节点文献
IL-6与动脉粥样硬化斑块易损性的关系及影响胶原代谢的机制
Relationship between Interleukin 6 and Vunerableatherosclerotic Plaque and the Mechanism of Collagen
【作者】 冯民；
【导师】 张梅；
【作者基本信息】 山东大学 ， 内科学， 2009， 博士
【摘要】 ⅠIL-6与动脉粥样硬化斑块易损性及P4Hα1/MMP-14关系的研究背景动脉粥样硬化（Atherosclerosis,AS）斑块不稳定性导致的斑块破裂、血栓形成和血管阻塞是引起急性心脑血管事件的主要原因。易损斑块的典型特点是:薄的纤维帽,具有大的脂核,伴有胶原含量减少,平滑肌细胞密度降低,巨噬细胞密度及活性增加,活化T细胞浸润增加;血管外膜可见新生血管及炎症反应（巨噬细胞、T细胞、肥大细胞增多）。明确易损斑块的发生机制对于防止心脑血管事件的发生具有十分重要的理论及临床意义。因此,关于易损性斑块的研究是心血管病的研究热点之一。动脉粥样硬化斑块纤维帽的结构与斑块易损性关系较大。纤维帽的完整性和对破裂的抵抗力主要依赖于细胞外基质,其中最主要的结构是胶原纤维。纤维帽中胶原的含量主要由胶原合成与降解来调节的。斑块内胶原的代谢失调如胶原合成减少,降解增加均可导致斑块表面纤维帽变薄,斑块容易破裂。因此,明确斑块细胞外基质的代谢及调节机制对于稳定斑块具有重要意义。许多因素调节胶原的合成与降解,任何因素的失调均可引起胶原代谢的平衡紊乱。胶原合成过程受基因转录水平,翻译水平和翻译后修饰等调节,其中翻译后修饰有重要的作用。脯氨酸4羟化酶（Prolyl-4-hydroxylase,P4H）是重要的修饰酶,P4H酶的亚型P4Hα1是起作用的关键酶。P4Hα1在大多数组织包括平滑肌细胞、内皮细胞中表达。关于P4Hα1与斑块易损性的关系尚未见报道。基质金属蛋白酶中的基质金属蛋白酶-14（MMP-14）与心血管疾病的形成有关。MMP-14是一个有效的ECM降解酶,它负责许多种细胞外及细胞膜相关的物质的蛋白水解作用,包括胶原和其他ECM蛋白。炎症反应是导致动脉粥样硬化斑块破裂的主要因素之一。大量研究证实炎性细胞分泌大量细胞因子如白细胞介素6（interleukin 6,IL-6）、白细胞介素8（interleukin 8,IL-8）等可导致内皮细胞的活化、平滑肌细胞的增生调亡及粥样病变的进展,最终结果是导致纤维帽的胶原成分减少、变薄,易于破裂。IL-6是一个强的炎症因子,而有关炎性因子IL-6与动脉粥样硬化斑块的易损性及斑块内P4Hα1、MMP-14关系的研究未见报道。目的1.观察IL-6与动脉粥样硬化斑块易损性的关系。2.观察IL-6与易损斑块中胶原合成酶P4Hα1及胶原降解酶MMP-14的变化。方法1.易损斑块动物模型的构建通过颈动脉套管、限制性应激的方法构建AS易损斑块动物模型。80只10-12周龄雄性apoE一小鼠分为IL-6刺激组和对照组,每组40只,均给予高脂饮食（含0.25%胆固醇和15%脂肪）。给予实验动物施行右颈总动脉套管术以诱发AS病变。颈动脉套管手术4周后,将小鼠放在限制其活动的小笼子中,每次1小时,每天1次,持续8周。2.pLVX-AcGFP-N1-IL6重组病毒载体的构建在平滑肌细胞中提取RNA,逆转录成cDNA,然后合成DNA,电泳鉴定并纯化,回收目的片段,目的片段与Feasy载体连接,然后扩增、吸收目的片段,使目的片段与病毒载体（GFP）连接,再扩增、提取质粒,鉴定后脂质体转染293细胞,最后包装产毒,-80℃保存。3.将pLVX-AcGFP-N1-IL6转入动脉粥样硬化斑块:暴露动脉粥样硬化模型的局部颈动脉,然后将IL-6-HIV滴于局部,孵育20分钟,将局部缝合。对照组给予局部滴HIV载体。4.观察IL-6与动脉粥样硬化斑块易损指数、P4Hα1及MMP-14的关系4.1病理学检测:对颈动脉斑块进行H&E染色以观察斑块的形态结构,测量斑块面积和纤维帽的厚度,计算斑块的破裂率;对颈动脉斑块进行油红0染色以检测斑块内脂质含量;对颈动脉斑块进行天狼猩红染色以检测斑块内胶原含量。并通过免疫组织化学方法检测斑块内巨噬细胞（MOMA-2）、平滑肌肌动蛋白（a-actin）、P4Hα1及MMP-14的蛋白水平。检测斑块内胶原、脂质、巨噬细胞、血管平滑肌细胞的含量,计算易损指数:易损指数=（巨噬细胞+脂质）阳性面积百分比/（平滑肌细胞十胶原）阳性面积百分比。4.2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反应检测P4Hα1和MMP-14 mRNA的表达量。4.3 Western blot检测:取新鲜颈动脉斑块组织,提取蛋白质,Western blot检测斑块内P4Hαl和MMP-14蛋白质水平。结果1.pLVX-AcGFP-N1-IL6病毒载体的构建:制备载体编码的携带GFP和小鼠IL-6基因的质粒载体,转染HK293细胞,48小时后荧光显微镜下观察。通过基因重组技术构建pLVX-AcGFP-N1-IL6病毒载体,PCR反应鉴定,证实反应产物正确。2.pLVX-AcGFP-N1-IL6病毒载体体内转染:pLVX-AcGFP-N1-IL6病毒载体转染3天后颈动脉AS斑块内GFP的表达量为36.07%;转染14天后斑块内GFP的表达量减少18.81%。3.血液生化指标检测:IL-6组和对照组比较血清IL-6浓度增高（分别为81.37±10.79 pg/ml与60.27±9.54pg/ml,P=0.023）,差异有统计学意义,血清TC,TG,HDL-C,LDL-C差异无统计学意义。4.IL-6对易损动脉粥样硬化斑块的影响4.1病理学检测:IL-6组和对照组比较,斑块面积无显著性差异（分别为71000±8000 um2和82000±11000um2,P=0.073）,IL-6组纤维帽厚度明显减少（分别为6.73±0.96 um和7.25±1.35um,P=0.029）,帽/核比值明显减少（分别为0.06±0.02和0.09±0.02,P=0.045）,胶原含量明显减少（分别为11.23%±3.57%和16.34%±4.72%,P=0.005）,脂质含量明显增加（分别为21.23%±3.56%和12.25%±2.01%,P=0.0011）。IL-6组有1例斑块破裂并血栓形成,1例斑块内出血,1例斑块内埋藏的纤维帽,2例纤维帽中断;对照组有1例斑块内埋藏的纤维帽,1例纤维帽中断,未见血栓和出血现象。IL-6组和对照组比较,a-SMC-actin明显减少（分别为5.77%±1.09%和11.97%±1.92%,P=0.017）,巨噬细胞明显增加（分别为15.32%±3.01%和7.67%±2.87%,P=0.004）,IL-6明显增加（分别为12.32%±1.44%和6.89%±0.57%,P=0.021）,P4Hα1明显减少（分别为5.01%±0.46%和9.35%±1.24%,P=0.028）,MMP-14明显增加（分别为9.78%±0.98%和4.29%±0.31%,P=0.019）。与对照组比较,IL-6组易损指数明显增加（分别为2.83±1.02和0.81±0.43,P=0.009）。4.2实时定量RT-PCR检测与对照组相比,IL-6组颈动脉斑块组织中P4Hα1 mRNA的表达量减少69.3%,两组间统计学差异具有显著性意义（P=0.03）。与对照组相比,IL-6组颈动脉斑块组织中MMP-14 mRNA的表达量增加77.2%,两组间统计学差异具有显著性意义（P=0.028）。4.3免疫印迹（Westenr blot）检测:与对照组相比,IL-6组颈动脉斑块组织中P4Hα1蛋白的表达量减少61.9%,两组间存在显著的统计学差异（P＜0.01）;与对照组相比,IL-6组颈动脉斑块组织中MMP-14蛋白的表达量增加56.3%,两组间存在显著的统计学差异（P＜0.05）。结论1.体内实验结果表明,pLVX-AcGFP-N1-IL6显著增加斑块内巨噬细胞和脂质的含量,降低斑块内平滑肌细胞及胶原的表达,从而增加斑块的易损性。2.体内实验结果表明,pLVX-AcGFP-N1-IL6能抑制斑块局部P4Hα1 mRNA的表达和减少P4Hα1蛋白的含量;pLVX-AcGFP-N1-IL6能增强斑块局部MMP-14 mRNA的表达和增加MMP-14蛋白的含量。ⅡIL-6对P4Hα1表达的影响及信号传导通路的研究背景动脉粥样硬化易损斑块导致的斑块破裂和血栓形成是引起急性心脑血管事件的主要原因。薄的纤维帽,且伴有胶原含量减少是易损斑块的典型特点。动脉粥样硬化斑块纤维帽的完整性和高强度主要依赖于其细胞外基质的支持,其中最主要的成分是胶原,而纤维帽中胶原合成减少是导致易损斑块破裂的最主要原因。因此,研究胶原代谢和动脉粥样硬化斑块易损性的关系,明确动脉粥样硬化易损斑块的发生的分子生物学机制对于防止心脑血管事件的发生具有十分重要的理论及临床意义。胶原代谢平衡主要通过胶原合成与降解代谢来实现,而分布在动脉内膜的血管平滑肌细胞是动脉粥样斑块内胶原的主要来源。许多因素均可调节胶原的合成与降解代谢,其中任何因素的失调均可引起胶原代谢平衡的紊乱。胶原合成过程受基因转录水平,翻译水平和翻译后修饰等调节,其中翻译后修饰具有重要的作用。脯氨酸4羟化酶（P4H）是重要的修饰酶,是一组由21个胶原合成和形成需要的细胞内酶构成,其中起关键作用的是P4Hα1。P4Hα1酶的活性被抑制可产生不稳定胶原,并在细胞内降解,不能被分泌,从而减少胶原生成。有大量研究表明,P4Hα1酶活性的变化在胶原代谢中起到非常重要的作用,而多种细胞因子均可调节P4Hα1酶的活性。因此,本研究将P4Hα1作为与胶原合成有关的主要研究靶点。炎症反应是导致动脉粥样硬化易损斑块破裂主要因素之一。IL-6是公认的强烈的炎症因子,通过其膜受体上的信号传导蛋白gp130激活RAF-MAPK通路,并进一步激活激活蛋白-1（activator protein-1,AP-1）、核因子κB（nuclearfactor-κB,NF-κB）等转录因子,从而在转录水平调节多种蛋白质的表达,发挥其生物学作用。国内外学者对细胞因子与胶原降解酶MMPs的关系进行了大量的研究,但IL-6与影响胶原合成的关键酶P4Hα1的有关传导通路的研究尚未见报道。