【作者】 王琪；

【导师】 张铭湘； 葛志明；

【作者基本信息】 山东大学 ， 内科学， 2018， 博士

【摘要】 1研究背景中国心血管病患病率处于持续上升阶段。《中国心血管病报告2017》推算心血管病现患人数2.9亿,2015年心血管病死亡率仍居首位,高于肿瘤及其他疾病。动脉粥样硬化(atherosclerosis,AS)作为心血管疾病的最主要病理学基础以及急性心血管事件的最重要原因,严重威胁人类的生命与健康。动脉粥样硬化斑块的形成和发展主要包括炎症细胞的异常聚集,泡沫细胞形成,平滑肌细胞增殖,细胞外基质合成增多及伴随而来的动脉血管重构。最近研究表明,动脉粥样硬化患者的血管壁自始至终都处于一种慢性炎症性反应状态,在动脉粥样硬化斑块内存在大量炎症细胞尤其是巨噬细胞,激活的免疫细胞以及各种细胞因子。越来越多证据表明炎症因子和与之相关的信号通路与动脉粥样硬化的进展密切相关,但是,确切的分子机制仍然不明确。研究动脉粥样硬化的发生发展及其机制要用到动物模型,现在实验所用的动物模型主要有:大鼠、小鼠、豚鼠、小型猪、新西兰兔、狗和非人灵长类动物等。我们通过改变实验动物的基因型,给予特定的手术干预,调整喂养动物的饮食结构,控制喂养时间等手段获得预期的AS动物模型,进而采用各种实验技术研究AS的病理生理机制。动物模型方便了临床实验研究与假设验证,但也存在不同模型发病机制不同、病理进程各异、AS斑块成分及好发部位有所区别的差异性,每一种动物模型都有其各自的优缺点,至今尚没有一种动物模型能完全模拟人类AS的发生发展特点。许多研究者往往使用一种模型来研究AS的科学机制,却忽视了不同AS模型可能会产生不同的结果。因此,研究者必须了解这些差异并根据研究手段、靶点、目的等的不同选择合适的模型,同时采用多种动物模型开展研究,以获得真实可靠的实验结果。ApoE-/-小鼠体积小,繁殖快,成本低,在普通饮食条件下可以自发性产生AS斑块,是目前研究AS应用最广泛的物种。ApoE在脂蛋白的代谢中发挥重要作用,除了低密度脂蛋白(low density lipoprotein,LDL),其他所有脂蛋白中都含有ApoE,不同饮食的ApoE-/-小鼠表现为血总胆固醇升高,且不随性别与年龄改变,普通饲料饮食10周时就可在主动脉根部出现黄白色结节,20周时就可出现纤维斑块,而高脂饲料能加快AS病变的进展。但是小鼠的生理特点与解剖结构与人类差异大,其中最主要的是富含ApoB48的乳糜微粒和VLDL,而人类血浆中主要以LDL为主。兔类对外源性胆固醇吸收率高,也是目前用于建立动脉粥样硬化模型最常用的动物之一,兔类较鼠类动物体型更大,可以进行的手术操作范围更广,并且解剖结构上与人类更接近,血浆中的脂蛋白成分以LDL为主,但是由于兔子没有肝酯酶,只有在血清胆固醇水平很高的时候才能形成斑块,这种做法会使兔子内脏脂质沉着,免疫力下降,极易因感染死亡,兔子作为食草性动物,脂质代谢方式和人类也存在差异。ILF3是一种双链RNA连接蛋白,作为活化T淋巴细胞核因子(NFAT)转录因子家族成员之一,在激活的淋巴细胞中与控制早期炎症反应有关。ILF3参与到一系列细胞反应过程中,包括DNA代谢,转录,翻译,RNA稳定以及microRNA的生物合成。以往对ILF3的研究主要集中在肿瘤方面。近年来有研究发现ILF3可能与血脂调节,血栓形成,心肌梗死,创伤后应激障碍介导的心脏病等心血管系统方面疾病有关。ILF3与动脉粥样硬化的发生发展是否有关,至今在国内外没有相关报道。本文拟通过检测ILF3在不同动脉粥样硬化斑块中的表达情况,明确ILF3是否和动脉粥样硬化的发生发展有关。2目的(1)明确ILF3在小鼠和兔动脉粥样硬化易损斑块中的表达变化情况;(2)检测小鼠动脉粥样硬化模型血浆中的ILF3浓度改变;(3)阐明ILF3的变化与AS斑块易损性的关系;3方法3.1动物动脉粥样硬化斑块模型的建立第一部分:小鼠颈动脉套管动脉粥样硬化模型75只8周龄雄性ApoE-/-小鼠,随机分为以下组:A:对照组(Control,n=15):给予普通饲料饮食,12周末结束实验;B:颈动脉套管模型组(Cannula,n=15):给予颈动脉套管手术,当天开始高脂饲料饮食,至12周末结束实验;C:颈动脉套管模型+辛伐他汀组(Statin+Cannula,n=15):给予颈动脉套管手术,当天开始高脂饲料饮食,从第8周开始给予辛伐他汀(9.4mg/kg/d)治疗,至12周末结束实验;D:颈动脉套管模型+联合触发组(Cannula+Triggering,n=15):给予颈动脉套管手术,当天开始高脂饲料饮食,至12周末结束实验,实验结束前给予联合触发以刺激斑块破裂;E:颈动脉套管模型+辛伐他汀+联合触发组(Statin+Cannula+Triggering,n=15):给予颈动脉套管手术,当天开始高脂饲料饮食,从第8周开始给予辛伐他汀(9.4mg/kg/d)治疗,至12周末结束实验,实验结束前给予联合触发以刺激斑块破裂。第二部分:兔腹主动脉球囊拉伤动脉粥样硬化模型50只三月龄雄性纯种新西兰大白兔,随机分为以下组:A:对照组(Control,n=10):给予普通饲料,12周末结束实验;B:球囊损伤模型组(Balloon-injury,n=10):行腹主动脉球囊拉伤,当天开始给予含1%胆固醇的高脂饮食,12周末实验结束;C:球囊损伤模型+辛伐他汀组(Statin+Balloon-injury,n=10):行腹主动脉球囊拉伤,当天开始给予含1%胆固醇的高脂饮食,于8周末开始给予辛伐他汀治疗(3.7mg/kg/d),12周末实验结束;D:球囊损伤+药物触发组(Balloon-injury+triggering,n=10):行腹主动脉球囊拉伤,当天开始给予含1%胆固醇的高脂饮食,于12周末实验结束,实验结束前24h和48h给予两次药物触发注射造成斑块破裂及血栓形成;E:球囊损伤模型+辛伐他汀+药物触发组(Statin+Bal loon injury+triggering,n=10)行腹主动脉球囊拉伤,当天开始给予含1%胆固醇的高脂饮食,于8周末开始给予辛伐他汀治疗(3.7mg/kg/d),12周末实验结束,实验结束前24h和48h给予两次药物触发注射造成斑块破裂及血栓形成。3.2体重及血液学指标检测所有动物造模开始时称量体重,处死之前称量体重并心尖取血,检测血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)以及血糖水平。3.3组织病理学与免疫组化检测分别对小鼠套管侧颈动脉以及兔腹主动脉行油红0染色检测脂质沉积情况,天狼猩红染色检测胶原纤维含量,免疫组化CD68和α-SMA染色分布检测巨噬细胞及平滑肌细胞数量多少,计算斑块易损指数,易损指数=(巨噬细胞+脂质)阳性面积%/(平滑肌细胞+胶原)阳性面积%。免疫组化ILF3、IL-6和TNF-α染色检测炎症因子表达情况;免疫组化方法检测正常小鼠心尖,肝脏,脾,肺,肾,血管中ILF3表达情况。3.4免疫荧光化学检测免疫荧光双标检测ILF3在人主动脉斑块内巨噬细胞和平滑肌细胞中的表达情况。3.5小鼠血清ELISA检测小鼠血清ELISA检测不同模型组血清中ILF3的表达情况。3.6统计分析所有统计数据学均使用SPSS19.0软件包分析,所有实验至少重复3次。计量资料以均数土标准差表示,计数资料以数值和百分比表示。两组比较采用采用独立样本t检验。P<0.05被认为差异有统计学意义。4结果4.1动物一般情况对各组小鼠以及新西兰大白兔处死前称量体重,测量血清TC、TG、LDL-C、HDL-C浓度,结果显示相比正常饮食对照组,体重有所升高,TC、TG、LDL-C、HDL-C浓度均升高(P<0.05)。至实验结束,共1只小鼠死亡,6只新西兰大白兔死亡。最终74只小鼠完成实验,44只兔子完成实验。4.2斑块易损性情况与对照组相比,套管/球囊拉伤组斑块内脂质成分,胶原含量,巨噬细胞以及平滑肌细胞数量均增加;与未治疗组相比,他汀治疗能降低斑块内脂质成分,增加胶原含量,减少巨噬细胞数量,增加平滑肌细胞数量,降低斑块易损性(P<0.05);与未破裂组比较,斑块破裂组斑块内脂质成分,增加胶原含量,减少巨噬细胞数量,增加平滑肌细胞数量,降低斑块易损性(P<0.05);套管/球囊拉伤组,2/15只小鼠发生斑块破裂,无兔子发生斑块破裂,套管/球囊拉伤加触发组,7/14只小鼠发生斑块破裂,4/8只兔子发生斑块破裂,说明药物触发增加斑块破裂;套管/球囊拉伤加他汀治疗组1/15只小鼠发生斑块破裂,无兔子发生斑块破裂,套管/球囊拉伤加他汀治疗加触发组5/15只小鼠发生斑块破裂,2/9只兔子发生斑块破裂,进一步说明他汀治疗能增加斑块稳定性。4.3斑块中炎症因子表达情况与对照组相比,套管/球囊拉伤组斑块内ILF3、IL-6、TNF-α表达增加;与套管/球囊拉伤组相比,他汀治疗能降低斑块内ILF3、IL-6、TNF-α表达(P<0.05);与套管/球囊拉伤组相比,套管/球囊拉伤加触发组斑块内ILF3、IL-6、TNF-α表达增加(P<0.05);与球囊拉伤加他汀治疗组相比,套管/球囊拉伤加他汀治疗加触发组,ILF3、IL-6、TNF-α表达增加(P<0.05)。4.4 ILF3在小鼠各正常组织中的表达情况对正常小鼠心尖,肝脏,脾,肺脏,肾组织进行ILF3免疫组化染色,发现ILF3表达于心肌,肝脏,肾中,血管中少量表达,脾和肺中不表达。4.5小鼠血清ILF3浓度检测与对照组相比,套管组血清ILF3浓度显著增加;与套管组相比,他汀治疗能降低血清ILF3浓度(P<0.05)。4.6人主动脉斑块中ILF3的表达情况对人主动脉斑块进行免疫荧光染色,发现ILF3在斑块中大量表达。ILF3与巨噬细胞的标志物CD68免疫荧光双标染色发现ILF3主要表达于巨噬细胞中;ILF3与平滑肌细胞标志物α-SMA免疫荧光双标染色发现ILF3在平滑肌细胞中表达很少。5结论(1)ILF3在正常小鼠的心肌、肝、肾组织中表达丰富,在肺和脾中不表达,血管中少量表达;(2)ILF3在AS斑块中高表达,且主要表达于巨噬细胞中;(3)他汀治疗能降低ILF3在AS斑块中的表达,增加斑块稳定性;1研究背景动脉粥样硬化作为冠状动脉疾病的主要病理过程,近年来备受关注。在动脉粥样硬化发展的早期阶段,氧化修饰的低密度脂蛋白(ox-LDL)导致单核细胞来源的巨噬细胞向血管壁聚集,这些巨噬细胞过度吞噬胆固醇,导致泡沫细胞的形成,促进动脉壁的炎症反应,导致多种致命性的病理表现,比如斑块内出血,斑块破裂,钙化等。炎症是动脉粥样硬化发展的关键因素,动脉粥样硬化斑块中的脂蛋白代谢以及炎症状态的调节已经成为研究动脉粥样硬化的焦点领域。