【作者】 张若楠；

【导师】 温进坤；

【作者基本信息】 河北医科大学 ， 中西医结合基础， 2014， 博士

【摘要】 病理性血管重塑（vascular remodeling）是指血管壁结构对血流变化、机械负荷或者血管损伤所发生的失代偿性改变，进而可导致许多血管疾病的发生和发展，比如动脉粥样硬化、血管成形术后再狭窄以及血管旁路移植失败等。新生内膜形成是血管重塑的主要病理特征之一，病理学基础主要为局部炎性细胞的浸润和中膜平滑肌细胞的增殖和迁移。大量研究表明，炎症应答在引发和加剧血管重塑中起关键作用，表现为单核/巨噬细胞浸润以及炎性介质的释放，由此触发血管平滑肌细胞（vascular smoothmuscle cells, VSMCs）向内膜下迁移和增殖，最终导致管腔狭窄甚至闭塞。MicroRNAs（miRNAs）是体内存在的一类非编码小RNA，长度通常不超过22个核苷酸，主要通过抑制翻译或促进mRNA降解在转录后水平调控基因表达。研究证实，许多miRNAs都参与了血管重塑过程。MicroRNA-155（miR-155）是一个炎症相关miRNA，在巨噬细胞和动脉粥样硬化斑块中表达上调。miR-155通过靶向抑制巨噬细胞炎症应答来发挥促炎或抗炎的作用。近期研究表明，miR-155在动脉粥样硬化中同样具有抗斑块形成和促斑块形成的双重作用，但其在血管重塑及新生内膜形成中的作用还尚不清楚。通心络是一种中药复方提取物，由人参、赤芍、酸枣仁、檀香、降香、土鳖虫、蜈蚣、水蛭、蝉蜕、全蝎、乳香及冰片组成。该药已显示出多种血管保护样作用，包括降血脂、抗氧化、抗血栓形成、改善内皮细胞功能、促进血管新生以及抑制炎症反应等。抗炎是通心络发挥血管保护作用的主要机制之一，miRNA是否介导或参与通心络抑制血管炎症反应的过程目前还尚不清楚。因此，本研究利用小鼠颈动脉结扎模型诱导血管内膜增生，探讨通心络是否通过调控miR-155的表达而发挥抑制血管炎症及血管重塑的作用，旨在为阐明通心络的血管保护分子机制以及扩大临床应用范围提供实验依据。第一部分通心络抑制颈动脉结扎诱导的内膜增生和血管炎症反应目的：观察通心络对颈动脉结扎诱导的内膜增生和局部炎症应答中的抑制作用。方法：1于左侧颈总动脉近分叉处结扎C57BL/6小鼠颈总动脉诱导内膜增生。2应用苏木精-伊红染色和Image-Pro Plus Analyzer version5.1软件观察和评价内膜增生的形态学改变。3qRT-PCR检测VEGF-α、FGF-b、TGF-β、PDGF-BB、TNF-α、IL-1β和IL-6的mRNA表达水平。4应用mac-2和SMA免疫荧光双重染色观察巨噬细胞浸润和血管平滑肌细胞增殖。5免疫组化法观察TNF-α和IL-1β的表达量。结果：1通心络抑制颈动脉结扎诱导的内膜增生形态学分析显示，颈动脉结扎7天后既有新生内膜形成，到结扎后14天内膜增生进一步加重，到结扎后21天，新生内膜大量增加至血管壁厚度的70%以上。与结扎组相比，通心络低剂量、中剂量和高剂量灌胃组（于术前3天开始，每天灌胃给药一次）结扎血管的内膜面积和I/M比值在3个时间点（7、14和21天）均显著减少。同时，通心络中剂量和高剂量灌胃组对内膜增生的抑制作用比通心络低剂量灌胃组更显著。以上结果说明，通心络呈剂量依赖性抑制颈动脉结扎诱导的新生内膜形成。2通心络抑制颈动脉结扎诱导的炎性因子的表达qRT-PCR结果显示，在颈动脉结扎后21天，结扎血管中的TGF-β、PDGF-BB、TNF-α、IL-1β的mRNA水平均比对照组血管显著升高。尤其是TNF-α的mRNA水平达对照组的17倍。与结扎组相比，通心络中剂量灌胃组显著降低了PDGF-BB、TNF-α、IL-1β的mRNA水平，但没有影响TGF-β的mRNA水平。这些结果说明，通心络对颈动脉结扎诱导的炎性因子的上调有显著的抑制作用。3通心络抑制颈动脉结扎诱导的巨噬细胞浸润和血管平滑肌细胞增殖，减少结扎血管中TNF-α和IL-1β的产生免疫荧光结果显示，在颈动脉结扎后21天的血管新生内膜中有大量巨噬细胞浸润，同时伴有血管平滑肌细胞的大量增殖。然而，在通心络中剂量灌胃组的结扎血管中，几乎观察不到巨噬细胞的浸润，中膜平滑肌细胞的增殖和迁移也不明显。免疫组化显示，与对照组相比，损伤21天后结扎血管的新生内膜中有大量TNF-α、IL-1β的产生，而通心络中剂量灌胃显著抑制了结扎血管中TNF-α和IL-1β的表达。4结扎后再给予通心络干预仍然可以显著抑制颈动脉结扎诱导的内膜增生形态学结果显示，于颈动脉结扎后3天再给予通心络中剂量灌胃，与结扎组相比，在术后21天仍然抑制了血管内膜增生，但抑制作用与术前预干预组比较有所减弱。小结：通心络可以显著抑制小鼠颈动脉结扎诱导的内膜增生，其作用与减少巨噬细胞浸润从而减轻局部炎症反应有关。第二部分通心络通过下调miR-155的表达发挥抑制内膜增生的作用目的：观测miR-155是否参与颈动脉结扎诱导的新生内膜形成过程，以及通心络是否通过调节miR-155的表达来发挥抑制内膜增生和血管炎症反应的作用。方法：1对miR-155基因敲除（miR-155-/-）小鼠和野生型（WT）小鼠行左侧颈总动脉结扎术。苏木精-伊红染色和Image-Pro Plus Analyzerversion5.1软件观察和评价内膜增生的形态学改变。2将腺病毒载体Ad-miR-155（空载体Ad-null作为对照）经尾静脉注射导入WT小鼠体内过表达miR-155。3qRT-PCR检测miR-155、TNF-α和IL-1β的表达水平。4mac-2免疫组化染色观察巨噬细胞浸润；免疫组化染色观察TNF-α的表达量。结果：1通心络抑制颈动脉结扎诱导的miR-155的表达与未结扎的对照组相比，miR-155在颈动脉结扎14天和21天后的小鼠血管中均显著上调，通心络中剂量灌胃在结扎后14天和21天均明显抑制了结扎血管中miR-155的表达。在颈动脉结扎7天后的结扎组和通心络中剂量灌胃组的血管中miR-155的表达没有明显变化。2miR-155缺失抑制颈动脉结扎诱导的内膜增生，而miR-155过表达部分拮抗了通心络对内膜增生的抑制作用形态学分析显示，颈动脉结扎21天后，WT小鼠血管中有大量新生内膜形成，而miR-155-/-小鼠的结扎血管，无论是I/M比值还是内膜面积均显著低于WT小鼠，说明miR-155的缺失抑制了颈动脉结扎诱导的内膜增生。同时，在miR-155-/-小鼠给予通心络中剂量灌胃后，其结扎血管中的内膜增生进一步减少。给通心络中剂量灌胃的WT小鼠尾静脉注射Ad-miR-155（于术前1天开始注射，每隔7天注射一次，共注射3次），与Ad-null注射组比较，在结扎21天后的血管中miR-155上调3.5倍。