柴胡總皂甙對戊四氮慢性點燃大鼠海馬γ

1、柴胡總皂甙對戊四氮慢性點燃大鼠海馬 【關(guān)鍵詞】 柴胡總皂甙；戊四氮；癲癇；摘要：目的 研究柴胡總皂甙對戊四氮(PTZ)慢性點燃癲癇模型大鼠海馬區(qū)氨基丁酸(GABA)表達(dá)的影響。方法 48只健康SD大鼠被隨機(jī)均分為6組，即空白對照組(A組)、生理鹽水組(B組)、丙戊酸鈉(VPA)組(C組)和柴胡總皂甙高、中、低三種劑量組(D組、E組、F組)。除A組不做處理外，其他各組采用腹腔注射PTZ慢性點燃造模，造模同時給予VPA、柴胡總皂甙等不同處理因素，連續(xù)4周后取腦組織切片進(jìn)行GABA免疫組化染色，從陽性細(xì)胞數(shù)、灰度值分析結(jié)果。結(jié)果 B組海馬CA1、CA2、DG區(qū)的陽性細(xì)胞數(shù)高于其他各組(P0.05)，

2、而各區(qū)陽性細(xì)胞灰度值低于其他各組(P0.05)。結(jié)論 柴胡總皂甙可以影響PTZ點燃大鼠海馬的GABA水平，發(fā)揮抑制PTZ對大鼠點燃的作用。關(guān)鍵詞：柴胡總皂甙；戊四氮；癲癇；氨基丁酸；大鼠Effect of saikosaponin on GABA of hippocampus of chronickindling induced by pentylenetetrazol in ratsABSTRACT: Objective To study the effect of active ingredients (saikosaponin) of bupleurum chinensis D C. o

3、n GABA of hippocampus of chronic kindling induced by pentylenetetrazol (PTZ) in rats. Methods Fortyeight healthy SpragueDawley rats were randomly divided into six groups with eight in each group: normal control (Group A), normal saline (NS) group(Group B), sodium valproate (VPA) group(Group C), grou

4、p of largedose saikosaponin (Group D), group of mediumdose saikosaponin (Group E), and group of smalldose saikosaponin (Group F). Except Group A to be controlled, the other groups were given different treatments when chronically kindled by PTZ. The process of the test lasted for 4 weeks. After 4 wee

5、ks administration the brain tissues were sampled, sliced and stained by enzyme immunohistochemistry(EIH). The results were analyzed according to the positive cell population and gray value. Results The positive cell population of CA1, CA2 and DG in group B was significantly different from that in th

6、e other groups(P0.05). The gray value of CA, CA2 and DG in group B was significantly different from that in the other groups (P0.05). Conclusion Saikosaponin can influence GABA of hippocampus of rats ,and inhibit chronic kindling effect induced by PTZ.KEY WORDS: saikosaponin; pentylenetetrazol; epil

7、epsy; GABA; rat人類癲癇及癇性疾病大約占人口的1%-5%，其中約20%的患者對現(xiàn)使用的藥物存在耐藥性1，給治療帶來很大難度，臨床需要新的抗癲癇藥物。本課題組前期提出柴胡總皂甙能有效對抗戊四氮(PTZ)慢性點燃致癇作用，影響致癇大鼠腦電活動，并能抑制大鼠海馬星形膠質(zhì)細(xì)胞的異常激活。在本研究中我們繼續(xù)探討柴胡總皂甙對慢性點燃大鼠海馬氨基丁酸(GABA)陽性細(xì)胞變化的影響，為柴胡總皂甙抗癲癇機(jī)制提供1.1 實驗動物及分組 SPF級SpagueDawley(SD)大鼠48只，體重(20020)g，雌雄各半,購自南方醫(yī)科大學(xué)實驗動物中心(動物合格證號：2004B023)，飼養(yǎng)于南方醫(yī)院動物

8、所。大鼠隨機(jī)分為6組，每組8只(雌雄各半)：A組為空白對照組，B組為生理鹽水組，C組為丙戊酸鈉(VPA)組，D、E、F組分別為柴胡總皂甙高、中、低劑量組。1.2 藥品、試劑與儀器 柴胡總皂甙粉購自上海醫(yī)藥工業(yè)研究院，批號050106； VPA為湖南湘中制藥有限公司生產(chǎn)，批號050123；PTZ為Sigma公司生產(chǎn)，批號 064K0686；兔抗GABA及SABC免疫組化染色試劑盒購自博士德公司；切片機(jī)為Leica公司產(chǎn)品。采用PLA6XX Camera kit圖像獲取系統(tǒng)及Image tool 3.0圖像分析軟件。1.3 方法1.3.1 藥液配制 應(yīng)用生理鹽水準(zhǔn)確配制PTZ溶液(32.50g/L

9、)及柴胡總皂甙高、中、低3種質(zhì)量濃度溶液(分別為15.60g/L、7.80g/L、3.90g/L)和VPA溶液(17.50g/L)，冷藏備用。40g/L多聚甲醛-0.1mol/L磷酸鹽緩沖液(pH 7.3)和PBS溶液臨用前配制。1.3.2 PTZ慢性點燃癲癇大鼠造模及處理 各組均選用灌胃法給予處理。A組與B組給予等容積生理鹽水；C組按87.5mg/kg給予VPA溶液；D組、E組和F組分別按照156、78、39mg/kg給予柴胡總皂甙溶液。每日上午8時左右給藥，給藥1h后，A組按10mL/kg生理鹽水腹腔注射，其他各組按32.5mg/kg腹腔注射PTZ溶液，每日1次，連續(xù)4周。腹腔注射PTZ溶

