【作者】 张红兵；

【导师】 韩梅；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 当血管内皮损伤时,局部募集的炎性细胞可产生大量的活性氧分子(reactive oxygen species,ROS),而ROS又可作为刺激因素,进一步激活血管平滑肌细胞(vascular smooth muscle cell,VSMC)的迁移、增殖、凋亡及细胞外基质分泌等一系列病理事件,导致血管内膜发生肥厚和硬化等病变。因此,寻找新的有效药物,抑制损伤诱导的血管氧化应激反应以及由此引发的心血管疾病已成为药物研发的热点之一。黄酮类化合物(flavonoids)是自然界中广泛存在的一类酚类物质,是药用植物中的主要活性成分。流行病学研究表明,Flavonoids具有抗炎、抗氧化、抗凝血等生理活性,适量摄取可以降低心血管疾病的发生概率。欧亚旋覆花(Inula britannica L.)是菊科(compositae)、旋覆花属多年野生草本植物,性味咸、温;入肺、肾经。旋覆花总黄酮(total flavonoid extracts,TFE)是旋覆花中含量最多的一类活性成分,其含量约占旋覆花干重的10.9%,主要成分包括槲皮素、木犀草素、芦丁、槲皮苷和异槲皮苷等。其在心血管系统中的抗氧化功效未见报道。本研究观察TFE对内皮剥脱诱导的血管组织氧化应激和新生内膜形成的干预效应,并在细胞和分子水平上探讨其作用机制,旨在挖掘TFE在防治心血管疾病中的药用价值。1旋覆花总黄酮抑制损伤诱导的血管组织氧化应激和新生内膜形成研究证实,ROS是内皮损伤诱导的血管内膜增生的一个重要诱发因素,具有抗氧化作用的药物均具有较好的抗血管硬化的临床疗效。TFE具有抗菌、抗炎、抗氧化、抗凝血、抗衰老、抗肿瘤、降血脂等多种生理活性。然而,TFE对损伤诱导的血管氧化应激和内膜增生的干预效应尚不清楚。本部分研究,以大鼠颈外动脉和腹主动脉球囊剥脱内膜增生模型为对象,观察TFE对损伤诱导的血管氧化应激和内膜增生的抑制效应和相互关系。1.1球囊损伤诱导血管内膜增生形态学分析显示,球囊剥脱内皮3天后,血管损伤局部有大量血小板聚集,第7天时内膜出现增厚,14天时增生达到峰值,可见血管内膜向腔内凸起,细胞增多且排列紊乱,内膜呈现弥散性增厚。形态测量学分析显示,术后第7天,I/M比值开始升高;14天时,I/M比值较对照组增加50倍左右(p<0.01)。表明本实验复制的球囊剥脱内膜增生模型是成功的。1.2 TFE剂量依赖性抑制球囊损伤诱导的血管内膜增生为了观察TFE抗内膜增生效应,将制备的TFE乳液于术前3天至术后14天以不同剂量(12.5,25,50 mg/kg/d)灌服大鼠。结果显示,无论是颈总动脉还是腹主动脉,给予高剂量(50 mg/kg/d)TFE组的血管内膜增生程度较模型组明显减轻;抑制效应与阳性对照药物阿托伐他汀(ATO)相似,两组动物的血管腔内径比模型组均明显扩大,而中、低剂量TFE的抗内膜增生效应不明显。形态测量学结果分析表明,与对照组比较,模型组血管I/M比值明显增加,具有显著性差异(P<0.01)。不同剂量TFE组的颈总动脉和腹主动脉I/M比值均有不同程度的降低,而高剂量TFE组I/M比值明显低于模型组(P<0.01),其抑制内膜增生的功效与ATO相近。1.3 TFE抑制球囊损伤诱导的血管氧化应激血浆MDA和SOD活性分析显示,血管内皮剥脱后,血浆MDA含量明显高于假手术组,而血浆总SOD活性则显著下降(P<0.05)。提示血管剥脱后,机体处于氧化应激状态。给予TFE药物处理的动物,其血浆MDA含量较模型组有显著降低,而血浆总SOD活性回升(P<0.05),机体的氧化应激状态得到明显改善。提示该药物可提高血管组织的抗氧化能力。1.4 TFE降低大鼠血管组织中超氧阴离子(O2?-)的含量取不同处理组的大鼠颈总动脉进行冷冻切片和DHE染色以检测O2?-的生成情况,荧光显微镜下可见,假手术组的血管壁仅存在少量的红色荧光染色颗粒;内皮损伤后,新生内膜区中可见大量密集分布的O2?-红色荧光颗粒,表明损伤诱导血管细胞产生大量的O2?-;给予TFE后,O2?-的生成明显减少,血管内膜处的阳性染色明显减弱。血管组织O2?-的生成量变化与内膜增生程度具有正相关关系。1.5 TFE上调血管组织中SOD蛋白的表达Western blot分析和免疫组化染色结果显示,从假手术组的血管组织中可检测到较高水平的SOD蛋白;而球囊损伤14天后,血管中SOD含量明显降低,约为对照组的60%左右;给予TFE干预后,血管中的SOD含量有所增加,接近对照组水平,尤其在新生内膜的管腔侧,可见大量的SOD阳性染色颗粒。血管组织中SOD水平与O2?-的含量呈负相关关系。提示该药物可通过促进SOD的表达,提高血管组织清除氧自由基的能力,降低O2?-的含量,从而消除超氧化物对血管组织的病理性刺激。1.6 TFE抑制球囊剥脱诱导的VSMC的表型转化对分化型VSMC标志基因SM22α的Western blot分析显示,球囊损伤不同时间后,SM22α表达量呈进行性下降,表明VSMC由分化型向去分化型转化;给予TFE干预治疗后,损伤诱导的SM22α表达活性降低受到明显抑制,表明该药物可抑制VSMC表型转化。2旋覆花总黄酮体外抑制血管平滑肌细胞氧化应激在动物整体水平上证明TFE能够抑制球囊损伤诱导的血管内膜增生和氧化应激的基础上,为进一步探讨TFE调制血管氧化应激的作用机制,本部分以H2O2为诱导剂,建立VSMC氧化应激模型,给与TFE干预,同时以槲皮素(TFE的主要成分之一)为对照,比较观察两种制剂对VSMC氧化应激的调制效应。2.1 H2O2对VSMC生长的影响MTT分析结果表明,低浓度的H2O2(100-400μmol/L)处理VSMC后,不影响细胞活力和生长速度,高浓度的H2O2(>800μmol/L)具有明显的细胞毒性作用。本研究采用200μmol/L H2O2的终浓度用于后续实验。2.2不同剂量TFE和槲皮素对VSMC生长的影响MTT分析显示,用不同浓度(0、1、5、10、20、50、100 mg/L)TFE处理培养的VSMC,对细胞生长无明显影响,而用不同浓度(0、10、50、100、150、200、250μmol/L)槲皮素处理VSMC 24 h后,其对细胞生长活性具有显著抑制作用,该抑制作用具有剂量依赖性。结果表明,TFE对细胞增殖的直接抑制效应较弱。2.3 TFE抑制VSMC中O2?-的生成DHE染色结果显示,200μmol/L H2O2刺激VSMC 24 h后,与对照组相比,细胞内红色荧光的亮度明显增强,提示细胞内O2?-生成明显增多。而用20 mg/L的TFE预孵育的VSMC,在同浓度的H2O2刺激下,其细胞荧光着色的强度明显减弱,提示O2?