中国科技论文统计源期刊　中文核心期刊  
美国《化学文摘》《国际药学文摘》
《乌利希期刊指南》
WHO《西太平洋地区医学索引》来源期刊  
日本科学技术振兴机构数据库（JST）
第七届湖北十大名刊提名奖  
医药导报
HERALD OF MEDICINE, 2018, 37(6): 648-653
doi: 10.3870/j.issn.1004-0781.2018.06.002
漆黄素对大鼠体外心肌缺血-再灌注损伤的影响
Effect of Fisetin on Myocardial Ischemia-reperfusion Injury in Isolated Rats
张秀娟, 郝雯瑾, 李德芳, 王博, 韩吉春, 郑秋生
滨州医学院中西医结合学院,烟台 264003
ZHANG Xiujuan,, HAO Wenjin, LI Defang, WANG Bo, HAN Jichun, ZHENG Qiusheng,
College of Integrated Traditional and Western Medicine, Binzhou Medical University, Yantai 264003, China

作者简介 张秀娟(1989-),女,山东滨州人,在读硕士,研究方向:心血管药理研究。E-mail:291626103@qq.com。

通信作者 郑秋生(1967-),男,山东潍坊人,教授,博士,研究方向:心血管药理研究。电话:0535-6913186,E-mail:zqsyt@souhu.com。
基金资助: 滨州医学院科研启动基金项目(BY2014KYQD01)
中图分类号: R965     文献标识码: A     文章编号: 1004-0781(2018)06-0648-06
摘要:

目的 探讨漆黄素抗大鼠体外心肌缺血-再灌注(I/R)损伤作用及作用机制。方法 通过Langendorff体外心脏灌流系统建立心肌I/R损伤模型,32只SPF级SD大鼠随机分为4组:正常对照组、模型对照组、漆黄素小剂量组(0.5 μmol·L-1)和漆黄素大剂量组(5 μmol·L-1),每组8只。使用BL420S生物机能实验系统测定心功能参数;通过氯化三苯基四氮唑(TTC)染色检测心肌梗死面积;酶联免疫吸附测定(ELISA)法检验灌流液中肌酸激酶同工酶(CK-MB)含量;ELISA法检测心肌组织中丙二醛(MDA)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的水平;苏木精-伊红(HE)染色法观察心肌组织的变化;通过TUNEL染色检测心肌细胞凋亡程度。结果 与正常对照组比较,模型对照组心功能参数显著降低,心肌组织严重梗死和坏死,心肌细胞中CK-MB的出量显著升高,心肌组织中MDA,GSSG,IL-6和TNF-α含量显著升高,SOD和GSH的活性显著降低,模型对照组心肌细胞严重凋亡。与模型对照组比较,漆黄素小、大剂量组可以明显逆转I/R诱导的心肌损伤变化,大剂量漆黄素的心肌保护作用更为明显。结论 漆黄素可显著减轻I/R诱导的心肌损伤,并具有一定的剂量依赖性;作用机制可能与其抗氧化、抗炎和抗凋亡作用相关。
关键词: 漆黄素 ; 损伤 ; 心肌缺血-再灌注 ; 抗氧化 ; 抗炎作用 ; 抗凋亡

Abstract:

Objective To investigate the effect of fisetin on myocardial ischemia reperfusion (I/R) injury in isolated rats and its mechanism. Methods The myocardial I/R injury model was established through Langendorff system. A total of 32 SPF grade SD rats were randomly divided into 4 groups (n=8): normal control group, model control group, low dose fisetin group (0.5 μmol·L-1) and high dose fisetin group (5 μmol·L-1). The myocardial function was measured by TTC staining. The content of CK-MB was measured by enzyme-linked immunosorbent assay (ELISA). The levels of SOD, MDA, GSH, GSSG, TNF-α and IL-6 in the myocardium were measured by ELISA. The changes of myocardial tissue were observed by HE staining. The degree of apoptosis was detected by TUNEL staining. Results Compared with the normal control group, the cardiac function parameters of the model control group were decreased significantly, serious infarction and necrosis developed in myocardium, and the leaking amount of CK-MB was significantly increased. The contents of MDA, GSSG, IL-6 and TNF-α in myocardium were significantly increased, and the activities of SOD and GSH significantly decreased. Severe apoptosis appeared in the cardiomyocytes of the model control group. Compared with the model control group, the pretreatment of fisetin could significantly reverse the changes of myocardial injury induced by I/R, and the protective effect of high dose of fisetin was more obvious. Conclusion Fisetin can significantly alleviate I/R-induced myocardial injury and exerts a dose-dependent effect. The protective effect of fisetin may be related to its anti-oxidation, anti-inflammatory and anti-apoptotic effects.
Key words: Fisetin ; Injury ; myocardial ischemia-reperfusion ; Antioxidant ; Anti-inflammatory ; Anti-apoptosis

心肌缺血-再灌注损伤(myocardial ischemia-reperfusion injury,MIRI)指缺血心肌在恢复血液再通时损伤加重的现象,表现在可逆的再灌注诱发心律失常、心肌顿抑损伤及不可逆的微血管阻塞、致死性心肌再灌注损伤[1]。黄酮类化合物在心血管疾病方面有显著优势[2]。漆黄素存在很多植物中,是黄栌的主要化学成分之一[3],同时存在于日常膳食类食品中。研究发现,漆黄素具有抗炎、抗氧化、抗凋亡、抗增殖等作用[4],对心血管病、衰老类疾病、肿瘤类疾病有预防作用。漆黄素对心肌缺血-再灌注损伤的研究很少。因此,本课题组进行漆黄素对体外心肌缺血-再灌注损伤模型的作用研究,探讨漆黄素的可能作用机制。

1 材料与方法
1.1 实验动物

无特定病原体(SPF)级雄性SD大鼠,体质量260~280 g,购自济南朋悦实验动物繁育有限公司,实验动物生产许可证号:SCXK(鲁)2014-0006,适应性饲养3 d,12 h光/12 h暗,相对湿度40%~50%,温度20~23 ℃,自由进食。

