CN110464844A - Alox12抑制剂在制备心脏缺血再灌注损伤治疗药物中的应用 - Google Patents
Alox12抑制剂在制备心脏缺血再灌注损伤治疗药物中的应用 Download PDFInfo
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- CN110464844A CN110464844A CN201910802423.8A CN201910802423A CN110464844A CN 110464844 A CN110464844 A CN 110464844A CN 201910802423 A CN201910802423 A CN 201910802423A CN 110464844 A CN110464844 A CN 110464844A
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Abstract
本发明通过研究发现，在肝脏缺血再灌注损伤中，ALOX12蛋白表达量和mRNA表达量的变化显著，而ALOX5和ALOX15的变化没有差异，表明ALOX12与其它ALOX成员相比，对肝脏缺血再灌注损伤的作用更大。ALOX12过表达会加重缺氧和复氧处理引起的肝细胞、肾脏细胞的活性降低，促进肝脏细胞的炎症反应；并且，ALOX12低表达能缓解缺氧和复氧处理引起的心肌细胞活性降低，这些结果表明ALOX12可促进肝脏、心脏和肾脏等脏器的缺血再灌注损伤，以及这些脏器中其他炎症反应的发生发展。在此基础上，ALOX12可以作为缺血再灌注损伤及其相关疾病、以及炎症性疾病和细胞死亡相关疾病的治疗靶点。
Description
本申请为2017年8月21日提交的，发明名称为“ALOX12抑制剂在制备缺血再灌注损伤治疗药物中的应用”的中国专利申请201710719319.3的分案申请。
技术领域
本发明属于生物医药技术领域，具体涉及ALOX12抑制剂在制备药物中的用途，所述药物用于治疗缺血再灌注损伤及相关疾病，特别是肝脏、心脏、肾脏等脏器的缺血再灌注损伤，以及治疗这些脏器的炎症疾病和细胞死亡相关疾病。
背景技术
脂氧合酶(arachidonate lipoxygenase，ALOX)是一类能够催化花生四烯酸、亚麻油酸、脂肪酸及其他多不饱和脂肪酸生成有生物活性的代谢产物参与到炎症反应和免疫反应的酶类。哺乳动物的ALOX根据氧分子插入花生四烯酸的特异性位置不同，而分为四种亚型：ALOX5，ALOX8，ALOX12，ALOX15，其中ALOX12可分为12S-LOX和12R-LOX。
目前认为，ALOX5参与了肿瘤和炎症性疾病例如哮喘等的发病过程，FDA批准了唯一一个LOX抑制剂齐留通(ALOX5的抑制剂)上市。
另外，研究认为ALOX15参与了动脉粥样硬化、神经退行性疾病、急性缺血导致的神经损伤等病变过程。
而ALOX12能催化花生四烯酸生成12-HPETE和12-HETE，从而能催化亚油酸的新陈代谢生成羟十八碳二烯醇(HODEs)。目前研究认为ALOX12的活化参与了促瘤效应和抑瘤效应，ALOX12近年来成为抗肿瘤研究的热点。此外，还有研究认为ALOX12参与了皮肤疾病、血小板凝血、糖尿病、糖尿病性肾病、糖尿病性神经病变等疾病的发病过程。
缺血再灌注损伤(Ischemia-Reperfusion Injury，IRI)是1960年Jennings首先提出的概念，是指组织器官缺血后血液再灌注，不仅不能使组织器官功能恢复，反而加重组织器官的功能障碍和结构损伤。缺血再灌注损伤在许多重要器官包括心、肝、肺、肾、胃肠道等均可发生。
肝脏缺血再灌注损伤(Hepatic Ischemia Reperfusion Injury，HIRI)是肝脏外科手术中常见的病理过程，多见于休克、需要阻断肝脏血流的肝外科手术以及肝移植术等病理生理过程中。近年来，随着临床治疗技术的发展，肝移植、溶栓治疗以及肝门阻断术等手术的开展越来越多，尽管肝脏保护、外科技巧及术中监护不断改进，但缺血再灌注所致肝损伤依然是引起术后脏器无功能，移植失败甚至患者死亡的主要原因。肝脏经历缺血再灌注后，肝脏组织细胞发生一系列代谢、结构和功能的损伤，易诱发肝功能衰竭，是影响疾病预后、手术成功率和病人存活率的主要原因之一。
急性冠状动脉梗阻性疾病是目前心脑血管疾病的主要致死原因之一。尽管心脏搭桥术、介入及溶栓等治疗有了很大的进步，但急性心梗患者的死亡率依然较高，其中一个很重要的原因就是尚无有效办法抑制缺血心肌恢复血流时所引起的缺血再灌注损伤。心肌缺血一定时间后，重新恢复血供，会造成机体内炎症因子及氧自由基等大量释放，心肌细胞凋亡率增加，恶性心律失常如室颤、室速等发生增加，心肌能量代谢及结构上出现损伤。
肾脏也同样是高灌注器官，对缺血以及缺血再灌注均敏感。肾脏缺血再灌注损伤是缺血性急性肾功能衰竭的重要损伤环节，也是肾移植中影响移植肾早期功能恢复的制约因素。
因此，如何减轻和消除缺血再灌注损伤以及阐明这种损伤的机制，有重要的临床实用价值。目前认为有多个机制参与了器官的缺血再灌注损伤：如致炎性细胞因子(TNF-α和IL等)、氧自由基、钙超载、微循环障碍、能量代谢紊乱等，还受缺血的时间、组织对氧的需求、侧枝循环的建立及电解质浓度等因素影响。
发明内容
本发明通过实验研究发现了ALOX12，而不是ALOX5或者ALOX15，在肝脏的缺血再灌注损伤中起重要作用，通过抑制ALOX12能显著改善肝脏缺血再灌注损伤，而完成了本发明。
本发明的实验研究发现，在肝脏缺血再灌注损伤中，ALOX12蛋白表达量和mRNA表达量的变化显著，而ALOX5和ALOX15的变化没有差异，表明ALOX12与其它ALOX成员相比，对肝脏缺血再灌注损伤的作用更大。
