罗布麻叶提取物预处理对缺血再灌注大鼠心肌损伤的作用*

屈小玲 王文清 梁向艳 铁 茹 刘芳娥 李 榕 张海锋，#

罗布麻叶提取物预处理对缺血再灌注大鼠心肌损伤的作用*

屈小玲1,2王文清3梁向艳1铁 茹1刘芳娥1李 榕4张海锋1，#

目的：观察罗布麻叶提取物(AVLE)预处理对心肌缺血再灌注(MI/R)大鼠心肌细胞凋亡和细胞信号转导系统中丝(苏)氨酸激酶(Akt)和细胞外信号调节激酶(ERK1/2)的影响。方法：采用SD大鼠MI/R模型(结扎冠脉左前降支起始部30min后再灌注4h)，随机分为Sham组(假手术)、MI/R组、AVLE预处理组[AVLE (500mg/kg)灌胃，每日一次，连续灌胃7天后再行MI/R]。用TUNEL法检测各组心肌细胞凋亡，计算凋亡指数(AI)；荧光免疫分析法检测各组心肌组织凋亡蛋白Caspase-3活性；Western Blotting测定心肌组织Akt和ERK1/2的表达水平和磷酸化水平。结果：与Sham组比较，MI/R组心肌细胞AI显著升高(P<0.05)，心肌组织Caspase-3活性明显升高(P<0.05)，心肌Akt和ERK1/2的磷酸化水平显著降低(P<0.05)；与MI/R组比较，AVLE预处理组AI明显下降(P<0.05)，心肌组织Caspase-3活性显著降低(P<0.05)，Akt和ERK1/2磷酸化水平显著升高(P<0.05)。结论：AVLE能够抑制缺血再灌注所致心肌细胞损伤，其作用与生存信号通路蛋白PI3K/Akt和ERK1/2的活化水平有关。

罗布麻叶提取物；心肌缺血再灌注损伤；丝(苏)氨酸激酶；细胞外信号调节激酶；大鼠

1 材料与方法

1.1 实验动物、药物和主要试剂

健康6周龄清洁级雄性SD大鼠(体重200-220g)，由第四军医大学动物中心提供。自制AVLE提取物：取干燥罗布麻叶(由第四军医大学药物研究所提供)100g浸泡于60ml 70%乙醇1h，连续两次后，提取上层蒸发至干(28g)。取13.5g提取物溶解于200ml蒸馏水中，用硫酸调整pH值为3.0后，采用Diaion HP20层析柱(3.6cm i.d.×18cm;美国Sigma-Aldrich公司)过滤。滤过液用200ml超净水和200ml 70%乙醇按常规方法洗脱。收集含水乙醇，蒸发至干，得到AVLE粉剂4.2g，用生理盐水配制为5%溶液。心肌细胞凋亡检测试剂盒购自德国Roche公司(批号：10770900)；Caspase-3试剂盒购自美国Abcam公司(批号：GR182122)；总Akt(tAkt)、磷酸化Akt(p-Akt)、总ERK1/2(tERK1/2)和磷酸化ERK1/2(p-ERK1/2)检测试剂均购自美国Cell Signaling公司。

1.2 实验分组处理和MI/R

54只SD大鼠适应性饲养2周之后，用随机数字表法分为：假手术组(Sham组)；MI/R组；AVLE预处理组；每组18只。根据文献[10,11]，AVLE预处理组给予5%AVLE溶液(500mg/kg)每天灌胃1次，连续7天后行假手术或复制MI/R模型。另两组给予等量蒸馏水灌胃，7天后行假手术处理或复制MI/R模型。

7天后大鼠禁食过夜，以2%戊巴比妥钠(60mg/kg)腹膜内注射麻醉，按参考文献[12]复制MI/R模型：大鼠仰卧于实验台上，分离左侧股静脉并插管用于药物注射，同步记录肢体II导联心电图；紧贴胸骨左缘开胸，切断左侧2、3、4肋软骨，在左心耳下缘与肺动脉圆锥间冠脉左前降支起始部，穿6-0号丝线(进针深度约0.1cm，宽度为0.1—0.2cm，橡皮筋垫底)。除假手术组外，另两组以双线结扎冠脉左前降支，见心电图S-T段明显上抬、T波高耸、结扎线以下心肌颜色变暗和收缩减弱(为心肌缺血标志)，30min后松开结扎线，恢复血流4h(再灌注)。Sham组只手术不结扎。行MI/R后大鼠使用断头法迅速处死，同时同法处死Sham组大鼠。