1.2 试剂

漆黄素(Solarbio公司,含量98%,批号:SF8050-20),水合氯醛(成都科龙化工试剂厂,批号:2015090601),苏木精-伊红染试剂盒(Solarbio公司,批号:2016030601),氯化三苯基四氮唑(triphenyltetrazolium chloride,TTC,Sigma公司,超氧化物歧化酶(SOD,批号:20160822)、丙二醛(MDA,批号:20160816)、还原型谷胱甘肽(GSH,批号:20161124)、氧化型谷胱甘肽(oxidized glutathione,GSSG,批号:20161108)、肌酸激酶同工酶(creatine kinase-MB,CK-MB,批号:20160816)、白细胞介素-6(IL-6,批号:20160826)、肿瘤坏死因子-α(TNF-α,批号:20160805)、In Situ Cell Death Detection Kit POD试剂盒(批号:20170314)购自Tsz公司,4-0线,其他试剂均为国产分析纯。

1.3 实验仪器

Langendorff体外心脏灌流系统、BL420S生物机能实验系统、恒温水浴器(成都泰盟软件有限公司),超低温冰箱(海尔有限公司),恒温箱(上海博讯实业有限公司),数码相机(佳能公司,型号:Canon EOS 600D),电子分析天平(赛多利斯公司,型号:SQP型,感量:1 mg),酶标仪(Thermo公司,型号:infinite 200Pro),切片机(Leica公司,型号:RM2235),显微镜(Leica公司),离心机(Leica公司)。

1.4 MIRI模型建立,分组及给药方法

参照Langendorff法建立MIRI模型[5]。称定大鼠体质量,给予10%水合氯醛3 mL·kg-1麻醉,舌下静脉注射6 250 U·(100 mL)-1肝素钠0.9 mL·kg-1肝素化,固定,开胸取心,保留主动脉约1 cm,眼科剪剪掉其他动脉、静脉等组织,迅速放入预冷的K-H缓冲液(配方:磷酸二氢钾1.2 mmol·L-1、葡萄糖11.0 mmol·L-1、氯化钙1.2 mmol·L-1、氯化钠118 mmol·L-1、氯化镁1.2 mmol·L-1、碳酸氢钠 24 mmol·L-1、氯化钾4.5 mmol·L-1,盐酸、氢氧化钠调pH值)中,洗净积血,迅速行将灌流装置插管,逆行插入主动脉约0.4 cm,固定,眼科剪于左心耳剪口将压力球囊置左心室、另一端接传感器,左心室舒张末期压成5~10 mmHg(1 mmHg=0.133 kPa)。灌注充分氧饱和的K-H缓冲液灌流系统,温度(37±0.5)℃,混合气体为95%氧气/5%二氧化碳,8.82 kPa(90 cmH2O)恒压恒温灌流。生物信号系统采集传感器将压力信号转换的电信号,测量灌流心脏的心功能指标:左心室收缩压(left ventricular systolic pressure,LVSP),左心室舒张末压(left ventricular end-diastolic pressure,LVEDP),左室压力最大升高/降低速率(±dp/dtmax),计算左心室的发展压 (left ventricular developed pressure,LVDP),LVDP=LVSP-LVEDP,计滴器测定冠脉流量(coronary flow,CF),统计再灌注结束血液动力学系数。灌流中用1.5 mL离心管收集各模型心脏停灌前10 min及再灌注20,40 min灌流液1 mL,冻存。恒压平衡灌注体外心脏期间排除管道内的气泡,剔除实验中心律不齐、LVDP<850 Pa及心率<150 次·min-1实验模型。将大鼠按随机数字表法分为4组:正常对照组、模型对照组、漆黄素大剂量组(5 μmol·L-1),漆黄素小剂量组(0.5 μmol·L-1),每组8只。漆黄素剂量根据预实验检测设定,设置漆黄素浓度为0.1,0.5,1.0,2.5,15.0,40.0 μmol·L-1,分别取两只SD大鼠,血液动力学检测及TTC染色检测确定给药浓度。将漆黄素0.014 3,0.143 mg分别溶解于95%乙醇40 μL中配置给药浓度,预实验中设同浓度乙醇给药组,结果显示灌流液100 mL溶解95%乙醇40 μL与正常对照组比较差异无统计学意义。灌注:正常对照组持续灌注K-H液95 min;模型对照组K-H缓冲液平衡灌流30 min,停灌20 min,再灌45 min;漆黄素小剂量组K-H液平衡灌流20 min,灌注含漆黄素0.5 μmol·L-1K-H液10 min,停灌20 min,再灌45 min。漆黄素大剂量组平衡灌流20 min,灌注含漆黄素5 μmol·L-1的K-H液10 min,停灌20 min,再灌K-H液45 min。

1.5 检测模型灌流液CK-MB

将置于-80 ℃冰箱灌流液样品于常温30 min,酶联免疫吸附测定(ELISA)法检测样品中CK-MB含量,实验按试剂盒说明操作,不用的灌流液样品,放4 ℃冰箱保存,避免反复冻融从而影响酶活性。

1.6 心脏梗死面积的检测

灌流操作后,取下心脏用常温磷酸盐缓冲液(PBS)冲洗,置于-80 ℃冷冻10 min,刀片垂直心脏纵轴均匀横切为厚度1 mm 8或9片,放入2%TTC染液中,水浴锅37 ℃中反应25 min,10%甲醛固定24 h,切片置于蓝色塑料板上,摄像,梗死区域呈灰白色,非梗死区域呈红色,Image-Pro Plus软件检测梗死面积,非梗死面积,心肌梗死率(%)=梗死区面积/心肌切片区总面积×100%。

1.7 心肌缺血心肌组织SOD,MDA,GSH,GSSG,TNF-α,IL-6检测

取左心室心尖心肌50 mg,倒入预冷的PBS 0.5 mL制成10%组织匀浆,3 000 r·min-1离心15 min,ELISA法检测匀浆上清液中SOD、MDA、GSH、GSSG、TNF-α、IL-6酶活性及含量,按照检测试剂盒说明操作。