在通心络中剂量灌胃的WT小鼠中，颈动脉结扎21天后，与Ad-null注射组相比，Ad-miR-155注射组的结扎血管中I/M比值和内膜面积均显著增加，说明miR-155过表达部分拮抗了通心络对内膜增生的抑制作用。3miR-155缺失抑制颈动脉结扎诱导的巨噬细胞浸润和炎性因子TNF-α和IL-1β的表达，而miR-155过表达部分拮抗了通心络对巨噬细胞浸润和TNF-α表达的抑制作用免疫组化结果显示，颈动脉结扎21天后，WT小鼠的结扎血管的新生内膜中有大量巨噬细胞浸润，而miR-155-/-小鼠的结扎血管中巨噬细胞显著减少。给予通心络中剂量灌胃的miR-155-/-小鼠的结扎血管中几乎观察不到浸润的巨噬细胞。同时，在通心络中剂量灌胃的WT小鼠中，与尾静脉注射Ad-null组相比，尾静脉注射Ad-miR-155组的结扎血管中浸润的巨噬细胞明显增多。qRT-PCR结果显示，在颈动脉结扎后21天，miR-155-/-小鼠结扎血管中TNF-α的mRNA水平减少到WT小鼠的11%。给予通心络中剂量灌胃的miR-155-/-小鼠的结扎血管中TNF-α的mRNA水平减少到给予通心络灌胃的WT小鼠的20%。此外，在通心络中剂量灌胃的WT小鼠中，与Ad-null注射组相比，Ad-miR-155注射组的结扎血管中TNF-α的mRNA水平升高了2.2倍。以上结果说明，miR-155介导了结扎血管中TNF-α的表达，而miR-155过表达拮抗了通心络对TNF-α表达的抑制作用。免疫组化显示，各组的TNF-α的表达水平与qRT-PCR结果一致。另外，与WT小鼠相比，miR-155-/-小鼠结扎血管中的IL-1β mRNA水平下降了50%，但给予通心络中剂量灌胃的miR-155-/-小鼠与给予通心络中剂量灌胃的WT小鼠组比较，IL-1β mRNA水平无明显变化，同时，在通心络中剂量灌胃的WT小鼠中，Ad-miR-155注射使miR-155过表达后并没影响结扎血管中IL-1β的mRNA水平。这些结果说明，通心络对IL-1β表达的抑制作用并不是通过调节miR-155的表达实现的。4在颈动脉结扎后期过表达miR-155仍可部分拮抗通心络对内膜增生的抑制作用在通心络中剂量灌胃的WT小鼠中，于颈动脉结扎术后第14天注射Ad-miR-155，与Ad-null注射组相比，Ad-miR-155注射组结扎血管的内膜增生加重，说明在血管重塑后期过表达miR-155仍可减弱通心络对内膜增生的抑制作用。小结：miR-155参与颈动脉结扎诱导的新生内膜的形成过程；通心络对颈动脉结扎诱导的内膜增生和血管炎症应答的抑制作用在一定程度上是通过抑制miR-155的表达实现的。第三部分通心络通过阻断miR-155与TNF-α之间的反馈环路抑制巨噬细胞炎症应答目的：揭示通心络调节miR-155表达以及抑制巨噬细胞炎症应答的分子机制。方法：1用不同因素处理原代培养的小鼠骨髓来源巨噬细胞（BMMs）。2腺病毒感染实验用于在BMMs中过表达miR-155。3Westernblot检测p-Akt和Akt1的表达。4siRNA转染实验用于在BMMs中敲低Akt1。5qRT-PCR检测miR-155、TNF-α和IL-1β的表达水平。6ELISA检测BMMs培养基中TNF-α和IL-1β的含量。7伤口愈合实验。8细胞粘附实验。结果：1通心络通过阻断TNF-α和miR-155之间形成的正反馈环路而抑制巨噬细胞炎症应答qRT-PCR结果显示，与对照组相比，TNF-α刺激使miR-155的表达水平上升2.5倍，通心络预孵育以剂量依赖性的方式抑制TNF-α诱导的miR-155表达上调。另一方面，在对照组、IL-1β刺激组和通心络预孵育+IL-1β刺激组之间miR-155的表达水平无明显差异。这些结果说明，TNF-α可以促进BMMs中miR-155的表达，但IL-1β对miR-155的表达无影响。与Ad-null感染组相比，Ad-miR-155感染的BMMs中TNF-α的mRNA水平升高6倍。来源于miR-155-/-小鼠的BMMs中TNF-α的mRNA水平比WT组显著降低。与qRT-PCR结果相一致，ELISA检测结果显示，BMMs中过表达miR-155显著增加培养基中TNF-α的含量，而miR-155-/-小鼠的BMMs培养基中TNF-α含量比WT组明显下降。另一方面，miR-155过表达或敲除均未影响BMMs及其培养基中IL-1β的表达水平。以上结果显示，TNF-α和miR-155之间形成正反馈环路促进巨噬细胞的炎症应答。qRT-PCR和ELISA结果显示，与单纯过表达miR-155组比较，通心络预孵育减少了过表达miR-155诱导的TNF-α的表达上调，TNF-α的mRNA水平和培养基中的蛋白水平分别减少到过表达miR-155组的31%和78%。同时，miR-155-/-BMMs无论给予或不给予通心络预孵育，其TNF-α的表达均处于较低水平。这些结果表明，通心络可以阻断TNF-α和miR-155之间形成的正反馈环路，这可能是其抑制炎症应答发挥抗炎作用的机制之一。2通心络通过上调Akt1抑制巨噬细胞中miR-155的表达Western blot结果显示，BMMs被TNF-α刺激10、20和40分钟后p-Akt均轻微下调，通心络预孵育（2h）+TNF-α刺激组的p-Akt在三个时间点（10、20和40分钟）均显著上调。但是，通心络预孵育（2h）+TNF-α刺激组的总Akt1的蛋白水平在这三个时间点也显著上调。这些结果说明，通心络预孵育后p-Akt水平上升是由于上调了总的Akt1蛋白表达引起的。qRT-PCR结果显示，在BMMs中敲低Akt1后，通心络对TNF-α诱导的miR-155表达上调的抑制被消除，说明通心络抑制BMMs中miR-155的表达是通过上调Akt1实现的。3miR-155缺失抑制巨噬细胞的迁移，但不影响巨噬细胞的粘附伤口愈合实验结果显示，来源于miR-155-/-小鼠的BMMs在划痕后迁移的距离比WT小鼠的BMMs有所下降，说明miR-155缺失在一定程度上抑制了巨噬细胞的迁移。细胞粘附实验显示，与WT组相比，miR-155-/-BMMs与内皮细胞的粘附数量没有明显变化，说明miR-155缺失不影响巨噬细胞的粘附。小结：通心络通过阻断TNF-α和miR-155之间形成的正反馈环路而抑制巨噬细胞炎症应答；Akt1介导通心络对巨噬细胞中miR-155表达的抑制作用；miR-155缺失抑制巨噬细胞的迁移，但不影响巨噬细胞的粘附。结论：1通心络抑制颈动脉结扎诱导的内膜增生。2通心络通过抑制炎性因子的表达和巨噬细胞的浸润而减轻血管炎症反应。3miR-155缺失抑制颈动脉结扎诱导的内膜增生；通心络对内膜增生的抑制作用是通过抑制miR-155的表达实现的。4TNF-α和miR-155之间形成正反馈环路，通心络阻断TNF-α和miR-155之间的正反馈环路，从而抑制巨噬细胞炎症应答。5Akt1介导通心络对巨噬细胞miR-155表达的抑制。更多还原