-的生成减少,与对照药物50μmol/L槲皮素处理组相似。表明TFE和槲皮素均可抑制H2O2诱导的O2?-的生成。2.4 TFE抑制H2O2诱导的MDA生成MDA是脂质过氧化产物,其水平的高低反映脂质过氧化损伤的程度,反映了VSMC氧化应激的状态。当用200μmol/L H2O2刺激VSMC 24 h后,培养液中MDA的生成与对照组相比明显升高,约为对照组的2.78倍,有显著性差异(P <0.01);用TFE预孵育VSMC后,再用相同浓度的H2O2刺激细胞,MDA的生成量较H2O2组降低了25.33%(P <0.01)。与对照药物槲皮素的效果(抑制率为30%)相近。表明TFE和槲皮素均可抑制H2O2诱导的MDA的生成,二者具有相同的功效。2.5 TFE上调SOD活性SOD作为一种可以分解O2?-的抗氧化酶,其活性高低反映了机体清除氧自由基的能力,与氧化应激状态密切相关。H2O2刺激VSMC可明显降低总SOD活性,比未处理的对照组下降了42.86%(P <0.01)。用TFE预孵育后再进行相同处理,则总SOD活性被部分恢复,为H2O2刺激组的1.46倍(P <0.01),与槲皮素具有相同的效果。表明TFE和槲皮素均可以部分逆转H2O2导致的SOD活性降低。3旋覆花总黄酮抑制血管平滑肌细胞p47phox表达和磷酸化在体内,催化生成O2?-的系统主要包括NAD(P)H氧化酶、线粒体氧化还原系统、细胞色素P450单加氧酶,黄嘌呤氧化酶和脂氧合酶等。已有的研究表明,在血管组织和VSMC中,NAD(P)H氧化酶是催化O2?-产生的主要酶类。NAD(P)H氧化酶的激活过程有赖于胞浆亚基p47phox的磷酸化和膜转位。p47phox的表达活性、磷酸化水平和膜转位速率的变化,均可直接影响细胞ROS的生成。本研究部分在已经确证TFE具有抑制血管ROS生成,加快O2?-清除的基础上,以p47phox为切入点,对TFE减少ROS产生的上游机制进行了探讨。3.1 H2O2诱导VSMC中p47phox的表达Western blot结果显示,不同浓度(0, 50, 100, 200, 400μmol/L)H2O2处理VSMC 12 h后,p47phox的表达随H2O2浓度的增加而升高,于200μmol/L时达到峰值,随后表达水平略有下降,但仍高于正常水平。当给予200μmol/L的H2O2处理VSMC不同时间后,p47phox蛋白的表达也是逐渐增加的,并于12 h达到峰值,在24 h时,仍维持在较高水平。结果提示,H2O2对p47phox表达的诱导效应具有浓度和时间依赖性。3.2 TFE抑制内皮损伤诱导的血管p47phox的表达免疫组化结果显示,球囊剥脱内皮14天后的血管新生内膜中,可见有大量p47phox阳性染色的VSMC,弥散性分布于新生内膜区;而给予TFE药物的血管,其新生内膜肥厚程度减轻的同时,p47phox阳性的细胞数也大大减少。Western blot分析得到了相似的结果,在球囊损伤3天时,p47phox水平开始升高,约为对照组的1.9倍;到第14天时达到高峰,约为对照组的4倍左右;预先给予TFE药物干预组,其损伤诱导的p47phox的表达上升被部分抑制,与14天的模型组相比降低50%以上。3.3 TFE和槲皮素抑制H2O2诱导的VSMC中p47phox的表达为了在细胞水平上进一步验证TFE对p47phox表达的抑制效应,用TFE处理VSMC,以槲皮素作为阳性对照。提取细胞总RNA,半定量RT-PCR结果显示,H2O2刺激VSMC 12 h后,p47phox mRNA水平明显升高,是对照组的1.7倍;而用TFE和槲皮素预处理VSMC 12 h后,再用H2O2处理,p47phox mRNA水平显著降低,与H2O2处理组比较,分别降低了17%和25%。但是,两种药物对静息状态VSMC的p47phox表达无明显影响,表明TFE和槲皮素可抑制H2O2诱导的VSMC中p47phox的基因转录。细胞免疫荧光分析也证实,TFE和槲皮素可抑制p47phox蛋白的表达。H2O2处理组免疫荧光阳性细胞数和荧光强度均比对照组明显增加,提示细胞内p47phox蛋白的表达水平升高;给予TFE和槲皮素的预处理组,荧光强度降低至接近对照组的水平,表明p47phox的上调被部分抑制;Western blot结果显示,H2O2刺激VSMC 12 h后,p47phox蛋白表达明显升高,达到对照组的1.79倍;而在TFE和槲皮素预处理的细胞,其H2O2诱导的p47phox蛋白表达分别降低了38.2%和39.4%。该结果与RT-PCR结果一致。3.4 TFE和槲皮素抑制H2O2诱导的p47phox的磷酸化免疫沉淀分析结果显示,对照组VSMC中,p47phox的磷酸化水平相对较低,H2O2刺激VSMC 15 min、30 min、1 h后,p47phox磷酸化水平快速、短暂升高,于刺激15 min时即达高峰,随后略有降低,但仍高于正常水平。TFE预处理VSMC 12 h后,再用H2O2刺激细胞,p47phox的磷酸化水平较H2O2组下降47.2%,采用槲皮素预处理的结果与之相似。说明TFE和槲皮素可部分消除H2O2诱导的p47phox的磷酸化活化,二者具有相同的功效。4旋覆花总黄酮对ERK1/2、AKT和NF-κB信号通道的调制作用血管内皮损伤局部募集的炎性细胞除释放多种致炎因子外,还可产生大量的ROS,后者可通过影响胞内信号分子的磷酸化修饰,而诱导细胞产生氧化应激。细胞内有多条信号通路参与介导ROS诱导的p47phox磷酸化过程。前三部分已从整体和细胞水平上证实,内皮损伤或H2O2处理可引起VSMC中p47phox的过表达和磷酸化,TFE和槲皮素能够有效阻断上述过程。本部分分别以细胞外调节蛋白激酶(extracellular regulated protein kinase,ERK1/2)、蛋白激酶B(protein kinase B,Akt)和核因子κB (nuclear factor–κB,NF-κB)通路为观测点,探讨TFE和槲皮素对三条信号途径的调制作用。4.1 TFE和槲皮素抑制内皮损伤和H2O2诱导的ERK1/2的磷酸化免疫组化和Western blot分析检测了TFE和槲皮素对氧化应激诱导的ERK1/2磷酸化的影响,结果显示,球囊剥脱损伤14天后,模型组大鼠血管新生内膜VSMC中有大量磷酸化ERK1/2阳性染色颗粒;而给药组大鼠新生内膜肥厚程度减轻的同时,血管组织中磷酸化ERK1/2阳性颗粒数量明显减少。而三组血管内膜中的总ERK蛋白阳性染色强度无明显差异。提示球囊剥脱损伤可激活血管组织中的ERK1/2信号通路,而对ERK1/2蛋白的表达活性影响较小;给予TFE干预后可明显抑制损伤诱导的ERK1/2激活,TFE的这种效应在细胞水平上得到验证。