RhoA/Rho激酶信号通路在肺动脉高压中作用及尼索地平对其影响

目的探讨Rho A/Rho激酶信号通路在大鼠肺动脉高压[5-羟色胺(HT)诱导]中对其肺动脉平滑肌细胞的作用及尼索地平(Nis)与此信号通路的关系。方法将原代培养大鼠肺动脉平滑肌细胞(PASMCs)随机分为空白对照组、5-HT(1μmol/L)组,Nis(1×10~(-5)、1×10~(-6)、1×10~(-7)及1×10~(-8)mol/L)组。噻唑蓝(MTT比色法)测定大鼠PASMCs增殖,Western印迹法及RT-PCR法检测PASMCs增殖细胞的Rho A、Rho激酶mRNA及Rho激酶的调节亚单位p-磷酸化肌球蛋白磷酸酶靶亚基(MYPT)1水平。结果不同浓度的Nis均能明显抑制5-HT诱导的PASMCs增殖(P0.05),呈现出一定的浓度依赖性;并且降低5-HT诱导的Rho A、Rho激酶mRNA表达及MYPT1磷酸化。结论5-HT作用下肺动脉高压大鼠PASMCs存在增殖及Rho A/Rho激酶信号通路异常表现,Nis能够抑制该细胞异常增殖并调节信号通路表达。     

霉酚酸对大鼠肺动脉平滑肌细胞增殖的影响

目的 在细胞水平研究霉酚酸对大鼠肺动脉平滑肌细胞增殖的影响.方法 采用生长曲线、甲基噻唑基四唑(MTT)方法、流式细胞仪检测生长细胞数、细胞的存活值、DNA含量,计算G1,S,G2M和增殖指数.结果 除低浓度(100 nmol/L)外,各霉酚酸组(1、10、100μmol/L)生长细胞数均较对照组减少,差异有统计学意义(p<0.01).MTT法检测霉酚酸组细胞的存活值降低,呈剂量依赖性(100 nmol/L～100 μmol/L).霉酚酸组G1期比例增加,S期比例减少:G2M期比例减少,增殖指数降低,呈剂量依赖性.结论 霉酚酸可有效地抑制大鼠肺动脉平滑肌细胞的增殖,主要作用于DNA合成期,并呈剂量依赖性.其有效浓度在临床可应用的范围内.     

新型KATP通道开放剂埃他卡林对人肺动脉平滑肌细胞增殖的影响

目的 研究新型KATP通道开放剂埃他卡林（iptakalim）对内皮素-1（ET-1）诱导培养的人肺动脉平滑肌细胞（HPASMC）增殖的影响。方法 内皮素-1诱导培养建立人肺动脉平滑肌细胞增殖模型；氚-胸腺嘧啶核苷（3H—TdR）掺入法观察脱氧核糖核酸（DNA）合成；流式细胞仪技术检测HPASMC细胞周期。结果埃他卡林能剂量依赖性抑制内皮素-1所致的3H—TdR掺入量增多，阻止HPASMC由静止期（G0／G1期）进入DNA合成期（S期）和有丝分裂期（G2／M期）。特异性KATP通道阻断剂格列本脲可拮抗埃他卡林对。H—TdR掺入的抑制作用。结论 埃他卡林可能通过激活KATP通道来抑制ET-1诱导入肺动脉平滑肌细胞的增殖作用，有望成为治疗肺动脉高压的新药。     

法舒地尔对VEGF诱导肺动脉平滑肌细胞增殖的抑制作用

肺动脉平滑肌细胞(PASMCs)增殖是肺动脉高压(PAH)中肺动脉重塑的一个重要因素.各种刺激均可促进PASMCs的移行和增殖,导致动脉壁增厚和PAH的形成[1].血管内皮生长因子(VEGF)可以促进PASMCs的增殖[2,3],在严重的PAH发病过程中,VEGF控制着毛细血管前动脉的重构[4].小G蛋白Rho及其效应物Rho激酶(ROCK)信号通路在各种细胞功能中,包括血管平滑肌细胞的收缩和增殖中都起着重要作用.     

