细胞色素P450表氧化酶2J2基因过表达对大鼠肺缺血再灌注损伤的保护作用及其机制
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摘要
研究背景及目的
花生四烯酸(arachidonic acid，AA)是人体内含量最丰富，性质最活跃的多不饱和脂肪酸之一，在体内主要通过三种不同的途径进行酶解代谢，⑴AA经环氧化酶(cyclooxygenases，COX)途径代谢；⑵AA经脂质氧化酶(lipoxygenase，LOX)途径代谢；⑶AA经细胞色素P450(cytochrome P450，CYP450)氧化酶途径代谢CYP450氧化酶包括CYP450表氧化酶(cytochrome P450epoxygenase)亦有称之为环加氧酶途径和ω-羟化酶途径，CYP450表氧化酶是一个巨大的氧化酶超家族，CYP450表氧化酶主要包括2C和2J两类，目前已发现CYP450表氧化酶6个克隆的2J类型，其中在人类中仅发现了CYP2J2，通过细胞色素P450表氧化酶途径，花生四烯酸代谢生成环氧-二十碳三烯酸（epoxyeicosatrienoic acids，EETs），EETs在生物体内，尤其是在人体内的病理生理学及生物学意义，尚未被完全认知和阐明
既往大量研究证据表明：CYP表氧化酶-EETs系统主要在体内各个系统发挥以下几方面的作用：⑴EETs具有强大的扩张血管和调节血压功能；⑵EETs可显著下调由肿瘤坏死因子（tumor necrosis factor-α，TNF-α）所诱导的内皮细胞和心肌细胞的凋亡；⑶EETs可以显著抑制炎症反应中由TNF-α所诱导的内皮细胞炎症黏附分子的表达，减轻损伤；⑷EETs可以促进内皮细胞增殖，抑制内皮细胞凋亡，促进组织新生血管形成，⑸EETs可以使纤溶酶原激活物（tissue plasminogen activator，tPA）活化，这在维持体内血栓形成/溶栓的平衡过程中可能发挥重要作用；⑹EETs可以抑制主动脉平滑肌细胞由转移生长因子-β（transforming growth factor-β，TGF-β)所诱导的迁移；⑺缺氧-复氧过程可明显下调CYP2J2基因在血管内皮细胞中的表达，通过增加外源性EETs或CYP2J2过表达途经可以改善细胞缺氧-复氧损伤
肺缺血再灌注损伤(lung ischemia reperfusion injury，LIRI)一直是胸外科面临的重要问题，它在多种临床情况下发生，如肺移植ǎ心肺联合移植ǎ袖状肺叶切除ǎ肺动脉成形术ǎ体外循环ǎ肺栓塞ǎ休克以及心肺复苏等，肺缺血再灌注损伤可导致组织炎性细胞浸润增加，产生并释放大量氧自由基，损害细胞，它对于保护肺功能ǎ降低术后死亡率，延长移植器官的存活时间，减轻全身炎症反应综合症及多器官功能障碍综合征的发生，尤其是减少急性肺损伤导致的急性呼吸窘迫综合征具有重要意义近年来，我国肺癌的发病率和死亡率逐年攀升，目前已排在各种恶性肿瘤发病的第一位，越来越多的肺癌患者需要外科手术治疗，切除病变肺组织加系统淋巴结清扫依旧是目前能够最为彻底治疗肺癌的手段肺外科手术逐年增多，如何最大限度地为患者保存健肺以改善其长期的生活质量，如何为一部分肺功能较差的患者争取手术机会，如何尽可能的减轻外科手术操作过程中缺血再灌注损伤对残留健康肺组织的损害，最大可能保护肺功能，减少并发症的发生，改善患者生活质量等问题，就显得更加有意义了
缺血再灌注损伤的发生机制十分复杂，既往的大量研究表明：缺血再灌注损伤的发生发展与白细胞的活化ǎ多种促炎症反应介质释放ǎ多种炎症相关蛋白酶的释放，氧化应激反应的发生ǎ氧自由基增多ǎ细胞内钙超载ǎ多种转录因子的激活ǎ细胞膜分子上调，生物膜损伤以及保护性物质如NO和肺表面活性物质减少等诸多因素相关，随着对肺缺血再灌注损伤本质认识的不断深入，炎症反应与氧化应激机制参与了肺缺血再灌注损伤与血管内皮功能的紊乱的发生ǎ发展，并影响结局，从本质上讲，缺血再灌注损伤是一种炎症反应，炎症趋化因子表达的增加导致肺组织局部炎症细胞大量浸润，浸润的炎症细胞又导致氧化应激反应的发生，氧化应激又可以促进炎症细胞的募集，又进一步炎症粘附因子的表达增加，加重细胞损伤，促进细胞凋亡
基于以上结果，在本研究中我们设想，能否通过激活内源性保护机制，改善肺缺血再灌注损伤？我们首先在动物体内过表达CYP2J2基因，再构建CYP2J2基因过表达动物肺缺血再灌注损伤的模型，观察CYP2J2基因对肺缺血再灌注损伤的影响，达到预防和改善肺缺血再灌注损伤的目的本研究中，将携带CYP2J2目的基因的质粒导入Wistar大鼠体内，确认CYP2J2基因在大鼠体内获得稳定表达并代谢AA生成EET后，构建大鼠单侧肺缺血再灌注损伤模型，研究细胞色素P450表氧化酶-EETs系统对大鼠肺缺血再灌注损伤的影响及其可能的作用机制，旨在从整体动物水平阐明细胞色素P450表氧化酶在肺缺血再灌注损伤中的保护作用及其作用机制，试图探索研究CYP2J2能否抑制或阻断肺缺血再灌注损伤过程中的炎症反应与氧化应激，以期达到预防和治疗肺缺血再灌注损伤的目的，以期通过本研究为临床防治肺缺血再灌注损伤及更深入的研究提供一定的实验基础和理论支持
实验方法和结果
实验方法
1.采用无内毒素质粒大提试剂盒自大肠杆菌DH5α菌液中大量提取携带目的基因2J2的pcDNA3.1(+)-2J2表达载体质粒及携带对照基因GFP的pcDNA3.1(+)-GFP表达载体的质粒
2.健康雄性Wistar大鼠50只，12周龄，体重320g～380g，饲养于华中科技大学同济医学院实验动物中SPF级标准动物饲养室，适应性喂养1周随机分为5组：Blank组：空白对照组（Blank，10只）ǎSham组：单纯开胸组（Sham，10只）ǎIR组：左肺缺血再灌注组（IR，10只）ǎIR+GFP组：GFP基因导入+左肺缺血再灌注组（IR+GFP，10只）ǎIR+2J2组：2J2基因导入+左肺缺血再灌注（IR+2J2，10只）在行左肺缺血再灌注损伤造模手术操作前2周，按照3mg/kg体重/次，1次/周，共注射2次，经大鼠尾静脉注射携带目的基因的质粒溶液，IR+GFP组给予注射pcDNA3.1(+)-GFP质粒溶液，IR+2J2组给予注射pcDNA3.1(+)-2J2质粒溶液，Blank组ǎSham组和IR组在相同时间点经尾静脉注射同等体积生理盐水，第2次注射完成后1周进行造模手术操作，按分组要求进行手术，操作完毕后，处死动物，留取肺组织ǎ血液ǎ肺泡灌洗液等样本所有的针对动物的实验操作程序均符合中华人民共和国国家科学技术委员会ǎ实验动物管理条例ǐ及ǎ湖北省实验动物管理条例ǐ所制定的标准及规范
3.具体造模实验方法如下：①Blank组：不执行任何手术操作，麻醉后处死动物，留取标本；②Sham组：麻醉，气管插管，单纯左侧开胸，仅显露左肺门，注射肝素，暂时合拢手术切口，不执行肺缺血再灌注操作，观察180min后处死动物，留取标本；③IR组：麻醉，气管插管，左侧开胸，暴露左侧肺门，注射肝素，在肺充气状态下，显微无创血管夹阻断左侧肺门，暂时合拢手术切口，阻断60min后(模拟缺血)，松开血管夹，关闭胸腔，继续呼吸机辅助呼吸，观察120min后（模拟再灌注），处死动物，留取标本；④IR+GFP组：给予注射携带GFP基因的质粒，造模手术操作过程同IR组；⑤IR+2J2组：给予注射携带2J2目的基因的质粒，造模手术操作过程同IR组
4. Western blot检测各组大鼠肺组织中CYP2J2蛋白表达水平，ELISA方法检测大鼠肺组织中14,15-EET含量
5.处死动物前每组随机将半数动物用Millar导管经右侧颈静脉插管至右心室，记录右心室血流动力学指标，检测CYP2J2过表达对大鼠右心舒缩功能的影响
6.检测大鼠左肺组织湿干重比，检测血清与左肺BALF蛋白浓度之比，检测CYP2J2过表达对肺毛细血管通透性的影响
7.观察大鼠肺组织缺血再灌注损伤后大体及切片HE染色光镜下病理改变
8.观察大鼠肺组织缺血再灌注损伤后切片TUNEL染色光镜下细胞凋亡情况
9.检测大鼠血清及肺组织中炎症因子及炎症相关粘附因子的水平及CYP2J2过表达对炎症的影响：ELISA方法检测血清TNF-α，IL-1β，IL-8，IL-10，sP-selectin，sE-selectin的水平Western blot及实时荧光定量PCR检测肺组织中ICAM-1，VCAM-1的表达
10.检测大鼠血清及肺组织中氧化应激相关指标及CYP2J2过表达对氧化应激的影响：实时荧光定量PCR方法检测大鼠肺组织中eNOS，XOD，NADPH P22phox，NADPH P67phoxmRNA表达；ELISA方法检测血清COX的水平
11. Western blot检测大鼠肺组织标本中PI3K，Akt，p-Akt及NF κB-p65蛋白质的表达水平及CYP2J2过表达对其表达的影响
实验结果
1.通过经尾静脉注射质粒导入的方法，成功将CYP2J2基因导入大鼠体内，经WesternBlot及ELISA方法验证：CYP2J2基因在大鼠肺组织中获得稳定表达，并代谢AA产生EET，显著提高了肺组织中EETs水平
2. CYP2J2过表达对大鼠缺血再灌注后左肺组织炎症反应及细胞凋亡影响
大鼠左肺组织大体及石蜡切片HE染色光镜观察结果显示：缺血再灌注后大鼠肺泡腔内可见较多浆液性炎性渗出物，大量炎性细胞浸润，肺泡纤维间隔明显水肿ǎ增厚，部分区域间隔组织破坏；小叶间动脉充血ǎ扩张；部分肺泡腔内可见肺泡巨噬细胞浸润，支气管壁周围可见炎症细胞浸润；部分区域可见点状ǎ片状肺组织坏死，显示大鼠左肺缺血再灌注损伤模型制作成功；CYP2J2过表达可显著抑制大鼠左肺缺血再灌注损伤后炎性渗出，可明显减轻缺血再灌注损伤后肺组织内中性粒细胞ǎ巨噬细胞等炎症反应细胞的浸润，提示CYP2J2过表达可明显减轻肺缺血再灌注损伤导致的炎症反应
各组大鼠肺组织切片TUNEL染色及凋亡指数计算结果显示：与Blank组（AI=0.188%±0.0896%）及Sham组（AI=0.182%±0.0464%）比较，IR组（AI=3.732%±0.4896%），IR+GFP组（AI=3.622%±0.3656%），IR+2J2组（AI=2.236%±0.4968%），均显著升高，缺血再灌注损伤可诱导大鼠肺组织细胞发生明显凋亡（P＜0.01）；与IR组和IR+GFP组比较，IR+2J2组AI明显减小（P＞0.05），CYP2J2过表达显著减少了缺血再灌注损伤诱发的细胞凋亡
3. CYP2J2过表达对大鼠左肺缺血再灌注损伤后肺毛细血管通透性的影响
IR+2J2组大鼠左肺湿干重比值（W/D（）5.215±0.860）明显低于IR组的（7.061±1.189）和IR+GFP组的（8.048±0.489）（P＜0.05），但均显著高于Blank组（4.075±0.729）及Sham组（4.700±0.645）的W/D（P＜0.05），Blank组和Sham组之间，IR组和IR+GFP组之间W/D差异无统计学意义（P＞0.05）