用H2O2刺激细胞后,ERK1/2磷酸化水平迅速升高,并于15 min达到高峰,而经TFE和槲皮素预处理的细胞, ERK1/2磷酸化水平的升高幅度明显减小,但二药物之间并没有明显的差异,提示TFE和槲皮素可通过抑制ERK1/2通路活化来降低H2O2诱导的VSMC的氧化应激。4.2 TFE和槲皮素抑制H2O2诱导的Akt的磷酸化Western blot结果显示,不同条件处理的VSMC,其总Akt表达量基本一致,但磷酸化Akt的水平存在明显差异。在正常培养的细胞中,Akt的磷酸化水平较低,H2O2刺激后Akt的磷酸化水平迅速升高,5 min达到峰值,约为正常组的2倍左右(p<0.01),并维持至15 min,然后逐渐下降。而用TFE和槲皮素预处理VSMC 12 h后,再用H2O2刺激细胞,Akt的磷酸化水平较H2O2组有明显下降,其中TFE组在5 min时较H2O2组下降约33.3%(p<0.01)。而槲皮素的抑制效应在15 min时最为明显,磷酸化Akt水平比H2O2组下降了37.2%。4.3 TFE和槲皮素能够抑制H2O2诱导的NF-κB p65亚基的核转位处于静止期的VSMC中,NF-κB p65在胞核的分布较少。给予H2O2刺激后,VSMC核内的NF-κB p65水平逐渐升高,于刺激15 min达到高峰,一直维持高水平至30 min,1 h后逐渐降低,说明H2O2处理可引起p65核转位加快。用TFE预处理VSMC 12 h后,对H2O2诱导的NF-κB p65的核转位有较弱抑制作用。而用槲皮素预处理则可明显抑制H2O2诱导的p65核转位。上述结果表明,TFE和槲皮素可不同程度地抑制H2O2诱导的NF-κB p65的核转位,而槲皮素单体具有更强的抑制效应。结论1. TFE通过上调总SOD蛋白表达和活性、促进O2?-的清除,降低球囊损伤诱导的血管氧化应激,进而抑制血管新生内膜形成和由此导致的血管狭窄。2. TFE可以部分逆转H2O2导致的SOD活性降低、降低MDA水平、促进O2?-的清除,从而抑制VSMC氧化应激。3. TFE可抑制p47phox基因的转录和蛋白表达及磷酸化活化,该效应是TFE减少ROS生成和抑制氧化应激的分子机制之一。4. TFE对ROS诱导的ERK1/2、Akt和NF-κB信号通路的激活具有不同程度的调制作用,这是该药物抗血管氧化应激反应的作用途径之一。5. TFE是一种有效的血管保护制剂,通过抗氧化而减轻血管内皮损伤和内膜增生程度。更多还原

【Abstract】 The use of percutaneous transluminal coronary angioplasty (PTCA) has greatly reduced the number of fatalities in patients who suffer myocardial infarction. Unfortunately, this technique is plagued by a high incidence of vessel renarrowing or restenosis, occurring in 30 to 40% of patients within 6 months of the procedure. Reactive oxygen species (ROS) are thought to play an important role in restenosis. ROS derived mainly from vascular smooth muscle cells (VSMCs) contribute to the proliferation and migration of medial VSMCs leading to neointimal hyperplasia and adverse remodeling and ultimately, vessel restenosis. NAD(P)H oxidases appear to be the major sources of superoxide production in vascular tissues. Association has been made between elevated superoxide levels and neointima formation. This is apparent in models of restenosis and balloon catheter injury. Particularly, the role of NAD(P)H oxidases in neointimal hyperplasia has been demonstrated. Also, sustained downregulation of the expression of extracellular superoxide dismutase (EC-SOD) has been shown to occur after balloon angioplasty, contributing to the imbalance between the production and disposal of ROS and constrictive remodeling.Flavonoids are a large group of polyphenolic compounds abundantly present in the human diet, first identified as plant pigments, but now also recognized as very potent antioxidants and immunomodulators. Large epidemiological studies have shown an inverse association between dietary flavonoid intake and mortality from coronary heart disease. Importantly, therapeutic approaches aiming at augmenting the resistance of tissue to oxidation, such as administration of flavonoids, inhibit vascular remodeling in animal models of hypertension. The beneficial effects of flavonoid consumption on cardiovascular risk are supported