内皮素-1在血管平滑肌细胞增殖作用中的研究进展

血管重构是血管壁功能和结构异常改变的过程,涉及血管内皮细胞、血管平滑肌细胞和细胞外基质的病理改变。血管平滑肌细胞增殖在血管重构中起关键性的作用。内皮素-1是一种活性多肽,参与调控血管平滑肌细胞收缩、增殖、迁移和基因表达等多项细胞功能。内皮素-1在平滑肌细胞增殖的作用机制可能是防止血管重构的关键靶点。     

1-磷酸鞘氨醇受体1参与晚期糖基化终产物引起的血管平滑肌细胞增殖和迁移

目的观察1-磷酸鞘氨醇受体1(S1PR1)在晚期糖基化终产物(AGE)引起的血管平滑肌细胞增殖和迁移中的作用。方法培养人脐动脉平滑肌细胞(HUASMC),葡萄糖与牛血清白蛋白(BSA)孵育法获得AGE-BSA,分为对照组、BSA组和AGE-BSA组,采用CCK-8实验检测平滑肌细胞增殖能力,采用细胞划痕和Transwell实验检测平滑肌细胞迁移能力,并进一步观察BSA和AGE-BSA在有或无S1PR1拮抗剂VPC23019/激动剂SEW2871预处理后的细胞增殖和迁移。结果与对照组比较,BSA和AGE-BSA均能诱导HUASMC增殖和迁移,AGE-BSA的作用比BSA更加显著(P0.05);S1PR1拮抗剂VPC23019可以明显抑制BSA和AGE-BSA诱导的HUASMC增殖和迁移;S1PR1激动剂SEW2871本身就可以促进HUASMC增殖和迁移,并且进一步促进BSA诱导的HUASMC增殖和迁移,而对AGE-BSA诱导的HUASMC增殖和迁移没有进一步的促进作用。结论血浆白蛋白本身对平滑肌细胞的增殖具有促进作用,糖基化修饰的白蛋白这一作用更加明显;S1PR1的激活参与了BSA和AGE-BSA促进平滑肌细胞增殖和迁移,AGE-BSA对S1PR1的激活作用更加显著。     

不同低氧时间诱导的大鼠肺动脉平滑肌细胞miR-199a表达的动态变化

目的 观察并分析microRNA-199a(miR-199a)在不同低氧时间诱导的大鼠肺动脉平滑肌细胞(PASMCs)中表达的变化,为探讨miR-199a对PASMCs增殖的调控作用及机制奠定基础.方法 原代培养的PASMCs分别于常氧和低氧下培养6h、12 h、24 h和48 h,采用四甲基偶氮唑蓝比色法(MTT)检测各组细胞增殖情况,实时荧光定量PCR检测各组细胞miR-199a的表达情况.结果 ①MTT检测结果显示低氧组各时间点PASMCs吸光度(A值)均显著高于相对应的常氧组(P＜0.05);②低氧组PASMCs的miR-199a表达随时间的延长呈持续降低趋势,N48 h组、H6 h组、H12 h组、H24 h组、H48 h组的PASMCs的miR-199a表达量分别为5.55±0.52、4.73±0.25、3.41±0.38、1.32±0.07、0.84±0.16.与N48 h组比较,H6 h组、H12 h组、H24 h组、H48 h组PASMCs miR-199a的表达均显著下降(P值均＜0.05);除低氧48 h组与低氧24 h组比较,miR-199a的表达差异无统计学意义(P =0.10)外,其余各组之间PASMCs miR-199a的表达差异均有统计学意义(P值均＜0.05).结论 低氧诱导后miR-199a在PASMCs中的表达下调,且随着低氧时间的延长,miR-199a的表达有降低的趋势,推测其可能参与了低氧诱导的PASMCs增殖的调控.     

BQ123对慢性低氧大鼠肺动脉平滑肌细胞KV1.5表达的影响

内皮素1(ET-1)可浓度依赖性的抑制大鼠肺动脉平滑肌细胞(PASMCs)电压门控钾电流(IKv)，但ET-1对PASMCs电压门控钾通道(Kv)基因表达的调节目前尚少见报道，本组研究拟从mRNA和蛋白水平，阐明内皮素受体拮抗剂BQ123对PASMCs Kv1．5表达的影响。     

