血清淀粉样蛋白A加速动脉粥样硬化斑块形成及其致炎作用的机制研究
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摘要
研究背景
心脑血管疾病已成为严重危害人类健康的头号杀手,其高发病率和高致残率带来了诸多的社会问题。而动脉粥样硬化(atherosclerosis, AS)是多种心脑血管疾病共同的病理基础,是临床上导致急性冠脉综合征(acute coronary syndrome,ACS)发生的最主要病因。经过多年的丛础和临床研究,Ross明确提出“AS是一种慢性炎症性疾病”,其发生发展是涉及到脂质代谢紊乱、免疫炎症反应、巨噬细胞浸润等多个因素和环节的复杂病理过程。因此,深入探讨影响AS病变形成的各种危险因素并寻找有效的干预靶点,预防甚至避免AS疾病的发生,成为了近年来心血管领域的研究热点。
人类血清淀粉样蛋白A (serum amyloid A, SAA)是一组由同一基因编码的多形性蛋白,包括SAA1-SAA4这四种蛋白亚型。SAA1和SAA2蛋白结构近93%
一致,在炎症的急性反应期中显著上调；SAA3基因则被认为是一种不能转录和表达的假基因；SAA4蛋白虽能够持续表达,但不参与急性炎症反应。研究表明,不论是在正常还是在炎症状态下,SAA1亚型始终是SAA的主要组分,决定着总SAA蛋白的作用水平。
SAA血清水平在机体急性炎症(如外伤、感染等)反应过程中上升极快,短时间内由肝脏细胞大量分泌,可超过正常值的1000倍以上,这种特性也使得SAA成为目前最敏感的炎症标志物之一。同时,众多的研究也发现,在包括肥胖、AS在内的各种慢性炎症病理状态下,人类的脂肪细胞、血管内皮细胞及单核-巨噬细胞等肝外组织也能合成分泌SAA蛋白并导致血清SAA水平呈一定程度的升高,从而时刻准确反映着机体的慢性炎症水平。
近年来,国内外多宗大规模临床试验显示血清SAA的水平可以作为评估和预测冠心病病情严重程度的敏感标志物,这提示SAA与AS的发生与流行之间似乎存在着密切的联系。随着对SAA研究的深入,SAA的众多生物学功能也逐渐为人们所了解。首先,SAA对单核-巨噬细胞具有较强的趋化性。其次,SAA可以作为载脂蛋白,参与胆固醇代谢并增加脂质与巨噬细胞的亲和力。再次,在一系列的体外细胞实验中发现SAA能够上调单核细胞趋化蛋白1(monocyte chemotactic protein-1, MCP-1)、肿瘤坏死因子(tumor necrosis factor, TNF)-α、白细胞介素(interleukin, IL)-6及IL-8等炎症相关因子的表达。上述关于SAA生物学效能的研究结果进一步提示我们,SAA应该在AS的发生发展过程中发挥了一定的作用。
然而,尽管近年来国内外学者在SAA与AS斑块形成两者关系的研究中已取得了较大进展,但仍存在一系列困扰我们的重大问题：(1)究竟SAA仅仅是AS慢性炎症状态的一个标志物,还是AS发生过程中的一个积极的参与者?(2)如果SAA直接或者间接地参与了AS的发生,那么它发挥了何种作用?(3)SAA在AS病程中所发挥的作用又是通过哪些途径和靶点来实现的?
针对上述这些亟待解决的关键问题,本课题构建了SAA1高表达慢病毒,成功实现了SAA1蛋白在实验动物体内的稳定高表达；同时采用ApoE-/-小鼠构建AS动物模型,探讨SAA在AS斑块形成过程中所发挥的作用及其可能的机制,旨在为有效控制AS疾病的发生发展提供全新的干预靶点与治疗策略。
研究目的
1.明确血清SAA浓度的升高在ApoE-/-小鼠AS斑块形成过程中所发挥的作用。
2.探讨SAA在AS斑块内的沉积及表达情况以及SAA对AS斑块内巨噬细胞水平及分布的影响。
3.探讨血清SAA浓度的升高对促AS发生的相关炎症因子表达水平的影响。
研究方法
1.动物模型的建立
90只8周龄雄性ApoE-/-小鼠随机分为SAA1高表达慢病毒干预组(lenti-SAA组)、空载体慢病毒对照组(lenti-null组)和生理盐水对照组(saline control组)三组。本课题所使用的小鼠SAA1高表达慢病毒购自上海Invitrogen公司。lenti-SAA组每只小鼠尾静脉注射107TU的慢病毒；作为对照,lenti-null组和saline control组分别注入相应体积的空载体慢病毒和生理盐水。为避免高脂饮食喂养对AS斑块形成所产生的影响,实验全程采用普通饮食(5%脂质,不添加胆固醇)喂养实验动物,期间使用小动物超声监测斑块形成时间、面积。普通饮食喂养14周后,小鼠隔夜禁食后称重,麻醉后处死并取血,离心后取血清冻存。随机处死各组中的小鼠经灌流和固定后取材,将小鼠心脏和主动脉与周围组织分离并冻存。
2.血液生化指标检测
检测血清甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、及低密度脂蛋白胆固醇(LDL-C)水平。同时,使用酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)测定血清总SAA、IL-6、TNF-α水平。
3.主动脉表面AS病变染色
收集自主动脉弓到腹主动脉分叉处的主动脉节段,去除结缔组织成分后,纵剖后展开固定于蜡板上并行油红O染色,AS斑块区域将被染成红色。AS的病变程度用油红O阳性染色区域占整个主动脉表面积的百分比表示。
4.主动脉根部AS病灶分析
对主动脉窦做平行于瓣环的横截位冰冻切片,对斑块内结构和成分进行染色,包括苏木素-伊红(H&E)染色、油红O染色等,并进行量化分析,AS的病变程度用总的斑块面积占整个主动脉瓣环面积的百分比表示。
5.免疫组织化学染色
取主动脉窦冰冻切片,进行巨噬细胞、MCP-1的免疫组织化学染色,观察两者在AS斑块内的表达水平。
6.免疫荧光染色
取主动脉窦冰冻切片,进行SAA、巨噬细胞及血管细胞粘附分子1(vascular cell adhesion molecule-1, VCAM-1)的免疫荧光染色,观察各个指标在AS斑块内的表达水平,并进一步明确SAA与巨噬细胞在斑块内分布的相关性。
7.实时定量PCR (real-time PCR)
通过Trizol法提取小鼠主动脉总RNA,逆转录后,以β-actin为参照,采用real-time PCR技术检测MCP-1和VCAM-1的mRNA表达水平。
研究结果
1.体重及血液生化指标检测
实验结束时lenti-SAA组、lenti-null组和saline control组三组之间的体重、TG、TC、HDL-C及LDL-C水平未见统计学差异(P>0.05),这说明采用普通饮食喂养实验动物,成功避免了高脂饮食喂养所造成的血脂异常对AS斑块形成所产生的影响。lenti-null组和saline control组的血清SAA、IL-6及TNF-α水平未见统计学差异(P>0.05),这说明SAA1高表达慢病毒的注射并未引起小鼠机体的炎症反应。因此,对本实验结果的观察重点聚焦于lenti-SAA组与lenti-null组之间实验结果的对比。lenti-SAA组较lenti-null组的SAA、IL-6及TNF-α水平显著升高(P<0.01),这证明了SAA1高表达慢病毒的注射在小鼠体内能够有效的高表达,保证了血清SAA水平的稳定升高；而SAA水平的升高又进一步导致了IL-6及TNF-α水平的升高,加重了机体的炎症水平。
2.AS病变严重程度分析
主动脉AS病变大体染色显示lenti-SAA组较lenti-null组的AS斑块面积显著增加(P<0.01),这与对主动脉窦AS病变区域行H&E及油红O染色所得到的结果一致(P<0.01),说明血清SAA水平的升高加速了AS斑块的形成。
3.AS斑块内SAA与巨噬细胞表达情况
对主动脉窦AS斑块内SAA、巨噬细胞行免疫组织化学和荧光染色发现,lenti-SAA组较lenti-null组的巨噬细胞数目显著增多(P<0.01),且巨噬细胞与SAA在斑块内部的分布位置相互重叠,密切相关。
4.AS斑块内MCP-1的表达情况
real-time PCR、免疫组织化学染色的结果显示,与lenti-null组相比,lenti-SAA组AS斑块区域MCP-1的mRNA及蛋白表达水平显著上调(P<0.01),这提示血清SAA水平的升高通过上调AS斑块内MCP-1的表达,增强了对巨噬细胞的趋化作用。
5.AS斑块内VCAM-1的表达情况
通过real-time PCR、免疫荧光染色技术显示,与lenti-null组相比,lenti-SAA组AS斑块区域VCAM-1的mRNA及蛋白表达水平显著上调(P<0.01),这说明血清SAA水平的升高增加了AS病变区域主动脉内皮细胞VCAM-1的表达,从而增强了对巨噬细胞的粘附作用。
结论
1.本研究应用SAA1高表达慢病毒首次成功地实现了SAA1蛋白在实验动物体内的持续稳定高表达。
2.本研究采用普通饮食喂养ApoE-/-小鼠,避免了高脂饮食喂养对研究AS斑块形成所产生的影响。
3.血清SAA水平的升高,通过上调IL-6及TNF-α水平,加重了机体的炎症反应,从而显著加速了AS斑块的形成。
4.SAA通过上调AS病变区域MCP-1及VCAM-1的表达,增加了病变区域对巨噬细胞的趋化及粘附,加重了斑块内巨噬细胞的浸润,从而促进了AS斑块的发生。
研究背景
动脉粥样硬化(atherosclerosis, AS)是导致多种心脑血管疾病发生的首要病理生理基础,其发生发展是以肥胖、高脂血症、高血压和吸烟为代表的多种危险因素共同作用的结果。Ross在“损伤-反应学说”基础上提出：各种炎症因子介导的免疫炎症连锁反应在AS发生发展过程中发挥着重要作用。这种免疫炎症反应往往伴随着血管内皮细胞的功能不良及动脉血管壁内的单.核-巨噬细胞和T淋巴细胞浸润等特征。
人类血清淀粉样蛋白A (serum amyloid A, SAA)通常被定义为一种敏感的炎症标志物。在机体急性炎症(如外伤、感染等)和各种慢性炎症病理状态(如肥胖、AS)下时刻准确反映着机体的炎症水平。最新的研究表明, SAA能够在体外细胞实验中上调肿瘤坏死因子(tumor necrosis factor, TNF)-α、白细胞介素(interleukin, IL)-6、IL-12及单核细胞趋化蛋白-1(monocyte chemotactic protein-1, MCP-1)等多种炎症相关因子的表达。并且,SAA上调机体TNF-α、IL-6及MCP-1等炎症因子表达的作用已在论文一中的体内动物实验中得以证实。这提示我们,SAA的生物学功能似乎不仅仅局限于作为单纯的炎症标志物,它本身就可以发挥致炎作用,刺激相关靶细胞(如单核-巨噬细胞、主动脉内皮细胞等),上调各种炎症因子的表达,进一步加重机体的炎症水平。