MAPKs是脊椎动物体内广泛存在的丝氨酸/苏氨酸蛋白调节激酶,是一组存在于胞浆的蛋白激酶,在一系列复杂的细胞程序中其重要作用,如细胞的增殖、分化、发育、转化和凋亡。哺乳动物中,MAPKs分为三个家族,细胞外信号调节激酶（extracellular-signal regulated proteinkinase,ERK）、c-jun氨基末端激酶（c-jun amino -terminal kinase,JNK）/应激激活蛋白激酶（stress-activated protein kinase,SAPK）、p38。MAPKs酶可被多种应激刺激活化,如LPS、肿瘤坏死因子（TNF）-α、白介素（IL）-1、渗透压改变以及紫外线辐射等。细胞被激活后,活化的MAPKs既能够磷酸化胞浆内的靶蛋白,又能够迅速进入细胞核内,作用于转录因子,从而调节基因的表达。但是,MAPKs通路是否介导IL-6对P4Hαl的影响尚不清楚。基于以上,我们提出如下假说:炎症因子IL-6通过MAPKs通路调节P4Hα1的表达水平,而激活的MAPKs通路是通过调节转录因子活性,在转录水平调节了P4Hα1的表达,从而影响易损斑块的胶原代谢,导致易损斑块的破裂。本研究针对这一假说进行研究。目的1.观察在HASMCs中IL-6对P4Hα1 mRNA和蛋白质表达的影响。2.明确在HASMCs中IL-6是否通过RAF-MEK-ERK1/2-AP-1通路影响P4Hαl的表达,并进一步研究其分子生物学机制。方法1.平滑肌细胞的培养:人主动脉平滑肌细胞（HASMCs,human aortic smooth muscle cells）购CELLSCIENCE公司,使用CELLSCIENCE公司的平滑肌培养基,在37℃和5%CO2孵育箱中进行培养。前4代冻存于液氮罐中保存,5-8代行细胞试验。2.观察不同剂量IL-6引起P4Hal的mRNA和蛋白质水平变化:浓度试验中以0、10、20、50、100ng/ml浓度刺激平滑肌细胞24h,然后检测P4Hα1的mRNA表达量和蛋白质的水平。观察使P4Hα1的mRNA和蛋白质水平变化最大的浓度。3.IL-6刺激HASMCs,观察IL-6不同时间引起P4Hα1的变化:以20ng/ml的IL-6在不同时间点刺激平滑肌细胞,分别在刺激0、6、12、24、48h后收集细胞,通过RT-PCR和Western blot分别检测平滑肌细胞中P4Hα1的mRNA表达量和蛋白质的水平,观察使P4Hα1变化最大的时间点。4.用AP-1siRNA干扰IL-6引起的P4Hα1变化:设立对照组,AP-1siRNA组,IL-6组,IL-6+AP-1siRNA组。经Western blot检测,与对照组比较,观察AP-1siRNA对IL-6引起的P4Hα1的影响。5.用NF-κBsiRNA干扰IL-6引起的P4Hα1变化:设立对照组,NF-κBsiRNA组,IL-6组,IL-6+NF-κBsiRNA组。经Westernblot检测,与对照组比较,观察NF-κBsiRNA对IL-6引起的P4Hα1的影响。6.加入ERK1/2抑制剂PD98059以观察IL-6诱导的平滑肌细胞P4Hα1的蛋白表达:将细胞分为4组,分别为:对照组,PD98059组,IL-6组和PD98059+IL-6组。与对照组相比较,观察抑制剂PD98059对P4Hα1表达的影响。7.加入ERK1/2抑制剂PD98059以观察IL-6诱导的平滑肌细胞AP-1蛋白质水平:将细胞分为4组,分别为:对照组,PD98059组,IL-6组和PD98059+IL-6组。与对照组相比较,观察抑制剂PD98059对AP-1蛋白质水平的影响。8.加入JNK抑制剂SP600125以观察IL-6诱导的平滑肌细胞P4Hα1蛋白质水平:将细胞分为4组,分别为:对照组,SP600125组,IL-6组和SP600125+IL-6组。与对照组相比较,观察抑制剂SP600125对P4Hα1蛋白质水平的影响。9.加入P38抑制剂SB203580以观察IL-6诱导的平滑肌细胞P4Hα1蛋白质水平:将细胞分为4组,分别为:对照组,SB203580组,IL-6组和SB203580+IL-6组。与对照组相比较,观察抑制剂SB203580对P4Hα1蛋白质水平的影响。10.IL-6对HASMCs的RAF、MEK和ERK1/2蛋白质水平的影响:以20ng/ml IL-6刺激平滑肌细胞24小时后,收集细胞,通过Westernblot分别测定细胞RAF、MEK和ERK1/2的蛋白水平,然后与对照组比较。结果1.浓度试验:以不同浓度的IL-6（0、10、20、50、100ng/ml）分别刺激平滑肌细胞24h后,检测P4Hα1 mRNA和蛋白质水平的表达,结果提示20ng/ml的IL-6抑制P4Hα1表达达到高峰。2.时间试验:以20ng/ml的IL-6在不同时间点刺激平滑肌细胞,分别在刺激0、6、12、24、48h后收集细胞,通过RT-PCR和Western Blot检测平滑肌细胞中P4Hα1 mRNA和蛋白质的表达水平,结果显示为IL-6刺激24h后的P4Hα1浓度最低。3.AP-1siRNA对IL-6引起的P4Hα1变化的影响:设立对照组,AP-1siRNA组,IL-6组,IL-6+AP-1siRNA组。经Western blot检测,与对照组比较,AP-1siRNA组的P4Hα1表达轻度下降,但两组差异无统计学意义（P＞0.05）,IL-6组和IL-6+AP-1siRNA组P4Hα1均明显下降,且IL-6+AP-1siRNA组P4Hα1的表达较IL-6组明显增高。4.NF-κBsiRNA对IL-6引起的P4Hα1变化的影响:设立对照组,NF-κBsiRNA组,IL-6组,IL-6+NF-κBsiRNA组。经Westernblot检测,与对照组比较,NF-κBsiRNA组的P4Hα1表达无变化,IL-6组和IL-6+NF-κBsiRNA组P4Hα1均明显下降,且此两组比较差异无统计学意义（P＞0.05）。5.ERK1/2抑制剂PD98059对IL-6诱导的平滑肌细胞P4Hα1蛋白表达的影响:将细胞分为4组,分别为:对照组,PD98059组,IL-6组和PD98059+IL-6组。与对照组相比较,PD98059组P4Hα1表达无明显变化,IL-6组和PD98059+IL-6组P4Hα1表达均明显降低。与IL-6组比较,PD98059+IL-6组P4Hα1表达明显增加。6.ERK1/2抑制剂PD98059对IL-6诱导的平滑肌细胞AP-1蛋白表达的影响:将细胞分为4组,分别为:对照组,PD98059组,IL-6组和PD98059+IL-6组。与对照组相比较,PD98059组AP-1表达无明显变化,IL-6组和PD98059+IL-6组AP-1表达均明显增高。与IL-6组比较,PD98059+IL-6组AP-1表达明显减低。7.p38抑制剂SB203580对IL-6诱导的平滑肌细胞P4Hα1蛋白表达的影响:将细胞分为4组,分别为:对照组,SB203580组,IL-6组和SB203580+IL-6组。与对照组相比较,SB203580组P4Hα1表达无明显变化,IL-6组和SB203580+IL-6组P4Hα1表达均明显降低。与IL-6组比较,PD98059+IL-6组P4Hα1表达无明显变化。8.JNK抑制剂SP600125对IL-6诱导的平滑肌细胞P4Hα1蛋白表达的影响:将细胞分为4组,分别为:对照组,SP600125组,IL-6组和SP600125+IL-6组。与对照组相比较,SP600125组P4Hα1表达无明显变化,IL-6组和SP600125+IL-6组P4Hα1表达均明显降低。与IL-6组比较,SP600125+IL-6组P4Hα1表达无明显变化。9.IL-6对HASMCs的RAF、MEK、ERK1/2蛋白表达的影响:以20ug/ml IL-6刺激平滑肌细胞24小时后,收集细胞,通过western blot分别测定细胞RAF,MEK、ERK1/2的蛋白水平,结果发现,与对照组比较,IL-6刺激后血管平滑肌细胞的RAF,MEK、ERK1/2的蛋白水平均较对照组显著增高。结论1.IL-6刺激HASMCs引起P4Hα1蛋白质及mRNA水平降低,且20ng/ml IL-6刺激24h达到最佳效果。2.IL-6可通过RAF-MEK-ERR1/2-AP-1途径下调P4Hα1的表达,进一步明确了IL-6影响胶原合成的机制。3.在HASMCs中,JNK、p38和NF-κB不参与IL-6下调P4Hα1的表达过程。ⅢIL-6对MMP-14表达的影响及信号传导通路的研究背景急性心脑血管事件的发生和发展很大程度上受到AS斑块易损性的影响。研究发现典型的易损斑块的主要特点是较大的脂核和较薄的纤维帽。其中斑块纤维帽的厚薄主要由细胞外基质和血管胶原的代谢平衡所决定的。细胞外基质是人体组织结构的重要成分,细胞外基质产生与降解的平衡是维持组织正常结构的必要条件。在AS血管组织中,细胞外基质的增多与减少都可以直接造成血管壁的病变,如较少的细胞外基质会增加动脉粥样硬化斑块的易损性,而过多的细胞外基质会使动脉粥样硬化斑块增厚,从而使管腔狭窄和阻塞。胶原是构成血管细胞外基质的主要成分,胶原的降解既可以引起斑块纤维帽变薄,增加斑块的易损性,又可引起动脉瘤的产生,从而增加急性心脑血管事件的发生率。因此斑块内细胞外基质,特别是胶原的代谢平衡是近年来研究的热点。炎症反应是导致动脉粥样硬化斑块破裂主要因素之一,MMPs也参与了动脉粥样硬化斑块的这一重塑过程。有研究表明,基质金属蛋白酶中的基质金属蛋白酶-14（MMP-14）与心血管疾病的形成有密切关系。且近年的研究表明,多种致动脉粥样硬化因子可以促进MMP-14的表达,而MMP-14缺乏小鼠斑块内的胶原含量明显高于野生型对照。这些研究表明,MMP-14是影响斑块稳定性的一个重要因素。国内外对细胞因子与MMPs之间的相互关系进行了大量的研究,但尚未有对细胞因子与MMP-14在胶原代谢中的分子生物学机制展开系统研究。MMP-14是一个有效的ECM降解酶,它可以水解多种细胞外及细胞膜相关物质的蛋白,包括胶原和其他ECM蛋白,它与血管疾病的发生和发展过程有关。然而,MMP-14参与动脉粥样硬化斑块重塑过程的具体机制尚不明确。新近研究表明,细胞外信号调节激酶（extracellular-signal regulatedproteinkinase,ERK）酶促级联反应在胶原代谢的调节方面具有重要的作用。