屈小玲 王文清 梁向艷 鐵 茹 劉芳娥 李 榕 張海鋒，#

羅布麻葉提取物預(yù)處理對缺血再灌注大鼠心肌損傷的作用*

屈小玲1,2王文清3梁向艷1鐵 茹1劉芳娥1李 榕4張海鋒1，#

目的：觀察羅布麻葉提取物(AVLE)預(yù)處理對心肌缺血再灌注(MI/R)大鼠心肌細(xì)胞凋亡和細(xì)胞信號轉(zhuǎn)導(dǎo)系統(tǒng)中絲(蘇)氨酸激酶(Akt)和細(xì)胞外信號調(diào)節(jié)激酶(ERK1/2)的影響。方法：采用SD大鼠MI/R模型(結(jié)扎冠脈左前降支起始部30min后再灌注4h)，隨機(jī)分為Sham組(假手術(shù))、MI/R組、AVLE預(yù)處理組[AVLE (500mg/kg)灌胃，每日一次，連續(xù)灌胃7天后再行MI/R]。用TUNEL法檢測各組心肌細(xì)胞凋亡，計算凋亡指數(shù)(AI)；熒光免疫分析法檢測各組心肌組織凋亡蛋白Caspase-3活性；Western Blotting測定心肌組織Akt和ERK1/2的表達(dá)水平和磷酸化水平。結(jié)果：與Sham組比較，MI/R組心肌細(xì)胞AI顯著升高(P<0.05)，心肌組織Caspase-3活性明顯升高(P<0.05)，心肌Akt和ERK1/2的磷酸化水平顯著降低(P<0.05)；與MI/R組比較，AVLE預(yù)處理組AI明顯下降(P<0.05)，心肌組織Caspase-3活性顯著降低(P<0.05)，Akt和ERK1/2磷酸化水平顯著升高(P<0.05)。結(jié)論：AVLE能夠抑制缺血再灌注所致心肌細(xì)胞損傷，其作用與生存信號通路蛋白PI3K/Akt和ERK1/2的活化水平有關(guān)。

羅布麻葉提取物；心肌缺血再灌注損傷；絲(蘇)氨酸激酶；細(xì)胞外信號調(diào)節(jié)激酶；大鼠

1 材料與方法

1.1 實驗動物、藥物和主要試劑

健康6周齡清潔級雄性SD大鼠(體重200-220g)，由第四軍醫(yī)大學(xué)動物中心提供。自制AVLE提取物：取干燥羅布麻葉(由第四軍醫(yī)大學(xué)藥物研究所提供)100g浸泡于60ml 70%乙醇1h，連續(xù)兩次后，提取上層蒸發(fā)至干(28g)。取13.5g提取物溶解于200ml蒸餾水中，用硫酸調(diào)整pH值為3.0后，采用Diaion HP20層析柱(3.6cm i.d.×18cm;美國Sigma-Aldrich公司)過濾。濾過液用200ml超凈水和200ml 70%乙醇按常規(guī)方法洗脫。收集含水乙醇，蒸發(fā)至干，得到AVLE粉劑4.2g，用生理鹽水配制為5%溶液。心肌細(xì)胞凋亡檢測試劑盒購自德國Roche公司(批號：10770900)；Caspase-3試劑盒購自美國Abcam公司(批號：GR182122)；總Akt(tAkt)、磷酸化Akt(p-Akt)、總ERK1/2(tERK1/2)和磷酸化ERK1/2(p-ERK1/2)檢測試劑均購自美國Cell Signaling公司。

1.2 實驗分組處理和MI/R

54只SD大鼠適應(yīng)性飼養(yǎng)2周之后，用隨機(jī)數(shù)字表法分為：假手術(shù)組(Sham組)；MI/R組；AVLE預(yù)處理組；每組18只。根據(jù)文獻(xiàn)[10,11]，AVLE預(yù)處理組給予5%AVLE溶液(500mg/kg)每天灌胃1次，連續(xù)7天后行假手術(shù)或復(fù)制MI/R模型。另兩組給予等量蒸餾水灌胃，7天后行假手術(shù)處理或復(fù)制MI/R模型。

7天后大鼠禁食過夜，以2%戊巴比妥鈉(60mg/kg)腹膜內(nèi)注射麻醉，按參考文獻(xiàn)[12]復(fù)制MI/R模型：大鼠仰臥于實驗臺上，分離左側(cè)股靜脈并插管用于藥物注射，同步記錄肢體II導(dǎo)聯(lián)心電圖；緊貼胸骨左緣開胸，切斷左側(cè)2、3、4肋軟骨，在左心耳下緣與肺動脈圓錐間冠脈左前降支起始部，穿6-0號絲線(進(jìn)針深度約0.1cm，寬度為0.1—0.2cm，橡皮筋墊底)。除假手術(shù)組外，另兩組以雙線結(jié)扎冠脈左前降支，見心電圖S-T段明顯上抬、T波高聳、結(jié)扎線以下心肌顏色變暗和收縮減弱(為心肌缺血標(biāo)志)，30min后松開結(jié)扎線，恢復(fù)血流4h(再灌注)。Sham組只手術(shù)不結(jié)扎。行MI/R后大鼠使用斷頭法迅速處死，同時同法處死Sham組大鼠。