正五聚蛋白-3(pentraxin3, PTX3)是进化过程中一个很保守的五聚体蛋白质,主要由单核-巨噬细胞、内皮细胞、树突状细胞和脂肪细胞等在IL-1、TNF-α等炎症因子的刺激下分泌。PTX3与C反应蛋白(C reactive protein, CRP)、血清淀粉样成分P (serum amyloid P, SAP)共同隶属于五聚体蛋白家族(pentraxin)。一方面,PTX3与CRP、SAA相似,也属于炎症反应的急性时相蛋白家族。在创伤、炎症、感染、肿瘤等情况下血清水平迅速升高,从而能够敏感地反应机体的炎症水平。众多的研究已经证实,监测血清PTX3的水平对评估急性心血管事件和心衰患者的预后具有重要的预测价值。但由于CRP、SAA主要由肝脏细胞分泌,反映机体系统性炎症水平,而PTX3主要由血管内皮细胞、单核-巨噬细胞等在脉管系统病变的局部产生,因而被称为“脉管系统的CRP”,能够更加直接反映病变局部及血管的炎症状态。另一方面,PTX3还在炎症反应与固有免疫(innate immunity)之间起到了桥梁和纽带作用,它能够与补体(complement,C)分子Clq结合,激活补体经典途径,参与炎症反应和固有免疫,在介导细胞凋亡过程中发挥着重要的调节作用。
然而,SAA的致炎作用尚需要更多的实验证据来加以证明,并且其致炎作用的具体信号转导通路尚未完全明了。针对上述亟待解决的问题,本课题采用人重组SAA蛋白,干预人主动脉内皮细胞(human aortic endothelial cells, HAECs),观察SAA上调另一种炎症因子PTX3的表达,并且深入探讨了SAA发挥作用的具体信号转导通路,为进一步证明SAA的致炎作用和及明确其作用机制提供更为坚实的理论基础。
研究目的
1.探讨SAA刺激HAECs诱导PTX3表达上调,进一步证明SAA的致炎作用；
2.明确SAA上调PTX3表达的具体信号转导通路。
研究方法
1.实验设计
HAECs购自美国模式培养物集存库(American type culture collection, ATCC),采用专用的内皮细胞培养基(endothelial culture medium, ECM)进行培养。取代数在3-5代,处于对数生长期的细胞进行实验。
1)分别给予HAECs不同浓度梯度(0、0.1、1、10、20μg/ml)的人重组SAA蛋白及采用不同干预时间(0、3、6、9、12、15、24h)进行刺激,采用酶联免疫吸附测定(enzyme-linked immuno sorbent assay, ELISA)检测细胞上清中PTX3的分泌水平,观察SAA诱导HAECs上调PTX3表达的浓度-时间依赖性。
2)分别对HAECs给予甲酰肽样受体1(formyl peptide receptor like-1, FPRL1)的小干扰RNA (small interfering RNA, siRNA)预处理48h,或给予FPRL1受体的特异性抑制剂WRW4以及G蛋白偶联受体的抑制剂pertussis toxin预处理1h,再给予最佳剂量的人重组SAA蛋白刺激24h后,采用ELISA法,检测细胞上清中PTX3的分泌水平,探讨FPRL1受体在SAA诱导HAECs增加PTX3表达过程中所发挥的作用。
3)分别对HAECs给予c-Jun氨基末端激酶(c-Jun NH2-terminal kinase, JNK)1、JNK2的siRNA预处理48h,再给予最佳剂量的人重组SAA蛋白刺激24h后,采用ELISA法,检测细胞上清中PTX3的分泌水平,探讨JNK通路在SAA诱导HAECs增加PTX3表达过程中所发挥的作用。
4)分别对HAECs给予转录激活蛋白1(activator protein, AP-1)的抑制剂丹参酮ⅡA (Tanshinone ⅡA)及核转录因子-κB (nuclear factor-kappa B, NF-κB)的抑制剂BAY11-7082和SC-514预处理1h,再给予最佳剂量的人重组SAA蛋白刺激24h后,采用ELISA法,检测细胞上清中PTX3的分泌水平,探讨AP-1及NF-κB的激活在这一过程中的作用。
2. ELISA
分别给予HAECs上述处理后,取细胞上清液,离心去除细胞碎屑及杂质,采用ELISA法检测其中PTX3的蛋白水平。
3.细胞免疫荧光染色
将HAECs种植在放有载玻片的24孔中培养,待细胞生长至70%的融合状态,给予NF-κB抑制剂BAY11-7082和SC-514预处理1h,再给予最佳剂量的人重组SAA蛋白刺激24h后,取出载玻片,固定,封闭,NF-κB一抗孵育,FITC标记的二抗显色后,DAPI复染细胞核,荧光显微镜下观察BAY11-7082和SC-514对SAA介导的NF-κB激活的抑制作用。
4. Western blot检测蛋白质的表达水平
在对HAECs给予相关处理之后,使用细胞刮刀收集贴壁的HAECs,提取细胞总蛋白,进行SDS-PAGE电泳分离,转膜,蛋白印迹和免疫反应后检测FPRL1及JNK1和JNK2的蛋白质表达水平。
5.实时定量PCR (real-time PCR)检测mRNA的表达变化
在对HAECs给予相关处理之后,利用Trizol法提取HAECs的总RNA,并以β-actin作为参照,采用real-time PCR技术检测PTX3、FPRL1及JNK1和JNK2的mRNA表达水平。
研究结果
1.SAA呈浓度和时间依赖性上调HAECs中PTX3的表达水平
给予HAECs一定浓度梯度(0、0.1、1、10、20μg/ml)的SAA蛋白干预24h,收集上清ELISA检测PTX3表达水平,结果显示,SAA能够呈浓度依赖性的诱导HAECs中PTX3的表达,且最佳刺激浓度为10μg/ml。随后,对HAECs给予10μg/ml的SAA蛋白干预0、3、6、9、12、15、24h后发现,SAA可以呈时间依赖性的上调HAECs中PTX3的表达,且在干预24h后达到蛋白表达的最高水平。同时,real-time PCR的实验结果也显示SAA能够以时间依赖性的方式上调HAECs中PTX3的mRNA的表达水平。
2.SAA通过FPRL1受体上调PTX3的表达
给予FPRL1的siRNA预处理HAECs后,使用real-time PCR及Western blot的方法验证siRNA的干扰表达作用,结果显示,FPRL1的siRNA可以有效的降低FPRL1的表达水平。随后,对HAECs给予FPRL1的siRNA预处理48h,再给予10μg/ml的SAA蛋白刺激24h后,检测细胞上清PTX3的表达,结果发现,对HAECs行FPRL1的siRNA预处理后,SAA诱导的PTX3表达水平显著下调,说明FPRL1参与了这一过程。
3.SAA上调PTX3的表达受JNK通路的调控
分别对HAECs给予JNK1和JNK2的siRNA预处理48h,再给予10μg/ml的SAA蛋白刺激24h后,检测细胞上清PTX3的表达,结果显示,SAA诱导的PTX3表达水平显著下调,这提示SAA介导的PTX3表达上调受JNK1和JNK2的调控。
4.SAA上调PTX3的表达需要AP-1的激活
在给予HAECs一定浓度梯度(0、5、10、20μg/ml)的AP-1抑制剂TanshinoneⅡA预处理1h后,再给予10μg/ml的SAA蛋白刺激24h后,检测细胞上清PTX3的表达,结果显示,SAA诱导的PTX3表达水平呈浓度依赖性的被Tanshinone ⅡA下调,这提示SAA介导的PTX3表达上调需要AP-1的激活。
5.SAA上调PTX3的表达需要NF-κB的激活
对HAECs给予NF-κB的抑制剂BAY11-7082和SC-514预处理1h,再给予10μg/ml的SAA蛋白刺激24h后,免疫荧光染色显示SAA介导的NF-κB激活被BAY11-7082和SC-514显著下调。随后,在分别给予HAECs一定浓度梯度(0、1、5、10、20μM)的BAY11-7082和SC-514预处理1h后,再给予10μg/ml的SAA蛋白刺激24h后,检测细胞上清PTX3的表达,结果显示,SAA诱导的PTX3表达被NF-κB的抑制剂BAY11-7082和SC-514显著下调,这提示NF-κB的激活参与SAA诱导的PTX3表达这一过程。
结论
1.SAA能够显著上调HAECs中PTX3的表达。
2.SAA诱导的PTX3的表达上调是通过FPRL1受体发挥作用的。
3.SAA诱导的PTX3的表达上调受JNK通路的调节。
4.SAA诱导的PTX3的表达上调需要AP-1及NF-κB的激活。
Background
Atherosclerosis is an important underlying pathologic condition of many cardio-cerebro-vascular diseases, the leading cause of morbidity and mortality worldwide. Basing on the long-term experimental and clinical research, the theory that atherosclerosis is a chronic inflammatory disease has been proposed by Ross explicitly. The occurrence and development of atherosclerosis is a complex pathological process involving a series of influencing factors such as lipid metabolism, immuno-inflammatory response, macrophages infiltration and so on. Therefore, seeking the effective targets to avoid the formation of atherosclerosis plaque has already become a hot topic in the cardiovascular field.
