meprin-α介导血管活性氧生成促进动脉粥样硬化形成的作用及机制研究
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摘要
随着社会经济水平提高和人口老龄化形势的加重，动脉粥样硬化（Atherosclerosis，AS）导致的心脑血管疾病已经成为严重危害人类健康并导致死亡的最主要的疾病之一。动脉粥样硬化是通过脂质沉积、平滑肌细胞增殖和钙化以及炎症细胞浸润进而引起的一种以反应性损伤-修复为特征的动脉血管疾病。动脉血管硬化和血管内粥样斑块形成是AS的主要病理改变，可逐渐导致动脉管腔狭窄，造成心脏、脑及远端肢体缺血。近年来，对AS形成的机制研究的不断深入，氧化应激反应在AS形成中的作用受到越来越多的重视，但抗氧化药物在防治AS进展方面并没有取得预期的效果，氧化应激与AS形成之间的关系值得进一步深入探讨。
金属蛋白酶meprin是哺乳动物细胞表面的一种含锌指结构的蛋白水解酶(Met-Zincin），在包括血管内皮细胞、巨噬细胞在内的多种细胞表面广泛分布。meprin包含α、β两种亚基，在体内的通过活化或者灭活细胞因子发挥生理作用，研究发现meprin-α在体内与肿瘤发生和炎症反应密切相关。我们在前期研究中发现meprin-α参与AS形成，用meprin抑制剂actinonin（AC）慢性干预高脂饲养的ApoE-/-小鼠可显著抑制其动脉血管壁的AS形成及进展，同时伴随粥样斑块内巨噬细胞数目的减少和血管壁原位的活性氧（Reactive oxygen species，ROS）生成减少，提示meprin-α可能通过调控血管壁的氧化应激反应参与AS的进展。但由于meprin-α在ApoE-/-小鼠的小鼠血管壁的表达较低，未能进一步明确其具体作用机制。
表皮生长因子受体（epidermal growth factor receptor，EGFR)是一种广泛分别在多种动物组织细胞表面的跨膜糖蛋白受体，主要通过配体作用被激活进而发挥生理功能，促进细胞内ROS生成。研究发现Met-Zincin蛋白水解酶家族成员可以在体内活化EGFR的三个主要配体，包括表皮生长因子（epidermal growth factor，EGF），肝素结合的表皮生长因子（heparin-binding EGF-like growth factor，HB-EGF）和转化生长因子-ɑ(transforming growth factor alpha,TGF-ɑ)，被活化的EGFR配体由于细胞类型和作用底物的不同而不同。meprin-α属于Met-Zincin蛋白水解酶家族，我们推测其可能通过活化巨噬细胞的EGFR的配体激活EGFR，进而介导巨噬细胞ROS生成，参与AS进展。
基于以上假设，本实验拟构建含有meprin-α的慢病毒载体对高脂饲养的ApoE-/-小鼠进行在体干预，运用分子生物学的技术从动物及细胞两方面观察meprin-α对AS形成和巨噬细胞ROS生成的影响并探讨其作用机制。通过本研究可初步阐明meprin-α介导血管ROS生成促进动脉粥样硬化形成的作用及机制，以及EGFR在巨噬细胞ROS生成过程中的作用及下游信号通路。有助于从一个新的角度揭示氧化应激反应与AS形成的关系，为进一步研究调控AS形成中的氧化应激反应提供实验基础，为研究新的药物靶点提供理论支持。
方法：
一、构建meprin-α慢病毒载体
1.委托上海捷瑞生物工程有限公司合成MEP1A基因。
2.以合成的MEP1A基因为模板PCR扩增目的基因。表达载体酶切后进行胶回收载体片段。目的基因与载体片段同源重组后转化E.coli感受态细胞。用菌落PCR鉴定转化子，阳性克隆送测序。测序无误的克隆进行质粒抽提。
3.使用293T细胞进行病毒颗粒的包装并进行滴度测定。
二、动物实验
1、实验动物及分组：150只雄性ApoE-/-小鼠（C57/BL6基因背景），6周龄，体重18-20g，随机平均分为6组，每组25只。对照组（CON组）一直用普通饲料；其余5组高脂饲料饲养，根据不同的干预方式分为：高脂组（HF组）：11周开始予无菌生理盐水（0.5ml/天）腹腔注射，连续注射4周。高脂+L-meprin-α组（HF+LM组）：第11周经尾静脉注射L-meprin-α（1.84x108TU）,之后予无菌生理盐水（0.5ml/天）腹腔注射，连续注射4周。高脂+L-meprin-α+AC组（HF+LM+AC组）：第11周经尾静脉注射L-meprin-α（1.84x108TU）,之后予AC（5mg/kg/day）腹腔注射，连续注射4周。高脂+L-meprin-α+AG1478组（HF+LM+AG组）：第11周经尾静脉注射L-meprin-α（1.84x108TU）,之后予AG1478（20mg/kg/day）腹腔注射，连续注射4周。高脂+L-EGFP组（HF+LGFP组）：第11周经尾静脉注射L-EGFP（1.92x108TU）,之后予无菌生理盐水（0.5ml/天）腹腔注射，连续注射4周。该组分别有3只小鼠在尾静脉注射L-EGFP后的第1周、4周被处死，以确认病毒在粥样斑块内转染的效率。所有各组ApoE-/-小鼠于第14周处死。
2、组织病理学观察斑块大小和数量的检测：ApoE-/-小鼠用过量的戊巴比妥钠腹腔麻醉后处死，取小鼠胸主动脉固定、包埋，切成5um厚的小片。组织制片后，常规采用苏木素-伊红(hematoxylin and eosin，HE)染色，镜下观察粥样斑块形成，油红O染色，大体水平上确定粥样斑块的数量和大小。
3、血管内膜原位ROS生成的检测：小鼠胸主动脉血管切片脱蜡处理后用DHE处理，通过共聚焦显微镜下可检测并获得图像。用IPP6.0图像分析软件分析结果。
4、蛋白印迹法检测(Western Blot,WB)检测血管内膜的meprin-α的表达：提取-80℃冰冻保存的小鼠胸主动脉粥样斑块的组织克，蛋白通过SDS-PAGE法提取出来并转位到PVDF膜上，一抗封闭并且孵育过夜。辣根过氧化物酶结合的第二抗体检测到条带并化学发光。
5、免疫组织化学的方法检测血管内膜EGFR的表达：小鼠胸主动脉血管切片用一抗包埋并于4℃孵育过夜，然后孵育生物素化的第二抗体30min，加入DAB直观在显微镜下观察。
三、细胞实验
1、DHE荧光探针标记细胞内ROS：在不同因素预处理J774A.1细胞的情况下，检测细胞内ROS生成的情况，明确影响细胞内ROS生成的因素及其相互关系。
2、ELISA法检测细胞培养液中蛋白浓度：在不同因素预处理J774A.1细胞的情况下，吸取J774A.1细胞培养液，用ELISA试剂盒检测培养液中HB-EGF蛋白浓度，明确影响J774A.1细胞释放HB-EGF的因素。
3、WB法检测细胞内蛋白表达水平：不同因素预处理J774A.1细胞的情况下，检测EGFR及其下游信号蛋白分子的表达水平，明确影响信号蛋白分子表达的因素及其相互关系。
四、统计学分析
计量资料以均值±标准差（mean±SD）表示。统计数据采用单因素方差分析比较（One-way ANOVA），以P<0.01为有显著性差异。统计学处理采用SPSS13.0软件完成。
结果：
一、慢病毒载体构建
成功构建含有MEP1A基因的慢病毒载体和pSB1对照载体。
一、动物实验部分
1、组织病理学观察的情况
⑴HE染色：CON组血管壁三层结构较清晰，内膜光滑完整，平滑肌细胞排列整齐，动脉血管壁稍隆起形成非常少量的粥样斑块。内皮下可见少量脂质及炎性细胞浸润，中层及外膜结构清楚，未见病理改变；高脂饲料饲养的HF组可见血管壁弥漫性隆起，突向管腔面形成明显斑块,内膜明显增厚,内膜结构明显破坏。内膜下大量排列不规则平滑肌细胞增生，内有脂质沉积，形成大小不等、形态不规则的泡沫细胞，大量巨噬细胞浸润，中层结构紊乱，平滑肌增生、坏死，并有炎性细胞浸润。弹力板处及细胞间隙可见大量脂质沉积。HF+LM组与HF组相比，血管内膜形成的斑块面积更大，内膜增厚更明显，脂质沉积更多，斑块内巨噬细胞浸润更加明显；HF+LM+AG组和HF+LM+AC组与HF组相比，血管内膜形成的斑块面积减小，内膜增厚，脂质沉积减少，斑块内可见少量的巨噬细胞和炎症细胞；HF+LGFP组与HF组相比，血管壁的变化基本接近。⑵油红O染色：CON组胸主动脉柔软富于弹性，内膜光滑平整，仅有局部染色较深的损伤区域；高脂饲料饲养的HF组血管内膜可见片状颜色较深的损伤区域，内膜欠完整，大量内膜结构被破坏(P<0.01)。HF+LM组与HF组相比，损伤部位数量和面积均显著增加(P<0.01)；HF+LM+AG组和HF+LM+AC组与HF+LM组相比，损伤部位数量和面积均显著减少(P<0.01)相比；HF+LGFP组与HF组相比，损伤部位数量和面积无明显差异。
