【作者】 江波；

【导师】 陈建国；

【作者基本信息】 华中科技大学 ， 药理学， 2009， 硕士

【摘要】 抑郁症是一组以显著而持久的心境障碍为主要特征的综合症,已影响到全球17%的人口,在世界致残性疾病中排行第四,对社会危害极大[1]。研究探讨抑郁症的发病机理和开发有效抗抑郁药物十分必要,但目前临床上应用的抗抑郁药多为化学合成类药物,虽有一定效果,但使用周期长,毒副作用较多[2]。因此,以传统中药为基础开发药效显著且无副反应的新型抗抑郁药,正日益受到国内外的强烈关注。三七皂苷Rg1是中国传统中药三七的重要活性成分之一。已有的研究表明,该单体可以增加动物海马中的脑源性营养因子含量,对抗脑缺血再灌注;具有抗疲劳和耐缺氧作用;能够增强动物的学习记忆功能,故推测三七皂苷Rg1可能具有抗抑郁作用。本课题选用慢性温和不可预测刺激动物模型,研究三七皂苷Rg1是否可以对抗抑郁,并通过放射性免疫测定、RT-PCR、HE染色及WESTERN-BLOT技术初步探讨其作用机制。第一部分三七皂苷Rg1的抗抑郁作用目的:评价三七皂苷Rg1的抗抑郁作用方法:采用昆明种雄性小鼠(3周大小,15 g左右)制作模型,共设七个组别。空白组给予生理盐水,模型组给予生理盐水,阳性对照组给予盐酸丙米嗪(15 mg/kg),三七皂苷Rg1(用生理盐水溶解)所用剂量分别为2.5 mg/kg、5 mg/kg、10 mg/kg和20 mg/kg,以腹腔注射途径给药,每组20只。构建为期8周的慢性中度应激模型,于最后2周开始每天分别按组别和体重给药(10 ml/kg),给药同时应激照样进行。8周结束之后进行行为学测试,包括悬尾测试(TST)、强迫游泳测试(FST)和敞箱测试,采用盲法,各组平行进行,测试均在上午进行。结果:FST结果显示,游泳绝望状态下模型组小鼠的不动时间明显高出正常组小鼠三倍以上,证明模型制造成功;与模型组相比,阳性丙米嗪组小鼠的不动时间明显降低,与正常组差不多;三七皂苷Rg1四个剂量组可以呈剂量依赖性的降低小鼠的不动时间,其中20 mg/Kg剂量组的效应与阳性组等同,具有统计学意义。TST结果显示,模型组小鼠在悬尾状态下的不动时间数值为正常组小鼠的两倍以上,表明模型制造成功;阳性丙米嗪能有效的将不动时间降至正常组水平;三七皂苷Rg1四个剂量组都有显著的改善作用,且具有统计学意义。敞箱测试结果显示,CMS部分降低了昆明小鼠的自主活动性,15 mg/Kg盐酸丙米嗪,10 mg/Kg和20 mg/Kg剂量的三七皂苷Rg1有轻微的改善作用,2.5 mg/Kg和5 mg/Kg剂量的三七皂苷Rg1作用不明显。结论:在2.5～20 mg/Kg剂量范围内,三七皂苷Rg1具有抗抑郁作用。第二部分三七皂苷Rg1抗抑郁作用的机制目的:探讨三七皂苷Rg1的抗抑郁作用机制方法:8周的CMS造模给药结束后,每组随机取10只小鼠处死,分离海马组织并储存于-80℃备用;每组随机取5只小鼠处死后取出全脑储存于-80℃备用,并断头取躯干血液,取上层血清,储存于-20℃备用;每组随机取3只小鼠麻醉,用4%多聚甲醛溶液(4℃)进行心脏灌注、固定,分离海马制作石蜡切片。使用DFM96型免疫γ计数器对CMS实验中各组小鼠的血清皮质酮含量进行测定;使用RT-PCR技术手段检测CMS实验中各组小鼠的海马组织中脑源性营养因子(BDNF)的表达;通过HE染色观察CMS和药物对各组小鼠海马神经元形态的影响;通过WESTERN-BLOT技术检测CMS实验中各组小鼠海马神经元内一系列与抑郁相关的蛋白表达变化。结果:三七皂苷Rg1和丙米嗪能显著逆转由CMS引起的动物海马神经元内BDNF mRNA水平的降低,20 mg/kg和10 mg/kg剂量尤为明显;在CMS作用下,模型组小鼠血清皮质酮浓度远高于正常组,三七皂苷Rg1和丙米嗪均能逆转它;HE染色结果表明海马确为三七皂苷Rg1的作用靶点,恢复CMS引起神经元形态改变;WESTERN-BLOT方法证实三七皂苷Rg1确实能够上调与抑郁症相关的一系列蛋白,如p-ERK激酶。结论:三七皂苷Rg1可减少血清中的皮质酮浓度,通过促进BDNF、p-ERK、等一系列促存活蛋白,增加海马神经元生成来产生抗抑郁作用。更多还原