【Abstract】 Pathological vascular remodeling is defined as any maladaptivealterations of the vessel wall structure in response to altered blood flow,mechanical load, or after vascular injury. Neointima formation is one of thehallmarks of the remodeling process which occurs in several vascular diseasessuch as atherosclerosis, restenosis after angioplasty and bypass graft failuredriven by local infiltration of inflammatory cells and by migration andproliferation of resident medial vascular smooth muscle cells (VSMCs).Numerous studies have shown that inflammatory response is a key event forthe initiation and progression of vascular remodeling. The infiltration ofmonocytes/macrophages and production of cytokines trigger VSMC migrationand proliferation into the intima and cause arterial stenosis, even occlusions.MicroRNAs (miRNAs) are a class of endogenous, small, noncodingRNAs of~22nucleotides that control gene expression by translationalrepression or mRNA degradation. Several miRNAs have been identified to bethat are involved in vascular remodeling. MiR-155, aninflammation-related-miRNA, which is upregulated in inflammatorymacrophages and in macrophages in atherosclerotic lesions, has both pro-andanti-inflammatory effect by targeting several mediators of inflammatorysignaling in macrophages. Recent studies have also shown that miR-155hasopposite effects on lesion formation in atherosclerosis. For the contrary effectsof miR-155on macrophage inflammatory reponse and atherosclerosis, the neteffect of miR-155on vascular remodeling and neointima formation remainsunclear.Tongxinluo (TXL), a compound of traditional Chinese herbal medicine,is composed of ginseng, Hirudo, Buthus martensi, Eupolyphaga seusteleophaga, Periostracum cicadae, Scolopendra subspinipes, Radix paeoniae rubra, Lignum dalbergiae odoriferae, Semen ziziphi spinosae, Lignum santalialbi, and Borneolum syntheticum. Recent studies have shown that TXL haspleiotropic effects including lipid lowering, antioxidation, antithrombosis,improvement of endothelial function, enhancement of angiogenesis andanti-inflammation, which may be due to the synergistic effects of multiplecompounds present in the herbal extract. Although anti-inflammation is one ofthe major mechanisms underlying the beneficial effects of TXL, it remainsunclear whether anti-inflammation effects are mediated by miRNAs.Therefore, in this study, we used the carotid artery ligation model in mice toinvestigate whether and how TXL exerts a protective effect on carotidligation-induced vascular inflammation and remodeling by regulatingmiR-155expression.PartⅠ TXL inhibits neointima hyperplasia and local inflammatoryresponse induced by carotid ligationObjective: To observe the role of TXL in protecting against neointimahyperplasia and local inflammatory response induced by carotid ligation