CN111650380A - 5-oxo-ETE及其氧桥二十烷类受体在急性心肌梗塞中的应用 - Google Patents
5-oxo-ETE及其氧桥二十烷类受体在急性心肌梗塞中的应用 Download PDFInfo
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- CN111650380A CN111650380A CN202010488965.5A CN202010488965A CN111650380A CN 111650380 A CN111650380 A CN 111650380A CN 202010488965 A CN202010488965 A CN 202010488965A CN 111650380 A CN111650380 A CN 111650380A
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Abstract
本发明公开了一种代谢标志物在制备诊断急性心肌梗塞的药物中的应用，所述代谢标志物为5‑oxo‑ETE；本发明还公开了一种急性心肌梗塞诊断试剂盒，该试剂盒中包含上述代谢标志物的试剂；本发明同时公开了氧桥二十烷类受体作为治疗靶点在制备或筛选急性心肌梗塞的药物中的应用。本发明首次提出5‑oxo‑ETE作为代谢标志物用于急性心肌梗塞诊断药物中，并且将其氧桥二十烷类受体作为筛选预防、缓解和/或治疗心肌梗塞损伤药物的靶标。
Description
技术领域
本发明属于生物化学领域，具体涉及5-oxo-ETE及其氧桥二十烷类受体在急性心肌梗塞中的应用。
背景技术
急性心肌梗死(AMI)是一种世界范围内非常普遍的疾病，且有很高的死亡率和发病率，以心脏的氧气和血液供应减少为特征。目前心梗的诊断方法主要有高灵敏度的肌钙蛋白检测和心电图检测。高灵敏度的肌钙蛋白检测对于心梗的灵敏性更高但是特异性有所下降，因此心电图检测仍是心梗诊断的主要手段。在治疗方面，主要包括抗血小板和抗血栓治疗，并同时进行冠状动脉的侵入性评估，以便适时进行血管重建。尽管建立的急性心梗的临床诊断标准和治疗方法已被用于诊断和治愈患者，但急性心肌梗死仍然是全球范围内导致死亡和残疾的主要原因。
本发明旨在寻找可用于早期诊断的指标和更有效的靶点，并试图通过代谢物的异常变化发现急性心肌梗死相关药物的诊断指标和治疗方法。
发明内容
针对现有问题的不足，本发明的第一个目的是提供一种代谢标志物在制备诊断急性心肌梗塞的药物中的应用，所述代谢标志物为5-oxo-ETE；第二个目的是提供检测上述代谢标志物的试剂在制备诊断急性心肌梗塞药物中的应用；第三个目的是提供一种急性心肌梗塞诊断试剂盒；第四个目的是提供氧桥二十烷类受体作为治疗靶点在制备或筛选急性心肌梗塞的药物中的应用。
本发明解决其技术问题采用的技术方案是：
一种代谢标志物在制备诊断急性心肌梗塞的药物中的应用，所述代谢标志物为5-oxo-ETE。
本发明还保护检测上述代谢标志物的试剂在制备诊断急性心肌梗塞药物中的应用。
一种急性心肌梗塞诊断试剂盒，该试剂盒中含有检测上述代谢标志物的试剂。
本发明还保护上述代谢标志物在制备治疗或筛选急性心肌梗塞药物中的应用。
本发明还保护氧桥二十烷类(OXE)受体作为治疗靶点在制备治疗或筛选急性心肌梗塞的药物中的应用。
本发明保护氧桥二十烷类受体(OXE-R)作为靶点在制备筛选预防、缓解和 /或治疗心肌梗塞损伤药物的应用。
本发明还保护抑制氧桥二十烷类受体表达的物质在治疗急性心肌梗塞的药物中的应用。
优选的，所述的抑制氧桥二十烷类受体表达的物质为Gue1654。
更优选的，所述Gue1654的剂量为0.3-3mg/kg。