Serum amyloid A (SAA) is one of the most sensitive acute phase proteins in vertebrates. It is produced principally by the liver in response to acute inflammatory stimuli and its plasma concentration can increase by up to100-to1000-fold over the basal level. Meanwhile, accumulating evidences also support that under the chronic inflammatory status such as obesity and atherosclerosis, the secretion of SAA by human adipocytes, vascular endothelial cells and macrophages can cause a modest increase in the plasma and reflect the inflammatory level exactly.
SAA is a family of homologous proteins including4isoforms which are encoded by the same gene. SAA1and SAA2are the major acute phase reactants, with primary structures that are93%identical. The human SAA3gene is a pseudogene. SAA4 which is constitutively expressed in humans but not in mice do not participate in the acute inflammatory reaction. Many studies have already demonstrated that SAA1predominates in plasma, where it functions as a major isotype.
So far, many large clinical trials have demonstrated that increased plasma SAA level can be regarded as a useful indicator for identifying individuals at high risk of cardiovascular disease (CVD), suggesting that there may be a link between the SAA and prevalent CVD. With the further study, more and more biological function of SAA has been known by us. Firstly, SAA exhibits considerable chemoattractant activity for human monocytes and macrophages. Secondly, as the apolipoprotein of high-density lipoprotein (HDL) and low-density lipoprotein (LDL), SAA increases the binding affinity of cholesterol for macrophages and endothelial cells. In addition, a series of studies in vitro show that SAA can up-regulate the expression of many inflammatory factors such as monocyte chemotactic protein-1(MCP-1), tumor necrosis factor-α(TNF-α), interleukin (IL)-6and IL-8and so on. All the results mentioned above suggest that SAA should play an effective role in the progression of atherosclerosis.
Despite the numerous reported proatherogenic properties of SAA, we lack direct proof that SAA is an active participant in the atherosclerosis process in vivo. To investigate whether SAA is purely a risk marker for atherosclerosis or is also an active participant in vivo, we examine the effect of high-level expression of SAA on atherosclerosis development by using apolipoprotein E-deficient (ApoE-/-) mice transfected with lentivirus to induce SAA overexpression. Findings from this study can provide the first in vivo evidence that an elevated plasma level of SAA accelerates the progression of atherosclerosis directly and independently, and then may be helpful in designing novel therapeutic strategies against atherosclerosis.
Objectives
1. To investigate the role of increased SAA plasma level on the formation of atherosclerosis plaque in ApoE-/-mice.
2. To observe the deposition and the influence of SAA on the distribution of macrophages in atherosclerosis plaque.
3. To elucidate the role of increased SAA plasma level on the expression of proatherogenic factors.
Methods
1. Establishment of animal model
90male ApoE-/-mice (8weeks age) were randomly divided into3groups, and then were injected intravenously with lentivirus-expressing mouse SAA1(lenti-SAA group, n=30) at a total lentivirus dose of1×107TU/mouse, a null lentivirus (lenti-null group, n=30) or saline (saline control group, n=30). To avoid the influence of high plasma lipids on atherogenesis, all the animals were fed a chow diet (5%fat and no added cholesterol) throughout the entire experiment. At14weeks after lentivirus injection, mice were anesthetized with pentobarbital injected intraperitoneally, and then blood samples were taken. Serum was separated by centrifugation at4℃. Meanwhile, the hearts and aortas were removed and perfusion-fixed with4%paraform aldehyde for histological and morphological staining or with PBS for real-time polymerase chain reaction (real-time PCR).
2. Biological measurements
The levels of triglycerides (TG), total cholesterol (TC), HDL-C and LDL-C were measured by use of an automatic biochemistry analyzer. And serum levels of SAA, IL-6and TNF-α were detected by enzyme-linked immuno sorbent assay (ELISA).
3. En face analysis of the aorta
The aorta was stripped of adventitia and dissected longitudinally from the iliac arteries to the aortic root, then the branching vessels were removed. The paraformaldehyde-fixed aorta was pinned flat on a black surface, and the atherosclerotic lesion area was readily visualized with Oil-Red-O staining. Average lesion area was quantified by use of ImagePro-Plus soft ware. The ratio of total atherosclerotic lesion area to aorta intimal surface area was calculated as an indicator to evaluate the level of atherogenesis.
4. H&E and Oil-Red-O staining of aortic sinus Murine hearts were fixed in4%paraformaldehyde overnight and then embedded in optimal cutting temperature compound. At least50serial cryosections6-μm thick were cut, beginning at the junction of the left ventricle and the aorta. Sections were stained with hematoxylin and eosin (H&E). The lipid core was identified by Oil-Red-O staining. The atherogenesis level at the aortic sinus was evaluated by Oil-Red-O and H&E staining according to the ratio of total atherosclerotic lesion area to aortic lumen area.
5. Immunohistochemical analysis Immunohistochemical analysis was used to detect the expression of macrophages and MCP-1in lesions. Data were analyzed by use of ImagePro-Plus software.
6. Immunofluorescence
Cryosections of the aortic sinus were chosen to observe the expression of SAA, macrophages and vascular cell adhesion molecule-1(VCAM-1) through immunofluorescence analysis. The colocalization of SAA with macrophages was also examined in atherosclerosis plaque. Data were analyzed by use of ImagePro-Plus software.
7. Quantitative real-time PCR
Total RNA was extracted from murine frozen aortic specimens by use of trizol, and then reverse transcribed. Real-time PCR was used to detect the mRNA levels of MCP-1and VCAM-1.
Results
1. Body weight and measurement of plasma variables ApoE-/-mice in3groups fed a chow diet did not differ in body weight, TG, TC, HDL-C or LDL-C (P>0.05). Therefore, we excluded the influence of lipid levels on atherosclerosis in this study. The lenti-null and saline control groups did not differ in plasma levels of IL-6or TNF-α, so the injection of the SAA1lentivirus vector was safe and did not induce inflammatory responses (P>0.05). The plasma levels of SAA were higher for the lenti-SAA group than lenti-null and saline control groups, so the SAA1lentivirus was efficiently transfected in vivo (P<0.01). Most importantly, with elevated SAA level, the plasma levels of IL-6and TNF-a were significantly higher in the lenti-SAA than lenti-null group, suggesting that the increased SAA level may aggravate the inflammatory reflect (P<0.01).
2. Atherogenesis level analysis
By using en face analysis of the aorta, lesion area was significantly larger for the lenti-SAA than lenti-null group (P<0.01). Atherogenesis level at the aortic sinus was evaluated by Oil-Red-O and H&E staining by ratio of total atherosclerotic lesion area to aortic lumen area. The mean lesion size at the aortic sinus were greater for the lenti-SAA than lenti-null group (P<0.01). All the results demonstrated that increased plasma SAA level directly promotes atherosclerotic lesions.
3. Accumulation of SAA and macrophages in atherosclerosis plaque
Immunohistochemistry analysis showed a greater increase in accumulation of macrophages for the lenti-SAA than lenti-null group (P<0.01). Because SAA is a classic chemoattractant to peripheral blood leukocytes, we observed the colocalization of SAA with macrophages in lesions on aortic cryosections through immunofluorescence. The distribution of macrophages was consistent with SAA protein localization.
4. Expression of MCP-1in atherosclerosis plaque
Because MCP-1is a key molecule regulating chemotactic migration of macrophages, we observed the expression of MCP-1in vivo by immunohistochemistry. MCP-1secretion was increased with elevated level of plasma SAA, which was also confirmed by real-time PCR (P<0.01). Our data suggested that SAA could enhance the chemotaxis of macrophages through up-regulating the expression of MCP-1in lesions.
5. Expression of VCAM-lin atherosclerosis plaque
Immunofluorescence analysis revealed upregulated VCAM-1expression in vivo in the lenti-SAA group as compared with the lenti-null group (P<0.01). VCAM-1mRNA expression results agreed with protein level results. Our research suggested that SAA could promote the adhesion of macrophages in lesions through up-regulating the expression of VCAM-1.
Conclusions
1. In the present study, we achieved the persistent high expression of SAA protein by applying SAA1lentivirus in ApoE-/-mice effectively.
2. Throughout the entire experiment, all the animals were fed a chow diet, so that the influence of lipid levels on atherosclerosis formation was excluded in our study.