ALOX12过表达会加重缺氧和复氧处理引起的肝细胞、肾脏细胞的活性降低，促进肝脏细胞的炎症反应；并且，ALOX12低表达能缓解缺氧和复氧处理引起的心肌细胞活性降低，这些结果表明ALOX12可促进肝脏、心脏和肾脏等脏器的缺血再灌注损伤，以及这些脏器中其他炎症反应的发生发展。
在此基础上，ALOX12可以作为缺血再灌注损伤及其相关疾病、以及炎症性疾病和细胞死亡相关疾病的治疗靶点。
本发明的技术方案如下：
本发明的第一个方面是提供，ALOX12抑制剂在制备药物中的用途，所述药物用于治疗缺血再灌注损伤及相关疾病，或者治疗炎症疾病和细胞死亡相关疾病。
根据本发明，所述缺血再灌注损伤及相关疾病选自肝脏缺血再灌注损伤及其相关疾病，心脏缺血再灌注损伤及其相关疾病，肾脏缺血再灌注损伤及其相关疾病，和/或脑缺血再灌注损伤及其相关疾病。所述缺血再灌注损伤可以是由器官移植、组织部分或全部切除、血管栓塞导致组织缺血等多种原因引发的。
肝脏缺血再灌注损伤及相关疾病的引发因素包括但不限于：肝脏囊肿、肝脏移植、溶栓治疗、肝门阻断术、肝昏迷。
心脏缺血再灌注损伤及相关疾病的引发因素包括但不限于：心肌梗塞、心梗再通损伤、心脏移植、冠状动脉溶栓术、经皮冠状动脉成形术，冠状动脉内扩张术，冠状动脉旁路术。
肾脏缺血再灌注损伤及相关疾病的引发因素包括但不限于：肾脏移植、肾脏囊肿、肾脏血管手术。
脑缺血再灌注损伤及相关疾病的引发因素包括但不限于：脑卒中、脑血管手术等。
优选所述缺血再灌注损伤及相关疾病为肝脏缺血再灌注损伤，心脏缺血再灌注损伤，肾脏缺血再灌注损伤，和/或脑缺血再灌注损伤。特别优选为肝脏缺血再灌注损伤。
所述的炎症疾病和细胞死亡相关疾病包括但不限于：肝炎、心肌炎、心内膜炎、肾炎。
根据本发明，ALOX12抑制剂可以是抑制ALOX12蛋白质活性或蛋白质水平的抑制剂、或者抑制ALOX12的mRNA水平的抑制剂。所述抑制活性可以是可逆的或不可逆的。
抑制ALOX12蛋白质活性或蛋白质水平的抑制剂包括但不限于ALOX12的抗体、抑制ALOX12蛋白质活性或蛋白质水平的蛋白质、多肽、酶、小分子化合物。所述抑制ALOX12蛋白质活性或蛋白质水平的抑制剂是指可以结合ALOX12但在结合时不产生生物应答的物质。所述抑制剂可以阻断、抑制或减弱由激动剂介导的应答，并可与激动剂竞争结合ALOX12。
抑制ALOX12的mRNA水平的抑制剂可以是其反义核酸序列、siRNA、miRNA、shRNA、dsRNA，或者其它能够抑制ALOX12的mRNA水平的蛋白质、多肽、酶、化合物。
根据本发明，所述抗体包括但不限于单克隆抗体、合成抗体、多克隆抗体、多特异性抗体、人抗体、人源化抗体、嵌合抗体、单链Fv(scFv)(包括双特异性scFv)、单链抗体、Fab片段、F(ab')片段、二硫键连接的Fv(sdFv)和任何上述的表位结合片段。特别地，用于本发明的抗体包括免疫球蛋白分子和免疫球蛋白分子的免疫活性部分。用于本发明的免疫球蛋白分子可以是免疫球蛋白分子的任何类型(例如，IgG、IgE、IgM、IgD、IgA和IgY)、类别(例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类。优选地，抗体是人或人源化单克隆抗体。如本文所用，“人”抗体包括具有人免疫球蛋白的氨基酸序列的抗体，并且包括从人免疫球蛋白文库或从由人基因表达抗体的小鼠或其它动物分离的抗体。
根据本发明，所述抑制ALOX12蛋白质活性或蛋白质水平的小分子化合物可以是各种具有ALOX12抑制活性的化合物，包括但不限于baicalein、NDGA(去甲二氢愈创木酸)、US2017001955A1中公开的化合物(例如该专利申请中的化合物19、22、27、35-41、43、46-51、53、55-59、61-83，优选为化合物35，即ML355)，J Med Chem.2011August 11；54(15):5485–5497.Discoveryof Potent and Selective Inhibitors of Human Platelet type 12-Lipoxygenase中公开的化合物(例如该文献表1中的化合物1-17、22)。在此，所述文献全部引入参考。
在本发明的一个实施方式中，所述抑制剂为ML355
在本发明的另一个实施方式中，所述抑制剂为ALOX12的mRNA的shRNA，其干扰的靶向序列为GCATCGAGAGAAGGAACTGAA。
优选所述shRNA序列为：正向寡聚核苷酸:5’-CCGGGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGCTTTTTG-3’；反向寡聚核苷酸:5’-AATTCAAAAAGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGC-3’。
根据本发明，所述药物进一步包含药学上可接受的辅料。
所述药学上可接受的辅料是制药领域中常用或已知的各种辅料，包括但不限于：稀释剂、粘合剂、抗氧化剂、pH调节剂、防腐剂、润滑剂、崩解剂等。
所述稀释剂例如：乳糖、淀粉、纤维素衍生物、无机钙盐、山梨醇等。所述粘合剂例如：淀粉、明胶、羧甲基纤维素钠、聚乙烯吡咯烷酮等。所述抗氧化剂例如：维生素E、亚硫酸氢钠、亚硫酸钠、丁羟基茴香醚等。所述pH调节剂例如：盐酸、氢氧化钠、柠檬酸、酒石酸、Tris、乙酸、磷酸二氢钠、磷酸氢二钠等。所述防腐剂例如：对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、间甲酚、苯扎氯铵等。所述润滑剂例如：硬脂酸镁、微粉硅胶、滑石粉等。所述崩解剂例如：淀粉、甲基纤维素、黄原胶、交联羧甲基纤维素钠等。
