【摘要】 背景:中药补肾益智方对阿尔茨海默病大鼠模型学习记忆能力有一定的保护作用,该方含药血清也能减轻神经瘤细胞NG108-15细胞对β淀粉样蛋白的神经毒性反应,为了进一步了解该方的作用机制及其配伍规律,需对该方进行拆方研究。目的:研究补肾益智方拆方亚组的含药血清对阿尔茨海默病细胞模型生长、分化等方面的影响,从血清药理学的角度探讨该方的配伍规律。设计:随机对照实验。单位:广州中医药大学临床药理研究所DME中心。对象:实验于2003-01/08在广州中医药大学临床药理研究所DME中心实验室进行。实验对象为3月龄SD雄性健康大鼠40只和冻存的NG108-15细胞株。方法:①制备药物血清:将40只SD大鼠随机分为对照组、原方组(蛇床子、枸杞子、人参、何首乌、丹皮、冰片)、补肾组(蛇床子、枸杞等)、益气养血组(人参、制首乌等)、去冰片组(蛇床子、枸杞子、人参、何首乌、丹皮),每组8只。将各组中药浓缩液按按10μL/g(相当于生药6g/kg)分别进行灌胃,连续1个月,对照组灌等量生量盐水。各组大鼠最后1次灌胃2h,麻醉后心脏采血,分离血清备用。②细胞增殖、培养及分化:将体外培养的NG108-15细胞分为6组,对照组、模型组孔中的增殖培养液含正常大鼠血清,其余4组分别为原方组及3个亚组的大鼠含药血清;同时每个孔加淀粉样β蛋白25～35至终浓度5umol/L(对照组除外),继续培养48h。主要观察指标:用MTT法测定细胞增殖数和存活率;同时检测分化突起细胞占总细胞数的比率及突起平均长度。结果:①细胞增殖情况:模型组A值显著低于对照组(0.520±0.022,0.665±0.037,P<0.01),各含药血清组A值均高于模型组,但以去冰片组效果最显著(0.636±0.035,P<0.01)。②分化细胞存活率:模型组显著低于对照组(58.4%,100%),各含药血清组均高于模型组,但以去冰片组效果最显著(75.8%,P<0.01)。③分化突起细胞占总细胞数的比率:模型组显著低于对照组犤(42.95±11.42)%,(58.75±12.84)%,P<0.01犦,各含药血清组均高于模型组,但以去冰片组效果最显著犤(58.20±8.40)%,P<0.01犦。④突起平均长度:模型组显著低于对照组犤(356.0±109.0),(493.8±133.0)μm,P<0.01犦,各含药血清组均高于模型组,但以去冰片组效果最显著犤(486.8±79.2)μm,P<0.01犦。结论:补肾益智方及各亚组含药血清都能在一定程度上抑制淀粉样β蛋白25-35对NG108-15细胞的损伤作用,但各组情况有所不同。各组含药血清对细胞的保护作用由强至弱依次是去冰片组>原方组>补肾组>益气养血组。故方中冰片的功效有待进一步研究。更多还原

【Abstract】 BACKGROUND:Chinese herb, bushen yizhi formula protects at certain extent learning and memory in rat model of Alzheimer disease. The drug serum in this formula can alleviate neurotoxic reaction of nerve tumor cell NG 108-15 to beta-amyloid protein. In order to understand further the mechanism and compatibility of the formula, it is necessary to carry on the study on the broken formulas.OBJECTIVE:To study the effect of drug serum in subgroups of broken bushen yizhi formulas on growth and differentiation of cell model of Alzheimer disease and probe into the compatibility rule of bushen yizhi formula in view of serum pharmacology.DESIGN: Randomized controlled experiment.SETTING: DME Center of Clinical Pharmacological Institute Affiliated to Guangzhou University of Traditional Chinese Medicine.PARTICIPANTS:The experiment was performed in DME Center of Clinical Pharmacological Institute Affiliated to Guangzhou University of Traditional Chinese Medicine from January to August 2003, in which, 40 healthy male SD rats of 3 months old were employed and NG108-15 cell line was frozen-preserved.METHODS:①Preparation of drug serum: Forty SD rats were randomized into the control, original formula group (No.1 group) [shechuanzi (Cnidium monnieri (L.) Cuss), gouqizi (Lycium barbarum L.), renshen (Panax ginseng C.A.Mey.), heshouwu (Polygonum multiflorum Thunb.), danpi (Paeonia Suf-fruticisa Andr.) and bingpian (Borneolum)], kidney replenishment group (No.2 group) [shechuanzi (Cnidium monnieri (L.) Cuss), gouqizi (Lycium barbarum L.), etc.], group for benefiting qi and nourishing blood (No.3 group) [renshen (Panaz ginseng C.A.Mey), zhishouwu (Polygonum multiflorum Thunb.), etc.] and group with bingpian (Borneolum) removed (No.4 group) [Panax ginseng C.A.Mey.], heshouwu (Polygonum multiflorum Thunb.) and danpi (Paeonia Suffruticisa Andr.)], 8 rats in each group. The concentrated Chinese herbal solutions of every group were applied at 10 μL/g (equal to 6 g/kg of raw herbs) for gastric infusion successively,continuously for 1 month. In the control, the physical saline solution of equal dosage was used for infusion. Two hours after the last gastric infusion in rats of each group, the blood was collected from heart after anesthesia and the serum was separated for preparation.②Cell proliferation, culture and differentiation: NG108-15 cell cultured in vitro was divided into 6 groups. In the control and model group, normal rat serum was contained in proliferated culture solution. In the rest 4 groups, the drug serum of No.1 group and 3 sub-groups was contained.Simultaneously, beta-amyloid protein 25-35 in each hole was prepared to the terminal concentration 5 μmol/L (except in the control) and the culture went on for 48 hours.MAIN OUTCOME MEASURES:MTT method was used to determine proliferated number and survival rate of cells. Simultaneously, the ratio of neurite cells to total cell count and average length of neurit were determined.RESULTS: ① Cell proliferation: A value in model group was lower significantly than the control (0.520±0.022, 0.665±0.037, P < 0.01), and that in every drug serum group was higher than model group, of which, the result in No. 4 group was the most significant (0.636±0.035, P < 0.01). ② Survival rate of differentiated cells: That in model group was lower significantly than the control (58.4%, 100%) and that in every drug serum group was higher than model group, of which, the result in No.4 group was the most significant (75.8%, P < 0.01). ③ Ratio of differentiated neurite cells to total cell count: That in model group was lower significantly than the control [(42.95±11.42)%, (58.75±12.84)%, P < 0.01] and that in every drug serum group was higher than model group, of which, the result in No.4 group was the most significant [(58.20±8.40)%, P < 0.01]. ④ Average length of neurite: That in model group was shorter significantly than the control [(356.0 ±109.0), (493.8±133.0) μm, P < 0.01] and that in every drug serum group was longer than model group, of which, the result in No.4 group was the most significant [(486.8±79.2) μm, P < 0.01].CONCLUSION: The drug serum in all of bushen yizhi formula and every subgroup inhibits at certain extent the injury of beta-amyloid protein 25-35 to NG108-15 cell, but the results of each group are various. The protection of drug serum to the cell in every group is in the sequence from strong to weak as group with bingpian removed > original formula group > kidney replenishment group > group for benefiting qi and nourishing blood. It is to expect a further study on the efficacy of group with bingpian removed.更多还原