【作者】 李晶晶；

【导师】 张运；

【作者基本信息】 山东大学 ， 内科学， 2012， 博士

【摘要】 背景动脉粥样硬化(atherosclerosis, AS)斑块形成是心脑血管疾病的共同病理学基础。大量证据表明,铁在动脉粥样硬化的发生发展中发挥重要作用。在人及新西兰大白兔的动脉粥样硬化斑块处,均发现铁沉积明显增多,而在给予铁螯合剂治疗后,动脉粥样硬化的情况有明显好转。斑块内的铁沉积总是与巨噬细胞浸润和泡沫细胞的出现密切相关。在动脉粥样硬化发生发展的过程中,铁作为一种强氧化剂,可以通过Haber-Weiss反应催化产生大量活性氧,促进细胞内脂质过氧化反应,损伤蛋白质和(或)核酸,促进巨噬细胞凋亡并释放出大量细胞内容物,这些物质可以进一步促进巨噬细胞的侵润,使脂质过氧化反应不断放大。铁调素(hepcidin, Hep)是近年发现的一种在肝脏合成并富含半胱氨酸的抗菌多肽,具有抑制肠道铁吸收和单核巨噬细胞系统铁释放的作用,是一种重要的负性铁调节激素。膜铁转运蛋白1(ferroportinl, FP1)是巨噬细胞膜上已知唯一的铁输出蛋白。Hep与巨噬细胞表面的FP1结合后,促进其内吞和降解,使巨噬细胞内的铁增多,氧化作用增强。Hep的表达受炎性因子的调节,发生炎症或感染时,一些细胞因子表达量增加促进铁调素基因表达,进而调节体内铁平衡。LPS、Interleukin-6(IL-6)、Interleukin-1(IL-1)均可促进体内Hep水平的升高。2007年,Sullivan JL等人提出了铁调素与动脉粥样硬化之间具有相关性的假说。新近的研究证实,铁调素与代谢综合征具有相关性,认为在大约15%的代谢综合征患者中存在铁的超负荷,而这些患者中,大约50%的患者同时患有非酒精源性脂肪肝。近期,ValentiL等人的临床研究发现,在代谢综合征患者中,血清铁调素水平与血清MCP-1水平及血管损害程度相关,但对其发生机制未作进一步探讨。综上所述,尽管大量数据支持铁代谢与动脉粥样硬化的关系以及铁调素对体内铁代谢的调控作用,当尚未获得铁调素与动脉粥样硬化发生发展具有相关性的直接证据。本论文拟通过建立ApoE-/-小鼠动脉粥样硬化模型,并分别在动物体内过表达和干扰铁调素基因,观察铁调素水平上调及干扰对动脉粥样硬化斑块稳定性的影响并探讨其分子机制,从而明确铁调素在动脉粥样硬化发生发展及斑块不稳定中的作用。目的1.观察铁调素在ApoE-/-小鼠动脉粥样硬化斑块中的表达水平,明确斑块内铁调素表达的细胞类型；2.在ApoE-/-小鼠动脉粥样硬化模型中,以基因干预的方法,明确铁调素对动脉粥样硬化斑块稳定性的影响；3.探讨铁调素导致斑块不稳定的可能机制。方法1重组腺病毒干扰载体与表达载体的的构建及扩增扩增小鼠hepcidin基因,PCR产物与pMD18T simple载体连接,以EcoRI和BamH I为酶切位点,连接pMD18T simple-hamp和pIRES2-EGFP载体,保留测序验证正确的pIRES2-EGFP-Hamp；设计针对hepcidin的shRNA,插入到miRNA表达载体pcDNA TM6.2-GW/EmGFPmiR中,保留测序验证正确的pcDNA TM6.2-GW/EmGFPmiR-hamp.将pIRES2-EGFP-Hamp和pcDNATM6.2-GW/EmGFPmiR-hamp分别与腺病毒表达的目的载体pAD/CMV/V5-DEST进行LR重组反应,以获得含目的基因序列或目的基因干扰片段—GFP的腺病毒表达载体。将含目的基因序列的腺病毒表达载体转染293A细胞,包装并纯化病毒。通过超速离心及层析的方法浓缩及纯化病毒,获得病毒浓缩液。仅表达EGFP的腺病毒载体作为对照。2动物模型的建立及腺病毒转染动物实验分为两部分进行。在第1部分动物实验中,40只6周龄的雄性ApoE-/-小鼠随机分为对照组和模型组,对照组给予全程普通饮食,模型组全程高脂饮食喂养并在第2周末行左颈总动脉套管术。两组动物均于第13周末处死。在第2部分动物实验中,75只6周龄的雄性ApoE-/-小鼠全程给予高脂饮食喂养,第2周末行左颈总动脉套管术,术后8周随机分为3组并分别转染携带EGFP, EFP-hepcidin,和EGFP-hepcidin shRNA的重组腺病毒。3组动物继续高脂喂养3周后于第13周末处死。3血清指标检测第2部分动物处死前心脏采血,检测血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)及血糖水平；同时检测3组动物血清中铁及铁调素的水平。4组织病理与免疫组化检测对左侧颈动脉斑块分别进行H&E染色、油红0染色、天狼猩红染色,并通过免疫组织化学方法检测斑块内胶原、脂质、巨噬细胞(MOMA-2)、平滑肌肌动蛋白(α-actin)含量。测量斑块面积,观察斑块的形态结构,计算易损指数。易损指数=(巨噬细胞+脂质)阳性面积百分比/(平滑肌细胞+胶原)阳性面积百分比。通过免疫组织化学方法检测斑块内炎性因子IL-6、MCP-1、TNFα、MMP-2以及铁相关蛋白hepcidin、H-Ferritin、L-Ferritin的表达水平。5免疫荧光化学检测以免疫双标的方法检测Hepcidin在斑块巨噬细胞及平滑肌细胞的表达；同样方法检测ox-LDL在3组斑块巨噬细胞内的表达。6non-Heme铁检测取新鲜颈动脉斑块组织,以原子吸收分光光度仪法检测3组斑块内non-heme铁含量。7实时定量RT-PCR检测取新鲜颈动脉斑块组织,提取RNA,实时荧光定量RT-PCR检测Hepcidin、 H-Ferritin、L-Ferritin、IL-6、MCP-1、TNF-a、MMP-2等指标在mRNA水平的表达情况；取新鲜肝脏组织,提取RNA,实时荧光定量RT-PCR检测3组动物肝脏Hepcidin mRNA水平的表达情况。8统计分析所有统计学数据均使用SPSS16.0软件分析。计量资料以均数±标准差表示,计数资料以数值和百分比表示。统计分析采用t检验、Oneway ANOVA或LSD post-hoc分析。P<0.05被认为差异具有统计学意义。结果1成功构建重组腺病毒干扰载体与表达载体经重组腺病毒载体测序验证,Hep腺病毒表达载体及干扰载体构建正确,病毒包装成功,表达载体的病毒滴度为3.11×1012ifu/ml,干扰载体的病毒滴度为9.12×1012ifu/ml。2铁调素在ApopE-/-小鼠动脉粥样硬化斑块中的表达情况及细胞定位动物实验的第1部分结果显示,Hepcidin在对照组颈动脉血管内几乎不表达,但在模型组颈动脉斑块内存在mRNA和蛋白水平的高表达。两组之间有显著统计学差异。铁调素主要在斑块内的巨噬细胞和平滑肌细胞表达,内皮细胞未见铁调素表达。3腺病毒体内转染效率腺病毒局部转染1周末GFP表达量明显增加,两周达到高峰,然后开始衰减,3周末实验结束时,斑块内仍可见GFP表达；转染两周后,GFP阳性面积与斑块面积的平均比值在EGFP组、EGFP-Hep组及EGFP-Hep-shRNA组间没有统计学差异。4腺病毒转染前后小鼠体重测量转染前后各组间小鼠体重无明显差异。5血液生化指标检测结果各组小鼠血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、血糖未见统计学差异,铁调素基因干预对血脂、血糖水平无明显影响。6血清铁水平检测结果实验结束时,血清铁水平在三组间无显著统计学差异。7血清及肝脏Hepcidin水平检测结果实验结束时,ELISA检测血清hepcidin水平,三组间无显著统计学差异；各组小鼠肝脏Hepcidin在mRNA水平的表达无显著统计学差异。8斑块内Hepcidin表达检测结果EGFP-Hep组与对照组相比,hepcidin的表达在蛋白和mRNA水平均升高,EGFP-Hep-shRNA组与对照组相比,hepcidin的表达在蛋白和mRNA水平均降低。9斑块面积检测结果三组小鼠颈动脉斑块面积平均值无显著差异。10斑块成分检测及易损指数测量结果油红O染色检测斑块内脂质成分,EGFP、EGFP-Hep和EGFP-Hep-shRNA三组脂质含量无显著差异；天狼猩红染色检测斑块内胶原成分,EGFP-Hep组较对照组胶原含量显著减少,EGFP-Hep-shRNA组较对照组胶原含量显著增加；MOMA-2免疫组化检测斑块内巨噬细胞,EGFP-Hep组较对照组巨噬细胞含量显著增加,EGFP-Hep-shRNA组较对照组巨噬细胞含量显著减少；a-SM actin免疫组化检测斑块内平滑肌细胞,EGFP-Hep组较对照组平滑肌细胞含量显著减少EGFP-Hep-shRNA组较对照组平滑肌细胞含量显著增多；EGFP-Hep组易损指数较对照组显著增加,EGFP-Hep-shRNA组较对照组易损指数显著降低。11.斑块内炎性因子检测结果EGFP-Hep组IL-6、MCP-1、TNF-α和MMP-2的表达,无论在蛋白水平还是mRNA水平,较对照组均明显升高(P<0.05)；反之,EGFP-Hep-shRNA组IL-6、MCP-1、TNF-α和MMP-2的表达,无论在蛋白水平还是mRNA水平,较对照组均明显降低(P<0.05)。12.ox-LDL在巨噬细胞内表达水平检测结果免疫荧光双标及激光共聚焦显微镜观察ox-LDL在EGFP、EGFP-Hep和EGFP-Hep-shRNA三组巨噬细胞内的表达百分比,EGFP-Hep组较对照组显著升高,EGFP-Hep-shRNA组较对照组显著降低。13斑块内铁蛋白表达水平检测结果EGFP-Hep组L-Ferritin和H-Ferritin的表达,无论在蛋白水平还是]mRNA水平,较对照组均明显升高；EGFP-Hep-shRNA组L-Ferritin和H-Ferritin的表达,无论在蛋白水平还是mRNA水平,较对照组均明显降低。14.斑块内non-heme铁检测结果原子吸收分光光度仪检测斑块内non-heme铁的水平。EGFP-Hep组较对照组明显升高；EGFP-Hep-shRNA组较对照组明显降低。结论1.铁调素在动脉粥样硬化斑块中高表达,巨噬细胞和平滑肌细胞是表达铁调素的主要细胞类型；2.铁调素促进动脉粥样硬化斑块不稳定；3.铁调素促进动脉粥样硬化斑块内铁沉积、炎症反应及巨噬细胞内脂质积聚,这可能是铁调素导致斑块不稳定的主要原因。背景斑块稳定性研究是近年国内外研究的热点。众所周知,白细胞和血小板在动脉粥样硬化进程中起着重要作用,然而红细胞在此进程中的作用却一直少为人知。近年来,越来越多的证据表明红细胞可能是一种强大的AS刺激物,可增加斑块不稳定的危险性。研究发现,纤维帽薄和脂核大的斑块中红细胞和铁的含量以及巨噬细胞数量显著增多。有趣的是,那些斑块内出血的斑块更易在18个月内再次出血,反复出血可作为一新的不稳定因素刺激AS的进展。前期我们以改良的斑块内出血动物模型研究了红细胞与AS斑块不稳定之间的关系,研究发现,斑块内出血后斑块不稳定性增高,且与出血量呈剂量依赖关系；斑块内出血后脂质核增大、炎症浸润增加、纤维帽变薄,这三个方面最终导致斑块不稳定性增加,斑块更趋于破裂。红细胞导致斑块不稳定的机制可能涉及红细胞降解的几种物质。其中,大量证据表明,铁在动脉粥样硬化的发生发展中发挥重要作用。在人及新西兰大白兔的动脉粥样硬化斑块处,均发现铁沉积明显增多,而在给予铁螯合剂治疗后,动脉粥样硬化的情况有明显好转。大量研究表明,斑块内的铁沉积总是与巨噬细胞浸润和泡沫细胞的出现密切相关。巨噬细胞是动脉粥样硬化发生发展中的关键细胞,它吞噬氧化低密度脂蛋白(ox-LDL)后变为泡沫细胞。研究发现,在人的颈动脉斑块处,巨噬细胞向斑块的浸润与铁蛋白及转铁蛋白受体的高表达密切相关。巨噬细胞内铁含量增高可促进巨噬细胞清道夫受体-1的表达,从而使细胞内脂质含量进一步增高。铁作为一种强氧化剂,通过Haber-Weiss反应产生大量的氧自由基,促进细胞内脂质发生过氧化反应,从而导致巨噬细胞凋亡并释放出细胞内容物,这些物质可以进一步促进巨噬细胞的侵润,使脂质过氧化反应不断放大。铁调素(Hepcidin,Hep)是近年发现的一种重要的负性铁调节激素。铁调素体内表达的调控非常复杂,缺血缺氧状态、铁水平、红细胞生成率、炎症等均会对铁调素的表达产生影响。值得注意的是,炎症或感染时,包括LPS、IL-6、IL-1在内的一些炎性物质可以增加铁调素基因表达,其中,人们对于IL-6与铁调素的关系研究最充分。然而,在动脉粥样硬化发生发展过程中,关于ox-LDL自身是否可诱导hepcidin的表达,尚未见相关研究报道。膜铁转运蛋白1(Ferroportin1, FP1)是巨噬细胞膜上已知唯一的铁输出蛋白。