本发明药物的剂型可以是口服剂的形式，例如片剂、胶囊、丸剂、粉剂、颗粒剂、悬浮剂、糖浆剂等；也可以是注射给药的剂型，例如注射液、粉针剂等，通过静脉内、腹膜内、皮下或肌肉内的途径。所有使用的剂型形式都是药学领域普通技术人员所熟知的。
本发明的药物可施用于会发生或已经发生缺血再灌注损伤的任何动物。这些动物包括人类和非人类的动物，例如宠物或牲畜等。
本发明的药物可以本领域已知的途径施用于受试者，包括但不限于口服，胃肠外，皮下，肌内，静脉内，腹腔内，肝内，心肌内，肾内，阴道，直肠，颊，舌下，鼻内，透皮方式等。
施用的剂量将取决于接受者的年龄、健康和体重，联用药物的种类，治疗频率，给药途径等。药物可以单一日剂量施用，或者总日剂量以每天两次，三次或四次的分开剂量施用。药物可以手术前、手术中、手术后施用。剂量可以施用一次或多次，施药时间可以单日至几个月或更长时间。
根据本发明，所述药物还可以与其他能改善或抑制缺血再灌注损伤、炎症反应的药物联合使用。
本发明的第二个方面是提供，表达靶向于ALOX12的mRNA的shRNA的载体在制备药物中的应用，所述药物用于治疗缺血再灌注损伤及相关疾病，或者治疗炎症疾病和细胞死亡相关疾病。
所述疾病的定义同前。
所述shRNA干扰的靶向序列为GCATCGAGAGAAGGAACTGAA或其他可干扰ALOX12表达的靶向序列。
优选所述shRNA序列为：正向寡聚核苷酸:5’-CCGGGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGCTTTTTG-3’；反向寡聚核苷酸:5’-AATTCAAAAAGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGC-3’。
所述载体可以是表达载体。表达载体中可以包含与上述shRNA序列可操作地连接的启动子和转录终止序列。
所述表达载体可以是真核细胞表达载体。
所述真核细胞表达载体可以是质粒表达载体或病毒表达载体。
所述质粒表达载体可以是但不限于pcDNA3.1+/-，pcDNA4/HisMax B，pSecTag2 A，pVAX1，pBudCE4.1，pTracer CMV2，pcDNA3.1(-)/myc-His A，pcDNA6-Myc/His B，pCEP4，pIRES，pIRESneo，pIRES hyg3，pCMV-myc，pCMV-HA，pIRES-puro3，pIRES-neo3，pCAGGS，pSilencer1.0，pSilencer2.1-U6 hygro，pSilencer3.1-H1 hygro，pSilencer3.1-H1 neo，pSilencer4.1-CMV neo。
所述病毒表达载体可以为慢病毒载体、腺病毒载体、腺相关病毒表达载体或其他类型病毒载体，包括但不限于pLKO.1，pLVX-IRES-ZsGreen1，pCDH-EF1-Luc2-T2A-tdTomato，pCDH-MSCV-MCS-EF1-Puro，pCDH-MSCV-MCS-EF1-copGFP，pLVX-ZsGreen1-C1，pAdEasy-1，pShuttle-CMV，pShuttle，pAdTrack，pAdTrack-CMV，pShuttle-IRES-hrGFP-1，pShuttle-IRES-hrGFP-2，pShuttle-CMV-lacZ，pShuttle-CMV-EGFP-C，pXC1，pBHGE3，pAAV-MCS，pAAV-RC，pHelper，pAAV-LacZ，优选pLKO.1载体。
本发明的第三个方面是提供，含有靶向于ALOX12的mRNA的shRNA的慢病毒载体在制备药物中的应用，所述药物用于治疗缺血再灌注损伤及相关疾病，或者治疗炎症疾病和细胞死亡相关疾病。
所述疾病的定义同前。
所述shRNA干扰的靶向序列为GCATCGAGAGAAGGAACTGAA或其他可干扰ALOX12表达的靶向序列。
优选所述shRNA序列为：正向寡聚核苷酸:5’-CCGGGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGCTTTTTG-3’；反向寡聚核苷酸:5’-AATTCAAAAAGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGC-3’。
优选所述慢病毒载体为pLKO.1载体。
能用于制备所述第二和第三个方面所述的药物的药用载体，可以是本领域中常规使用的注射液载体，如等渗的NaCl溶液，等渗的葡萄糖溶液，或等渗的含有缓冲体系的溶液，如PBS溶液等。也可以根据制剂需要，选择性的添加防止慢病毒发生物理或化学变化而失活的保护剂，例如二价阳离子盐或表面活性剂等。
附图说明
图1A：肝脏缺血再灌注损伤中，ALOX12、ALOX5、ALOX15的mRNA表达量RT-PCR的检测结果图(n.s.表示P≥0.05，**表示P<0.01)。
图1B：肝脏缺血再灌注损伤中，ALOX12、ALOX5、ALOX15的蛋白表达量western-blot的检测结果图。图中GAPDH为对照标准品。
图2：L02细胞经GFP及ALOX12过表达慢病毒转染后ALOX12蛋白表达情况鉴定图。图中GAPDH为对照标准品。
图3：ALOX12过表达对H/R处理诱导的L02细胞损伤中，LDH释放检测结果(n.s.表示P≥0.05，*表示0.01≤P<0.05，**表示P<0.01)。