ERK分为ERK1和ERK2（分别为p44和p42）两个亚型,主要参与细胞内增生、转化及分化的信号转导。在静止的细胞中,ERK1/2主要存在于胞浆中,细胞被激活后,活化的ERK1/2既能够磷酸化胞浆内的靶蛋白,又能够迅速进入细胞核,作用于转录因子,调节基因的表达。激活蛋白-1（AP-1）是生物体内最为重要的转录因子之一,AP-1对多种基因的转录活性有重要的作用,AP-1可以被PKC和各种细胞因子激活,这些细胞因子包括TNFα和白介素6,激活是通过几种蛋白酪氨酸激酶（PTK）和细胞外信号调节激酶（MAPK）,这些激酶可以激活一系列细胞内激酶从而引起其下游底物的变化。转录因子能使基因转录增加或减少,基因转录的多少将导致蛋白合成的多少和改变细胞的功能。已有研究提示,AP-1参与了胶原代谢的调节。但是,AP-1是否为MMP-14的调节因子,以及ERK-MAPK通路对AP-1的转录活性的影响尚不清楚。针对以上问题,我们提出如下假说:IL-6能够通过ERK-MAPK通路上调MMP-14的表达,而AP-1可能作为转录因子参与了该过程的调节。ERK-MAPK-AP-1通路可能是动脉粥样硬硬化斑块的重塑过程的重要机制之一。目的1.阐明在HASMCs中IL-6对MMP-14 mRNA、蛋白质水平的影响。2.阐明在HASMCs中IL-6通过RAF-MEK-ERK1/2-AP-1途径上调MMP-14表达的机制。方法1.平滑肌细胞的培养:购买HASMCs（CELLSCIENCE）细胞株,细胞于培养箱中培养,培养箱条件为37℃,5%CO2。培养基为购自CELLSCIENCE公司的平滑肌培养基。前4代冻存于液氮罐中保存,5-8代行细胞试验。2.IL-6刺激HASMCs,观察IL-6不同剂量引起的MMP-14的变化:浓度试验中以0、5、10、20、50ng/ml浓度刺激平滑肌细胞12h,通过RT-PCR和Western Blot检测平滑肌细胞中MMP-14的mRNA和蛋白表达水平。3.IL-6刺激HASMC,观察IL-6刺激不同时间MMP-14的变化:以10ng/ml的IL-6在不同时间点刺激平滑肌细胞,分别在刺激0、6、12、24h后收集细胞,通过RT-PCR和Western Blot检测平滑肌细胞中MMP-14的mRNA和蛋白表达水平。4.用AP-1siRNA干预IL-6引起的MMP-14变化:设立对照组,siRNA组,IL-6组,IL-6+siRNA组。经Western blot检测,与对照组比较,观察AP-1siRNA对MMP-14的影响。5.加入ERK1/2抑制剂PD98059以观察IL-6诱导的平滑肌细胞MMP-14的蛋白表达:将细胞分为4组,分别为:对照组,PD98059组,IL-6组和PD98059+IL-6组。与对照组相比较,观察抑制剂PD98059对MMP-14的表达的影响。6.加入ERK1/2抑制剂PD98059以观察IL-6诱导的平滑肌细胞AP-1的蛋白表达:将细胞分为4组,分别为:对照组,PD98059组,IL-6组和PD98059+IL-6组。与对照组相比较,观察抑制剂PD98059对AP-1的表达的影响。7.IL-6对HASMCs的RAF、MEK和ERK1/2表达的影响:以10ng/ml IL-6刺激平滑肌细胞12小时后,收集细胞,通过Westernblot分别测定细胞RAF、MEK和ERK1/2的蛋白水平,然后与对照组比较。结果1.浓度-效应关系:应用不同浓度（0、5、10、20、50ng/ml）的IL-6刺激平滑肌细胞12小时,通过RT-PCR和Western Blot检测平滑肌细胞中MMP-14的mRNA和蛋白质的表达水平,结果提示10ng/ml的IL-6上调MMP-14表达达到高峰。2.时间-效应关系:以10ng/ml的IL-6在不同时间点刺激平滑肌细胞,分别在刺激0、6、12、24h后收集细胞,通过RT-PCR和Western Blot检测平滑肌细胞中MMP-14的mRNA和蛋白表达水平,结果显示刺激12小时平滑肌细胞中MMP-14的mRNA和蛋白质表达水平最高。3.AP-1siRNA对IL-6引起的MMP-14变化的影响:本实验分为:对照组,AP-1siRNA组,IL-6组,IL-6+AP-1siRNA组。经Westernblot检测,与对照组比较,AP-1siRNA组的MMP-14表达轻度增加,但差异无统计学意义,IL-6组和IL-6+AP-1siRNA组MMP-14均明显增加,且IL-6+AP-1siRNA组的MMP-14表达较IL-6组明显减少。4.ERK1/2抑制剂PD98059对IL-6诱导的平滑肌细胞MMP-14表达的影响:将细胞分为4组,分别为:对照组,PD98059组,IL-6组和PD98059+IL-6组。与对照组相比较,PD98059组MMP-14表达无显著性差异,IL-6组和PD98059+IL-6组MMP-14表达均明显升高。与IL-6组比较,PD98059+IL-6组MMP-14表达明显降低。5.ERK1/2抑制剂PD98059对IL-6诱导的平滑肌细胞AP-1表达的影响:将细胞分为4组,分别为:对照组,PD98059组,IL-6组和PD98059+IL-6组。与对照组相比较,PD98059组AP-1表达无明显变化,IL-6组和PD98059+IL-6组AP-1表达均明显升高。与IL-6组比较,PD98059+IL-6组AP-1表达明显降低。6.IL-6对HASMCs的RAF、MEK、ERK1/2表达的影响:以10ng/ml IL-6刺激平滑肌细胞12小时后,收集细胞,通过western blot分别测定细胞RAF,MEK、ERK1/2的蛋白水平,结果发现,与对照组比较,IL-6刺激后血管平滑肌细胞的RAF、MEK、ERK1/2的蛋白质表达水平均较对照组显著增加。结论1.IL-6刺激HASMCs后,能够增加MMP-14的蛋白质及mRNA表达水平,且10ng/mlIL刺激12小时达最佳效果。2.在HASMCs中,IL-6通过RAF-MEK-ERK1/2-AP-1途径上调MMP-14的表达,进一步明确了IL-6促进胶原降解的部分机制。
【Abstract】 Ⅰstudy of the relationship between IL-6 and vulnerability of vulnerable plaque and P4Hα1/MMP-14BackgroundAtherosclerosis,which causes ischemic cardiopathy and stroke,is the most common cause of mortality and morbidity in developed countries.Atherosclerotic disease of arteries leading to formation of the atherosclerotic plaque involves modulation of the normal functioning of the vessel wall endothelium,the entrapment of low-density lipoprotein(LDL) within the vessel wall and itsoxidative modification, the migration into the wall of monocytes,their conversion to activated macrophages by oxidized LDL and the release from macrophages of cytokines and proteases and the uptake of lipid by macrophages in the vessel wall to form the "foam" cells characteristic of the early lesion,the "fatty streak." The most dangerous plaque is rupture -prone plaques,also named vulnerable plaque which are characterized by possessing an easily disrupted/fissured and occlusive thrombus have a large lipid core and a thin fibromuscular cap accompanied by many macrophages and other inflammatory cells on or beneath the cap surface.Finding the mechanism of the formation of vulnerable plaque is a hot study recently years.In atherosclerotic arteries collagen is crucial for plaque stability and its removal from the plaque’s fibrous cap area may result in plaque rupture.Since collagen plays key roles in plaque stability and cell migration properties,a comprehensive understanding of collagen expression and organization during the progression of atherosclerosis is essential.Inflammation directly contributes to the pathogenesis of most cardiovascular diseases,including heart failure,cardiomyopathy,atherosclerotic plaque rupture,and aortic aneurysm and degradation of extracellular matrix. Extracellular