2、各组ApoE-/-小鼠血管内膜原位ROS生成的情况
CON组胸主动脉血管内膜原位仅有非常少量的ROS生成，与CON组比较，HF组原位ROS生成增加(P<0.01)。与HF组相比，HF+LM组血管内膜原位ROS生成显著增加(P<0.01)；与HF+LM组比较，HF+LM+AC组血管内膜原位ROS生成显著减少(P<0.01)。
3、各组ApoE-/-小鼠血管壁meprin-α表达的情况
CON组仅有少量meprin-α表达，HF组血管内膜meprin-α表达显著增高(P<0.01)。
4、各组ApoE-/-小鼠血管内膜EGFR表达的情况
CON组血管内膜几乎无EGFR表达，HF组血管内膜可见EGFR表达，与HF组比较，HF+LM组EGFR表达显著增加(P<0.01)，而HF+LM+AC组几乎没有EGFR的表达。
二、细胞实验部分
1、不同干预因素处理的情况下J774A.1细胞内ROS生成的情况
⑴在各组的比较中，与control组比较，OxLDL处理的细胞内ROS生成均显著增多。⑵与OxLDL处理的各组细胞比较：OxLDL+AC、OxLDL+meprin-α siRNA、OxLDL+AG1478、OxLDL+抗HB-EGF和p38抑制剂SB203580处理的细胞内ROS生成明显减少(P<0.01)，OxLDL+meprin-α处理的细胞内ROS生成增多(P<0.01)。⑶与OxLDL+meprin-α处理的细胞比较，OxLDL+meprin-α+AG1478处理的细胞内ROS明显减少(P<0.01)。
2、不同干预因素处理的情况下J774A.1细胞释放HB-EGF的情况：
⑴与control组比较，OxLDL和OxLDL+meprin-α处理的细胞培养液中HB-EGF浓度增加(P<0.01)。⑵与OxLDL处理的细胞比较， OxLDL+AC和OxLDL+meprin-αsiRNA处理的细胞培养液中HB-EGF浓度减少(P<0.01)。
3、不同干预因素处理的情况下J774A.1细胞内蛋白表达的情况
⑴与control组比较，OxLDL处理的细胞EGFR、p38和PI3K-Akt磷酸化水平增多，GTP-rac1表达增高(P<0.01)；⑵与OxLDL处理的细胞比较，抗HB-EGF抗体、AC或者meprin-αsiRNA处理的细胞EGFR磷酸化水平减少(P<0.01)， AC、meprin-αsiRNA、Rac1抑制剂EHT1864和PI3K抑制剂wortmsnin处理的细胞内p38磷酸化水平降低(P<0.01)， wortmsnin处理的细胞内GTP-rac1表达水平显著降低(P<0.01)，AC和meprin-αsiRNA，以及AG1486处理的细胞内PI3K-Akt磷酸化水平显著降低(P<0.01)。
结论：
1、meprin-ɑ通过介导氧化应激促进高脂饲养的Ape-/-小鼠胸主动脉粥样斑块形成，meprin-ɑ抑制剂AC和EGFR抑制剂AG均可抵消meprin-ɑ的作用。meprin-ɑ是EGFR的上游信号分子，通过影响EGFR的表达和活性发挥作用。
2、meprin-α促进具有跨膜结构的HB-EGF胞外部分释放到巨噬细胞外，作为配体与同样具有跨膜结构的EGFR的胞外域结构结合，通过促进酪氨酸激酶磷酸化的方式激活EGFR，启动下游信号通路并促进细胞内ROS生成。
3、 meprin-α在巨噬细胞中反式激活EGFR启动受体后的信号传导，通过PI3K-Rac1-p38的途径促进了细胞内ROS生成。
With the increasing improvement of the socio-economic level and the population agingsituation，Cardiovascular and cerebrovascular disease caused by atherosclerosis（AS）havebecome a serious threat to human health and one of the most important diseases lead todeath. AS is one vascular diseases characterized by reactive damage repair which caused bythe depositon of lipid，proliferation and calcification of smooth muscle cells and theinfiltration of inflammatory cells. The hardening of the arteries and atherosclerotic plaqueformation are the main pathologic changes of AS, can gradually lead to stenosis of theartery lumen, is the most major cause of heart, brain and distal limb ischemia. In recentyears, researchers understood more about the mechanism of formation of atherosclerosis,oxidative stress response in the form of AS function is attracting more and more attention,but the antioxidant drugs has not achieved the expected effect to prevent the development ofAS, the relationship between oxidative stress and the development of AS is need furtherresearch
Metalloprotease meprin is the surface protein of mammalian cells containing a zincfinger protein hydrolase structure, contains α and β two subtype，widely distributed in avariety of cells including endothelial cells, macrophages and tumor cells. meprin usuallyplay a physiological role by activation or inactivation of cytokines in vivo. Studies havefound that meprin-α in closely related to the occurrence of tumor and chronic inflammatoryreaction. In our previous research we found that meprin-α play a role in AS formation anddevelopment, he chronic intervention of meprin inhibitor actinonin (AC)，the high fat-fedApoE-/-mice present less worse formation and progression of AS to control mice in arterywall, accompanied with the reduction in the number of macrophages in atherosclerotic andless generation of active oxygen species(ROS) in vessel wall in situ. These results suggestthat meprin-α may be involved in regulation of AS by involved in oxidative stress reaction.But because of the expression of meprin-α in ApoE-/-mice vascular wall was too low to make experiment to clarify the specific mechanism further.