1.3 檢測指標(biāo)和方法

1.3.1 心肌細(xì)胞凋亡和Caspase-3活性檢測：(1)TUNEL法檢測心肌細(xì)胞的凋亡指數(shù)(AI)：迅速摘取大鼠心臟，置于冰鎮(zhèn)PBS中洗凈殘血，分離左心室前壁缺血區(qū)，于4%多聚甲醛中固定12h后常規(guī)方法石蠟包埋，連續(xù)切片(4μm)，脫蠟至水后，嚴(yán)格按照TUNEL試劑盒說明書進(jìn)行染色。每只大鼠各選2張切片于高倍鏡(×100)下選擇3個不重復(fù)視野，計數(shù)該視野內(nèi)的凋亡細(xì)胞(呈綠色熒光)和心肌細(xì)胞總數(shù)，以凋亡指數(shù)(Apoptotic Index，AI)反映各組心肌細(xì)胞凋亡情況。(2)熒光分析法檢測Caspase-3活性：根據(jù)文獻(xiàn)[13]，取心肌組織100g勻漿，離心后取上層樣本0.5ml加入Caspase-3檢測試劑盒，用DTT和2×反應(yīng)緩沖液的混合液(DTT 10μl、2×反應(yīng)緩沖液1ml)50μl，再加Caspase-3底物DEVD-AFC 5μl，37℃孵育60min，以激發(fā)光波長400nm，發(fā)射光波長505nm，測Caspase-3孵育前后熒光值，按試劑盒說明繪制標(biāo)準(zhǔn)曲線測得其斜率為133，并通過公式計算其活性，Caspase-3活性=(Δ熒光值/h)×1/133。

1.3.2 Western Blotting測定心肌組織中tAkt、tERK1/2、p-Akt和p-ERK1/2蛋白表達(dá)：取大鼠左心室前壁，按常規(guī)方法制備組織勻漿、BCA法蛋白定量，將樣品(含蛋白150μg)加樣于SDS-PAGE凝膠進(jìn)行電泳。轉(zhuǎn)膜、封閉后，分別加入抗P-Akt、抗tAkt、抗P-ERK1/2和抗tERK1/2多克隆抗體(均為1∶500)進(jìn)行反應(yīng)，再加二抗雜交，化學(xué)發(fā)光顯色。β-肌動蛋白(β-actin)作為內(nèi)參照。采用Image-Pro Plus(Version 4.1)軟件分析各種靶蛋白電泳條帶的積分吸光度值IA(=各條帶吸光度值×面積)，以靶蛋白IA與β-actin IA比值表示各種靶蛋白水平。

1.4 統(tǒng)計學(xué)處理

2 結(jié) 果

2.1 各組大鼠心肌細(xì)胞凋亡率和Caspase-3活性

2.1.1 各組心肌細(xì)胞凋亡率：與Sham組相比，MI/R組可見大量凋亡細(xì)胞；AVLE預(yù)處理組凋亡細(xì)胞較MI/R組明顯減少，見圖1。各組AI差異有統(tǒng)計學(xué)意義(F=9.005，P<0.05)，MI/R組較Sham組顯著增加(t=3.3，P<0.01)，ALVE預(yù)處理組較MI/R組顯著減少(t=2.1，P<0.05)，見圖2。

2.1.2 各組大鼠心肌組織Caspase-3活性：各組Caspase-3活性差異有統(tǒng)計學(xué)意義(F=11.024，P<0.05)。與Sham組相比，MI/R組顯著升高(t=4.2，P<0.01)；AVLE預(yù)處理組較MI/R組顯著降低(t=2.3，P<0.05)。見圖3。

2.2 各組Akt和ERK1/2及其磷酸化蛋白表達(dá)

各組tAkt和tERK1/2的表達(dá)差異無統(tǒng)計學(xué)意義(F值分別為2.35、1.58，P均>0.05)，而各組p-Akt和p-ERK1/2的表達(dá)差異均有統(tǒng)計學(xué)意義(F分別為11.205和13.069，P均<0.05)。與Sham組比較，MI/R組p-Akt和p-ERK1/2顯著增加(t分別為1.9和2.3，P均<0.05)。與MI/R組比較，AVLE預(yù)處理組p-Akt和p-ERK1/2明顯增加(t分別為2.1和1.7，P均<0.05)。見圖4。

注：與Sham組比較,*P<0.01; 與MI/R組比較,#P<0.05

注：與Sham組比較,*P<0.01; 與MI/R組比較,#P<0.05

[本文圖1見插1反面]

