三七皂苷R1对肺高压大鼠模型肺动脉的舒张作用

目的探讨三七皂苷R1(notoginsenoside R1)对慢性低氧(chronic hypoxia,CH)和野百合碱(monocrotaline,MCT)致肺高压(pulmonary hypertension,PH)大鼠模型肺动脉(pulmonary arteries,PAs)收缩效应的作用及其机制。方法清洁级♂SD大鼠随机分为3组。正常对照组;CH组:大鼠饲养于氧分压为10.0%±0.5%的密封有机玻璃箱内;MCT组:大鼠按60 mg·kg-1(BW)一次性腹腔注射20 mg·L-1的MCT。观察三七皂苷R1对两种肺高压模型大鼠PAs收缩效应的作用。结果可成功制备CH和MCT致肺高压大鼠;三七皂苷R1(0.1～100μmol·L-1)对10 nmol·L-1内皮素-1(endothelin-1,ET-1)诱发正常大鼠PAs收缩效应呈剂量依赖性舒张,EC50为(5.25±0.14)μmol·L-1;3 nmol·L-1Gd3+阻断ET-1诱发两种肺高压模型大鼠PAs收缩效应后,再加入三七皂苷R1无进一步抑制作用;6μmol·L-1三七皂苷R1对环匹阿尼酸(cyclopiazonic acid,CPA)诱发两种肺高压模型大鼠PAs收缩效应均有明显的抑制作用。结论三七皂苷R1可能通过阻断钙池操纵性钙内流介导的PAs收缩产生治疗肺高压作用。     

钾通道阻滞剂四乙基铵敏感性钾电流在肺高压大鼠肺动脉平滑肌细胞中的变化

目的研究大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)中电压依赖性钾通道(voltagedepending potassium channel,K_V)电流在肺高压(pulmonary hypertension,PH)中的变化,及钾通道阻滞剂四乙基铵(tetraethylammonium,TEA)对K_V电流的影响。方法采用全细胞膜片钳技术记录PASMCs的K_V电流,观察PH大鼠中K_V电流的变化,以及TEA对K_V的影响。结果野百合碱(monocrotaline,MCT)、慢性低氧(chronic hypoxia,CH)明显减小了大鼠PASMCs上记录到的K_V电流密度;TEA干预能够明显降低正常和PH大鼠PASMCs的K_V电流,并且TEA对K_V电流的抑制作用在PH大鼠中明显减小。结论在CH、MCT诱导的两种PH大鼠模型中,PASMCs的K_V开放程度被明显抑制,而且其对应的TEA敏感性K_V电流明显减小。     

肺动脉高压大鼠肺动脉平滑肌细胞Ryanodine受体亚型的变化

目的　查明大鼠肺动脉平滑肌细胞 (PASMC)上Ryanodine受体 (RyR)的亚型并检测其在肺动脉高压 (PAH)时表达水平的变化。方法　采用大鼠一次性腹腔注射野百合碱 (monocrotaline ,MCT ,6 0mg·kg-1)复制PAH动物模型 ,RyR亚型的确定及其mRNA水平变化用RT PCR法测定 ,并应用Westernblot检测PAH大鼠RyR蛋白表达的变化。结果　大鼠PASMC只表达RyR 3个亚型中的Ⅱ亚型(RyR2 ) ,在PAH时RyR2的mRNA表达量 (以RyR2区带与β actin区带的密度百分比表示 )为 85 18%± 11 17% ,高于对照组 36 87%± 7 30 % (P <0 0 1) ,且RyR2蛋白水平亦升高 ,为对照组的 170 2 3 %± 45 34% ,(P <0 0 1)。结论　肺动脉平滑肌细胞存在RyR2亚型 ,其表达增强可能参与了PAH的发生。     