10、液后觀察記錄大鼠癇性發(fā)作級別。癇性發(fā)作分級采用Racine標(biāo)準(zhǔn)，即0級：無發(fā)作反應(yīng)；1級：節(jié)律性口角、耳或面部肌肉抽動陣攣；2級：點頭并伴隨更嚴(yán)重的面部肌肉抽動陣攣；3級：出現(xiàn)前肢陣攣但不伴隨直立；4級：前肢陣攣伴隨直立；5級：全身強(qiáng)直陣攣發(fā)作而跌倒。凡連續(xù)至少5次2級以上癇性發(fā)作大鼠被認(rèn)為慢性點燃致癇成功。1.3.3 GABA免疫組化染色 4周實驗結(jié)束時，按常規(guī)制備大鼠海馬石蠟切片，片厚3m。切片常規(guī)脫蠟、水化，3%(體積分?jǐn)?shù))H2O2去離子水孵育10min。熱修復(fù)后，滴加抗原修復(fù)液1(ARS1)，室溫10min，蒸餾水沖洗2min3；滴加5%(體積分?jǐn)?shù))BSA(bovine serum a

11、lbumin)封閉液，室溫10min，滴加一抗(兔抗GABA稀釋度為1100)4過夜。滴加生物素化山羊抗兔IgG，37 20min。上兩步之間用PBS沖洗，2min3次。滴加試劑SABC(鏈霉親和素生物素過氧化物酶復(fù)合物)，20-37 20min，PBS沖洗，5min4次；應(yīng)用新鮮配制DAB顯色，10min后水沖洗、復(fù)染、脫水、透明、封片。1.3.4 GABA陽性細(xì)胞計數(shù)及灰度值檢測 應(yīng)用OLYMPUS BH2型光學(xué)顯微鏡，高倍視野下(400)隨機(jī)觀察CA1、CA2、齒狀回(DG)區(qū)的GABA陽性細(xì)胞數(shù)(每個區(qū)選擇3個視野，計算均值)，運用PLA6XX Camera kit系統(tǒng)獲取圖片，并進(jìn)一

12、步用Image tool 3.0圖像分析軟件分析細(xì)胞平均灰度值(每個陽性表達(dá)細(xì)胞的平均灰度值分為256度，最黑者為0度，最亮者為255度)。1.4 統(tǒng)計學(xué)處理 數(shù)據(jù)采用s表示，應(yīng)用SPSS 10.0統(tǒng)計軟件。計量資料的比較用方差分析，以P0.05為差異有統(tǒng)計學(xué)意義。2 結(jié)果2.1 各組海馬CA1、CA2、DG區(qū)GABA陽性細(xì)胞數(shù)的比較 在CA1區(qū)，GABA陽性細(xì)胞數(shù)以B組最多，與其他5組比較有顯著性差異(P0.05或P0.01)。在CA2區(qū)和DG區(qū)，GABA陽性細(xì)胞數(shù)也以B組最多，除F組外其余各組與其比較均有顯著性差異(P0.05或P0.01，表1、圖1)。提示在大鼠海馬CA1、CA2、DG各

13、區(qū)，經(jīng)PTZ慢性點燃后GABA陽性細(xì)胞數(shù)量明顯增多，經(jīng)柴胡總皂甙和VPA處理后，大鼠海馬各區(qū)的GABA陽性細(xì)胞數(shù)量較模型鼠(生理鹽水組)明顯減少，接近于空白對照組，柴胡總皂甙高劑量組尤為明顯。表1 柴胡總皂甙對海馬CA1、CA2、DG區(qū)GABA陽性細(xì)胞數(shù)的影響（略）Table 1 The effect of saikosaponin on positive cells of GABA in CA1, CA2 and DG of hippocampusA: normal control group; B: normal saline group; C: sodium valproate grou

14、p; D: group of largedose saikosaponins; E: group of mediumdose saikosaponin; F:group of smalldose saikosaponin The cases less than eight were lost during the experiment of EEG*P0.05, *P0.01 vs. group B2.2 各組海馬CA1、CA2、DG區(qū)GABA陽性細(xì)胞平均灰度值的比較及圖片分析 在CA1和CA2區(qū)，GABA陽性細(xì)胞的灰度值以B組最低，與A、C、D、E組比較有顯著性差異(P0.05)。在DG區(qū)也

15、以B組灰度值最低，與A、C、D組比較有顯著性差異(P0.01，表2)。結(jié)合圖1，大鼠海馬GABA免疫反應(yīng)陽性細(xì)胞分布廣泛，細(xì)胞胞漿均勻染色，在各組均有表達(dá)。B組大鼠海馬GABA陽性細(xì)胞呈線狀排列，胞漿深染，說明B組陽性細(xì)胞染色深于其他各組，提示胞漿內(nèi)GABA含量增加。圖1 柴胡總皂甙對海馬區(qū)GABA陽性細(xì)胞表達(dá)的影響（略）Fig.1 The effect of saikosaponin on positive cells of GABA in hippocampus (400)A: group A (normal control group); B: group B (normal salin

16、e group); C: group C (sodium valproate group); D: group D (group of largedose saikosaponin); E:group E (group of mediumdose saikosaponin); F: group F (group of smalldose saikosaponin) 表2 柴胡總皂甙對海馬CA1、CA2、DG區(qū)GABA陽性細(xì)胞平均灰度值的影響（略）Table 2 The effect of saikosaponin on gray value of positive cells of GABA

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