根据动脉粥样硬化斑块的组织形态学特点,又可以将斑块分为稳定性斑块和易损斑块,20世纪末,“易损斑块”用于描述导致多数急性心血管事件发生的易于发生破裂的斑块,又称不稳定斑块;易损斑块是急性血栓形成的最常见原因,被认为是导致急性冠脉综合征(acute coronary syndrome,ACS)的罪犯血管。易损斑块的特征包括:①体积相对比较大②薄的纤维帽(平滑肌和胶原纤维少)③大的脂质核(≥斑块体积的40%)④大量炎性细胞浸润(主要为单核-巨噬细胞)⑤正性重构(outward remodeling)⑥增多的斑块内血管新生。易损斑块肩部浸润大量免疫炎症细胞,产生促炎因子和水解酶类,会加重斑块炎症反应,加剧动脉粥样硬化斑块破裂。ILF3是一种双链RNA连接蛋白,此前有研究表明ILF3蛋白参与了细胞周期调控,在RNA转录、剪切、编辑、RNA出核转运,亚细胞定位等方面都可能发挥作用,涉及细胞发育,细胞周期和病毒感染等多重细胞功能。特别值得注意的是,近年有研究表明ILF3可能与血脂调节,血栓形成,心肌梗死,创伤后应激障碍介导的心脏病等心血管系统方面疾病有关,而这些研究都集中在基因水平,在蛋白功能水平是否有关还未知,ILF3在动脉粥样硬化斑块易损性中是否扮演重要角色,还没有相关探索。2目的(1)建立ApoE-/-小鼠AS模型,观察病毒抑制ILF3表达对AS斑块易损性中的作用;(2)探讨ILF3在巨噬细胞脂质沉积及炎症因子分泌中的作用。3方法3.1慢病毒shRNA-ILF3载体构建设计3对ILF3基因的shRNA质粒,用RT-PCR和蛋白印记法检测干扰效率,选取干扰效率最高的一对序列包装包含绿色荧光蛋白(GFP)报告基因的慢病毒载体。ILF3 的目标干扰序列为:5’-GCCAATGGACTGAAGTCATGT-3’,阴性对照序列为:5’-TTCTCCGAACGTGTCACGT-3,。3.2巨噬细胞特异性过表达小鼠的构建巨噬细胞条件性过表达Rosa26-ILF3小鼠,购于北京唯尚立德生物科技有限公司。3.2动脉粥样硬化动物模型的建立45只8周龄雄性ApoE-/-小鼠,行左侧颈总动脉套管手术建立动脉粥样硬化不稳定模型,高脂喂养8周后,经尾静脉给予不同慢病毒注射,根据转染病毒的不同,随机分为以下几组:(1)对照组(NS组,n=15),不加干预(2)空载体组(NC组,n=15),转染只携带GFP的空载体(3)病毒干预组(LV-ILF3(-)组,n=15),转染shRNA-ILF3慢病毒转染后继续高脂喂养至12周末,安乐死处死小鼠,留取组织,一部分存于液氮中保存,一部分固定于4%多聚甲醛中至少24小时。3.3体重及血脂指标检测实验结束前称量小鼠体重,取材时留取小鼠心尖血,检测血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)以及血糖水平。3.4主动脉大体油红0检测将主动脉从4%多聚甲醛中取出,流水冲洗后在大体显微镜下剖除血管周围组织,将主动脉沿长轴剖开,进行油红0染色,染色后将血管固定在白板上,拍照留取图像。3.5组织病理与免疫组化检测将左侧颈动脉从4%多聚甲醛中取出,流水冲洗后包埋OTC,进行冰冻切片。对切片进行油红0染色,天狼猩红染色,CD68和αα-SMA免疫组化染色,分别检测斑块中脂质,胶原,巨噬细胞,平滑肌细胞的含量。计算斑块易损指数,易损指数=(巨噬细胞+脂质)阳性面积%/(平滑肌细胞+胶原)阳性面积%。3.6实时定量RT-PCR检测取新鲜颈动脉组织,提取RNA,进行实时荧光定量RT-PCR检测ILF3,IL-6,TNF-α在mRNA水平上的表达情况。3.7小鼠腹腔巨噬细胞的提取和培养6周龄Rosa26-ILF3小鼠和6-8周龄C57/B6小鼠腹腔注射6%淀粉,72h后提取诱导产生的原代腹腔巨噬细胞,经过CD68细胞免疫荧光染色鉴定腹腔巨噬细胞。3.8细胞分组及刺激体外实验1:明确ox-LDL在诱导小鼠腹腔巨噬细胞产生ILF3中的作用。给予不同浓度 ox-LDL(Oug/ml,25ug/ml,50ug/ml,75ug/ml,100ug/ml)刺激C57/B6小鼠腹腔巨噬细胞24h,观察ILF3蛋白水平的表达情况;以50ug/ml ox-LDL浓度刺激小鼠腹腔巨噬细胞不同时间(Oh,4h,8h,12h,24h,48h),观察ILF3蛋白水平的表达情况。体外实验2:探讨ILF3在ox-LDL诱导的巨噬细胞泡沫化,炎症因子分泌及相关信号通路中发挥的作用:(1)提取C57/B6小鼠腹腔巨噬细胞,sh-ILF3慢病毒载体转染细胞后再加ox-LDL刺激,分为以下三组:NC组;NC+ox-LDL刺激组;SI+ox-LDL刺激组;(2)提取C57/B6小鼠腹腔巨噬细胞以及Rosa26-ILF3过表达小鼠腹腔巨噬细胞,分为以下两组:WT组;ILF3 overexpression组;体外实验3:探讨ILF3在LPS诱导的巨噬细胞炎症反应中发挥的作用:提取C57/B6小鼠腹腔巨噬细胞,sh-ILF3慢病毒载体转染细胞后再加LPS刺激,分为以下三组:NC组;NC+LPS刺激组;SI+LPS刺激组;3.6实时定量RT-PCR检测收集各组的巨噬细胞,提取RNA,实时荧光定量RT-PCR检测ILF3、Arg1、iNOS、CD86、IL-6、TNF-α、IL-1β、IL-10在 mRNA水平的表达。3.7 Western Blot检测收集各组的巨噬细胞,提取蛋白,Western Blot方法检测ILF3、ABCA1、ABCG1、SRA1、LOX1、ACAT1、IL-6、IL-10、NF-κB、NF-κB(p65)、AKT、p-AKT 在蛋白水平的表达。强弱以目的蛋白和β-actin条带积分光密度的比值表示。3.6免疫细胞荧光染色干预后的细胞收集后,免疫细胞荧光染色检测ILF3在细胞中的表达情况。3.7细胞油红0染色各组细胞刺激后,进行油0染色检测细胞中脂质含量。3.8统计分析所有统计数据学均使用SPSS19.0软件包分析,所有实验至少重复3次。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。两组比较采用采用独立样本t检验。P<0.05被认为差异有统计学意义。4结果4.1小鼠一般情况对各组小鼠处死前称量体重,测量血清TC、TG、LDL-C、HDL-C浓度,结果显示各组小鼠之间各项指标没有差异,说明ILF3病毒转染不影响小鼠体内脂质代谢。4.2慢病毒转染效率及病毒转染后ILF3在斑块中的表达荧光显微镜下观察病毒转染后斑块组织中GFP的表达,可见有明显的转染绿色荧光蛋白;RT-PCR结果结果显示,NC组ILF3水平和NS组没有统计学差异,LV-ILF3(-)组ILF3表达比NC组显著降低(P<0.05)。4.3 ILF3对动脉粥样硬化斑块成分及易损性的影响与对照组相比,sh-ILF3可以降低斑块内脂质和巨噬细胞含量,增加平滑肌和胶原含量,降低斑块易损性(P<0.05)。4.4 ILF3对AS斑块内炎症因子表达水平的影响RT-PCR结果显示,LV-ILF3(-)组的IL-6和TNF-α水平显著降低(P<0.05)。4.5小鼠腹腔巨噬细胞的提取(cd68染色)成功提取小鼠腹腔巨噬细胞,对其进行巨噬细胞标志物CD68免疫细胞荧光染色,鉴定所提取细胞纯度,>99%的细胞均为巨噬细胞。4.6 ox-LDL对小鼠腹腔巨噬细胞ILF3表达的影响分别加入 Oug/ml,25ug/ml,50ug/ml,75ug/ml,100ug/ml 的 ox-LDL,培养 C57/B6小鼠腹腔巨噬细胞24h后发现,ox-LDL能浓度依赖性的上调ILF3蛋白的表达。与正常对照组相比,25ug/ml ox-LDL刺激细胞24h后ILF3就可以明显增加(P<0.01),100ug/ml 作用最强(P<0.01)。以50ug/mlox-LDL分别刺激小鼠腹腔巨噬细胞Oh,4h,8h,12h,24h,48h后,发现ox-LDL能时间依赖性的上调ILF3蛋白的表达。与正常对照组相比,处理4h后ILF3即有明显增加(P<0.01),24h达到峰值(P<0.01),并持续至48h。4.7巨噬细胞Sh-ILF3转染效率检测shRNA-NC和shRNA-ILF3分别转染巨噬细胞,实时定量PCR和Western Blot检测巨噬细胞转染前后ILF3的表达水平变化,结果显示与NC组相比,SI组ILF3在mRNA水平及蛋白水平都显著降低(P<0.05)。4.8 Rosa26-ILF3小鼠腹腔巨噬细胞ILF3表达水平检测Western Blot检测C57/B6小鼠腹腔巨噬细胞和Rosa26-ILF3小鼠腹腔巨噬细胞ILF3的表达水平,结果显示ILF3过表达小鼠ILF3水平比C57小鼠显著升高。4.9 ILF3促进ox-LDL诱导的巨噬细胞泡沫化将C57/B6小鼠腹腔巨噬细胞分为NC组,NC+ox-LDL刺激组,SI+ox-LDL刺激组,检测各组细胞脂ABCA1,ABCG1,SRA1,L0X1,ACAT1在蛋白水平的表达情况。结果显示,ox-LDL刺激巨噬细胞后,与脂质转出相关蛋白ABCA1,ABCG1以及与脂质转入相关蛋白LOX1,ACAT1均有所升高,其中LOX1和ACAT1有统计学意义(P<0.05),转染sh-ILF3以后,ABCA1和ABCG1进一步升高但ABCG1达不到统计学差异,LOX1和ACAT1则表达降低(P<0.05)。对各组细胞刺激后进行油红0染色,结果显示ox-LDL刺激巨噬细胞后脂质沉积增多,转染sh-ILF3后,脂质沉积明显减少。提取C57/B6小鼠以及Rosa26-ILF3小鼠腹腔巨噬细胞,ox-LDL刺激24h后进行油红0染色,结果显示ILF3过表达组脂质沉积明显增多,以上结果说明ILF3具有促进胆固醇沉积的作用。4.10 ILF3促进ox-LDL诱导的巨噬细胞炎症因子分泌将C57/B6小鼠腹腔巨噬细胞分为NC组,NC+ox-LDL刺激组,SI+ox-LDL刺激组,检测各组细胞IL-6,iNOS,IL-10,Arg1蛋白水平的表达情况。结果显示,ox-LDL刺激巨噬细胞后,IL-6,iNOS,IL-10均有所升高,,转染sh-ILF3以后,促炎因子IL-6,iNOS表达降低(P<0.05),而抑炎因子Arg1和IL-10表达进一步升高(P<0.05),证明ILF3有促进炎症因子分泌的作用。4.11 ILF3促进LPS诱导的巨噬细胞炎症因子分泌将C57/B6小鼠腹腔巨噬细胞分为NC组,NC+ox-LDL刺激组,SI+ox-LDL刺激组,检测各组细胞 ILF3、Arg1、iNOS、CD86、IL-6、TNF-α、IL-1β、IL-10 在 mRNA水平的表达情况。结果显示,LPS刺激巨噬细胞后,ILF3、iNOS、CD86、IL-6、TNF-α、IL-11、IL-10均有所升高(P<0.05),转染sh-ILF3以后,促炎因子ILF3、iNOS、CD86、IL-6、TNF-α、IL-1 β、IL-6 表达降低,而抑炎因子 Arg1和IL-10表达进一步升高(P<0.05),证明ILF3有促进炎症因子分泌的作用。4.12 ILF3在信号通路中的作用将C57/B6小鼠腹腔巨噬细胞分为NC组,NC+LPS刺激组,SI+LPS刺激组,检测各组细胞NF-κB(p-p65)和p-AKT蛋白水平的表达情况。结果显示,ox-LDL刺激巨噬细胞后,NF-κB(p-p65)和p-AKT表达升高,转染sh-ILF3以后,表达降低(P<0.05)。说明ILF3调节炎症因子分泌和胆固醇沉积的作用可能是通过NF-κ B信号通路和AKT信号通路。5结论(1)敲除ILF3通过调节巨噬细胞向M2转化,抑制斑块中炎症因子表达,增加斑块稳定性(2)敲除ILF3能降低巨噬细胞脂质吞噬,增加脂质外流,(3)ILF3对巨噬细胞的调节作用是通过NF-κ B和AKT信号通路来实现的更多还原