Hep与巨噬细胞表面的FP1结合后,促进其内吞和降解,使巨噬细胞内的铁增多,氧化作用增强。巨噬细胞吞噬红细胞后,铁调素是否通过促进巨噬细胞内铁沉积而在噬红巨噬细胞的活化和凋亡中发挥作用,尚无直接证据。本轮子在实验室前期研究以及本课题体内部分研究基础上,体外探讨ox-LDL诱导hepcidin表达的情况以及铁调素在噬红巨噬细胞活化及凋亡中的作用,从而进一步探讨铁调素及红细胞促进AS斑块不稳定的相关机制。目的1.明确ox-LDL在诱导巨噬细胞表达hepcidin中的作用；2.探讨红细胞在ox-LDL诱导的巨噬细胞泡沫化、氧化应激,炎症反应和细胞凋亡中的作用；3.探讨铁调素在ox-LDL诱导的巨噬细胞泡沫化、氧化应激,炎症反应和细胞凋亡中的作用；4.探讨铁调素在ox-LDL诱导的噬红巨噬细胞泡沫化、氧化应激,炎症反应和细胞凋亡中发挥的作用；5.阐明铁调素在噬红巨噬细胞活化及凋亡中作用的相关机制。方法1.巨噬细胞培养及吞噬红细胞培养小鼠J774A.1巨噬细胞；将小鼠红细胞分离并清洗后,包被抗红细胞的IgG,将调理过的红细胞置入巨噬细胞培养皿内共同抚育1.5-2小时,倒置显微镜观察红细胞吞噬情况；红细胞裂解液去除未吞噬的红细胞。按照实验目的进行干预,检测相关指标。2.siRNA的合成、筛选及转染构建针对小鼠铁调素基因的siRNA和control siRNA,并转染J774A.1巨噬细胞。3转染效率检测分别将不同浓度的Hepcidin siRNA及对照control siRNA,转染巨噬细胞24h,然后观察细胞的形态变化,并采用实时定量RT-PCR及细胞免疫荧光化学法检测GAPDH和Hepcidin在蛋白和mRNA水平的表达,评价抑制效率,筛选最佳浓度。4.细胞分组及刺激体外实验1：明确ox-LDL在诱导巨噬细胞表达hepcidin中的作用。将巨噬细胞分为对照组和ox-LDL刺激组,以不同浓度的ox-LDL刺激巨噬细胞不同时间后,观察hepcidin的表达情况。体外实验2：探讨红细胞在ox-LDL诱导的巨噬细胞氧化应激和细胞凋亡中发挥的作用。将细胞分为巨噬细胞组,巨噬细胞+ox-LDL组,噬红细胞组,噬红细胞+ox-LDL组；检测各组细胞内脂质水平、ROS产量、凋亡率及炎性因子水平。体外实验3：探讨铁调素在ox-LDL诱导的巨噬细胞氧化应激及细胞凋亡过程中发挥的作用。将巨噬细胞分为对照组,oxLDL刺激组；hepcidin刺激组,oxLDL+hepcidin刺激组；检测各组细胞内脂质水平、ROS产量、凋亡率及炎性因子水平。体外实验4：探讨铁调素在ox-LDL诱导的噬红巨噬细胞氧化应激及细胞凋亡过程中发挥的作用。巨噬细胞吞噬红细胞后,分为对照组,噬红细胞+oxLDl组：噬红细胞+hepcidin组,噬红细胞+oxLDL+hepcidin组；噬红巨噬细胞转染hepcidin siRNA或ccontrol siRNA后,分别给予或不给予oxLDL刺激,从而将噬红细胞分为hepcidin siRNA组,control siRNA组：hepcidin siRNA+ox-LDL组及control siRNA+ox-LDL组；检测各组细胞内脂质水平、ROS产量、凋亡率及炎性因子水平。体外实验5：探讨铁调素在噬红巨噬细胞活化及凋亡中发挥作用的相关机制。将细胞分为巨噬细胞组,噬红细胞组,噬红细胞+hepcidin组；噬红巨噬细胞转染hepcidin siRNA或control siRNA后,分别给予或不给予ox-LDL刺激,从而将噬红细胞分为hepcidin siRNA组,control siRNA组：hepcidin siRNA+ox-LDL组及control siRNA+ox-LDL组；检测各组Ferroportin1、H-Ferritin和L-Ferritin的表达水平。体外实验6：为进一步明确铁调素在噬红巨噬细胞活化及凋亡中发挥作用与铁沉积有关,将细胞分为巨噬细胞组、噬红巨噬细胞组、噬红巨噬细胞+螯合剂(BPDL+DFO)组,观察铁螫合剂对噬红巨噬细胞氧化应激、炎症反应及细胞凋亡的影响；将细胞分为巨噬细胞组、噬红巨噬细胞组、噬红巨噬细胞+ox-LDL组,噬红巨噬细胞+ox-LDL+螯合剂(BPDL+DFO)组,观察铁螯合剂对巨噬细胞内脂质聚集的影响。5.实时定量RT-PCR检测收集不同干预的巨噬细胞或噬红巨噬细胞,提取RNA,实时荧光定量RT-PCR检测Hepcidin、Ferroportin1、H-Ferritin、L-Ferritin、IL-6、MCP-1、TNF-a等指标在mRNA水平的表达。6. Western Blot检测收集不同干预的巨噬细胞或噬红巨噬细胞,提取蛋白,Western Blot方法检测Ferroportin1、H-Ferritin、L-Ferritin、IL-6、MCP-1、TNF-a等指标在蛋白水平的表达。强弱以表达蛋白和β-actin条带积分光密度的比值表示。7.免疫细胞荧光染色不同干预的巨噬细胞和噬红巨噬细胞收集后,免疫细胞荧光染色检测hepcidin和Ferroportin1在巨噬细胞的表达情况。8.TUNEL凋亡检测不同干预的巨噬细胞和噬红巨噬细胞收集后,Tunel法观察细胞凋亡情况。9.活性氧簇检测不同处理的巨噬细胞和噬红巨噬细胞收集后,流式细胞仪检测ROS荧光强度,评价各组ROS产量。10.细胞内脂质水平检测不同处理的巨噬细胞和噬红巨噬细胞收集后,Folch法提取细胞内脂质,酶法检测细胞内总胆固醇、甘油三酯、低密度胆固醇水平。11.统计分析所有统计学数据均使用SPSS16.0软件包分析。计量资料以均数±标准差表示,计数资料以数值和百分比表示。统计分析采用t检验、Oneway ANOVA或LSD post-hoc分析。P<0.05被认为差异有统计学意义。结果1.巨噬细胞吞噬红细胞的检测洗涤包被后的红细胞与巨噬细胞37℃共同孵育2小时并去除未吞噬红细胞后,镜下观察到巨噬细胞成功吞噬红细胞,每个巨噬细胞平均大约吞噬9个红细胞(红细胞：巨噬细胞≈9：1)。2.巨噬细胞hepcidin siRNA转染效率检测Hepcidin siRNA和control siRNA分别转染巨噬细胞,实时定量RT-PCR和免疫细胞荧光染色法检测巨噬细胞转染前后hepcidin的表达水平,hepcidin siRNA的转染效率为70%～80%,control siRNA对巨噬细胞铁调素表达水平无影响。3.Ox-LDL对巨噬细胞hepcidin表达的影响将巨噬细胞分为生理盐水对照组及ox-LDL刺激组,以50ug/ml的ox-LDL刺激巨噬细胞0,1,2,4,8,16,24小时,RT-PCR检测Hepcidin在mRNA水平的表达,结果显示ox-LDL刺激2小时后Hepcidin表达最强；以0ug/ml,25ug/ml,50ug/ml,75ug/ml,100ug/ml的ox-LDL刺激巨噬细胞2小时,RT-PCR检测显示50ug/ml的ox-LDL刺激巨噬细胞时hepcidin表达最强。以50ug/ml的ox-LDL刺激巨噬细胞2小时后,细胞免疫荧光检测刺激组铁调素表达较对照组显著增强。4.红细胞促进ox-LDL诱导的巨噬细胞脂质聚集、氧化应激、炎症反应和凋亡的作用将细胞分为对照组,巨噬细胞+ox-LDL组,噬红巨噬细胞组,噬红细胞+ox-LDL组,检测各组细胞内脂质水平、ROS产量,凋亡发生率及IL-6、MCP-1、TNF-a在mRNA和蛋白水平的表达情况。结果显示,噬红细胞+ox-LDL组的细胞内脂质水平、ROS产量,凋亡发生率及IL-6、MCP-1、TNF-a的表达水平不仅明显高于对照组,且与巨噬细胞+ox-LDL组、噬红巨噬细胞组相比亦有显著统计学差异。巨噬细胞+ox-LDL组,以及噬红巨噬细胞组的ROS产量、凋亡发生率及炎性因子水平较对照组明显升高,但两者间无统计学差异。证实红细胞本身具有促进巨噬细胞氧化应激、炎症反应和凋亡的作用,且促进ox-LDL诱导的巨噬细胞氧化应激、炎症反应和凋亡过程。ox-LDL刺激巨噬细胞后细胞内脂质水平显著升高,吞噬红细胞本身并不能显著提高细胞内脂质水平,,但对ox-LDL诱导的细胞内脂质水平升高有显著促进作用。5.铁调素对ox-LDL诱导的巨噬细胞脂质聚集、氧化应激、炎症反应和凋亡的作用将细胞分为巨噬细胞对照组,巨噬细胞+ox-LDL组,巨噬细胞+hepcidin组,巨噬细胞+ox-LD+hepcidin组,检测各组细胞内脂质水平、ROS产量,凋亡发生率及IL-6、MCP-1、TNF-a在1mRNA和蛋白水平的表达情况。结果显示,巨噬细胞+hepcidin组各项指标较对照组并无明显变化,巨噬细胞+ox-LDL+hepcidin组的各项指标显著高于对照组,但较巨噬细胞+ox-LDL组并无显著变化,提示铁调素对ox-LDL诱导的巨噬细胞脂质聚集、氧化应激、炎症反应和凋亡无显著影响。6.铁调素对ox-LDL诱导的噬红巨噬细胞脂质水平、氧化应激、炎性反应及细胞凋亡的作用巨噬细胞吞噬红细胞后,将噬红巨噬细胞分为对照组,噬红细胞+oxLDL组：噬红细胞+hepcidin组,噬红细胞+oxLDL+hepcidin组,分别检测各组细胞内脂质水平,ROS产量,凋亡发生率及IL-6、MCP-1、TNF-a在mRNA和蛋白水平的表达情况。结果显示噬红细胞+ox-LDL+hepcidin组的细胞内脂质水平、ROS产量,凋亡发生率及IL-6、MCP-1、TNF-a的表达水平不仅明显高于对照组,且与噬红细胞+oxLDl组及噬红细胞+hepcidin组相比亦有显著统计学差异；噬红细胞+oxLDL组和噬红细胞+hepcidin组的ROS产量、凋亡发生率及IL-6、MCP-1、TNF-a表达水平较对照组显著升高,但两组间无统计学差异。噬红细胞+oxLDL组细胞内的脂质水平显著低于噬红细胞+oxLDL+hepcidin组,但明显高于噬红细胞+Hepcidin组,证实hepcidin本身并不上调噬红巨噬细胞内脂质,但促进ox-LDL诱导的噬红巨噬细胞内脂质聚集。噬红巨噬细胞转染hepcidin siRNA或control siRNA后,分别给予或不给予oxLDL刺激,从而将噬红细胞分为hepcidin siRNA组,control siRNA组；hepcidin siRNA+ox-LDL组及control siRNA+ox-LDL组,分别检测各组细胞内脂质水平、ROS产量,凋亡发生率及IL-6、MCP-1、TNF-a在mRNA和蛋白水平的表达情况。Hepcidin siRNA组的各项指标较control siRNA组显著下降,给予ox-LDL刺激后,hepcidin siRNA+ox-LDL组的各项指标仍显著低于control siRNA+ox-LDL组,两组各项检测指标较无ox-LDL刺激组显著升高。7.巨噬细胞吞噬红细胞及铁调素干预对巨噬细胞膜铁转运蛋白1(Ferroportin1)及细胞内铁蛋白表达水平的影响巨噬细胞吞噬红细胞后,RT-PCR结果显示,巨噬细胞Fpnl的表达升高,在4小时达到高峰,然后逐渐下降,24小时基本恢复到基线水平；而细胞内H-Ferritin和L-Ferrtin的表达水平分别在噬红后4小时和6小时达到高峰,并一直持续到24小时。铁调素刺激噬红巨噬细胞1小时后,H-Ferritin和L-Ferritin的表达水平显著升高,而Fpnl的水平明显下降。转染hepcidin siRNA后,噬红巨噬细胞H-Ferritin和L-Ferritin的表达,无论有无ox-LDL刺激,较control siRNA组均明显下降。8.铁鳌合剂对噬红巨噬细胞的活化和凋亡的作用在给予铁螯合剂(BPDL+DFO)后,噬红巨噬细胞内H-Ferritin及L-Ferritin表达水平较刺激前明显下降,同时,给予铁螯合剂(BPDL+DFO)后,噬红巨噬细胞内脂质水平、ROS产量、炎性因子表达及细胞凋亡率均明显下降。结论1. Ox-LDL刺激巨噬细胞诱导hepcidin表达,并呈浓度和时间依赖性；2.红细胞本身具有促进巨噬细胞氧化应激、炎症反应和凋亡的作用,并可上调ox-LDL诱导的巨噬细胞内脂质沉积、氧化应激、炎症反应和凋亡过程；3.铁调素只有在巨噬细胞吞噬红细胞后,才能上调噬红巨噬细胞内脂质积聚、氧化应激、炎症反应和凋亡,并在ox-LDL诱导的噬红巨噬细胞内脂质积聚、氧化应激、炎症反应和凋亡中发挥促进作用；4.铁调素在ox-LDL诱导的噬红巨噬细胞的活化与凋亡过程中的作用是通过降解巨噬细胞膜上的膜铁转运蛋白,上调巨噬细胞内铁沉积而实现的。更多还原