inmouse.Methods:1The left common carotid artery was ligated near the carotidbifurcation to induce intima formation.2Morphology of neointimahyperplasia was assessed by hematoxylin/eosin staining and Image-Pro PlusAnalyzer version5.1software.3The expression of VEGF-α, FGF-b, TGF-β,PDGF-BB, TNF-α, IL-1β, and IL-6was observed at mRNA level by qRT-PCR.4Macrophage infiltration and VSMC proliferation were examined byimmunofluorescence histochemical double-staining for mac-2and SMA.5Immunohistochemical staining was performed to examine TNF-α and IL-1βproduction.Results:1TXL inhibits neointima hyperplasia induced by carotid ligationMorphometric analysis showed that neointima hyperplasia was observedat days7after vessel ligation and well developed by14days. By21days,neointima accounted for over70%of the carotid arterial wall thickness. When compared with that of the ligated group, carotid arterial wall thickness wassignificantly decreased in the three TXL-treated groups (TXL power wasadministered by intragastric administration beginning3days before ligation),both I/M ratio and intima area were lower than that observed at the ligatedgroup at all three time points. Moreover, the inhibitory effect of TXL onneointima formation was more significant in the moderate-and high-doseTXL groups than that in low-dose TXL group. These results suggest that TXLdose-dependently inhibits neointima formation induced by carotid ligation.2TXL inhibits inflammatory cytokine expression induced by carotidligationqRT-PCR showed a significant increase in TGF-β, PDGF-BB, TNF-α andIL-1β mRNA in ligated vessels at21days after ligation. Especially, TNF-αmRNA in ligated vessels reached16.78-fold above the control levels.Moderate-dose TXL markedly suppressed carotid ligation-inducedup-regulation of PDGF-BB, TNF-α and IL-1β mRNA in ligated artery.Whereas，TXL had no effect on IL-1β mRNA expression after ligation. Theseresults show that TXL markedly suppressed carotid ligation-inducedup-regulation of inflammatory cytokine expression in ligated artery.3TXL inhibits macrophage infiltration and VSMC proliferation inducedby carotid ligation, and reduces the production of TNF-α and IL-1β inligated arteryMacrophages infiltrated into the neointima of ligated arteries were readilydetectable at21days after carotid ligation with a large number of VSMCproliferation, whereas macrophages was barely observed in the neointima inmoderate-dose TXL-treated group similar to that in the unligated carotid artery.Consistent to the mRNA expression, the protein expression of TNF-α andIL-1β assessed by immunohistochemical staining was significantly elevated inthe neointima at21days after ligation compared with unligated arteries, butmoderate-dose TXL treatment inhibited carotid ligation-induced up-regulationof TNF-α and IL-1β production.4TXL post-treatment still inhibits neointima hyperplasia