IR+2J2组大鼠血清与左肺BALF蛋白浓度比值（30.306±2.4956）明显高于IR组（17.727±1.870）和IR+GFP组（18.414±1.993）（P＜0.05），但均小于Blank组（44.463±4.023）及Sham组（43.005±1.703），Blank组和Sham组之间，IR组和IR+GFP组之间血清与左肺BALF蛋白浓度比值差异无统计学意义（P＞0.05）
缺血再灌注损伤可导致肺毛细血管通透性明显增大，表现为肺湿干重比值增加及血清与BALF中蛋白浓度比值降低，CYP2J2过表达可明显降低肺毛细血管通透性，减轻缺血再灌注肺组织内蛋白液体的渗出
4.血清ǎ肺组织中炎症相关因子检测结果
ELISA方法检测大鼠血清中相关炎症因子水平的结果表明：CYP2J2基因过表达显著降低了缺血再灌注损伤后大鼠血清中炎症因子的水平IR+2J2组血清TNF-α水平（124.182±12.734pg/ml）显著低于IR组（191.554±19.603pg/ml）和IR+GFP组（183.246±14.443pg/ml），但均高于Blank组（41.224±12.443pg/ml）和Sham组（93.004±12.701pg/ml）；IR+2J2组血清IL-1β水平（74.393±9.029pg/ml）显著低于IR组（110.516±24.519pg/ml）和IR+GFP组（103.704±15.872pg/ml），但均高于Blank组（50.039±12.318pg/ml）和Sham组（62.018±12.319pg/ml）；IR+2J2组血清IL-8水平（55.330±6.608pg/ml）显著低于IR组（67.754±8.797pg/ml）和IR+GFP组（65.008±7.670pg/ml），但均高于Blank组（36.757±6.051pg/ml）；IR+2J2组血清IL-10水平（191.473±4.639pg/ml）显著高于IR组（125.792±6.256pg/ml）和IR+GFP组（123.279±7.453pg/ml），以上三组均高于Blank组（46.828±5.010pg/ml）和Sham组（53.987±4.925pg/ml）；IR+2J2组血清sP-Selectin水平（38.297±3.3527ng/ml）显著低于IR组（56.732±5.985ng/ml）和IR+GFP组（55.616±5.455ng/ml），但均高于Blank组（6.259±1.631ng/ml）和Sham组（19.951±4.166ng/ml） IR+2J2组血清sE-Selectin水平（1312.109±211.164pg/ml）显著低于IR组（1779.279±322.441pg/ml）和IR+GFP组（1775.263±261.194pg/ml），但均高于Blank组（500.193±162.362pg/ml）和Sham组（930.488±192.240pg/ml）
RT-PCR检测及western blot检测结果同样表明：缺血再灌注导致肺组织内ICAM-1ǎVACM-1表达水平显著上调；CYP2J2基因过表达显著抑制了缺血再灌注后ICAM-1ǎVACM-1表达水平的上调，从而抑制了单核巨噬细胞向内皮细胞的粘附以及以及向血管组织的迁移，减轻炎症损伤
5.血清ǎ肺组织中氧化应激相关指标检测结果
缺血再灌注损伤可导致血清中COX水平显著升高，CYP2J2基因过表达未能明显降低LIRI大鼠血清中COX水平；肺缺血再灌注损伤可导致大鼠肺组织中XODǎeNOSǎNADPH氧化酶P22phoxǎP67phox的表达上调，CYP2J2基因过表达显著抑制了肺缺血再灌注后XOD，eNOS，NADPH氧化酶P22phoxǎP67phox表达的上调
6. Western blot检测结果显示：左肺缺血再灌注损伤导致大鼠肺组织内PI3Kǎp-Akt的表达显著下调，而CYP2J2过表达的IR+2J2组PI3Kǎp-Akt的表达较IR组和IR+GFP组明显升高，但仍然低于未发生缺血再灌注损伤的Blank组和Sham组
左肺缺血再灌注损伤导致大鼠肺组织内NF-κB的表达显著上调，而CYP2J2过表达的IR+2J2组NF-κB的表达较IR组和IR+GFP组明显降低，但仍然高于未发生缺血再灌注损伤的Blank组和Sham组
7.左肺缺血再灌注可使心率明显减慢，CYP2J2基因过表达对大鼠缺血再灌注后心率减慢无显著影响；IR组ǎIR+GFP组及IR+2J2组大鼠右心室收缩压（RVSP）明显低于Blank组和Sham组（P<0.01），IR+2J2组RVSP显著高于IR组与IR+GFP组（P<0.05）；IR+2J2组右心室舒张期末压力（RVEDP）显著低于其余四组大鼠（P<0.05）；IR组ǎIR+GFP组及IR+2J2组大鼠右心室等容收缩期室内压上升最大速率（RV+dp/dt max，mmHg/s）ǎ右心室等容舒张期室内压下降最大速率（RV-dp/dt max，mmHg/s）明显低于Blank组及Sham组（P<0.01），IR+2J2组±dp/dt max显著高于IR组和IR+GFP组（P<0.05）
统计学分析
用SPSS19.0软件进行统计学分析，比较两组间差别计量资料用均数±标准差（x SD）进行统计学描述，采用均数t检验和单因素方差分析（ANOVA）比较两组组内ǎ组间的差别所有统计学检验均为双侧检验，P<0.05为差异有统计学意义
结论
1.成功构建了Wister大鼠在体左肺缺血再灌注损伤的动物模型
2.通过尾静脉注射质粒的方法，成功在Wister大鼠体内表达CYP2J2基因，并将花生四烯酸代谢为EETs，发挥生物学作用
3. CYP2J2基因过表达显著减少细胞凋亡，减轻了大鼠左肺缺血再灌注损伤
4. CYP2J2基因过表达明显改善了左肺缺血再灌注损伤所导致的大鼠右心室收缩和舒张功能的损害
5. CYP2J2基因过表达明显抑制了左肺缺血再灌注损伤所导致的血清促炎症因子及趋化因子水平的升高
6. CYP2J2基因过表达明显抑制了左肺缺血再灌注所导致的促氧化应激相关指标的升高，抑制了促氧化应激相关蛋白的表达
7. CYP2J2基因抗炎症和抑制氧化应激的作用可能和PI3K/AKT/NF-κB信号通路有关
Background and purpose
Arachidonic acid (AA) is the most widely distributed and the most abundantpolyunsaturated fatty in human body, in vivo, AA metabolic network is an important part ofthe multiple metabolic pathways and of endogenous bioactive substances, including oxidativestress network and inflammation signaling pathway. AA mainly metabolized through threedifferent enzymatic pathways, including the cyclooxygenase (COX) pathway, thelipoxygenase (LOX) pathway and the third important pathway of cytochrome P450(CYP)pathway which play an important roles in numerous physiological&pathophysiologicalprocesses.
Cytochrome P450epoxygenases(CYP2J and CYP2C) metabolize arachidonic acid (AA)to four different epoxyeicosatrienoic acids (EETs), namely5,6-EET,8,9-EET,11,12-EETand14,15-EET, which are converted to dihydroxyeicosatrienoic acids (DHETs) by solubleepoxide hydrolase(sEH).The cytochrome P450enzymes comprise a large superfamily ofproteins. CYP widely distributed in the body, the CYP epoxygenase including two types2Cand2J, CYP has been found for6clones type of oxidase2J, which the type CYP2J2is onlyfound in the human. After the years of research on CYP-EET system, evidence shows that CYP-EET system plays an important role in the human body.⑴EETs are with a strongexpansion effects of Cardiovascular function and regulate blood pressure;⑵EETs cansignificantly alleviate the apoptosis in endothelial cells and cardiomyocytes induced by tumornecrosis factor-α(TNF-α);⑶EETs can inhibit inflammation, inhibiting endothelial cellexpression of inflammatory adhesion molecules and protect the biological function ofendothelial cells;⑷EETs can promote endothelial cell proliferation, inhibits apoptosis ofendothelial cells and promotes tissue neovascularization,⑸EETs can activate plasminogenactivator(tPA), which may play an important role to maintain thrombosis/thrombolysisbalancing in vivo;⑹EETs can inhibit aortic smooth muscle cell migration induced by thetransforming growth factor-β(TGF-β);⑺hypoxia-reoxygenation process can significantlyinhibit CYP2J2gene expression in endothelial cells, By providing the exogenous EETs orCYP2J2overexpression in vivo can improve hypoxia-reoxygenation injury.
