血管紧张素Ⅱ上调自发性高血压大鼠和Wistar-Kyoto大鼠血管平滑

-1及MKP-1 mRNA水平均明显高于对照组(P<0.05); SHR和WKY大鼠AngⅡ＋缬沙坦组和AngⅡ+PD98059组的上述指标与对照组比较均无显著性差异。(2) SHR大鼠VSMCs中ERK活性、p-ERK、MKP-1及MKP-1 mRNA均显著高于相同干预的WKY大鼠(P<0.01)。 (3) SHR和WKY大鼠之间以及对照组、AngⅡ刺激组、AngⅡ+缬沙坦组和AngⅡ+PD98059组间VSMCs中t-ERK水平均无显著性差异。以上结果表明，AngⅡ可能主要通过其1型(AngⅡ type 1，AT1)受体激活SHR和WKY大鼠VSMCs中ERK途径，增加ERK活性和p-ERK蛋白水平，继而引起MKP-1及 MKP-1 mRNA水平升高。关键词：血管紧张素Ⅱ；丝裂素活化蛋白激酶；细胞外信号调节激酶；血管平滑肌细胞；大鼠；自发性高血压中图分类号：Q254; R544.1 Effects of angiotensin Ⅱ on extracellular signal-regulated protein kinases signaling pathway in cultured vascular smooth muscle cells from Wistar-Kyoto rats and spontaneously hypertensive rats ZHU Jian-Hua, LIU Zhong, HUANG Zhao-Yang*, LI Shan Department of Cardiology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China Abstract: The aim of this study was to investigate the effects of angiotensinⅡ (Ang Ⅱ) on extracellular signal-regulated protein kinase(ERK) signaling pathway in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY)rats. VSMCs from SHR and WKYrats were treated with 1×10-7mmol/L AngⅡ for 24h in the absence or presence of 30 min of pre-treatment of valsartan (1×10-5mmol/L) or PD98059 (1×10-5mmol/L), selective inhibitor of ERKs- dependent pathways, when they were cultured in 20% calf serum medium. VSMCs of SHR and WKY cultured in serum-free medium were used as control groups. Among the different treatments, VSMCs from the SHR and WKY were devided into four groups: (1) control, (2) AngⅡ, (3) Ang Ⅱ+valsartan, (4) Ang Ⅱ +PD98059. ERK activity in VSMCs was measured by immuno-precipitation. Proteins of total ERK (t-ERK), phosphorylated-ERK (p-ERK) and mitogen-activated protein kinases phosphatase-1 (MKP-1) in VSMCs were detected by Western blot. MKP-1 mRNA in VSMCs was measured by RT-PCR. In VSMCs from WKY or SHR rats, ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in AngⅡ group were higher than those in control group (P<0.05). In both SHRs and WKYs, there were no significant differences in ERK activity, p-ERK, MKP-1 and MKP-1 mRNA among the control group, AngⅡ+valsartan group and AngⅡ+PD98059 group. ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in SHRs were significantly higher than those in WKYs with same treatments (P<0.01). There was no significant difference in t-ERK among different groups and no difference in t-ERK between SHRs and WKYs (P>0.05). Our results show that AngⅡ activates VSMCs ERK signaling pathways via AngⅡ type 1 (AT1) receptors. Ang Ⅱ increased ERK activity and p-ERK, but not t-ERK, accompanied by an increase in MKP-1 mRNA expression and protein. Among the different treatments, ERK activity and p-ERK were higher in SHR than in WKY. Valsartan and PD98059 blocked AngⅡ-stimulated ERK activation. These results suggest that ERK signaling pathway plays an important role in the pathogenesis of hypertension. The effect of AngⅡ on SHR and WKY VSMCs?ERK signaling pathway may be mediated by AT1 receptors, enhancing ERK activity and the amount of p-ERK, and then increasing MKP-1 mRNA and its expression. Key words: angiotensinⅡ; mitogen-activated protein kinases; extracellular signal-regulated protein kinase; vascular smooth muscle cells; rats; spontaneously hypertensiveReceived 2005-01-05 Accepted 2005-04-25 This work was supported by the Science and Technology Committee of Zhejiang Province (No. 021107057) and the Natural Science Foundation of Zhejiang Province (No. M303874). *Corresponding author. Tel: +86-571-88967874; E-mail: [email protected] 高血压时的血管重构、弹性降低等病理改变与血管平滑肌细胞(vascular smooth muscle cells, VSMCs)增殖密切相关。丝裂素活化蛋白激酶(mitogen-acti vated protein kinases，MAPK)信号途径在机体的细胞生长、分化和增殖过程中发挥重要的调控作用[1]。 MAPK 由p38-MAPK，C-jun N末端激酶(C-jun N-terminal kinases，JNKs)和细胞外信号调节激酶(extra- cellular signal-regulated kinases，ERKs)三类同功酶组成，在高血压的病理生理改变中又以 ERKs 的作用最为相[2]。 ERKs主要有ERK1和ERK2两种同工酶，磷酸化ERK (phosphorylated-ERK，p-ERK)是其活性形式。具有双效性的丝裂素活化蛋白激酶磷酸酶-1 (mitogen-activated protein kinases phosphatase-1, MKP-1)被p-ERK活化，活化的 MKP-1又通过去磷酸化作用使 p-ERK 失活以保持 ERK 信号途径的平衡[3]。血管紧张素Ⅱ (angiotensinⅡ，AngⅡ)具有刺激血管收缩、VSMCs增殖和胶原合成等广泛的生物学效应，在高血压的发生和发展过程中起着重要的作用。在体实验表明，血管紧张素转换酶抑制剂和 AngⅡ受体阻滞剂可以通过降低ERK活性而抑制血管重塑[4]。离体实验也表明，AngⅡ对VSMCs增殖及DNA合成有促进作用，且其作用与MAPK信号途径激活有关[5]。但AngⅡ在激活VSMCs内ERK 信号途径时，是ERK蛋白总量增加还是仅有p-ERK增加，MKP-1在VSMCs增殖中的作用及AngⅡ对MKP-1的影响如何，以及AngⅡ对不同种系大鼠ERK信号途径有何不同影响等问题尚不明确。缬沙坦是一种AngⅡ 1型(AngⅡ type 1，AT1)受体阻滞剂，PD98059则可通过抑制ERK上游分子 MAPK/ERK激酶(MAPK/ERK kinase, MEK)的活化以阻断ERK信号途径。本文拟通过观察Wistar-Kyoto (WKY)大鼠和自发性高血压大鼠(spontaneously hypertensive rat，SHR) VSMCs在Ang Ⅱ刺激下ERK活性、总ERK(total ERK，t-ERK)、p-ERK、 MKP-1及MKP-1 mRNA水平的变化，以及缬沙坦和PD98059对 AngⅡ刺激下的ERK信号途径的作用，了解ERK信号途径在高血压发生发展过程中的作用机制及AngⅡ对此信号途径的影响。 