图4：ALOX12过表达对H/R处理诱导的L02细胞损伤中，炎症因子Il-6、Tnf-α及趋化因子Ccl2、Cxcl10的mRNA的检测结果(n.s.表示P≥0.05，*表示0.01≤P<0.05，**表示P<0.01)。
图5：H9C2细胞经shRNA及shALOX12慢病毒转染后ALOX12蛋白表达情况鉴定图。图中GAPDH为对照标准品。
图6：ALOX12敲低对H/R处理后H9C2细胞活性的影响(n.s.表示P≥0.05，**表示P<0.01)。
图7：HK2细胞经GFP及ALOX12过表达慢病毒转染后ALOX12蛋白表达情况鉴定图。图中GAPDH为对照标准品。
图8：ALOX12过表达对H/R处理诱导的HK2细胞损伤中，LDH释放检测结果(n.s.表示P≥0.05，*表示0.01≤P<0.05，**表示P<0.01)。
图9A和9B：肝脏缺血再灌注损伤中，给药ML355 3mg/kg后，随时间推移小鼠血清中ALT、AST的检测结果(n.s.代表P≥0.05，*代表0.01≤P<0.05，**代表P<0.01)。
图10：肝脏缺血再灌注损伤中，给药ML355 3mg/kg后，随时间推移肝脏组织TUNEL染色结果，图中白色细胞代表凋亡的细胞。
图11A和11B：肝脏缺血再灌注损伤中，给药ML355 3mg/kg后，随时间推移肝脏Mac1和Ly6G阳性炎症细胞免疫荧光染色结果，图中浅灰色细胞代表炎症细胞。
具体实施方式
以下结合实施例对本发明做进一步描述。需要说明的是，实施例不能作为对本发明保护范围的限制，本领域的技术人员理解，任何在本发明基础上所作的改进和变化都在本发明的保护范围之内。
以下实施例所用化学试剂都是常规试剂，均可商购获得。未做特殊说明的实验方法都是采用本领域已知的常规方法。
在以下实施例中所采用的动物模型及各研究指标的检测方法：
实验动物：选用8-10周龄、体重在24g-27g、背景为雄性C57BL/6品系的野生型小鼠(购自北京华阜康生物科技股份有限公司)为实验对象。
动物饲养:所有实验小鼠均饲养在武汉大学SPF级实验动物中心。饲养条件：室温在22-24℃之间，湿度在40-70％之间，明暗交替照明时间为12h，自由饮水摄食。
HEK293T，人胚肾细胞，购自中国科学院细胞库，目录号GNHu43。
L02，人肝细胞系，购自中国科学院细胞库，目录号GNHu6。
H9C2，大鼠心肌细胞，购自中国科学院细胞库，目录号GNR5。
HK2，人肾近曲小管细胞HK-2，购自中国科学院细胞库，目录号SCSP-511。
细胞均培养于DMEM高糖培养基(含10％FBS，1％青霉素-链霉素)中。培养环境：37℃，5％CO2。
小鼠肝缺血再灌注(ischemia/reperfusion，I/R)损伤模型构建：
1)手术前12h给小鼠禁食，可自由饮水。
2)手术前用3％戊巴比妥钠成功麻醉小鼠后，将其平卧固定肢体，用剃毛器将小鼠腹部术区毛刮净，用10％碘酒和75％乙醇对术区消毒。
3)取腹正中切口进腹，暴露肝脏左、中叶之肝蒂。
4)用无创血管夹夹闭中叶和左叶的门静脉和肝动脉，使约70％的肝脏缺血，以防止发生严重肠系膜静脉淤血。0.5min后，与非阻断的右叶相比，可见阻断叶变白，说明阻断成功。此时，注意记录缺血开始时间，维持缺血60min，期间用湿的盐水纱布覆盖切口，并注意小鼠的保温(Sham组的小鼠与手术组小鼠平行操作，但不进行血流阻断)。
5)缺血60min后去除血管夹，恢复缺血的肝脏血流，然后关闭腹腔。手术后小鼠置单独饲养，观察。
取材：于术后1h取假手术组(Sham)以及缺血再灌注组小鼠，3％戊巴比妥钠过量麻醉，取缺血区肝组织，立即放入液氮中30min以上，之后保存于-80℃冰箱中，用于RT-PCR及Western blot分析。
此外，分别于术后0h、1h、3h、6h、12h、24h取假手术组(Sham组)以及缺血再灌注组小鼠，3％戊巴比妥钠麻醉，眼眶静脉丛取血1mL，分离血清。同时统一取缺血区肝左叶组织分别置于液氮中进行速冻或于10％中性福尔马林中固定24h后脱水，包埋，制作石蜡切片。
分离血清：收集血液的EP管于室温下静置1-2h使血液自然凝固。4℃、4000rpm/min离心30min，充分分离血清，保存于-80℃冰箱备用。
肝脏缺血再灌注损伤严重程度的评估指标主要包括肝功能指标(AST、ALT)、炎症反应、细胞死亡等，均与肝脏缺血再灌注损伤严重程度正相关。
采用全自动生化分析仪(Sysmex，Chemix 180i)测定小鼠血清的ALT、AST含量。
对石蜡切片采用HE染色，显微镜拍照，观察肝脏病理改变。
采用用TUNEL试剂盒染色石蜡切片检测肝细胞凋亡情况：TUNEL试剂盒：Plus In Situ Apoptosis Fluorescein Detection Kit(S7111，Chemicon)。按照试剂盒说明书进行操作。
用Ly6G和MAC1免疫荧光染色检测缺血再灌注后肝脏炎症细胞浸润情况，具体步骤如下：
1)将石蜡切片置于烤箱中，60℃烤片30分钟；
2)二甲苯，5分钟×3；
3)100％乙醇，5分钟×2次；95％乙醇，5分钟；70％乙醇，5分钟；
4)ddH2O漂洗，5分钟×2次；
5)柠檬酸盐组织抗原修复液(100×，pH6.0，福州迈新)高压修复5min；
6)ddH2O漂洗5分钟×2次，PBS漂洗5分钟×2次；
7)组化笔划圈，滴加10％羊血清(GTX27481，GeneTex)封闭，于湿盒中37℃封闭60min；
8)弃封闭液，滴加适当比例稀释的一抗：兔抗Mac1(CD11b，1:100稀释，ab75476，Abcam)；大鼠抗Ly6G(1:100稀释，551459，BD Biosciences)，4℃孵育过夜；
9)37℃复温30min；
10)弃去一抗，PBS洗10min×3；