induced by carotid ligation.TXL was administrated intragastrically at moderate dose beginning3days after ligation injury and continuing for21days thereafter. TXLpost-treatment still inhibited neointima hyperplasia induced by carotid ligation,although the suppressive effect of TXL become less robust.PartⅡ TXL exerts its inhibitory action on neointima formation partly bysuppressing miR-155expressionObjective: To examine whether miR-155is required for neointimaformation induced by carotid ligation and whether TXL exerts its inhibitoryaction on neointima formation by regulating miR-155expression.Methods:1Left carotid artery ligation was performed in miR-155–/–andwild-type (WT) mice, and morphology of neointima hyperplasia was assessedby hematoxylin/eosin staining and Image-Pro Plus Analyzer version5.1software.2Ad-miR-155（Ad-null as a control） was introduced into WTmice through tail vein injection to overexpress miR-155.3qRT-PCR wasperformed to examine miR-155, TNF-α and IL-1β expression.4Immunohistochemical staining was performed to examine mac-2and TNF-αexpression.Results:1TXL suppresses miR-155expression induced by carotid ligationMiR-155was up-regulated in ligated artery at14and21days comparedwith the unligated vessel. Moderate-dose TXL treatment significantlysuppressed the expression of miR-155induced by carotid ligation at14and21days. There was no significant difference between unligated, ligated andmoderate-dose TXL treatment group at7days after carotid ligation.2MiR-155deletion protects against neointimal hyperplasia induced bycarotid ligation, and miR-155overexpression partly reverses theinhibitory effect of TXL on neointimal hyperplasiaLigated carotid artery caused significant neointimal formation in WTmice after21days, whereas the I/M ratio and intima area in miR-155–/–micewere significantly lower than WT mice on21days after ligation，suggesting that deletion of miR-155protects against neointimal hyperplasia induced bycarotid ligation. Furthermore, neointima formation was further reduced in theligated carotid artery of miR-155–/–mice treated with TXL.MiR-155expression was increased by3.5-fold after Ad-miR-155injection (every7days beginning1day before ligation injury until21days) inTXL-treated mice, and neointima thickness in the ligated carotid artery wassignificantly increased after Ad-miR-155was injected and overexpressedcompared with that in Ad-null-introduced mice treated with TXL，indicatingthat overexpression of miR-155partly reverses the inhibitory effect of TXL onneointimal hyperplasia3MiR-155deletion inhibits macrophage infiltration and inflammatorycytokine production induced by carotid ligation, and miR-155overexpression partly reverses the inhibitory effect of TXL onmacrophage infiltration and TNF-α productiona marked macrophage infiltration was observed in the ligated carotidartery of WT mice on21days after