【Abstract】 1 BackgroundThe prevalence of cardiovascular disease in China is continuing to rise.The"China Cardiovascular Diseases Report 2017" estimates that the current number of cardiovascular diseases is 290 million.In 2015,cardiovascular disease mortality still ranked first,higher than cancer and other diseases.Atherosclerosis(AS)is the most important pathological basis of cardiovascular disease and the primary pathology underlying cardiovascular disease development is atherosclerosis.Atherosclerosi’s morbidity and mortality are increasing day by day.The formation and development of atherosclerotic plaques mainly include abnormal accumulation of inflammatory cells,formation of foam cells,proliferation of smooth muscle cells,increased synthesis of extracellular matrix,and concomitant arterial vascular remodeling.Recent studies have shown that atherosclerosis is a chronic inflammatory response condition of the blood vessel wall.There are abundant inflammatory cells,activated immune cells,and cytokines in the atherosclerotic plaque.There is increasing evidence that inflammatory factors and their associated signaling pathways are closely related to the progression of atherosclerosis,but the exact molecular mechanism remains unclear.ILF3 protein is a kind of double-stranded RNA(dsRNA)-binding protein.As one of the members of the activated T lymphocyte nuclear factor(NFAT)transcription factor family and is involved in the control of early inflammatory responses in activated lymphocytes.ILF3 participates in a series of cellular processes including DNA metabolism,transcription,translation,RNA stabilization,and microRNA biosynthesis.Previous studies of ILF3 have focused on tumors.In recent years,studies have found that ILF3 may be associated with diseases of the cardiovascular system,such as regulation of bloodlipid,thrombosis,myocardial infarction,and post-traumatic stress disorder-mediated heart disease.Whether ILF3 is related to the formation and development of atherosclerosis or not has not been reported at home and abroad.This article intends to detect the expression of ILF3 in different atherosclerotic plaques and to determine whether ILF3 is related to the development of atherosclerosis.2 Objectives(1)Expression and distribution detection of ILF3 in normal heart,liver,spleen,lung,kidney in mice.(2)To clarify the expression of ILF3 in vulnerable plaques of atherosclerosis in mice,rabbits and humans,respectively.(3)Detection of ILF3 expression level in mouse peripheral blood.3 Method3.1 Establishment of Animal Atherosclerotic ModelPart one:Mouse carotid atherosclerosis modelEight-week-old male ApoE-/-mice(n=75)were randomly divided into the following groups:A:Control(n=15):Give a normal diet for 12 weeks;B:Cannula(n=15):The surgery was performed with the silastic tube placed around the left common carotid artery and given a high-fat diet from the same day to 12 week;C:Statin+Cannula(n=15):The surgery was performed with the silastic tube placed around the left common carotid artery and given a high-fat diet from the same day to 12 week;from the eighth week,simvastatin(9.4mg/kg/d)was given to the mice tntil the 12 week;D:Cannula+Triggering(n=15):The surgery was performed with the silastic tube placed around the left common carotid artery and given a high-fat diet from the same day to 12 week,before the end of the experience,jointly triggered experiments was performed to stimulate the plaque to rupture;E:Statin+Cannula+Triggering(n=15):The surgery was performed with the silastic tube placed around the left common carotid artery and given a high-fat diet from the same day to 12 week;from the eighth week,simvastatin(9.4mg/kg/d)was given to the mice tntil the 12 week.,before the end of the experience,jointly triggered experiments was performed to stimulate the plaque to rupture.Part two:Rabbit abdominal aorta atherosclerosis modelthree-month-old