5-oxo-ETE是花生四烯酸与白三烯通过5-脂氧合酶途径产生的代谢物，在 NADP+存在下，由5-羟基二十烷酸脱氢酶选择性氧化5-HETE而产生的。我们的研究结果表明急性心梗小鼠血清中5-oxo-ETE的含量有所升高。5-oxo-ETE主要通过介导OXE-R从而诱导嗜酸性细胞、中性粒细胞、嗜碱性细胞和单核细胞迁移。基于OXE-R对5-oxo-ETE的高度选择性，既往研究表明OXE-R的激活可能是5-oxo-ETE发挥功能的主要途径。
有益效果
本发明首次提出5-oxo-ETE作为代谢标志物用于急性心肌梗塞诊断药物中，并且将氧桥二十烷类受体作为筛选预防、缓解和/或治疗心肌梗塞损伤药物的靶标。
附图说明
图1为采用LC-MS法测定假手术和急性心梗模型小鼠血清中5-oxo-ETE的浓度(n＝9)；
图2为假手术组与给予5-oxo-ETE急性心梗组小鼠的心，肝，脾，肺，肾组织HE染色代表性结果(n＝3)(放大倍数200倍，图中横线作为50μm标尺)；
图3为假手术组与给予5-oxo-ETE急性心梗组小鼠的心，肝，脾，肺，肾组织Masson染色代表性结果(n＝3)(放大倍数200倍，图中横线作为50μm标尺)；
图4为酶联免疫吸附法测定假手术和急性心梗模型小鼠血清中OXE-R的含量(n＝10)；
图5为酶联免疫吸附法测定假手术和急性心梗模型小鼠心脏组织中OXE-R 的含量(n＝14)；
图6为免疫印迹法分析假手术和急性心梗模型小鼠心脏组织中OXE-R的表达(n＝4)；
图7为免疫组化法分析假手术和急性心梗模型小鼠心脏组织中OXE-R的表达(n＝3)；
图8为酶联免疫吸附法测定正常人和急性心梗患者血清中OXE-R的含量 (n＝20)；
图9为TTC染色法反映给予OXE-R抑制剂—Gue1654后小鼠的心肌梗死区域与心肌梗死大小(腹腔注射，剂量分别为0.3mg/kg,1mg/kg和3mg/kg)(n＝5)；
图10为酶联免疫吸附法测定给予Gue1654后小鼠血清中肌酸激酶和乳酸脱氢酶含量(腹腔注射，剂量分别为0.3mg/kg,1mg/kg和3mg/kg)(n＝6-8)；
图11为假手术组和给予Gue1654急性心梗组小鼠心脏组织HE和Masson 染色代表性结果(腹腔注射，剂量分别为0.3mg/kg,1mg/kg和3mg/kg)(n＝3)(放大倍数200倍，图中横线作为50μm标尺)；
图12为酶联免疫吸附法测定给予Gue1654小鼠血清中心肌肌钙蛋白I，C 反应蛋白和肿瘤坏死因子的含量(腹腔注射，3mg/kg)(n＝8-12)；
图13为给予Gue1654小鼠超声心动图代表性图像以及左心室射血分数、左心室短轴缩短分数、每搏输出量的统计结果(腹腔注射，3mg/kg)(n＝5)；
图14为给予Gue1654小鼠心脏组织透射电镜代表性图像(腹腔注射， 3mg/kg)(n＝3)；
图15为给予Gue1654小鼠心脏组TUNEL染色的代表性显微图像以及凋亡率的统计结果(腹腔注射，3mg/kg)(n＝3)。
具体实施方式
以下结合实施例对本发明做进一步详细说明。所用试剂或者仪器设备未注明生产厂商的，均视为可以通过市场购买的常规产品；所用方法未具体阐述的均为现有技术中的常规方法。
1实验方法：
1.1动物及急性心梗模型
实验动物为C57BL/6J小鼠，体重22-25g，由扬州大学模型动物研究中心提供。小鼠分笼饲养，可自由饮水和摄食。所有程序均按照美国国立卫生研究院关于实验室动物的护理和使用指南进行，并得到中国药科大学动物伦理委员会的批准。采用1％戊巴比妥钠腹腔注射麻醉小鼠，取仰卧位，左胸除毛，涂碘伏消毒，连接人工呼吸机(潮气量3mL，呼吸比2:1，心率110)。纵行切开皮层后，逐层钝性分离胸前肌肉直至肋骨显露，在肋间隙用弯钳扎破胸腔，撕开心包，暴露心脏，轻压胸廓即可将心脏挤出胸腔外。快速用6-0号丝线，在冠状动脉左前降支起源出下方3mm处连同线穿过的心肌一并结扎(假手术组只进线不结扎)，结扎完成迅速放回胸腔，挤出气体并缝合。造模完成后，进行心电图测试，心电图上ST段抬高表明造模成功。小鼠分组情况如下：