三七皂苷单体R1对低氧高二氧化碳肺动脉平滑肌细胞ERK1/2表达的影响

目的:探讨三七皂苷单体R1减轻低氧高二氧化碳性肺动脉收缩(hypoxia hypercapnia-in-duced pulmonary vasoconstriction,HHPV)的作用及其与细胞外信号调节激酶(ERK)1/2信号通路的关系。方法:原代培养雄性SD大鼠肺动脉平滑肌细胞(PASMCs),随机分为6组:常氧组(N组),低氧高二氧化碳组(H组),DMSO对照组(HD组),R1干预组(R8、R40、R100组)。采用免疫印迹法测定ERK1/2磷酸化蛋白表达,半定量逆转录-聚合酶链反应技术检测ERK1、ERK2基因表达水平。结果:p-ERK蛋白在N组表达弱,与H、HD组比较,R8、R40、R100组均不同程度下调,以R8组为著,差异有统计学意义(P<0.01);ERK1mRNA、ERK2mRNA在N组弱表达,与H、HD组比较,R8、R40、R100组表达均不同程度降低(P<0.01和P<0.05),以R8组为著。结论:ERK1/2信号通路可能介导大鼠低氧高二氧化碳性肺动脉收缩;三七皂苷单体R1可能通过抑制ERK1/2通路减轻低氧高二氧化碳性肺动脉收缩。     

慢性低氧大鼠TRPC1表达与肺动脉收缩变化时间曲线关系

目的探讨慢性低氧(chronic hypoxia,CH)上调TRPC1表达和SOCE介导肺动脉(pulmonary arteris,PAs)收缩的时间曲线。方法清洁级♂SD大鼠,常压低氧(氧分压为9.5%¹0.5%)饲养,观察在不同时间点CH对TRPC1表达量和SOCE介导的PAs收缩的影响,描记其时间依赖性曲线。结果 1 CH上调平均右心室收缩压(mRVSP),低氧1 d压力增高明显,7 d达到最大值,21 d后达到稳定;右心室质量指数(RVMI)低氧3 d后开始增高,此后一直呈持续增高状态,直到21 d达到稳定;2半定量RT-PCR检测显示,CH上调TRPC1 mRNA的表达量,低氧1 d上调明显,3 d达到最大值一直维持到7 d,21 d后达到稳定;3 CH上调SOCE介导的PAs收缩,低氧3 d开始上调,7 d达到最大值,21 d后达到稳定。结论 CH早期TRPC1/SOCE明显上调,两者的时间曲线具有相关性,表明TRPC1和SOCE的上调在慢性低氧致肺高压的机制中发挥着重要作用。     

三七总皂苷调控SIRT1/FOXO3a/p27通路抑制大鼠肺动脉平滑肌细胞增殖

目的 探讨三七总皂苷(PNS)通过SIRT1/FOXO3a/p27通路抑制大鼠肺动脉平滑肌细胞(PASMCs)增殖的作用。方法 首先将PASMCs随机分为正常(Control)组和三七总皂苷(PNS)组、野百合碱(MCT)组和野百合碱+三七总皂苷(MCT+PNS)组,CCK8检测PNS对正常细胞的安全浓度,进一步检测PNS抑制增殖的最适浓度;为研究作用机制将PASMCs分为Control组、溶剂(DMSO组)组、MCT组、MCT+PNS组、MCT+PNS+SIRT1抑制剂(MCT+PNS+EX-527)组。造模结束,Edu检测细胞增殖;免疫荧光检测SIRT1、FOXO3a表达;qPCR检测细胞SIRT1、FOXO3a、p27、PCNA的表达。结果 CCK8显示：0～400 mg·L^-1的PNS对正常细胞无毒(P>0.05),100 mg·L^-1的PNS可显著抑制MCT诱导的细胞增殖(P<0.01);Edu显示：MCT组较Control组增殖增多;MCT+PNS组较MCT组增殖减少;MCT+PNS+EX-527组较MCT+PNS组增...     

