补肾益智方对老年性痴呆模型大鼠神经递质释放的影响(英文)

Effect of bushen yizhi formula on neurotransmitter release in rat models with Alzheimer disease

背景:胆碱乙酰基转移酶活性水平的降低和神经递质囊泡膜蛋白的减少是老年性痴呆的主要生化指标,中药补肾益智方具有补肾、益气、生髓、养脑作用,其含药血清能有效地保护由淀粉样蛋白引起的这些改变。目的:观察补肾益智方含药血清能否改善由淀粉样蛋白产物诱导神经细胞显示的老年性痴呆样病理损害,以此评价补肾益智方治疗老年性痴呆的作用。设计:随机对照实验。单位:广西中医学院第二附属医院神经科学研究所,广州中医药大学老年脑病研究所。材料:实验于2003-09/2004-03在广西中医学院新药研究开发中心完成。选取健康清洁级3月龄Wistar大鼠60只,随机分为空白对照组与中药灌胃组,各30只。补肾益智方主要由枸杞、蛇床子、人参、首乌、丹皮、冰片等组成,临用前配置为成药0.176g/mL(生药1.13g/mL)液体。方法:①中药灌胃组每只大鼠每天灌胃成药液1.0～1.5mL,连续灌胃1个月,于末次给药后4h内,5000r/min离心5min分离血清,无菌过滤,56℃灭活补体,即为含药血清。空白对照组给予等量生理盐水灌胃,同样方法提取分离血清。②对于NG108-15细胞,当淀粉样蛋白25～35片段在培养液浓度为5μmol/L或淀粉样蛋白1-42的浓度为200nmol/L时较为合适。③NG108-15细胞以Dulbecco改良的Eagle培养基在37℃,体积分数为0.1的CO2培养箱中培养1d,然后加入浓度为5μmol/L淀粉样蛋白25-35或200nmol/L淀粉样蛋白1-42片段,继续培养1d后,即为老年性痴呆细胞模型。以含50g/L中药灌胃组血清或空白对照组血清的Dulbecco改良的Eagle培养基更换原培养液,仍加入上述浓度的淀粉样蛋白片段以维持老年性痴呆细胞模型,分化培养5d后进行各项指标观察,并设立无淀粉样蛋白片段对照。④利用免疫蛋白印迹检查、免疫放射活性测定及电生理学测定方法观察补肾益智方含药血清处理NG108-15细胞老年性痴呆模型后的胆碱乙酰基转移酶活性、突触素蛋白水平及功能性突触形成率。主要观察指标:补肾益智方含药血清对胆碱乙酰基转移酶活性、突触素蛋白水平、NG108-15细胞功能性突触形成、NG108-15细胞微小终板电位频率的影响。结果:实验纳入大鼠60只,全部进入结果分析。①补肾益智方含药血清对胆碱乙酰基转移酶活性的影响:在无淀粉样蛋白或淀粉样蛋白1-42最终浓度200nmol/L时,中药灌胃组胆碱乙酰基转移酶活性均明显高于空白对照组[(1651.2±134.5),(1336.1±268.2);(1586.1±223.4),(1290.7±381.5)μmol/(g·h);P<0.05]。②补肾益智方含药血清对突触素蛋白水平的影响:中药灌胃组突触素Ⅰa和Ⅰb积分吸光度总值在无淀粉样蛋白、淀粉样蛋白25-35、淀粉样蛋白1-42时分别为5.02,1.36和2.46,空白对照组分别为3.18,0.57和0.71,中药灌胃组分别是空白对照组的1.6,2.4,3.5倍。③补肾益智方含药血清对NG108-15细胞功能性突触形成的影响:当无淀粉样蛋白或淀粉样蛋白1-42时,中药灌胃组功能性突触形成水平均高于空白对照组[(90.6±6.0)%,(63.2±17.0)%;(58.0±13.1)%,(34.2±13.0)%;P<0.05]。④补肾益智方血清对NG108-15细胞微小终板电位频率的影响:无淀粉样蛋白或淀粉样蛋白1-42时,中药灌胃组微小终板电位频率均高于空白对照组[(9.28±4.1),(5.48±5.14);(5.55±5.85),(3.05±4.46)个/min;P<0.01,P<0.05]。结论:补肾益智方剂含药血清在无淀粉样蛋白或淀粉样蛋白存在的情况下均能提高胆碱乙酰基转移酶的活性,不仅能保护由淀粉样蛋白引起的细胞突触素水平降低,在无淀粉样蛋白的情况下还可以提高突触素蛋白的表达,提高功能性突触的形成率和递质含量的释放。表明该方能拮抗老年性痴呆的病理发展,并通过提高神经递质的释放能力发挥对老年性痴呆的治疗作用。