by mechanistic and epidemiologic evidence. Inula Britannica L. is a traditional Chinese medicinal herb that has been used to treat bronchitis and inflammation. We have previously demonstrated that 1-O-acetylbritannilactone (ABL) from Inula Britannica L. inhibits NF-κB activation and decreases the level of iNOS and COX-2 gene expression in a dose-dependent manner in RAW 264.7 macrophages stimulated by LPS/IFN-γ, and suppresses neointimal formation induced by balloon injury. Recently, the total flavonoid extracts (TFE) were prepared from Inula Britannica L. Based on a protective effect of antioxidants on neointimal thickening or remodeling after balloon injury, we speculate that the TFE could inhibit the neointimal formation after balloon injury by suppressing oxidative stress generation. In this study, we have investigated whether and how TFE blocks the neointimal hyperplasia in balloon-injured rat carotid arteries.1 TFE suppresses oxidative stress generation and neointimal formation induced by balloon injury in ratTFE was extracted from Inula Britannica L. and identified by HPLC. The yield obtained was 4% relative to the original crude drugs. TFE used in this experiment contained 45.5% quercitrin, 25.85% luteolin, 12.96% 6-methoxyluteolin, 4.33% spinacetin and 1.85% isorhamnetin. The model of rat carotid artery balloon-injury was established as described previously. TFE freshly dissolved in 10% polyglycol-400, was administered orally at doses of 12.5, 25, and 50 mg/kg/d by gastric gavage from 3 days before balloon injury to 2 weeks after the injury.1.1 TFE inhibits injured-induced neointimal hyperplasia of carotid artery in ratAt 14 days after balloon injury, six cross-sections from the middle of each common carotid artery were stained with hematoxylin and eosin (HE). The neointimal and medial areas were calculated using the Image-Pro Plus Analyzer version 5.1 software in a blind manner. Neointimal formation was induced by balloon injury, and increasing I/M ratio of carotid arteries was observed after balloon injury. The injury-induced neoinimal hyperplasia started to increase at day 3 after balloon injury and reached the peak level at day 14. To explore the effect of TFE on neoinimal hyperplasia, the rats were administered orally at doses of TFE 12.5, 25, and 50 mg/kg/d by gastric gavage from 3 days before balloon injury to 2 weeks after the injury, respectively. The animals treated with high dose of TFE (50 mg/kg/d) showed reduction in neointimal hyperplasia, and the ratio of I/M was significantly reduced by over 70% in balloon injured-carotid arteries, compared with injured group. The inhibitory effect of TFE (50 mg/kg/d) on neointimal hyperplasia was almost consistent with atorvastatin that is a positive control. However, the lower doses of TFE (12.5 and 25 mg/kg/d) did not significantly inhibit the injury-induced neointimal formation.1.2 TFE suppresses oxidative stress generation induced by balloon injuryTo characterize O2?