肺动脉高压模型大鼠肺动脉平滑肌细胞内钙的改变

目的分析肺动脉高压时肺动脉平滑肌细胞Ryanod-ine受体[Ca2+]i释放功能的改变。方法腹腔注射野百合碱建立大鼠肺动脉高压模型,原代培养肺动脉平滑肌细胞,Fura-2/AM负载培养细胞,荧光测钙技术测量Ryanodine受体激动剂对[Ca2+]i变化的影响。结果10nmol.L-1Ry-anodine使对照组[Ca2+]i平均增加(93.31±12.41)nmol.L-1,使PAH组[Ca2+]i平均增加(141.71±13.59)nmol.L-1。两组样本[Ca2+]i增加的数值差异有显著性(P<0.01);10mmol.L-1Caffine使对照组[Ca2+]i平均增加(149.02±13.02)nmol.L-1,使PAH大鼠PASMC的[Ca2+]i平均增加(191.2±21.26)nmol.L-1,两组样本[Ca2+]i数值的变化差异有显著性(P<0.01)。结论肺动脉高压大鼠原代培养的肺动脉平滑肌细胞对Ryanodine受体激动剂的敏感性增强,提示肺动脉高压时Ryanodine受体释放[Ca2+]i的功能发生了异常改变。     

HPLC-ELSD测定三七通脉片中三七皂苷R1、人参皂苷Rg1及Rb1的含量

目的:用HPLC—FLSD测定三七通脉片中三七皂苷R1、人参皂苷Rg1及Rb1的含量。方法:色谱柱填料为十八烷基硅烷键合硅胶,流动相为乙腈-水,梯度洗脱,漂移管温度105℃,载气流速3．0L·min-1。结果:三七皂苷R1、人参皂苷Rg1及Rb1分别在0．300-3．000μg,1．01-10．100μg,0．976-9．760μg之间呈良好的线性关系;制剂中3种成分的平均回收率分别为96．5％(RSD 1．2％),98．3％(RSD 1．5％),97．6％(RSD 2．1％)。结论:该方法简便、准确、分离效果好,无干扰,可用于三七通脉片的质量评价。     

Carvedilol inhibits proliferation of cultured pulmonary artery smooth muscle cells of patients with idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is associated with proliferation of smooth muscle cells (SMCs) in small pulmonary arteries. Inhibition of proliferation of pulmonary artery smooth muscle cells (PASMCs) may be an effective treatment of patients with idiopathic pulmonary arterial hypertension. Recent studies have shown that carvedilol, an alpha- and beta-blocker with antioxidant and calcium channel blocking properties, inhibits the proliferation of cultured normal human pulmonary artery smooth muscle cells. In this study, we tested the hypothesis that carvedilol has antiproliferative effects on pulmonary artery smooth muscle cells of patients with idiopathic pulmonary arterial hypertension. Pulmonary artery smooth muscle cells from six idiopathic pulmonary arterial hypertension patients who had undergone lung transplantation were cultured. To determine cell proliferation, H-thymidine incorporation was measured. Platelet-derived growth factor-induced proliferation of IPAH-PASMCs was significantly greater than that of normal control pulmonary artery smooth muscle cells. Carvedilol (0.1 microM to 10 microM) inhibited the proliferation of idiopathic pulmonary arterial hypertension-pulmonary artery smooth muscle cells in a concentration-dependent manner. Prazosin (an alpha-blocker) and N-acetyl L cysteine (an antioxidant agent) (0.1 microM to 10 microM) did not inhibit their proliferation, but the high concentration of propranolol (a beta-blocker) and nifedipine (a calcium channel blocker) (10 microM) inhibited the proliferation. The combination of propranolol and nifedipine inhibited the proliferation but only at a high concentration (10 microM) combination. Cell cycle analysis revealed that carvedilol (10 microM) significantly decreased the number of cells in S and G2/M phases. These results indicate that carvedilol inhibits the exaggerated proliferation of pulmonary artery smooth muscle cells of patients with idiopathic pulmonary arterial hypertension partially via its beta-blocking [corrected] and calcium channel blocking effects in vitro.     

Decrease in the Ca2+-activated K+ current of pulmonary arterial smooth muscle in pulmonary hypertension rats