male purebred New Zealand white rabbits(n=50)were randomly divided into the following groups:A:Control(n=10):Give a normal diet for 12 weeks;B:Balloon-injury(n=10):Abdominal aorta balloon injury surgery was performed and given a high-fat diet from the same day to 12 week;C:Statin+Balloon-injury(n=10):Abdominal aorta balloon injury surgery was performed and given a high-fat diet from the same day to 12 week,from the eighth week,simvastatin(9.4mg/kg/d)was given to the mice tntil the 12 week;D:Balloon-injury+triggering(n=10):Abdominal aorta balloon injury surgery was performed and given a high-fat diet from the same day to 12 week,before the end of the experience,two drug-triggered injections was performed at 24h and 48h before the end of the experiment to stimulate plaque rupture;E:Statin+Balloon injury+triggering(n=10):Abdominal aorta balloon injury surgery was performed and given a high-fat diet from the same day to 12 week,from the eighth week,simvastatin(9.4mg/kg/d)was given to the mice tntil the 12 week,two drug-triggered injections was performed at 24h and 48h before the end of the experiment to stimulate plaque rupture.3.2 Body weight and serum index measurementAll animals were weighed before sacrifice and blood was collected from the apex cordis.Serum total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL),high-density lipoprotein(HDL)were measured.3.3 Histopathological and immunohistochemical detectionCannulated carotid arteries of the mice and the injuried abdominal aorta of the rabbits were individually stained by oil red lipids to detect lipid deposition.Sirius red staining was used to detect collagen.Immunohistochemical staining of CD68 and a-SMA was performed to detect macrophages and smooth muscles.The vulnerability index of the plaque was calculated,and the vulnerability index =(macrophage + lipid)positive area%/(smooth muscle cell + collagen)positive area%.Immunohistochemical staining of ILF3,IL-6 and TNF-a was used to detect the expression of inflammatory factors.Immunohistochemistry was used to detect the expression of ILF3 in the heart,liver,spleen,lung,kidney of normal mice.3.4 Immunofluorescencethe expression of ILF3 in macrophages and smooth muscle cells in human aortic plaque was detected by double primary antibodies which including those against CD68 and ILF3,a-SMA and ILF3.3.5 ELISA assay of serum ILF3 in miceELISA assay was used to detecte the concentration level of ILF3 in different models.3.6 Statistical analysisAll statistics were analyzed using the SPSS 19.0 software package.All experiments were repeated at least 3 times.The measurement data is expressed as mean ± standard deviation,and the count data is expressed as a numerical value and a percentage.An independent sample t test was used for comparison between the two groups.P<0.05 was considered to be statistically significant.4 Results4.1 The general conditions of animalsEach group of mice and New Zealand white rabbits were weighed before sacrifice,and serum concentration of TC,TG,LDL-C,and HDL-C were measured.The results showed that compared with the normal diet group,body weight increased,The concentration of TC,TG,LDL-C and HDL-C increased(P<0.05).There were no statistical differences among each model groups.By the end of the experiment,a total of 1 mouse died and 6 New Zealand white rabbits died.Eventually.74 mice completed the experiment and 44 rabbits completed the experiment.4.2 Plaque vulnerability of each groupsCompared with the control group,the lipid content,collagen content,macrophage and smooth muscle cells in the plaque of the cannula/balloon injury group increased.Compared with the untreated group,statin treatment can reduce the lipid content in the plaque,increased collagen content,decreased the number of macrophages,increased the number of smooth muscle cells,reduced plaque vulnerability(P<0.05);compared with the unruptured group,the rupture group showed increased lipid cotnet and reduced collagen content,increased macrophages and reduced smooth muscle cells,and the plaque vulnerability is increased(P<0.05);in the cannula/balloon injury group,2/15 mice developed plaque rupture,no rabbit plaque rupture,In the cannula/balloon strain plus trigger group,plaque rupture occurred in 7/14 mice,and plaque rupture occurred in 4/8 rabbits,indicating that drug triggering increased plaque rupture;cannula/balloon strain plus statin treatment Plaque rupture occurred in 1/15 