Epidermal growth factor receptor (EGFR) is a widely distributed cell surfacetransmembrane glycoprotein receptors in a variety of animal tissue, mainly is activatedthrough the ligand effect and then play a physiological function, promote the generation ofROS in cells. The study found the metalloproteinase family of zinc metalloprotease couldactivate the three main ligand in vivo which could activate EGFR, including epidermalgrowth factor (EGF), heparin binding epidermal growth factor (HB-EGF) and transforminggrowth factor alpha (TGF-α). EGFR ligands are activated vary in different cell types anddifferent substrate. Metalloproteinase meprin-α belongs to zinc metalloprotease, we supposethat the meprin-α maybe activated the EGFR through activated the ligand of EGFR inmacrophages，induce ROS production in macrophage and participate in AS development.
Based on the above assumptions, in this study we plan to construct a lentiviral vectorcontaining meprin-α and chronic intervene the high fat-fed ApoE-/-mice，using molecularbiology techniques to observe the impact of meprin-α in AS formation and macrophageROS production from animal and cell level，further explore its mechanism. Through thisstudy we can initial clarify the effect and mechanism of meprin-α induced vascular ROSproduction to promote the AS development，and the role of EGFR in macrophages in theformation process of ROS and its downstream signaling pathways. There results shouldhelp us understanding the relationship between oxidative stress and AS from a new angleand provide the experimental basis for further study on the regulation of oxidative stressresponse in AS formation, provides the theory support for the new drug target research.
Method
1. Construct MEP1A lentiviral vector
⑴Shanghai Jierui Biotechnology Co was commissioned to synthesis the MEP1Agene.
⑵The MEP1A gene synthesized was PCR amplified of target gene as PCR template.Expression vector digested plastic recycling after the vector fragment. Objective genefragment homologous recombination and transformed into E.coli competent cells.Transformants with colony PCR identification, the positive clones were sequenced. Cloningand sequencing of the correct plasmid.
⑶Virus particles was packed using HEK-293T cells and titer determination.
2.animal experiment
⑴the experimental animal and grouping:150male ApoE-/-mice (C57/BL6genebackground),6weeks old, weighing18-20g, were randomly divided into6groups,25ratsin each group. The control group (group CON) was under the ordinary feed feeding. Theother5groups were under high-fat diet, according to the different intervention: high fatgroup (HF group): treated with sterile saline (0.5ml/days) intraperitoneal injection fromeleventh weeks， continuous injection for4weeks. High fat+L-meprin-α group (HF+LMgroup): intravenous injection of L-meprin-α (1.84x108TU) through caudal vein at eleventhweeks, then treated with sterile saline (0.5ml/days) intraperitoneal injection, continuousinjection for4weeks. High fat and+L-meprin-α+AC group (group HF+LM+AC):intravenous injection of L-meprin-α (1.84x108TU) at eleventh weeks through caudal vein,then treated with AC (5mg/kg/day) intraperitoneal injection from eleventh weeks,continuous injection for4weeks. High fat and+L-meprin-α+AG1478group (groupHF+LM+AG): intravenous injection of L-meprin-α (1.84x108TU) at eleventh weeksthrough caudal vein, then treated with AG1478(20mg/kg/day) intraperitoneal injectionfrom eleventh weeks， continuous injection for4weeks. High fat group+L-EGFP(HF+LGFP group): intravenous injection of L-EGFP (1.92x108TU) at eleventh weeksthrough caudal vein, then treated with sterile saline (0.5ml/days) intraperitoneal injection,continuous injection for4weeks. The HF+LGFP group has3mice were executed beforethe tail vein injection of L-EGFP and after the injection one or four weeks，in order toconfirm the efficiency of transfection of lentiviral vector in atherosclerotic plaques. Allmice were killed after fourteenth weeks for each group.
⑵Observe the size and number of atherosclerotic plaque in pathology: ApoE-/-mousewere executed by intraperitoneal anesthesia with overdose of pentobarbital sodium. thoracicaortic were peeled and removed from body and then fixed，embeddinged and cutted into5um thick slices. After conventional hematoxylin and eosin (hematoxylin and eosin, HE)staining， tissue section were observed under microscope to observe formation ofatherosclerotic plaque；oil red O staining, observe the number and size of atheroscleroticplaque in the general level.
⑶Detection of the generation of ROS in vascular intima in situ by DHE: mousethoracic aorta section treatment with DHE after dewaxing, confocal microscopy can detect the fluorescence of ROS and obtain image. using IPP6image analysis software was used toanalysis results.
⑷Detection of meprin-α expression in endothelium by Western blot assay (WesternBlot, WB): extraction of-80℃frozen mouse thoracic aortic atherosclerotic plaquepreservation organization50micrograms of protein by SDS-PAGE method, extracted andindexed to the PVDF membrane, antibody closed and incubation overnight. Secondantibodies with horseradish peroxidase detection protein strips and chemiluminescence.