- production and localization within the vascular wall, ethidium red fluorescence was analyzed in sections of carotid arteries incubated with DHE that is converted into ethidium by O2?--induced oxidation. Positive red nuclei could be observed in adventitial, medial, and endothelial cells. At 14 days after balloon injury, carotid arteries wall showed an increase in O2?- production that was most evident in the neointimal and medial layer of the vessel. TFE (50 mg/kg/d) significantly prevented injury-induced increase in O2?- production.1.3 Effect of TFE on plasma SOD activity and MDA content after balloon injuryTo test whether the inhibiting neointimal hyperplasia effect of TFE is related to its anti-oxidative properties, the level of MDA and the activity of SOD in plasma were measured before and after balloon injury with or without TFE treatment. The data showed that the plasma MDA content were significantly higher in injured-group than those in sham group. The activity of SOD in plasma was significantly reduced after balloon injury. The animal treated by TFE (50 mg/kg/d) provoked a significant reduction of plasma MDA content, and increment of plasma SOD activity as compared with model group (p<0.01), respectively. Results showed that plasma SOD activity was obviously increased by TFE (50 mg/kg/d) treatment (p<0.01), and plasma MDA production was markedly decreased by TFE (50 mg/kg/d) treatment (p<0.01). Furthermore, the expression and distribution of SOD protein in neointima was detected by Western blot and immunohistochemical analysis, respectively. TFE treatment abolished the decrease in SOD expression induced by balloon injury, and the level of SOD protein approached that of sham group after TFE (50 mg/kg/d) treatment, and increased SOD protein localized in endothelial side of neointima.1.4 TFE inhibits VSMC phenotypic remodeling induced by balloon injuryTo determine whether TFE affects VSMC phenotype, SM22α, a marker of differentiated VSMCs, was detected. The results showed that the expression of SM22αprotein was reduced in neointimal formation induced by balloon injury, indicating that VSMCs change from differentiated state to dedifferentiated state. However, in TFE treatment group, the level of SM22αprotein was almost similar to the level observed in sham group. These results suggest that TFE inhibits VSMC phenotypic remodeling, in consistent with inhibiting neointimal hyperplasia.2 TFE reduces oxidative stress in VSMCs induced by H2O2The present study was designed to investigate crucial mechanisms of quercetin suppressing the oxidative stress in vascular smooth muscle cells (VSMCs).2.1 TFE and quercetin inhibit the formation of O2?- induced by H2O2The production of O2?