11)滴加二抗(羊抗兔IgG，Invitrogen；羊抗大鼠IgG，Carlsbad)，于湿盒中37℃孵育60min；
12)弃去二抗，PBS浸洗5min×3；
13)SlowFade Gold antifade reagent with DAPI封片
14)荧光镜(OLMPUS DX51)下观察，拍照，用Image Pro Plus(version 6.0)软件进行图片分析。
RT-PCR：
组织中RNA的提取
①取100mg组织，放入1ml玻璃匀浆器中，加入1ml TRizol，在冰浴中研磨，悬液转入1.5ml离心管中，室温静置5min，使核蛋白完全从核酸上解离；
②4℃12000r/min离心5min，取上清液，加氯仿200μl，漩涡混匀器震荡30s，冰盒上静置10min；
③4℃12000r/min离心15min，取上清液，加入0.5ml异丙醇，充分混匀，冰盒上静置10min，使RNA充分沉淀；
④4℃12000r/min离心15min，弃去上清，加入1ml预冷的75％乙醇，漩涡混匀器震荡30s洗涤RNA沉淀；
⑤4℃12000r/min离心5min，弃去上清液，沉淀快速风干。提取RNA加适量的DEPC去离子水溶解。
细胞中RNA的提取
收集细胞并用PBS缓冲液洗涤2次，完成后加入1mlTRizol，用加样器吹打均匀，吸入1.5ml离心管中，漩涡混匀器震荡30s，室温静置5min，使核蛋白完全从核酸上解离。其余操作步骤同组织中RNA提取②-⑤。
反转录
使用Transcriptor First Strand cDNA Synthesis Kit(04896866001,Roche,Basel,Switzerland)反转录试剂盒根据试剂盒说明书进行反转录实验。
Western blot：
1)组织蛋白提取
①向于干冰中预冷的EP管中放入3-4颗钢珠，并加入称重定量之后的组织样本。
②向裂解液中加入PMSF，混匀后加至样品中，迅速摇匀。
③于-80℃预冷研磨仪适配器中研磨样品，研磨参数设置为30Hz/s，90s。
④研磨结束后，冰上放置10min，取出钢珠。
⑤超声裂解仪裂解样本(5KHz/次，每次1s，间隔1s，重复10次)，超声完成后冰上放置10min。
⑥样本放入4℃预冷的离心机中，12000rpm/min离心30min。
⑦吸取上清转移到新的EP管中，4℃，14000rpm/min离心10min。
⑧吸取上清转移到新的EP管中继续离心，4℃，14000rpm/min离心5min。
⑨准确吸取上清液并利用BCA Protein Assay Kit(PierecTM，23225)进行蛋白定量。
2)细胞中蛋白提取
细胞加入裂解液，裂解完成后离心取上清，运用BCA Protein Assay Kit定量收集蛋白样品。
3)上样与电泳
配置好电泳凝胶，并在电泳槽内加入电泳液。把蛋白样品上样到SDS-PAGE胶加样孔内，点样完成后开始电泳。
4)转膜
①配制转膜液，于4℃预冷。
②将PVDF在甲醇中浸泡15s后放入转膜液中备用。
③取出凝胶板中的凝胶，用转膜液洗涤凝胶，将凝胶平铺在负极的滤纸上，将PVDF膜覆盖其上，夹上夹板。
④将夹板放入转膜槽中，灌满转膜液以淹没凝胶。
⑤转膜槽接通电源，电压设为250V，电流设为0.2A。转移1.5h。
⑥转移结束后，取出PVDF膜。
5)封闭
把蛋白膜放置到预先准备好的TBST中，洗去膜上的转膜液。蛋白膜放入封闭液中，在摇床上缓慢摇动，室温封闭1-4h。
6)一抗孵育
①用TBST洗涤蛋白膜3次，每次5min。
②封口机将薄膜封入杂交袋中，加上一抗。
③将杂交袋放入4℃摇床中，过夜。
7)二抗孵育
①将薄膜取出用TBST洗涤3次，每次5min，回收一抗。
②将膜放入对应的加有二抗的二抗稀释液中，避光孵育1h。
8)蛋白检测
孵育后用TBST洗涤3次，每次5min。利用Bio-Rad Chemi Doc XRS+凝胶成像系统检测目的条带。
ALOX12过表达质粒构建：
1)PCR扩增ALOX12基因，引物为：
正向：5’-TCGGGTTTAAACGGATCCATGGGCCGCTACCGCATCCG-3’；
反向；5’-GGGCCCTCTAGACTCGAGTCAGATGGTGACACTGTTCT-3’；
2)PCR产物进行琼脂糖凝胶电泳，随后使用DNA凝胶回收试剂盒(天根)进行DNA片段的回收；
3)将所得DNA产物和限制性内切核酸酶FastDigest restriction enzymes(Thermo)、buffer orGreen buffer、ddH2O混合均匀(50μl体系)，置于37℃条件下反应。使用AxyPrepTM PCR Clean-Up Kit(Axygen)回收酶切产物；
4)使用PCR一步定向克隆试剂盒(Novoprotein)按照试剂盒说明书进行重组反应；
5)制作大肠杆菌感受态细胞，将上述连接产物进行转化实验，涂板，置于37℃培养箱，过夜培养；
6)从37℃培养箱中取出过夜培养的平板，挑克隆摇菌，并检测菌落PCR阳性克隆；
7)将PCR鉴定为阳性的菌液吸取5-10μl接种至5ml LB(含抗性)培养基中，在220rpm，37℃摇床中过夜培养；
8)取出过夜培养的菌液，对已经混浊的菌液进行质粒提取(天根质粒DNA小提试剂盒)；
9)提取后的质粒可直接用于ALOX12瞬转或构建慢病毒稳转细胞系。
ALOX12干扰质粒构建：