⑸Detection of EGFR expression in endothelium by immunohistochemistry: mouseaorta sections with antibodies embedding and incubation at4℃overnight, and thenincubated with biotinylated second antibody30min, adding DAB directly under themicroscope.
3. Cell experiment
⑴Detection of generation of ROS in cell by DHE fluorescent probe labeled: J774A.1cells were pretreat with different factors，intracellular ROS generation were detected，theresults should make it clear that the factors affected the intracellular ROS generation andtheir relationship.
⑵Detection of protein concentration in cell culture fluid by ELISA: J774A.1cellswere pretreat with different factors，HB-EGF protein concentration in cell culture fluidwere detected by ELISA，the results should make it clear that the influence factors ofJ774A.1cells to release HB-EGF.
⑶Detection of intracellular protein expression by: J774A.1cells were pretreat withdifferent factors，EGFR and its downstream signal factors were detection by WB, the resultsshould make it clear that the factors affected the protein expression and their relationship.
4.Statistical analysis
Measurement data as mean±standard deviation (mean±SD) said. Statistical datausing single factor analysis of variance (One-way ANOVA), there were significantdifferences in P<0.01. Statistical analysis using SPSS13.0software.
Result
1. Construction of Lentivirus vector
Lentivirus vector containing Merin1A gene and pSB1control vector were successfullyconstructed.
2.animal experiment
⑴Results of histopathological observation
①HE staining: The three layer structure of vascular wall in CON group is clear, innermembrane is smooth, smooth muscle cells arranged in neat rows, the artery wall slightlyuplift and form very small amount atherosclerotic plaque. A small amount of lipid andinflammatory cells infiltration under the endothelial. Middle and outer membrane structureare clear, no pathological change. Vessel wall were diffuse swelling in high fat-diet HFgroup, protruding into the lumen surface formed obvious plaque, the inner membrane wasthickening and structure was serious damaged. A large number of irregular arrangement ofsmooth muscle cell proliferation under inner membrane, with lipid deposition, the formationof unequal size, irregular shape of the foam cells, a massive infiltration of macrophages,disorder of middle structure, smooth muscle hyperplasia, necrosis, and inflammatory cellinfiltration. Clearance elastic plate and cells showed large amounts of lipid deposition.Compared with HF+LM group and HF group, plaque area, neonatal formation of larger,intimal thickening was more obvious, more lipid deposition, plaque macrophage infiltrationis more obvious; HF+LM+AG group and HF+LM+AC group compared with HF+LMgroup, the plaque area decreased, inner membrane thickening, plaque decreases lipiddeposition, a small amount of macrophages and inflammatory cells; compared with HFgroup, the change of vessel wall in HF+LGFP group is similar.
②The oil red O staining: The thoracic aorta in CON group were soft elastic, innermembrane were smooth, with only a local dyeing damage area. The visible flake darkerdamage area of vascular inner membrane in high fat-diet HF group, inner membrane werenot complete and large amounts of membrane structure was destroyed (P<0.01). Comparedwith HF group, the number and size of lesions were significantly increased in HF+LMgroup (P<0.01); HF+LM+AG group and HF+LM+AC group compared with HF group, thenumber and size of lesions were significantly reduced (P<0.01); compared with HF+LGFPgroup and HF group, the number and size of the injury site has no obvious difference.
⑵Each group ofApoE-/-mouse vascular intima in situ ROS generation
There were very small amounts of ROS generation thoracic aortic intimal in situ inCON group.Compared with CON group, HF group have increased ROS generation (P<0.01)in situ. Compared with HF group, HF+LM group ROS generation in situ increased significantly (P<0.01).Compared with HF+LM group, HF+LM+AC group ROS generationin situ was significantly reduced (P<0.01).
⑶Each group ofApoE-/-mouse expression of meprin-α in vascular intimal
Only a small amount of the expression of meprin-α in CON group.Compared withCON group, expression of meprin-α increased significantly in HF group (P<0.01).
⑷Each group ofApoE-/-mouse expression of EGFR in vascular intimal
There were almost no expression of EGFR in CON group thoracic aortic vascularintimal and a small amount expression of EGFR was visible in HF group. Compared withHF group, the expression of EGFR in HF+LM group was significantly increased (P<0.01).Compared with HF+LM group, the expression of EGFR in HF+LM+AC group wassignificantly reduced (P<0.01).
3.cell experiment
⑴Different intervention treatment impact on J774A.1intracellular ROS generation.
①In the comparison of the groups, compared with the control group, OxLDLtreatment significantly increased the intracellular ROS generation at all time.
②Compared with OxLDL treated cells: OxLDL+AC, Ox-LDL+meprin-α siRNA,OxLDL+AG1478, Ox-LDL+HB-EGF and p38inhibitor SB203580treatment ofintracellular ROS generation was significantly reduced（P<0.01），while ROS generatesOxLDL+meprin-α treated cells increased (P<0.01).
③Compared with OxLDL+meprin-α treated cells, ROS significantly reduced inOxLDL+meprin-α+AG1478treated cell (P<0.01).
⑵Different intervention treatment impact on J774A.1release HB-EGF
①Compared with group control，the cell culture fluid of OxLDL and OxLDL+meprin-α treatment cells had increased concentration of HB-EGF (P<0.01).
②Compared with cells treated with OxLDL, the cell culture fluid of OxLDL+AC andOxLDL+meprin-α siRNA treated cells had reduced concentration of HB-EGF(P<0.01).
⑶Different intervention treatment impact on J774A.1intracellular proteinexpression.
Compared with control group, OxLDL treated cells had increased expression of EGFR,p38and PI3K-Akt phosphorylation(P<0.01), increased expression of GTP-rac1(P<0.01).Compared with OxLDL treated cells, expression of EGFR phosphorylation were reduced in cell treated with antibodies against HB-EGF, AC or meprin-α siRNA (P<0.01), expressionof p38phosphorylation were reduced in cell treated with AC, meprin-α siRNA, Rac1inhibitor EHT1864and PI3K inhibitor wortmsnin (P<0.01), intracellular expression ofGTP-rac1were significantly reduced in cells treated with wortmsnin (P<0.01), intracellularexpression of PI3K-Akt phosphorylation in cell treated with AC，meprin-α siRNA andAG1486were decreased significantly (P<0.01).
Conclusion
1.Meprin-α promote formation and development of thoracic aortic atheroscleroticplaque in high fat-fed ApoE-/-mouse through induce oxidative stress. meprin-α inhibitor ACand EGFR inhibitor AG1478can counteract the effect of meprin-α. meprin-α is theupstream signal molecule of EGFR, affecting the expression and activity of EGFR.
