劉洛同,明揚(yáng),陳禮剛Δ,周杰,彭里磊,孫霞

(1.瀘州醫(yī)學(xué)院附屬醫(yī)院 神經(jīng)外科，四川 瀘州 646000；2.山東大學(xué) 分子藥理學(xué)系，山東 濟(jì)南 250100)

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當(dāng)歸多糖與川芎嗪不同配比對大鼠腦缺血再灌注損傷神經(jīng)可塑性的影響

劉洛同1,明揚(yáng)1,陳禮剛1Δ,周杰1,彭里磊1,孫霞2

(1.瀘州醫(yī)學(xué)院附屬醫(yī)院 神經(jīng)外科，四川 瀘州 646000；2.山東大學(xué) 分子藥理學(xué)系，山東 濟(jì)南 250100)

目的 研究低分子量當(dāng)歸多糖-川芎嗪(LMW-ASP)不同配比對大鼠腦缺血再灌注損傷后神經(jīng)可塑性的影響。方法 將42只實(shí)驗(yàn)大鼠隨機(jī)分為對照組(N)、假手術(shù)組(S)、模型組(M)、50:10組(mg/kg:mg/kg)(E1)、25:10組(E2)、50:20組(E3)、25:20組(E4)。N組不進(jìn)行任何手術(shù)處理，S組除不插線栓外，余同模型組，M、E1、E2、E3、E4組大鼠建立MCAO再灌注模型，其中N、S、M組不給藥治療，腹腔注射相同劑量的生理鹽水，E1-E4分別腹腔注射相應(yīng)比例的低分子量當(dāng)歸多糖和川芎嗪，于再灌注即刻開始注藥，1次/日，共7次。分別在再灌注后4 h、24 h、3 d、7 d進(jìn)行大鼠神經(jīng)功能缺損評分，并于第7 d多聚甲醛固定后斷頭取腦進(jìn)行免疫組織化學(xué)方法檢測腦梗死周圍皮質(zhì)MAP-2、SYP的表達(dá)。結(jié)果 免疫組織化學(xué)方法檢測腦梗死灶周圍皮質(zhì)MAP-2、SYP表達(dá)顯示，在第7d，建立腦缺血再灌注損傷模型的各組MAP-2、SYP的免疫活性較N組、S組強(qiáng)，藥物干預(yù)組MAP-2、SYP的免疫活性高于M組，且差異均具有統(tǒng)計(jì)學(xué)意義(P<0.05)；低分子量當(dāng)歸多糖-川芎嗪不同配比組間MAP-2的免疫活性差異有統(tǒng)計(jì)學(xué)意義，其活性分別為E3>E1>E4>E2；低分子量當(dāng)歸多糖-川芎嗪不同配比組間SYP的免疫活性差異均有統(tǒng)計(jì)學(xué)意義，其活性分別為E3>E1>E4>E2。其比例為50 mg/kg:20 mg/kg時(shí)最適宜。結(jié)論 大鼠腦缺血再灌注損傷后神經(jīng)功能部分可自行恢復(fù)，低分子量當(dāng)歸多糖-川芎嗪配伍干預(yù)后可促進(jìn)大鼠腦缺血再灌注損傷腦梗死灶周圍皮質(zhì)MAP-2、SYP的表達(dá)，恢復(fù)效果更顯著。

當(dāng)歸多糖；川芎嗪；大腦缺血再灌注模型

缺血性腦卒中是腦血管疾病發(fā)病率最高的一類疾病，其致殘率也居首位[1-3]。本課題以線栓法大腦中動(dòng)脈阻塞(middle cerebral artery occlusion, MCAO)再灌注大鼠為實(shí)驗(yàn)對象，通過觀察MCAO再灌注大鼠腦缺血后神經(jīng)功能損傷行為學(xué)變化、缺血腦組織周圍MAP-2和SYP的表達(dá)，探討低分子量當(dāng)歸多糖與川芎嗪不同配比對腦缺血再灌注損傷的神經(jīng)可塑性的影響，尋找2者最佳配比。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物及術(shù)前準(zhǔn)備 成年健康雄性Sprague-Dawley大鼠，SPF級，體質(zhì)量(220 ± 20)g，由上海大學(xué)動(dòng)物實(shí)驗(yàn)中心提供，合格證號4200500164。飼養(yǎng)于上海大學(xué)生物安全三級動(dòng)物實(shí)驗(yàn)室(animalBiosafety Level 3 Laboratory, ABSL-3)，飼養(yǎng)環(huán)境保持室溫22 ℃，濕度72%，12 h晝夜交替。術(shù)前大鼠環(huán)境適應(yīng)性喂養(yǎng)1周，自由進(jìn)食、飲水。術(shù)前禁食12 h，不禁水。本實(shí)驗(yàn)遵循《實(shí)驗(yàn)動(dòng)物保護(hù)條例》。線栓總長度40 mm，線頭直徑(0.32±0.02)mm，線身直徑0.24 mm，術(shù)前用肝素鈉溶液浸潤備用。