【Abstract】 BackgroundAtherosclerosis formation and plaque rupture are the common pathological basis of most of cardio-cerebrovascular diseases. It has been documented for decades that a state of sustained iron depletion or mild iron deficiency protects against atherosclerosis. Ferritin are highly expressed in human and rabbit atherosclerotic plaques and iron chelation is benefitial to the endothelial function in patients with coronary artery disease. Accumulation of ferritin and redox-active iron may contribute to the oxidative stress in atherogenesis. In macrophage, in the presence of iron, the oxidants can be activated to form ROS via the iron-catalyzed Haber-Weiss reaction or Fenton reaction. ROS are cytotoxic because of its ability to initiate lipid peroxidation, damage membranes,oxidize sulfhydryl compounds, and inactivate enzymes and transporters. Oxidative reactions associated with the over-loaded iron and lipids facilitate macrophage apoptosis with the release of cellular contents into the lesion, which further enhances inflammatory responses such as recruitment of more monocytes to amply this process.Recently, hepcidin has been demonstrated to be a key peptide in the regulation of iron homeostasis. Hepcidin binds to the iron transporter ferroportin1(Fp1) on the cell surface, and induces Fpn internalization and degradation. As a result, the intracellular iron level is elevated. Hepcidin is produced by a wide variety of cells including macrophages and hepcidin expression is increased in response to inflammation. Some inflammatory cytokines, including IL-6, LPS and IL-1were proven to stimulate the expression of hepcidin.Notably, hepcidin is a major determinant of the amount of iron retained in macrophages. Therefore, it has been proposed in2007by Sullivan, et al. that hepcidin promoted progression of atherosclerotic plaque by slowing or preventing the mobilization of iron from macrophages within atherosclerotic plaque. Hepcidin and metabolic syndrome are positively correlated. Recent research found that about15%of the metabolic syndrome patients were with iron overload, while about50%of the15%patients got NAFLD at the same time. Another report demonstrated recently that there was a positive correlation between hepcidin, macrophage iron, MCP-1and vascular damage in patients with metabolic syndrome.In summary, it has been proven that iron is associated with atherosclerosis and hepcidin is a key hormone in the regulation of iron balance and iron recycling, however, a direct, clear, and causal relationship between hepcidin and atherosclerotic lesion formation or plaque stability has not yet been established. In the present study, we studied the potential role of hepcidin in atherogenesis and plaque stability by hepcidin gain-and loss-of-function approaches in a mouse model of accelerated atherosclerosis and explored the underlying mechanisms.Objectives1. To observe the expression of hepcidin in the ApoE-/-mice atherosclerotic model.2. To explore the potential role of hepcidin in atherogenesis and plaque stability by hepcidin gain-and loss-of-function approaches in a ApoE-/-mice atherosclerotic model.3. To explore the underlying mechanisms of hepcidin on the development and progreesion of atherosclerosis.Methods1. Preparation of Adenoviral VectorsThe murine hepcidin cDNA was amplified by RT-PCR, cloned into pMD18-T vector, and then sub-cloned into pIRES2-EGFP vector using the EcoRI and BamH I sites. The hepcidin cDNA sequence was confirmed by sequencing. A shRNA sequence that is used to target hepcidin was cloned into the pcDNATM6.2-GW/EmGFPmiR vector and confirmed by sequencing. Both hepcidin and its shRNA IRES2-EGFP cassettes were cloned into the adenoviral expression vector pAd/CMV/V5-DEST using the Gateway Technology. Recombinant viruses were packaged and amplified in HEK293cells and purified by anion chromatography. The titer of the viral vectors was determined by TCID50method. The adenoviral vectors expressing EGFP alone (Ad-EGFP) was used as control.2. Animal Model and Gene TransferOur study in animal models consisted of two parts. In the first part of the in vivo study, forty male apoE-/-mice (6weeks of age) were randomly divided into a control group (n=20) and a model group (n=20). Mice in the control group were given a normal diet for13weeks and then euthanized. Mice in the model group received a high-fat diet for2weeks and then a constrictive silastic tube was placed around the left common carotid artery near its bifurcation. These mice were maintained on a high-fat diet for additional11weeks. The left common carotid arteries in both groups of mice were collected for histological and mRNA analysis.In the second part of the in vivo study, seventy-five male apoE-/-mice (6weeks of age) were given a high-fat diet for2weeks. Then a constrictive silastic tube was placed around the left common carotid artery near its bifurcation. Eight weeks after the collar placement, mice were randomly divided into three groups (n=25, each) for adenoviral gene delivery of EGFP, EGFP-hepcidin, and EGFP-hepcidin shRNA. These mice were maintained on a high-fat diet for additional3weeks, and then euthanized.3. Serum Index measurementAt the end point of the second part of the in vivo study, blood was collected from the inferior vena cava before