2.Meprin-α could promote macrophages release the extracellular part of thetransmembrane structure of HB-EGF out of cell, as a combination of ligand oftransmembrane structure of the extracellular domain of EGFR structure, activation of EGFRby promoting and tyrosine kinase phosphorylation patterns, initiate downstream signalingpathways and promote the generation of ROS in cells.
3. Meprin-α transactivation EGFR start receptor signaling in macrophages, via thePI3K-Rac1-p38pathway induce the generation of ROS in cells.
金属蛋白酶meprin是哺乳动物细胞表面的一种含锌指结构的蛋白水解酶(Met-Zincin），在包括血管内皮细胞、巨噬细胞在内的多种细胞表面广泛分布。meprin包含α、β两种亚基，在体内的通过活化或者灭活细胞因子发挥生理作用，研究发现meprin-α在体内与肿瘤发生和炎症反应密切相关。我们在前期研究中发现meprin-α参与AS形成，用meprin抑制剂actinonin（AC）慢性干预高脂饲养的ApoE-/-小鼠可显著抑制其动脉血管壁的AS形成及进展，同时伴随粥样斑块内巨噬细胞数目的减少和血管壁原位的活性氧（Reactive oxygen species，ROS）生成减少，提示meprin-α可能通过调控血管壁的氧化应激反应参与AS的进展。但由于meprin-α在ApoE-/-小鼠的小鼠血管壁的表达较低，未能进一步明确其具体作用机制。
表皮生长因子受体（epidermal growth factor receptor，EGFR)是一种广泛分别在多种动物组织细胞表面的跨膜糖蛋白受体，主要通过配体作用被激活进而发挥生理功能，促进细胞内ROS生成。研究发现Met-Zincin蛋白水解酶家族成员可以在体内活化EGFR的三个主要配体，包括表皮生长因子（epidermal growth factor，EGF），肝素结合的表皮生长因子（heparin-binding EGF-like growth factor，HB-EGF）和转化生长因子-ɑ(transforming growth factor alpha,TGF-ɑ)，被活化的EGFR配体由于细胞类型和作用底物的不同而不同。meprin-α属于Met-Zincin蛋白水解酶家族，我们推测其可能通过活化巨噬细胞的EGFR的配体激活EGFR，进而介导巨噬细胞ROS生成，参与AS进展。
基于以上假设，本实验拟构建含有meprin-α的慢病毒载体对高脂饲养的ApoE-/-小鼠进行在体干预，运用分子生物学的技术从动物及细胞两方面观察meprin-α对AS形成和巨噬细胞ROS生成的影响并探讨其作用机制。通过本研究可初步阐明meprin-α介导血管ROS生成促进动脉粥样硬化形成的作用及机制，以及EGFR在巨噬细胞ROS生成过程中的作用及下游信号通路。有助于从一个新的角度揭示氧化应激反应与AS形成的关系，为进一步研究调控AS形成中的氧化应激反应提供实验基础，为研究新的药物靶点提供理论支持。
方法：
一、构建meprin-α慢病毒载体
1.委托上海捷瑞生物工程有限公司合成MEP1A基因。
2.以合成的MEP1A基因为模板PCR扩增目的基因。表达载体酶切后进行胶回收载体片段。目的基因与载体片段同源重组后转化E.coli感受态细胞。用菌落PCR鉴定转化子，阳性克隆送测序。测序无误的克隆进行质粒抽提。
3.使用293T细胞进行病毒颗粒的包装并进行滴度测定。
二、动物实验
1、实验动物及分组：150只雄性ApoE-/-小鼠（C57/BL6基因背景），6周龄，体重18-20g，随机平均分为6组，每组25只。对照组（CON组）一直用普通饲料；其余5组高脂饲料饲养，根据不同的干预方式分为：高脂组（HF组）：11周开始予无菌生理盐水（0.5ml/天）腹腔注射，连续注射4周。高脂+L-meprin-α组（HF+LM组）：第11周经尾静脉注射L-meprin-α（1.84x108TU）,之后予无菌生理盐水（0.5ml/天）腹腔注射，连续注射4周。高脂+L-meprin-α+AC组（HF+LM+AC组）：第11周经尾静脉注射L-meprin-α（1.84x108TU）,之后予AC（5mg/kg/day）腹腔注射，连续注射4周。高脂+L-meprin-α+AG1478组（HF+LM+AG组）：第11周经尾静脉注射L-meprin-α（1.84x108TU）,之后予AG1478（20mg/kg/day）腹腔注射，连续注射4周。高脂+L-EGFP组（HF+LGFP组）：第11周经尾静脉注射L-EGFP（1.92x108TU）,之后予无菌生理盐水（0.5ml/天）腹腔注射，连续注射4周。该组分别有3只小鼠在尾静脉注射L-EGFP后的第1周、4周被处死，以确认病毒在粥样斑块内转染的效率。所有各组ApoE-/-小鼠于第14周处死。
2、组织病理学观察斑块大小和数量的检测：ApoE-/-小鼠用过量的戊巴比妥钠腹腔麻醉后处死，取小鼠胸主动脉固定、包埋，切成5um厚的小片。组织制片后，常规采用苏木素-伊红(hematoxylin and eosin，HE)染色，镜下观察粥样斑块形成，油红O染色，大体水平上确定粥样斑块的数量和大小。
3、血管内膜原位ROS生成的检测：小鼠胸主动脉血管切片脱蜡处理后用DHE处理，通过共聚焦显微镜下可检测并获得图像。用IPP6.0图像分析软件分析结果。
4、蛋白印迹法检测(Western Blot,WB)检测血管内膜的meprin-α的表达：提取-80℃冰冻保存的小鼠胸主动脉粥样斑块的组织克，蛋白通过SDS-PAGE法提取出来并转位到PVDF膜上，一抗封闭并且孵育过夜。辣根过氧化物酶结合的第二抗体检测到条带并化学发光。
5、免疫组织化学的方法检测血管内膜EGFR的表达：小鼠胸主动脉血管切片用一抗包埋并于4℃孵育过夜，然后孵育生物素化的第二抗体30min，加入DAB直观在显微镜下观察。
三、细胞实验
1、DHE荧光探针标记细胞内ROS：在不同因素预处理J774A.1细胞的情况下，检测细胞内ROS生成的情况，明确影响细胞内ROS生成的因素及其相互关系。
2、ELISA法检测细胞培养液中蛋白浓度：在不同因素预处理J774A.1细胞的情况下，吸取J774A.1细胞培养液，用ELISA试剂盒检测培养液中HB-EGF蛋白浓度，明确影响J774A.1细胞释放HB-EGF的因素。
3、WB法检测细胞内蛋白表达水平：不同因素预处理J774A.1细胞的情况下，检测EGFR及其下游信号蛋白分子的表达水平，明确影响信号蛋白分子表达的因素及其相互关系。
四、统计学分析
计量资料以均值±标准差（mean±SD）表示。统计数据采用单因素方差分析比较（One-way ANOVA），以P<0.01为有显著性差异。统计学处理采用SPSS13.0软件完成。
结果：
一、慢病毒载体构建
成功构建含有MEP1A基因的慢病毒载体和pSB1对照载体。
一、动物实验部分
1、组织病理学观察的情况
⑴HE染色：CON组血管壁三层结构较清晰，内膜光滑完整，平滑肌细胞排列整齐，动脉血管壁稍隆起形成非常少量的粥样斑块。内皮下可见少量脂质及炎性细胞浸润，中层及外膜结构清楚，未见病理改变；高脂饲料饲养的HF组可见血管壁弥漫性隆起，突向管腔面形成明显斑块,内膜明显增厚,内膜结构明显破坏。内膜下大量排列不规则平滑肌细胞增生，内有脂质沉积，形成大小不等、形态不规则的泡沫细胞，大量巨噬细胞浸润，中层结构紊乱，平滑肌增生、坏死，并有炎性细胞浸润。弹力板处及细胞间隙可见大量脂质沉积。HF+LM组与HF组相比，血管内膜形成的斑块面积更大，内膜增厚更明显，脂质沉积更多，斑块内巨噬细胞浸润更加明显；HF+LM+AG组和HF+LM+AC组与HF组相比，血管内膜形成的斑块面积减小，内膜增厚，脂质沉积减少，斑块内可见少量的巨噬细胞和炎症细胞；HF+LGFP组与HF组相比，血管壁的变化基本接近。⑵油红O染色：CON组胸主动脉柔软富于弹性，内膜光滑平整，仅有局部染色较深的损伤区域；高脂饲料饲养的HF组血管内膜可见片状颜色较深的损伤区域，内膜欠完整，大量内膜结构被破坏(P<0.01)。HF+LM组与HF组相比，损伤部位数量和面积均显著增加(P<0.01)；HF+LM+AG组和HF+LM+AC组与HF+LM组相比，损伤部位数量和面积均显著减少(P<0.01)相比；HF+LGFP组与HF组相比，损伤部位数量和面积无明显差异。
