三七总皂苷对阿尔茨海默病大鼠脑胆碱能神经的保护作用(英文)

Protective effects of panax notoginseng saponins on cholinergic neurons in rats with Alzheimer disease

背景:现代医学目前对阿尔茨海默病尚无有效的治疗方法,三七总皂苷治疗阿尔茨海默病已引起重视和关注,但其作用机制仍不十分清楚。目的:观察具有化瘀止血、活血定痛功效的三七总皂苷对阿尔茨海默病动物模型大脑胆碱能神经病理损害的保护作用。设计:完全随机分组设计,对照试验。单位:广西中医学院神经科学研究所。材料:实验于2003-06/2005-04在广西中医学院中药药效研究实验室完成,选用清洁级健康Wistar大鼠90只,雌雄各半。其中15月龄老年鼠75只,3月龄青年鼠15只。方法:实验于2003-06/2005-04在广西中医学院中药药效研究实验室(广西重点实验室)完成。①选用清洁级健康Wistar大鼠90只,雌雄各半。其中15月龄老年鼠75只,3月龄青年鼠15只。设3月龄15只为青年对照组。于15月龄75只中随机抽取15只作为老年对照组。对其余60只大鼠进行造模。以D-半乳糖腹腔注射致亚急性损伤合并鹅膏蕈氨酸损毁双侧大脑Meynert基底核建立阿尔茨海默病大鼠模型。青年对照组和老年对照组均在相同条件下以生理盐水作平行对照。②2周后将存活的造模大鼠随机分为4组:模型组,三七总皂苷高、低剂量组和石杉碱甲组,每组12只。三七总皂苷高、低剂量组分别按200和100mg/kg灌胃三七总皂苷(云南玉溪维和制药厂生产),1次/d;石杉碱甲组按0.3mg/kg灌胃给药,1次/d,连续4周;模型组、青年对照组和老年对照组以等体积的生理盐水灌胃,连续4周。③给药结束后行大脑组织病理切片,利用免疫组织化学方法检测大脑切片胆碱乙酰基转移酶免疫反应活性及阳性神经元数量及形态学改变。应用IBAS图像分析系统对胆碱乙酰转移酶免疫阳性神经元进行图像分析处理,测定胆碱乙酰转移酶免疫阳性细胞平均截面积和平均吸光度(A值);用显微镜测微尺测定计算胆碱乙酰转移酶阳性神经元数目。④采用单因素方差分析进行计量资料间差异比较。主要观察指标:三七总皂苷对阿尔茨海默病大脑胆碱能神经元分布和胆碱乙酰基转移酶的影响。结果:老年大鼠75只和青年大鼠15只均进入结果分析。①青年对照组胆碱乙酰转移酶免疫阳性神经元最多,免疫阳性物质染色最深;三七总皂苷高剂量组免疫阳性神经元明显多于石杉碱甲组和模型组;模型组免疫阳性神经元较其他各组的神经元小,且数目明显减少,胞体的轴突及树突变短。②基底前脑胆碱乙酰转移酶免疫阳性细胞数:模型组明显少于其他各组(P<0.05),三七总皂苷低剂量组、石杉碱甲组、模型组明显少于老年对照组(P<0.05),石杉碱甲组、模型组明显少于三七总皂苷高、低剂量组(P<0.05),青年对照组明显高于其他各组(P<0.05)。基底前脑胆碱乙酰转移酶免疫阳性细胞平均A值:变化与各组免疫阳性细胞数变化基本相同。基底前脑胆碱乙酰转移酶免疫阳性细胞平均截面积:三七总皂苷低剂量组和模型组明显小于青年对照组(P<0.05),其他各组间差异不明显(P>0.05)。结论:三七总皂苷对老年性痴呆大鼠动物模型大脑胆碱能神经元具有较强的保护作用,通过改善和修复受损神经元而提高细胞存活的数量和质量、提高胆碱乙酰转移酶的含量和活性,从而保护和改善中枢胆碱能系统的功能,发挥抗老化、抗痴呆的作用。