1.8 心肌组织形态检测

灌流操作后,常温PBS冲洗心脏,10%甲醛固定24 h,自来水冲洗24 h。心肌组织依次50%,60%,70%,80%的乙醇各1 h,95%乙醇45 min,100%乙醇Ⅰ30 min,100%乙醇Ⅱ20 min脱水,乙醇-二甲苯(1:1)中30 min,二甲苯液Ⅰ、Ⅱ各20 min,观察至充分透明。浸入62 ℃恒温箱软蜡杯甲苯混合杯Ⅰ,软蜡硬蜡Ⅱ、Ⅲ,硬蜡Ⅳ中各浸蜡1 h,整修蜡块至上下蜡面两边平行切片6~8 μm,捞片,将烤箱设置62 ℃烘烤心脏切片30 min,依次放入二甲苯液Ⅰ,二甲苯液Ⅱ中各10 min脱蜡,100%乙醇Ⅰ、Ⅱ各5 min脱苯,95%,85%,75%,60%,50%乙醇各5 min,水化,苏木精染色5 min,自来水冲洗染液2 s,1%盐酸乙醇2 s分化,流水1~3 s,0.5%伊红3 min,水洗1~2 s,50%,60%,70%,80%乙醇各5 min,95%乙醇2 min,100%乙醇Ⅰ、Ⅱ各1 min梯度脱水,二甲苯Ⅰ、Ⅱ各10 min透化,封片,摄像。

1.9 检测心肌切片细胞凋亡情况

TUNEL染色检测心肌切片细胞的凋亡,据In Situ Cell Death Detection Kit POD试剂盒说明操作。心肌组织切片制作方法同苏木精-伊红染色标本制作方法。脱蜡,水化,DAB显色后,苏木精染色标记正常细胞核,10 mmol·L-1蛋白酶K将标本切片处理15 min,避光置恒温水浴锅37 ℃下TUNEL液中温育1 h,水浴锅37 ℃POD液中30 min,处理的标本载玻片用光学显微镜观察,正常细胞核显示蓝色,凋亡细胞呈棕黄色。选取标本中8个区域计数凋亡细胞,心肌细胞凋亡指数(apoptotic index,AI)=阳性细胞总数/总细胞数×100%,计算凋亡指数平均值。

1.10 统计学方法

采用SPSS13.0版统计软件进行统计分析,数据以均数±标准差( x ¯ ±s)表示,多组间均数用单因素方差分析,两组间均数比较采用t检验,以P<0.05为差异有统计学意义。

2 结果
2.1 漆黄素对MIRI心脏血流动力学系数的影响

与正常对照组比较,模型对照组LVDP、+dp/dtmax、-dp/dtmax及CF降低(P<0.05);与模型对照组比较,漆黄素大、小剂量组LVDP、+dp/dtmax、-dp/dtmax、CF升高(P<0.01或P<0.05),心脏左室发展压力、心肌舒缩功能、冠脉灌流量显著恢复;与漆黄素小剂量组比较,漆黄素大剂量组LVDP、+dp/dtmax、-dp/dtmax、CF进一步恢复(P<0.05)。结果表明漆黄素降低体外大鼠心脏损伤、恢复心功能呈剂量依赖性。见表1。

表1 4组大鼠血流动力学系数
Tab.1 Hemodynamics of four groups of rats x¯±s,n=8
组别 LVDP +dp/dtmax -dp/dtmax CF/
mL
mmHg
正常对照组 95.72±5.85 94.39±5.86 92.71±6.94 94.36±7.19
模型对照组 61.23±7.55*1 68.31±4.86*1 60.45±5.80*1 65.21±2.53*1
漆黄素
小剂量组 71.23±4.39*3 77.63±5.90*3 78.26±5.99*3 75.31±5.79*3
大剂量组 86.50±5.28*2 88.35±4.65*2 89.76±6.37*2 89.25±5.64*2

Compared with normal control group,*1P<0.01;Compared with model control group,*2P<0.01,*3P<0.05

与正常对照组比较,*1P<0.01;与模型对照组比较,*2P<0.01,*3P<0.05

表1 4组大鼠血流动力学系数

Tab.1 Hemodynamics of four groups of rats x¯±s,n=8

2.2 漆黄素降低心冠脉流出液中CK-MB的含量

与正常对照组比较,模型对照组20,40 min灌流液CK-MB表达量升高(t=3.15,9.71,P<0.05);与模型对照组比较,漆黄素大、小剂量组CK-MB表达量降低(t=1.31,2.86,P<0.01或P<0.05)。与再灌注20 min比较,再灌注40 min的CK-MB表达量降低,结果证明漆黄素可能有较长的半衰期。与漆黄素小剂量组比较,漆黄素大剂量组再灌注20及40 min CK-MB表达量明显降低(t=3.39,4.14,P<0.01或P<0.05)。结果表明漆黄素降低体外大鼠心脏冠脉流液CK-MB表达量,降低作用有剂量依赖性。见表2。

表2 4组大鼠不同时间灌流液CK-MB的检测值
Tab.2 CK-MB in perfusate of four groups of rats at different time points ng·mL-1,x¯±s,n=8
组别 停灌前10 min 再灌注20 min 再灌注40 min
正常对照组 127.61±27.47 137.16±11.24 128.26±24.73
模型对照组 129.01±15.03 227.58±25.58*1 243.05±10.60*1
漆黄素
小剂量组 128.76±7.47 182.67±1.56*3 165.89±12.17*2
大剂量组 130.09±12.58 168.06±3.64*3*4 151.72±23.53*2*5

Compared with normal control group,*1P<0.05;Compared with model control group,*2P<0.01,*3P<0.05;Compared with low-dose fisetin group,*4P<0.01,*5P<0.05