mice,plaque rupture occurred in rabbits,plaque rupture occurred in 5/15 mice in cannula/balloon injury plus statin plus trigger group,and 2/9 rabbits developed plaque rupture,furtherlly indicates that statin treatment can increase plaque stability.4.3 Expression of inflammatory factors in plaqueCompared with the control group,the expression of ILF3,IL-6,and TNF-a in the plaque of the cannula/balloon injury group increased;compared with the cannula/balloon injury group,statin treatment can reduce the expression of ILF3,IL-6,TNF-a in plaque(P<0.05);Compared with the cannula/balloon injury group,the expression of ILF3,IL-6,and TNF-a in the cannula/balloon injury plus trigger group increased(P<0.05);Compared with the balloon-injured plus statin group,the expression of ILF3,IL-6,and TNF-a increased in the cannula/balloon injury plus statin plus trigger group(P<0.05).4.4 Expression of ILF3 in mouse normal tissuesThe immunohistochemical staining of ILF3 in normal mice hearts,livers,spleens,lungs,and renal revealed that ILF3 is expressed in myocardium,liver,kidney.3.5 ELISA assay of serum ILF3 in miceCompared with the control group,serum ILF3 concentration in the cannula group increased significantly(P<0.05);compared with the cannula group,statin treatment can reduce the serum ILF3 concentration(P<0.05);the serum ILF3 concentration in the joint trigger group was increased,but Not reach statistical significance.4.6 Expression of ILF3 in human’s plaquesImmunofluorescence staining of human’s aortic plaques revealed that ILF3 was abundantly expressed in the plaque.immunofluorescence double-label staining of ILF3 and CD68 suggested that ILF3 is mainly expressed in macrophages;immunofluorescence double-label staining of ILF3 and α-SMA suggested that ILF3 expression in smooth muscle cells limited.5 Conclusion:(1)ILF3 is normally expressed in the heart,liver,and kidney tissues of mice.The expression of ILF3 is increased in plaques and further increased in vulnerable plaques.(2)ILF3 is elevated in peripheral blood of atherosclerotic mice,and statin can reduce ILF3 concentration in peripheral blood.(3)ILF3 is mainly expressed in macrophages in the atherosclerosis plaque.1 BackgroundAtherosclerosis,as the main pathological process of coronary artery disease,has received much attention in recent years.In the early stages of the development of atherosclerosis,oxidized low-density lipoprotein(ox-ldl)causes mononuclear-derived macrophages to aggregate into the blood vessel wall,These macrophages excessively phagocytose cholesterol,leading to the formation of foam cells and promoting the inflammatory response of the arterial wall,and this condition induces a variety of fatal pathological outcomes,such as intra-plaque hemorrhage,plaque rupture,calcification and so on.Inflammation is a key factor in the development of atherosclerosis.lipoprotein metabolism and inflammatory regulation in atherosclerotic plaques have become the focus of research on atherosclerosis.According to the histomorphological features of atherosclerotic plaque,plaques could be divided into stable plaques and vulnerable plaques.At the end of the 20th century,"vulnerable plaques" were used to describe plaques which are prone to rupture.It is the most common cause of acute thrombosis and is considered to be the culprit blood vessel leading to acute coronary syndrome(ACS).The characteristics of vulnerable plaque include:1 volume is relatively large 2 thin fiber cap(smooth muscle and collagen fiber less)3 large lipid nucleus(≥ 40%of plaque volume)4 a large number of inflammatory cell infiltration(mainly single Nuclear-macrophage)5 positive remodeling 6 increased angiogenesis in plaques.The vulnerable plaque infiltrates a large number of immune inflammatory cells,producing pro-inflammatory factors and hydrolases,which will aggravate the plaque inflammatory response and aggravate the rupture of atherosclerotic plaque.ILF3 is a double-stranded RNA connexin.Previous studies have shown that ILF3 protein is involved in cell cycle regulation and may play a role in RNA transcription,cleavage,editing,RNA export,and subcellular localization,involving cell development.Multiple cellular functions such as cell cycle and viral infection.It is particularly noteworthy that studies in recent years have shown that ILF3 may be involved in cardiovascular system diseases such as blood lipid regulation,thrombosis,myocardial infarction,post-traumatic stress disorder-mediated heart disease,and these studies are concentrated at the genetic level.Whether the level of protein function is unknown or not,whether ILF3 plays an important role in the vulnerability of atherosclerotic plaque has not been explored.2 Objectives(1)illuminate the effect of ILF3 on the atherosclerosic palque vulnerable and its regulatory mechanism in vivo experiments.