3. SAA accelerated the progression of atherosclerosis in ApoE-/-mice through aggravate the inflammatory level.
4. SAA enhanced the chemotaxis and adhesion of macrophages through up-regulating the expression of MCP-1and VCAM-1, thus promoting the formation of atherosclerosis plaque
Background
Atherosclerosis is the primary pathophysiological basis leading to the many cardio-cerebro-vascular diseases. A series of risk factors represented by obesity, hyperlipemia, hypertension and smoking participate in the occurrence of atherosclerosis. On the basis of the response-to-injury hypothesis, the theory that the chain reaction induced by inflammatory factors play an important role in the process of atherogenesis has been proposed by Ross explicitly. This chronic inflammation is characterized by the disfunction of vascular endothelial cell and the accumulation of macrophages and T lymphocytes in the arterial vascular walls.
Serum amyloid A (SAA) is one of the most sensitive acute phase proteins in vertebrates. And it can reflect the inflammatory levels exactly under the status of the acute and chronic inflammations. Recently, a series of studies in vitro have demonstrated that SAA can up-regulate the expression of many inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6and IL-12and so on. And these findings suggest that SAA may be not only a prognostic indicator but also a pro-inflammatory mediator by inducing the expression of the inflammatory factors in mononuclear phagocytes and aortic endothelial cells.
Pentraxins is an acute immunological response family of proteins that consists of three members including C reactive protein (CRP), serum amyloid P (SAP), and pentraxin3(PTX3). PTX3is one of the conserved proteins in evolution and produced by mononuclear phagocytes, dendritic cells (DCs), adipocytes and so on under the stimulation of IL-1and TNF-α. For one thing, just like CRP and SAA, PTX3belongs to classic acute phase reactants that can reflect the levels of inflammation exactly. Many studies have already proved that it is profound to estimate the levels of serum PTX3in the aspect of predicting the prognosis of acute cardiovascular events and hear failure. PTX3, as a cytokine-inducible factor, is mainly produced in vascular endothelial cells, fibroblasts and some other extrahepatic tissues and can reflect the inflammatory levels of local impaired tissues which make it distinguished from CRP and SAA that are synthesized majorly in the liver. For another, PTX3can activate the classical complement pathway through specific recognition and interaction with the complement component Clq and regulate the clearance of apoptotic cells, thus providing a link between the inflammation and innate immunity.
However, the experimental evidence about the pro-inflammatory role of SAA is still inadequate. And the signaling pathway involved in this process is still poor understanding. Therefore, we investigate the capacity of human aortic endothelial cells (HAECs) to express PTX3, another sensitive inflammatory biomarker, after pro-incubation with SAA. Then the signaling pathway involved in this process has also been further discussed. Our research may be helpful in understanding the pro-inflammatory role of SAA and will provide a new theoretical support on elucidating the relative mechanism.
Objectives
1. To investigate the effect of SAA on up-regulating the expression of PTX3in HAECs and further discuss the pro-inflammatory role of SAA.
2. To elucidate the relative signaling pathway involved in this process.
Methods
1. Experiment design
Human aortic endothelial cell line was obtained from American type culture collection (ATCC) and was cultured in endothelial culture medium (ECM) supplemented with5%fetal bovine serum. These cells were used for experiments between3rd and5th passage.
1) HAECs were treated with different concentration gradients (0,0.1,1,10,20μg/ml) of recombinant human SAA for24h or different checkpoints (0,3,6,9,12,15and24h) with10μg/ml of SAA respectively. The capacity of SAA on PTX3secretion in HAECs was observed through detecting the levels of PTX3secretion in cell-free supernatants by using Enzyme-linked immunosorbent assay (ELISA).
2) HAECs were pretreated by the specific siRNA sequences of formyl peptide receptor like-1(FPRL1), or WRW4, an effective inhibitor of FPRL1, or pertussis toxin, an antagonist of G protein-coupled receptor. And then the cells were challenged by SAA (10mg/ml) for another24h. ELISA was used to investigate the role of FPRL1on SAA-induced expression of PTX3by detecting the levels of PTX3secretion in cell-free supernatants.
3) HAECs were pretreated by the specific siRNA sequences c-Jun NH2-terminal kinase (JNK)1and JNK2for48h respectively, and then were challenged by SAA (10mg/ml) for another24h. ELISA was used to investigate the role of JNK1and JNK2on SAA-induced expression of PTX3by detecting the levels of PTX3secretion in cell-free supernatants.
4) HAECs were pretreated by Tanshinone Ⅱ A, the specific inhibitor of activator protein (AP-1), or two effective nuclear factor-kappa B (NF-κB) inhibitors (BAY11-7082and SC-514) for1h respectively, and then were stimulated by SAA (10mg/ml) for another24h. ELISA was used to evaluate the role of AP-1and NF-κB activation in this process by detecting the levels of PTX3secretion in cell-free supernatants.
2. ELISA
HAECs were placed in ECM containing5%FBS in96-well plates and kept in a5%CO2incubator at37℃. After stimulation, cell-free supernatants were collected, centrifuged, and assayed for PTX3by ELISA according to the manufacturer's instructions.
3. Immunofluorescence
HAECs cultured on glass cover slips were pretreated by BAY11-7082and SC-514, two effective NF-κB inhibitors for1h respectively, and then were stimulated by SAA (10mg/ml) for another24h. After that, cells were fixed in2%paraformaldehyde and blocked in3%BSA, then incubated with anti-phospho-NF-kBp65antibody overnight. After being incubated with FITC conjugated secondary antibody, immunolabeled cells were counterstained with DAPI to detect cell nuclei. Then inhibitory role of BAY11-7082and SC-514on SAA-mediated NF-κB activation was observed by visualizing with a fluorescence microscope equipped with a digital camera.
4. Western blot
After being stimulated respectively, HAECs were harvested for protein extraction. Western blot was used to detect the protein expression levels of FPRL1, JNK1and JNK2.
5. Quantitative real-time PCR
Total RNA was extracted from the harvested HAECs by the use of Trizol, and then reverse transcribed. Real-time PCR was used to detect the mRNA levels of PTX3, FPRL1,JNK1and JNK2.
Results
1. SAA up-regulates the PTX3expression in a time-and dose-dependent manner in HAECs
HAECs were treated with different concentration gradients (0,0.1,1,10,20μg/ml) of SAA for24h and the supernatant concentration of PTX3was detected by ELISA. As a result, we observed that SAA could induce PTX3secretion in a concentration-dependent manner. Then cells were stimulated with SAA (10μg/ml) at various time points (0,3,6,9,12,15, and24h). The results demonstrated that SAA could induce PTX3accumulation in a time-dependent manner and reached its maximal activity at24h after stimulation. These results altogether indicate that the induction of PTX3by SAA is time-and dose-dependent. At the same time, the effect of SAA on PTX3at mRNA transcription level was examined via real-time PCR. Our data demonstrated a similar expression in compliance with its protein level, as the mRNA expression of PTX3increased by the time of stimulation after3-9h treatment with SAA which suggested that SAA-induced PTX3protein synthesis required transcriptional activation (P<0.05).
2. SAA induces PTX3production via FPRL1
Human FPRL1siRNA (Santa Cruz, sc-40123) was a convenient tool designed for FPRL1gene silencing and its effect had been examined carefully. Then, human FPRL1siRNA was used to confirm whether SAA-induced PTX3production in HAECs was mediated by FPRL1. After being preincubated with the siRNA sequences of FPRL1for48h and stimulated with10μg/ml SAA for another24h, both HAECs and supernatants were harvested for real-time PCR and Western blot to check the effect of the siRNA sequences. The data from real-time PCR and Western blot indicated that the SAA-induced PTX3secretion could be inhibited significantly by siRNA-mediated FPRL1gene silencing (P<0.05). All these results strongly support that FPRL1is likely to be one of the receptors through which SAA mediates its effects.
3. SAA-induced PTX3production in HAECs is mediated through JNK pathway
After being preincubated with the siRNA sequences of JNK1and JNK2for48h, HAECs were stimulated with10μg/ml SAA for another24h. SAA-induced PTX3production levels were detected by ELISA. Our data showed that SAA-induced PTX3expression could be partially but significantly reduced (P<0.05). These results strongly suggest that JNK pathway is crucial for SAA-induced PTX3expression in HAECs, and activations of both JNK1and JNK2are involved in mediating PTX3production.
4. SAA stimulates PTX3production in HAECs via AP-1activation
After being incubated with Tanshinone Ⅱ A, an AP-1inhibitor, at various dose levels (0,5,10and20μg/ml) for24h, HAECs was exposed to SAA(10μg/ml) for additional24h. We found that Tanshinone Ⅱ A could inhibit SAA-induced PTX3expression dramatically in a dose-dependent manner (P<0.05), which suggested that the up-regulation of PTX3need the activation of AP-1.
5. SAA induces PTX3production in HAECs via NF-κB activation
We examined the effect of SAA on NF-κB activity in HAECs by using two NF-κB inhibitors, BAY11-7082and SC-514. The results from immunofluorescence showed that both BAY11-7082and SC-514are effective in inhibiting the activation of NF-κB. After being incubated with BAY11-7082and SC-514at various dose levels (0,1,5,10and20μM) for1h, HAECs was exposed to SAA (10μg/ml) for additional24h. And both of them can block SAA-induced PTX3production significantly in dose-dependent manners (P<0.05). These results indicate that NF-κB activation is important for the SAA-induced PTX3production in HAECs.
