间尼索地平对野百合碱诱导的肺动脉高压的防治作用及机制研究
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摘要
肺动脉高压(PAH)是以肺动脉压力升高以及血管痉挛、内膜增生和重构为主要特征的一种疾病。其发病机制尚未完全明确,目前观点认为,肺动脉高压形成的关键在于肺血管收缩和重构。肺动脉高压时,肺血管发生重构主要是由于血管平滑肌细胞增殖凋亡失衡引起的,因此抑制肺血管收缩,改善肺循环血流动力学以及阻止肺血管平滑肌细胞异常增殖是治疗肺动脉高压的关键。
钙信号转导在肺动脉高压发生发展过程中起着非常重要的作用,细胞内钙离子浓度([Ca2+]i)的变化是触发细胞增殖相关信号转导的始动因素之一。在PAH发生过程中,5-HT等细胞因子激动相应的细胞表面受体后,都要通过胞质内游离钙浓度增高这一信号转导途径发挥作用,因此,[Ca2+]i的变化在PAH的形成过程中具有重要作用。
钙拮抗剂是近年来临床常用的抗肺动脉高压药物,但关于其逆转肺动脉高压的确切机制尚不甚明了。间尼索地平(m-Nis)是我校药学院合成的二氢吡啶类钙拮抗剂,前期研究表明其具有良好的稳定性以及抑制血管收缩的作用。本实验以野百合碱(MCT)诱导的大鼠肺动脉高压为模型,观察m-Nis对肺动脉高压的防治作用及肺血管重构的改善作用。还通过细胞免疫化学、流式细胞术、激光共聚焦及细胞分子生物学等技术研究m-Nis对5-HT诱导的肺动脉平滑肌细胞增殖、迁移的影响,深入探讨其作用机制。
第一部分m-Nis对野百合碱诱导的大鼠肺动脉高压的防治作用
目的:研究m-Nis对野百合碱诱导的大鼠肺动脉高压的防治作用及其作用机制。
方法:大鼠单剂量皮下注射MCT(60mg/kg)制备肺动脉高压模型。导管法测定肺血流动力学指标。光镜观察肺小动脉结构的改变。按试剂盒步骤测定血清中MDA含量及SOD活性。免疫印迹法测定PCNA、ERK1/2和p-ERK1/2的表达水平。免疫组化法观察5-HT和PCNA的表达。
结果:
1 m-Nis对野百合碱诱导的肺动脉高压大鼠平均肺动脉压(PAP)及其它血流动力学指标的影响
模型组PAP(17.4±1.6 mmHg)比对照组(10.5±1.6 mmHg)显著升高(P<0.01);与模型组相比,m-Nis 0.5, 1.0和2.0 mg/kg组的PAP分别降至14.4±1.1,14.0±1.3及13.6±0.9mmHg(P<0.01)。而平均颈动脉压(SAP)及心率在各组之间并无显著差异。
2 m-Nis对肺动脉高压大鼠右室肥厚的影响
与对照组相比,模型组大鼠体重显著降低(P<0.05),右心室重量/体重(RV/BW)明显升高(从0.79±0.09升至1.05±0.09,P<0.01),m-Nis 0.5, 1.0和2.0mg/kg对大鼠体重并无改善作用,却使RV/BW分别降至0.90±0.04,0.83±0.07,0.85±0.09(P<0.05或P<0.01,与模型组相比)。RV/(LV+SP)测定结果表明:与对照组相比,模型组大鼠RV/(LV+SP)明显升高(从0.307±0.007升至0.408±0.015,P<0.01),而m-Nis 0.5, 1.0, 2.0mg/kg组的RV/(LV+SP)分别降至0.365±0.013, 0.341±0.023和0.353±0.021(P<0.01,与模型组相比)。
3 m-Nis对肺动脉高压大鼠肺小动脉构型改变的影响
光镜下观察HE染色切片,发现模型组肺小动脉中膜厚度增加,血管管腔狭窄甚至闭塞。与对照组相比,MT%显著增加(P<0.01),VA%明显减少(P<0.01),而m-Nis各组均不同程度地减轻肺组织损伤程度,肺小动脉中膜增厚程度也有明显减轻,与模型组相比, MT%明显降低(P < 0.05或P < 0.01),而VA%明显增加(P < 0.01)。
4 m-Nis对肺动脉高压大鼠血清中SOD活性及MDA含量的影响
与对照组相比,模型组大鼠血清中MDA含量显著升高(从6.32±0.27升至7.62±0.63nmol/ml,P<0.01),SOD活性明显降低(从163±10降至122±11 U/ml,P < 0.01)。而m-Nis 0.5,1.0,2.0mg/kg组均不同程度地逆转了上述反应,MDA含量分别降至6.49±0.82,6.76±0.38及6.40±0.60(P< 0.05),SOD活性相应升至142±12(P<0.05),149±8和146±11(P<0.01)。
5 m-Nis对肺动脉高压大鼠肺组织中5-HT表达的影响
与对照组相比,模型组大鼠肺组织中5-HT阳性细胞百分率显著增加(从4.2±2.1%增至14.2±3.0%,P<0.01)。m-Nis 0.5,1.0,2.0mg/kg组5-HT阳性细胞百分率分别降至8.6±1.3%,7.1±1.9%和7.2±1.0%(P<0.01,与模型组相比)。
6 m-Nis对细胞增殖核抗原(PCNA)表达的影响