perfusion-fixation. Serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein cholesterol (HDL), glucose, iron and hepcidin concentrations were measured.4. Histopathological and immunohistochemical measurementSections were stained with hematoxylin and eosin. Collagen was visualized by sirius red staining. Lipid deposition was identified by Oil-red O staining. Corresponding sections on separate slides were immunostained with anti-mouse monocyte/macrophage monoclonal antibodies, anti-a-smooth muscle (SM) actin monoclonal antibodies, anti-human interleukin-6(IL-6) polyclonal antibodies, anti-human tumor necrosis factor-a (TNF-a) monoclonal antibodies, anti-mouse monocyte chemoattractant protein-1(MCP-1) antibodies, anti-human matrix metalloproteinase (MMP-2) monoclonal antibodies, anti-mouse hepcidin monoclonal antibodies and anti-mouse ferritin antibodies. Positive staining areas of smooth muscle cells (SMCs), macrophages, lipids, collagen, IL-6, MCP-1, TNF-a, MMP-2, hepcidin, H-Ferritin and L-Ferritin were quantified by computer-assisted color-gated measurement, and the ratios of the positive staining area to the arterial cross sectional area (part1in vivo study) or plaque area (part2in vivo study) were calculated. The vulnerable index was calculated by the following formula:the relative positive staining area of (macrophages%+lipid%)/the relative positive staining area of (a-SMCs%+collagen%).5. ImmunofluorescenceTissue sections of the carotid arteries were incubated with double primary antibodies, including those against macrophages and hepcidin, SMCs and hepcidin, macrophages and ox-LDL as well as SMCs and ox-LDL. Fluorescent images were obtained by a laser scanning confocal microscopy.6. Measurement of non-heme iron by Atomic absorption spectrometryThe content of non-heme iron in atherosclerotic plaques were measured by flame atomic absorption spectrometry with a Solar M-6Atomic absorption spectrophotometer.7. Quantitative real-time RT-PCR analysisThe mRNA levels of Hepcidin、H-Ferritin、L-Ferritin、IL-6、MCP-1、TNF-a and MMP-2in the atherosclerotic plaques and the mRNA level of hepcidin in liver were quantified by RT-PCR.8. Statistical analysisAll analyses were performed using SPSS16.0(SPSS Inc., Chicago, IL). Data were expressed as mean±SE. An independent-samples t-test was used to compare continuous data for between-group differences and comparisons among groups involved the use of ANOVA with LSD post hoc test used for multiple comparisons. P<0.05was considered statistically significant.Results1. Detection of the restructuring adenovirus interference vectorsBoth the restructuring adenovirus expressing vector and interference vector were verified to be correct by sequencing validation. The virus tilter of Ad-hep is3.11×1012ifu/ml and the verus tilter of Ad-hep-shRNA is9.12×1012ifu/ml.2. The experssion of hepcidin in Atherosclerotic PlaqueRelative to the homolateral carotid arteries in the control group without atherosclerotic lesions, immunochemical staining and RT-PCR analysis revealed that both mRNA and protein expression levels of hepcidin were up-regulated in the carotid atherosclerotic plaques indicating the potential role of hepcidin in the pathogenesis of atherosclerosis. Hepcidin was located in macrophages ans SMCs.3. The result of transfection efficiency measurementElevated and comparable levels of fluorescent densities were observed in plaques of these infected carotid arteries, appearing at one week after infection and sustaining for additional two weeks. The ratio of the GFP positive staining area to the plaque area in the three treatment groups of mice two weeks after the infection was similar among these3groups.4. Effects of hepcidin on weightIn contrast, the local adenoviral gene delivery for three weeks hardly affected the weight.5. Effects of hepcidin on blood lipids and glucoseThe local adenoviral gene delivery for three weeks hardly affected the serum levels of total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL),glucose.6. Effects of hepcidin on serum ironNeither the overexpression nor knockdown of hepcidin altered the serum iron by the collar placement in ApoE-/-mice relative to the control group.7. Effects of hepcidin on serum and liver hepcidinIn contrast, the local adenoviral gene delivery for three weeks hardly affected the serum hepcidin and the hepcidin mRNA expression in liver. 8. The expression of hepcidin in three groups after adenoviral deliveryImmunochemical staining and RT-PCR analysis confirmed the up-regulation of hepcidin in the carotid arteries of the Ad-hepcidine group and down-regulation of hepcidin in those of the Ad-hepcidin shRNA group.9. Effects of hepcidin overexpression or knockdown on lesion area in the three groupsNeither the overexpression nor knockdown of hepcidin altered the plaque size induced by the collar placement in ApoE-/-mice relative to the control group.10. Effects of hepcidin overexpression or knockdown on the plaque composition and stability of ApoE-/-mice in the three groupsThe plaque composition including macrophages, smooth muscle cells (SMCs), and collagen was significantly affected by hepcidin overexpression or knockdown. In the plaques of the Ad-hepcidin group, the relative contents of macrophages were increased whereas those of SMCs and collagen were decreased. In