半边莲生物碱抑制内皮素诱导的大鼠主动脉平滑肌细胞增殖

目的研究半边莲生物碱对内皮素诱导的血管平滑肌细胞增殖的作用。方法用内皮素1剌激大鼠主动脉平滑肌细胞增殖,以细胞计数试剂盒计数细胞个数、氚标胸腺嘧啶脱氧核苷掺入检测DNA的合成量、免疫细胞化学技术观察增殖细胞核抗原的表达活性作为细胞增殖的指标,并应用台盼兰拒染、乳酸脱氢酶检测观察半边莲生物碱的细胞毒性反应。结果内皮素1(10-7mol/L)可明显促进细胞增殖(与对照组相比,P<0.01);半边莲生物碱和BQ-123均可抑制内皮素1所诱导的细胞增殖(与内皮素1组相比,P<0.05);半边莲生物碱的抑制作用与浓度存在明显的依赖关系,但对活细胞数目和乳酸脱氢酶释放量均没有影响(P>0.05)。结论半边莲生物碱(50~200 mg/L)可浓度依赖性地抑制内皮素1所诱导的大鼠主动脉平滑肌细胞增殖,且此抑制作用并非是通过细胞毒性作用实现的。     

阿魏酸钠对血小板源生长因子和内皮素1诱导的血管平滑肌细胞迁移的影响

为探讨阿魏酸钠对血小板源生长因子二聚体和内皮素 1诱导的血管平滑肌细胞迁移的影响 ,采用组织块外生法体外培养血管平滑肌细胞 ,采用改良的Boyden微孔膜双槽法进行细胞迁移实验 ,荧光染料Fura 2 /AM法测定细胞内游离钙离子浓度。结果发现 ,血小板源生长因子二聚体和内皮素 1均可诱导血管平滑肌细胞迁移 ,作用峰值浓度分别为 10 μg/L和 10 - 7mol/L。阿魏酸钠 (10 - 7～ 10 - 3mol/L)呈浓度依赖性抑制上述物质诱导的细胞迁移 ,10 - 3mol/L阿魏酸钠对血小板源生长因子二聚体和内皮素 1诱导的细胞迁移的抑制率分别为 85 .0 4 %和 81.92 %。血小板源生长因子二聚体和内皮素 1促进细胞内游离钙离子浓度升高 (P <0 .0 5 ) ,作用峰值浓度分别为 10μg/L和 10 - 8mol/L。阿魏酸钠明显抑制该作用 ,峰抑制率分别为 80 .14 %和 76 .6 9%。以上提示 ,阿魏酸钠可能通过抑制细胞内游离钙离子浓度的升高来抑制上述物质诱导的血管平滑肌细胞迁移。     

内皮素受体拮抗剂GF-063和BQ-485对低氧培养大鼠肺动脉平滑肌细胞增殖的影响

目的:探讨新型内皮素受体拮抗剂GF-063和BQ-485对低氧培养的大鼠肺动脉平滑肌细胞(PASMCs)增殖的影响。方法:贴壁原代培养PASMCs。实验分为4组:常氧组(氧浓度210ml/L)、低氧组(氧浓度20ml/L)、低氧+内皮素A(ETA)受体拮抗剂BQ-485组(BQ-485的终浓度分别为1×10-6、1×10-7、1×10-8和1×10-9mo1/L)和低氧+GF-063组(GF-063的终浓度分别为1×10-6、1×10-7、1×10-8和1×10-9mo1/L)。上述4组细胞均分别培养24、48和72h。采用MTT比色法(波长492nm)检测BQ-485和GF-063对PASMCs增殖的影响(A值)。用流式细胞仪测定细胞周期;放射免疫检查法测定细胞培养上清液中内皮素-1(ET-1)的含量。结果:培养24h时,各实验组的A值差异不明显;培养48h时,低氧组的A值明显增加(P0.01),低氧+BQ-485组和低氧+GF-063组在二者的终浓度为1×10-8、1×10-7、1×10-6mo1/L时A值下降,与低氧组比较具有统计学意义(P0.01);在培养72h时,低氧组的A值仍高于常氧组,但较培养48h显著下降(P0.05)。培养48h时,低氧组G2、S期细胞的比率及DNA合成增加,与常氧组比较有统计学意义(分别为P0.01,P0.05);与低氧组比较,低氧+BQ-485组和低氧+GF-063组G2、S期细胞的比率下降(P0.05),G1期细胞增多(P0.05),DNA合成减少。在培养48h时,与常氧组比,低氧组ET-1的水平增高(P0.01),给予BQ-485、GF-063后ET-1的水平降低,BQ-485为1×10-7mol/L、GF-063为1×10-9mol/L时,可明显降低ET-1的水平(P0.01)。结论:GF-063和BQ-485作为两种新型的内皮素受体拮抗都剂能抑制低氧培养的PASMCs增殖,减少ET-1的生成,且GF-063的其作用较BQ-485强。     