(2)illuminate the effect of ILF3 on the inflammatory factor secreted bymacrophages and lipid phagocytosis and study the relevant mechanism.3 Method3.1 Construction of Ientiviral vectorsThree pairs of shRNA plasmids of ILF3 gene were designed,and the interference efficiency was detected by RT-PCR and Western blotting.A shRNA sequence with the highest interference efficiency was cloned into the lentiviral vector containing the green fluorescent protein(GFP)reporter gene.The target interference sequence(Sh-ILF3)of ILF3 is:5’-GCCAATGGACTGAAGTCATGT-3’,and the negative control sequence(Sh-NC)is:5’-TTCTCCGAACGTGTCACGT-3’.3.2 Construction of macrophage-specific knockout miceMacrophage conditional overexpression of Rosa26-ILF3 mice were purchased from Beijing viewsolid biological technology co,ltd.3.3 Establishment of mice atherosclerotic modelEight-week-old male ApoE-/-mice(n=45)underwent the cannula surgery of left common carotidartery to establish the atherosclerosic plaque vunerable model.After eight weeks’ high-fat diet,different lentiviral injections were given via the tail vein,according to different transfection,mice were randomly divided into the following groups:(1)NS group(n=15),without intervention(2)Empty vector group(NC group,n=15),transfected with empty vector carrying only GFP(3)ShRNA-ILF3 intervention group(LV-ILF3(-)group,n= 15),transfected with ShRNA-ILF3 lentivirusAfter transfection,the high-fat diet was continued until 12 weeks.The mice were killed by euthanasia,the tissues were taken,some were stored in liquid nitrogen,and some were fixed in 4%paraformaldehyde for at least 24 hours.3.4 Body weight and blood lipid index testThe weight of the mice were measured before the end of the experiment.The apical blood of the mice were taken and the serum total cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL),high density lipoprotein(HDL)and Blood sugar levels were measured.3.5 aorta oil red O detectionThe aorta was removed from 4%paraformaldehyde.After washing with running water,the perivascular tissue was dissected under microscope.The aorta was dissected along the long axis for oil red O staining.After staining,the blood vessels were fixed on the white plate and photographed.3.6 Histopathology and immunohistochemistryThe left carotid artery was taken out from 4%paraformaldehyde,washing with running water,and embedded in OTC,and frozen sections were taken.The sections were staining with oil red O to identify the lipid deposition,Sirius red to visualize the collagen,CD68 and α-SMA immunohistochemical staining to identify the macrophages and the amooth muscle cells(SMC).The plaque vulnerability index was calculated,and the vulnerability index =(macrophage + lipid)positive area%/(smooth muscle cells + collagen)positive area%.Immunohistochemistry was used to detect the expression of IL-6 and TNF-α in plaques.3.7 Real-time quantitative RT-PCR detectionFresh carotid tissue was taken,RNA was extracted,and real-time quantitative RT-PCR was used to detect the expression of IL-6 and TNF-a at the level of mRNA.3.8 Extraction and culture of mouse peritoneal macrophagesSix-week-old Rosa26-ILF3 mice and 6-8-week-old C57/B6 mice were injected with 6%starch intraperitoneally.After 72 hours,the induced primary peritoneal macrophages were extracted and the peritoneal macrophages were identified by immunofluorescence staining of CD68.3.9 Cell grouping and stimulationExperiment I:Defining the role of ox-LDL in inducing ILF3 production in mouse peritoneal macrophages.C57/B6 mouse peritoneal macrophages were stimulated with different concentrations of ox-LDL(Oug/ml,25ug/ml,50ug/ml,75ug/ml,100ug/ml)for 24h,and the expression of ILF3 mRNA and protein was observed;50ug/ml ox-LDL were used to stimulated mouse peritoneal macrophages at different times(Oh,4h,8h,12h,24h,48h),and the expression of ILF3 mRNA and protein levels was boserved.Experiment 2:To investigate the role of ILF3 in ox-LDL-induced macrophage foaming,inflammatory factor secretion and related signaling pathways.