PCNA免疫组化染色结果表明,与对照组相比,模型组大鼠肺组织中PCNA阳性细胞百分数显著增加(P<0.01),胞核染色较深。而m-Nis各组PCNA阳性细胞百分数均明显减少,与模型组相比有明显差异(P<0.01)。western blot结果分析表明,与对照组相比,模型组大鼠肺动脉中PCNA蛋白表达显著增加(P<0.01),而m-Nis 0.5,1.0和2.0mg/kg组PCNA蛋白表达水平明显降低(P <0.05,与模型组相比)。
7 m-Nis对ERK1/2磷酸化水平的影响
western blot结果表明,与对照组相比,模型组大鼠肺动脉中p-ERK1/2蛋白表达显著增加(P<0.01),各浓度m-Nis均能明显抑制其表达(P<0.01)。而总ERK1/2的表达水平在各组之间并无显著性差异。各组条带灰度值计算结果表明,模型组p-ERK1/2/ ERK1/2的比值明显增加(P<0.01,与对照组相比),而m-Nis可明显降低此比值(P<0.05,与模型组相比)。
结论:m-Nis能明显降低肺动脉高压大鼠的平均肺动脉压,改善右室肥厚程度及肺小动脉构型改变,对野百合碱诱导的大鼠肺动脉高压有一定的防治作用,可能与其抗氧化作用,降低5-HT的表达及抑制ERK1/2 MAPK信号通路有关。
第二部分m-Nis对5-HT诱导大鼠肺动脉平滑肌细胞增殖、迁移及细胞内游离钙浓度变化的影响
目的:研究m-Nis对5-HT诱导大鼠肺动脉平滑肌细胞增殖、迁移及细胞内游离钙浓度变化的影响
方法:组织块种植法原代培养大鼠PASMCs。MTT法测定PASMCs增殖。Transwell迁移小室(8μm孔径)测定PASMCs的迁移能力。流式细胞仪测定细胞周期。免疫细胞化学染色及western blot法测定PCNA蛋白表达。激光共聚焦显微镜检测平滑肌细胞内Ca2+的变化。定磷法测定CaN活性。western blot法测定ERK1/2及JNK的磷酸化水平。RT-PCR法检测c-fos、c-jun、CaM及CaN mRNA的表达。
结果:
1 m-Nis平对5-HT诱导的PASMCs增殖的影响
MTT结果表明,与对照组比,5-HT组A值明显升高(从0.48±0.04升至0.74±0.09,P <0.01)。与5-HT组比,m-Nis 10-5,10-6,10-7,10-8 mol/L组A值分别下降至0.54±0.05,0.57±0.07,0.59±0.06(P <0.01),0.62±0.07 (P <0.05)。western blot结果显示,与对照组相比,5-HT组PASMCs的PCNA表达明显增加(P<0.01)。不同浓度m-Nis预处理,均明显降低5-HT诱导的PCNA的表达(P<0.05或P<0.01)。PCNA免疫细胞化学染色结果表明,5-HT处理组PCNA阳性细胞百分率明显高于对照组(P<0.01)。不同浓度m-Nis预处理后,均明显降低5-HT诱导的PCNA的阳性表达(P<0.01)。
2 m-Nis对5-HT诱导的PASMCs细胞迁移的影响
与对照组比,5-HT(1μmol/L)能明显促进PASMCs迁移,平均每个视野下的迁移细胞数从对照组的34±7增加至95±13(P<0.01)。与5-HT组比,m-Nis 10-5,10-6,10-7,10-8 mol/L组平均每个视野下的迁移细胞数分别下降至43±9,44±10,58±9,62±9(P <0.01)。
3 m-Nis对PASMCs细胞周期的影响
与对照组比,5-HT组G0/G1期PASMCs百分率明显减少(从84%降至55%,P < 0.01),S期PASMCs百分率明显增加(从4%增至21%,P < 0.01),PI值也相应增加(从16%增至45%,P <0.01),提示5-HT能诱导PASMCs增殖。与5-HT组比,m-Nis 10-5,10-6,10-7 mol/L组G0/ G1期PASMCs百分率分别增至78%(P < 0.01),71%和64%(P < 0.05),S期PASMCs百分率也分别降至6%,10%和11%(P < 0.05或P < 0.01),G2/M期细胞无显著变化,说明m-Nis作用后使细胞停滞于G0/G1期。PI值分别降至22%,29%和36%(P <0.05或P <0.01)。而m-Nis 10-8 mol/L对细胞周期及PI值均无明显影响。
4 5-HT对培养的大鼠PASMCs内游离钙离子浓度的影响
Flou-3/AM负载的肺动脉平滑肌细胞图象清晰,可较好地显示细胞内游离钙离子浓度的变化过程,在加入1μmol/L 5-HT后,荧光强度增强,表明细胞内钙离子浓度迅速升高,在90s左右达到峰值,然后减弱,在200s左右荧光强度达到稳定的状态,而后有一个较长的平台期。
5 m-Nis对5-HT诱导大鼠PASMCs内[Ca2+]i升高的影响