Conclusions
1. In the present study, SAA can up-regulate the expression of PTX3in HAECs.
2. SAA induces PTX3production via FPRL1.
3. SAA-induced PTX3production in HAECs is mediated through JNK pathway
4. SAA stimulates PTX3production in HAECs via AP-1and NF-κB activation
心脑血管疾病已成为严重危害人类健康的头号杀手,其高发病率和高致残率带来了诸多的社会问题。而动脉粥样硬化(atherosclerosis, AS)是多种心脑血管疾病共同的病理基础,是临床上导致急性冠脉综合征(acute coronary syndrome,ACS)发生的最主要病因。经过多年的丛础和临床研究,Ross明确提出“AS是一种慢性炎症性疾病”,其发生发展是涉及到脂质代谢紊乱、免疫炎症反应、巨噬细胞浸润等多个因素和环节的复杂病理过程。因此,深入探讨影响AS病变形成的各种危险因素并寻找有效的干预靶点,预防甚至避免AS疾病的发生,成为了近年来心血管领域的研究热点。
人类血清淀粉样蛋白A (serum amyloid A, SAA)是一组由同一基因编码的多形性蛋白,包括SAA1-SAA4这四种蛋白亚型。SAA1和SAA2蛋白结构近93%
一致,在炎症的急性反应期中显著上调；SAA3基因则被认为是一种不能转录和表达的假基因；SAA4蛋白虽能够持续表达,但不参与急性炎症反应。研究表明,不论是在正常还是在炎症状态下,SAA1亚型始终是SAA的主要组分,决定着总SAA蛋白的作用水平。
SAA血清水平在机体急性炎症(如外伤、感染等)反应过程中上升极快,短时间内由肝脏细胞大量分泌,可超过正常值的1000倍以上,这种特性也使得SAA成为目前最敏感的炎症标志物之一。同时,众多的研究也发现,在包括肥胖、AS在内的各种慢性炎症病理状态下,人类的脂肪细胞、血管内皮细胞及单核-巨噬细胞等肝外组织也能合成分泌SAA蛋白并导致血清SAA水平呈一定程度的升高,从而时刻准确反映着机体的慢性炎症水平。
近年来,国内外多宗大规模临床试验显示血清SAA的水平可以作为评估和预测冠心病病情严重程度的敏感标志物,这提示SAA与AS的发生与流行之间似乎存在着密切的联系。随着对SAA研究的深入,SAA的众多生物学功能也逐渐为人们所了解。首先,SAA对单核-巨噬细胞具有较强的趋化性。其次,SAA可以作为载脂蛋白,参与胆固醇代谢并增加脂质与巨噬细胞的亲和力。再次,在一系列的体外细胞实验中发现SAA能够上调单核细胞趋化蛋白1(monocyte chemotactic protein-1, MCP-1)、肿瘤坏死因子(tumor necrosis factor, TNF)-α、白细胞介素(interleukin, IL)-6及IL-8等炎症相关因子的表达。上述关于SAA生物学效能的研究结果进一步提示我们,SAA应该在AS的发生发展过程中发挥了一定的作用。
然而,尽管近年来国内外学者在SAA与AS斑块形成两者关系的研究中已取得了较大进展,但仍存在一系列困扰我们的重大问题：(1)究竟SAA仅仅是AS慢性炎症状态的一个标志物,还是AS发生过程中的一个积极的参与者?(2)如果SAA直接或者间接地参与了AS的发生,那么它发挥了何种作用?(3)SAA在AS病程中所发挥的作用又是通过哪些途径和靶点来实现的?
针对上述这些亟待解决的关键问题,本课题构建了SAA1高表达慢病毒,成功实现了SAA1蛋白在实验动物体内的稳定高表达；同时采用ApoE-/-小鼠构建AS动物模型,探讨SAA在AS斑块形成过程中所发挥的作用及其可能的机制,旨在为有效控制AS疾病的发生发展提供全新的干预靶点与治疗策略。
研究目的
1.明确血清SAA浓度的升高在ApoE-/-小鼠AS斑块形成过程中所发挥的作用。
2.探讨SAA在AS斑块内的沉积及表达情况以及SAA对AS斑块内巨噬细胞水平及分布的影响。
3.探讨血清SAA浓度的升高对促AS发生的相关炎症因子表达水平的影响。
研究方法
1.动物模型的建立
90只8周龄雄性ApoE-/-小鼠随机分为SAA1高表达慢病毒干预组(lenti-SAA组)、空载体慢病毒对照组(lenti-null组)和生理盐水对照组(saline control组)三组。本课题所使用的小鼠SAA1高表达慢病毒购自上海Invitrogen公司。lenti-SAA组每只小鼠尾静脉注射107TU的慢病毒；作为对照,lenti-null组和saline control组分别注入相应体积的空载体慢病毒和生理盐水。为避免高脂饮食喂养对AS斑块形成所产生的影响,实验全程采用普通饮食(5%脂质,不添加胆固醇)喂养实验动物,期间使用小动物超声监测斑块形成时间、面积。普通饮食喂养14周后,小鼠隔夜禁食后称重,麻醉后处死并取血,离心后取血清冻存。随机处死各组中的小鼠经灌流和固定后取材,将小鼠心脏和主动脉与周围组织分离并冻存。
2.血液生化指标检测
检测血清甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、及低密度脂蛋白胆固醇(LDL-C)水平。同时,使用酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)测定血清总SAA、IL-6、TNF-α水平。
3.主动脉表面AS病变染色
收集自主动脉弓到腹主动脉分叉处的主动脉节段,去除结缔组织成分后,纵剖后展开固定于蜡板上并行油红O染色,AS斑块区域将被染成红色。AS的病变程度用油红O阳性染色区域占整个主动脉表面积的百分比表示。
4.主动脉根部AS病灶分析
对主动脉窦做平行于瓣环的横截位冰冻切片,对斑块内结构和成分进行染色,包括苏木素-伊红(H&E)染色、油红O染色等,并进行量化分析,AS的病变程度用总的斑块面积占整个主动脉瓣环面积的百分比表示。
5.免疫组织化学染色
取主动脉窦冰冻切片,进行巨噬细胞、MCP-1的免疫组织化学染色,观察两者在AS斑块内的表达水平。
6.免疫荧光染色
取主动脉窦冰冻切片,进行SAA、巨噬细胞及血管细胞粘附分子1(vascular cell adhesion molecule-1, VCAM-1)的免疫荧光染色,观察各个指标在AS斑块内的表达水平,并进一步明确SAA与巨噬细胞在斑块内分布的相关性。
7.实时定量PCR (real-time PCR)
通过Trizol法提取小鼠主动脉总RNA,逆转录后,以β-actin为参照,采用real-time PCR技术检测MCP-1和VCAM-1的mRNA表达水平。
研究结果
1.体重及血液生化指标检测
实验结束时lenti-SAA组、lenti-null组和saline control组三组之间的体重、TG、TC、HDL-C及LDL-C水平未见统计学差异(P>0.05),这说明采用普通饮食喂养实验动物,成功避免了高脂饮食喂养所造成的血脂异常对AS斑块形成所产生的影响。lenti-null组和saline control组的血清SAA、IL-6及TNF-α水平未见统计学差异(P>0.05),这说明SAA1高表达慢病毒的注射并未引起小鼠机体的炎症反应。因此,对本实验结果的观察重点聚焦于lenti-SAA组与lenti-null组之间实验结果的对比。lenti-SAA组较lenti-null组的SAA、IL-6及TNF-α水平显著升高(P<0.01),这证明了SAA1高表达慢病毒的注射在小鼠体内能够有效的高表达,保证了血清SAA水平的稳定升高；而SAA水平的升高又进一步导致了IL-6及TNF-α水平的升高,加重了机体的炎症水平。
2.AS病变严重程度分析
主动脉AS病变大体染色显示lenti-SAA组较lenti-null组的AS斑块面积显著增加(P<0.01),这与对主动脉窦AS病变区域行H&E及油红O染色所得到的结果一致(P<0.01),说明血清SAA水平的升高加速了AS斑块的形成。
3.AS斑块内SAA与巨噬细胞表达情况
对主动脉窦AS斑块内SAA、巨噬细胞行免疫组织化学和荧光染色发现,lenti-SAA组较lenti-null组的巨噬细胞数目显著增多(P<0.01),且巨噬细胞与SAA在斑块内部的分布位置相互重叠,密切相关。
4.AS斑块内MCP-1的表达情况
real-time PCR、免疫组织化学染色的结果显示,与lenti-null组相比,lenti-SAA组AS斑块区域MCP-1的mRNA及蛋白表达水平显著上调(P<0.01),这提示血清SAA水平的升高通过上调AS斑块内MCP-1的表达,增强了对巨噬细胞的趋化作用。
5.AS斑块内VCAM-1的表达情况
通过real-time PCR、免疫荧光染色技术显示,与lenti-null组相比,lenti-SAA组AS斑块区域VCAM-1的mRNA及蛋白表达水平显著上调(P<0.01),这说明血清SAA水平的升高增加了AS病变区域主动脉内皮细胞VCAM-1的表达,从而增强了对巨噬细胞的粘附作用。
结论
1.本研究应用SAA1高表达慢病毒首次成功地实现了SAA1蛋白在实验动物体内的持续稳定高表达。
2.本研究采用普通饮食喂养ApoE-/-小鼠,避免了高脂饮食喂养对研究AS斑块形成所产生的影响。
3.血清SAA水平的升高,通过上调IL-6及TNF-α水平,加重了机体的炎症反应,从而显著加速了AS斑块的形成。
4.SAA通过上调AS病变区域MCP-1及VCAM-1的表达,增加了病变区域对巨噬细胞的趋化及粘附,加重了斑块内巨噬细胞的浸润,从而促进了AS斑块的发生。
研究背景
动脉粥样硬化(atherosclerosis, AS)是导致多种心脑血管疾病发生的首要病理生理基础,其发生发展是以肥胖、高脂血症、高血压和吸烟为代表的多种危险因素共同作用的结果。Ross在“损伤-反应学说”基础上提出：各种炎症因子介导的免疫炎症连锁反应在AS发生发展过程中发挥着重要作用。这种免疫炎症反应往往伴随着血管内皮细胞的功能不良及动脉血管壁内的单.核-巨噬细胞和T淋巴细胞浸润等特征。
人类血清淀粉样蛋白A (serum amyloid A, SAA)通常被定义为一种敏感的炎症标志物。在机体急性炎症(如外伤、感染等)和各种慢性炎症病理状态(如肥胖、AS)下时刻准确反映着机体的炎症水平。最新的研究表明, SAA能够在体外细胞实验中上调肿瘤坏死因子(tumor necrosis factor, TNF)-α、白细胞介素(interleukin, IL)-6、IL-12及单核细胞趋化蛋白-1(monocyte chemotactic protein-1, MCP-1)等多种炎症相关因子的表达。并且,SAA上调机体TNF-α、IL-6及MCP-1等炎症因子表达的作用已在论文一中的体内动物实验中得以证实。这提示我们,SAA的生物学功能似乎不仅仅局限于作为单纯的炎症标志物,它本身就可以发挥致炎作用,刺激相关靶细胞(如单核-巨噬细胞、主动脉内皮细胞等),上调各种炎症因子的表达,进一步加重机体的炎症水平。