Pulmonary hypertension exhibits acute elevation of vascular tone and hyperreactivity of pulmonary vasculature, which are closely related to patient mortality. In the present study, we investigated the characteristics of membrane currents of isolated pulmonary artery smooth muscle cells taken from rats with monocrotaline-induced pulmonary hypertension. Male Wistar rats were given a single subcutaneous injection of monocrotaline or saline, and then sacrificed between 18 to 21 days after the injection. The membrane currents in the smooth muscle cells from both groups of rats were compared using the whole-cell patch clamp technique. With 0.1 mM EGTA in the pipette, the densities of outward currents in monocrotaline-injected rats were smaller than those in control rats. When EGTA in patch pipettes was increased to 10 mM, the densities of the outward currents in monocrotaline-injected rats were equal to those of control rats. The Ca2+-activated K+ channel blockers (TEA, iberiotoxin) and nisoldipine were less effective on the outward currents of monocrotaline-injected rats. In the current clamp mode, a depolarization of membrane potential induced by 4-aminopyridine was greater in monocrotaline-injected rats than in control rats because of the reduced activity of the Ca2+-activated K+ channels. The Ca2+-activated K+ channels were decreased in pulmonary hypertension. The reduced activity of the currents may be related to the vascular hyperreactivity in pulmonary hypertension.     

Endothelial cell dysfunction and cross talk between endothelium and smooth muscle cells in pulmonary arterial hypertension

The pathogenesis of pulmonary arterial hypertension (PAH) involves a complex and multifactorial process in which endothelial cell dysfunction appears to play an integral role in mediating the structural changes in the pulmonary vasculature. Disordered endothelial cell proliferation along with concurrent neoangiogenesis, when exuberant, results in the formation of glomeruloid structures known as the plexiform lesions, which are common pathological features of the pulmonary vessels of patients with PAH. In addition, an altered production of various endothelial vasoactive mediators, such as nitric oxide, prostacyclin, endothelin-1, serotonin, chemokines and thromboxane, has been increasingly recognized in patients with PAH. Because most of these mediators affect the growth of the smooth muscle cells, an alteration in their production may facilitate the development of pulmonary vascular hypertrophy and structural remodeling characteristic of PAH. It is conceivable that the beneficial effects of many of the treatments currently available for PAH, such as the use of prostacyclin, nitric oxide, and endothelin receptor antagonists, result at least in part from restoring the balance between these mediators. A greater understanding of the role of the endothelium in PAH will presumably facilitate the evolution of newer, targeted therapies.     

Endothelial cell dysfunction and cross talk between endothelium and smooth muscle cells in pulmonary arterial hypertension

The pathogenesis of pulmonary arterial hypertension (PAH) involves a complex and multifactorial process in which endothelial cell dysfunction appears to play an integral role in mediating the structural changes in the pulmonary vasculature. Disordered endothelial cell proliferation along with concurrent neoangiogenesis, when exuberant, results in the formation of glomeruloid structures known as the plexiform lesions, which are common pathological features of the pulmonary vessels of patients with PAH. In addition, an altered production of various endothelial vasoactive mediators, such as nitric oxide, prostacyclin, endothelin-1, serotonin, chemokines and thromboxane, has been increasingly recognized in patients with PAH. Because most of these mediators affect the growth of the smooth muscle cells, an alteration in their production may facilitate the development of pulmonary vascular hypertrophy and structural remodeling characteristic of PAH. It is conceivable that the beneficial effects of many of the treatments currently available for PAH, such as the use of prostacyclin, nitric oxide, and endothelin receptor antagonists, result at least in part from restoring the balance between these mediators. A greater understanding of the role of the endothelium in PAH will presumably facilitate the evolution of newer, targeted therapies.     

肺动脉高压大鼠肺动脉平滑肌上calponin和TGFβ1的变化

各种病因所致的肺动脉高压(pulmonary artery hyperten-sion,PAH)是临床的常见病症,它是慢性阻塞性肺疾患(chronic obstructive pulmonary disease,COPD)发展成肺心病的中心环节[1]。PAH的发病机制相当复杂,但COPD所致的PAH主要是由于肺血管阻力增加所引起[2]。血管内皮细胞通过合成、代谢、分泌多种因子,调控血管平滑肌的舒缩和增殖。调宁蛋白(calponin)为一种平滑肌特有的调控蛋白,它的功能包括抑制平滑肌收缩、参与细胞信号转导和维持细胞骨架等[3]。转化生长因子β1(TGFβ1)是调控细胞增殖、分化的重要因子,能促进血管平滑肌细…     