在5-HT组,达峰值时其相对荧光强度(FI-F0)/F0为132.4±4.3%,与对照组相比明显升高(P<0.01)。与5-HT组相比,m-Nis 10-5,10-6,10-7,10-8 mol/L均能明显抑制5-HT诱导的细胞内钙离子浓度的升高,达峰值时相对荧光强度(FI-F0)/F0分别为20.1±4.1%,33.6±9.0%,41.5±2.3%和54.7±2.2%(P<0.01)。
6 m-Nis对5-HT诱导大鼠肺动脉平滑肌细胞CaN活性的影响
与对照组相比,5-HT组CaN活性明显升高(从1.7±0.5升至3.7±0.8μmol Pi/mg pro/h,P < 0. 01),m-Nis 10-5, 10-6,10-7,10-8mol/L均不同程度地降低了5-HT诱导的PASMCs中CaN活性的升高(分别降至2.4±0.6,2.9±0.6,3.0±0.7和3.1±0.5,P< 0. 05或P<0.01)。
7 m-Nis对5-HT诱导大鼠PASMCs内CaM、CaN mRNA表达的影响
与对照组相比,1μmol/L 5-HT作用24小时后明显诱导大鼠PASMCs内CaM及CaN mRNA表达水平升高(P < 0.01),加入m-Nis 10-5,10-6,10-7 mol/L预处理均明显抑制了5-HT诱导的CaM mRNA表达水平的升高(P < 0.05或P < 0.01),而m-Nis 10-8 mol/L抑制作用不明显。m-Nis 10-5,10-6,10-7,10-8 mol/L均明显抑制了5-HT诱导的CaN mRNA表达水平的升高(P < 0.05或P < 0.01)。
8 m-Nis对5-HT诱导的大鼠PASMCs中ERK1/2及JNK磷酸化的影响
western blot结果显示,1μmol/L 5-HT在5至30分钟内诱导大鼠动脉平滑肌细胞ERK1/2及JNK磷酸化水平明显升高,在15分钟时磷酸化水平最高。而m-Nis可浓度依赖性地拮抗5-HT的作用。m-Nis 10-5,10-6,10-7 mol/L可明显抑制5-HT诱导的ERK1/2磷酸化水平的升高(P < 0.05或P < 0.01),而m-Nis 10-8 mol/L的抑制作用并不明显。10-8 ~10-5 mol/L m-Nis均可抑制5-HT诱导的JNK磷酸化水平的升高(P < 0.05或P < 0.01)。
9 m-Nis对5-HT诱导的大鼠PASMCs中c-fos及c-jun mRNA表达的影响
1μmol/L 5-HT在5至60分钟内能明显诱导大鼠肺动脉平滑肌细胞c-fos及c-jun mRNA表达水平升高,在30分钟时表达水平达最高。而不同浓度m-Nis均明显抑制了5-HT诱导的c-fos及c-jun mRNA表达水平的升高(P < 0.05或P < 0.01)。
结论:(1)m-Nis对5-HT诱导的肺动脉平滑肌细胞增殖和迁移有明显的抑制作用,可能与其抑制增殖细胞核抗原(PCNA)表达及细胞周期进程有关。(2)m-Nis明显抑制了5-HT引起的[Ca2+]i的升高、CaM和CaNmRNA的表达以及CaN活性的升高;对5-HT诱导的ERK1/2, JNK磷酸化水平也有不同程度的抑制,还明显抑制了增殖相关基因c-fos, c-jun mRNA的表达水平。由此,我们推测m-Nis抗5-HT诱导的PASMCs增殖作用可能还与抑制细胞内[Ca2+]i的增高从而阻断了5-HT所介导的ERK1/2, JNK MAPK信号通路有关。
第三部分RhoA/Rho激酶信号通路在5-HT刺激PASMCs增殖中的作用及m-Nis对此信号通路的影响
目的:研究RhoA/Rho激酶信号通路在5-HT刺激PASMCs增殖中的作用及m-Nis对此信号通路的抑制作用。
方法:组织块种植法原代培养大鼠PASMCs。MTT法测定PASMCs增殖。流式细胞仪测定细胞周期。western blot法测定PCNA蛋白表达及ERK1/2、JNK、MYPT1磷酸化水平。RT-PCR法检测RhoA、ROCK1、c-fos及c-jun mRNA的表达。结果:
1法舒地尔对5-HT诱导的PASMCs增殖的影响
MTT实验结果表明,与对照组比,5-HT组A值明显升高(从0.37±0.05升至0.61±0.03,P <0.01)。法舒地尔25、50、100μmol/L各组A值分别降至0.48±0.05,0.46±0.03和0.43±0.04(P <0.01)。western blot结果显示,与对照组比,5-HT组PCNA蛋白表达明显增多(P <0.01),而25、50、100μmol/L法舒地尔不同程度地降低了PCNA的蛋白表达(P<0.05或P<0.01)。
2法舒地尔对5-HT作用下PASMCs细胞周期的影响