1 材料和方法 1.1 材料与试剂　　雄性8周龄的SHR和WKY大鼠各4只，重量在180~200 g，购自中国科学院上海实验动物中心。兔抗鼠t-ERK多克隆抗体、小鼠抗大鼠p-ERK单克隆抗体、辣根酶标记山羊抗兔和山羊抗鼠IgG二抗以及强化的化学发光试剂(enhanced chemiluminescence, ECL)均购自Santa Cruz公司；兔抗鼠MKP-1单克隆抗体、苯甲磺酰氟(phenyl- methylsulfonyl fluoride，PMSF)、抑肽酶(apro-tinin)、亮肽素(leupeptin)、AngⅡ和5-溴脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine，BrdU)购自Sigma公司，DMEM培养基购自Gibco公司，TRIzol购自Invitrogen公司，M-MLV逆转录酶和PD98059购自Promega公司，PVDF膜和蛋白A的琼脂糖珠子购自Amersham公司，ERK1/2活性测定试剂盒购自Chemicon公司，ERK标准蛋白购自New England Biolab公司，引物由上海生物工程公司合成，胎牛血清购自杭州四季青公司，缬沙坦由诺华公司馈赠，其余试剂均购自上海生物工程公司。 1.2 大鼠VSMCs的原代培养　　采用文献报道的组织贴块法[6]，在无菌条件下分离大鼠胸主动脉，剪碎后加入含有20%胎牛血清的DMEM 培养液，在37℃、5% CO2孵育箱中静置培养。待细胞生长融合至75%~95%培养皿时，用0.25%胰蛋白酶消化传代增殖。用平滑肌a-actin免疫组化染色鉴定细胞，胞浆有棕-颗粒者为阳性，证明为VSMCs，3~5代细胞用于本实验。在传代过程中，细胞以5×05 个/ml接种于6孔板培养板上。实验前48 h换成不含血清的DMEM，使细胞处于静止状态。在培养基中加入终浓度为1×0-5 mmol/L的缬沙坦或1×0-5 mmol/L的PD98059或不加药物预处理30 min，再予1×0-7 mmol/L的Ang Ⅱ刺激24 h后收集细胞，以无血清培养基培养的VSMCs作对照，每组实验重复4次。SHR和WKY大鼠的VSMCs各分4组：(1)对照组；(2) Ang Ⅱ刺激组；(3) Ang Ⅱ+缬沙坦组；4) Ang II+PD98059组。 1.3 样品的获取 1.3.1 蛋白提取　　用冰冷的PBS洗VSMCs 2次，倒净PBS。加入0.4 ml裂解液冰上裂解30 min，其组成为：Tris-Hcl 50 mmol/L，pH 8.0；NaCl 150 mmol/L；EDTA 0.5 mmol/L；DTT 1 mmol/L；NP-40 1%；脱氧胆酸钠0.5%；SDS 0.1%；PMSF 100 mg/L；抑肽酶1 mg/L；亮肽素2 mg/L；钒酸钠100 mmol/L。用塑料刮勺刮下细胞，吹打后将细胞悬液和碎片移入1.5 ml Eppendorf管中，4℃，12 000 g离心10 min，取上清，用Folin酚法进行蛋白定量后保存于-70℃。 1.3.2 总RNA的提取　　用TRIzol试剂一步法提取总RNA，溶解于20 ml DEPC水中，在Smart Spec 3000分光光度计上测定A260/280比值为1.8~2.0，并分别测定浓度后保存于-70℃。 1.4 ERK1/2活性测定免疫沉淀法纯化蛋白并用ERK1/2试剂盒测定ERK的活性。取0.5 ml蛋白样本，加入抗p-ERK抗体10 ml，在4℃孵育12 h。加入20 ml 50% (V/V)蛋白A的琼脂糖珠子，4℃孵育2 h。2 000 g，4℃离心1 min，弃去上清液，加入1 ml裂解液重悬珠子，4℃孵育10 min后离心；重复2次，其中第3次用适量的激酶活性测定缓冲液代替裂解液重悬。弃去上清液，沉淀的蛋白进行激酶活性的测定，在酶标仪450 nm处读取灰度(optical density，OD)值，并根据ERK标准蛋白OD值绘制的标准曲线计算样本的ERK活性。 1.5 Western blot法检测t-ERK、p-ERK和MKP-1蛋白表达　　t-ERK检测取20 ml蛋白，p-ERK和MKP-1检测取40 ml蛋白，加入上样缓冲液煮沸3 min变性后按文献报道的Western blot方法[7]，完成十二磺基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE) 和蛋白转膜操作，膜片分别与一抗(兔抗鼠t-ERK多克隆抗体和小鼠抗大鼠p-ERK单克隆抗体，稀释浓度为1:1 000；兔抗鼠MKP-1单克隆抗体，稀释浓度为1:5 000)进行抗原抗体结合反应，再与辣根酶标记二抗(山羊抗兔或山羊抗鼠IgG，稀释浓度均为 1:10 000)反应，经适当洗涤，膜片与ECL温浴1 min，保鲜膜包裹后，经X光片曝光，显影和定影，显现特异的蛋白信号。用GS-800 Calibrated Densitometer激光光密度仪扫描，结果用计算机Meta Morph程序分析。设内对照，同一胶上被测蛋白与内对照所测OD值的比值，即相对OD值代表蛋白的相对表达量。 1.6 RT-PCR法检测MKP-1 mRNA的表达 1.6.1 逆转录反应(reverse transcription，RT)两步法：RNA样品2 μg、0.01 mmol/L Oligo(T)18 1 μl、加DEPC水至12 μl体积，70℃水浴变性5 min，再加入M-MLV逆转录酶200 U、5×缓冲液5μl、10 mmol/L dNTP 1.25 μl、RNA酶抑制剂20 U、补DEPC水至25 μl体积，37℃水浴1 h。 