- in VSMCs was detected by dihydroethidium (DHE) staining. DHE staining showed that the intensity of red fluorescence increased significantly after treatment with H2O2 (200μmol/L) for 12 h in VSMCs. The preincubation with 20 mg/L of TFE and 50μmol/L quercetin resulted in reduction in O2?- production induced by H2O2, respectively. This result suggested that TFE and quercetin can inhibit the formation of O2?- induced by H2O2 in VSMCs.2.2 TFE decreases the generation of MDA induced by H2O2MDA, a production of lipid peroxidation, can be used as a marker of vessel oxidative stress state and oxidative injury. When being treated with 200μmol/L H2O2 for 24 h, the level of MDA in culture medium of VSMCs was increased by 2.78-fold compared with control group (p<0.01). However, the amount of MDA was significantly reduced by 25.33% (p<0.01) in VSMCs pretreated by TFE before H2O2 stimulation compared to H2O2 group, almost similar to that of quercetin that is positive control, suggesting that both TFE and quercetin can inhibit the MDA production induced by H2O2.2.3 TFE enhances SOD activity in VSMCs treated by H2O2SOD is an anti-oxidant enzyme to scavenge oxygen-free radical production. SOD activity in culture medium of VSMCs treated with H2O2 decreased significantly by 42.86%, comparing with control cells (p<0.01). However, the activity of SOD in medium of VSMCs pretreated with TFE increased by 1.46-fold (P<0.01), compared with H2O2 group, suggesting that both TFE and quercetin can restore SOD activity, which is associated with its anti-oxidative activity.3 TFE inhibits p47phox expression and phosphorylationNAD(P)H oxidases are the major sources of superoxide production in vascular tissues. NAD(P)H oxidase system is composed of two cytosolic subunits p47phox and p67phox. The translocation of cytosolic p47phox to the membrane is essential in the assembly process of this complex and plays a major role in NAD(P)H oxidase activity in cardiovascular cells. Other results suggest that decreased NAD(P)H oxidase derived O2?- may be an important mechanism contributing to the prevention of endothelial dysfunction and neointimal hyperplasia by flavonoids.3.1 H2O2 induces the expression of p47phox in VSMCsWestern blot analysis showed that H2O2 triggered p47phox expression. The expression of p47phox increased dramatically in VSMCs treated with H2O2 at different doses (0, 50, 100, 200, 400μmol/L) for 12 h. After treatment by 200μmol/L H2O2 from 0 to 24 h, p47phox protein increased by 1.08-, 1.21-, 1.43-, 1.79- and 1.62-fold compared with control group (P<0.01), respectively. These findings indicated that H2O2 induces the expression of p47phox in a dose- and time-dependent manner. 