matrix(ECM) of the vascular is a major target of inflammatory cytokines.The ECM,which includes fibrillar(collagen and elastin) and adhesive proteins(laminin and fibronectin),serves as the structural framework for all 3 layers of the arterial wall.Of the more than subtypes of collagens,typesⅠandⅢare the most common in thearterial wall IL-6(a 26 kDa cytokine),produced by lymphocytes, monocytes,fibroblasts,vascular smooth muscle cells,and endothelial cells has been identified as a local and circulating marker of coronary plaque inflammation.IL-6 is a pleiotropic cytokine and its role in the modulation of inflammation-related processes, particularly cytokine responses and tissue inflammatory cell infiltration.IL-6 deficiency was reported to result in an enhanced formation of atherosclerotic lesions, reduced collagen metabolism,and elevated levels of serum cholesterolin an ApoE-/-model of atherosclerosisCollagen synthesis and secretion require posttranslational modifications of procollagens,including hydroxylation and glycosylation.Prolyl-4-hydroxylase(P4H), an essential enzyme involved in collagen modification,catalyzes the formation of hydroxyproline from proline residues locatedin repeating X-Pro-Gly triplets in the procollagens.This step is required for folding the polypeptide chains into stable triple-helical molecules.Therefore,adequate P4H expression is essential for the formation of functional collagen in the arterial wall and pathogenesis of vascular diseases.Some studies have examined inflammatory cytokines,such as TNF alpha,inducing suppression of prolyl-4 hydroxylase alphal(P4Halphal),the rate-limiting isoform of P4H responsible for procollagen hydroxylation,maturation,and organization.In the present study,we aim to find the effection of IL-6 to vulnerable plaque and the interaction between IL-6 and P4Halphal in the plaque.Objectives 1.Elucidate the interaction between MMP-14 and P4Halphal and the corresponding mechanisms during the process of the Atherosclerosis.2.Elucidate the effects of IL-6 on vulnerable plaque and the corresponding mechanisms.3.Elucidate the interaction between IL-6 and P4Halphal in the vulnerable plaque and the corresponding mechanisms.4.Elucidate the interaction between IL-6 and MMP-14 in the vulnerable plaque and the corresponding mechanisms.Methods1.Animal modelAnimal stress and received a model with AS vulnerable plaque was established LPS stimulation.Eighty male apoE-/- mice were divided into two groups,with Westem-type diet(0.25%cholesterol and 15%by combination mental 12 weeks of age,were cocoa butter) from the beginning of the study.Carotid atherosclerotic lesions were induced perivascular constrictive collars placement on the left common carotid arteries.Four weeks after surgery,mice were put into boxes independly to limited these actions,one hour a day,one day a week.2.Establishment of pLVX-AcGFP-N1-IL6 virus vectorSeprate RNA from smooth muscle cells,througeh reverse transcripting it into cDNA to synthetize DNA.Obtain the target segment by electrophoresis identification. Make combination of target segment and GFP.3.pLVX-AcGFP-N1-IL6 virus vector transfection into the vulnerable plaque4.1 Histological and Morphology AnalysesSections were stained with hematoxylin and eosin(H&E),Masson’s trichrome,sirius red staining,Verhoeff staining,oil red O staining,Perl’s staining. Immunostaining were performed for detecting MOMA-2,a-actin,P4Hα1、IL-6 expression in the vulnerable plaque.Plaque area,vessel area and cap thickness were measured.Smooth muscle cell,macrophage,lipid and collagen positive areas werequantified and the ratios correlated to the entimal areas were calculated.Plaque rupture rate and vulnerable index were calculated.4.2.Real-Time RT PCRThe mRNA levels of P4Hα1 mRNA in the fresh carotid lesions.were quantified by real-time reverse-transcriptase polymerase chain reaction(RT-PCR) using SYBR Green technology.4.3.Western-blotWestern-blot was performed for examining P4Hα1 protein expression in the fresh carotid lesions.Results1.Establishment of pLVX-AcGFP-N1-IL6 virus vectorPlasmid of pLVX-AcGFP-N1-IL6 virus vector,which binded the the sequence of GFP and IL-6 gene of mouse,was transfected into HK293 cells.Forty-eight hours later,pLVX-AcGFP-N1-IL6 virus vector was successfully constructed confirmed by PCR reaction.2.pLVX-AcGFP-N1-IL6 virus vector transfection in