Exhaled breath condensate in pulmonary arterial hypertension

Over the last decade, several new agents have been developed for the treatment of pulmonary arterial hypertension (PAH), and blood biomarkers have been developed which aim to monitor such treatment, and which correlate well with physiological parameters, symptoms and mortality. However, little is known regarding biomarkers collected using non-invasive methods such as exhaled breath condensate (EBC). EBC biomarkers show potential as a rapid, repeatable and easy method of sampling the pulmonary vasculature in severely ill patients. The current study aimed to investigate EBC biomarkers in patients with PAH of different aetiologies. We studied 89 patients in four groups: pulmonary arterial hypertension (PAH, n = 30), PAH associated with COPD (COPD/PAH, n = 14), COPD but no PAH (n = 16) and healthy controls (n = 29). Levels of the following EBC markers were measured: amino-terminal pro-brain natriuretic peptide (NT-proBNP), endothelin-1 (ET-1), 6-keto prostaglandin (PG)F(1α), hydrogen peroxide (H(2)O(2)), total oxides of nitrogen (NO(x)), total protein and pH. ET-1 and NT-proBNP were measured in plasma concurrently. Data were analysed with ANOVA or Kruskal Wallis tests where appropriate. Correlations were performed using Pearson's correlation coefficient. NT-proBNP was detectable in EBC and was highest in the PAH group, significantly higher than the COPD/PAH group (194.1 ± 23.3 versus 80.8 ± 22.2 fmol ml(-1), p < 0.05). EBC ET-1 was significantly higher in subjects with PAH (1.53 ± 0.32 fmol ml(-1)) compared to those with COPD/PAH (0.25 ± 0.03 fmol ml(-1), p < 0.05) and controls (0.66 ± 0.18 fmol ml(-1), p < 0.05). 6-keto PGF(1α) was low in the PAH group, significantly lower than the COPD/PAH group (4027 ± 445 versus 8381 ± 1024 pg ml(-1), p < 0.01). EBC biomarkers are measurable in PAH. EBC ET-1 was raised in PAH compared with controls and patients with PAH secondary to COPD, whereas 6-keto PGF(1α) was low. EBC biomarkers may be useful in detection and monitoring of PAH.     

钌红、丁卡因对肺动脉高压模型大鼠肺动脉平滑肌细胞[Ca2+]i释放功能的影响

目的:分析肺动脉高压时肺动脉平滑肌细胞雷诺定(Ryanodine)受体[Ca2+]i释放功能的改变及钌红(Rutheniumred)、丁卡因对肺动脉高压模型大鼠肺动脉平滑肌细胞[Ca2+]i释放功能的影响.方法:清洁级Wister大白鼠25只,腹腔注射野百合碱,建立大鼠肺动脉高压模型,死亡2只,最终23只;原代培养肺动脉平滑肌细胞;Fura-2/AM(钙离于荧光指示剂)负载培养细胞;荧光测钙技术比较雷诺定诱导的野百合碱组(n=15)及对照组(n=8)[Ca2+]i数值,观测钌红、丁卡因对雷诺定受体激动剂雷诺定诱导的[Ca2+]i变化的影响.结果:10 nmol/L雷诺定使对照组[Ca2+]i平均增加(93.31±12.41)nmoL/L,使野百合碱组[Ca2+]i平均增加(141.71±13,59) nmol/L;两组样本[Ca2+]i增加的数值比较差异有统计学意义(P＜0.01).钌红浓度为o.5～ 10 μmol/L、丁卡因浓度为2～40 μnoL/L时,可使雷诺定受体激动剂雷诺定诱导的[Ca2+]i值最大下降(115.32±7.47) nmol/L和(107.97±7.11) nmol/L.结论:肺动脉高压大鼠肺动脉平滑肌细胞对雷诺定受体激动剂雷诺定的敏感性增强;钌红、丁卡因对雷诺定诱导的肺动脉高压大鼠肺动脉平滑肌细胞[Ca2+]i上升有明显的抑制作用.     