3.2 TFE and quercetin inhibits the expression of p47phox in vascular tissue induced by balloon injury.Immunohistochemistry assays showed that the increased positive cells for p47phox mainly localized in endothelial side of neointima in model group. However, the number of p47phox positive cells is decreased in TFE group, compare with that in model group. Similarly, Western blot analysis showed that balloon injury induced the expression of p47phox in rat carotid artery, and that the expression of p47phox increased at 3 days after balloon injury and was maximal at 14 days. Administration with TFE (50 mg/kg/d) inhibited the injury-induced increase in p47phox expression in carotid arteries. Immunohistochemical analysis revealed that overexpression of p47phox occurred mainly in the neointima layer and was also inhibited by treatment with TFE (50 mg/kg/d).3.3 TFE and quercetin inhibits the expression of p47phox in VSMCs induced by H2O2In order to confirm the inhibitory effect of TFE on the expression of p47phox, total RNA was extracted from VSMCs by Trizol reagent and then the mRNA of p47phox in VSMCs were detected by RT-PCR. The result showed that treatment with H2O2 resulted in 1.7-fold increase in the level of p47phox, compared with control group. After the cells were pre-incubated with TFE or quercetin, the gene transcriptional activities of p47phox were significantly reduced, and the level of mRNA decreased by 17% or 25%, respectively. However, both agents have no effect on the expression of p47phox in quiescent VSMCs. The data suggested that both TFE and quercetin can inhibit the transcriptional activities of p47phox gene induced by H2O2 in VSMCs.Immunofluorescence analysis confirmed that TFE and quercetin can inhibit the expression of p47phox protein in VSMCs. The p47phox positive cells were increased in H2O2 group. When preincubate with TFE or quercetin, the fluorescence intensities were decreased, indicating that the up-regulation of p47phox expression was inhibited. Western blot analysis showed that treatment with 200μmol/L H2O2 for 12 h resulted in 1.79-fold increase in the level of p47phox protein, compared with control group. After the cells were pre-incubated with TFE or quercetin, p47phox protein was decreased by 38.2% or 39.4% compared with H2O2 group, respectively, coincidence with those in RT-PCR.3.4 TFE and quercetin inhibits the H2O2-induced phosphorylation of p47phox in VSMCsImmunoprecipitation analysis showed that H2O2 triggered p47phox phosphorylation, which reached to peak at 15 min and kept high level up to 30 min and decreased slowly. The pretreating of the cells with TFE resulted in 47.2% decrease in the level of p47phox phosphorylation (P<0.01).4 TFE modulates ERK1/2, Akt and NF-κB signaling pathwaysProtein phosphorylation plays a major role in modulating the activity of transcription factors. The mitogen-activated protein kinase (MAPK) signaling cascades are involved in transducting extracellular signals to nucleus by phosphorylating and activating a variety of transcription factors. ROS