2、各组ApoE-/-小鼠血管内膜原位ROS生成的情况
CON组胸主动脉血管内膜原位仅有非常少量的ROS生成，与CON组比较，HF组原位ROS生成增加(P<0.01)。与HF组相比，HF+LM组血管内膜原位ROS生成显著增加(P<0.01)；与HF+LM组比较，HF+LM+AC组血管内膜原位ROS生成显著减少(P<0.01)。
3、各组ApoE-/-小鼠血管壁meprin-α表达的情况
CON组仅有少量meprin-α表达，HF组血管内膜meprin-α表达显著增高(P<0.01)。
4、各组ApoE-/-小鼠血管内膜EGFR表达的情况
CON组血管内膜几乎无EGFR表达，HF组血管内膜可见EGFR表达，与HF组比较，HF+LM组EGFR表达显著增加(P<0.01)，而HF+LM+AC组几乎没有EGFR的表达。
二、细胞实验部分
1、不同干预因素处理的情况下J774A.1细胞内ROS生成的情况
⑴在各组的比较中，与control组比较，OxLDL处理的细胞内ROS生成均显著增多。⑵与OxLDL处理的各组细胞比较：OxLDL+AC、OxLDL+meprin-α siRNA、OxLDL+AG1478、OxLDL+抗HB-EGF和p38抑制剂SB203580处理的细胞内ROS生成明显减少(P<0.01)，OxLDL+meprin-α处理的细胞内ROS生成增多(P<0.01)。⑶与OxLDL+meprin-α处理的细胞比较，OxLDL+meprin-α+AG1478处理的细胞内ROS明显减少(P<0.01)。
2、不同干预因素处理的情况下J774A.1细胞释放HB-EGF的情况：
⑴与control组比较，OxLDL和OxLDL+meprin-α处理的细胞培养液中HB-EGF浓度增加(P<0.01)。⑵与OxLDL处理的细胞比较， OxLDL+AC和OxLDL+meprin-αsiRNA处理的细胞培养液中HB-EGF浓度减少(P<0.01)。
3、不同干预因素处理的情况下J774A.1细胞内蛋白表达的情况
⑴与control组比较，OxLDL处理的细胞EGFR、p38和PI3K-Akt磷酸化水平增多，GTP-rac1表达增高(P<0.01)；⑵与OxLDL处理的细胞比较，抗HB-EGF抗体、AC或者meprin-αsiRNA处理的细胞EGFR磷酸化水平减少(P<0.01)， AC、meprin-αsiRNA、Rac1抑制剂EHT1864和PI3K抑制剂wortmsnin处理的细胞内p38磷酸化水平降低(P<0.01)， wortmsnin处理的细胞内GTP-rac1表达水平显著降低(P<0.01)，AC和meprin-αsiRNA，以及AG1486处理的细胞内PI3K-Akt磷酸化水平显著降低(P<0.01)。
结论：
1、meprin-ɑ通过介导氧化应激促进高脂饲养的Ape-/-小鼠胸主动脉粥样斑块形成，meprin-ɑ抑制剂AC和EGFR抑制剂AG均可抵消meprin-ɑ的作用。meprin-ɑ是EGFR的上游信号分子，通过影响EGFR的表达和活性发挥作用。
2、meprin-α促进具有跨膜结构的HB-EGF胞外部分释放到巨噬细胞外，作为配体与同样具有跨膜结构的EGFR的胞外域结构结合，通过促进酪氨酸激酶磷酸化的方式激活EGFR，启动下游信号通路并促进细胞内ROS生成。
3、 meprin-α在巨噬细胞中反式激活EGFR启动受体后的信号传导，通过PI3K-Rac1-p38的途径促进了细胞内ROS生成。
With the increasing improvement of the socio-economic level and the population agingsituation，Cardiovascular and cerebrovascular disease caused by atherosclerosis（AS）havebecome a serious threat to human health and one of the most important diseases lead todeath. AS is one vascular diseases characterized by reactive damage repair which caused bythe depositon of lipid，proliferation and calcification of smooth muscle cells and theinfiltration of inflammatory cells. The hardening of the arteries and atherosclerotic plaqueformation are the main pathologic changes of AS, can gradually lead to stenosis of theartery lumen, is the most major cause of heart, brain and distal limb ischemia. In recentyears, researchers understood more about the mechanism of formation of atherosclerosis,oxidative stress response in the form of AS function is attracting more and more attention,but the antioxidant drugs has not achieved the expected effect to prevent the development ofAS, the relationship between oxidative stress and the development of AS is need furtherresearch
Metalloprotease meprin is the surface protein of mammalian cells containing a zincfinger protein hydrolase structure, contains α and β two subtype，widely distributed in avariety of cells including endothelial cells, macrophages and tumor cells. meprin usuallyplay a physiological role by activation or inactivation of cytokines in vivo. Studies havefound that meprin-α in closely related to the occurrence of tumor and chronic inflammatoryreaction. In our previous research we found that meprin-α play a role in AS formation anddevelopment, he chronic intervention of meprin inhibitor actinonin (AC)，the high fat-fedApoE-/-mice present less worse formation and progression of AS to control mice in arterywall, accompanied with the reduction in the number of macrophages in atherosclerotic andless generation of active oxygen species(ROS) in vessel wall in situ. These results suggestthat meprin-α may be involved in regulation of AS by involved in oxidative stress reaction.But because of the expression of meprin-α in ApoE-/-mice vascular wall was too low to make experiment to clarify the specific mechanism further.