the vulnerable plaqueThree days after transfection,expression of green fluresence protein was 36.07%in plaque,and decreased to 18.81%fourteen days after transfection.3.Blood lipid profileNo statistic differences of serum lipid profiles were found brtween IL-6 group and control.4.1 Histological and Morphology AnalysesAtherosclerotic plaques of 10 animals of each group were analyzed for histology. No statistic difference of plaque area was found brtween IL-6 group and control,(71000±8000 vs 82000±11000um~2,P=0.073).Compared with control,mean fibrous cap thickness(6.73±0.96 vs 7.25±1.35um,P=0.029),cap/core ratio(0.06±0.02 vs 0.09±0.02,P=0.045) and collagen(11.23%±3.57%vs 16.34%±4.72%,P=0.005) were significantly decreased,and lipid content was significantly increased (21.23%±3.56%vs 12.25%±2.01%,P=0.0011) in IL-6group.Plaque rupture rates were 20%,significantly higher in IL-6 group(P=0.017).Immunostainning results showed that there more macrophages in the IL-6 group(15.32%±3.01%vs 7.67%±2.87%,P=0.004) than those in control.IL-6 expression increased by 72.33%in IL-6group compared with that in control(12.54%±2.76%vs63%±1.18%,P＜0.005).In addition,P4Hα1 expression were all significantly decreased in IL-6 group than those in control(all P＜0.05).Vulnerable index of IL-6 group was statistically higher than that of control(2.83±1.02 vs 0.81±0.43,P=0.009).4.2 Real time RT PCRThe mRNA expression of P4Hα1 in the vulnerable carotid plaques decreased 69.3%compared with that in controls(P=0.03).4.3 Westem-blot analysisThe proteins expression of P4Hα1 in the vulnerable carotid plaques decreased 61.9%compared with that in controls(P＜0.001).Conclusions1.pLVX-AcGFP-N1-IL6 virus vector was successfully established.2.In vivo,pLVX-AcGFP-N1-IL6 virus vector can reduced P4Hα1 expression in some reagion of vulnerable plaques,increased lipid content and the number of macrophage,decreased the expression of mooth muscle cells and collagens significantly.So IL-6 validated to increase the vulnerability of plaques markably.3.In vivo,pLVX-AcGFP-N1-IL6 virus vector can reduce P4Hα1 expression and increase MMP-14 expression in some reagion of vulnerable plaques. ⅡInterleukin-6 Decreases Proly-4-hydroxylaseα1 Expression via the Raf-Mek-Erk1/2-Ap-1 pathwayBackgroundInflammation plays a pivotal role in the pathogenesis of cardiovascular diseases, including heart failure and atherosclerosis.An advanced atherosclerotic lesion typically has a fibrous cap,which prevents the necrotic core from having direct contact with flowing blood.In the event of cap rupture,thrombosis occurs and causes acute coronary syndrome.Arterial collagen is the main extracellular matrix(ECM) constituent of the fibrous cap.Arterial interstitial collagen confers tensile strength on the fibrous cap and thus determines plaque stability and vulnerability to ruputure. Synthesis of collagen in the fibrous cap therefore may be directly responsible for the plaque rupture.ECM is the structural framework of all tissues,in which fibrillar proteins (collagen and elastin) and adhesive proteins(eg,laminin and fibronectin) form the structural backbone.In the arterial wall,various cells including smooth muscle cells, endothelial cells,fibroblasts and certain inflammatory cells,are the major cell source regulating ECM metabolism.Collagen,as one of the most metabolically active ECM components,has at least 39 subtypes.TypeⅠandⅢcollagens are most commonly found in the arterial wall.Although ECM degradation is important for balanced ECM metabolism,ECM synthesis,mainly modified by Prolyl-4-hydroxylaseα1(P4Hα1),is also crucial.Considerable evidence suggests that P4Hα1 is involved in plaque remodeling.P4Hα1 is one of the key intracellular enzymes required for the synthesis of all known types of collagens.It is essential for folding the procollagen polypeptide chains into stable triple helical molecules.Inhibition of P4Hα1produces unstable collagen associated with collagen decrease.P4Hα1is regulated by various cytokines, such as TNF-A and cigarette smoking.Among many regulatory factors of ECM,cytokines such as interleukin 6(IL-6) may directly inhibit P4Hα1 and therefore participate in ECM metabolism.IL-6,a 26-kDa protein,is released by T cells,tumour-associated macrophages and fibroblasts, especially from activated CD4+T cells.It is one of the most crucial cytokines involved in cardiovascular