contrast, in the plaques of the Ad-hepcidin shRNA group, the relative contents of macrophages were reduced while those of SMCs and collagen were increased. The relative contents of lipids in plaques did not differ significantly among the Ad-EGFP, Ad-hepcidin and Ad-hepcidin shRNA groups. The plaque vulnerability index was elevated by the hepcidin overexpression but reduced by the hepcidin knockdown, respectively, suggesting that hepcidin plays a critical role in plaque destabilization rather than plaque formation.11. Effects of hepcidin overexpression or knockdown on the inflammatory cytokine expression in three groups of ApoE-/-miceThe expression levels of interleukin-6(IL-6), monocyte chemotactic protein-1(MCP-1), tumor necrosis factor-alpha (TNF-α) and metalloproteinase-2(MMP-2) were substantially enhanced in both by hepcidin overexpression but were dramatically suppressed by hepcidin knockdown, suggesting that hepcidin destabilizes atherosclerotic plaques at least partly via exaggerating inflammatory responses in atherosclerotic lesions.12. Effects of hepcidin overexpression or knockdown on the levels of intracellular lipid in three groups of ApoE-/-miceThe expression level of ox-LDL in intraplaque macrophages was substantially enhanced by hepcidin over-expression but was dramatically suppressed by hepcidin knockdown.13. Effects of hepcidin overexpression or knockdown on the ferritin expression in three groups of ApoE-/-miceThe the expression of ferritin mRNA and protein in the atherosclerotic plaque was up-regulated or down-regulated by the local adenoviral over-expression of hepcidin or by its shRNA, respectively. These results demonstrated that hepcidin controls iron trapping in e atherosclerotic lesions.14. Effects of hepcidin overexpression or knockdown on the non-heme iron in three groups of ApoE-/-miceThe non-heme iron level in the atherosclerotic plaque was up-regulated or down-regulated by the local adenoviral over-expression of hepcidin or by its shRNA, respectively. These results demonstrated that hepcidin controls iron trapping in e atherosclerotic lesions.Conclusions1. In a mouse model of atherosclerosis, the expression of hepcidin was upregulated in atherosclerotic plaque and located on macrophages and SMCs;2. Hepcidin plays a critical role in plaque destabilization.3. Hepcidin destabilizes atherosclerotic plaques via exaggerating inflammatory responses, promoting iron trapping and intracellular lipids accumaulation in atherosclerotic lesions. BackgroundThe stability of atherosclerotic plaque has been a hot topic in recent years. It has been recognized for years that leukocytes and platelets play an important role in the pathogenesis of atherosclerosis and erythrocytes have been traditionally deemed as an innocent bystander in the process of atherosclerosis. However, recent studies have suggested that erythrocytes are important factors contributing to atherosclerotic plaque growth and destabilization. More iron deposition and macrophages accumulation had been found in the atherosclerotic plaques which were characterized by accumulation of lipid contents and thin fribrous cap of plaque. Moreover, plaques within intraplaque hemorrhage are vulnerable to new plaque hemorrhage, which not only stimulates the progression of atherosclerosis but also promotes the transition from a stable to an unstable lesion. Our lab had established a rabbit model of intraplaque hemorrhage and demonstrated that erythrocytes induce plaque vulnerability in a dose-dependent way. However, the exact mechanisms underlying the erythrocyte-induced atherosclerosis remain obscure. Intraplaque hemorrhage can lead to accumulation of lipid contents, augmentation of inflammation and thin fribrous cap of plaque, which may be the main mechanisms of erythrocyte-induced plaque vunerability.Some substances released from erythrocytes should be related to the erythrocyte-induced vulnerability of plaque. Several lines of evidence have suggested that cellular iron signaling and iron-mediated oxidative damage are related to cardiovascular diseases. Iron plays important roles in the initiation and progression of atherosclerosis and a state of sustained iron depletion or mild iron deficiency protects against atherosclerosis. Ferritin are highly expressed in human and rabbit atherosclerotic plaques and iron chelation is benefitial to the endothelial function in patients with coronary artery disease. Accumulation of ferritin and redox-active iron may contribute to the oxidative stress in atherogenesis. In macrophage, with the presence of iron, the oxidants can be activated to form ROS via the iron-catalyzed Haber-Weiss reaction or Fenton reaction. ROS are cytotoxic because of its ability to initiate lipid peroxidation, damage membranes, oxidize sulfhydryl compounds, and inactivate enzymes and transporters. Oxidative reactions associated with the over-loaded iron and lipids facilitate macrophage apoptosis with the release of cellular contents into the lesion, which further enhances inflammatory responses such as recruitment of more monocytes to amply this process. We found in our earlier studies that postive erythrocyte staining paralleled with not only the iron deposition but also macrophage infiltration.Hepcidin is a recently discovered key