Lung ischemia-reperfusion injury (LIRI) is an important issues in the face of thoracicsurgery, It occurs in a variety of contexts such as lung transplantation, heart and lungtransplantation, lobe sleeve resection, pulmonary artery angioplasty, cardiopulmonary bypass,pulmonary embolism, shock and cardiopulmonary resuscitation, Lung ischemia-reperfusioninjury can result in the infiltration of inflammatory cells to increase, and Inflammatory cellsproduce and release large amounts of oxygen free radicals and thus damaging cells. relievesthe ischemia-reperfusion injury can protect lung function, reduce the post-operative mortalityand prolong the survival time of the transplanted organs, can reduce the occurrence ofsystemic inflammatory response syndrome and multiple organ dysfunction syndrome,especially to reduce acute respiratory distress syndrome(ARDS) caused by acute lunginjury(ALI).In recent years, the incidence and mortality rates of lung cancer are rising rapidlyin China; the incidence of lung cancer among malignant tumors has been ranked in the first.Removal of lesion tissue by surgical procedures is still the most thorough way to treat lungcancer. How to lessen IRI is a constantly focal point in lung cancer surgery study.
LIRI is a complex phenomenon involving not only intracellular injury processes, but also injurious inflammatory responses and biochemical changes, During ischemia-reperfusion,such as leukocyte activation, a variety of promoting the release of inflammatory reactionmedium, the release of inflammation-related protease, the occurrence of oxidative stress,intracellular calcium overload, activation of multiple transcription factors, membranemolecular raised, the biofilm injury and protective substances such as NO and decrease inpulmonary surfactant and other relevant factors, endothelial cells, and other immune cells togenerate ROS Calcium/calmodulin dependent nitric oxide synthases(NOS), nuclearfactor-κB (NF-κB), nicotinamide adenine dinucleotide phosphate (NADPH), andproinflammatory cytokines are activated, causing an upregulation of cell surface adhesionmolecules on the endothelial of the lung, along with lung ischemia-reperfusion injury in thenature of deepening understanding, inflammation and oxidative stress mechanisms involvedin lung ischemia-reperfusion injury and endothelial dysfunction, many studies have shownthat IRI and in essence, is an inflammatory response to ischemia-reperfusion injury,inflammatory chemokine expression resulting in lung tissue inflammatory cell infiltration,infiltration a large number of inflammatory cells and lead to the occurrence of oxidativestress, these are lung ischemia-reperfusion injury formed an important start contributingfactor.
So, can CYP2J2be used for the prevention of lung ischemia-reperfusion injury? Wesuppose that through over-expression the CYP2J2gene in animals model of lungischemia-reperfusion injury in order to increases the level of endogenous EETs;it couldactivate endogenous protective mechanisms of the vascular wall through the inhibition ofinflammation and oxidative stress to relieve lung ischemia-reperfusion injury. Thus, weconducted experiments to confirm the effects of CYP2J2overexpression on the animal’smodel of lung ischemia-reperfusion injury in rats, to explore CYP2J2ability to inhibit orblock the lung ischemia-reperfusion injury in the inflammatory response and oxidative stressand the possible mechanisms which exist in the protective effects of CYP2J2overexpression.
Methods and results
Methods
1Plasmid pcDNA3.1(+)-2J2and pcDNA3.1(+)-GFP were extracted and purificated byusing endotoxin-free plasmid mention kit.
2.50male Wistar rats,8weeks old, weight300-350g, subjected to a one-week adaptationperiod, were randomly divided into five groups, and each group has10rats: Blank group(Blank), Sham group (Sham), IR group (IR), IR+GFP group (IR+GFP), IR+2J2group(IR+2J2). The target gene pcDNA3.1-2J2and pcDNA3.1(+)-GFP as well as the equalvolume of saline were injected into the bodies of rats via tail veins, and the dose of plasmidinjection was3mg/kg body weight, injected once a week, a total of two injections. One weekafter the gene delivery, modeling surgery was performed. After the surgery was completed,the rats were sacrificed, Blood samples, bronchoalveolar lavage fluid(BALF) lung tissuesamples were collected and part of lung tissue were placed in liquid nitrogen immediately, allsamples transferred into the-80℃refrigerator, some of the lung tissues were fixed withformaldehyde solution, embedded in paraffin after dehydration at room temperature.