1.2 實(shí)驗(yàn)方法 將42只實(shí)驗(yàn)大鼠隨機(jī)分為對照組(N)、假手術(shù)組(S)、模型組(M)、50:10組(mg/kg:mg/kg)(E1)、25:10組(E2)、50:20組(E3)、25:20組(E4)。N組不進(jìn)行任何手術(shù)處理，S組除不插線栓外，余同模型組，M、E1、E2、E3、E4組大鼠建立MCAO再灌注模型，其中N、S、M組不給藥治療，腹腔注射相同劑量的生理鹽水，E1-E4分別腹腔注射相應(yīng)比例的低分子量當(dāng)歸多糖和川芎嗪，于再灌注即刻開始注藥，1次/天，共7次。分別在再灌注后4 h、24 h、3 d、7 d進(jìn)行大鼠神經(jīng)功能缺損評分，并于第7 d多聚甲醛固定后斷頭取腦進(jìn)行免疫組織化學(xué)方法檢測腦梗死周圍皮質(zhì)MAP-2、SYP的表達(dá)。

2 結(jié)果

2.1 大腦中動(dòng)脈缺血再灌注模型的大鼠的基本狀況 納入實(shí)驗(yàn)的SD大鼠共42只，制作MCAO再灌注模型的大鼠30只，整個(gè)實(shí)驗(yàn)中制作MCAO再灌注模型實(shí)際使用的SD 大鼠42 只，總體造模成功率71.4%，具體情況如下：M：9只，1只在手術(shù)后24 h內(nèi)死亡，1只第2 天死亡，1只在第4 天死亡；E1：8只，1只蘇醒神經(jīng)功能評分為0 分而剔除實(shí)驗(yàn)，1只在術(shù)后24 h內(nèi)死亡；E2：8只，1只在術(shù)中死亡，1只在第2天死亡；E3：9只，1只蘇醒后神經(jīng)功能評分為4 分剔除實(shí)驗(yàn)，1 只在術(shù)后24 h內(nèi)死亡，1只第7天死亡；E4：8只，1只術(shù)后拔線栓時(shí)大出血而死亡，1只術(shù)后24 h內(nèi)死亡；對照組，假手術(shù)組未出現(xiàn)發(fā)生死亡。

2.2 免疫組化測定腦梗死灶周圍皮質(zhì)MAP-2 表達(dá) 采用免疫組化SP 法檢測梗死灶周圍皮質(zhì)MAP-2表達(dá)。在大鼠腦缺血再灌注后第7 d，腦梗死灶周圍皮質(zhì)MAP-2表達(dá)的灰度值明顯低于正常組(N)和假手術(shù)組(S)，即MAP-2免疫活性較正常組和假手術(shù)組強(qiáng)，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；實(shí)驗(yàn)組E1-E4 MAP-2 表達(dá)的灰度值低于模型組M(209.54±1.62)，差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)；低分子量當(dāng)歸多糖-川芎嗪不同配比干預(yù)組中，實(shí)驗(yàn)組E3(200.64 ± 0.95)灰度值最低，MAP-2 的免疫活性最強(qiáng)，實(shí)驗(yàn)組E2(206.49±1.23)灰度值最高，MAP-2免疫活性最弱，MAP-2免疫活性強(qiáng)弱為E3(200.64±0.95)>E1(202.43±1.66)>E4(204.67±1.44)>E2(206.49±1.23)，見圖1。

圖1 各組大鼠在腦缺血再灌注后第7 d腦梗死灶周圍皮質(zhì)MAP-2的表達(dá)(×400)Fig.1 Expression of MAP-2 in cerebral infarction around rats cortex at 7th after cerebral ischemia reperfusion(×400)

2.3 免疫組化測定腦梗死灶周圍皮質(zhì)SYP 的表達(dá) 在大鼠腦缺血再灌注后第7d，建立MCAO 再灌注模型的大鼠腦梗死灶周圍皮質(zhì)SYP 表達(dá)的灰度值明顯低于正常組(N)和假手術(shù)組(S)，差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)；實(shí)驗(yàn)組E1-E4 SYP 表達(dá)的灰度值低于模型組M(208.41±1.77)，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)；低分子量當(dāng)歸多糖與川芎嗪不同配比干預(yù)組中，實(shí)驗(yàn)組E3(197.07±0.63)灰度值最低，SYP 的免疫活性最強(qiáng)，兩者成反比關(guān)系，實(shí)驗(yàn)組E2(206.29±0.98)灰度值最高，SYP 免疫活性最弱，在藥物干預(yù)組中SYP 免疫活性強(qiáng)弱為E3(197.07±0.63)>E1(199.12±1.31)>E4(203.93±1.74)>E2(206.29±0.98)，見圖2。