A)将小鼠随机分为4组，每组14只：
1)假手术组(Sham)：腹腔注射(ip)等容积量的氯化钠注射液；
2)假手术给5-oxo-ETE组(Sham+5-oxo-ETE)：静脉注射(iv)5-oxo-ETE 0.1mg/kg；
3)模型组(Model)：腹腔注射(ip)等容积量的氯化钠注射液；
4)模型给5-oxo-ETE组(Model+5-oxo-ETE)：静脉注射(iv)5-oxo-ETE 0.1mg/kg。
B)将小鼠随机分为6组，每组12只：
1)假手术组(Sham)：腹腔注射(ip)等容积量的氯化钠注射液；
2)模型组(Model)：腹腔注射(ip)等容积量的氯化钠注射液；
3)Gue1654低剂量组(Low-dose of Gue1654)：腹腔注射(ip)Gue1654 0.3mg/kg；
4)Gue1654中剂量组(Middle-dose of Gue1654)：腹腔注射(ip)Gue1654 1mg/kg；
5)Gue1654高剂量组(High-dose of Gue1654)：腹腔注射(ip)Gue1654 3mg/kg；
6)美托洛尔组(Met)：灌胃(ig)美托洛尔5.14mg/kg。
所有药物均于左冠状动脉前降支结扎(CAL)30分钟后给予，并于结扎24 小时后处死小鼠。
1.2临床样本
急性心肌梗死患者和正常人样本均来自江苏省中医院。临床医生根据双源 CT、心电图及极性心梗相关生化指标(如：肌酸激酶、乳酸脱氢酶、心肌肌钙蛋白I)进行综合评估以排除其他疾病的影响从而筛选急性心肌梗死患者。所有参与患者均知情同意。本研究在《赫尔辛基宣言》指导下实行。
1.3 5-oxo-ETE靶向定量分析
1.3.1小鼠样本前处理
将冻存样本常温解冻，取生物样本50μL，内标工作溶液10μL，甲醇140μL，涡旋混匀1min。4℃13000rpm低温离心30min沉淀蛋白，取160μL上清液，再次4℃13000r/min离心30min，取130ul上清，使用0.22μm有机滤膜进行过滤，置入进样瓶4℃冷藏备用，等待上机检测。
1.3.2色谱条件
采用安捷伦1260高效液相系统进行色谱分离，使用SynergiTM Fusion-RP C18色谱柱(50×2mm i.d.,2.5μm)，柱温为25℃，自动进样器温度为4℃，进样量为5μL。流动相组成：A相为含0.1％甲酸的水溶液，B相为含0.1％甲酸的乙腈。梯度洗脱条件：0-5min为5％B，5-6min为5-40％B，6-10min为40-80％B， 10-14min为80-90％B，14-15min为90％B，之后回到初始状态，平衡2min；流速0.4mL/min。
1.3.3质谱条件
经色谱分离后，使用Agilent Ultivo三重四极杆质谱仪进行质谱分析和数据采集，采用电喷雾离子源。在正离子模式下采用多反应监测采集5-oxo-ETE并通过与标准品质谱信息和保留时间的对比进行确认和量化。多反应监测模式参数如下：母离子选择319.2，子离子为189.3，单次扫描时间为1.2s。离子源参数经优化后，结果如下：毛细管与锥管电压分别为3500V，1500V；干燥气温度为30℃，流速为7L/min；喷雾器压力为50psi；鞘气温度为350℃，流速为12L/min；碰撞能为5V，碎裂电压为105V。采用Masshunter软件进行数据分析和处理。
对靶向定量方法进行线性度、精密度、准确度、加样回收率和基质效应的考察。精密度与准确度实验结果表明相对标准偏差均低于15％，该方法可靠性高。
1.4 TTC染色