pathogenesis and actively regulates ECM metabolism.IL-6 acts through a membrane receptor composed of two subunits:anαchain,the IL-6 binding protein gp80;and aβchain,the signal-transducing protein gp130. Subsequently,gp130 can activate the Raf-dependent mitogen-activated protein kinase (Raf-MAPK) pathway,which leads to the activation of transcription factors such as AP-1 and NF-IL-6 The relation between IL-6 and P4Hα1 activity is unclear.In the current study,we investigated the effect of IL-6 on P4Hα1 expression and the underlying molecular mechanisms.IL-6 reduced P4Hα1 expression at the RNA and protein levels,with the RAF-MEK-ERK1/2 pathway involved in the process. Furthermore,we also showed that AP-1 was involved in the IL-6-mediated downregulation.Our study describes the molecular pathway responsible for IL-6-induced ECM disturbance in cardiovascular disease.Objectives1.Describes the effect of IL-6 on P4Hαexpression during the process of HASMCs.2.To investigate if IL-6 reduced P4Hαexpression through the molecular pathway RAF-MEK-ERK1/2-AP-1 during the process of HASMCs,and to elucidate the corresponding mechanisms.Methods1.Cell CultureHuman aortic smooth muscle cells(HASMCs) were obtained from ScienCell (Carlsbad,CA) and cultured in smooth-muscle-cell culture medium(Cat#311,Cell Application,San Diego,CA) containing 10%fetal bovine serum.Cells were cultured up to passage 4 before the experiments were conducted.2.Different Dose to P4Hα1 ExpressionHASMCs were treated with 0,10,20,50,and 100 ng/mL recombinant human IL-6(Cat#206-IL/CF,R&D Systems,Minneapolis,MN) for 24 hr before being harvested for measurement of target gene mRNA and protein levels.And find the expression of P4Hα1 was mostly induced at the level of 20ng/ml IL-6.3.different time point to P4Hα1 ExpressionMeasurement of target gene mRNA and protein levels were harvested at the time point of 0、6、12、24、48h independtly with 20ng/ml IL-6 treating the HASMCs).And find that the expression of P4Hα1 was mostly induced at time point of 24 hr.4.the effect of AP-1siRNAFour groups were detected as control group,AP-1siRNA group,IL-6 group, IL-6+ AP-1siRNA group with Western blot.To investigate the effect of AP-1siRNA.5.the effect of NF-κBsiRNAFour groups were detected as control group,NF-κBsiRNA group,IL-6 group,IL-6+ NF-κBsiRNA group with Western blot.To investigate the effect of NF-κBsiRNA6.PD98059,the inhibiter of ERK1/2 to P4Hα1All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.To investigate the effect of PD98059.7.PD98059,the inhibiter of ERK1/2 to AP-1All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.To investigate the effect of PD98059.8.SB203580,the inhibiter of p38All the cells were devided into four groups as control group,SB203580 group,IL-6 group,IL-6+ SB203580 group.To investigate the effect of SB203580.9.SP600125,the inhibiter of JNKAll the cells were devided into four groups as control group,SP600125 group,IL-6 group,IL-6+ SP600125 group.To investigate the effect of SP600125.10.Effect of IL-6 to expression of protein RAF,MEK,ERK1/2 of HASMCsTo find compared with the control group,effection of IL-6 group to expression of protein RAF,MEK,ERK1/2 of HASMCs.Results1.At different dose of IL-6HASMCs were treated with 0,10,20,50,and 100 ng/mL recombinant human IL-6(Cat#206-IL/CF,R&D Systems,Minneapolis,MN) for 24 hr before being harvested for measurement of target gene mRNA and protein levels.And find the expression of P4Hα1 was mostly induced at the level of 20ng/ml IL-6.2.At different point of timeMeasurement of target gene mRNA and protein levels were harvested at the time point of 0、6、12、24、48h independtly with 20ng/ml IL-6 treating the HASMCs).And find that the expression of P4Hα1 was mostly induced at time point of 24 hr.3.the effect of AP-1siRNAFour groups were detected as control group,AP-1siRNA group,IL-6 group, IL-6+ AP-1siRNA group with Western blot.Find that compared with the control group,protein levels of P4Hα1 in AP-1siRNA group decreased slightly but no significant,IL-6 group,IL-6+ AP-1siRNA group decreased significantly,and IL-6+ AP-1siRNA group is superior than IL-6 group.4.the effect of NF-κBsiRNAFour groups were detected as control group,NF-κBsiRNA group,IL-6 group,IL-6+ NF-κBsiRNA group with Western blot.Find that compared with the control group,protein levels of P4Hα1 in NF-κBsiRNA group have no changes, IL-6 group,IL-6+ NF-κBsiRNA group decreased significantly,and the two groups have no diffenence.5.PD98059,the inhibiter of ERK1/2 to P4Hα1All the cells were devided into four groups as control group,PD98059 group,IL-6 group,IL-6+ PD98059 group.Find that compared with the control group,protein levels of P4Hα1 in PD98059 group have no changes, IL-6 group,IL-6+ PD98059 group decreased significantly.IL-6 group is superior than IL-6+ PD98059 group.6.PD98059,the inhibiter of ERK1/2 to AP-1All the cells were devided into four groups as control group,PD98059 group,IL-6 group,IL-6+ PD98059 group.Find that compared with the control group,protein levels of AP-1 in PD98059 group have no changes, IL-6 group,IL-6+ PD98059 group increased significantly.IL-6 group is superior than IL-6+ PD98059 group.7.SB203580,the inhibiter of p38All the cells were devided into four groups as control group,SB203580 group,IL-6 group,IL-6+ SB203580 group.Find that compared with the control group,protein levels of P4Hα1 in SB203580 group have no changes,IL-6 group, IL-6+ SB203580 group decreased significantly,and the two groups have no diffenence.8.SP600125,the inhibiter of JNKAll the cells were devided into four groups as control group,SP600125 group,IL-6 group,IL-6+ SP600125 group.Find that compared with the control group,protein levels of P4Hα1 in SP600125 group have no changes, IL-6 group,IL-6+ SP600125 group decreased significantly,and the two groups have no diffenence.9.Effect of IL-6 to expression of protein RAF,MEK,ERK1/2 of HASMCsFind that compared with the control group,I protein levels of P4Hα1 in L-6 group increased significantly. Conclusions1.IL-6 decrease P4Hαexpression at the level of mRNA and protein through the process of HASMCs.2.RAF-MEK-ERK1/2-AP-1 pathway responsible for IL-6 reduced P4Hα1 expression during the process of HASMCs.3.JNK,p38 and NF-κB have nothing to do with the process of IL-6 reducing P4Hα1 expression ⅢInterleukin-6 Enhances Matrix Metalloproteinase-14 Expression via the Raf-Mek-Erk1/2-Ap-1 pathwayBackgroundInflammation plays a pivotal role in the pathogenesis of cardiovascular diseases, including heart failure and atherosclerosis.An advanced atherosclerotic lesion typically has a fibrous cap,which prevents the necrotic core from having direct contact with flowing blood.In the event of cap rupture,thrombosis occurs and causes acute coronary syndrome.Arterial collagen is the main extracellular matrix(ECM) constituent of the fibrous cap.Arterial interstitial collagen confers tensile strength on the fibrous cap and thus determines plaque stability and vulnerability to ruputure. Synthesis of collagen in the fibrous cap therefore may be directly responsible for the plaque rupture.ECM is the structural framework of all tissues,in which fibrillar proteins(collagen and elastin) and adhesive proteins(eg,laminin and fibronectin) form the structural backbone.In the arterial wall,various cells including smooth muscle cells,endothelial cells,fibroblasts and certain inflammatory cells,are the major cell source regulating ECM metabolism.Collagen,as one of the most metabolically active ECM components, has at least 39 subtypes.TypeⅠandⅢcollagens are most commonly found in the arterial wall.Although ECM synthesis is important for balanced ECM metabolism, ECM degradation,mainly catalyzed by matrix metalloproteinases(MMPs),is also crucial.Considerable evidence suggests that MMPs are involved in plaque remodeling. As one of the MMP family proteins,MT1-matrix metalloproteinase(MMP-14) is associated with the pathogenesis of cardiovascular disease.MMP-14 is an efficient ECM-degrading enzyme and is responsible for the proteolytic cleavage of multiple pericellular and membrane-associated substrates,including collagen and other ECM proteins,and is implicated in the development of vascular diseases.Among many regulatory factors of ECM,cytokines such as interleukin 6(IL-6) may directly inhibit P4Hα1 and therefore participate in ECM metabolism.IL-6,a 26-kDa protein,is released by T cells,tumour-associated macrophages and fibroblasts, especially from activated CD4+ T cells.It is one of the most crucial cytokines involved in cardiovascular pathogenesis and actively regulates ECM metabolism.IL-6 acts through a membrane receptor composed of two subunits:anαchain,the IL-6 binding protein gp80;and aβchain,the signal-transducing protein gp130. Subsequently,gp130 