3. Blank group: do not perform any surgical procedure, the animals were sacrificed afteranesthesia; Sham group: left thoracotomymodel, before animals were sacrificed, we observethe animals180min; IR group:rats lung IRI model, the model were established by obstructingthe left hilum of lung, ischemia1h and2h of reperfusion，then animals were sacrificed;IR+2J2group: same perform just like the IR group; IR+GFP group: same perform just likethe IR group. Before sacrificed, half of animals were invented Millar catheter into rightventricle, in order to record right ventricular hemodynamic parameters.
4. Proteins from frozen lung tissue were extracted for western blot methods to detect proteinexpression levels of CYP2J2protein. And ELISA method was used to detect content of thelung tissue14,15-DHET.
5.The ratio of lung wet/dry（W/D） weight were measured to assess lung edema after IRI;Serum/BALF protein concentration ratio was detection; the paraffin sections of lung wereprepared to carry out HE stain, Immunohistochemical TUNEL staining was used to detect apoptosis of alveolar epithelial cell.
6. Detection of serum and lung tissue Inflammatory-related cytokines: ELISA was used todetect TNF-α, IL-1β, IL-8, IL-10, sP-selectin, sE-selectin levels in serum; western blot todetect protein expression levels of ICAM-1and VCAM-1inlung tissue.
7. Detection of oxidative stress in rat blood and lungtissue: Real-time quantitative PCRwasused to detect mRNA expression of oxidative stress related cytokines eNOS, XOD, NADPHp22phoxand NADPH P67phox; ELISA method to detect the serum levels of COX.
8. Western blot to detect protein expression levels of PI3K, Akt, p-Akt and NF κB-p65in ratlung tissue.
Results:
1. Western blot and ELISA results showed that after2weeks/2times CYP2J2genedelivery through tail vein into rat,the CYP2J2overexpression in rat lung;14,15-EET contentin lung of CYP2J2overexpression rats were significantly higher than the control group(P<0.05).
2. The results of TUNEL staining to detect apoptosis showed that, compared with Blankgroup and Sham group, the apoptotic index(AI) was significantly higher in IR group,IR+GFP group and IR+2J2group (P<0.01); The AI of IR+2J2group was significantly lessthan the IR group and IR+GFP group (P<0.05).CYP2J2overexpression significantlyinhibited ischemia-reperfusion injury induced apoptosis.
3. After1h ischemia and2h reperfusion, the ratio of W/D in IR+2J2group is (5.215±0.860),significantly lower than that (8.061±1.189) of IR group and (8.048±0.489) of IR+GFP group(P<0.05); both of three group were significantly higher than that in (4.075±0.729) of Blankgroup and (4.700±0.645) of Sham group (P<0.01), there was no significant differencebetween Blank group and Sham group, but also between IR group and IR+GFP group.After1h ischemia and2h reperfusion, the ratio of Serum/BALF protein concentrationwas significantly higher in IR+2J2(30.306±2.4956) than that in IR(17.727±1.870) andIR+GFP(18.414±1.993)(P<0.05); both of three group were significantly lower than that in Blank(44.463±4.023) and Sham (43.005±1.703)(P<0.01), there was no significant differencebetween Blank group and Sham group, also between IR group and IR+GFP group.
4. Results of Inflammatory-related cytokines test:
ELISA: detection of inflammatory cytokines in serum shows that CYP2J2overexpression significantly inhibited the serum levels of pro-inflammatory cytokines
The serum levels of TNF-α in IR+2J2group (124.182±12.734pg/ml) is significantlylower than that in IR group(191.554±19.603pg/ml) and IR+GFP group(183.246±14.443pg/ml), all these three group are significantly higher than that Blank group(41.224±12.443pg/ml) and Sham group (93.004±12.701pg/ml);
The serum levels of IL-1β in IR+2J2group (74.393±9.029pg/ml) is significantly lowerthan that in IR group(110.516±24.519pg/ml) and IR+GFP group(103.704±15.872pg/ml), allthese three group are significantly higher than that Blank group(50.039±12.318pg/ml) andSham group (62.018±12.319pg/ml);
The serum levels of IL-8in IR+2J2group(55.330±6.608pg/ml) is significantly lowerthan that in IR group(67.754±8.797pg/ml) and IR+GFP group(65.008±7.670pg/ml), but arehigher than that in Blank group(36.757±6.051pg/ml).
The serum levels of IL-10in IR+2J2group (191.473±4.639pg/ml) is significantlyhigher than that in IR group(125.792±6.256pg/ml) and IR+GFP group(123.279±7.453pg/ml), all these three group are significantly higher than that in Blank group (46.828±5.010pg/ml) and Sham group(53.987±4.925pg/ml).
The serum levels of sP-Selectin in IR+2J2group (38.297±3.3527ng/ml) is significantlylower than that in IR group (56.732±5.985ng/ml) and IR+GFP group (55.616±5.455ng/ml),all these three group are significantly higher than that in Blank group (6.259±1.631ng/ml)and Sham group (19.951±4.166ng/ml).
The serum levels of sE-Selectin in IR+2J2group (1312.109±211.164pg/ml) issignificantly lower than that in IR group (1779.279±322.441pg/ml) and IR+GFP group(1775.263±261.194pg/ml), all these three group are significantly higher than that in Blank group (500.193±162.362pg/ml) and Sham group (930.488±192.240pg/ml).
Western blot test results and Real-time quantitative PCR data of the mRNA expressionlevel showed that ICAM-1and VACM-1expression level was significantly upregulated afterlung ischemia-reperfusion; CYP2J2gene overexpression significantly inhibited the effects.
5. Results of oxidative stress related cytokines test
Real-time quantitative PCR result showed that XOD, eNOS, NADPH P22phox, NADPHP67phoxmRNA expression level was significantly upregulated after lung ischemia-reperfusion,CYP2J2gene overexpression can inhibited the effects.
6. Western blot test results showed that: Compared with Sham and Sham group, PI3K, p-Aktprotein expression level was obviously decreased after LIR, and NFκB-p65protein expressionlevel was obviously increased after LIR. CYP2J2gene overexpression significantly inhibitedthe changes of related protein expression above-mentioned.7. Hemodynamic records showed that LIRI induced significant decreased the right ventricleSystolic blood pressure (RVSP)and the maximum rate of right ventricular pressure (±dp/dtmin); CYP2J2overexpression reversed this effects induced by LIRIsignificantly; comparedwith other groups, CYP2J2overexpression decrease right ventricular end-diastolic pressure(RVEDP); LIRIinduced significant decrease in heart rate (HR), the CYP2J2overexpressiondid not show significant changes.
Statistical Analysis
All data were analyzed by SPSS19.0statistical software(IBM). Enumeration data wereexpressed as frequency or composition ratio， and the Chi-square test were used to evaluatethe differences between two groups. Values were present as mean standard deviation （x
SD）.Analysis of variance (ANOVA) was used to compare group differences. Student’s ttest was used to assess whether differences in the values of two groups were statisticallysignificant. The level for statistical differences was set at P<0.05.
Conclusion:
1. Successfully constructed an animal model in vivo of left lung ischemia-reperfusion injury in Wister rats
2. CYP2J2gene delivery in rat by plasmid tail vein injection received a stable expression,and increased the content of EETs in lung tissuesignificantly.
3. CYP2J2overexpression significantly reduces the left lung ischemia-reperfusion injuryand apoptosis in rats.
4. CYP2J2gene overexpression significantly improves right ventricular systolic anddiastolic function damage caused by the left lung ischemia-reperfusion injury.
5. CYP2J2overexpression significantly decreased the serum levels of proinflammationcytokines and increased the levels of anti-inflammatory cytokines in the LIRI rats.
6. CYP2J2overexpression inhibited the oxidative stress associated indicators (XOD,NADPH subunits) in blood, lung tissue significantly, and significantly upregulatedexpression of eNOS,
7. A possible mechanism that CYP2J2anti-inflammatory and inhibit oxidative stress byactivating PI3K/AKT/NF-κB signaling pathway.