BACKGROUND： There are no effective methods to cure Alzheimer disease （AD）. Now, researches have shown that panax notoginseng saponins （PNS） play an important role in improving AD, but its mechanism is unclear. OBJECTIVE： To observe the protective effect of PNS characterized by removing blood stasis to stop bleeding and promoting blood circulation to relieve pain on pathological lesion of cholinergic neuron in rat with AD. DESIGN： Completely randomized grouping design and controlled study. SETTING： Neuroseience Institute of Guangxi Traditional Chinese Medical University. MATERIALS： This experiment was completed in the Chinese Herb Pharmacodynamic Laboratory of Guangxi Traditional Chinese Medical University between June 2003 and April 2005. A total of 90 health Wistar rats of clean grade and half gender were selected in this study. Among them, there were 75 old rats with 15 months old and 15 young rats with 3 months old. METHODS： This experiment was completed in the Chinese herb Pharmacodynamie Laboratory （Key Laboratory） of Guangxi Traditional Chinese Medical University between June 2003 and April 2005. ① A total of 90 healthy Wistar rats of clean grade and half gender were selected in this study. Among them, there were 75 old rats with 15 months old and 15 young rats with 3 months old. Fifteen young rats with 3 months old were regarded as young control group, and other 15 selected from 75 rats with 15 months old were regarded as old control group, The rest 60 rats were modeled on the basis of subacute injury induced by intravenous injection of D-galactose and bilateral cerebral Meynert basal nuclei injured by ibotenic acid. Parallel control was performed with saline on rats in young control group and old control group under the same condition. ② Two weeks later, survival modeling rats were divided randomly into 4 groups： model group, high-dosage PNS group, low-dosage PNS group and huperzine A group with 12 in each group. Rats in high-and low-dosage PNS groups were perfused with 200 and 100 mg/kg PNS （provided by Yunnan Yuxi Weihe Pharmaceutical Factory）, respectively, once a day; rats in huperzine A group were perfused with 0.3 mg/kg huperzine A once a day for 4 weeks; rats in mod- el group, young control group and old control group were perfused with the same volume of saline for 4 weeks. ③After administration, pathological sections of brain tissue were cut, and immunologic-reaction activity of choline acetyltransferase （CHAT）, morphological changes and numbers of positive neuron in cerebral sections were determined by immunohistochemistry analysis, ChAT immuno-positive neurons were analyzed with IBAS imaging analysis system to assay average area of section and average absorbance （A）, and amount of ChAT immuno-positive neurons was calculated with microscope micrometer. ④ Measurement data were compared with single-factor analysis of variance. MAIN OUTCOME MEASURESt Effect of PNS on distribution of cholinergie neuron and ChAT content in cerebral tissue of AD rat models. RESULTS： A total of 75 old rats and 15 young rats entered the final analysis. ① Amount of ChAT immuno-positive neurons was the most, and the color was the deepest in young control group; amount of ChAT immuno-positive neurons was higher in high-dosage PNS group than that in huperzine A group and model group; ChAT immuno-positive neurons were smaller in model group than those in other goups, and the amount was decreased obviously. Axis-cylinder and dendrite of soma were shortened remarkably. ② Amounts of ChAT immuno-positive neurons in basal forebrain were less in model group than those in other groups （P 〈 0.05）, less in lowdosage PNS group, huperzine A group and model group than those in old control group （P 〈 0.05）, less in huperzine A group and model group than those in high- and low-dosage PNS group （P 〈 0.05）, and less in young control group than those in other groups （P 〈 0.05）. The mean A value of ChAT immuno-positive neurons in basal forebrain was similar to amounts in each group. Average area of section of ChAT immuno-positive neurons in basal forebrain was smaller in low-dosage PNS group and model group than that in young control group （P 〈 0.05）, and differences in other groups were not significant （P 〉 0.05）. CONCLUSION： PNS plays a protective role in pathological lesion of cholinergic neuron in AD rat models. PNS can also increase survival amount and quality of cell and increase content and activity of ChAT so as to protect and improve central cholinergic system, and inhibit aging and dementia through improving and repairing injured cholinergic neurons.