調(diào)心方中β淀粉樣蛋白結(jié)合成分的初步探討

1、調(diào)心方中淀粉樣蛋白結(jié)合成分的初步探討                    作者：程敏, 馮瓊, 錢淑文, 高慧, 朱粹青【關(guān)鍵詞】  淀粉樣蛋白; 調(diào)心方; 乳酸脫氫酶; 老年性癡呆   Objective: To explore whether there are -amyloid protein (A) binding elements in heart-beneficial re

2、cipe (HBR, a compound traditional Chinese herbal medicine), which can ameliorate the cytotoxicity of A.   Methods: The extract of HBR and A1-40 were co-precipitated, and the A1-40 in pellets was detected by immunoblotting. Affi-gel-A1-40 was constructed, and Affi-gel-A1-40 affinity element

3、s 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 A1-42, and the protection effects of the HBR serum and the Affi-gel-A1-40 tr

4、eated HBR serum.   Results: Immunoblotting examination showed A1-40 could be co-precipitated with components of HBR following co-incubation, and the amount of A1-40 within pellets decreased when the HBR extract was diluted. A1-40 affinity elements from the extract of HBR, eluted from Affi-

5、gel-A1-40 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 mol/L A1-42 for 48 h caused a significant increase of LDH release in either a serum free or 10% serum contained culture condition (P0.01). T

6、he rat HBR serum was able to suppress A1-42 induced LDH release (P0.05), whereas Affi-gel-A1-40 treated HBR serum still maintained the ability to attenuate A1-42 induced LDH release although the effect was somewhat decreased compared with Affi-gel treated HBR serum.   Conclusion: There are

7、 A affinity components in HBR, which could not increase the A cytotoxicity, but might be able to inhibit the cytotoxicity of A. The results implied that the exploration of A affinity elements from Chinese medicinal recipe which is effective for Alzheimer disease, might be an important direction in A

8、lzheimer disease therapeutic research area.   Keywords: -amyloid protein; heart-beneficial recipe; lactic dehydrogenase; Alzheimer disease   老年性癡呆（A1zheimer disease, AD）是一種與增齡相關(guān)的神經(jīng)系統(tǒng)退行性疾病。隨著社會(huì)人口的老齡化，其發(fā)病率呈上升趨勢(shì)。淀粉樣蛋白（-amyloid protein, A）不僅是AD腦病理結(jié)構(gòu)之一，即老年斑的主要組分，而且A所介導(dǎo)的神經(jīng)毒性在AD腦病理改變中起非

9、常重要的作用，它可激活膠質(zhì)細(xì)胞分泌多種細(xì)胞因子，誘導(dǎo)與神經(jīng)纖維纏結(jié)相關(guān)的tau蛋白磷酸化，誘導(dǎo)神經(jīng)元凋亡等1。因此，目前抗A的神經(jīng)毒性作用已成為防治AD研究的重要方向之一2。調(diào)心方的臨床研究提示其有改善AD癡呆癥狀的作用3，4，而實(shí)驗(yàn)研究已經(jīng)顯示其可減少A誘導(dǎo)的神經(jīng)毒性5-7。本實(shí)驗(yàn)試圖探索調(diào)心方中是否有直接與A作用并抑制A神經(jīng)毒性的成分。   1  材料與方法   1.1  調(diào)心方提取物A結(jié)合分析   1.2  調(diào)心方抑制A對(duì)原代培養(yǎng)神經(jīng)元的毒性作用   1.3  統(tǒng)計(jì)學(xué)方

10、法  數(shù)據(jù)用Excel 2000的Students t檢驗(yàn)進(jìn)行各組間分析。P0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。   2  結(jié)  果   2.1  調(diào)心方共沉淀A1-40  調(diào)心方提取液中加入A，相互作用后，在離心沉淀中可檢出A。隨調(diào)心方的稀釋，沉淀減少；同樣，隨調(diào)心方的稀釋，可見(jiàn)沉淀的A量也減少。另外還可見(jiàn)調(diào)心方沉淀中A的電泳遷移并沒(méi)有明顯的改變。見(jiàn)圖1。   2.2  HPLC檢測(cè)Affi-gel-A1-40吸附的調(diào)心方成分  被Affi-gel-A1-40吸附的調(diào)心

11、方成分，如果不經(jīng)OPA柱前衍生處理，本實(shí)驗(yàn)的HPLC分離檢測(cè)系統(tǒng)則無(wú)法檢測(cè)到陽(yáng)性信號(hào)，見(jiàn)圖2A；而經(jīng)柱前衍生可檢測(cè)到一些陽(yáng)性信號(hào)，第1、3峰為特異峰，見(jiàn)圖2B和D；圖2C和E中的2號(hào)峰為洗脫液中的甘氨酸峰，而4號(hào)峰由于在對(duì)照Affi-gel吸附成分中也能被檢測(cè)到，因此認(rèn)為是非特異峰。   2.3  Affi-gel-A1-40吸附處理的調(diào)心方藥物血清對(duì)A1-42誘導(dǎo)的神經(jīng)元損害的影響  實(shí)驗(yàn)觀察了較低濃度的A1-42對(duì)原代培養(yǎng)神經(jīng)元的損傷作用。結(jié)果顯示，1 mol/L和5 mol/L A1-42在無(wú)血清環(huán)境中處理48 h后均可導(dǎo)致明顯神經(jīng)元損害，LDH漏