1)ALOX12靶向干扰序列为GCATCGAGAGAAGGAACTGAA，设计适合pLKO.1载体的寡聚核苷酸；正向寡聚核苷酸:5’CCGGGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGCTTTTTG 3’；反向寡聚核苷酸:5’AATTCAAAAAGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGC 3’；阴性对照siRNA序列为：CAACAAGATGAAGAGCACCAA；
2)将上述两条寡聚核苷酸分半加无菌水溶解，终浓度为100mM，进行融合；
3)根据“ALOX12表达质粒构建”步骤进行酶切反应、酶切产物的回收、连接反应、转化、挑单克隆、测序和提取质粒；
4)所得质粒可用于慢病毒介导的ALOX12敲低细胞系构建；
慢病毒载体构建和包装：
1)用胰酶消化并记数293T细胞，按1×106个293T/孔传至6孔板中；
2)第二天细胞汇合度至80％时开始转染；
3)取1.5ml灭菌EP管，加入2个包装质粒(pSpax和pMD2G)和过表达或干扰质粒各1μg溶于100μl的无血清培养基。轻柔混匀，室温孵育5min。
4)取1.5ml灭菌EP管，取3μl PEI(1.6μg/μl)溶于100μl无血清培养基中。轻柔混匀，室温孵育5min。
5)将DNA溶液和PEI溶液轻柔混匀，室温孵育15min；
6)将上述DNA-PEI混合液，逐滴加入6孔板中；
7)转染6h后，换新鲜培养基；
8)转染后48-72h收获含病毒的上清，3000rpm离心10min，去除沉淀，并用0.45μm的滤膜过滤；
9)过滤后的病毒可立即用于感染或-80℃贮存。
细胞缺氧复氧(H/R)：
1)细胞培养至对数期，预温PBS洗2次，弃去；
2)将细胞分成正常对照组和H/R实验组，对照组换完全培养基，放37℃，5％CO2培养，实验组换无糖无血清的DMEM培养基，并放O2/CO2细胞培养系统的培养箱内(37℃，5％CO2，5％O2)缺氧培养，1h后，实验组换完全培养基复氧培养；
3)复氧至预定的复氧培养时间后，弃去上清，用PBS洗2次，保存；
LDH释放及细胞活性(cell viability)检测：
使用LDH细胞毒性比色测试试剂盒(G1782,Promega,Madison,WI,USA)检测LDH的释放量。使用非放射性CCK-8试剂盒(CK04；Dojindo,Kumamoto,Japan)检测细胞活性。按照说明书进行相关检测。
实施例1不同ALOX在缺血肝脏组织中表达变化
C57小鼠随机分为2组，分别为Sham组及手术组，取缺血1h后手术组小鼠以及Sham组小鼠肝脏组织，Western blot及RT-RCR检测肝脏组织中ALOX12、ALOX5、ALOX15蛋白含量以及mRNA含量。其中WB所用一抗为：12-LO Antibody(C-5)(sc-365194；Santa Cruz),15-LOAntibody(B-7)(sc-133085；Santa Cruz),5-Lipoxygenase(C49G1)Rabbit mAb(#3289；CST)，二抗为：Peroxidase AffiniPure goat anti-rabbit-IgG(H+L)(#111-035-003；Jackson Laboratory)和goat anti-mouse-IgG(H+L)(#115-035-003；JacksonLaboratory)；RT-RCR所用引物序列如下：
| 基因 | 正向引物 | 反向引物 |
| ALOX12 | TCCCTCAACCTAGTGCGTTTG | GTTGCAGCTCCAGTTTCGC |
| ALOX5 | AACGATCACCCACCTTCTGC | TCGCAGATAAGCTGTTCCCG |
| ALOX15 | GCTGCCCAATCCTAATCAGTC | TTCCTTATCCAAGGCAGCCAG |
结果如图1A和1B所示，肝脏缺血1h后，肝脏组织中ALOX12基因mRNA含量相对于Sham组显著上升，约为Sham组的2.6倍，而ALOX5及ALOX15的mRNA含量相比于Sham组则上升不明显(图1A)。与RT-PCR结果相似，WB分析三种不同ALOX蛋白表达量，结果显示ALOX12蛋白在缺血1h后表达量相比于Sham组显著增加，而ALOX5及ALOX15蛋白表达量则增加不明显(图1B)。
上述RT-PCR及WB结果一致地表明了，在肝脏缺血后，肝脏组织中不同ALOX表达量变化间存在差异，且ALOX12表达量增加最明显。表明相比于ALOX的其他主要成员，肝脏组织缺血再灌注损伤和ALOX12之间关联更明显。
实施例2ALOX12过表达对H/R处理诱导的L02细胞损伤及炎症反应的影响
L02细胞分为4组：GFP过表达对照组、ALOX12过表达对照组、GFP过表达H/R组、ALOX12过表达H/R组。对应质粒分别转染贴壁L02细胞(融合度约80％)，24h后进行H/R处理(缺氧6h，复氧6h)。质粒转染完成后提取细胞总蛋白，进行WB分析(3次独立重复实验，每次2个重复)，检测ALOX12的过表达情况。H/R处理完成后，检测培养基中LDH的释放量(每组6个重复)，以评价ALOX12过表达对H/R诱导的肝细胞损伤的影响；提取RNA进行RT-PCR分析(2次独立重复实验，每次3个技术重复)，检测炎症相关细胞因子及趋化因子mRNA含量变化，以评价ALOX12过表达对H/R诱导的肝细胞炎症反应的影响。以GFP过表达对照组LDH释放检测结果以及炎症相关因子mRNA含量为1，计算其余各组相比于该组的比值。
RT-RCR所用引物序列如下：
| 基因 | 正向引物 | 反向引物 |
| Il6 | TCTGGATTCAATGAGGAGACTTG | GTTGGGTCAGGGGTGGTTAT |
| Tnfα | TACTCCCAGGTCCTCTTCAAGG | TTGATGGCAGAGAGGAGGTTG |