1.3 检测指标和方法

1.3.1 心肌细胞凋亡和Caspase-3活性检测：(1)TUNEL法检测心肌细胞的凋亡指数(AI)：迅速摘取大鼠心脏，置于冰镇PBS中洗净残血，分离左心室前壁缺血区，于4%多聚甲醛中固定12h后常规方法石蜡包埋，连续切片(4μm)，脱蜡至水后，严格按照TUNEL试剂盒说明书进行染色。每只大鼠各选2张切片于高倍镜(×100)下选择3个不重复视野，计数该视野内的凋亡细胞(呈绿色荧光)和心肌细胞总数，以凋亡指数(Apoptotic Index，AI)反映各组心肌细胞凋亡情况。(2)荧光分析法检测Caspase-3活性：根据文献[13]，取心肌组织100g匀浆，离心后取上层样本0.5ml加入Caspase-3检测试剂盒，用DTT和2×反应缓冲液的混合液(DTT 10μl、2×反应缓冲液1ml)50μl，再加Caspase-3底物DEVD-AFC 5μl，37℃孵育60min，以激发光波长400nm，发射光波长505nm，测Caspase-3孵育前后荧光值，按试剂盒说明绘制标准曲线测得其斜率为133，并通过公式计算其活性，Caspase-3活性=(Δ荧光值/h)×1/133。

1.3.2 Western Blotting测定心肌组织中tAkt、tERK1/2、p-Akt和p-ERK1/2蛋白表达：取大鼠左心室前壁，按常规方法制备组织匀浆、BCA法蛋白定量，将样品(含蛋白150μg)加样于SDS-PAGE凝胶进行电泳。转膜、封闭后，分别加入抗P-Akt、抗tAkt、抗P-ERK1/2和抗tERK1/2多克隆抗体(均为1∶500)进行反应，再加二抗杂交，化学发光显色。β-肌动蛋白(β-actin)作为内参照。采用Image-Pro Plus(Version 4.1)软件分析各种靶蛋白电泳条带的积分吸光度值IA(=各条带吸光度值×面积)，以靶蛋白IA与β-actin IA比值表示各种靶蛋白水平。

1.4 统计学处理

2 结 果

2.1 各组大鼠心肌细胞凋亡率和Caspase-3活性

2.1.1 各组心肌细胞凋亡率：与Sham组相比，MI/R组可见大量凋亡细胞；AVLE预处理组凋亡细胞较MI/R组明显减少，见图1。各组AI差异有统计学意义(F=9.005，P<0.05)，MI/R组较Sham组显著增加(t=3.3，P<0.01)，ALVE预处理组较MI/R组显著减少(t=2.1，P<0.05)，见图2。

2.1.2 各组大鼠心肌组织Caspase-3活性：各组Caspase-3活性差异有统计学意义(F=11.024，P<0.05)。与Sham组相比，MI/R组显著升高(t=4.2，P<0.01)；AVLE预处理组较MI/R组显著降低(t=2.3，P<0.05)。见图3。

2.2 各组Akt和ERK1/2及其磷酸化蛋白表达

各组tAkt和tERK1/2的表达差异无统计学意义(F值分别为2.35、1.58，P均>0.05)，而各组p-Akt和p-ERK1/2的表达差异均有统计学意义(F分别为11.205和13.069，P均<0.05)。与Sham组比较，MI/R组p-Akt和p-ERK1/2显著增加(t分别为1.9和2.3，P均<0.05)。与MI/R组比较，AVLE预处理组p-Akt和p-ERK1/2明显增加(t分别为2.1和1.7，P均<0.05)。见图4。

注：与Sham组比较,*P<0.01; 与MI/R组比较,#P<0.05

注：与Sham组比较,*P<0.01; 与MI/R组比较,#P<0.05

[本文图1见插1反面]

3 讨 论

大量实验证明，急性I/R促使心脏产生大量的ROS[14-16]，引起氧化应激，是MI/R损伤和心肌细胞凋亡主要原因[3，17]。抑制氧化应激可减少ROS产生，对MI/R损伤起保护作用。本文中MI/R大鼠心肌组织Caspase-3活性和AI显著升高，而采用AVLE预处理大鼠心肌Caspase-3活性和AI明显下降，与上述结论相一致。

Akt和ERK1/2被认为是细胞存活的关键调节蛋白[18]，激活Akt和ERK1/2可减轻心脏的氧化应激和抑制心肌细胞凋亡，防止心肌的缺血性损伤[3,4，17]。本文首次采用AVLE对Akt和ERK1/2活性进行I/R前干预，结果发现，AVLE预处理能显著提高I/R心肌组织Akt和ERK1/2的磷酸化水平。即AVLE可激活PI3K/Akt和MEK/ERK1/2信号途径，使之通过调节抗凋亡和促凋亡蛋白以及转录因子活性，如触发Akt Ser136磷酸化及ERK1/2 Ser112磷酸化灭活促凋亡Bcl-2家族成员BAD[19,20]等发挥心脏保护作用。

综上，AVLE可激活Akt和ERK1/2信号途径，抑制I/R心肌的氧化应激和心肌细胞凋亡。对于PI3K/Akt和MEK/MRK1/2这两种信号途径在调节AVLE心脏保护作用的相互关系，尚待进一步研究。

注：与Sham组比较,*P<0.05; 与MI/R组比较,#P<0.05

◀

本文第一作者简介：

屈小玲(1973-)，女，汉族，主治医师，主要从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

国家自然科学基金(81270330, 81470413, 81300190);陕西省科学技术研究发展计划项目(2013KJXX-89)

第四军医大学 ，西安710032；1教学实验中心；2校直门诊部；3唐都医院血液科，西安 710038；4西京医院老年病科，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