3 討 論

大量實驗證明，急性I/R促使心臟產(chǎn)生大量的ROS[14-16]，引起氧化應(yīng)激，是MI/R損傷和心肌細(xì)胞凋亡主要原因[3，17]。抑制氧化應(yīng)激可減少ROS產(chǎn)生，對MI/R損傷起保護(hù)作用。本文中MI/R大鼠心肌組織Caspase-3活性和AI顯著升高，而采用AVLE預(yù)處理大鼠心肌Caspase-3活性和AI明顯下降，與上述結(jié)論相一致。

Akt和ERK1/2被認(rèn)為是細(xì)胞存活的關(guān)鍵調(diào)節(jié)蛋白[18]，激活A(yù)kt和ERK1/2可減輕心臟的氧化應(yīng)激和抑制心肌細(xì)胞凋亡，防止心肌的缺血性損傷[3,4，17]。本文首次采用AVLE對Akt和ERK1/2活性進(jìn)行I/R前干預(yù)，結(jié)果發(fā)現(xiàn)，AVLE預(yù)處理能顯著提高I/R心肌組織Akt和ERK1/2的磷酸化水平。即AVLE可激活PI3K/Akt和MEK/ERK1/2信號途徑，使之通過調(diào)節(jié)抗凋亡和促凋亡蛋白以及轉(zhuǎn)錄因子活性，如觸發(fā)Akt Ser136磷酸化及ERK1/2 Ser112磷酸化滅活促凋亡Bcl-2家族成員BAD[19,20]等發(fā)揮心臟保護(hù)作用。

綜上，AVLE可激活A(yù)kt和ERK1/2信號途徑，抑制I/R心肌的氧化應(yīng)激和心肌細(xì)胞凋亡。對于PI3K/Akt和MEK/MRK1/2這兩種信號途徑在調(diào)節(jié)AVLE心臟保護(hù)作用的相互關(guān)系，尚待進(jìn)一步研究。

注：與Sham組比較,*P<0.05; 與MI/R組比較,#P<0.05

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本文第一作者簡介：

屈小玲(1973-)，女，漢族，主治醫(yī)師，主要從事心肌缺血再灌注方面的研究

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Effects of Apocynum Venetum Leaf Extract on Myocardial Ischemia Reperfusion Injury Rat

QU Xiao-ling1,2，WANG Wen-qing3，LIANG Xiang-yan1，TIE Ru1，LIU Fang-e1，LI Rong4，ZHANG Hai-feng1#

1Experiment Teaching Center；2Outpatient Department；3Department of Hematology,Tangdu Hospital；4Department of Senile Disease, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China;#

Objective: To investigate the effect of apocynum venetum leaf extract (AVLE) on myocardium ischemia reperfusion(MI/R) injury rat and signal pathway serine/threonine kinase (Akt) and the mitogen-activated protein kinase p42/44extra-cellular signa-l regulated kinases (ERK1/2).Method: Male Sprague-Dawley rats were divided into 3 groups randomly: sham, ischemia reperfusion, AVLE preconditioned[(500mg/kg/d, o.g.) once daily for one week]+MI/R. The model of myocardial I/R injury in vivo was made by ligating the left anterior descending artery for 30 min followed by 4 h of reperfusion in SD rats. Cardiomyocyte apoptosis was detected using in situ TDT-mediated dUTP nick end labeling (TUNEL). The activity of Caspase-3 was determined by fluorescent assay. The expressions and phosphorylations of Akt and ERK1/2 were measured using Western blot. Results: Compared with Sham group, the apoptosis index and the expressions of Caspase-3 were significantly increased and the phosphorylations of both Akt and ERK1/2 in I/R cardiac issue were significantly attenuated in MI/R group (P<0.05). Compared with I/R group, the apoptosis index and the expressions of Caspase-3 were significantly attenuated in AVLE preconditioned animals (P<0.05). AVLE preconditioning significantly increased the phosphorylations of both Akt and ERK1/2 in I/R cardiac issue (P<0.05).Conclusion: The results suggest that AVLE preconditioned may be able to protect the heart against reperfusion-induced injury mediated by the increase of phosphorylating Akt and ERK1/2.

Apocynum venetum leaf extract; Myocardial ischemia/reperfusion injury; Akt; ERK1/2; Rat

國家自然科學(xué)基金(81270330, 81470413, 81300190);陜西省科學(xué)技術(shù)研究發(fā)展計劃項目(2013KJXX-89)

第四軍醫(yī)大學(xué) ，西安710032；1教學(xué)實驗中心；2校直門診部；3唐都醫(yī)院血液科，西安 710038；4西京醫(yī)院老年病科，西安 710032；#

，E-mail: hfzhang@fmmu.edu.cn

本文2015-02-13收到，2015-04-12修回

R285.5

A

1005-1740(2015)02-0015-05