与正常对照组比较,*1P<0.05;与模型对照组比较,*2P<0.01,*3P<0.05;与漆黄素小剂量比较,*4P<0.01,*5P<0.05

表2 4组大鼠不同时间灌流液CK-MB的检测值

Tab.2 CK-MB in perfusate of four groups of rats at different time points ng·mL-1,x¯±s,n=8

2.3 漆黄素对MIRI心脏梗死面积的影响

与正常对照组比较,模型对照组心肌梗死率显著升高。与模型对照组比较,漆黄素小、大剂量组心肌梗死率降低(P<0.01);与漆黄素小剂量组比较,漆黄素大剂量组心肌梗死率明显降低(P<0.01)。漆黄素保护缺血-再灌注心肌,显著降低体外大鼠心脏梗死率,降低作用有剂量依赖性。见图1。


图1 4组大鼠MIRI心肌梗死率(x¯±s,n=8)
与正常对照组比较,*1P<0.01;与模型对照组比较,*2P<0.01

Fig.1 Rate of myocardial infarct in four groups of rats(x¯±s,n=8)
Compared with normal control group,*1P<0.01;Compared with model control group,*2P<0.01

2.4 ELISA检测MIRI组织SOD、GSH、GSSG、MDA、IL-6、TNF-α表达

与正常对照组比较,模型对照组SOD(t=10.34)、GSH(t=5.65)表达量降低,MDA(t=5.14)升高,IL-6(t=4.86)、TNF-α升高(t=4.32)。与模型对照组比较,漆黄素小、大剂量组SOD(t=13.11,16.51)、GSH(t=3.30,18.01)表达量升高,MDA(t=4.11,8.73)降低,IL-6(t=3.47,7.13)、TNF-α降低(t=2.64,3.78),表明漆黄素显著升高体外大鼠心脏组织抗氧化酶表达量,降低心脏组织中损伤指标、炎症指标的表达量。与漆黄素小剂量组比较,漆黄素大剂量组SOD(t=5.19)、GSH(t=5.13)表达量升高、MDA(t=5.49)降低、IL-6(t=4.93)、TNF-α(t=2.81)及GSSG(t=4.08)表达。结果表明漆黄素显著减轻心肌再灌注损伤,保护作用有剂量依赖性。见表3。

表3 4组心肌组织SOD、GSH、GSSG、MDA、IL-6、TNF-α测定值
Tab.3 The level of SOD, GSH, GSSG, MDA, IL-6 and TNF-α in myocardium of four groups of rats x¯±s,n=8
组别 SOD/
(pg·mL-1)		 GSH/
(ng·L-1)		 GSSG/
(nmol·mL-1)		 MDA/
(nmol·L-1)		 IL-6/
(pg·mL-1)		 TNF-α/
(ng·L-1)
正常对照组 38.43±0.83 10.35±0.47 36.29±1.45 100.93±3.73 142.55±7.60 82.52±3.67
模型对照组 35.94±2.05 9.35±1.41*1 47.41±2.09*1 114.45±1.29*1 177.25±2.47*1 91.71±4.60*1
漆黄素
小剂量组 40.72±0.11*2 10.38±0.62*2 43.11±1.58*2 102.59±6.15*2 148.81±16.33*2 80.00±4.56*2
大剂量组 43.25±2.12*2*3 11.87±2.45*2*3 39.50±1.72*2*3 93.06±4.17*2*3 128.09±14.79*4*5 74.48±5.00*3*4

Compared with normal control group,*1P<0.05;Compared with model control group,*2P<0.05,*4P<0.01;Compared with low-dose fisetin group,*3P<0.05,*5P<0.01

与正常对照组比较,*1P<0.05;与模型对照组比较,*2P<0.05,*4P<0.01;与漆黄素小剂量比较,*3P<0.05,*5P<0.01

表3 4组心肌组织SOD、GSH、GSSG、MDA、IL-6、TNF-α测定值

Tab.3 The level of SOD, GSH, GSSG, MDA, IL-6 and TNF-α in myocardium of four groups of rats x¯±s,n=8

2.5 HE染色检测MIRI组织形态情况

与正常对照组比较,模型对照组心肌染色不均匀,细胞肿胀,肌纤维排列紊乱,断裂明显。与模型对照组比较,漆黄素小、大剂量组心肌染色均匀,细胞肿胀不明显,肌纤维排列较规则,断裂情况减少。与漆黄素小剂量组比较,漆黄素大剂量组形态损伤减轻。结果表明漆黄素可以显著减轻心肌再灌注损伤,作用结果表现为剂量依赖性。见图2。


图2 4组MIRI组织病理特征(HE,×400)

Fig.2 Pathological feature of myocardium in four groups of rats (HE,×400)

2.6 TUNEL染色检测细胞凋亡情况

正常对照组平衡灌流细胞凋亡率低,染色切片棕黄色细胞不明显,说明心肌细胞损伤不明显。与正常对照组比较,模型对照组心肌细胞棕黄色细胞明显增多,凋亡率增高(P<0.01)。与模型对照组比较,漆黄素小、大剂量组棕黄色细胞明显增多,细胞凋亡率降低(P<0.01或P<0.05),漆黄素显著降低体外大鼠心脏组织细胞凋亡。与漆黄素小剂量组比较,漆黄素大剂量组棕黄色细胞明显减少,细胞凋亡率降低,差异有统计学意义(P<0.05)。结果表明漆黄素可以显著降低心肌再灌注损伤细胞凋亡率,保护作用有剂量依赖性。见图3。


图3 4组心肌凋亡情况(×400)
与正常对照组比较,*1P<0.01;与模型对照组比较,*2P<0.05,*3P<0.01

Fig.3 Apoptosis of myocardium in four groups of rats (×400)
Compared with normal control group,*1P<0.01;Compared with model control group,*2P<0.05,*3P<0.01

