调心方中β淀粉样蛋白结合成分的初步探讨  

Preliminary assay of β-amyloid binding elements in heart-beneficial recipe

作　　者：程敏[1] 冯琼[1] 钱淑文[1] 高慧[1] 朱粹青[1] 

机构地区：[1]复旦大学上海医学院医学神经生物学国家重点实验室,上海200032

出　　处：《中西医结合学报》2008年第1期68-72,共5页Journal of Chinese Integrative Medicine

基　　金：国家自然科学基金资助项目(No39830450)

摘　　要：目的:探讨调心方中是否具有通过结合β淀粉样蛋白(β-amyloid protein,Aβ)而减少其神经元毒性的成分。方法:调心方提取物与Aβ共同沉淀并对沉淀物中的Aβ进行免疫印迹检测,制备小量Affi-gel-Aβ,用高效液相色谱(high-performance liquid chromatography,HPLC)检测调心方中Affi-gel-Aβ结合组分;大鼠原代皮层神经元培养,以乳酸脱氢酶(lactic dehydrogenase,LDH)测定法反映细胞损害程度。结果:免疫印迹检测显示Aβ能与调心方中的成分一起沉淀,且沉淀中的Aβ会随调心方的稀释而减少;调心方与Affi-gel-Aβ反应后,用甘氨酸液(pH2.5)洗脱的组分中有可被HPLC荧光检测器所检测的物质。LDH漏出检测结果显示,在无血清和含血清的培养体系中5μmol/LAβ1-42处理48h均可导致原代培养神经元损害,LDH漏出率显著增加(P<0.01),而大鼠调心方药物血清可抑制Aβ诱导的LDH漏出(P<0.05);经Affi-gel-Aβ吸附的调心方药物血清对Aβ毒性仍有抑制作用(P<0.05),但较经Affi-gel吸附的药物血清有减弱的趋势。结论:调心方中有Aβ结合成分,这类成分不促进Aβ的毒性,而可能具有抑制Aβ毒性的作用。提示在对老年性痴呆有效的中药方剂中探寻Aβ结合成分可能是探寻治疗AD药物的一个手段。Objective： To explore whether there are β-amyloid protein （Aβ） binding elements in heart-beneficial recipe （HBR, a compound traditional Chinese herbal medicine）, which can ameliorate the cytotoxicity of Aβ. Methods： The extract of HBR and Aβ1-40 were co-precipitated, and the Aβ1-10 in pellets was detected by immunoblotting. Affi-gel-Aβ1-40 was constructed, and Affi-gel-Aβ1-40 affinity elements from the extract of HBR were analyzed by high-performance liquid chromatography （HPLC）. The assay of lactic dehydrogenase （LDH） release from the primary cultured rat cortex neurons was used to evaluate the cytotoxicity of Aβ1-42, and the protection effects of the HBR serum and the Affi-geI-Aβ1-40 treated HBR serum. Results. Immunoblotting examination showed Aβ1-40 could be co-precipitated with components of HBR following co-incubation, and the amount of Aβ1-40 within pellets decreased when the HBR extract was diluted. Aβ1-40 affinity elements from the extract of HBR, eluted from Affi-gel-Aβ1-40 by glycine solution （pH=2.5）, could be detected by HPLC-fluorescent detector system. The analysis of LDH release showed that exposure of neurons to 5 μmol/L Aβ1-12 for 48 h caused a significant increase of LDH release in either a serum free or 10% serum contained culture condition （P〈0.01）. The rat HBR serum was able to suppress Aβ1-12 induced LDH release （P〈0.05）, whereas Affi-gel-Aβ1-40; treated HBR serum still maintained the ability to attenuate Aβ1-42 induced LDH release although the effect was somewhat decreased compared with Affi-gel treated HBR serum.Conclusion： There are Aβ affinity components in HBR, which could not increase the Aβcytotoxicity, but might be able to inhibit the cytotoxicity of Aβ. The results implied that the exploration of Aβ affinity elements from Chinese medicinal recipe which is effective for Alzheimer disease, might be an important direction in Alzheimer disease therapeutic research area.

关 键 词：Β淀粉样蛋白 调心方 乳酸脱氢酶 老年性痴呆 

分 类 号：R284[医药卫生—中药学]