| Ccl2 | GTCTCTGCCGCCCTTCTG | ACTTGCTGCTGGTGATTCTTCT |
| Cxcl10 | GTGGCATTCAAGGAGTACCTC | TGATGGCCTTCGATTCTGGATT |
ALOX12过表达WB检测结果如图2所示，相比于GFP组，ALOX12过表达组蛋白条带显著增强，即L02细胞中ALOX12过表达显著。
LDH释放检测结果如图3所示，ALOX12过表达对照组LDH释放与GFP过表达对照组相比无显著差异，表明ALOX12的过表达对正常培养的L02细胞无影响。当进行H/R处理后，LDH的释放量显著增加，且ALOX12过表达组LDH的释放量的增加程度显著高于GFP组。这一结果表明，ALOX12过表达加重H/R处理引起的肝细胞损伤及肝细胞毒性。
炎症因子及趋化因子mRNA检测结果如图4所示，与LDH释放检测结果相同，ALOX12过表达对照组炎症因子Il-6、Tnf-α，趋化因子Ccl2、Cxcl10的mRNA含量与GFP过表达对照组相比无显著差异，表明ALOX12的过表达对正常培养的L02细胞炎症反应无影响。当进行H/R处理后，各因子mRNA含量显著增加，且ALOX12组的增加量显著大于GFP组。这一结果表明，ALOX12过表达加重H/R处理引起的肝细胞炎症反应。
实施例3ALOX12敲低(shALOX12)对H/R处理后H9C2细胞活性的影响
H9C2细胞分为4组：shRNA对照组、shALOX12对照组、shRNA H/R组、shALOX12 H/R组。对应的重组慢病毒病毒液分别感染培养的H9C2细胞，24h后进行H/R处理(缺氧1h，复氧6h)。质粒转染完成后提取细胞总蛋白，进行WB分析(3次独立重复实验)，检测ALOX12的敲低情况。H/R完成后检测细胞活性(每组6个重复)。以shRNA对照组检测结果为1，计算其余各组相比于该组的比值。
ALOX12敲低WB检测结果如图5所示，相比于shRNA组，shALOX12组蛋白条带显著减弱，即H9C2细胞中ALOX12表达被敲低。
细胞活性检测结果如图6所示，shALOX12对照组细胞活性相比于shRNA对照组无显著差异。当对H/R的两组细胞进行H/R处理后，shRNA组细胞活性相比于对照组显著降低。而当ALOX12的表达被敲低后，shALOX12 H/R组细胞活性的降低程度显著低于shRNA H/R组。这一结果表明ALOX12表达的降低可显著缓解H/R诱导的心肌细胞损伤，维持心肌细胞的活性。即ALOX12可促进心肌细胞损伤相关疾病的发生发展。
实施例4ALOX12过表达对H/R处理后HK2细胞损伤的影响
HK2细胞分为4组：GFP对照组、ALOX12对照组、GFP H/R组、ALOX12 H/R组。对应的慢病毒病毒液分别感染培养的HK2细胞，24h后进行H/R处理(缺氧3h，复氧24h)。质粒转染完成后提取细胞总蛋白，进行WB分析(3次独立重复实验)，检测ALOX12的过表达情况。H/R完成后检测培养基中LDH的释放量(每组6个重复)，以评价ALOX12过表达对H/R诱导的肾细胞损伤的影响。以GFP对照组LDH释放检测结果为1，计算其余各组相比于该组的比值。
ALOX12过表达WB检测结果如图7所示，相比于GFP组，ALOX12组蛋白条带显著增强，即HK2细胞中ALOX12过表达显著。
肾脏细胞LDH释放量检测结果如图8所示，ALOX12对照组LDH释放与GFP对照组相比无显著差异，表明ALOX12的过表达对正常培养的HK2细胞无影响。当进行H/R处理后，LDH的释放量显著增加，且ALOX12过表达组LDH的释放量的增加程度显著高于GFP组。这一结果表明，与ALOX12在肝脏、心肌细胞中作用相同，ALOX12的过表达可加重H/R处理引起的肾细胞毒性。
上述结果说明ALOX12在肝脏、心肌及肾脏细胞损伤及炎症相关疾病中发挥着重要的作用，其可显著促进相关缺血再灌注及其它可引起肝、心及肾细胞损伤的疾病的发生发展。
实施例5ML355有效减轻缺血再灌注引起的肝功能损伤
C57小鼠随机分为7组，每组20只。每组的其中10只小鼠通过尾静脉注射给以3mg/kg的溶于溶剂中的ML355(HY-12341，MCE公司)，另外10只给以溶剂(对照组，DMSO:Solutol:PEG400:水＝5:10:20:65(v:v:v:v))。给药完成后对7组小鼠进行I/R手术(其中一组为Sham对照组，剩余6组为I/R实验组)，分别取Sham对照组以及术后0h、1h、3h、6h、12h、24h I/R实验组小鼠血清，进行ALT、AST检测，以评价肝功能损伤程度；取Sham对照组以及术后0h、6hI/R实验组小鼠肝脏组织，制作石蜡切片，进行TUNEL染色、Mac1免疫荧光染色和Ly6G免疫荧光染色，以评价肝脏细胞的凋亡情况以及肝脏炎症细胞浸润情况。
ALT、AST检测结果如图9A和9B所示，Sham假手术组ALT、AST含量均较低，且ML355组与溶剂组之间无显著性差异。ML355组与溶剂组在I/R术后，随时间点的延长，ALT、AST含量逐渐升高，并在术后6h达到最高点，随后ALT、AST含量缓慢降低。而ML355组在术后1h、3h、6h、12h、24h，小鼠血清中ALT、AST含量均较溶剂组显著降低。
TUNEL染色结果如图10所示，随I/R术后时间的延长，肝脏细胞凋亡数量逐渐增多，在同一时间点，ML355组凋亡肝细胞数量显著低于溶剂组。