ligation, which was strongly reduced inmiR-155–/–mice, and macrophage infiltration was barely observed in theligated carotid artery of miR-155–/–mice treated with TXL. Meanwhile, in theTXL-treated WT mice, Ad-miR-155-infected arteries showned increasedmacrophage infiltration compared with Ad-null-injected group. In addition,mRNA level of TNF-α in ligated arteries of miR-155–/–mice was reduced to11%of WT mice. In TXL-treated miR-155–/–mice, TNF-α mRNA levels werereduced to20%of TXL-treated WT mice. Importantly, TNF-α mRNA levelswere increased2.2-fold in the ligated arteries of Ad-miR-155-infected micecompared with that of Ad-null-injected mice regardless of TXL treatment，suggesting that miR-155might mediate TNF-α mRNA expression, and thatmiR-155overexpression declines the inhibitory effect of TXL on TNF-αexpression. In contrast, although a50%reduction of the IL-1β mRNA wasobserved in ligated arteries of miR-155–/–mice, no significant difference inIL-1β mRNA levels was detected between miR-155–/–and WT mice whenthese mice were treated with TXL. Moreover, after carotid artery ligation, miR-155overexpression did not affect IL-1β mRNA expression in the ligatedarteries regardless of TXL treatment. These results suggested that inhibitoryeffect of TXL on IL-1β expression is not through suppressing miR-155expression.4Overexpression of miR-155in the late stage of carotid ligation alsopartly reverses the inhibitory effect of TXL on neointimal hyperplasiaNeointima thickness in the ligated carotid artery was increased afterAd-miR-155was injected on days14after carotid ligation compared with thatin Ad-null-introduced mice treated with TXL, indicating that Ad-miR-155injection initiated on days14after carotid ligation also partly reversed theinhibitory effect of TXL on neointimal hyperplasia.Part Ⅲ TXL suppresses macrophage inflammatory response by blockingthe feedback loop between miR-155and TNF-αObjective: To explore the molecular mechanisms whereby TXL regulatesmiR-155expression and the mechanism of the prevention effect of TXL onmacrophage inflammatory response.Methods:1Primary cultured bone marrow-derived macrophages(BMMs) were used in vitro study.2Adenovirus infection experiments wereperformed in BMMs to overexpressing miR-155.3p-Akt and Akt1wereassessed by Western Blot.4siRNA transfection experiments were performedin BMMs to knocked down Akt1.5The expression of miR-155, TNF-α andIL-1β was examined by qRT-PCR.6The productions of TNF-α and IL-1βwere determined in BMMs cell culture medium by Elisa.7Wound Healingassay.8Cell adhesion assay.Results:1MiR-155and TNF-α form a positive feedback loop to promotemacrophage inflammatory response, and TXL blocks this feedbackpathwayqRT-PCR showed that miR-155expression was increased by2.5-foldafter TNF-α treatment, whereas TXL treatment suppressed the expression ofmiR-155induced by TNF-α in a