正五聚蛋白-3(pentraxin3, PTX3)是进化过程中一个很保守的五聚体蛋白质,主要由单核-巨噬细胞、内皮细胞、树突状细胞和脂肪细胞等在IL-1、TNF-α等炎症因子的刺激下分泌。PTX3与C反应蛋白(C reactive protein, CRP)、血清淀粉样成分P (serum amyloid P, SAP)共同隶属于五聚体蛋白家族(pentraxin)。一方面,PTX3与CRP、SAA相似,也属于炎症反应的急性时相蛋白家族。在创伤、炎症、感染、肿瘤等情况下血清水平迅速升高,从而能够敏感地反应机体的炎症水平。众多的研究已经证实,监测血清PTX3的水平对评估急性心血管事件和心衰患者的预后具有重要的预测价值。但由于CRP、SAA主要由肝脏细胞分泌,反映机体系统性炎症水平,而PTX3主要由血管内皮细胞、单核-巨噬细胞等在脉管系统病变的局部产生,因而被称为“脉管系统的CRP”,能够更加直接反映病变局部及血管的炎症状态。另一方面,PTX3还在炎症反应与固有免疫(innate immunity)之间起到了桥梁和纽带作用,它能够与补体(complement,C)分子Clq结合,激活补体经典途径,参与炎症反应和固有免疫,在介导细胞凋亡过程中发挥着重要的调节作用。
然而,SAA的致炎作用尚需要更多的实验证据来加以证明,并且其致炎作用的具体信号转导通路尚未完全明了。针对上述亟待解决的问题,本课题采用人重组SAA蛋白,干预人主动脉内皮细胞(human aortic endothelial cells, HAECs),观察SAA上调另一种炎症因子PTX3的表达,并且深入探讨了SAA发挥作用的具体信号转导通路,为进一步证明SAA的致炎作用和及明确其作用机制提供更为坚实的理论基础。
研究目的
1.探讨SAA刺激HAECs诱导PTX3表达上调,进一步证明SAA的致炎作用；
2.明确SAA上调PTX3表达的具体信号转导通路。
研究方法
1.实验设计
HAECs购自美国模式培养物集存库(American type culture collection, ATCC),采用专用的内皮细胞培养基(endothelial culture medium, ECM)进行培养。取代数在3-5代,处于对数生长期的细胞进行实验。
1)分别给予HAECs不同浓度梯度(0、0.1、1、10、20μg/ml)的人重组SAA蛋白及采用不同干预时间(0、3、6、9、12、15、24h)进行刺激,采用酶联免疫吸附测定(enzyme-linked immuno sorbent assay, ELISA)检测细胞上清中PTX3的分泌水平,观察SAA诱导HAECs上调PTX3表达的浓度-时间依赖性。
2)分别对HAECs给予甲酰肽样受体1(formyl peptide receptor like-1, FPRL1)的小干扰RNA (small interfering RNA, siRNA)预处理48h,或给予FPRL1受体的特异性抑制剂WRW4以及G蛋白偶联受体的抑制剂pertussis toxin预处理1h,再给予最佳剂量的人重组SAA蛋白刺激24h后,采用ELISA法,检测细胞上清中PTX3的分泌水平,探讨FPRL1受体在SAA诱导HAECs增加PTX3表达过程中所发挥的作用。
3)分别对HAECs给予c-Jun氨基末端激酶(c-Jun NH2-terminal kinase, JNK)1、JNK2的siRNA预处理48h,再给予最佳剂量的人重组SAA蛋白刺激24h后,采用ELISA法,检测细胞上清中PTX3的分泌水平,探讨JNK通路在SAA诱导HAECs增加PTX3表达过程中所发挥的作用。
4)分别对HAECs给予转录激活蛋白1(activator protein, AP-1)的抑制剂丹参酮ⅡA (Tanshinone ⅡA)及核转录因子-κB (nuclear factor-kappa B, NF-κB)的抑制剂BAY11-7082和SC-514预处理1h,再给予最佳剂量的人重组SAA蛋白刺激24h后,采用ELISA法,检测细胞上清中PTX3的分泌水平,探讨AP-1及NF-κB的激活在这一过程中的作用。
2. ELISA
分别给予HAECs上述处理后,取细胞上清液,离心去除细胞碎屑及杂质,采用ELISA法检测其中PTX3的蛋白水平。
3.细胞免疫荧光染色
将HAECs种植在放有载玻片的24孔中培养,待细胞生长至70%的融合状态,给予NF-κB抑制剂BAY11-7082和SC-514预处理1h,再给予最佳剂量的人重组SAA蛋白刺激24h后,取出载玻片,固定,封闭,NF-κB一抗孵育,FITC标记的二抗显色后,DAPI复染细胞核,荧光显微镜下观察BAY11-7082和SC-514对SAA介导的NF-κB激活的抑制作用。
4. Western blot检测蛋白质的表达水平
在对HAECs给予相关处理之后,使用细胞刮刀收集贴壁的HAECs,提取细胞总蛋白,进行SDS-PAGE电泳分离,转膜,蛋白印迹和免疫反应后检测FPRL1及JNK1和JNK2的蛋白质表达水平。
5.实时定量PCR (real-time PCR)检测mRNA的表达变化
在对HAECs给予相关处理之后,利用Trizol法提取HAECs的总RNA,并以β-actin作为参照,采用real-time PCR技术检测PTX3、FPRL1及JNK1和JNK2的mRNA表达水平。
研究结果
1.SAA呈浓度和时间依赖性上调HAECs中PTX3的表达水平
给予HAECs一定浓度梯度(0、0.1、1、10、20μg/ml)的SAA蛋白干预24h,收集上清ELISA检测PTX3表达水平,结果显示,SAA能够呈浓度依赖性的诱导HAECs中PTX3的表达,且最佳刺激浓度为10μg/ml。随后,对HAECs给予10μg/ml的SAA蛋白干预0、3、6、9、12、15、24h后发现,SAA可以呈时间依赖性的上调HAECs中PTX3的表达,且在干预24h后达到蛋白表达的最高水平。同时,real-time PCR的实验结果也显示SAA能够以时间依赖性的方式上调HAECs中PTX3的mRNA的表达水平。
2.SAA通过FPRL1受体上调PTX3的表达
给予FPRL1的siRNA预处理HAECs后,使用real-time PCR及Western blot的方法验证siRNA的干扰表达作用,结果显示,FPRL1的siRNA可以有效的降低FPRL1的表达水平。随后,对HAECs给予FPRL1的siRNA预处理48h,再给予10μg/ml的SAA蛋白刺激24h后,检测细胞上清PTX3的表达,结果发现,对HAECs行FPRL1的siRNA预处理后,SAA诱导的PTX3表达水平显著下调,说明FPRL1参与了这一过程。
3.SAA上调PTX3的表达受JNK通路的调控
分别对HAECs给予JNK1和JNK2的siRNA预处理48h,再给予10μg/ml的SAA蛋白刺激24h后,检测细胞上清PTX3的表达,结果显示,SAA诱导的PTX3表达水平显著下调,这提示SAA介导的PTX3表达上调受JNK1和JNK2的调控。
4.SAA上调PTX3的表达需要AP-1的激活
在给予HAECs一定浓度梯度(0、5、10、20μg/ml)的AP-1抑制剂TanshinoneⅡA预处理1h后,再给予10μg/ml的SAA蛋白刺激24h后,检测细胞上清PTX3的表达,结果显示,SAA诱导的PTX3表达水平呈浓度依赖性的被Tanshinone ⅡA下调,这提示SAA介导的PTX3表达上调需要AP-1的激活。
5.SAA上调PTX3的表达需要NF-κB的激活
对HAECs给予NF-κB的抑制剂BAY11-7082和SC-514预处理1h,再给予10μg/ml的SAA蛋白刺激24h后,免疫荧光染色显示SAA介导的NF-κB激活被BAY11-7082和SC-514显著下调。随后,在分别给予HAECs一定浓度梯度(0、1、5、10、20μM)的BAY11-7082和SC-514预处理1h后,再给予10μg/ml的SAA蛋白刺激24h后,检测细胞上清PTX3的表达,结果显示,SAA诱导的PTX3表达被NF-κB的抑制剂BAY11-7082和SC-514显著下调,这提示NF-κB的激活参与SAA诱导的PTX3表达这一过程。
结论
1.SAA能够显著上调HAECs中PTX3的表达。
2.SAA诱导的PTX3的表达上调是通过FPRL1受体发挥作用的。
3.SAA诱导的PTX3的表达上调受JNK通路的调节。
4.SAA诱导的PTX3的表达上调需要AP-1及NF-κB的激活。
Background
Atherosclerosis is an important underlying pathologic condition of many cardio-cerebro-vascular diseases, the leading cause of morbidity and mortality worldwide. Basing on the long-term experimental and clinical research, the theory that atherosclerosis is a chronic inflammatory disease has been proposed by Ross explicitly. The occurrence and development of atherosclerosis is a complex pathological process involving a series of influencing factors such as lipid metabolism, immuno-inflammatory response, macrophages infiltration and so on. Therefore, seeking the effective targets to avoid the formation of atherosclerosis plaque has already become a hot topic in the cardiovascular field.