结扎24小时后，迅速取出心脏，于-70℃冷冻，垂直于心脏长轴将心室组织切成5片。将心脏切片和1％TTC溶液在24孔培养板中室温孵育15分钟，然后拍照。用计算机平面测量法测量梗死面积。梗死面积大小以梗死部分占左心室总面积的百分比表示。
1.5超声心动图
结扎24小时后，小鼠送至南京医科大学动物实验中心进行超声心动图检测。异氟烷吸入式麻醉小鼠，仰位，利用Visual Sonics Vevo2100小动物专用高频彩色超声仪，测定各组小鼠左室射血分数(EF)、左室缩短分数(FS)、每搏输出量(SV) 作为心功能评价指标。
1.6组织病理学检查
小鼠取血后，摘除心脏。用10％多聚甲醛的缓冲溶液固定心脏组织，然后石蜡包埋并切成5μm的薄片，采用HE染色和Masson染色。HE染色的操作步骤：将石蜡切片置于烘箱中60℃烤1～2h；石蜡切片常规二甲苯，乙醇脱蜡至水，苏木素染10分钟，流水冲洗，去余色，0.7％盐酸乙醇分化数秒钟，流水冲洗，切片变蓝约15分钟，7.95％乙醇30秒钟，8.酒精性伊红染30秒，I 95％乙醇30秒钟，II 95％乙醇30秒钟，I 100％乙醇30秒钟，II100％乙醇30秒钟，石碳酸二甲苯30秒钟，(1：4石碳酸1-二甲苯4)I二甲苯30秒钟，II二甲苯30秒钟，中性树胶封片。Masson染色的操作步骤：石蜡切片脱蜡至水；铬化处理或去汞盐沉淀；依次自来水和蒸馏水洗；用Harris氏苏木素染液或Weigert苏木素液染核1-2min；流水稍洗；0.5％盐酸酒精分化15s；流水冲洗3min；丽春红酸性品红液染8min；蒸馏水稍冲洗；1％磷钼酸水溶液处理约5min；不用水洗，直接用苯胺蓝液或亮绿液复染5min；1％冰醋酸处理1min；95％乙醇脱水5min×2次，用吸水纸吸干液体；100％乙醇5min×2次，用吸水纸吸干液体；二甲苯中透明 5min×2次，用吸水纸吸干液体；中性树胶封片。最后使用光学显微镜观察组织病理学变化。
1.7 Tunel染色
取缺血心肌组织，用4％多聚甲醛溶液固定。使用荧光素原位细胞死亡检测试剂盒进行末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法评估心肌细胞凋亡。具体操作参照产品使用说明。使用共聚焦扫描显微镜(LSM700,Zeiss, USA)进行拍摄，每个切片随机拍取5个视野。细胞凋亡的程度以TUNEL染色阳性细胞核与DAPI染色细胞核的比值来表示。
1.8免疫组化
取各脏器组织，利用免疫组化分析氧桥二十烷类受体(OXE-R)表达情况。心脏取出后置于4％多聚甲醛固定，石蜡包埋，切成4μm的切片。切片用PBS水合，置于3％过氧化氢溶液中封闭过氧化物酶。切片取出后置于37℃封闭液进封闭1小时，4℃下孵育一抗(OXE-R稀释比例为1:200)过夜。PBS洗涤后，切片37℃下孵育1小时的HRP-Conjugated Secondary抗体(1：200)。在DAB染色，苏木精复染和分段脱水后封片，于400X显微镜下观察。
1.9酶联免疫吸附试验
摘眼球法取血，血样在3500rpm下离心10min，取上清液，获得血清样本并保存在-70℃。用ELISA试剂盒(双抗体夹心酶联免疫吸附法)测定血清中肌酸激酶(CK)、乳酸脱氢酶(LDH)、OXE-R、C-反应蛋白(CRP)、肿瘤坏死因子α(TNF-α)和肌钙蛋白I(cTn-I)的含量，具体操作步骤参照试剂盒使用说明书。
1.10透射电镜
心脏样本切片用2.5％戊二醛固定，在含0.3％单宁酸的0.1M二甲胂酸钠缓冲液中于4℃下浸泡4小时。随后，样本在含1％四氧化锇的0.1M二甲胂酸钠缓冲液中进行后固定，运用系列丙酮在温室下脱水，环氧树脂包埋。超薄切片用醋酸铀酰和柠檬酸铅进行染色，在LEO 906电子显微镜下进行观察。为了观察破碎的肌节，在5000X和1700X的放大倍数下，每组随机拍摄以获取数码电子显微图片。
1.11蛋白质印迹分析