花生四烯酸(arachidonic acid，AA)是人体内含量最丰富，性质最活跃的多不饱和脂肪酸之一，在体内主要通过三种不同的途径进行酶解代谢，⑴AA经环氧化酶(cyclooxygenases，COX)途径代谢；⑵AA经脂质氧化酶(lipoxygenase，LOX)途径代谢；⑶AA经细胞色素P450(cytochrome P450，CYP450)氧化酶途径代谢CYP450氧化酶包括CYP450表氧化酶(cytochrome P450epoxygenase)亦有称之为环加氧酶途径和ω-羟化酶途径，CYP450表氧化酶是一个巨大的氧化酶超家族，CYP450表氧化酶主要包括2C和2J两类，目前已发现CYP450表氧化酶6个克隆的2J类型，其中在人类中仅发现了CYP2J2，通过细胞色素P450表氧化酶途径，花生四烯酸代谢生成环氧-二十碳三烯酸（epoxyeicosatrienoic acids，EETs），EETs在生物体内，尤其是在人体内的病理生理学及生物学意义，尚未被完全认知和阐明
既往大量研究证据表明：CYP表氧化酶-EETs系统主要在体内各个系统发挥以下几方面的作用：⑴EETs具有强大的扩张血管和调节血压功能；⑵EETs可显著下调由肿瘤坏死因子（tumor necrosis factor-α，TNF-α）所诱导的内皮细胞和心肌细胞的凋亡；⑶EETs可以显著抑制炎症反应中由TNF-α所诱导的内皮细胞炎症黏附分子的表达，减轻损伤；⑷EETs可以促进内皮细胞增殖，抑制内皮细胞凋亡，促进组织新生血管形成，⑸EETs可以使纤溶酶原激活物（tissue plasminogen activator，tPA）活化，这在维持体内血栓形成/溶栓的平衡过程中可能发挥重要作用；⑹EETs可以抑制主动脉平滑肌细胞由转移生长因子-β（transforming growth factor-β，TGF-β)所诱导的迁移；⑺缺氧-复氧过程可明显下调CYP2J2基因在血管内皮细胞中的表达，通过增加外源性EETs或CYP2J2过表达途经可以改善细胞缺氧-复氧损伤
肺缺血再灌注损伤(lung ischemia reperfusion injury，LIRI)一直是胸外科面临的重要问题，它在多种临床情况下发生，如肺移植ǎ心肺联合移植ǎ袖状肺叶切除ǎ肺动脉成形术ǎ体外循环ǎ肺栓塞ǎ休克以及心肺复苏等，肺缺血再灌注损伤可导致组织炎性细胞浸润增加，产生并释放大量氧自由基，损害细胞，它对于保护肺功能ǎ降低术后死亡率，延长移植器官的存活时间，减轻全身炎症反应综合症及多器官功能障碍综合征的发生，尤其是减少急性肺损伤导致的急性呼吸窘迫综合征具有重要意义近年来，我国肺癌的发病率和死亡率逐年攀升，目前已排在各种恶性肿瘤发病的第一位，越来越多的肺癌患者需要外科手术治疗，切除病变肺组织加系统淋巴结清扫依旧是目前能够最为彻底治疗肺癌的手段肺外科手术逐年增多，如何最大限度地为患者保存健肺以改善其长期的生活质量，如何为一部分肺功能较差的患者争取手术机会，如何尽可能的减轻外科手术操作过程中缺血再灌注损伤对残留健康肺组织的损害，最大可能保护肺功能，减少并发症的发生，改善患者生活质量等问题，就显得更加有意义了
缺血再灌注损伤的发生机制十分复杂，既往的大量研究表明：缺血再灌注损伤的发生发展与白细胞的活化ǎ多种促炎症反应介质释放ǎ多种炎症相关蛋白酶的释放，氧化应激反应的发生ǎ氧自由基增多ǎ细胞内钙超载ǎ多种转录因子的激活ǎ细胞膜分子上调，生物膜损伤以及保护性物质如NO和肺表面活性物质减少等诸多因素相关，随着对肺缺血再灌注损伤本质认识的不断深入，炎症反应与氧化应激机制参与了肺缺血再灌注损伤与血管内皮功能的紊乱的发生ǎ发展，并影响结局，从本质上讲，缺血再灌注损伤是一种炎症反应，炎症趋化因子表达的增加导致肺组织局部炎症细胞大量浸润，浸润的炎症细胞又导致氧化应激反应的发生，氧化应激又可以促进炎症细胞的募集，又进一步炎症粘附因子的表达增加，加重细胞损伤，促进细胞凋亡
基于以上结果，在本研究中我们设想，能否通过激活内源性保护机制，改善肺缺血再灌注损伤？我们首先在动物体内过表达CYP2J2基因，再构建CYP2J2基因过表达动物肺缺血再灌注损伤的模型，观察CYP2J2基因对肺缺血再灌注损伤的影响，达到预防和改善肺缺血再灌注损伤的目的本研究中，将携带CYP2J2目的基因的质粒导入Wistar大鼠体内，确认CYP2J2基因在大鼠体内获得稳定表达并代谢AA生成EET后，构建大鼠单侧肺缺血再灌注损伤模型，研究细胞色素P450表氧化酶-EETs系统对大鼠肺缺血再灌注损伤的影响及其可能的作用机制，旨在从整体动物水平阐明细胞色素P450表氧化酶在肺缺血再灌注损伤中的保护作用及其作用机制，试图探索研究CYP2J2能否抑制或阻断肺缺血再灌注损伤过程中的炎症反应与氧化应激，以期达到预防和治疗肺缺血再灌注损伤的目的，以期通过本研究为临床防治肺缺血再灌注损伤及更深入的研究提供一定的实验基础和理论支持
实验方法和结果
实验方法
1.采用无内毒素质粒大提试剂盒自大肠杆菌DH5α菌液中大量提取携带目的基因2J2的pcDNA3.1(+)-2J2表达载体质粒及携带对照基因GFP的pcDNA3.1(+)-GFP表达载体的质粒
2.健康雄性Wistar大鼠50只，12周龄，体重320g～380g，饲养于华中科技大学同济医学院实验动物中SPF级标准动物饲养室，适应性喂养1周随机分为5组：Blank组：空白对照组（Blank，10只）ǎSham组：单纯开胸组（Sham，10只）ǎIR组：左肺缺血再灌注组（IR，10只）ǎIR+GFP组：GFP基因导入+左肺缺血再灌注组（IR+GFP，10只）ǎIR+2J2组：2J2基因导入+左肺缺血再灌注（IR+2J2，10只）在行左肺缺血再灌注损伤造模手术操作前2周，按照3mg/kg体重/次，1次/周，共注射2次，经大鼠尾静脉注射携带目的基因的质粒溶液，IR+GFP组给予注射pcDNA3.1(+)-GFP质粒溶液，IR+2J2组给予注射pcDNA3.1(+)-2J2质粒溶液，Blank组ǎSham组和IR组在相同时间点经尾静脉注射同等体积生理盐水，第2次注射完成后1周进行造模手术操作，按分组要求进行手术，操作完毕后，处死动物，留取肺组织ǎ血液ǎ肺泡灌洗液等样本所有的针对动物的实验操作程序均符合中华人民共和国国家科学技术委员会ǎ实验动物管理条例ǐ及ǎ湖北省实验动物管理条例ǐ所制定的标准及规范
3.具体造模实验方法如下：①Blank组：不执行任何手术操作，麻醉后处死动物，留取标本；②Sham组：麻醉，气管插管，单纯左侧开胸，仅显露左肺门，注射肝素，暂时合拢手术切口，不执行肺缺血再灌注操作，观察180min后处死动物，留取标本；③IR组：麻醉，气管插管，左侧开胸，暴露左侧肺门，注射肝素，在肺充气状态下，显微无创血管夹阻断左侧肺门，暂时合拢手术切口，阻断60min后(模拟缺血)，松开血管夹，关闭胸腔，继续呼吸机辅助呼吸，观察120min后（模拟再灌注），处死动物，留取标本；④IR+GFP组：给予注射携带GFP基因的质粒，造模手术操作过程同IR组；⑤IR+2J2组：给予注射携带2J2目的基因的质粒，造模手术操作过程同IR组
4. Western blot检测各组大鼠肺组织中CYP2J2蛋白表达水平，ELISA方法检测大鼠肺组织中14,15-EET含量
5.处死动物前每组随机将半数动物用Millar导管经右侧颈静脉插管至右心室，记录右心室血流动力学指标，检测CYP2J2过表达对大鼠右心舒缩功能的影响
6.检测大鼠左肺组织湿干重比，检测血清与左肺BALF蛋白浓度之比，检测CYP2J2过表达对肺毛细血管通透性的影响
7.观察大鼠肺组织缺血再灌注损伤后大体及切片HE染色光镜下病理改变
8.观察大鼠肺组织缺血再灌注损伤后切片TUNEL染色光镜下细胞凋亡情况
9.检测大鼠血清及肺组织中炎症因子及炎症相关粘附因子的水平及CYP2J2过表达对炎症的影响：ELISA方法检测血清TNF-α，IL-1β，IL-8，IL-10，sP-selectin，sE-selectin的水平Western blot及实时荧光定量PCR检测肺组织中ICAM-1，VCAM-1的表达
10.检测大鼠血清及肺组织中氧化应激相关指标及CYP2J2过表达对氧化应激的影响：实时荧光定量PCR方法检测大鼠肺组织中eNOS，XOD，NADPH P22phox，NADPH P67phoxmRNA表达；ELISA方法检测血清COX的水平
11. Western blot检测大鼠肺组织标本中PI3K，Akt，p-Akt及NF κB-p65蛋白质的表达水平及CYP2J2过表达对其表达的影响
实验结果
1.通过经尾静脉注射质粒导入的方法，成功将CYP2J2基因导入大鼠体内，经WesternBlot及ELISA方法验证：CYP2J2基因在大鼠肺组织中获得稳定表达，并代谢AA产生EET，显著提高了肺组织中EETs水平
2. CYP2J2过表达对大鼠缺血再灌注后左肺组织炎症反应及细胞凋亡影响
大鼠左肺组织大体及石蜡切片HE染色光镜观察结果显示：缺血再灌注后大鼠肺泡腔内可见较多浆液性炎性渗出物，大量炎性细胞浸润，肺泡纤维间隔明显水肿ǎ增厚，部分区域间隔组织破坏；小叶间动脉充血ǎ扩张；部分肺泡腔内可见肺泡巨噬细胞浸润，支气管壁周围可见炎症细胞浸润；部分区域可见点状ǎ片状肺组织坏死，显示大鼠左肺缺血再灌注损伤模型制作成功；CYP2J2过表达可显著抑制大鼠左肺缺血再灌注损伤后炎性渗出，可明显减轻缺血再灌注损伤后肺组织内中性粒细胞ǎ巨噬细胞等炎症反应细胞的浸润，提示CYP2J2过表达可明显减轻肺缺血再灌注损伤导致的炎症反应
各组大鼠肺组织切片TUNEL染色及凋亡指数计算结果显示：与Blank组（AI=0.188%±0.0896%）及Sham组（AI=0.182%±0.0464%）比较，IR组（AI=3.732%±0.4896%），IR+GFP组（AI=3.622%±0.3656%），IR+2J2组（AI=2.236%±0.4968%），均显著升高，缺血再灌注损伤可诱导大鼠肺组织细胞发生明显凋亡（P＜0.01）；与IR组和IR+GFP组比较，IR+2J2组AI明显减小（P＞0.05），CYP2J2过表达显著减少了缺血再灌注损伤诱发的细胞凋亡
3. CYP2J2过表达对大鼠左肺缺血再灌注损伤后肺毛细血管通透性的影响
IR+2J2组大鼠左肺湿干重比值（W/D（）5.215±0.860）明显低于IR组的（7.061±1.189）和IR+GFP组的（8.048±0.489）（P＜0.05），但均显著高于Blank组（4.075±0.729）及Sham组（4.700±0.645）的W/D（P＜0.05），Blank组和Sham组之间，IR组和IR+GFP组之间W/D差异无统计学意义（P＞0.05）
IR+2J2组大鼠血清与左肺BALF蛋白浓度比值（30.306±2.4956）明显高于IR组（17.727±1.870）和IR+GFP组（18.414±1.993）（P＜0.05），但均小于Blank组（44.463±4.023）及Sham组（43.005±1.703），Blank组和Sham组之间，IR组和IR+GFP组之间血清与左肺BALF蛋白浓度比值差异无统计学意义（P＞0.05）