Serum amyloid A (SAA) is one of the most sensitive acute phase proteins in vertebrates. It is produced principally by the liver in response to acute inflammatory stimuli and its plasma concentration can increase by up to100-to1000-fold over the basal level. Meanwhile, accumulating evidences also support that under the chronic inflammatory status such as obesity and atherosclerosis, the secretion of SAA by human adipocytes, vascular endothelial cells and macrophages can cause a modest increase in the plasma and reflect the inflammatory level exactly.
SAA is a family of homologous proteins including4isoforms which are encoded by the same gene. SAA1and SAA2are the major acute phase reactants, with primary structures that are93%identical. The human SAA3gene is a pseudogene. SAA4 which is constitutively expressed in humans but not in mice do not participate in the acute inflammatory reaction. Many studies have already demonstrated that SAA1predominates in plasma, where it functions as a major isotype.
So far, many large clinical trials have demonstrated that increased plasma SAA level can be regarded as a useful indicator for identifying individuals at high risk of cardiovascular disease (CVD), suggesting that there may be a link between the SAA and prevalent CVD. With the further study, more and more biological function of SAA has been known by us. Firstly, SAA exhibits considerable chemoattractant activity for human monocytes and macrophages. Secondly, as the apolipoprotein of high-density lipoprotein (HDL) and low-density lipoprotein (LDL), SAA increases the binding affinity of cholesterol for macrophages and endothelial cells. In addition, a series of studies in vitro show that SAA can up-regulate the expression of many inflammatory factors such as monocyte chemotactic protein-1(MCP-1), tumor necrosis factor-α(TNF-α), interleukin (IL)-6and IL-8and so on. All the results mentioned above suggest that SAA should play an effective role in the progression of atherosclerosis.
Despite the numerous reported proatherogenic properties of SAA, we lack direct proof that SAA is an active participant in the atherosclerosis process in vivo. To investigate whether SAA is purely a risk marker for atherosclerosis or is also an active participant in vivo, we examine the effect of high-level expression of SAA on atherosclerosis development by using apolipoprotein E-deficient (ApoE-/-) mice transfected with lentivirus to induce SAA overexpression. Findings from this study can provide the first in vivo evidence that an elevated plasma level of SAA accelerates the progression of atherosclerosis directly and independently, and then may be helpful in designing novel therapeutic strategies against atherosclerosis.
Objectives
1. To investigate the role of increased SAA plasma level on the formation of atherosclerosis plaque in ApoE-/-mice.
2. To observe the deposition and the influence of SAA on the distribution of macrophages in atherosclerosis plaque.
3. To elucidate the role of increased SAA plasma level on the expression of proatherogenic factors.
Methods
1. Establishment of animal model
90male ApoE-/-mice (8weeks age) were randomly divided into3groups, and then were injected intravenously with lentivirus-expressing mouse SAA1(lenti-SAA group, n=30) at a total lentivirus dose of1×107TU/mouse, a null lentivirus (lenti-null group, n=30) or saline (saline control group, n=30). To avoid the influence of high plasma lipids on atherogenesis, all the animals were fed a chow diet (5%fat and no added cholesterol) throughout the entire experiment. At14weeks after lentivirus injection, mice were anesthetized with pentobarbital injected intraperitoneally, and then blood samples were taken. Serum was separated by centrifugation at4℃. Meanwhile, the hearts and aortas were removed and perfusion-fixed with4%paraform aldehyde for histological and morphological staining or with PBS for real-time polymerase chain reaction (real-time PCR).
2. Biological measurements
The levels of triglycerides (TG), total cholesterol (TC), HDL-C and LDL-C were measured by use of an automatic biochemistry analyzer. And serum levels of SAA, IL-6and TNF-α were detected by enzyme-linked immuno sorbent assay (ELISA).
3. En face analysis of the aorta
The aorta was stripped of adventitia and dissected longitudinally from the iliac arteries to the aortic root, then the branching vessels were removed. The paraformaldehyde-fixed aorta was pinned flat on a black surface, and the atherosclerotic lesion area was readily visualized with Oil-Red-O staining. Average lesion area was quantified by use of ImagePro-Plus soft ware. The ratio of total atherosclerotic lesion area to aorta intimal surface area was calculated as an indicator to evaluate the level of atherogenesis.
4. H&E and Oil-Red-O staining of aortic sinus Murine hearts were fixed in4%paraformaldehyde overnight and then embedded in optimal cutting temperature compound. At least50serial cryosections6-μm thick were cut, beginning at the junction of the left ventricle and the aorta. Sections were stained with hematoxylin and eosin (H&E). The lipid core was identified by Oil-Red-O staining. The atherogenesis level at the aortic sinus was evaluated by Oil-Red-O and H&E staining according to the ratio of total atherosclerotic lesion area to aortic lumen area.
5. Immunohistochemical analysis Immunohistochemical analysis was used to detect the expression of macrophages and MCP-1in lesions. Data were analyzed by use of ImagePro-Plus software.
6. Immunofluorescence
Cryosections of the aortic sinus were chosen to observe the expression of SAA, macrophages and vascular cell adhesion molecule-1(VCAM-1) through immunofluorescence analysis. The colocalization of SAA with macrophages was also examined in atherosclerosis plaque. Data were analyzed by use of ImagePro-Plus software.
7. Quantitative real-time PCR
Total RNA was extracted from murine frozen aortic specimens by use of trizol, and then reverse transcribed. Real-time PCR was used to detect the mRNA levels of MCP-1and VCAM-1.
Results
1. Body weight and measurement of plasma variables ApoE-/-mice in3groups fed a chow diet did not differ in body weight, TG, TC, HDL-C or LDL-C (P>0.05). Therefore, we excluded the influence of lipid levels on atherosclerosis in this study. The lenti-null and saline control groups did not differ in plasma levels of IL-6or TNF-α, so the injection of the SAA1lentivirus vector was safe and did not induce inflammatory responses (P>0.05). The plasma levels of SAA were higher for the lenti-SAA group than lenti-null and saline control groups, so the SAA1lentivirus was efficiently transfected in vivo (P<0.01). Most importantly, with elevated SAA level, the plasma levels of IL-6and TNF-a were significantly higher in the lenti-SAA than lenti-null group, suggesting that the increased SAA level may aggravate the inflammatory reflect (P<0.01).
2. Atherogenesis level analysis
By using en face analysis of the aorta, lesion area was significantly larger for the lenti-SAA than lenti-null group (P<0.01). Atherogenesis level at the aortic sinus was evaluated by Oil-Red-O and H&E staining by ratio of total atherosclerotic lesion area to aortic lumen area. The mean lesion size at the aortic sinus were greater for the lenti-SAA than lenti-null group (P<0.01). All the results demonstrated that increased plasma SAA level directly promotes atherosclerotic lesions.
3. Accumulation of SAA and macrophages in atherosclerosis plaque
Immunohistochemistry analysis showed a greater increase in accumulation of macrophages for the lenti-SAA than lenti-null group (P<0.01). Because SAA is a classic chemoattractant to peripheral blood leukocytes, we observed the colocalization of SAA with macrophages in lesions on aortic cryosections through immunofluorescence. The distribution of macrophages was consistent with SAA protein localization.
4. Expression of MCP-1in atherosclerosis plaque
Because MCP-1is a key molecule regulating chemotactic migration of macrophages, we observed the expression of MCP-1in vivo by immunohistochemistry. MCP-1secretion was increased with elevated level of plasma SAA, which was also confirmed by real-time PCR (P<0.01). Our data suggested that SAA could enhance the chemotaxis of macrophages through up-regulating the expression of MCP-1in lesions.
5. Expression of VCAM-lin atherosclerosis plaque
Immunofluorescence analysis revealed upregulated VCAM-1expression in vivo in the lenti-SAA group as compared with the lenti-null group (P<0.01). VCAM-1mRNA expression results agreed with protein level results. Our research suggested that SAA could promote the adhesion of macrophages in lesions through up-regulating the expression of VCAM-1.
Conclusions
1. In the present study, we achieved the persistent high expression of SAA protein by applying SAA1lentivirus in ApoE-/-mice effectively.
2. Throughout the entire experiment, all the animals were fed a chow diet, so that the influence of lipid levels on atherosclerosis formation was excluded in our study.