【Abstract】 Depression is a group of syndromes characterized by notable and persistent mood disorders, which influences 17% of the population in the world. It is the fourth crippled disease, and has brought great harm to sufferers and society. So, it is necessary to investigate the definite pathogenesis and available drugs of depression. Recently, there are many antidepressants used in clinic, most of which are chemosynthesis drugs. Although these antidepressants have good effects, they also cause many toxic effects. Therefore, researching new antidepressants with greater effectiveness and without (or with lower) side effects and drug dependency from traditional herb has drawn people’s attention in China and even foreign countries.Notoginsenoside-Rg1 is one of the major bioactive ingredients of traditional Chinese medicine-Notoginseng. By now, many studies have shown that this component has many pharmacological effects, including increasing brain concentration of brain-derived neurotrophic factor of animal, effect against focal cerebral ischemia reperfusion, effect against fatigue and hypoxia, strengthening learning and memory of animal, and so on. So we presume that notoginsenoside-Rg1 may exert antidepressant-like effects. This study consists of two parts. Background: To evaluate the antidepressant-like effect of Notoginsenoside-Rg1.Methods: Adult male Kunming mice(3 weeks, 18～22 g) were used for chronic mild stress (CMS) model, they were randomized into 7 groups with 20 mice per group. Animals in control and model group were administrated with 0.9% normal saline, imipramine hydrochloride was used as positive control drug and four doses of notoginsenoside-Rg1 (2.5, 5, 10 and 20 mg/kg) were used in the experiment, all drugs were solved in 0.9% normal saline and administrated by intraperitoneal (i.p) injection with a volume of 10ml/kg. The CMS procedure lasted for 8 weeks and repeated drug treatment of CMS mice was performed once daily during the last two weeks. Twenty-four hours after the last stressor and drug exposure, the open field test、tail suspension test (TST) and forced swim test (FST) were performed in the morning.Results: The results of the tail suspension test and the forced swim test showed that the CMS model was very successful, the immobility time of the stressed group was 2-3 times higher than the control group. The positive control treatment with imipramine (15 mg/kg) decreased the immobility time in the TST and FST tests of mice significantly to the level of vehicle-treated group. Notoginsenoside-Rg1 (2.5, 5, 10 and 20 mg/kg) also had the similar effect, decreased the immobility time statistically in a dose-dependent manner (P<0.01, respectively). Locomotion of mice were slightly decreased by CMS compared with un-stressed animals, Imipramine and Notoginsenoside-Rg1 at dose of either 20 mg/kg or 10 mg/kg could reverse it, while neither 5 mg/kg nor 2.5 mg/kg of this drug had the same effect.Conclusion: Notoginsenoside-Rg1 had antidepressant effect on mice’behavior between 2.5 to 20 mg/kg. Background: To explore the possible antidepressant mechanisms of notoginsenoside-Rg1. Methods: After 8 weeks of CMS, ten mice in each group were decapitated, hippocampus were separated out and stored at -80℃for use;five animals in each group were decapitated and serum were separated and stored at -20℃, brains were carefully dissected and stored at -80℃for use; the other 3 mice were perfused and the brains were fixed by 4% paraformaldehyde solution at 4℃. Radioimmunoassay (RIA) was used to assay the serum corticosterone level; reverse transcription-polymerase chain reaction (RT-PCR) were applied to observe the mRNA expression of BDNF in hippocampus of mice after CMS; HE stain was applied to observe the morphology of hippocampal neurons; western blotting technology was also used to observe the effect of notoginsenoside-Rg1 on several proteins related to depression.Results: CMS induced the decreasing of mRNA expression of BDNF, Notoginsen- oside-Rg1 significantly reversed BDNF level, especially at doses of 20 and 10mg/kg, similar to Imipramine; The serum corticosterone level was increased in CMS Kunming mice, Notoginsenoside-Rg1, as well as Imipramine, reduced the augmentation; The result of HE stain indicated that notoginsenoside-Rg1 was able to reverse the change of morphology of hippocampal neurons induced by CMS; We also proved that this herb could increase the expression of some proteins related to depression, such as p-ERK, by western-blotting method.Conclusion: Notoginsenoside-Rg1 possessed a certain antidepressant-like effect via down-regulating of serum corticosterone level. In addition, this herb can promote neurogenesis by increasing the expression of neurotrophic factor and p-ERK.更多还原