缺血再灌注损伤可导致肺毛细血管通透性明显增大，表现为肺湿干重比值增加及血清与BALF中蛋白浓度比值降低，CYP2J2过表达可明显降低肺毛细血管通透性，减轻缺血再灌注肺组织内蛋白液体的渗出
4.血清ǎ肺组织中炎症相关因子检测结果
ELISA方法检测大鼠血清中相关炎症因子水平的结果表明：CYP2J2基因过表达显著降低了缺血再灌注损伤后大鼠血清中炎症因子的水平IR+2J2组血清TNF-α水平（124.182±12.734pg/ml）显著低于IR组（191.554±19.603pg/ml）和IR+GFP组（183.246±14.443pg/ml），但均高于Blank组（41.224±12.443pg/ml）和Sham组（93.004±12.701pg/ml）；IR+2J2组血清IL-1β水平（74.393±9.029pg/ml）显著低于IR组（110.516±24.519pg/ml）和IR+GFP组（103.704±15.872pg/ml），但均高于Blank组（50.039±12.318pg/ml）和Sham组（62.018±12.319pg/ml）；IR+2J2组血清IL-8水平（55.330±6.608pg/ml）显著低于IR组（67.754±8.797pg/ml）和IR+GFP组（65.008±7.670pg/ml），但均高于Blank组（36.757±6.051pg/ml）；IR+2J2组血清IL-10水平（191.473±4.639pg/ml）显著高于IR组（125.792±6.256pg/ml）和IR+GFP组（123.279±7.453pg/ml），以上三组均高于Blank组（46.828±5.010pg/ml）和Sham组（53.987±4.925pg/ml）；IR+2J2组血清sP-Selectin水平（38.297±3.3527ng/ml）显著低于IR组（56.732±5.985ng/ml）和IR+GFP组（55.616±5.455ng/ml），但均高于Blank组（6.259±1.631ng/ml）和Sham组（19.951±4.166ng/ml） IR+2J2组血清sE-Selectin水平（1312.109±211.164pg/ml）显著低于IR组（1779.279±322.441pg/ml）和IR+GFP组（1775.263±261.194pg/ml），但均高于Blank组（500.193±162.362pg/ml）和Sham组（930.488±192.240pg/ml）
RT-PCR检测及western blot检测结果同样表明：缺血再灌注导致肺组织内ICAM-1ǎVACM-1表达水平显著上调；CYP2J2基因过表达显著抑制了缺血再灌注后ICAM-1ǎVACM-1表达水平的上调，从而抑制了单核巨噬细胞向内皮细胞的粘附以及以及向血管组织的迁移，减轻炎症损伤
5.血清ǎ肺组织中氧化应激相关指标检测结果
缺血再灌注损伤可导致血清中COX水平显著升高，CYP2J2基因过表达未能明显降低LIRI大鼠血清中COX水平；肺缺血再灌注损伤可导致大鼠肺组织中XODǎeNOSǎNADPH氧化酶P22phoxǎP67phox的表达上调，CYP2J2基因过表达显著抑制了肺缺血再灌注后XOD，eNOS，NADPH氧化酶P22phoxǎP67phox表达的上调
6. Western blot检测结果显示：左肺缺血再灌注损伤导致大鼠肺组织内PI3Kǎp-Akt的表达显著下调，而CYP2J2过表达的IR+2J2组PI3Kǎp-Akt的表达较IR组和IR+GFP组明显升高，但仍然低于未发生缺血再灌注损伤的Blank组和Sham组
左肺缺血再灌注损伤导致大鼠肺组织内NF-κB的表达显著上调，而CYP2J2过表达的IR+2J2组NF-κB的表达较IR组和IR+GFP组明显降低，但仍然高于未发生缺血再灌注损伤的Blank组和Sham组
7.左肺缺血再灌注可使心率明显减慢，CYP2J2基因过表达对大鼠缺血再灌注后心率减慢无显著影响；IR组ǎIR+GFP组及IR+2J2组大鼠右心室收缩压（RVSP）明显低于Blank组和Sham组（P<0.01），IR+2J2组RVSP显著高于IR组与IR+GFP组（P<0.05）；IR+2J2组右心室舒张期末压力（RVEDP）显著低于其余四组大鼠（P<0.05）；IR组ǎIR+GFP组及IR+2J2组大鼠右心室等容收缩期室内压上升最大速率（RV+dp/dt max，mmHg/s）ǎ右心室等容舒张期室内压下降最大速率（RV-dp/dt max，mmHg/s）明显低于Blank组及Sham组（P<0.01），IR+2J2组±dp/dt max显著高于IR组和IR+GFP组（P<0.05）
统计学分析
用SPSS19.0软件进行统计学分析，比较两组间差别计量资料用均数±标准差（x SD）进行统计学描述，采用均数t检验和单因素方差分析（ANOVA）比较两组组内ǎ组间的差别所有统计学检验均为双侧检验，P<0.05为差异有统计学意义
结论
1.成功构建了Wister大鼠在体左肺缺血再灌注损伤的动物模型
2.通过尾静脉注射质粒的方法，成功在Wister大鼠体内表达CYP2J2基因，并将花生四烯酸代谢为EETs，发挥生物学作用
3. CYP2J2基因过表达显著减少细胞凋亡，减轻了大鼠左肺缺血再灌注损伤
4. CYP2J2基因过表达明显改善了左肺缺血再灌注损伤所导致的大鼠右心室收缩和舒张功能的损害
5. CYP2J2基因过表达明显抑制了左肺缺血再灌注损伤所导致的血清促炎症因子及趋化因子水平的升高
6. CYP2J2基因过表达明显抑制了左肺缺血再灌注所导致的促氧化应激相关指标的升高，抑制了促氧化应激相关蛋白的表达
7. CYP2J2基因抗炎症和抑制氧化应激的作用可能和PI3K/AKT/NF-κB信号通路有关
Background and purpose
Arachidonic acid (AA) is the most widely distributed and the most abundantpolyunsaturated fatty in human body, in vivo, AA metabolic network is an important part ofthe multiple metabolic pathways and of endogenous bioactive substances, including oxidativestress network and inflammation signaling pathway. AA mainly metabolized through threedifferent enzymatic pathways, including the cyclooxygenase (COX) pathway, thelipoxygenase (LOX) pathway and the third important pathway of cytochrome P450(CYP)pathway which play an important roles in numerous physiological&pathophysiologicalprocesses.
Cytochrome P450epoxygenases(CYP2J and CYP2C) metabolize arachidonic acid (AA)to four different epoxyeicosatrienoic acids (EETs), namely5,6-EET,8,9-EET,11,12-EETand14,15-EET, which are converted to dihydroxyeicosatrienoic acids (DHETs) by solubleepoxide hydrolase(sEH).The cytochrome P450enzymes comprise a large superfamily ofproteins. CYP widely distributed in the body, the CYP epoxygenase including two types2Cand2J, CYP has been found for6clones type of oxidase2J, which the type CYP2J2is onlyfound in the human. After the years of research on CYP-EET system, evidence shows that CYP-EET system plays an important role in the human body.⑴EETs are with a strongexpansion effects of Cardiovascular function and regulate blood pressure;⑵EETs cansignificantly alleviate the apoptosis in endothelial cells and cardiomyocytes induced by tumornecrosis factor-α(TNF-α);⑶EETs can inhibit inflammation, inhibiting endothelial cellexpression of inflammatory adhesion molecules and protect the biological function ofendothelial cells;⑷EETs can promote endothelial cell proliferation, inhibits apoptosis ofendothelial cells and promotes tissue neovascularization,⑸EETs can activate plasminogenactivator(tPA), which may play an important role to maintain thrombosis/thrombolysisbalancing in vivo;⑹EETs can inhibit aortic smooth muscle cell migration induced by thetransforming growth factor-β(TGF-β);⑺hypoxia-reoxygenation process can significantlyinhibit CYP2J2gene expression in endothelial cells, By providing the exogenous EETs orCYP2J2overexpression in vivo can improve hypoxia-reoxygenation injury.