(1)C57/B6 mouse peritoneal macrophages were extracted and Sh-ILF3 lentiviral vector transfected and then cells were stimulated with ox-LDL and divided into the following three groups:NC group;NC +ox-LDL stimulation group;SI+ ox-LDL stimulation group;(2)Extraction of mouse peritoneal macrophages,divided into:C57/B6 group(WT group)and Rosa26-ILF3 group(ILF3 group).experiment 3:To investigate the role of ILF3 in lps-induced macrophage inflammatory response:C57/B6 mouse peritoneal macrophages were extracted and transfected with Sh-ILF3 lentiviral vector and then stimulated with LPS.The cells were divided into the following three groups:NC group;NC+LPS stimulation group;SI+LPS stimulation group;3.7 Real-time quantitative RT-PCR detection Macrophages of each group were collected,RNA was extracted,and real-time quantitative RT-PCR detection ILF3、Arg1、iNOS、CD86、IL-6、TNF-α、IL-1β、IL-10 expression of mRNA.3.8 Western Blot detectionCollect macrophages from each group,extract proteins,and detect the expression of ILF3.ABCAI、ABCGI、SRA1、LOX1、ACAT1、IL-6、IL-10、NF-κB、NF-κB(p65)、AKT、p-AKT proteins by Western Blot at the protein level.The strength is expressed as the ratio of the target protein to the integrated optical density of theβ-actin band.3.9 Immunofluorescence staining After the cells were collected,the expression of il3in the cells was detected by immunofluorescence staining.3.10 oil red O stainingAfter stimulation of each group,oil O staining was performed to detect the lipid eposition in the cells.3.11 Statistical analysisAll statistics were analyzed using the SPSS 19.0 software package.All experiments were repeated at least 3 times.The measurement data is expressed as mean ± standard deviation,and the count data is expressed as a numerical value and a percentage.An independent sample t test was used for comparison between the two groups.P<0.05 was considered to be statistically significant.4 Results4.1 The general conditions of animalsThe weight of each group of mice was weighed before sacrifice.The concentrations of serum TC,TG,LDL-C and HDL-C were measured.The results showed that there was no difference between groups,indicating that ILF3 virus transfection did not affect the lipid metabolisim of mice.4.2 Expression of ILF3 in plaque after lentivirus transfectionThe results of immunohistochemistry showed that there was no statistical difference between the ILF3 level in the sh-nc group and the control group.The expression of ILF3 in the sh-ILF3 group was significantly lower than that in the sh-nc group(P<0.05).The RT-PCR results were consistent with the immunohistochemistry results.This indicates that the ILF3 virus can effectively interfere with the expression of ILF3 in mouse plaques.4.2 Expression level of ILF3 in plaque after lentivirus transfectionThe results of immunohistochemistry showed that there was no statistical difference between the ILF3 level in the sh-nc group and the control group.The expression of ILF3 in the sh-ILF3 group was significantly lower than that in the sh-nc group(P<0.05).The RT-PCR results were consistent with the immunohistochemistry results.This indicates that the ILF3 virus can effectively interfere with the expression of ILF3 in mouse plaques.4.3 The effect of ILF3 on the composition and vulnerability of atherosclerotic plaqueCompared with the control group,sh-ILF3 can reduce the content of lipids and macrophages in plaque,increase the content of smooth muscle and collagen,and reduce plaque vulnerability(P<0.05).4.4 Effect of ILF3 on the expression level of inflammatory cytokines in AS plaque results of Immunohistochemistry and RT-PCR showed that the levels of IL-6 and TNF-a in sh-ILF3 group were significantly lower(P<0.05).4.5 Extraction of mouse peritoneal macrophagesThe mouse peritoneal macrophages were extracted successfully,and the immunofluorescent staining of macrophage marker CD68 was performed to identify the purity of the extracted cells.