1.6.2 PCR反应体系　　cDNA 3 μl、Taq酶 1.5 U、10 mmol/L dNTP 1 μl、25 mmol/L MgCl2 3 μl、10×缓冲液4.7 μl、MKP-1的上游引物5-ATCTCC-CCCAACTTCAGCTT-3和下游引物 5-TGATGGG-GCTTTGAAGGTAG-3 各4μl(浓度均为0.01 mmol/L)、β-actin的上游引物5-CACGGCATTGTAACC-AACTG-3和下游引物5-TCTCA-GCTGTGGTGGT-GAAG-3 各4μl(浓度均为0.01 mmol/L)，补DEPC水至50 μl体积。PCR反应条件为：预变性94℃ 5 min；变性94℃ 30 s、退火55℃ 30 s、延伸72℃ 1 min，循环26次；72℃延伸7 min。 1.6.3 琼脂糖电泳　　PCR产物于2%琼脂糖凝胶电泳，MKP-1 202 bp、β-actin 400 bp，用凝胶图像分析系统进行灰度扫描，以β-actin为内参照作半定量分析，比较不同年龄组的SHR和WKY大鼠VSMCs的MKP-1/β-actin灰度比值。 1.7 统计处理　　实验数据以mean±SD表示，用SPSS 10.0统计软件包进行成组t检验和单因素方差分析(ANOVA)及Student-Newman-Keuls法作显著性分析。P<0.05为具有统计学差异。 2 结果 2.1 各组VSMCs中ERK活性水平比较 SHR和WKY大鼠VSMCs AngⅡ刺激组ERK活性水平均高于对照组、AngⅡ+缬沙坦组和AngⅡ +PD98059组(P<0.01); Ang Ⅱ+缬沙坦组及AngⅡ+PD98059组ERK活性与对照组比较无明显差异。各组SHR大鼠VSMCs中ERK活性水平均显著高于相同干预的WKY大鼠( P<0.01)，SHR对照组VSMCs ERK活性比WKY对照组增加1.7倍(P<0.01)(图1)。2.2 各组VSMCs中t-ERK水平比较 SHR和WKY大鼠4组VSMCs中t-ERK水平无明显差异，提示不同干预对大鼠VSMCs的t-ERK水平无明显影响；SHR和WKY大鼠间VSMCs中t-ERK水平也无差异(表1)。2.3 各组VSMCs中p-ERK水平比较 SHR和WKY大鼠VSMCs AngⅡ刺激组p-ERK水平均高于对照组、AngⅡ+缬沙坦组和AngⅡ +PD98059组(P<0.05); AngⅡ+缬沙坦组及AngⅡ+PD98059组，p-ERK水平与对照组比较无显著差异。另外，各组SHR大鼠VSMCs中p-ERK水平均显著高于相同干预的WKY大鼠( P<0.01)(图2)。2.4 各组VSMCs中MKP-1水平比较 SHR和WKY大鼠VSMCs AngⅡ刺激组MKP-1水平均高于对照组、AngⅡ+缬沙坦组和AngⅡ +PD98059组(P<0.05); Ang Ⅱ+缬沙坦组及AngⅡ+PD98059组MKP-1水平与对照组比较无显著差异。各组SHR大鼠VSMCs中MKP-1水平均显著高于相同干预的WKY大鼠( P<0.01)(图3)。2.5 各组VSMCs中MKP-1 mRNA水平比较 SHR和WKY大鼠VSMCs AngⅡ刺激组MKP-1 mRNA水平均高于对照组、AngⅡ+缬沙坦组和AngⅡ+PD98059组(P<0.05); AngⅡ+缬沙坦组及Ang Ⅱ+PD98059组MKP-1 mRNA水平与对照组比较无显著性差异(P>0.05)。另外，各组SHR大鼠VSMCs 中MKP-1 mRNA水平均显著高于相同干预的WKY大鼠(P<0.01)(图4)。3 讨论原发性高血压的生理病理过程主要包括VSMCs的增生和肥大，以及细胞外基质合成增加和重排等，越来越多的研究表明MAPKs的激活可能在VSMCs增殖过程中起重要作用[8]。ERK信号途径则被认为是经典的MAPK信号途径，与VSMCs增殖关系最为密切。 AngⅡ是肾素-血管紧张素系统的重要作用因子，不仅是一种血管收缩剂，还是一种促生长因子 [6]。我们的研究表明，经AngⅡ刺激后, SHR和WKY大鼠VSMCs中ERK活性、p-ERK水平均增加，但t-ERK水平却没有变化。这提示在AngⅡ激活大鼠 VSMCs的ERK信号途径过程中，ERK的总量并未变化，而是其活化形式增加了。各组SHR大鼠VSMCs 中ERK活性和p-ERK水平均显著高于相同干预的WKY大鼠，这可能是因为基础状态下SHR有较高的AngⅡ，AngⅡ激活了ERK途径。缬沙坦和PD98059可以抑制AngⅡ对ERK信号途径的激活，提示AngⅡ主要通过AT1受体激活ERK信号途径。在基础情况下，细胞缺乏MKP-1，激活的MAPK可以诱导MKP-1表达，MKP-1又反馈调节 MAPK的活性，这对于维持心血管组织稳态平衡有重要作用[9]。本实验表明，经AngⅡ刺激后, WKY和SHR大鼠VSMCs中MKP-1和MKP-1 mRNA水平均较对照组增加，其机制可能是AngⅡ使大鼠VSMCs 中ERK活性增加，从而反馈引起MKP-1的升高；另外，p-ERK还能增加MKP-1稳定性，减少MKP-1 降解[10]。缬沙坦和PD98059抑制了ERK活性程度后，同时也抑制了MKP-1的水平。