圖2 各組大鼠在腦缺血再灌注后第7d 腦梗死灶周圍皮質(zhì)SYP 的表達(dá)(×400)Fig.2 Expression of SYP in cerebral infarction around rats cortex at 7th after cerebral ischemia reperfusion(×400)

3 討論

本實(shí)驗(yàn)通過制備低分子量當(dāng)歸多糖和研究低分子量當(dāng)歸多糖-川芎嗪不同配比對大鼠腦缺血再灌注損傷神經(jīng)功能和腦梗死灶周圍皮質(zhì)MAP-2、SYP 表達(dá)水平的影響，可得到如下結(jié)論：

大鼠腦缺血再灌注損傷后神經(jīng)功能可自行恢復(fù)[4-8]，中藥低分子量當(dāng)歸多糖-川芎嗪配伍干預(yù)后恢復(fù)效果更顯著。低分子量當(dāng)歸多糖-川芎嗪不同配比可上調(diào)大鼠腦缺血再灌注損傷腦梗死灶周圍皮質(zhì)MAP-2、SYP 的表達(dá)[9]，促進(jìn)神經(jīng)的可塑性，比例為50 mg/kg:20 mg/kg 時(shí)效果最顯著。

本次研究對于進(jìn)一步探索低分子量當(dāng)歸多糖-川芎嗪配伍對大鼠腦缺血再灌注損傷的神經(jīng)保護(hù)作用的最佳比例具有重要意義。

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(編校：譚 玲)

Neuroplasticity effects of compatibility of angelica sinensis polysaccharide and tetramethylpyrazine on cerebral ischemia/reperfusion injury in rats

LIU Luo-tong1,MING Yang1,CHEN Li-gang1Δ,ZHOU Jie1,PENG Li-lei1,SUN Xia2

(1.Department of Neurosurgery, The Affiliated Hospital of Luzhou Medical College, Luzhou 646000, China; 2.Department of Molecular Pharmacology, Shandong University, Ji’nan 250100,China)

ObjectiveTo construct a middle cerebral artery occlusion(MCAO)/reperfusion rat model and study the effect of the different compatibility ratios of LMW-ASP and tetramethylpyrazine on the effects of neuroplasticity, and explore the best compatibility ratio between LMW-ASP and tetramethylpyrazine.MethodsIn the study, 42 experimental rats were divided into seven groups, they were normal group(N), sham operation group(S), model group(M),50:10 group(E1),25:10 group(E2),50:20 group(E3) and 25:20 group(E4).N group would not be carried out any operation, while S group was performed in the same way with M group but without inserting the intraluminal suture.MCAO reperfusion models were established in rats of the M,E1,E2,E3,E4 groups.The corresponding compatibility ratios of LMW-ASP and tetramethylpyrazine were injected to the E1-E4 groups by intraperitoneal injection in reperfusion instantly,once a day,a total of seven times.Neurological severity scores in rats were done in 4 h,24 h,3 d,7 d after reperfusion.In 7 d, The rats brains were removed after fixed using paraformaldehyde and then the immunohistochemistry method was used to detect the expression of MAP-2 and SYP in the cortex around cerebral infarction.ResultsIn 7 d, using the immunohistochemical methods detected the expression of MAP-2 and SYP in the cortex around cerebral infarction.The immune activity of MAP-2 and SYP in groups of cerebral ischemia/reperfusion injury model was stronger than the N group, S group and the difference was significant(P<0.05), and the immune activity of MAP-2 and SYP in the cortex around cerebral infarction in drug intervention groups were stronger than the M group and they had a significant difference(P<0.05); the immune activity of MAP-2 between the different compatibility ratios groups of LMW-ASP and tetramethylpyrazine was statistical difference(P<0.05), and the activity was E3>E1>E4>E2;the immune activity of SYP between the different compatibility ratios groups of LMW-ASP and tetramethylpyrazine was statistical difference(P<0.05), and the activity was E3>E1>E4>E2, and the compatibility ratio of 50 mg/kg:20 mg/kg was best effect. ConclusionsIn this study, we found that neurological function after cerebral ischemia/reperfusion injury can be partially self-recovery and the recovery effect after the drug intervention of the different compatibility ratios between the LMW-ASP and Tetramethylpyrazine is more pronounced.The compatibility of LMW-ASP and tetramethylpyrazine can promote the expression of MAP-2 and SYP in the cortex around cerebral infarction.

angelica sinensis polysaccharide; tetramethylpyrazine; middle cerebral artery occlusion/reperfusion

山東省醫(yī)藥衛(wèi)生科技發(fā)展計(jì)劃項(xiàng)目(2009QW003)

劉洛同，男，碩士，副主任醫(yī)師，研究方向：神經(jīng)外科，E-mail：qch18214600123@163.com；陳禮剛，通訊作者，男，博士，副主任醫(yī)師，研究方向：神經(jīng)外科，E-mail：qch18214600124@163.com。

R285.5

A

1005-1678(2015)03-0036-03