与对照组比,5-HT组G0/G1期PASMCs百分率明显减少(从83%降至55%,P < 0.01), S期PASMCs百分率明显增加(从4%增至21%,P < 0.01),PI值也相应增加(从17%增至45%,P <0.01),提示5-HT能诱导PASMCs增殖。与5-HT组比,法舒地尔25,50,100μmol/L组G0/ G1期PASMCs百分率分别增至65%,70%和78%(P<0.05或P<0.01),S期PASMCs百分率也分别降至12%,11%和9%(P < 0.05或P < 0.01),PI值分别降至35%,30%和22%(P <0.05或P <0.01)。
3法舒地尔对5-HT诱导的ROCK激活的影响
与对照组比,1μmol/L 5-HT明显诱导了ROCK1 mRNA的表达(P < 0.01),法舒地尔25、50、100μmol/L预处理不同程度地降低了5-HT诱导的ROCK1 mRNA的表达(P < 0.05或P < 0.01)。western blot结果显示,1μmol/L 5-HT作用后明显升高了MYPT1的磷酸化水平,10分钟左右磷酸化水平最高(P < 0.01),法舒地尔25、50、100μmol/L不同程度地降低了5-HT诱导的MYPT1的磷酸化水平(P < 0.05或P < 0.01)。
4法舒地尔对5-HT作用下ERK1/2及JNK磷酸化水平的影响
与对照组比,5-HT作用15分钟明显诱导了ERK1/2的磷酸化(P<0.01),法舒地尔25、50、100μmol/L预处理对ERK1/2的磷酸化水平没有明显影响。而对于JNK的磷酸化水平,5-HT作用15分钟能明显诱导其升高(P < 0.01),法舒地尔25、50、100μmol/L预处理明显降低了JNK的磷酸化水平(P < 0.01)。另外,5-HT明显增加了核蛋白中p-ERK1/2的表达,15分钟左右表达水平最高(P <0.01),法舒地尔25、50、100μmol/L预处理明显降低了核蛋白中p-ERK1/2的表达(P < 0.01)。
5法舒地尔对5-HT作用下c-fos和c-jun mRNA表达的影响
与对照组比,5-HT作用30分钟明显诱导了c-fos及c-jun mRNA表达(P < 0.01)。法舒地尔25、50、100μmol/L预处理均明显抑制了5-HT诱导的c-fos (P < 0.01)及c-jun mRNA的表达(P < 0.05或P < 0.01)。
6 m-Nis对5-HT作用下PASMCs中RhoA/ROCK通路的影响
与对照组比,5-HT明显诱导了RhoA、ROCK1 mRNA的表达(P < 0.01),m-Nis10-5 mol/L明显降低5-HT诱导的RhoA mRNA的表达(P < 0.05)。m-Nis 10-5,10-6 mol/L还不同程度地降低了5-HT诱导的ROCK1 mRNA的表达(P < 0.05或P < 0.01)。western blot结果显示,5-HT作用10分钟明显升高了MYPT1的磷酸化水平(P < 0.01),m-Nis 10-5,10-6,10-7 mol/L预处理不同程度地降低了5-HT诱导的MYPT1的磷酸化水平(P < 0.05或P < 0.01),而m-Nis 10-8 mol/L对MYPT1的磷酸化水平无明显影响。
结论:(1)5-HT明显诱导了大鼠PASMCs中Rho激酶的表达及激活;Rho激酶抑制剂法舒地尔对5-HT诱导的PASMCs的增殖,细胞周期进展有明显的抑制作用,还不同程度地抑制了JNK的磷酸化,ERK1/2的核转位以及增殖相关基因c-fos、c-jun mRNA的表达。说明RhoA/ROCK通路在5-HT诱导的PASMCs增殖中起着重要作用。(2)m-Nis不同程度地抑制了5-HT诱导的RhoA, ROCK1 mRNA的表达以及Rho激酶的活化标志p-MYPT1的蛋白表达。说明m-Nis抗5-HT诱导的PASMCs的增殖作用可能与抑制RhoA/ROCK信号通路有关。
第四部分活性氧在5-HT刺激PASMCs增殖中的作用及m-Nis对此通路的影响
目的:研究活性氧(ROS)在5-HT刺激PASMCs增殖中的作用和m-Nis对ROS的产生及其下游信号通路的影响。
方法:组织块种植法原代培养大鼠PASMCs。激光共聚焦检测ROS生成量的变化。TAB法测定MDA含量。黄嘌呤氧化酶法测定SOD活性。MTT法测定PASMCs增殖。western blot法测定PCNA蛋白表达及ERK1/2、JNK磷酸化水平。
结果:
1 m-Nis对5-HT诱导的大鼠PASMCs内ROS生成的影响
1μmol/L的5-HT处理不同时间组,荧光强度均增强,5-HT处理15分钟组荧光强度最强,达对照组的288±54%(P<0.01),然后减弱,在5-HT处理120和240分钟组荧光强度达到稳定的状态,但仍明显高于对照组(P<0.01)。m-Nis 10-5,10-6,10-7,10-8mol/L均能明显抑制5-HT诱导的ROS的生成,在5-HT处理15分钟时荧光强度分别降为对照组的157±29%,173±38%,178±43%和215±45%(P<0.01)。
2 m-Nis对5-HT作用下大鼠PASMCs内SOD活性和MDA含量的影响