hormone in the regulation of iron homeostasis and iron recycling. It has been proved that autocrine hepcidin leads to iron sequestration in human monocytes. The expression of hepcidin is stimulated by inflammation. Hepcidin binds to the iron transporter ferroportin1(Fpn1) on the cell surface, and induces Fpn internalization and degradation. As a result, the intracellular iron level is elevated.It has been proposed in2007that hepcidin promoted progression of atherosclerotic plaque by slowing or preventing the mobilization of iron from macrophages within atherosclerotic plaque. Hepcidin and metabolic syndrome are positively correlated. Recent research found that about15%of the metabolic syndrome patients were with iron overload, while about50%of the15%patients got NAFLD at the same time. Another report demonstrated recently that there was a positive correlation between hepcidin, macrophage iron, MCP-1and vascular damage in patients with metabolic syndrome.In summary, it has been proven that erythrocytes and iron are associated with atherosclerosis and hepcidin is a key hormone in the regulation of iron balance and iron recycling, however, the role of hepcidin in the erythrocytosis-induced atherosclerotic progression is still unclear. In the present study, we studied the relationship between erythrocytes and the activation of macrophage in vitro and explored the potential role of hepcidin in this process and the underlying mechanisms. Objectives1. To observe the role of ox-LDL on the expression of hepcidin in macrophage.2. To explore the role of RBC in ox-LDL induced intracellular lipids accumulation, oxidative stress, inflammation and apoptosis in macrophage.3. To explore the role of hepcidin in ox-LDL induced intracellular lipids accumulation, oxidative stress, inflammation and apoptosis in macrophage.4. To explore the role of hepcidin in ox-LDL induced intracellular lipids accumulation, oxidative stress, inflammation and apoptosis in erythrophagocytosed macrophage.5. To explore the underlying mechanisms on hepcidin’s role in the activation and apoptosis of macrophage after erythrophgocytosis.Methods1. Macrophage cultivation and erythrophagocytosisJ774macrophages were chosen for erythrophagocytosis and cultured in DMEM medium Erythrophagocytosis was performed as previously described with slight modifications, erythrocytes (2×109) were opsonized with goat anti-mouse IgG.The opsonized erythrocytes (2×107) were added to J774macrophage monolayers (2×106) and incubated for2hours. After uptake, noningested opsonized erythrocytes were removed using Red Blood Cell Lysis Buffer Control cells were subjected to the same lysis and washing steps as cells treated with erythrocytes.2. Transfection of siRNA targeting mouse hepcidin and the control siRNAHepcidin silence in cultured macrophages was achieved by hepcidin RNA interference approach.3. Quantification of transfection efficiencyMeasured the expression of hepcidin in both mRNA and protein levels and evaluted the transfection efficiency24hours after transfection.4. Cell grouping and treatmentPart Ⅰ. To explore the role of ox-LDL on the expression of hepcidin in macrophage, divide macrophages into the control group and ox-LDL stimulation group. Observe the expression of hepcidin in macrophage after different times of ox-LDL’s stimulating with different concentrations.Part2. To investigate the role of RBC on intracellular lipids accumulation, oxidative stress, inflammation and apoptosis in macrophage.The cells were divided into groups of macrophages, macrophages+ox-LDL, erythrophgocytosed macrophges, erythrophgocytosed macrophges+ox-LDL. Detect the levels of intracellular lipid, ROS production, apoptosis rate, and the levels of inflammatory factors in each group.Part3. To investigate the role of hepcidin on ox-LDL-induced macrophage oxidative stress and apoptosis process. Macrophages will be divided into groups of control, oxLDL stimulation, hepcidin stimulation, and oxLDL+hepcidin stimulation. Detect the levels of intracellular lipid, ROS production, apoptosis rate, and the levels of inflammatory factors in each group.Part4. To investigate the role of hepcidin on ox-LDL-induced oxidative stress and apoptosis in erythrophgocytosed macrophge, the erythrophgocytosed macrophge were divided into groups of control, oxLDL stimulation, hepcidin stimulation, and oxLDL+hepcidin stimulation. The erythrophgocytosed macrophge were divided into into groups of hepcidin siRNA, control siRNA, hepcidin siRNA+ox-LDL, and control siRNA+ox-LDL. Detect the levels of intracellular lipid, ROS production, apoptosis rate, and the levels of inflammatory factors in each group.Part5. To investigate the mechanism of hepcidin’s role on the activation and apoptosis of erythrophgocytosed macrophge. Cells were divided into groups of macrophages, erythrophgocytosed macrophge and erythrophgocytosed macrophge+hepcidin. After erythrophgocytosed macrophge were transfected the hepcidin siRNA or the control siRNA, treated them with or without ox-LDL stimulation, and accordingly divide erythrophgocytosed macrophge into groups of hepcidin siRNA, control siRNA, hepcidin siRNA+ox-LDL, and control siRNA+ox-LDL. Detect the expression level of Ferroportinl and Ferritin in each group.Part6. To further clarify that hepcidin is