has been shown to activation of ERK 1/2. To determine mechanism of TFE inhibiting phosphorylation of p47phox, the role of ERK 1/2, Akt and NF-κB p65 in the signal transduction responses to H2O2 was analyzed by Western blot and immunohistochemistry, respectively.4.1 TFE and quercetin inhibit the phosphorylation of ERK1/2 activated by endothelial injury or H2O2Immunohistochemistry assays and Western blot were used to determine the effect of TFE and quercetin on phosphorylation of ERK1/2 induced by oxidative stress. The results showed that phosphorylated ERK1/2 incresed and localized in neointima 14 days after balloon injury. The phosphorylation of ERK1/2 increased drastically, and reached the peak at 15 min after H2O2 treatment in VSMCs. This data suggest that ERK1/2 pathway can be activated by balloon injury or H2O2. The activation of ERK1/2 signaling pathway can be inhibited significantly by pro-treatment with TFE in vivo and in vitro.4.2 TFE and quercetin inhibit the phosphorylation of Akt activated by H2O2 Western blot analysis showed that total Akt protein among different treatment groups was not different. The level of Akt phosphorylation was increased by 2 folds in VSMCs after H2O2 treatment. However, pre-incubation of TFE or quercetin decreased in H2O2-induced Akt phosphorylation by 33.3% or 37.2%, respectively.4.3 TFE and quercetin inhibit H2O2-induced nuclear translocation of NF-κB p65The nuclear translocation of NF-κB is characterized as the NF-κB activation. We first used Western blot to show the location of NF-κB in nuclei of H2O2-induced VSMCs. The results showed that there was low level of NF-κB p65 in nucleus of resting cells. After induction with H2O2 for different times, p65 was gradually localized to the nuclei of VSMCs, indicating translocation of p65 to the nuclei in VSMCs induced by H2O2. In TFE-preincubated VSMCs, H2O2-induced increase in nuclear p65 was reduced.Conclusions1. TFE inhibits neointimal formation through up-regulating SOD protein expression and activity, and suppressing ROS generation induced by balloon injury. The inhibitory effect of TFE (50 mg) on neointimal hyperplasia is almost consistent with atorvastatin that is a positive control.2. TFE treatment abolishes the decrease in SOD activity, reduces MDA level in plasma and significantly prevents injury-induced increase in O2?- production in rats after balloon injury.3. TFE inhibits the expression and phosphorylation of p47phox induced by ROS in vascular cells, in vivo and in vitro.4. TFE modulate the activation of ERK1/2, Akt and NF-κB signaling pathway in VSMCs treated by H2O2.5. TFE is a very potent antioxidant and inhibits neointimal formation through antioxidant. The decreased NAD(P)H oxidase derived O2?- may be an important mechanism contributing to the prevention of endothelial dysfunction and neointimal hyperplasia by TFE.更多还原