舒马曲坦对大鼠肺动脉平滑肌细胞的促有丝分裂作用

目的　观察 5 HT1B/1D受体激动剂舒马曲坦对肺动脉平滑肌细胞 (PASMC)的促有丝分裂作用 ,探讨其作用机制和诱发肺血管构型重建的可能性。方法　分离培养大鼠PASMC ,用噻唑蓝 (MTT)法检测细胞增殖 ,用流式细胞术法分析细胞周期和DNA合成。结果　MTT法检测 5 羟色胺 (5 HT) 0 .0 1,0 .1,1.0 μmol·L- 1可促进PASMC增殖 ,增殖率分别增加2 0 1% ,2 2 8%和 2 5 6 %。舒马曲坦 0 .0 1,0 .1,1.0μmol·L- 1可促进PASMC增殖 ,增殖率分别增加199% ,2 2 0 %和 2 4 5 %。流式细胞术分析 5 HT 0 .1和1.0 μmol·L- 1组的细胞增殖指数分别为 2 2 .2 %和2 5 .9% ,S期细胞分数分别为 7.2 %和 9.8%。舒马曲坦 0 .1和 1.0 μmol·L- 1组的增殖指数分别为2 1.2 %和 2 3.9% ,S期细胞分数分别为 6 .6 %和8.8% ,均较对照组明显增高 (P <0 .0 5 )。 5 HT和舒马曲坦能够促进PASMC从G0 /G1期进入S期 ,5 HT的促有丝分裂作用可能有 5 HT1B/1D受体的参与。结论　舒马曲坦能促进PASMC的增殖。 5 HT1B/1D 受体在PASMC增殖和肺血管构型重建中有重要作用。舒马曲坦有致肺动脉高压的风险     

KATP通道开放剂吡那地尔对兔肺动脉平滑肌细胞增殖的影响

目的 :研究吡那地尔 (pinacidil,Pin)对内皮素 1(ET 1)诱导培养的兔肺动脉平滑肌细胞 (PASMC)增殖的影响。方法 :内皮素 1刺激培养兔PASMC增殖模型 ;以氚 胸腺嘧啶核苷 ([3 H] TdR)掺入法观察细胞增殖及脱氧核糖核苷酸 (DNA)合成 ;流式细胞仪技术检测兔PASMC细胞周期。结果 :吡那地尔可剂量依赖性的抑制内皮素 1所致的 [3 H] TdR掺入量增多 ,阻止兔PASMC由静止期 (G0 G1期 )进入DNA合成期 (S期 )和有丝分裂期 (G2 M期 )。ATP敏感性钾通道 (KATP)阻断剂格列本脲可拮抗吡那地尔对 [3 H] TdR掺入的抑制作用。结论 :吡那地尔可能通过激活KATP通道抑制内皮素 1诱导兔肺动脉平滑肌细胞的增殖 ,可望用于治疗肺动脉高压时所致的肺动脉重构。     

Endothelin-1 and IP3 induced Ca2+ sparks in pulmonary arterial smooth muscle cells

Endothelin-1 has been implicated as an important modulator or mediator of acute and chronic hypoxic pulmonary hypertension. It has been shown that endothelin-1 increases [Ca2+]i and contraction in pulmonary arterial smooth muscle cells. Recently, we have identified local Ca2+ release transients or Ca2+ sparks, which represent Ca2+ release from clusters of ryanodine receptors on the sarcoplasmic reticulum, in pulmonary arterial smooth muscle cells. These pulmonary Ca2+ sparks were associated with membrane depolarization, and activated specifically by endothelin-1 via endothelin-A receptor activation of phospholipase C and inositol trisphosphate production, possibly through local Ca2+ signaling between inositol trisphosphate receptors and ryanodine receptors. To test this hypothesis, we measured Ca2+ sparks in intralobar pulmonary arterial smooth muscle cells using laser scanning microscopy, and compared the spatiotemporal properties of Ca2+ sparks activated by endothelin-1 and by photorelease of inositol trisphosphate. We found that both endothelin-1 and inositol trisphosphate had similar effects on Ca2+ sparks. They both increased spark frequency, elevated spark amplitude and prolonged duration, without any significant effect on the spatial spread or size of Ca2+ sparks. These results provide further support to the suggestion that inositol trisphosphate production stimulated by endothelin-1 can account for the activation of Ca2+ sparks in pulmonary arterial smooth muscle cells.