Mac1以及Ly6G免疫荧光染色结果如图11A和11B所示，I/R术后6h，相比于Sham组，Mac1阳性细胞数以及Ly6G阳性细胞数显著增多，说明I/R术后6h，小鼠肝脏中出现了明显的炎症细胞浸润情况；而相比于溶剂组，ML355组小鼠炎症细胞浸润情况显著降低。上述结果说明ML355可显著抑制缺血再灌注导致的肝脏损伤，减轻肝细胞凋亡，抑制炎症细胞浸润，保护肝功能。
由上述实验可见，ML355对肝脏缺血再灌注损伤的保护作用不仅起效快，并在足够长的时间内均有显著作用。
Claims (8)
1.ALOX12抑制剂在制备药物中的用途，所述药物用于治疗心脏缺血再灌注损伤及其相关疾病；
优选所述心脏缺血再灌注损伤及相关疾病的引发因素选自心肌梗塞、心梗再通损伤、心脏移植、冠状动脉溶栓术、经皮冠状动脉成形术，冠状动脉内扩张术和/或冠状动脉旁路术。
2.如权利要求1所述的用途，所述ALOX12抑制剂是抑制ALOX12蛋白质活性或蛋白质水平的抑制剂、或者抑制ALOX12的mRNA水平的抑制剂；
优选抑制ALOX12蛋白质活性或蛋白质水平的抑制剂选自ALOX12的抗体、抑制ALOX12蛋白质活性或蛋白质水平的蛋白质、多肽、酶、小分子化合物；
优选抑制ALOX12的mRNA水平的抑制剂是其反义核酸序列、siRNA、miRNA、shRNA、dsRNA，或者能够抑制ALOX12的mRNA水平的蛋白质、多肽、酶、化合物。
3.如权利要求2所述的用途，所述抑制剂为ALOX12的mRNA的shRNA；
优选所述shRNA干扰的靶向序列为GCATCGAGAGAAGGAACTGAA；
优选所述shRNA序列为：正向寡聚核苷酸:5’-CCGGGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGCTTTTTG-3’；反向寡聚核苷酸:5’-AATTCAAAAAGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGC-3’。
4.表达靶向于ALOX12的mRNA的shRNA的载体在制备药物中的应用，所述药物用于治疗心脏缺血再灌注损伤及相关疾病；
优选所述心脏缺血再灌注损伤及相关疾病的引发因素选自心肌梗塞、心梗再通损伤、心脏移植、冠状动脉溶栓术、经皮冠状动脉成形术，冠状动脉内扩张术和/或冠状动脉旁路术；
优选所述shRNA干扰的靶向序列为GCATCGAGAGAAGGAACTGAA；
优选所述shRNA序列为：正向寡聚核苷酸:5’-CCGGGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGCTTTTTG-3’；反向寡聚核苷酸:5’-AATTCAAAAAGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGC-3’。
5.如权利要求4所述的应用，所述载体是表达载体，包含与所述shRNA序列可操作地连接的启动子和转录终止序列；
优选所述表达载体是真核细胞表达载体；
优选所述真核细胞表达载体是质粒表达载体或病毒表达载体；
优选所述质粒表达载体选自pcDNA3.1+/-，pcDNA4/HisMax B，pSecTag2 A，pVAX1，pBudCE4.1，pTracer CMV2，pcDNA3.1(-)/myc-His A，pcDNA6-Myc/His B，pCEP4，pIRES，pIRESneo，pIRES hyg3，pCMV-myc，pCMV-HA，pIRES-puro3，pIRES-neo3，pCAGGS，pSilencer1.0，pSilencer2.1-U6 hygro，pSilencer3.1-H1 hygro，pSilencer3.1-H1 neo，pSilencer4.1-CMV neo；
优选所述病毒表达载体选自慢病毒载体、腺病毒载体、腺相关病毒表达载体，进一步优选为pLKO.1，pLVX-IRES-ZsGreen1，pCDH-EF1-Luc2-T2A-tdTomato，pCDH-MSCV-MCS-EF1-Puro，pCDH-MSCV-MCS-EF1-copGFP，pLVX-ZsGreen1-C1，pAdEasy-1，pShuttle-CMV，pShuttle，pAdTrack，pAdTrack-CMV，pShuttle-IRES-hrGFP-1，pShuttle-IRES-hrGFP-2，pShuttle-CMV-lacZ，pShuttle-CMV-EGFP-C，pXC1，pBHGE3，pAAV-MCS，pAAV-RC，pHelper或pAAV-LacZ。
6.含有靶向于ALOX12的mRNA的shRNA的慢病毒载体在制备药物中的应用，所述药物用于治疗心脏缺血再灌注损伤及相关疾病；
优选所述心脏缺血再灌注损伤及相关疾病的引发因素选自心肌梗塞、心梗再通损伤、心脏移植、冠状动脉溶栓术、经皮冠状动脉成形术，冠状动脉内扩张术和/或冠状动脉旁路术；
优选所述shRNA干扰的靶向序列为GCATCGAGAGAAGGAACTGAA；
优选所述shRNA序列为：正向寡聚核苷酸:5’-CCGGGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGCTTTTTG-3’；反向寡聚核苷酸:5’-AATTCAAAAAGCATCGAGAGAAGGAACTGAACTCGAGTTCAGTTCCTTCTCTCGATGC-3’。
7.如权利要求6所述的应用，所述慢病毒载体为pLKO.1载体。
8.如权利要求4-7任一项所述的应用，所述药物进一步含有药用载体；所述药用载体优选为注射液载体；优选所述载体为等渗的NaCl溶液，等渗的葡萄糖溶液，或等渗的含有缓冲体系的溶液。
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