用预冷的含1mM PMSF的RIPA缓冲液裂解细胞。为了测定梗塞区域边缘心脏组织的蛋白表达，将组织在RIPA缓冲液中匀浆。在4℃下以12000rpm离心10分钟获得蛋白质，并通过BCA法测定其浓度。将等量蛋白(35μg)加入到12.5％SDS-PAGE上，并通过电印迹转移至PVDF膜。用含3％BSA的TBS/T进行封闭，并用caspase-3，Bax，Bcl-2，β-actin相应一抗(稀释比例分别为1：1000、 1：1000、1：1000、1：1000)在4℃下孵育过夜。用过氧化物酶偶联的二抗以1： 8000的比例稀释后进行孵育，然后用ECL试剂检测抗原-抗体复合物，通过ChemiDocTMMP System观察蛋白表达，并使用Image LabTM软件进行分析。
2.实验结果
2.1心肌梗塞小鼠血清中5-oxo-ETE及其氧桥二十烷类(OXE)受体含量异常
通过靶向代谢组学，我们发现急性心梗患者血清中5-oxo-ETE的含量显著增加，如图1。为进一步探究5-oxo-ETE增加与急性心梗的关系，在小鼠体内注射 5-oxo-ETE。HE和Masson染色结果显示，给予5-oxo-ETE 24小时后，心和肺的组织形态学损伤加重，进而加重了急性心梗所致的心肺损伤(图2-3)，而 5-oxo-ETE对肝、脾、肾、脑等脏器的作用效果不显著。此外，急性心梗使血清和心脏组织中OXE-R的含量显著增加(图4-5)，心脏组织中OXE-R的表达也显著升高(图6-7)。在急性心梗患者中证实了OXE-R血清含量的增加(图8)。
2.2抑制OXE受体可有效改善急性心肌梗塞
采用梗死面积、血液生化指标、组织病理学检查、心肌超微结构检测及TUNEL染色探究抑制OXE受体对急性心梗的影响。如图2所示，OXE-R的抑制剂—Gue1654在0.3-3mg/kg剂量下，可显著减少扩大的梗死面积(图9)。同时，抑制OXE受体可以降低CK和LDH活性(图10)并改善组织学特征(图11)。急性心梗造模后，给予Gue1654(3mg/kg)可显著降低血清中CRP、cTn-I和TNF-α的含量(图12)。此外，使用超声心动图以考察抑制OXE受体对心脏功能的影响。从图13可得，Gue1654(3mg/kg)显著减轻了急性心梗所致的左心室射血分数 (LVEF)、左心室短轴缩短率(LVFS)和每搏输出量(SV)的减小。透射电镜观察发现，Gue1654处理的小鼠出现轻度心肌超微结构损伤，并伴有细胞水肿程度减轻和线粒体基质维持(图14)。在给予Gue1654小鼠(3mg/kg)的心脏切片中，凋亡细胞的比例也显著降低(图15)。总之，这些数据表明，在急性心梗模型中，抑制OXE受体可以有效减少梗死面积从而改善心脏功能。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。
Claims (8)
1.一种代谢标志物在制备诊断急性心肌梗塞的药物中的应用，其特征在于，所述代谢标志物为5-oxo-ETE。
2.检测权利要求1所述代谢标志物的试剂在制备诊断急性心肌梗塞药物中的应用。
3.一种急性心肌梗塞诊断试剂盒，其特征在于，该试剂盒中含有检测权利要求1所述的代谢标志物的试剂。
4.权利要求1所述的代谢标志物在制备治疗或筛选急性心肌梗塞药物中的应用。
5.氧桥二十烷类受体作为治疗靶点在制备治疗或筛选急性心肌梗塞的药物中的应用。
6.抑制氧桥二十烷类受体表达的物质在治疗急性心肌梗塞的药物中的应用。
7.根据权利要求6所述的应用，其特征在于，所述的抑制氧桥二十烷类受体表达的物质为Gue1654。
8.根据权利要求7所述的应用，其特征在于，所述Gue1654的剂量为0.3-3mg/kg。
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