与对照组相比,5-HT处理组SOD活性明显降低(P < 0. 01),MDA含量明显升高(P < 0. 01)。m-Nis 10-5,10-6,10-7mol/L预处理均不同程度地逆转了5-HT诱导的PASMCs SOD活性的降低(P < 0. 05或P < 0. 01),同时也降低了MDA的含量(P < 0. 05或P < 0. 01),而m-Nis 10-8mol/L对SOD的活性没有明显影响,但可降低MDA的含量(P < 0.05)。
3 m-Nis平对H2O2诱导的大鼠PASMCs增殖的影响
MTT实验结果表明,100μmol/L H2O2能明显促进大鼠PASMCs增殖,与对照组比,H2O2处理组A值明显提高(P <0.01)。与H2O2处理组比,m-Nis各组A值均不同程度降低(P <0.05或P <0.01)。western blot结果显示,与对照组相比,H2O2处理组PASMCs的PCNA表达明显增加(P<0.01)。不同浓度m-Nis预处理后,均明显降低H2O2诱导的PCNA的表达(P<0.05或P<0.01)。
4 m-Nis对H2O2诱导的PASMCs中ERK1/2及JNK磷酸化水平的影响
与对照组相比,100μmol/L H2O2作用15分钟时能明显升高大鼠脉平滑肌细胞ERK1/2及JNK磷酸化水平(P<0.01)。m-Nis 10-5,10-6,10-7 mol/L可明显抑制H2O2诱导的ERK1/2及JNK磷酸化水平的升高(P < 0.05或P < 0.01),而m-Nis 10-8mol/L仅明显抑制了ERK1/2磷酸化水平的升高(P < 0.05),对JNK磷酸化水平无明显影响。
结论:(1)5-HT明显诱导了大鼠PASMCs中ROS的生成,H2O2不仅诱导了PASMCs增殖,还提高了ERK1/2及JNK的磷酸化水平。说明ROS在5-HT诱导的PASMCs增殖中起重要作用。(2)m-Nis不仅抑制了ROS的生成,还明显降低了H2O2诱导的PASMCs增殖及ERK1/2、JNK的磷酸化水平。说明m-Nis抗5-HT诱导的PASMCs增殖作用不仅与其抑制ROS的产生有关,还与其抑制ROS所介导的ERK1/2,JNK MAPK信号通路有关。
总结
1 m-Nis对野百合碱诱导的大鼠肺动脉高压有一定的防治作用,可能与其抗氧化作用,降低5-HT的表达及抑制ERK1/2 MAPK信号通路有关。
2 m-Nis对5-HT诱导的肺动脉平滑肌细胞增殖和迁移有明显的抑制作用,可能与其抑制细胞内[Ca2+]i的增高从而阻断了5-HT所介导的ERK1/2,JNK MAPK信号通路有关。
3 Rho激酶抑制剂法舒地尔明显抑制了5-HT诱导的PASMCs增殖,说明RhoA/ROCK通路在5-HT诱导的PASMCs增殖中起重要作用。
4 m-Nis不同程度地抑制了5-HT诱导的RhoA, ROCK1 mRNA的表达以及Rho激酶的活化标志p-MYPT1的蛋白表达。说明m-Nis抗5-HT诱导的PASMCs的增殖作用可能与抑制RhoA/ROCK信号通路有关。
5 ROS在5-HT诱导的PASMCs增殖中起重要作用。m-Nis抗5-HT诱导的PASMCs增殖作用不仅与其抑制ROS的产生有关,还与其抑制ROS所介导的ERK1/2,JNK MAPK信号通路有关。
Pulmonary artery hypertension (PAH) is a progressive and fatal disease characterized by an increase of vascular resistance, abnormal sustained pulmonary vasoconstriction, and progressive structural remodeling of pulmonary arteries, for which no clear pathogenesy has been developed. Recent studies suggest that constriction and remodeling of pulmonary arteries are the key point of PAH formation. The disequilibrium of proliferation of PASMCs and apoptosis results in pulmonary arteries remodeling. Therefore, the key points of PAH therapy include inhibiting the pulmonary artery constriction, improving the pulmonary circulation and preventing the proliferation of PASMCs.