related to iron deposition in its role of erythrophgocytosed macrophges’ activation and apoptosis, divide the cells into groups of macrophages, erythrophgocytosed macrophges, erythrophgocytosed macrophge+chelator (BPDL+DFO), and observe BPDL+DFO’s effect upon erythrophgocytosed macrophges’oxidative stress, inflammation and apoptosis. Divide the cells into groups of macrophages, erythrophgocytosed macrophges, erythrophgocytosed macrophges+ox-LDL, and erythrophgocytosed macrophges+ox-LDL+chelator (BPDL+DFO), and observe the iron chelator’s effect upon intracellular lipids accumulation in erythrophgocytosed macrophges.5. Quantitative Real-time-PCRThe J774macrophage with different treatments were extracted with TriZol Reagent and detected by RT-PCR.Hepcidin,Il-6, MCP-1, TNF-α, MMP-2, FPN1, L-ferritin and H-ferritin expression was normalized to that of β-actin.6. Western Blot AnalysisCollect the J774macrophage with different treatments, extract proteins, and detect protein expression levels of ferroportinl, H-Ferritin, L-Ferritin with Western Blot. IL-6, MCP-1and TNF-a. Sample loadings were normalized to β-actin expression.7. Immunofluorescence StainingExpression and localization of hepcidin and FPN1in J774macrophages or erythrophagocytosed macrophage were examined by immunofluorescent staining. The immunofluorescent staining of J774macrophages was then observed on a fluorescent microscopy.8.Detection of ApoptosisAfter the collection of the differently treated macrophages and erythrophgocytosed macrophges,apoptosis was assessed by terminal deoxynucleotidyl transferase end-labelling (TUNEL staining. The number of TUNEL-positive cells was counted three times in randomly selected fields from each treatment.9.Quantification of ROS productionAfter the collection of the differently treated macrophages and erythrophgocytosed macrophges, fluorescence measurement of ROS was performed with Flow Cytometerequipped with a488nm argon laser using conventional methods.10. Quantification of Intracellular LipidsAfter the collection of the differently treated macrophages and erythrophgocytosed macrophges., the lipids of macrophages with different treatments were extracted with the Folch method and the intracellular TC, TG and LDL-C were measured by enzymatic assay.11. Statistical analysisAll analyses were performed using SPSS16.0(SPSS Inc., Chicago, IL). Data were expressed as mean±SE. An independent-samples t-test was used to compare continuous data for between-group differences and comparisons among groups involved the use of ANOVA with LSD post hoc test used for multiple comparisons. P<0.05was considered statistically significant. All experiments were repeated for at least3times.Results1. Macrophage swallowed the erythrocytesThe opsonized erythrocytes were added to J774macrophage monolayers (2×106) and incubated for2hours at37℃with a final phagocytosis ratio of red blood cells to J774cells at about9:1.2. Detection of hepcidin siRNA transfection efficiencyDesign the sequence of hepcidin siRNA and control siRNA.transfect macrophages and detect the hepcidin expression level before and after the macrophages transfection by real-time quantitative RT-PCR and immunofluorescence staining method. The hepcidin siRNA transfection efficiency was70%-80%, while the control siRNA had no effect on macrophage hepcidin expression.3. The expression of hepcidin was up-regulated in macrophages treated with ox-LDL stimulationDivide macrophages into the control group and ox-LDL stimulation group. Use50ug/ml ox-LDL to stimulate macrophages for0,1,2,4,8,16, and24hours. We found that the ox-LDL-induced up-regulation of hepcidin was transient, peaked at2hour after the stimulation and thereafter declined to the basal level within24hours, Using0ug/ml,25ug/ml,50ug/ml,75ug/ml,100ug/ml of ox-LDL to stimulate macrophages for2hours and we found that the maximum effective dose was50μg/ml4. RBC enhanced the intracellular lipids accumulation, oxidative stress, inflammation and apoptosis of macrophage.Cells were divided into groups of control macrophages, macrophage+ox-LDL, erythrophagocytosed macrophages, and erythrophagocytosed macrophages+ox-LDL group. Detect the intracellular lipid levels, ROS production, apoptosis incidence and the expression of IL-6, MCP-1and TNF-a in mRNA and protein levels in each group. We found that erythrophagocytosed macrophages+ox-LDL group not only had a much higher level of intracellular lipid, ROS production, apoptosis and IL-6, MCP-1, and TNF-a expressions than that of the control group, but also was statistically different from the macrophages+ox-LDL group and the erythrophagocytosed macrophages group. ROS production, apoptosis rate and the level of cytokines in the macrophages+ox-LDL group and the erythrophagocytosed macrophages group became much higher than the control group, but there was no statistical difference between the two grouops. This confirmed that RBC itself has the effect on enhancing the oxidative stress, inflammation and apoptosis in macrophage, and has the effect of promoting the ox-LDL-induced oxidative stress, inflammation and apoptosis of macrophage. With the ox-LDL stimulating, intracellular lipid levels of macrophage were significantly elevated. Though RBC itself did not significantly improve the levels of