dose-dependent manner. No significant up-regulation of miR-155expression was observed after IL-1β treatment, andTXL treatment did not significantly affect miR-155expression. These resultssuggested that TNF-α, but not IL-1β, can markedly induce miR-155expression in BMMs. On the other hand, overexpression of miR-155mediatedby an adenoviral vector in BMMs increased the expression of TNF-α by5-foldover that of Ad-null-infected BMMs, whereas BMMs from miR-155–/–micehad a much lower level of TNF-α expression compared with that of BMMsfrom WT mice. Similarly, overexpression of miR-155in BMMs significantlyincreased the release of TNF-α into the medium, but BMMs from miR-155–/–mice released less TNF-α into the culture medium compared with WT mice.However, overexpression or deletion of miR-155did not affect the expressionof IL-1β. TXL pretreatment reduced the up-regulation of TNF-α expressioninduced by miR-155overexpression, with TNF-α mRNA level being reducedto31%of the control and TNF-α protein level being decreased to78%of thecontrol. Moreover, BMMs from miR-155–/–mice showed also a low level ofTNF-α expression regardless TXL treatment. Together, these results suggestthat miR-155and TNF-α form a positive feedback loop to promotemacrophage inflammatory response in mice and that TXL has a inhibitoryeffect on this feedback pathway.2TXL inhibits miR-155expression by up-regulating Akt1in BMMsWestern blot results showed that there was a slight decrease in the levelsof phospho-Akt upon TNF-α stimulation but TXL pretreatment markedlyincreased Akt phosphorylation. Meanwhile, the total Akt1also markedlyincreased by TXL pretreatment for2h, suggesting that the increasedphospho-Akt levels may be due to the up-regulation of total Akt1proteinlevels by TXL pretreatment. qRT-PCR revealed that knockdown of Akt1abrogated the inhibitory effect of TXL on miR-155expression induced byTNF-α. These data demonstrate that TXL inhibits miR-155expression byup-regulating Akt1in BMMs.3miR-155deletion reduces macrophage migration, but has no effect onadhesion. We have examined the functional effects of miR-155on macrophagemigration or adhesion. Wound healing assay and cell adhesion assay show thatmiR-155deletion reduced macrophage migration, but had no effect onadhesion.Conclusions:1TXL dose-dependently inhibited neointima formation induced bycarotid ligation.2TXL reduced local inflammatory response in ligated vessels byinhibiting inflammatory cytokine production and macrophage infiltration.3MiR-155deletion protected against neointimal hyperplasia induced bycarotid ligation, and TXL exerted its inhibitory action on neointima formationby suppressing miR-155expression.4MiR-155and TNF-α formed a positive feedback loop to promotemacrophage inflammatory response, and TXL blocked this feedback pathway.5Akt1mediated TXL-induced suppression of miR-155expression.更多还原