Calcium signals play an important role in the formation of PAH. Studies suggest that the elevation of [Ca2+]i is one of the most important initiation agents to trigger proliferation of PASMCs. In the progress of PAH, many cell factors including 5-HT can elevate the [Ca2+]i after they bind to their receptors. Therefore, the elevation of [Ca2+]i is important to PAH.
Recently, calcium antagonists are frequently used in the treatment of PAH in clinic, but the clear mechanisms have not been elucidated. M-nisoldipine (m-Nis), a Ca2+ channel blocker of dihydropyridines (DHPs), is developed by Hebei Medical University. Early studies suggested that m-Nis had favourable stability and could inhibit vasoconstriction. Therefore, in the present study, monocrotaline (MCT) was use to induce PAH in rats, then the effect of m-Nis on PAH and pulmonary artery remodeling were studied. The methods such as immunhistochemistry, flow cytometry, confocal laser scanning microscope and molecular biology techniques were applied to explore the effect of m-Nis on 5-HT induced proliferation and migration of PASMCs and the mechanisms. Part 1 The effect of m-Nis on MCT-induced PAH in Rats
Objective: To study the effect of m-Nis on MCT-induced PAH in rats.
Methods: Rats were injected with a single dose (60mg/kg) of MCT subcutaneously to induce PAH. Pulmonary haemodynamic measurement and lung tissue morphological investigations were undertaken. The MDA production and SOD activity in the serum were tested. Expression of PCNA, ERK1 and p-ERK1/2 expression in pulmonary blood vessels was analyzed by western blot. The expression of 5-HT and PCNA in pulmonary tissues was observed by immunohistochemistry.
Results:
1 Effect of m-Nis on PAP and hemodynamic variables in MCT-induced PAH rats
Compared with the control group, mean PAP in the MCT group was significantly elevated (from 10.5±1.6 to 17.4±1.6mmHg, P<0.01). Compared with the MCT group, mean PAP in m-Nis 0.5, 1.0 and 2.0 mg/kg groups was decreased to 14.4±1.1,14.0±1.3 and 13.6±0.9 mmHg(P<0.01), respectively。Yet, SAP and heart rate did not differ significantly among the five groups.
2 Effect of m-Nis on right ventricle hypertrophy in MCT-induced PAH rats
Body weight was significantly decreased by MCT injection (P<0.05). The ratio of right ventricular weight to body weight (RV/BW) was increased by MCT from 0.79±0.09 to 1.05±0.09 (P<0.01 vs control) and reduced by m-Nis 0.5, 1.0, 2.0mg/kg to 0.90±0.04 (P<0.05 vs MCT), 0.83±0.07 and 0.85±0.09 (P<0.01 vs MCT) respectively. Meanwhile, the right ventricular index RV/(LV+SP) was increased by MCT from 0.307±0.007 to 0.408±0.015 (P<0.01 vs control) and reduced obviously by m-Nis 0.5, 1.0, 2.0mg/kg to 0.365±0.013, 0.341±0.023 and 0.353±0.021 respectively (P<0.01 vs MCT).
3 Effect of m-Nis on the morphological alterations of small pulmonary arteries
Small pulmonary arteries in MCT group exhibited obvious medial wall thickening and lumen transverse area reducing, the MT% markedly increased, whereas the VA% decreased compared with those in control group (P<0.01). In m-Nis-treated groups, the pulmonary artery wall thickening was attenuated in different degrees, the MT% was decreased in m-Nis 0.5 mg/kg group (P<0.05) and m-Nis 1.0, 2.0 mg/kg groups (P<0.01) compared with that in MCT group. Meanwhile, the VA% was significantly increased in all the m-Nis groups compared with that in MCT group (P<0.01).
4 Effect of m-Nis on the activity of SOD and the content of MDA
Compared with control group, the content of MDA in MCT group was significantly elevated from 6.32±0.27 to 7.62±0.63 (P<0.01), and the activity of SOD was decreased from 163±10 to 122±11 (P<0.01). Compared with the MCT group, a significant reduction in MDA production was observed in all three m-Nis 0.5, 1.0 and 2.0mg/kg groups (from 7.62±0.63 to 6.49±0.82, 6.76±0.38 and 6.40±0.60 respectively) (P<0.05), which was associated with the increase of SOD activity (from 122±11 to 142±12, 149±8 and 146±11 respectively) (P<0.05 or P<0.01).
5 5-HT immunohistochemistry assessment
In MCT group, the percentage of 5-HT-positive cells (14.2±3.0%) was higher than that in control group (4.2±2.1%) (P<0.01), which was reduced to 8.6±1.3%,7.1±1.9% and 7.2±1.0% by m-Nis 0.5, 1.0 and 2.0mg/kg treatment (P<0.01 vs MCT group).
6 Effect of m-Nis on the expression of PCNA
From the result of immunohistological staining, we found that in MCT group the percentage of PCNA-positive cells was remarkably increased compared with the control group (P<0.01), which was decreased by pretreatment with m-Nis (P<0.01). Western blot analysis indicated that the expression of PCNA was significantly higher in MCT group than that in control group (P<0.01), which was markedly decreased (P<0.05 vs MCT group) by m-Nis 0.5, 1.0 and 2.0mg/kg pretreatment.