BACKGROUND： The decrease of activity level of choline acetyltransferase （ChAT） and reduction of membrane protein of synaptic vesicles are the main biochemical indexes in Alzheimer disease. Traditional Chinese bushen yizhi formula is of the effects of strengthening the kidney, replenishing qi, generating marrow and nourishing the brain, its medicine-containing serum can effectively protect these changes resulting from amyloid protein.OBJECTIVE： To investigate whether the medicine-containing serum of bushen yizhi formula can improve the pathological injury of neurons induced by amyloid protein metabolites in Alzheimer disease, thus to estimate the therapeutic effect of bushen yizhi formula on Alzheimer disease.DESIGN：A randomized and controlled trial. SETTING：Institute of Neuroscience, Second Affiliated Hospital, Guangxi College of Traditional Chinese Medicine, Nanning 530011, Guangxi Zhuang Nationality Autonomous Region, China; Encephalopathic Institute of Guangzhou University of Traditional Chinese Medicine.MATERIALS：The experiment was conducted from September 2003 to March 2004 at the Research and Development Center of New Drugs of Guangxi College of TCM. Totally 60 healthy Wistar rats of clean grade,three months, were at random divided as blank control and bushen yizhi groups, with 30 in each group. The bushen yizhi formula consisted of Fructus Lycii, Fructus Cnidii, Radix Gingeng, Radix Polygoni Multiflori,Cortex Moutan Radicis, Borneolum, etc. The medicine was prepared before use as 0.176 g/Ml liquid（including 1.13 g/Ml rude drugs）.METHODS：① Each of the rats in bushen yizhi group was by garage given 1.0-1.5 Ml prepared liquid per day for consecutive 30 days. Within 4hours after the last administration the serum was isolated at 5 000 r/min for 5 minutes, filtered aseptically, the compliment was inactivated at 56 ℃,then the medicine-containing serum was obtained. The rats in blank control group were given saline of the same volume, and the serum was prepared with the same method. ② For NG108-15 cells, the concentration of amyloid protein 25-35 segments in the culture liquid was 5 μ mol/L, or 1-42 segments, 200 nmol/L, was suitable. ③ NG108-15 cells was first in cubated for a day in CO2 incubator with Dulbecco-improved Eagle medium of 0.1 molarity at 37 ℃, then 5 μmol/L amyloid protein 25-35 segment or 200 nmol/L segment 1-42 was added to continuously incubate for a day, after that the cell models of Alzheimer disease was ready. Then 50 g/L medicine-containing serum or Dulbecco-improved Eagle medium of blank control group serum was taken to replace the original culture liquid, and the amyloid protein segments of the above-mentioned concentration was still added for maintaining the cell models of Alzheimer disease, after 5 days＇differentiation incubation, the indexes were detected with those in non-amyloid protein as control. ④ Western blotting, radioimmunoassay and electrophysiological examination were used to investigate the ChAT activity,synapsin protein level and rate of functional synapse formation of NG108-15cells in Alzheimer disease after treatment with the medicine-containing serum of bushen yizhi formula.MAIN OUTCOME MEASURES ：The effects of the medicine-containing serum of bushen yizhi formula on ChAT activity, synapsin protein level,rate of functional synapse formation of NG108-15 cells and frequency of miniature plate potential in NG108-15 cells.RESULTS：Totally 60 rats involved all entered the final result analysis.① The effect of the medicine-containing serum of bushen yizhi formula on ChAT activity： In the conditions when there was no amyloid protein or the final concentration of amyloid protein 1- 42 was 200 nmol/L, the ChAT activity in bushen yizhi group was obviously higher than that in blank control group [（1651.2±134.5）, （1336.1±268.2）, （ 586.1±223.4）,（1290.7±381.5）μmol/（g·h）; P ＜ 0.05] . ② The effect of the medicinecontaining serum of bushen yizhi formula on synapsin protein level： The absorbance values of synapsin Ⅰa and Ⅰb, in the conditions when there was no amyloid protein, amyloid protein 25-35 and 1-42, were respectively 5.02, 1.36 and 2.46 in bushen yizhi group; and 3.18, 0.57 and 0.71 in blank control group. The values in bushen yizhi group were respectively 1.6,2.4 and 3.5times those in blank control group. ③ The effect of the medicine-containing serum of bushen yizhi formula on functional synapse formation of NG108-15 cells： In the conditions when there was no amyloid protein or amyloid protein 1-42, the levels of functional synapse formation in bushen yizhi group wereall higher than those in blank control group [（90.6±6.0）%, （63.2±17.0）%, （58.0±13.1）%, （34.2±13.0）% ;P＜0.05].④ The effect of the medicine-containing serum of bushen yizhi formula on frequency of miniature plate potential in NG108-15 cells： In the conditions when there was no amyloid protein or amyloid protein 1-42, the frequencies of miniature plate potential in bushen yizhi group were all higher than those in blank control group [（9.28±4.1）, （5.48 ±5.14）, （5.55±5.85）,（3.05±4.46）/min; P ＜ 0.01, P＜ 0.05].CONCLUSION：In the conditions when there existed no amyloid protein or amyloid protein, the medicine-containing serum of bushen yizhi formula can raise ChAT activity, not only correct the decrease of cellular synapsin caused by amyloid protein, but also raise the expression of synapsin protein, the rate of functional synapse formation and neurotransmitter release under the condition of no amyloid protein. It was shown that the prescription can antagonize the pathological development of Alzheimer disease, and play the therapeutic effect through enhancing the release power of neurotransmitter.