Regulation of endothelin-1 synthesis in human pulmonary arterial smooth muscle cells. Effects of transforming growth factor-beta and hypoxia

OBJECTIVE: Endothelin-1 (ET-1) potently regulates pulmonary vascular tone and promotes vascular smooth muscle cell growth. Clinical and animal studies implicate increased ET-1 production in the pathogenesis of primary and secondary pulmonary hypertension. Although pulmonary arterial smooth muscle cells (PASMCs) synthesize ET-1 under basal conditions, it is unknown whether factors that may be important in pulmonary hypertension, such as transforming growth factor-beta (TGF-beta) or hypoxia, augment ET-1 production by these cells. METHODS: We determined the effect of TGF-beta and hypoxia on ET-1 release and preproET-1 mRNA from cultured rat and human PASMCs. RESULTS: In the basal state, rat and human PASMCs synthesize, on average (mean+/-S.E.M.), 872+/-114 and 563+/-57 pg ET-1/mg cell protein over 24 h, respectively, a level that causes autocrine and paracrine effects in other tissues. TGF-beta significantly increases the expression of preproET-1 mRNA and ET-1 production by both rat and human PASMCs. Hypoxia for 24 h, however, does not affect ET-1 release from rat or human PASMCs. CONCLUSIONS: Cultured rat and human PASMCs are a source of ET-1 production. Enhanced ET-1 release from PASMCs may contribute to the pathophysiology of TGF-beta-induced pulmonary hypertension. ET-1 production by PASMCs is unlikely to contribute to the role of ET-1 in hypoxia-induced pulmonary vasoconstriction.     

ET-1、VEGF、HIF-1α在COPD合并肺动脉高压形成中的意义

目的:探讨内皮素-1(ET-1)、血管内皮生长因子(VEGF)、缺氧诱导因子-1a(HIF-1a)在慢性阻塞性肺疾病(COPD)患者肺动脉高压形成中的意义。方法:选择40例合并肺动脉高压的COPD稳定期患者(实验组)和20例无肺动脉高压的COPD稳定期患者(对照组),予超声多普勒进行肺动脉压力检测、肺功能试验、动脉血气分析,并检测血浆ET-1、VEGF、HIF-1a水平。结果:COPD合并肺动脉高压组患者(实验组)第一秒用力呼气容积,第一秒用力呼气容积与用力肺活量比值、动脉血氧分压(60±6mmHg)均较对照组显著降低,有显著性差异,血浆ET-1(271.7±33.5pg/ml)、VEGF(261.4±49.2pg/ml)、HIF-1a(278.4±79.1ng/ml)浓度显著高于无合并肺动脉高压组(P0.05)。结论:COPD合并肺动脉高压患者均存在着ET-1、VEGF、HIF-1a等因子的升高,ET-1、VEGF、HIF-1a在COPD合并肺动脉高压形成中有着重要的意义。     

Decreased endothelin binding and [Ca2+]i signaling in microvessels of DOCA-salt hypertensive rats

OBJECTIVES AND DESIGN: The deoxycorticosterone acetate (DOCA)-salt model of hypertension is characterized by elevated vascular endothelin-1 (ET-1) and by reduced contraction to ET-1 in isolated mesenteric small arteries. The decreased contraction to ET-1 may be a compensatory mechanism caused by elevations in ET-1 and arterial pressure. The present study was designed to determine whether down-regulation of endothelin receptors or altered Ca2+ signaling contribute to the decreased contraction to ET-1. METHODS AND RESULTS: Contraction to ET-1 (10 to 10 mol/l) was significantly reduced in isolated mesenteric small arteries (87-286 microm intraluminal diameter) from DOCA-salt rats compared with placebo rats. Membrane protein was obtained for measurement of [125I]ET-1 receptor binding and ET receptor expression. Maximum binding was significantly reduced in vascular membranes from DOCA-salt rats (670 +/- 71 fmol/mg protein) compared with placebo rats (1165 +/- 75 fmol/mg protein), but binding affinity was unchanged. Conversely, ETA receptor protein was increased in DOCA-salt rat vessels. To assess Ca2+ signaling, freshly dissociated mesenteric small artery smooth muscle cells were loaded with fura-2 for measurement of the average myoplasmic free Ca2+ concentration ([Ca2+ ] ). The ET-1 (10 mol/l) induced increase in [Ca2+ ] was significantly less in cells from DOCA-salt rats compared with from placebo rats. This effect was not due to a loss of L-type Ca2+ channels since expression was increased in membrane protein from DOCA-salt rats compared with placebo rats, as measured by Western blot analysis. CONCLUSIONS: These findings indicate that decreases in receptor binding and Ca2+ signaling contribute to the impaired contraction to ET-1 in DOCA-salt hypertensive rats. However, these changes are not due to reduced expression of ETA receptors or L-type Ca2+ channels.     