can activate the Raf-dependent mitogen-activated protein kinase (Raf-MAPK) pathway,which leads to the activation of transcription factors such as AP-1 and NF-IL-6 The relation between IL-6 and MMP-14 activity is unclear.In the current study,we investigated the effect of IL-6 on MMP-14 expression and the underlying molecular mechanisms.IL-6 enhanced MMP-14 expression at the RNA and protein levels,with the RAF-MEK-ERK1/2 pathway involved in the process.Furthermore,we also showed that AP-1 was involved in the IL-6-mediated upregulation.Our study describes the molecular pathway responsible for IL-6-induced ECM disturbance in cardiovascular disease.Objectives1.Describes the effect of IL-6 on MMP-14expression during the process of HASMCs.2.To investigate if IL-6 reduced MMP-14expression through the molecular pathway RAF-MEK-ERK1/2-AP-1 during the process of HASMCs,and to elucidate the corresponding mechanisms.Methods1.Cell CultureHuman aortic smooth muscle cells(HASMCs) were obtained from ScienCell (Carlsbad,CA) and cultured in smooth-muscle-cell culture medium(Cat#311,Cell Application,San Diego,CA) containing 10%fetal bovine serum.Cells were cultured up to passage 4 before the experiments were conducted.2.Different Dose to MMP-14 ExpressionHASMCs were treated with 0,5,10,20,and50 ng/mL recombinant human IL-6(Cat#206-IL/CF,R&D Systems,Minneapolis,MN) for 12hr before being harvested for measurement of target gene mRNA and protein levels.And find the expression of MMP-14 was mostly induced at the level of 10ng/ml IL-6.3.different time point to MMP-14 ExpressionMeasurement of target gene mRNA and protein levels were harvested at the time point of 0、6、12、24 independtly with 10ng/ml IL-6 treating the HASMCs).And find that the expression of MMP-14 was mostly induced at time point of 12 hr.4.the effect of AP-1siRNAFour groups were detected as control group,AP-1siRNA group,IL-6 group, IL-6+ AP-1siRNA group with Western blot.To investigate the effect of AP-1siRNA.5.PD98059,the inhibiter of ERK1/2 to MMP-14All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.To investigate the effect of PD98059.6.PD98059,the inhibiter of ERK1/2 to AP-1All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.To investigate the effect of PD98059.7.Effect of IL-6 to expression of protein RAF,MEK,ERK1/2 of HASMCsTo find compared with the control group,effection of IL-6 group to expression of protein RAF,MEK,ERK1/2 of HASMCs.Results1.At different dose of IL-6HASMCs were treated with 0,5,10,20,and 50 ng/mL recombinant human IL-6(Cat#206-IL/CF,R&D Systems,Minneapolis,MN) for 12 hr before being harvested for measurement of target gene mRNA and protein levels.And find the expression of MMP-14 was mostly induced at the level of 10ng/ml IL-6.2.At different point of timeMeasurement of target gene mRNA and protein levels were harvested at the time point of 0、6、12、24 independtly with 20ng/ml IL-6 treating the HASMCs). And find that the expression of MMP-14 was mostly induced at time point of 12 hr.3.the effect of AP-1siRNAFour groups were detected as control group,AP-1siRNA group,IL-6 group, IL-6+ AP-1siRNA group with Western blot.Find that compared with the control group,expression of MMP-14 protein in AP-1siRNA group decreased slightly but no significant,IL-6 group,IL-6+ AP-1siRNA group decreased significantly, and IL-6+ AP-1siRNA group is superior than IL-6 group.4.PD98059,the inhibiter of ERK1/2 to MMP-14All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.Find that compared with the control group, expression of MMP-14 protein in PD98059 group have no changes,IL-6 group, IL-6+ PD98059 group decreased significantly.IL-6+ PD98059 group is superior than IL-6 group.5.PD98059,the inhibiter of ERK1/2 to AP-1All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.Find that compared with the control group, expression of AP-1 protein in PD98059 group have no changes,IL-6 group, IL-6+ PD98059 group decreased significantly.IL-6+ PD98059 group is superior than IL-6 group.6.Effect of IL-6 to expression of protein RAF,MEK,ERK1/2 of HASMCsFind that compared with the control group,expression of protein RAF,MEK, ERK1/2 in IL-6 group increased. significantly.Conclusions 1.IL-6 increase MMP-14 expression at the level of mRNA and protein through the process of HASMCs.2.RAF-MEK-ERK1/2-AP-1 pathway responsible for IL-6 enhanced MMP-14 expression.