清心开窍方抑制Aβ1-42纤维聚集的实验研究  

Experimental Study of Qingxin Kaiqiao Fang Inhibiting Aβ1-42 Fiber Aggregation

作　　者：胡海燕[1,2] 张益慧[2] 陈志裕[2] 席东泽[2] 王逸如[1,2] 徐冬梅[2] 王文花[1,2] 陈翔[2] 

机构地区：[1]温州医科大学第二临床学院,浙江温州325027 [2]温州医科大学附属第二医院育婴儿童医院,浙江温州325027

出　　处：《中华中医药学刊》2015年第7期1557-1560,I0004,I0005,共6页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(30973780);浙江省自然科学基金项目(Y2080273);温州市科技局项目(Y20080106)

摘　　要：目的:探讨清心开窍方抗Aβ1-42纤维聚集的机制。方法:实验分组:对照组、老化组单纯用Aβ1-42液,不同剂量清心开窍方组同时加入清心开窍方汤剂,老化组及不同剂量清心开窍方组均37℃恒温箱孵育7d,对照组Aβ1-42单独孵30 min,利用硫磺素T(Th-T)荧光分析法、透射电镜及扫描电镜观测Aβ1-42的聚集和纤维化程度。制备小量Affigel-Aβ,用高效液相色谱检测清心开窍方中与Affi-gel-Aβ结合的成分。结果:Th-T荧光分析示清心开窍方低、中、高剂量组对Aβ聚集与纤维形成的抑制率为分别为22.58%、75.51%、83.66%,组间比较P<0.01。透射电镜观察显示对照组Aβ1-42主要以无定型结构存在,聚集少或无明显聚集,电子密度较低,未见纤维结构形成;老化组Aβ1-42以淀粉样的纤维结构为主,交织成网状,呈针状或晶状聚集,无分枝,直径约10 nm;高、中剂量清心开窍方组Aβ1-42主要表现为无定型结构,未见明显Aβ纤维和Aβ聚集沉积,而低剂量清心开窍方组Aβ1-42主要表现为纤维型和无定型混合结构。扫描电镜观察显示对照组Aβ呈无定型,老化组纤维性结构明显增多,纤维粗,排列不规则,纵横交错,聚集形成片状结构,而清心开窍方与Aβ1-42共同孵育组,随清心开窍方浓度增加,纤维型明显减少,形成纤维束的直径逐渐变细。高效液相色谱(highperformance liquid chromatography,HPLC)检测显示:清心开窍方与Affigel-Aβ反应后,经甘氨酸(p H 2.5)洗脱的组分中存在能够被HPLC荧光检测仪检测到的物质。结论:清心开窍方具有抑制Aβ聚集和纤维形成的作用,且其中含有与Aβ结合的成分,这些成分不会促进Aβ的毒性,而可能具有抑制Aβ毒性的作用,提示在对于AD有效的中药方剂中探索与Aβ结合的成分,可能是探索AD治疗药物的一种手段。Objective：To study the mechanism of Qingxin Kaiqiao prescription anti Aβ1 -42 fiber aggregation. Meth- ods：Experimental groups ： Control group, aging group with pure Aβ1 -42 liquid,while the different dose groups join the corresponding dose of Qingxin Kaiqiao recipe medicinal broth at the same time, aging and the different dose groups of Qingxin Kaiqiao recipe are incubated at 37 ℃ for 7 days,while the control group Aβ1 -42 separately hatched 30 min. U- sing Thioflavine T fluorometric （ Th - T） fluorescence analysis methods, electronic microscopy negatively staining and scanning electron microscope observes the degree of Aβ1 -42 gathered and fibrosis formation. Preparation little dose of Affigel- Aβ ,with high -performance liquid chromatography （HPLC） detecting the composition combined with Affigel - Aβ. Results：The results of Thioflavine T fiuorometric fluorescence analysis methods show the inhibition rate of the low, me- dium and high dose groups of Qingxin Kaiqiao recipe for the degree of AIM -42 fibrosis is 22.58% ,75.51% and 83.66% respectively,P 〈0.01. The results of electronic microscopy negatively staining show that： the control group of AI31 -42 mainly exists in the amorphous structure,little or no obvious aggregation, electron density is low, no fiber struc- ture formation;the aging group of AIM -42 mainly exists in amyloid fiber structure, crisscross, a needle or crystalline gath- ered, unbranched, diameter about 10 nm;the high and medium dose groups of Qingxin Kaiqiao recipe groups of Aβ1 -42 mainly exist in the amorphous structure, there is no clearly Aβ fiber and aggregation deposit, while low dose of Qingxin Kaiqiao recipe group of Aβ1 - 42 mainly exists in the fiber type and amorphous composite structures. Scanning electron microscope shows that Aβ is amorphous in the control group, fibrous structure is obviously increased in the aging group, coarse fiber, irregular arrangement, crisscross, gathered to form lamellar structure,while the Aβ is incubated with Qingxin

关 键 词：清心开窍方 Aβ1-42聚集 阿尔茨海默病 

分 类 号：R285.5[医药卫生—中药学]