>99%of cells were macrophages.4.6 Effect of ox-LDL on the expression of ILF3 in mouse peritoneal macrophagesThe murine peritoneal macrophages were stimulated with ox-LDL at Oug/ml,25ug/ml,50ug/ml,75ug/ml,100ug/ml for 24h,it was found that ox-LDL could up-regulate ILF3 mRNA in a concentration-dependent manner.Protein expression.Compared with the normal control group,the ILF3 could be significantly increased after 24h of 25ug/ml ox-LDL stimulation(P<0.01),100ug/ml was the strongest(P<0.01).The murine peritoneal macrophages were stimulated with 50 ug/ml ox-LDL for 0h,4h,8h,12h,24h,48h,and it was found that ox-LDL could up-regulate the expression of ILF3 mRNA and protein in a time-dependent manner.Compared with the normal control group,ILF3 increased after treatment for 4h(P<0.01),peaked at 24h(P<0.01),and continued to 48h.4.7 Detection of transfection efficiency of macrophage Sh-ILF3After ShRNA-NC and ShRNA-ILF transfected into macrophages,the expression levels of ILF3 were detected by real-time quantitative PCR and Western Blot before and after macrophage transfection.4.8 Detection of ILF3 expression in peritoneal macrophages of Rosa26-ILF3 mice The expression levels of ILF3 in peritoneal macrophages of C57/B6 mice and Rosa26-ILF3 mice were detected by real-time quantitative PCR and Western Blot.The results showed that the level of ILF3 in Rosa26-ILF3 mice was significantly higher than that in C57 mice(P<0.01).4.9 ILF3 promotes ox-LDL-induced macrophage foamingC57/B6 mouse peritoneal macrophages were divided into Sh-NC group;ox-LDL stimulation + Sh-NC group;ox-LDL stimulation + Sh-ILF3 group;mRNA and protein expression levels of ABCA1,ABCG1,SRA1,LOX1,ACAT1 were detected in each group.The results showed that after ox-LDL stimulated macrophages,the lipid-transfer related proteins ABCA1,ABCG1 and lipid-transferred proteins LOX1 and ACAT1 were elevated,LOX1 and ACAT1 were statistically significant(P<0.05),after transfection of Sh-ILF3,ABCA1 and ABCG1 were further increased,but ABCG1 did not reach statistical significance,LOX1 and ACAT1 expression decreased(P<0.05).Oil red O staining was performed after stimulation of each group of cells.The results showed that ox-LDL stimulated macrophage and increased lipid deposition.After transfection of Sh-ILF3,lipid deposition was significantly reduced.It is shown that ILF3 has a role in promoting cholesterol deposition.4.10 ILF3 promotes ox-LDL-induced secretion of macrophage inflammatory factorsC57/B6 mouse peritoneal macrophages were divided into Sh-NC group;ox-LDL stimulation + Sh-NC group;ox-LDL stimulation + Sh-ILF3 group;mRNA and protein expression levels of IL-6,INOS,IL-10,ARG1 were detected in each group.The results showed that IL-6,INOS and IL-10 were increased after ox-LDL stimulation of macrophages.After transfection of Sh-ILF3,the expression of pro-inflammatory factors IL-6 and iNOS decreased(P<0.05),while anti-inflammatory factor ARG1 and IL-10 expression was further increased(P<0.05).It is proved that ILF3 has a role in promoting the secretion of inflammatory factors.Peritoneal macrophages were extracted from C57/B6 mice and Rosa26-ILF3 mice.After ox-LDL stimulation for 24 hours,oil red O staining was performed.The results showed that lipid deposition was significantly increased in the ILF3 overexpression group.Above results indicated that ILF3 promoted cholesterol deposition.effect.4.11 ILF3 promotes LPS-induced secretion of macrophage inflammatory factorsC57/B6 mouse peritoneal macrophages were divided into NC group,NC+LPS stimulation group and SI+LPS stimulation group.The mRNA levels of ILF3,Argl,iNOS,CD86,IL-6,TNF-α,IL-1β and IL-10 of each group were detected.The results showed that ILF3,iNOS,CD86,IL-6,TNF-α,IL-1β and IL-10 were increased after LPS stimulation of macrophages(P<0.05).After transfection of Sh-ILF3,proinflammatory cytokines of ILF3,iNOS,CD86,IL-6,TNF-a,IL-1β and IL-6 were decreased,while the expressions of anti-inflammatory factors ARG1 and IL-10 were further increased(P<0.05),which proved that ILF3 promoted the secretion of inflammatory factors.4.12 The role of ILF3 in the signaling pathwayC57/B6 mouse peritoneal macrophages were divided into Sh-NC group;ox-LDL stimulation + Sh-NC group;ox-LDL stimulation + Sh-ILF3 group;expression levels of NF-κB(p-p65)and p-AKT were detected in each group.The results showed that the expression of NF-κB(p-p65)and p-AKT was increased after ox-LDL stimulation of macrophages,and decreased after transfection with Sh-ILF3.This indicates that ILF3 can affect inflammatory factors and cholesterol deposition through the NF-κB signaling pathway and the AKT signaling pathway.5 Conclusion(1)In the APOE-/-mouse plaque vulnerability model,knockout of ILF3 by lentivirus can reduce the expression of inflammatory factors IL-6,TNF-a in plaque,thereby increasing plaque stability.(2)ILF3 could regulate macrophage inflammatory factor secretion and cholesterol deposition,which may be through NF-κB and AKT signaling pathways.更多还原