intraperitoneal injection of saline. At the same time of model establishment, rats in the rehmanniae decoction groups were treated with gastric perfusion of rehmanniae decoction containing crude drug of 5.4 and 2.7g/kg per day, and those in the Kangnaoshuai control group and huperzine A control group were given gastric perfusion of Kangnaoshuai capsule and huperzine A containing crude drugs of 2.403 and 0.018 mg/kg per day respectively, once a day for 5 weeks. The expression of apoptosis gene was detected with the reverse transcription-polymerase chain reaction (RT-PCR), and the ultrastructural changes of hippocampal neurons were observed under transmission electron microscope. RESULTS: Of the 120 animals, 20 died from model establishment including 5 in the model group, 6 in the huperzine A control group, 3 in the Kangnaoshuai control group, 3 in the rehmanniae decoction 5.4 g/kg per day group and 3 in the rehmanniae decoction 2.7 g/kg per day group respectively. Finally 200 rats entered the analysis of results. ① Expressions of caspase-3, nuclear factor-κB, hsp70 mRNA: The results of RT-PCR showed that amyloid β could induce the increase of the caspase-3 expression in hippocampus; Compared with the model group, the Kangnaoshuai control group, huperzine A control group, rehmanniae decoction 5.4 and 2.7 g/kg per day groups can decrease the expression in hippocampus of rats to different degree (P < 0.05), and it was down-regulated to the greatest extent in the rehmanniae decoction 5.4 g/kg per day group. Compared with the sham injury group, the levels of nuclear factor-κB and hsp70 gene mRNA in hippocampus were significantly decreased in the model group; Compared with the model group, the levels were obviously up-regulated in the Kangnaoshuai control group, huperzine A control group, rehmanniae decoction 5.4 and 2.7 g/kg per day groups (P < 0.05). Rehmanniae decoction 5.4 g/kg per day group had better effect in up-regulating the level of hsp70 gene mRNA than the Kangnaoshuai control group and huperzine A control group. The hsp70 mRNA electrophoresis intensity showed that: sham injury group > rehmanniae decoction 5.4 g/kg per day group > rehmanniae decoction 2.7 g/kg per day group > Kangnaoshuai control group > huperzine A control group > model group. ② Observation of the hippocampal neuron ultrastructure: In the model group, the hippocampal neurons showed the early morphological characters of apoptosis, the treatment groups and control groups all had inhibitory effects. CONCLUSION: The rehmanniae decoction has the inhibitory effects on apoptosis of hippocampal neurons in rats with Alzheimer disease in a dose-dependent manner, and its action mechanism may be associated with the effects of rehmanniae decoction in up-regulating the expressions of nuclear factor-κB and hsp70, restraining mitochondrial from releasing cytochrome C and controling the activation of caspase-3.更多还原