3 讨论

心肌梗死是冠心病演变的严重结果,药物溶栓、经皮冠状动脉再通、旁移植及心脏移植可以降低梗死患者病死率、致残率。研究发现,缺血-再灌注中缺血心肌会加重损伤,因此MIRI成为影响再通的重要因素。研究者对MIRI进行广泛研究,形成自由基损伤、氧化应激、钙超载、能量代谢障碍、凋亡及自噬等研究机制[6]。药物缺血预处理、机械缺血预处理、缺血后处理等处理方法可以降低MIRI。心血管病的不可预测性,现有药物的单靶点性及机械手段缺少人文关怀等弊端影响保护作用,急需开发药理作用更好的药物。

天然低毒的黄酮类化合物具有广谱药理作用,是植物药中多酚类抗氧化剂的研究热点,黄酮类化合物及活性成分在心肌梗死[7]、MIRI研究中作用显著。笔者对多种黄酮类化合物进行药理学研究,显示甘草[8,9],黄芩[10]等药材中的某些成分对MIRI有保护作用。漆黄素是本课题组进行网络药理学研究的重点药物成分。漆黄素又称非瑟酮、漆树黄酮,具有抗炎、抗氧化、抗凋亡及抗肿瘤等药理作用[11],是广泛存在于植物界的天然黄酮类化合物。

体外心脏灌注模型排除体液因子的干扰,可以通过血液动力学设备检测高效、快捷的筛选模型制备不成功的实验大鼠。缺血-再灌注降低心肌功能,左室末舒张压、左室压力升降速率及灌流量反应心功能恢复情况。实验结果表明,漆黄素明显升高左室压力升降速率及灌流量,明显降低缺血-再灌注心肌梗死率。

漆黄素灌流组织形态明显恢复,缺血-再灌注损伤减轻。心肌组织损伤会释放细胞内CK-MB,冠脉流出液中的CK-MB表达量可反映心肌损伤程度,结果表明,漆黄素可降低心肌细胞损伤中心肌酶含量。

研究表明,心肌缺血-再灌注氧化应激反应诱发的自由基[12],是MIRI的主要影响因素,自由基包括氧自由基,氮自由基。氧自由基损伤心肌组织的关键自由基,可过度激活脂质过氧化物,产生MDA等。正常情况下,人体内有少量自由基,同时体内存在清除自由基的抗氧化酶系统,如SOD等保护机体[13]。缺血缺氧组织恢复血液供应情况下,心肌组织氧化应激降低抗氧化酶的同时释放大量氧自由基[14],自由基促进氧化应激及一系列蛋白激酶及转录因子激活,介导损伤组织的生物学反应,如引发的链式脂质过氧化反应损伤细胞膜,细胞器膜,核酸的结构及功能,从而导致细胞凋亡、坏死和组织急慢性损伤,心肌组织氧化应激状态常用指标MDA,可以间接反映生物膜脂质过氧化损伤。漆黄素可降低MDA表达,提高SOD、GSH表达量,表明漆黄素有抑制自由基反应、提高MIRI抗氧化作用。

炎症反应是造成MIRI的重要因素[15]。心肌缺血缺氧,自由基造成内皮细胞损伤,抑制抗炎因子的产生,促进炎症因子如IL-6、TNF-α等释放,促进黏附分子的表达,诱导中性粒细胞对内皮细胞的黏附,加重冠状动脉内皮细胞,缺血心肌的损伤,是造成MIRI微循环障碍的重要因素。研究表明,中性粒细胞促进氧自由基如活性氧家族的产生,协同作用导致心功能降低。本研究结果显示,漆黄素有降低炎症物质IL-6、TNF-α表达量,抑制炎症反应,提高MIRI抗炎作用。

细胞凋亡是造成心肌缺血-再灌注损伤心肌的重要机制[16,17]。大量实验表明线粒体途径、内质网途径、死亡受体途径是介导细胞凋亡的主要通。自由基及炎症因素对组织,细胞结构功能的损伤,激活凋亡途径中相应的凋亡蛋白,转录因子,激活基因控制的细胞程序性死亡,造成心脏不可逆损伤[18]。实验结果表明,漆黄素有减轻MIRI组织形态损伤,恢复损伤心肌结构及功能的正常,减轻细胞核损伤及降低细胞凋亡率,提高MIRI抗凋亡作用。漆黄素对肿瘤细胞的作用研究发现,凋亡可能通过信号通NF-κB、MAPK,激活caspase、bcl2凋亡蛋白表达,漆黄素是否通过NF-κB、MAPK通对MIRI有作用需进一步研究。

本实验为中药成分漆黄素在MIRI的应用提供理论依据,发挥作用的信号转导机制有待进一步研究。实验研究证明,漆黄素对MIRI有恢复心功能、抑制氧化反应、降低炎症反应及抗凋亡作用,具有开发成保护MIRI药物的药理基础。