Epidermal growth factor receptor (EGFR) is a widely distributed cell surfacetransmembrane glycoprotein receptors in a variety of animal tissue, mainly is activatedthrough the ligand effect and then play a physiological function, promote the generation ofROS in cells. The study found the metalloproteinase family of zinc metalloprotease couldactivate the three main ligand in vivo which could activate EGFR, including epidermalgrowth factor (EGF), heparin binding epidermal growth factor (HB-EGF) and transforminggrowth factor alpha (TGF-α). EGFR ligands are activated vary in different cell types anddifferent substrate. Metalloproteinase meprin-α belongs to zinc metalloprotease, we supposethat the meprin-α maybe activated the EGFR through activated the ligand of EGFR inmacrophages，induce ROS production in macrophage and participate in AS development.
Based on the above assumptions, in this study we plan to construct a lentiviral vectorcontaining meprin-α and chronic intervene the high fat-fed ApoE-/-mice，using molecularbiology techniques to observe the impact of meprin-α in AS formation and macrophageROS production from animal and cell level，further explore its mechanism. Through thisstudy we can initial clarify the effect and mechanism of meprin-α induced vascular ROSproduction to promote the AS development，and the role of EGFR in macrophages in theformation process of ROS and its downstream signaling pathways. There results shouldhelp us understanding the relationship between oxidative stress and AS from a new angleand provide the experimental basis for further study on the regulation of oxidative stressresponse in AS formation, provides the theory support for the new drug target research.
Method
1. Construct MEP1A lentiviral vector
⑴Shanghai Jierui Biotechnology Co was commissioned to synthesis the MEP1Agene.
⑵The MEP1A gene synthesized was PCR amplified of target gene as PCR template.Expression vector digested plastic recycling after the vector fragment. Objective genefragment homologous recombination and transformed into E.coli competent cells.Transformants with colony PCR identification, the positive clones were sequenced. Cloningand sequencing of the correct plasmid.
⑶Virus particles was packed using HEK-293T cells and titer determination.
2.animal experiment
⑴the experimental animal and grouping:150male ApoE-/-mice (C57/BL6genebackground),6weeks old, weighing18-20g, were randomly divided into6groups,25ratsin each group. The control group (group CON) was under the ordinary feed feeding. Theother5groups were under high-fat diet, according to the different intervention: high fatgroup (HF group): treated with sterile saline (0.5ml/days) intraperitoneal injection fromeleventh weeks， continuous injection for4weeks. High fat+L-meprin-α group (HF+LMgroup): intravenous injection of L-meprin-α (1.84x108TU) through caudal vein at eleventhweeks, then treated with sterile saline (0.5ml/days) intraperitoneal injection, continuousinjection for4weeks. High fat and+L-meprin-α+AC group (group HF+LM+AC):intravenous injection of L-meprin-α (1.84x108TU) at eleventh weeks through caudal vein,then treated with AC (5mg/kg/day) intraperitoneal injection from eleventh weeks,continuous injection for4weeks. High fat and+L-meprin-α+AG1478group (groupHF+LM+AG): intravenous injection of L-meprin-α (1.84x108TU) at eleventh weeksthrough caudal vein, then treated with AG1478(20mg/kg/day) intraperitoneal injectionfrom eleventh weeks， continuous injection for4weeks. High fat group+L-EGFP(HF+LGFP group): intravenous injection of L-EGFP (1.92x108TU) at eleventh weeksthrough caudal vein, then treated with sterile saline (0.5ml/days) intraperitoneal injection,continuous injection for4weeks. The HF+LGFP group has3mice were executed beforethe tail vein injection of L-EGFP and after the injection one or four weeks，in order toconfirm the efficiency of transfection of lentiviral vector in atherosclerotic plaques. Allmice were killed after fourteenth weeks for each group.
⑵Observe the size and number of atherosclerotic plaque in pathology: ApoE-/-mousewere executed by intraperitoneal anesthesia with overdose of pentobarbital sodium. thoracicaortic were peeled and removed from body and then fixed，embeddinged and cutted into5um thick slices. After conventional hematoxylin and eosin (hematoxylin and eosin, HE)staining， tissue section were observed under microscope to observe formation ofatherosclerotic plaque；oil red O staining, observe the number and size of atheroscleroticplaque in the general level.
⑶Detection of the generation of ROS in vascular intima in situ by DHE: mousethoracic aorta section treatment with DHE after dewaxing, confocal microscopy can detect the fluorescence of ROS and obtain image. using IPP6image analysis software was used toanalysis results.
⑷Detection of meprin-α expression in endothelium by Western blot assay (WesternBlot, WB): extraction of-80℃frozen mouse thoracic aortic atherosclerotic plaquepreservation organization50micrograms of protein by SDS-PAGE method, extracted andindexed to the PVDF membrane, antibody closed and incubation overnight. Secondantibodies with horseradish peroxidase detection protein strips and chemiluminescence.
⑸Detection of EGFR expression in endothelium by immunohistochemistry: mouseaorta sections with antibodies embedding and incubation at4℃overnight, and thenincubated with biotinylated second antibody30min, adding DAB directly under themicroscope.
3. Cell experiment
⑴Detection of generation of ROS in cell by DHE fluorescent probe labeled: J774A.1cells were pretreat with different factors，intracellular ROS generation were detected，theresults should make it clear that the factors affected the intracellular ROS generation andtheir relationship.