3. SAA accelerated the progression of atherosclerosis in ApoE-/-mice through aggravate the inflammatory level.
4. SAA enhanced the chemotaxis and adhesion of macrophages through up-regulating the expression of MCP-1and VCAM-1, thus promoting the formation of atherosclerosis plaque
Background
Atherosclerosis is the primary pathophysiological basis leading to the many cardio-cerebro-vascular diseases. A series of risk factors represented by obesity, hyperlipemia, hypertension and smoking participate in the occurrence of atherosclerosis. On the basis of the response-to-injury hypothesis, the theory that the chain reaction induced by inflammatory factors play an important role in the process of atherogenesis has been proposed by Ross explicitly. This chronic inflammation is characterized by the disfunction of vascular endothelial cell and the accumulation of macrophages and T lymphocytes in the arterial vascular walls.
Serum amyloid A (SAA) is one of the most sensitive acute phase proteins in vertebrates. And it can reflect the inflammatory levels exactly under the status of the acute and chronic inflammations. Recently, a series of studies in vitro have demonstrated that SAA can up-regulate the expression of many inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6and IL-12and so on. And these findings suggest that SAA may be not only a prognostic indicator but also a pro-inflammatory mediator by inducing the expression of the inflammatory factors in mononuclear phagocytes and aortic endothelial cells.
Pentraxins is an acute immunological response family of proteins that consists of three members including C reactive protein (CRP), serum amyloid P (SAP), and pentraxin3(PTX3). PTX3is one of the conserved proteins in evolution and produced by mononuclear phagocytes, dendritic cells (DCs), adipocytes and so on under the stimulation of IL-1and TNF-α. For one thing, just like CRP and SAA, PTX3belongs to classic acute phase reactants that can reflect the levels of inflammation exactly. Many studies have already proved that it is profound to estimate the levels of serum PTX3in the aspect of predicting the prognosis of acute cardiovascular events and hear failure. PTX3, as a cytokine-inducible factor, is mainly produced in vascular endothelial cells, fibroblasts and some other extrahepatic tissues and can reflect the inflammatory levels of local impaired tissues which make it distinguished from CRP and SAA that are synthesized majorly in the liver. For another, PTX3can activate the classical complement pathway through specific recognition and interaction with the complement component Clq and regulate the clearance of apoptotic cells, thus providing a link between the inflammation and innate immunity.
However, the experimental evidence about the pro-inflammatory role of SAA is still inadequate. And the signaling pathway involved in this process is still poor understanding. Therefore, we investigate the capacity of human aortic endothelial cells (HAECs) to express PTX3, another sensitive inflammatory biomarker, after pro-incubation with SAA. Then the signaling pathway involved in this process has also been further discussed. Our research may be helpful in understanding the pro-inflammatory role of SAA and will provide a new theoretical support on elucidating the relative mechanism.
Objectives
1. To investigate the effect of SAA on up-regulating the expression of PTX3in HAECs and further discuss the pro-inflammatory role of SAA.
2. To elucidate the relative signaling pathway involved in this process.
Methods
1. Experiment design
Human aortic endothelial cell line was obtained from American type culture collection (ATCC) and was cultured in endothelial culture medium (ECM) supplemented with5%fetal bovine serum. These cells were used for experiments between3rd and5th passage.
1) HAECs were treated with different concentration gradients (0,0.1,1,10,20μg/ml) of recombinant human SAA for24h or different checkpoints (0,3,6,9,12,15and24h) with10μg/ml of SAA respectively. The capacity of SAA on PTX3secretion in HAECs was observed through detecting the levels of PTX3secretion in cell-free supernatants by using Enzyme-linked immunosorbent assay (ELISA).
2) HAECs were pretreated by the specific siRNA sequences of formyl peptide receptor like-1(FPRL1), or WRW4, an effective inhibitor of FPRL1, or pertussis toxin, an antagonist of G protein-coupled receptor. And then the cells were challenged by SAA (10mg/ml) for another24h. ELISA was used to investigate the role of FPRL1on SAA-induced expression of PTX3by detecting the levels of PTX3secretion in cell-free supernatants.
3) HAECs were pretreated by the specific siRNA sequences c-Jun NH2-terminal kinase (JNK)1and JNK2for48h respectively, and then were challenged by SAA (10mg/ml) for another24h. ELISA was used to investigate the role of JNK1and JNK2on SAA-induced expression of PTX3by detecting the levels of PTX3secretion in cell-free supernatants.
4) HAECs were pretreated by Tanshinone Ⅱ A, the specific inhibitor of activator protein (AP-1), or two effective nuclear factor-kappa B (NF-κB) inhibitors (BAY11-7082and SC-514) for1h respectively, and then were stimulated by SAA (10mg/ml) for another24h. ELISA was used to evaluate the role of AP-1and NF-κB activation in this process by detecting the levels of PTX3secretion in cell-free supernatants.
2. ELISA
HAECs were placed in ECM containing5%FBS in96-well plates and kept in a5%CO2incubator at37℃. After stimulation, cell-free supernatants were collected, centrifuged, and assayed for PTX3by ELISA according to the manufacturer's instructions.
3. Immunofluorescence
HAECs cultured on glass cover slips were pretreated by BAY11-7082and SC-514, two effective NF-κB inhibitors for1h respectively, and then were stimulated by SAA (10mg/ml) for another24h. After that, cells were fixed in2%paraformaldehyde and blocked in3%BSA, then incubated with anti-phospho-NF-kBp65antibody overnight. After being incubated with FITC conjugated secondary antibody, immunolabeled cells were counterstained with DAPI to detect cell nuclei. Then inhibitory role of BAY11-7082and SC-514on SAA-mediated NF-κB activation was observed by visualizing with a fluorescence microscope equipped with a digital camera.
4. Western blot
After being stimulated respectively, HAECs were harvested for protein extraction. Western blot was used to detect the protein expression levels of FPRL1, JNK1and JNK2.
5. Quantitative real-time PCR
Total RNA was extracted from the harvested HAECs by the use of Trizol, and then reverse transcribed. Real-time PCR was used to detect the mRNA levels of PTX3, FPRL1,JNK1and JNK2.
Results
1. SAA up-regulates the PTX3expression in a time-and dose-dependent manner in HAECs
HAECs were treated with different concentration gradients (0,0.1,1,10,20μg/ml) of SAA for24h and the supernatant concentration of PTX3was detected by ELISA. As a result, we observed that SAA could induce PTX3secretion in a concentration-dependent manner. Then cells were stimulated with SAA (10μg/ml) at various time points (0,3,6,9,12,15, and24h). The results demonstrated that SAA could induce PTX3accumulation in a time-dependent manner and reached its maximal activity at24h after stimulation. These results altogether indicate that the induction of PTX3by SAA is time-and dose-dependent. At the same time, the effect of SAA on PTX3at mRNA transcription level was examined via real-time PCR. Our data demonstrated a similar expression in compliance with its protein level, as the mRNA expression of PTX3increased by the time of stimulation after3-9h treatment with SAA which suggested that SAA-induced PTX3protein synthesis required transcriptional activation (P<0.05).
2. SAA induces PTX3production via FPRL1
Human FPRL1siRNA (Santa Cruz, sc-40123) was a convenient tool designed for FPRL1gene silencing and its effect had been examined carefully. Then, human FPRL1siRNA was used to confirm whether SAA-induced PTX3production in HAECs was mediated by FPRL1. After being preincubated with the siRNA sequences of FPRL1for48h and stimulated with10μg/ml SAA for another24h, both HAECs and supernatants were harvested for real-time PCR and Western blot to check the effect of the siRNA sequences. The data from real-time PCR and Western blot indicated that the SAA-induced PTX3secretion could be inhibited significantly by siRNA-mediated FPRL1gene silencing (P<0.05). All these results strongly support that FPRL1is likely to be one of the receptors through which SAA mediates its effects.
3. SAA-induced PTX3production in HAECs is mediated through JNK pathway
After being preincubated with the siRNA sequences of JNK1and JNK2for48h, HAECs were stimulated with10μg/ml SAA for another24h. SAA-induced PTX3production levels were detected by ELISA. Our data showed that SAA-induced PTX3expression could be partially but significantly reduced (P<0.05). These results strongly suggest that JNK pathway is crucial for SAA-induced PTX3expression in HAECs, and activations of both JNK1and JNK2are involved in mediating PTX3production.
4. SAA stimulates PTX3production in HAECs via AP-1activation
After being incubated with Tanshinone Ⅱ A, an AP-1inhibitor, at various dose levels (0,5,10and20μg/ml) for24h, HAECs was exposed to SAA(10μg/ml) for additional24h. We found that Tanshinone Ⅱ A could inhibit SAA-induced PTX3expression dramatically in a dose-dependent manner (P<0.05), which suggested that the up-regulation of PTX3need the activation of AP-1.
5. SAA induces PTX3production in HAECs via NF-κB activation
We examined the effect of SAA on NF-κB activity in HAECs by using two NF-κB inhibitors, BAY11-7082and SC-514. The results from immunofluorescence showed that both BAY11-7082and SC-514are effective in inhibiting the activation of NF-κB. After being incubated with BAY11-7082and SC-514at various dose levels (0,1,5,10and20μM) for1h, HAECs was exposed to SAA (10μg/ml) for additional24h. And both of them can block SAA-induced PTX3production significantly in dose-dependent manners (P<0.05). These results indicate that NF-κB activation is important for the SAA-induced PTX3production in HAECs.
Conclusions
1. In the present study, SAA can up-regulate the expression of PTX3in HAECs.
2. SAA induces PTX3production via FPRL1.
3. SAA-induced PTX3production in HAECs is mediated through JNK pathway
4. SAA stimulates PTX3production in HAECs via AP-1and NF-κB activation
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