Endothelial nitric oxide synthase-enhancing G-protein coupled receptor antagonist inhibits pulmonary artery hypertension by endothelin-1-dependent and endothelin-1-independent pathways in a monocrotaline model

This study investigates whether endothelin-1 (ET-1) mediates monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) and right ventricular hypertrophy (RVH), and if so, whether the G-protein coupled receptor antagonist KMUP-1 (7-{2-[4-(2-chlorobenzene)piperazinyl]ethyl}-1,3-dimethylxanthine) inhibits ET-1-mediated PA constriction and the aforementioned pathological changes. In a chronic rat model, intraperitoneal MCT (60 mg/kg) induced PAH and increased PA medial wall thickening and RV/left ventricle + septum weight ratio on Day 21 after MCT injection. Treatment with sublingual KMUP-1 (2.5 mg/kg/day) for 21 days prevented these changes and restored vascular endothelial nitric oxide synthase (eNOS) immunohistochemical staining of lung tissues. Western blotting analysis demonstrated that KMUP-1 enhanced eNOS, soluble guanylate cyclase, and protein kinase G levels, and reduced ET-1 expression and inactivated Rho kinase II (ROCKII) in MCT-treated lung tissue over long-term administration. In MCT-treated rats, KMUP-1 decreased plasma ET-1 on Day 21. KMUP-1 (3.6 mg/kg) maximally appeared at 0.25 hours in the plasma and declined to basal levels within 24 hours after sublingual administration. In isolated PA of MCT-treated rats, compared with control and pretreatment with l-NG-nitroarginine methyl ester (100 μM), KMUP-1 (0.1–100 μM) inhibited ET-1 (0.01 μM)-induced vasoconstriction. Endothelium-denuded PA sustained higher contractility in the presence of KMUP-1. In a 24-hour culture of smooth muscle cells (i.e., PA smooth muscle cells or PASMCs), KMUP-1 (0.1–10 μM) inhibited RhoA- and ET-1-induced RhoA activation. KMUP-1 prevented MCT-induced PAH, PA wall thickening, and RVH by enhancing eNOS and suppressing ET-1/ROCKII expression. In vitro, KMUP-1 inhibited ET-1-induced PA constriction and ET-1-dependent/independent RhoA activation of PASMCs. In summary, KMUP-1 attenuates ET-1-induced/ET-1-mediated PA constriction, and could thus aid in the treatment of PAH caused by MCT.     

PDCD4在野百合碱诱导肺动脉高压大鼠的表达及波生坦的干预机制

目的研究凋亡相关基因程序性细胞死亡因子4（PDCD4）蛋白在野百合碱诱导肺动脉高压大鼠中的表达并探讨波生坦对其干预机制。方法 SD大鼠正常对照组（N组,n=9）,不做任何处理。14只SD大鼠通过腹腔内注射野百合碱（60mg/kg）两周后,随机将大鼠分成2组,肺动脉高压模型对照组（M组,n=7）和波生坦组（B组,n=7）,分别通过胃管予以安慰剂或波生坦[200mg/（kg.d）]治疗,每天1次,共3周。测量血流动力学参数,观测肺血管和右室组织病理学改变,Western blot法测定肺组织中PDCD4和p-ERK1/2蛋白的表达。结果与N组相比,M组大鼠肺组织PDCD4蛋白表达明显减少、p-ERK1/2蛋白表达明显增加（P均〈0.001）。与M组相比,B组的平均肺动脉压和肺小动脉中膜平滑肌厚度均明显减少,肺组织PDCD4蛋白表达明显增加、p-ERK1/2蛋白表达明显减少（P均〈0.01）。结论 PDCD4蛋白在野百合碱诱导肺动脉高压大鼠中的表达减少,而波生坦可能经p-ERK1/2信号途径增加PDCD4蛋白表达从而降低肺动脉高压、减轻血管平滑肌细胞增殖,逆转肺血管重构的发生。     