⑵Detection of protein concentration in cell culture fluid by ELISA: J774A.1cellswere pretreat with different factors，HB-EGF protein concentration in cell culture fluidwere detected by ELISA，the results should make it clear that the influence factors ofJ774A.1cells to release HB-EGF.
⑶Detection of intracellular protein expression by: J774A.1cells were pretreat withdifferent factors，EGFR and its downstream signal factors were detection by WB, the resultsshould make it clear that the factors affected the protein expression and their relationship.
4.Statistical analysis
Measurement data as mean±standard deviation (mean±SD) said. Statistical datausing single factor analysis of variance (One-way ANOVA), there were significantdifferences in P<0.01. Statistical analysis using SPSS13.0software.
Result
1. Construction of Lentivirus vector
Lentivirus vector containing Merin1A gene and pSB1control vector were successfullyconstructed.
2.animal experiment
⑴Results of histopathological observation
①HE staining: The three layer structure of vascular wall in CON group is clear, innermembrane is smooth, smooth muscle cells arranged in neat rows, the artery wall slightlyuplift and form very small amount atherosclerotic plaque. A small amount of lipid andinflammatory cells infiltration under the endothelial. Middle and outer membrane structureare clear, no pathological change. Vessel wall were diffuse swelling in high fat-diet HFgroup, protruding into the lumen surface formed obvious plaque, the inner membrane wasthickening and structure was serious damaged. A large number of irregular arrangement ofsmooth muscle cell proliferation under inner membrane, with lipid deposition, the formationof unequal size, irregular shape of the foam cells, a massive infiltration of macrophages,disorder of middle structure, smooth muscle hyperplasia, necrosis, and inflammatory cellinfiltration. Clearance elastic plate and cells showed large amounts of lipid deposition.Compared with HF+LM group and HF group, plaque area, neonatal formation of larger,intimal thickening was more obvious, more lipid deposition, plaque macrophage infiltrationis more obvious; HF+LM+AG group and HF+LM+AC group compared with HF+LMgroup, the plaque area decreased, inner membrane thickening, plaque decreases lipiddeposition, a small amount of macrophages and inflammatory cells; compared with HFgroup, the change of vessel wall in HF+LGFP group is similar.
②The oil red O staining: The thoracic aorta in CON group were soft elastic, innermembrane were smooth, with only a local dyeing damage area. The visible flake darkerdamage area of vascular inner membrane in high fat-diet HF group, inner membrane werenot complete and large amounts of membrane structure was destroyed (P<0.01). Comparedwith HF group, the number and size of lesions were significantly increased in HF+LMgroup (P<0.01); HF+LM+AG group and HF+LM+AC group compared with HF group, thenumber and size of lesions were significantly reduced (P<0.01); compared with HF+LGFPgroup and HF group, the number and size of the injury site has no obvious difference.
⑵Each group ofApoE-/-mouse vascular intima in situ ROS generation
There were very small amounts of ROS generation thoracic aortic intimal in situ inCON group.Compared with CON group, HF group have increased ROS generation (P<0.01)in situ. Compared with HF group, HF+LM group ROS generation in situ increased significantly (P<0.01).Compared with HF+LM group, HF+LM+AC group ROS generationin situ was significantly reduced (P<0.01).
⑶Each group ofApoE-/-mouse expression of meprin-α in vascular intimal
Only a small amount of the expression of meprin-α in CON group.Compared withCON group, expression of meprin-α increased significantly in HF group (P<0.01).
⑷Each group ofApoE-/-mouse expression of EGFR in vascular intimal
There were almost no expression of EGFR in CON group thoracic aortic vascularintimal and a small amount expression of EGFR was visible in HF group. Compared withHF group, the expression of EGFR in HF+LM group was significantly increased (P<0.01).Compared with HF+LM group, the expression of EGFR in HF+LM+AC group wassignificantly reduced (P<0.01).
3.cell experiment
⑴Different intervention treatment impact on J774A.1intracellular ROS generation.
①In the comparison of the groups, compared with the control group, OxLDLtreatment significantly increased the intracellular ROS generation at all time.
②Compared with OxLDL treated cells: OxLDL+AC, Ox-LDL+meprin-α siRNA,OxLDL+AG1478, Ox-LDL+HB-EGF and p38inhibitor SB203580treatment ofintracellular ROS generation was significantly reduced（P<0.01），while ROS generatesOxLDL+meprin-α treated cells increased (P<0.01).
③Compared with OxLDL+meprin-α treated cells, ROS significantly reduced inOxLDL+meprin-α+AG1478treated cell (P<0.01).
⑵Different intervention treatment impact on J774A.1release HB-EGF
①Compared with group control，the cell culture fluid of OxLDL and OxLDL+meprin-α treatment cells had increased concentration of HB-EGF (P<0.01).
②Compared with cells treated with OxLDL, the cell culture fluid of OxLDL+AC andOxLDL+meprin-α siRNA treated cells had reduced concentration of HB-EGF(P<0.01).
⑶Different intervention treatment impact on J774A.1intracellular proteinexpression.
Compared with control group, OxLDL treated cells had increased expression of EGFR,p38and PI3K-Akt phosphorylation(P<0.01), increased expression of GTP-rac1(P<0.01).Compared with OxLDL treated cells, expression of EGFR phosphorylation were reduced in cell treated with antibodies against HB-EGF, AC or meprin-α siRNA (P<0.01), expressionof p38phosphorylation were reduced in cell treated with AC, meprin-α siRNA, Rac1inhibitor EHT1864and PI3K inhibitor wortmsnin (P<0.01), intracellular expression ofGTP-rac1were significantly reduced in cells treated with wortmsnin (P<0.01), intracellularexpression of PI3K-Akt phosphorylation in cell treated with AC，meprin-α siRNA andAG1486were decreased significantly (P<0.01).
Conclusion
1.Meprin-α promote formation and development of thoracic aortic atheroscleroticplaque in high fat-fed ApoE-/-mouse through induce oxidative stress. meprin-α inhibitor ACand EGFR inhibitor AG1478can counteract the effect of meprin-α. meprin-α is theupstream signal molecule of EGFR, affecting the expression and activity of EGFR.
2.Meprin-α could promote macrophages release the extracellular part of thetransmembrane structure of HB-EGF out of cell, as a combination of ligand oftransmembrane structure of the extracellular domain of EGFR structure, activation of EGFRby promoting and tyrosine kinase phosphorylation patterns, initiate downstream signalingpathways and promote the generation of ROS in cells.
3. Meprin-α transactivation EGFR start receptor signaling in macrophages, via thePI3K-Rac1-p38pathway induce the generation of ROS in cells.
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