12、出率分別為（124.9±21.3）%，n=6和（148.7±19.7）%，n=6，較溶劑假損傷對(duì)照（100.0±10.7）%，n=6顯著增加（分別為P0.05和P0.01）。同樣在含10%正常大鼠血清的系統(tǒng)中，5 mol/L A1-42處理48 h后LDH漏出率（134.4±13.9）%，n=6也較溶劑假損傷對(duì)照（100.0±9.1）%，n=6明顯增加（P0.01）；而10%調(diào)心方大鼠血清可顯著減少A1-42誘導(dǎo)的LDH漏出率（110.6±14.2）%，n=6），差異有統(tǒng)計(jì)學(xué)意義（P0.05）。經(jīng)Affi-gel-A吸附處理的調(diào)心方藥

13、物血清能顯著減少A1-42誘導(dǎo)的LDH漏出率（118.2±10.4）%，n=6；P0.05；用作為對(duì)照的Affi-gel吸附處理的調(diào)心方藥物血清亦能顯著減少A1-42誘導(dǎo)的LDH漏出率（107.9±11.8）%，n=6；P0.01）。經(jīng)Affi-gel-A吸附處理的藥物血清的效果較Affi-gel吸附處理藥物血清效果有所下降，但兩者之間的差異無(wú)統(tǒng)計(jì)學(xué)意義。見(jiàn)圖3。   3  討  論   目前對(duì)AD的治療是研究熱點(diǎn)之一，研究發(fā)現(xiàn)許多中藥制劑可改善AD的學(xué)習(xí)記憶能力，如調(diào)心方和腦還丹等3，4，9。調(diào)心方以黨參補(bǔ)心氣、

14、安神明；以遠(yuǎn)志、石菖蒲、茯苓化痰濁、通心竅，用之于臨床，的確能有效改善AD患者認(rèn)知功能和生活能力3，4。以往的實(shí)驗(yàn)研究主要集中在明確調(diào)心方抗A對(duì)神經(jīng)系統(tǒng)損害的效果以及調(diào)心方調(diào)節(jié)神經(jīng)元凋亡相關(guān)基因表達(dá)5，7。但調(diào)心方中的某藥物成分是否有可能通過(guò)直接與A作用而抑制A毒性，目前尚未見(jiàn)報(bào)道。            本實(shí)驗(yàn)用的是新鮮配置的A，一般認(rèn)為是單體型。實(shí)驗(yàn)顯示，隨提取液的稀釋，A與調(diào)心方共沉淀也明顯減少；提示A單體沉淀與調(diào)心方提取液中的成分有關(guān)，而不是A的非特異沉降。沉淀中的A電泳遷移沒(méi)有明顯變慢

15、，提示與A結(jié)合的成分在變性條件下可游離下來(lái)。為進(jìn)一步驗(yàn)證調(diào)心方中有A結(jié)合成分，利用HPLC分析系統(tǒng)對(duì)此進(jìn)行了分析，結(jié)果同樣提示調(diào)心方中有與A結(jié)合的物質(zhì)存在，但尚不知道這些是什么物質(zhì)。根據(jù)本實(shí)驗(yàn)室HPLC分析系統(tǒng)檢測(cè)的是小分子物質(zhì)的原理，提示調(diào)心方中存在與A結(jié)合的小分子物質(zhì)；由于實(shí)驗(yàn)條件限制，不能排除調(diào)心方中有未能檢測(cè)到的能與A結(jié)合的大分子存在。另外，由于這種方法需要樣品的柱前衍生，其原理是基于OPA與巰基試劑在堿性條件下與伯胺類反應(yīng)產(chǎn)生異吲哚衍生物，因此我們推測(cè)本實(shí)驗(yàn)HPLC檢測(cè)觀察到的A結(jié)合組分為含伯胺基團(tuán)的小分子；同樣，如果有能與A結(jié)合而不含伯胺基團(tuán)的物質(zhì)，這種方法也檢測(cè)不到。 

16、  能與A結(jié)合的物質(zhì)并不一定對(duì)抗A神經(jīng)毒性，有些結(jié)合物可能反而促進(jìn)A的神經(jīng)毒性，例如ApoE4 10。為了解調(diào)心方中與A結(jié)合的成分對(duì)A神經(jīng)毒性的影響，我們用小劑量（1 mol/L和5 mol/L）單體A1-42對(duì)原代培養(yǎng)神經(jīng)元作用48 h的模型進(jìn)行了觀察，這種模型可能涉及A在培養(yǎng)體系中對(duì)細(xì)胞本身A代謝的影響以及A在培養(yǎng)體系中的聚集11。結(jié)果顯示調(diào)心方藥物血清在Affi-gel-A1-40吸附處理后，其抗A1-42毒性作用有下降趨勢(shì)，但仍有效。提示調(diào)心方中的A結(jié)合成分可能不會(huì)促進(jìn)A1-42的毒性；同時(shí)也反映調(diào)心方藥物血清中除有A結(jié)合成分外，可能還有其他調(diào)節(jié)神經(jīng)元抗損傷的成分。以后的研究

17、還需要進(jìn)一步處理藥物血清中干擾觀察指標(biāo)的成分，因?yàn)檎Ｑ宄煞种幸埠蠥結(jié)合成分，如甲狀腺激素結(jié)合蛋白（transthyretin）12。   給予外源性A結(jié)合物質(zhì)來(lái)抑制A的神經(jīng)毒性是目前治療AD研究中的一個(gè)重要領(lǐng)域 2, 10。例如，原本用于老年斑檢測(cè)的剛果紅也被用于抗A神經(jīng)毒性的研究2，而剛果紅其實(shí)也是從植物中提取的物質(zhì)。從對(duì)AD有效的中藥方劑中提取、鑒定A結(jié)合物質(zhì)，研究它們的神經(jīng)保護(hù)作用，將是一個(gè)值得關(guān)注的研究方向。【參考文獻(xiàn)】  1 Selkoe DJ. Alzheimers disease: a central role for amyloid. J N

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