PD98059主要通过抑制ERK上游分子MEK的激活使ERK活性降低，不直接影响Ang II的作用，这提示AngⅡ主要不是直接调节MKP-1的水平。上述研究显示：AngⅡ主要通过AT1受体激活 WKY和SHR大鼠VSMCs中ERK途径，增加ERK活性和p-ERK蛋白水平，继而引起MKP-1及MKP-1 mRNA水平升高，但对ERK蛋白总量没有影响；缬沙坦和PD98059可以阻断AngⅡ对ERK信号途径的影响。SHR大鼠VSMCs中ERK活性、p-ERK、MKP-1及MKP-1 mRNA均显著高于WKY大鼠，提示ERK信号途径在高血压的病理过程中起重要的作用。参考文献 1 Aroor AR, Shuklar SD. MAP kinase signaling in diverse effects of ethanol. Life Sci 2004; 74(19): 2339-2364.[PubMed-abstract] 2 Purcell NH, Darwis D, Bueno OF, Muller JM, Schule R, Molkentin JD. Extracellular signal-regulated kinase 2 interacts with and is negatively regulated by the LIM-only protein FHL2 in cardiomyocytes. Mol Cell Biol 2004; 24(3): 1081-1095.[PubMed-abstract] 3 Communal C, Colucci WS, Remondino A, Sawyer DB, Port JD, Wichman SE, Bristow MR, Singh K. Reciprocal modulation of mitogen-activated protein kinases and mitogen-activated protein kinase phosphatase 1 and 2 in failing human myocardium. J Card Fail 2002; 8(2): 86-92.[PubMed-abstract] 4 El Mabrouk M, Touyz RM, Schiffrin EL. Differential ANGⅡ-induced growth activation pathways in mesenteric artery smooth muscle cells from SHR. Am J Physiol Heart Circ Physiol 2001; 281(1): H30-H39.[PubMed-abstract] 5 Hong HJ, Chan P, Liu JC, Juan SH, Huang MT, Lin JG, Cheng TH. AngiotensinⅡ induces endothlin-1 gene expression via extracellular signal-regulated kinase pathway in rat aortic smooth muscle cells. Cardiovasc Res 2004; 61(1): 159-168.[PubMed-abstract] 6 Touyz RM, Wu XH, He G, Salomon S, Schiffrin EL. Increased angiotensinⅡ-mediated Src signaling via epidermal growth factor receptor transactivation is associated with decreased C-terminal Src kinase activity in vascular smooth muscle cells from spontaneously hypertensive rats. Hypertension 2002; 39(2 Pt 2): 479-485.[PubMed-abstract] 7 Yin F (尹峰), Zhu Y, Li P, Han QD, Zhang YY. Isoproterenol-induced activation of MAPK, NFkB and JAK/STAT pathway in mouse myocardium. Acta Physiol Sin (生理学报) 2003; 55(4): 449-453 (Chinese, English abstract).[PubMed-abstract][Full-text PDF] 8 Wang TH (王庭槐), Tan Z, Fu XD, Yang D, Hu FX, Li YY. Effect of ERK on 17b-estradiol-induced inhibition of VSMC proliferation in rats after vascular injury. 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血管紧张素Ⅱ上调自发性高血压大鼠和Wistar-Kyoto大鼠 血管平滑

血管紧张素Ⅱ上调自发性高血压大鼠和Wistar-Kyoto大鼠血管平滑