7 Effect of m-Nis on the activation of ERK1/2
Western blot analysis showed that p-ERK1/2 expression in MCT group exhibited a significant increase compared with that in control group (P<0.01), which was obviously decreased by m-Nis 0.5, 1.0 and 2.0mg/kg (P<0.01). About the expression of ERK1/2, no statistical difference was found among the five groups. So, the ratio of p-ERK1/2/ERK1/2 increased significantly in MCT group compared with that in control group (P<0.01), which was decreased significantly by m-Nis (P<0.05).
Conclusion: M-Nis protects against MCT-induced pulmonary artery hypertension, which may be related to the antioxidation effect, the reduction of 5-HT exression and the suppression of the ERK/MAPK signal pathway. Part 2 The effect of m-Nis on 5-HT-induced proliferation, migration of PASMCs and change of [Ca2+]i
Objective: To study the effect of m-Nis on 5-HT-induced proliferation, migration of PASMCs and change of [Ca2+]i.
Methods: PASMCs were cultured with the explant technique. The proliferation of PASMCs was evaluated by MTT assay. Transwell chambers were used to detect the migration. Cell-cycle distribution was determined by flow cytometry assay. The expression of PCNA was evaluated by immunocytochemical stain and western blot. The change of [Ca2+]i was measured by confocal microscopy. The activity of CaN was measured using kits. The phosphorylation of ERK1/2 and JNK was detected by western blot. The mRNA expression of c-fos, c-jun, CaM and CaN was evaluated by RT-PCR.
Results:
1 Effect of m-Nis on 5-HT-induced proliferation of PASMCs
The results of MTT assay suggested that 5-HT significantly increased the A value from 0.48±0.04 to 0.74±0.09 (P<0.01), which was decreased to 0.54±0.05,0.57±0.07,0.59±0.06 (P<0.01) and 0.62±0.07 (P<0.05) by m-Nis 10-5,10-6,10-7,10-8 mol/L, respectively. Similarly, western blot analysis of PCNA indicated that the expression of PCNA was significantly higher in 5-HT group than that in control group (P <0.01). Whereas, in four m-Nis treated groups, the level of PCNA was markedly decreased (P<0.05 or P<0.01). Next, the results of immunocytochemical stain suggested that in 5-HT group the percentage of PCNA-positive cells was remarkably increased compared with that in control group (P < 0.01), which was decreased by pretreatment with m-Nis 10-5,10-6,10-7 and 10-8 mol/L (P < 0.01).
2 Effect of m-Nis on 5-HT-induced PASMCs migration
Compared with control group, the number of migration cells increased from 34±7 to 95±13 (P<0.01) after 5-HT treatment, which was decreased to 43±9,44±10,58±9 and 62±9 (P <0.01) by m-Nis 10-5,10-6,10-7 and 10-8 mol/L pretreatment, respectively.
3 Effect of m-Nis on cell-cycle progression
The percentage of cells in S phase increased dramatically from 4% to 21% after 5-HT stimulation (P<0.01) and was accompanied by a reduction in the percentage of cells in G1 phase from 84% to 55% (P<0.01). M-Nis 10-5,10-6,10-7 mol/L markedly blocked 5-HT-induced cell-cycle progression, with the percentages of PASMCs entering the S phase decreasing to 6%, 10% and 11% (P<0.05 or P<0.01), and those arrested in the G0/G1 phase increasing to 78%, 71% and 64% respectively (P<0.05 or P<0.01). Meanwhile, the proliferation index increased significantly from 16% to 45% after 5-HT stimulation (P<0.01) and was decreased to 22%, 29% and 36% respectively by preincubation with m-Nis 10-5,10-6,10-7 mol/L (P<0.05 or P<0.01).
4 Effect of 5-HT on fluorescence intensity of [Ca2+]i in rat PASMCs
The fluorescence intensity that represented the [Ca2+]i of PASMCs was significantly increased and reached the peak at 90 s after treated with 5-HT. After that, the [Ca2+]i decreased gradually and reached to a stable state at 200 s after 5-HT treatment.
5 Effect of m-Nis on [Ca2+]i in rat PASMCs
The relative fluorescence intensity of intracellular Ca2+ was significantly increased from 3.0±1.0% to 132.4±4.3% after perfusion with 5-HT (P <0.01 vs control), which was decreased to 20.1±4.1%,33.6±9.0%,41.5±2.3% and 54.7±2.2% (P<0.01) respectively by preincubation with m-Nis 10-5,10-6,10-7 and 10-8 mol/L.
6 Effect of m-Nis on the activity of CaN in rat PASMCs
The activity of CaN was significantly increased from 1.7±0.5 to 3.7±0.8 by 5-HT treatment (P <0.01 vs control), which was decreased to 2.4±0.6, 2.9±0.6 (P<0.01), 3.0±0.7 and 3.1±0.5 (P<0.05) respectively by preincubation with m-Nis 10-5, 10-6, 10-7 and 10-8 mol/L.