intracellular lipids, it has a significant role in promoting ox-LDL-induced intracellular lipids accumulation.5. Hepcidin has no effect on ox-LDL-induced macrophage lipid accumulation, oxidative stress, inflammation and apoptosisMacrophages were divided into control group, ox-LDL treatment group, hepcidin treatment group, and ox-LD+hepcidin treatment group. Detect the levels of intracellular lipid, ROS production, apoptosis incidence and IL-6, MCP-1and TNF-a expression in both mRNA and protein levels in each group. We found that all the detection targets in the hepcidin treatment group showed no significant difference from the control group. All detection targets of the ox-LDL+hepcidin treatment group were significantly higher than those of the control group, but showed no significant difference from those of the ox-LDL treatment group. This confirmed that hepcidin has no significant impact upon ox-LDL induced lipid accumulation, oxidative stress, inflammation and apoptosis in macrophage.6Hepcidin’s role in enhancing the erythrophagocytosed macrophages’s intracellular lipids accumulation, oxidative stress, inflammation and apoptosis with or without ox-LDL treatment The erythrophagocytosed macrophages were divided into groups of control, oxLDL treatment, hepcidin treatment, oxLDL+hepcidin treatment. Detect intracellular lipid levels, ROS production, apoptosis and IL-6, MCP-1and TNF-a expression in both mRNA and protein levels. We found that the ox-LDL+hepcidin treatment group’s intracellular lipid levels, ROS production, apoptosis and IL-6, MCP-1and TNF-a expression levels were not only significantly higher than those of the control group, but also also significantly different from those of the oxLDl treatment group and the hepcidin treatment group. The ROS production, apoptosis rate and IL-6, MCP-1and TNF-a expressions of the oxLDL treatment group and hepcidin treatment group were significantly higher than those of the control group, but no statistcal difference between the two groups was found. The intracellular lipid levels of the oxLDL treatment group were significantly lower than those of the oxLDL+hepcidin treatment group, but significantly higher than those of the Hepcidin treatment group, which confirmed that the hepcidin itself does not increase the intracellular lipid level of erythrophagocytosed macrophages, but significantly increase the accumulation of lipid in erythrophagocytosed macrophages induced by ox-LDL. erythrophagocytosed macrophages were treated with or without oxLDL-stimulated after they were transfected with hepcidin siRNA or control siRNA. And then erythrophagocytosed macrophages were divided into the hepcidin siRNA group, control siRNA group; hepcidin siRNA+ox-LDL group, and the control siRNA+ox-LDL-group. Detect respectively the intracellular lipid level, ROS production, apoptosis and IL-6, MCP-1and TNF-a expression in both mRNA and protein levels in each group.All the detected targets of the hepcidin siRNA group decreased significantly compared with the control siRNA group. With the ox-LDL stimulation, All the detected targets of the hepcidin siRNA group remained significantly lower than those of the control siRNA group, but all the detected targets of both the two groups were higher than those of the ox-LDL(-) group.7. The effect of erythrophagocytosis and hepcidin on the expression of Ferroportin1and ferritinAfter erythrophagocytosis, we observed a time-dependent up-regulation of L-ferritin and H-ferritin which are iron-storage proteins, and ferroportinl, an iron-export protein. The expression of Fpnl reached a peak at4hour, declined thereafter, and returned to the basal level by24hour after erythrophagocytosis, whereas the expression of H-ferritin and L-ferritin reached a peak at4hour and6hour, respectively, and sustained at least by24hour after erythrophagocytosis.Hepcidin further up-regulated the H-ferritin and L-ferritin expression whereas down-regulated the Fpn1expression in the erythrophagocytosed macrophages. After the transfection of hepcidin siRNA, erythrophagocytosed macrophages’s the H-Ferritin and L-Ferritin expression, with or without ox-LDL stimulation, significantly decreased compared with the control siRNA group.8. Iron chelator’s impact on the activation and apoptosis of erythrophagocytosed macrophagesIntracellular iron of macrophage was scavenged by iron chelators including desferrioxamine (DFO) and ferrous chelator2,2’-bipyridyl (BPDL). The increased pro-inflammatory cytokine production, ROS formation and apoptosis in macrophages after erythrophagocytosis were inhibited by adding of DFO and BPDL Meanwhile, the H-Ferritin and L-Ferritin expression levels in the iron chelators groups were significantly reduced in the erythrophagocytosed macrophages.Conclusions1. The expression of hepcidin was time-and dose-dependently up-regulated in macrophages treated with ox-LDL stimulation.2.The intracellular lipid levels, oxidative stress, inflammation and apoptosis could be promoted by RBC with or without ox-LDL treatment3. Hepcidin enhanced the ox-LDL-induced pro-atherogenic activation of macrophages only in the setting of erythrophagocytosis.4. Hepcidin plays an important enhancing role in the pro-atherogenic activation and apoptosis by promoting the intracellular iron depositin.更多还原