Lung ischemia-reperfusion injury (LIRI) is an important issues in the face of thoracicsurgery, It occurs in a variety of contexts such as lung transplantation, heart and lungtransplantation, lobe sleeve resection, pulmonary artery angioplasty, cardiopulmonary bypass,pulmonary embolism, shock and cardiopulmonary resuscitation, Lung ischemia-reperfusioninjury can result in the infiltration of inflammatory cells to increase, and Inflammatory cellsproduce and release large amounts of oxygen free radicals and thus damaging cells. relievesthe ischemia-reperfusion injury can protect lung function, reduce the post-operative mortalityand prolong the survival time of the transplanted organs, can reduce the occurrence ofsystemic inflammatory response syndrome and multiple organ dysfunction syndrome,especially to reduce acute respiratory distress syndrome(ARDS) caused by acute lunginjury(ALI).In recent years, the incidence and mortality rates of lung cancer are rising rapidlyin China; the incidence of lung cancer among malignant tumors has been ranked in the first.Removal of lesion tissue by surgical procedures is still the most thorough way to treat lungcancer. How to lessen IRI is a constantly focal point in lung cancer surgery study.
LIRI is a complex phenomenon involving not only intracellular injury processes, but also injurious inflammatory responses and biochemical changes, During ischemia-reperfusion,such as leukocyte activation, a variety of promoting the release of inflammatory reactionmedium, the release of inflammation-related protease, the occurrence of oxidative stress,intracellular calcium overload, activation of multiple transcription factors, membranemolecular raised, the biofilm injury and protective substances such as NO and decrease inpulmonary surfactant and other relevant factors, endothelial cells, and other immune cells togenerate ROS Calcium/calmodulin dependent nitric oxide synthases(NOS), nuclearfactor-κB (NF-κB), nicotinamide adenine dinucleotide phosphate (NADPH), andproinflammatory cytokines are activated, causing an upregulation of cell surface adhesionmolecules on the endothelial of the lung, along with lung ischemia-reperfusion injury in thenature of deepening understanding, inflammation and oxidative stress mechanisms involvedin lung ischemia-reperfusion injury and endothelial dysfunction, many studies have shownthat IRI and in essence, is an inflammatory response to ischemia-reperfusion injury,inflammatory chemokine expression resulting in lung tissue inflammatory cell infiltration,infiltration a large number of inflammatory cells and lead to the occurrence of oxidativestress, these are lung ischemia-reperfusion injury formed an important start contributingfactor.
So, can CYP2J2be used for the prevention of lung ischemia-reperfusion injury? Wesuppose that through over-expression the CYP2J2gene in animals model of lungischemia-reperfusion injury in order to increases the level of endogenous EETs;it couldactivate endogenous protective mechanisms of the vascular wall through the inhibition ofinflammation and oxidative stress to relieve lung ischemia-reperfusion injury. Thus, weconducted experiments to confirm the effects of CYP2J2overexpression on the animal’smodel of lung ischemia-reperfusion injury in rats, to explore CYP2J2ability to inhibit orblock the lung ischemia-reperfusion injury in the inflammatory response and oxidative stressand the possible mechanisms which exist in the protective effects of CYP2J2overexpression.
Methods and results
Methods
1Plasmid pcDNA3.1(+)-2J2and pcDNA3.1(+)-GFP were extracted and purificated byusing endotoxin-free plasmid mention kit.
2.50male Wistar rats,8weeks old, weight300-350g, subjected to a one-week adaptationperiod, were randomly divided into five groups, and each group has10rats: Blank group(Blank), Sham group (Sham), IR group (IR), IR+GFP group (IR+GFP), IR+2J2group(IR+2J2). The target gene pcDNA3.1-2J2and pcDNA3.1(+)-GFP as well as the equalvolume of saline were injected into the bodies of rats via tail veins, and the dose of plasmidinjection was3mg/kg body weight, injected once a week, a total of two injections. One weekafter the gene delivery, modeling surgery was performed. After the surgery was completed,the rats were sacrificed, Blood samples, bronchoalveolar lavage fluid(BALF) lung tissuesamples were collected and part of lung tissue were placed in liquid nitrogen immediately, allsamples transferred into the-80℃refrigerator, some of the lung tissues were fixed withformaldehyde solution, embedded in paraffin after dehydration at room temperature.