Notch3 signaling activation in smooth muscle cells promotes extrauterine growth restriction-induced pulmonary hypertension

Background and aimsEarly postnatal life is a critical developmental period that affects health of the whole life. Extrauterine growth restriction (EUGR) causes cardiovascular development problems and diseases, including pulmonary arterial hypertension (PAH). PAH is characterized by proliferation, migration, and anti-apoptosis of pulmonary artery smooth muscle cells (PASMCs). However, the role of PASMCs in EUGR has not been studied. Thus, we hypothesized that PASMCs dysfunction played a role in EUGR-induced pulmonary hypertension.Methods and resultsHere we identified that postnatal nutritional restriction-induced EUGR rats exhibited an elevated mean pulmonary arterial pressure and vascular remodeling at 12 weeks old. PASMCs of EUGR rats showed increased cell proliferation and migration features. In EUGR-induced PAH rats, Notch3 signaling was activated. Relative mRNA and protein expression levels of Notch3 intracellular domain (Notch3 ICD), and Notch target gene Hey1 in PASMCs were upregulated. We further demonstrated that pharmacological inhibition of Notch3 activity by using a γ-secretase inhibitor DAPT, which blocked the cleavage of Notch proteins to ICD peptides, could effectively inhibit PASMC proliferation. Specifically knocked down of Notch3 in rat PASMCs by shRNA restored the abnormal PASMC phenotype in vitro. We found that administration of Notch signaling inhibitor DAPT could successfully reduce mean pulmonary arterial pressure in EUGR rats.ConclusionsThe present study demonstrated that upregulation of Notch3 signaling in PASMCs was crucial for the development of EUGR-induced PAH. Blocking Notch3-Hey1 signaling pathway in PASMCs provides a potential therapeutic target for PAH.     

低氧致大鼠肺动脉平滑肌细胞中PTEN/Akt1表达的变化与细胞增殖的关系

目的 通过观察低氧致大鼠肺动脉平滑肌细胞(PASMC)与细胞张力蛋白同源在10号染色体有缺失的磷酸酶(PTEN)和丝氨酸/苏氨酸蛋白激酶1(Akt1)mRNA及其蛋白表达水平的变化与低氧PASMC增殖的关系,探讨PTEN/Akt1信号途径在低氧肺血管重建中的可能调控作用.方法 用组织块法培养PASMC.采用半定量逆转录-PCR技术检测常氧组、低氧2、8、12、24 h组PTEN、Akt1基因mRNA的表达水平,采用Western blot技术检测相应的蛋白表达水平.采用噻唑蓝比色法和氚-胸腺嘧啶脱氧核苷(3H-TdR)掺入法检测PASMC的增殖改变.数据用x±s表示,采用Excel 2003软件进行t检验,P<0.05为差异有统计学意义.结果低氧刺激PASMC不断增殖,3H-TdR法检测的吸光度值低氧12 h组(0.70±0.10)比常氧组(0.37±0.06)明显增高(t=14.29,P<0.01);噻唑蓝法检测的吸光度值低氧24 h组(11 208±679)比常氧组(8374±545)明显增高(t=19.56,P<0.01).各组均检测出PTEN、Akt1基因mRNA及蛋白表达的变化.常氧组Akt1的mRNA、总蛋白、磷酸化蛋白的吸光度值分别为0.76±0.09、25±6和48±8,随着低氧培养时间延长,吸光度值逐渐升高,低氧8 h组达到高峰,分别为1.05±0.09、41±7和79±14,与常氧组比较,差异均有统计学意义(t值为8.31～168.00,P<0.05和P<0.01),随后开始下降,低氧24 h组恢复至常氧组水平.常氧组PTEN的mRNA、磷酸化蛋白的吸光度值分别为0.25±0.06和98±8,并随着低氧培养时间延长而逐渐升高,低氧24 h组达到高峰,分别为0.38±0.05和232±12,与常氧组比较,差异均有统计学意义(t值分别为22.04和50.46,均P<0.01).结论 PTEN/Akt1的转录和激活与低氧肺血管重建的PASMC增殖密切相关.