The authors have declared that no competing interests exist.
参考文献
[1] KALOGERIS T,BAINES C P,KRENZ M.Ischemia/reperfusion[J].Compr Physiol,2016,7(1):113-170.
[本文引用:1]
[2] 周宗宝,王红,叶晓川,等.黄酮类化合物的结构修饰及生物活性研究进展[J].医药导报,2017,36(2):181-185.
[本文引用:1]
[3] 张天龙,高昂,巩江,等.黄栌药学研究概况[J].辽宁中医药大学学报,2011,13(4):75-76.
[本文引用:1]
[4] CHOU R H,HSIEH S C,YU Y L,et al.Fisetin inhibits migration and invasion of human cervical cancer cells by down- regulating urokinase plasminogen activator expression through suppressing the p38 MAPK- dependent NF-kappaB signaling pathway[J].PLoS One,2013,8(8):e71983.
DOI:10.1371/journal.pone.0071983      URL    
[本文引用:1]
[5] 黄健,周云,杨绍军,等.成功制作离体心脏灌注模型要点和经验[J].实验动物学,2011,28(4):60-62.
目的探讨并总结制作Langendorff离体心脏灌注模型的要点及成功经验。方法通过16只健康SD大鼠(体质量200～400 g),以HTK液作为心脏停搏液建立标准的Langendorff离体心脏灌注模型,将此模型与持续K-H液心肌灌注、持续心脏搏动组大鼠的同一时段的心肌酶学及血流动力学指标进行对比,使用SPSS 11.5作统计学重复测量资料的方差分析。结果 1例因操作不熟练致提取心脏时间延长,1例因灌注管插入深度过深导致主动脉瓣毁损,1例因室温过低,1例因灌注管管径过大无法插入而导致实验失败,其余12例在应用了室内加热器、不同管径灌注针、自制球囊测压管及加快操作速度后均获得实验成功。所制模型心肌酶学及血流动力学各指标与持续心脏搏动组无明显差异(P0.05)。结论掌握制作要点及注意事项后,Langendorff离体心脏灌注模型可顺利快速地成功制备。
DOI:10.3969/j.issn.1006-6179.2011.04.016      URL    
[本文引用:1]
[6] DONG Y,UNDYALA V V,GOTTLIEB R A,et al.Autoph-agy:definition,molecular,machinery,and potential role in myocardial ischemia-reperfusion injury[J].J Cardiov Pharmacol Therap,2010,15(3):220-230.
DOI:10.1177/1074248410370327      URL    
[本文引用:1]
[7] 王晓敏,高增光,席晓甜,等.红花黄色素注射液对急性心肌梗死模型大鼠低氧诱导因子和血管内皮生长因子的影响[J].医药导报,2016,35(6):570-573.
[本文引用:1]
[8] ZHOU M,LIU L,WANG W,et al.Role of licochalcone C in cardioprotection against ischemia/reperfusion injury of isolated rat heart via antioxidant,anti-inflammatory,and anti-apoptotic activities[J].Life Sci,2015,132:27-33.
This study aimed to evaluate the protective effect of licochalcone C against myocardial ischemia/reperfusion injury in rats. Left ventricular developed pressure (LVDP) and its maximum up/down rate (dp/dtmax) were recorded as myocardial function. Levels of creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione/glutathione disulfide (GSH/GSSG) ratio, and tumor necrosis factor-alpha (TNF-伪) were determined by using enzyme-linked immunosorbent assay. Cell morphology was observed and mitochondrial damage was assessed by HE coloration and transmission electron microscopy, respectively. Cardiomyocyte apoptosis was determined by using terminal deoxynucleotidyl transferased UTP nick-end labeling (TUNEL). Pretreatment with licochalcone C significantly improved the recovery of LVDP and dp/dtmax, and increased the levels of SOD and GSH/GSSG ratio. However, pretreatment with licochalcone C not only decreased the TUNEL-positive cell ratio and morphological changes, but also weaken the mitochondrial injury and the levels of CK, LDH, MDA, and TNF-伪. These results suggested an important function of licochalcone C extracted from traditional Chinese medicine in the cardioprotection via antioxidant, anti-inflammatory, and anti-apoptotic activities.
DOI:10.1016/j.lfs.2015.04.008      PMID:25921769      URL    
[本文引用:1]
[9] HAN J,WANG D,YU B,et al.Cardioprotection against ischemia/reperfusion by licochalcone B in isolated rat hearts[J].Oxid Med Cell Longev,2014,2014:134862.
Abstract The generation of reactive oxygen species (ROS) is a major cause of heart injury induced by ischemia-reperfusion. The left ventricular developed pressure (LVDP) and the maximum up/down rate of left ventricular pressure (00±dp/dt(max)) were documented by a physiological recorder. Myocardial infarct size was estimated macroscopically using 2,3,5-triphenyltetrazolium chloride staining. Coronary effluent was analyzed for lactate dehydrogenase (LDH) and creatine kinase (CK) release to assess the degree of cardiac injury. The levels of C-reactive protein (CRP), interleukin-8 (IL-8), tumor necrosis factor-02± (TNF-02±), and interleukin-6 (IL-6) were analyzed to determine the inflammation status of the myocardial tissue. Cardiomyocyte apoptosis analysis was performed using the In Situ Cell Death Detection Kit, POD. Accordingly, licochalcone B pretreatment improved the heart rate (HR), increased LVDP, and decreased CK and LDH levels in coronary flow. SOD level and GSH/GSSG ratio increased, whereas the levels of MDA, TNF-02±, and CRP and activities of IL-8 and IL-6 decreased in licochalcone B-treated groups. The infarct size and cell apoptosis in hearts from licochalcone B-treated group were lower than those in hearts from the I/R control group. Therefore, the cardioprotective effects of licochalcone B may be attributed to its antioxidant, antiapoptotic, and anti-inflammatory activities.
DOI:10.1155/2014/134862      PMID:25215172      URL    
[本文引用:1]
[10] 陈萌,韩吉春,王东,等.黄芩素对大鼠心肌缺血再灌注损伤的改善作用[J].中国药房,2016,27(1):23-26.
目的：研究黄芩素对大鼠心肌缺血/再灌注（I/R）损伤的改善作用。方法：采用Langendorff法建立心肌I/R损伤模型。取40只大鼠离体心脏随机均分为正常组（不停灌处理）、模型组（停灌20 min）和黄芩素高、中、低浓度组（停灌前分别灌注含40、10、2.5μmol/ml黄芩素的K-H液10 min）。检测心肌梗死率,冠脉流出液中肌酸激酶（CK）、乳酸脱氢酶（LDH）的活性,心肌组织中超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量、还原型谷胱甘肽/氧化型谷胱甘肽（GSH/GSSG）比值以及心肌细胞凋亡率。结果：与正常组比较,模型组大鼠离体心脏的心肌梗死率、心肌细胞凋亡率、CK和LDH活性升高,心肌组织中MDA含量增加、SOD活性、GSH/GSSG比值降低（P〈0.01）。与模型组比较,黄芩素低、中浓度组大鼠离体心脏的心肌梗死率、CK和LDH活性降低,心肌组织中SOD活性升高（P〈0.05或P〈0.01）,并且中浓度组心肌细胞凋亡率明显降低、心肌组织中MDA含量减少、GSH/GSSG比值升高（P〈0.05或P〈0.01）;高浓度组各指标均无明显变化（P〉0.05）。结论：黄芩素对大鼠心肌I/R损伤具有一定的改善作用,其机制可能与黄芩素的抗氧化和抗凋亡作用相关。
DOI:10.6039/j.issn.1001-0408.2016.01.07      URL    
[本文引用:1]
[11] MAHER P,DARGUSCH R,EHREN J L,et a1.Fisetin lowers methylglyoxal dependent protein glycation and limits the complications of diabetes[J].PLoS One,2011,6(6):e21226.