3. Blank group: do not perform any surgical procedure, the animals were sacrificed afteranesthesia; Sham group: left thoracotomymodel, before animals were sacrificed, we observethe animals180min; IR group:rats lung IRI model, the model were established by obstructingthe left hilum of lung, ischemia1h and2h of reperfusion，then animals were sacrificed;IR+2J2group: same perform just like the IR group; IR+GFP group: same perform just likethe IR group. Before sacrificed, half of animals were invented Millar catheter into rightventricle, in order to record right ventricular hemodynamic parameters.
4. Proteins from frozen lung tissue were extracted for western blot methods to detect proteinexpression levels of CYP2J2protein. And ELISA method was used to detect content of thelung tissue14,15-DHET.
5.The ratio of lung wet/dry（W/D） weight were measured to assess lung edema after IRI;Serum/BALF protein concentration ratio was detection; the paraffin sections of lung wereprepared to carry out HE stain, Immunohistochemical TUNEL staining was used to detect apoptosis of alveolar epithelial cell.
6. Detection of serum and lung tissue Inflammatory-related cytokines: ELISA was used todetect TNF-α, IL-1β, IL-8, IL-10, sP-selectin, sE-selectin levels in serum; western blot todetect protein expression levels of ICAM-1and VCAM-1inlung tissue.
7. Detection of oxidative stress in rat blood and lungtissue: Real-time quantitative PCRwasused to detect mRNA expression of oxidative stress related cytokines eNOS, XOD, NADPHp22phoxand NADPH P67phox; ELISA method to detect the serum levels of COX.
8. Western blot to detect protein expression levels of PI3K, Akt, p-Akt and NF κB-p65in ratlung tissue.
Results:
1. Western blot and ELISA results showed that after2weeks/2times CYP2J2genedelivery through tail vein into rat,the CYP2J2overexpression in rat lung;14,15-EET contentin lung of CYP2J2overexpression rats were significantly higher than the control group(P<0.05).
2. The results of TUNEL staining to detect apoptosis showed that, compared with Blankgroup and Sham group, the apoptotic index(AI) was significantly higher in IR group,IR+GFP group and IR+2J2group (P<0.01); The AI of IR+2J2group was significantly lessthan the IR group and IR+GFP group (P<0.05).CYP2J2overexpression significantlyinhibited ischemia-reperfusion injury induced apoptosis.
3. After1h ischemia and2h reperfusion, the ratio of W/D in IR+2J2group is (5.215±0.860),significantly lower than that (8.061±1.189) of IR group and (8.048±0.489) of IR+GFP group(P<0.05); both of three group were significantly higher than that in (4.075±0.729) of Blankgroup and (4.700±0.645) of Sham group (P<0.01), there was no significant differencebetween Blank group and Sham group, but also between IR group and IR+GFP group.After1h ischemia and2h reperfusion, the ratio of Serum/BALF protein concentrationwas significantly higher in IR+2J2(30.306±2.4956) than that in IR(17.727±1.870) andIR+GFP(18.414±1.993)(P<0.05); both of three group were significantly lower than that in Blank(44.463±4.023) and Sham (43.005±1.703)(P<0.01), there was no significant differencebetween Blank group and Sham group, also between IR group and IR+GFP group.
4. Results of Inflammatory-related cytokines test:
ELISA: detection of inflammatory cytokines in serum shows that CYP2J2overexpression significantly inhibited the serum levels of pro-inflammatory cytokines
The serum levels of TNF-α in IR+2J2group (124.182±12.734pg/ml) is significantlylower than that in IR group(191.554±19.603pg/ml) and IR+GFP group(183.246±14.443pg/ml), all these three group are significantly higher than that Blank group(41.224±12.443pg/ml) and Sham group (93.004±12.701pg/ml);
The serum levels of IL-1β in IR+2J2group (74.393±9.029pg/ml) is significantly lowerthan that in IR group(110.516±24.519pg/ml) and IR+GFP group(103.704±15.872pg/ml), allthese three group are significantly higher than that Blank group(50.039±12.318pg/ml) andSham group (62.018±12.319pg/ml);
The serum levels of IL-8in IR+2J2group(55.330±6.608pg/ml) is significantly lowerthan that in IR group(67.754±8.797pg/ml) and IR+GFP group(65.008±7.670pg/ml), but arehigher than that in Blank group(36.757±6.051pg/ml).
The serum levels of IL-10in IR+2J2group (191.473±4.639pg/ml) is significantlyhigher than that in IR group(125.792±6.256pg/ml) and IR+GFP group(123.279±7.453pg/ml), all these three group are significantly higher than that in Blank group (46.828±5.010pg/ml) and Sham group(53.987±4.925pg/ml).
The serum levels of sP-Selectin in IR+2J2group (38.297±3.3527ng/ml) is significantlylower than that in IR group (56.732±5.985ng/ml) and IR+GFP group (55.616±5.455ng/ml),all these three group are significantly higher than that in Blank group (6.259±1.631ng/ml)and Sham group (19.951±4.166ng/ml).
The serum levels of sE-Selectin in IR+2J2group (1312.109±211.164pg/ml) issignificantly lower than that in IR group (1779.279±322.441pg/ml) and IR+GFP group(1775.263±261.194pg/ml), all these three group are significantly higher than that in Blank group (500.193±162.362pg/ml) and Sham group (930.488±192.240pg/ml).
Western blot test results and Real-time quantitative PCR data of the mRNA expressionlevel showed that ICAM-1and VACM-1expression level was significantly upregulated afterlung ischemia-reperfusion; CYP2J2gene overexpression significantly inhibited the effects.
5. Results of oxidative stress related cytokines test
Real-time quantitative PCR result showed that XOD, eNOS, NADPH P22phox, NADPHP67phoxmRNA expression level was significantly upregulated after lung ischemia-reperfusion,CYP2J2gene overexpression can inhibited the effects.
6. Western blot test results showed that: Compared with Sham and Sham group, PI3K, p-Aktprotein expression level was obviously decreased after LIR, and NFκB-p65protein expressionlevel was obviously increased after LIR. CYP2J2gene overexpression significantly inhibitedthe changes of related protein expression above-mentioned.7. Hemodynamic records showed that LIRI induced significant decreased the right ventricleSystolic blood pressure (RVSP)and the maximum rate of right ventricular pressure (±dp/dtmin); CYP2J2overexpression reversed this effects induced by LIRIsignificantly; comparedwith other groups, CYP2J2overexpression decrease right ventricular end-diastolic pressure(RVEDP); LIRIinduced significant decrease in heart rate (HR), the CYP2J2overexpressiondid not show significant changes.
Statistical Analysis
All data were analyzed by SPSS19.0statistical software(IBM). Enumeration data wereexpressed as frequency or composition ratio， and the Chi-square test were used to evaluatethe differences between two groups. Values were present as mean standard deviation （x
SD）.Analysis of variance (ANOVA) was used to compare group differences. Student’s ttest was used to assess whether differences in the values of two groups were statisticallysignificant. The level for statistical differences was set at P<0.05.
Conclusion:
1. Successfully constructed an animal model in vivo of left lung ischemia-reperfusion injury in Wister rats
2. CYP2J2gene delivery in rat by plasmid tail vein injection received a stable expression,and increased the content of EETs in lung tissuesignificantly.
3. CYP2J2overexpression significantly reduces the left lung ischemia-reperfusion injuryand apoptosis in rats.
4. CYP2J2gene overexpression significantly improves right ventricular systolic anddiastolic function damage caused by the left lung ischemia-reperfusion injury.
5. CYP2J2overexpression significantly decreased the serum levels of proinflammationcytokines and increased the levels of anti-inflammatory cytokines in the LIRI rats.
6. CYP2J2overexpression inhibited the oxidative stress associated indicators (XOD,NADPH subunits) in blood, lung tissue significantly, and significantly upregulatedexpression of eNOS,
7. A possible mechanism that CYP2J2anti-inflammatory and inhibit oxidative stress byactivating PI3K/AKT/NF-κB signaling pathway.
引文
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