Abstract The elevated glycation of macromolecules by the reactive dicarbonyl and -oxoaldehyde methylglyoxal (MG) has been associated with diabetes and its complications. We have identified a rare flavone, fisetin, which increases the level and activity of glyoxalase 1, the enzyme required for the removal of MG, as well as the synthesis of its essential co-factor, glutathione. It is shown that fisetin reduces two major complications of diabetes in Akita mice, a model of type 1 diabetes. Although fisetin had no effect on the elevation of blood sugar, it reduced kidney hypertrophy and albuminuria and maintained normal levels of locomotion in the open field test. This correlated with a reduction in proteins glycated by MG in the blood, kidney and brain of fisetin-treated animals along with an increase in glyoxalase 1 enzyme activity and an elevation in the expression of the rate-limiting enzyme for the synthesis of glutathione, a co-factor for glyoxalase 1. The expression of the receptor for advanced glycation end products (RAGE), serum amyloid A and serum C-reactive protein, markers of protein oxidation, glycation and inflammation, were also increased in diabetic Akita mice and reduced by fisetin. It is concluded that fisetin lowers the elevation of MG-protein glycation that is associated with diabetes and ameliorates multiple complications of the disease. Therefore, fisetin or a synthetic derivative may have potential therapeutic use for the treatment of diabetic complications.
DOI:10.1371/journal.pone.0021226      PMID:3124487      URL    
[本文引用:1]
[12] DE VRIES D K,KORTEKAAS K A,TSIKAS D,et al.Oxidative damage in clinical ischemia/reperfusion injury:a reappraisal[J].Antioxid Redox Signal,2013,19(6):535-545.
Ischemia/reperfusion (I/R) injury is a common clinical problem. Although the pathophysiological mechanisms underlying I/R injury are unclear, oxidative damage is considered a key factor in the initiation of I/R injury. Findings from preclinical studies consistently show that quenching reactive oxygen and nitrogen species (RONS), thus limiting oxidative damage, alleviates I/R injury. Results from clinical intervention studies on the other hand are largely inconclusive. In this study, we systematically evaluated the release of established biomarkers of oxidative and nitrosative damage during planned I/R of the kidney and heart in a wide range of clinical conditions.Sequential arteriovenous concentration differences allowed specific measurements over the reperfused organ in time. None of the biomarkers of oxidative and nitrosative damage (i.e., malondialdehyde, 15(S)-8-iso-prostaglandin F2伪, nitrite, nitrate, and nitrotyrosine) were released upon reperfusion. Cumulative urinary measurements confirmed plasma findings. As of these negative findings, we tested for oxidative stress during I/R and found activation of the nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of oxidative stress signaling.This comprehensive, clinical study evaluates the role of RONS in I/R injury in two different human organs (kidney and heart). Results show oxidative stress, but do not provide evidence for oxidative damage during early reperfusion, thereby challenging the prevailing paradigm on RONS-mediated I/R injury.Findings from this study suggest that the contribution of oxidative damage to human I/R may be less than commonly thought and propose a re-evaluation of the mechanism of I/R.
DOI:10.1089/ars.2012.4580      PMID:23305329      URL    
[本文引用:1]
[13] WOZNIAK D,DRYS A,MATKOWSKI A.Antiradical and antioxidant activity of flavones from scutellariae baicalensis radix[J].Nat Prod Res,2015,29(16):1567-1570.
We evaluated the antioxidant properties of four main flavones from : baicalein, wogonin and their glucuronides – baicalin and wogonoside. We used three assays: free radical scavenging with 2,2′-diphenylpicrylhydrazyl radical, transition metal ions reducing power by phosphomolybdenum assay and inhibition of the hydroxyl radical-induced peroxidation of linoleic acid assay. All flavones have antioxidant capacity, which differs depending on the structure and mechanisms of activity. In all tests, only baicalein – the aglycone with three adjacent hydroxyl groups – exhibited consistent antioxidant effect. Wogonin protected linoleic acid against oxidation. Baicalin displayed less potent antioxidant properties whereas wogonoside did not have significant antioxidant activity.
DOI:10.1080/14786419.2014.983920      PMID:25427178      URL    
[本文引用:1]
[14] SYKIOTIS G B,BOHMANN D.Stress-activated cap'n'collar transcription factors in aging and human disease[J].Sci Signal,2010,3(112):re3.
Cap'n'collar (Cnc) transcription factors are conserved in metazoans and have important developmental and homeostatic functions. The vertebrate Nrf1, Nrf2, and Nrf3; the Caenorhabditis elegans SKN-1; and the Drosophila CncC comprise a subgroup of Cnc factors that mediate adaptive responses to cellular stress. The most studied stress-activated Cnc factor is Nrf2, which orchestrates the transcriptional response of cells to oxidative stressors and electrophilic xenobiotics. In rodent models, signaling by Nrf2 defends against oxidative stress and aging-associated disorders, such as neurodegeneration, respiratory diseases, and cancer. In humans, polymorphisms that decrease Nrf2 abundance have been associated with various pathologies of the skin, respiratory system, and digestive tract. In addition to preventing disease in rodents and humans, Cnc factors have life-span-extending and anti-aging functions in invertebrates. However, despite the pro-longevity and antioxidant roles of stress-activated Cnc factors, their activity paradoxically declines in aging model organisms and in humans suffering from progressive respiratory disease or neurodegeneration. We review the roles and regulation of stress-activated Cnc factors across species, present all reported instances in which their activity is paradoxically decreased in aging and disease, and discuss the possibility that the pharmacological restoration of Nrf2 signaling may be useful in the prevention and treatment of age-related diseases.
DOI:10.1126/scisignal.3112re3      PMID:2991085      URL    
[本文引用:1]
[15] AN F,YANG G D,TIAN J M,et al.Antioxidant effects of the orientin and vitexin in Trollius chinensis Bunge in D-galactose-aged mice[J].Neural Regen Res,2012,7(33):2565-2575.
Total flavonoids are the main pharmaceutical components of Trollius chinensis Bunge, and orientin and vitexin are the monomer components of total flavonoids in Trollius chinensis Bunge. In this study, an aged mouse model was established through intraperitoneal injection of D-galactose for 8 weeks, followed by treatment with 40, 20, or 10 mg/kg orientin, vitexin, or a positive control (vitamin E) via intragastric administration for an additional 8 weeks. Orientin, vitexin, and vitamin E improved the general medical status of the aging mice and significantly increased their brain weights. They also produced an obvious rise in total antioxidant capacity, superoxide dismutase, catalase, and glutathione peroxidase levels in the serum, and the levels of superoxide dismutase, catalase and glutathione peroxidase, Na+-K+-ATP enzyme, and Ca2+-Mg2+-ATP enzyme in the liver, brain and kidneys. In addition, they significantly reduced malondialdehyde levels in the liver, brain and kidney and lipofuscin levels in the brain. They also significantly improved the neuronal ultrastructure. The 40 mg/kg dose of orientin and vitexin had the same antioxidant capacity as vitamin E. These experimental findings indicate that orientin and vitexin engender anti-aging effects through their antioxidant capacities.
DOI:10.3969/j.issn.1673-5374.2012.33.001      PMID:4200723      URL    
[本文引用:1]
[16] GOTTLIEB R A.Cell death pathways in acute ischemia/reperfusion injury[J].J Cardiov Pharmacol Therap,2011,16(3/4):233-238.
Abstract The consequence of myocardial ischemia is energetic stress, while reperfusion is accompanied by abrupt ionic shifts and considerable oxidative stress. Cells die by apoptotic and necrotic pathways. After the acute injury, the healing myocardium is subject to biomechanical stress and inflammation, which can trigger a smaller but more sustained wave of cell death, as well as changes in the metabolic and functional characteristics of surviving cells. The goal of cardioprotection is to prevent cell death during the acute injury as well as to modulate the detrimental processes that ensue during remodeling. This review will focus on acute injury, and the central premise is that mitochondria are the key determinant of cardiomyocyte fate.
DOI:10.1177/1074248411409581      PMID:21821521      URL    
[本文引用:1]
[17] XIA J G,XU F F,QU Y,et al.Atorvastatin postconditioning attenuates myocardial ischemia reperfusion injury via inhibiting endoplasmic reticulum stress-related apoptosis[J].Shock,2014,42(4):365-371.
The present study examined whether atorvastatin, when used for pharmacological postconditioning, attenuated myocardial ischemia-reperfusion (I/R) injury in a manner similar to ischemic postconditioning (I-PostC), that is, by inhibition of endoplasmic reticulum (ER) stress-related apoptosis. In the present study, markers for myocardial injury, infarct area, and hemodynamics, and indicators of ER stress and apoptosis were compared in ischemic and atorvastatin-induced postconditioning as a means of evaluating the protective effect of atorvastatin postconditioning in I/R injury and whether, as in I-PostC, inhibition of ER stress is involved. Both ischemic and atorvastatin-mediated postconditioning significantly decreased indications of cardiac damage and reduced serum concentrations of markers for myocardial injury, reduced the infarct area seen at the end of reperfusion, and improved left ventricular systolic function. We found that high-dose atorvastatin- and I-PostC significantly downregulated expression of glucose-regulating protein 78 and calreticulin (CRT; ER stress markers), expression of C/EBP homologous protein (CHOP), and caspase 12 (markers for ER stress-related apoptosis), and Bax (downstream molecule of CHOP), in the myocardial area at risk. Atorvastatin and I-PostC have similar cardioprotective effects in I/R injury and inhibit the ER stress-related apoptotic pathway.
DOI:10.1097/SHK.0000000000000224      PMID:25004060      URL    
[本文引用:1]
[18] SINGH S S,KANG P M.Mechanisms and inhibitors of apoptosis in cardiovascular diseases[J].Curr Pharm Des,2011,17(18):1783-1793.
Apoptosis or progress of programmed cell death is a tightly regulated process which plays an important role in various cardiovascular diseases particularly in myocardial infarction, reperfusion injury, and heart failure. Over the past two decades, investigations of several pathways have broadened our understanding of programmed cell death. Many anti-apoptotic interventions have targeted ischemia- reperfusion, however only a limited number have been considered at the chronic stage of heart failure. Endogenous inhibitors, caspase inhibitors, PARP-1 inhibitors, as well as various other agents have been implicated as anti-apoptotic interventions. This review summarizes the apoptotic pathways associated with heart failure, discusses the current anti-apoptotic interventions available and reviews the clinical implications. <br/> <br/> <br/>
DOI:10.2174/138161211796390994      PMID:21631422      URL    
[本文引用:1]
资源
PDF (1968KB) | 摘要
PDF下载数    
RichHTML 浏览数    
摘要点击数    
分享
导出
EndNote | Ris | Bibtex
相关文章:
关键词（key words）
漆黄素
损伤
心肌缺血-再灌注
抗氧化
抗炎作用
抗凋亡
Fisetin
Injury
myocardial ischemia-reper...
Antioxidant
Anti-inflammatory
Anti-apoptosis
作者
张秀娟
郝雯瑾
李德芳
王博
韩吉春
郑秋生
ZHANG Xiujuan
HAO Wenjin
LI Defang
WANG Bo
HAN Jichun
ZHENG Qiusheng