7 Effect of m-Nis on mRNA expression of CaM and CaN in rat PASMCs
The mRNA expression of CaM and CaN increased obviously after 1μmol/L 5 -HT treatment for 24h (P<0.01), which was significantly reduced (P<0.05 or P<0.01) by pretreatment with m-Nis 10-5, 10-6 and 10-7 mol/L. M-Nis 10-8 mol/L also significantly reduced the CaN mRNA expression, but have no effect on CaM mRNA expression.
8 Effect of m-Nis on the phosphorylation of ERK1/2 and JNK in rat PASMCs
Treatment of cells with 1μmol/L 5-HT caused phosphorylation of ERK1/2 and JNK with a peak at 15 min. M-Nis 10-5, 10-6 and 10-7 mol/L pretreatment reduced 5-HT-induced phosphorylation of ERK1/2 and JNK obviously (P<0.05 or P<0.01), and m-Nis 10-8 mol/L also significantly reduced the JNK phosphorylation, but have no significant effect on ERK1/2 phosphorylation.
9 Effect of m-Nis on mRNA expression of c-fos and c-jun in rat PASMCs
Treatment of cells with 1μmol/L 5-HT increased mRNA expression of c-fos and c-jun with a peak at 30 min, which was decreased in different degree by m-Nis 10-5, 10-6, 10-7 and 10-8 mol/L pretreatment (P<0.05 or P<0.01).
Conclusion: (1) M-Nis inhibited 5-HT-induced proliferation and migration of PASMCs, which may be related to the inhibition of PCNA expression and cell-cycle progression. (2) M-Nis inhibited 5-HT induced elevation of [Ca2+]i, mRNA expression of CaM, CaN, c-fos and c-jun, phosphorylation of ERK1/2 and JNK. So, the inhibition of m-Nis on proliferation of PASMCs may be related to the blockage of ERK1/2, JNK MAPK signal pathway through inhibiting the elevation of [Ca2+]i.
Part 3 The activation of RhoA/ROCK signal pathway in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathway
Objective: To study the the activation of RhoA/ROCK signal pathway in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathway.
Methods: PASMCs were cultured with the explant technique. The proliferation of PASMCs was evaluated by MTT assay. Cell-cycle distribution was determined by flow cytometry assay. The expression of PCNA, the phosphorylation of ERK1/2, JNK and MYPT1 was detected by western blot. The mRNA expression of Rho A, ROCK1, c-fos and c-jun was evaluated by RT-PCR.
Results:
1 Effect of fasudil on 5-HT-induced proliferation of PASMCs
The results of MTT assay suggested that 1μmol/L 5-HT significantly increased the A value from 0.37±0.05 to 0.61±0.031 (P<0.01), which was decreased to 0.48±0.05 , 0.46±0.03 and 0.43±0.04 by fasudil 25, 50, 100μmol/L pretreatment respectively (P<0.01). Similarly, western blot analysis of PCNA indicated that the expression of PCNA was higher in 5-HT group than that in control group (P <0.01), which was inhibited in different degree (P<0.05 or P<0.01) by pretreatment of PASMCs with fasudil.
2 Effect of fasudil on cell-cycle progression
The percentage of cells in S phase increased dramatically from 4% to 21% after 5-HT stimulation (P<0.01) and was accompanied by a reduction in the percentage of cells in G1 phase from 83% to 55% (P<0.01). Fasudil 25, 50, 100μmol/L markedly blocked 5-HT-induced cell-cycle progression, with the percentages of PASMCs entering the S phase decreasing to 12%, 11% and 9% (P<0.05 or P<0.01), and those arrested in the G0/G1 phase increasing to 65%, 70% and 78% respectively (P<0.05 or P<0.01). Meanwhile, the proliferation index increased dramatically from 17% to 45% after 5-HT stimulation (P < 0.01) and was decreased to 35%, 30% and 22% (P<0.05 or P<0.01) respectively by preincubation with fasudil 25, 50 and 100μmol/L.
3 Fasudil inhibited 5-HT- mediated ROCK activation
5-HT induced a significant upregulation of ROCK1 mRNA expression compared with that in quiescent PASMCs (P<0.01), and pretreatment with fasudil 25, 50, 100μmol/L resulted in a significant reduction in ROCK1 mRNA expression (P<0.05 or P<0.01). 5-HT caused phosphorylation of MYPT1, with a peak at 10 min (P<0.01), which was inhibited by fasudil in a concentration-dependent manner (P<0.05 or P<0.01).
4 Effect of fasudil on ERK1/2 and JNK phosphorylation
5-HT caused obvious phosphorylation of ERK1/2 and JNK with a peak at 15 min (P < 0.01). Pretreatment with fasudil 25, 50 and 100μmol/L did not block ERK1/2 phosphorylation, but reduced JNK phosphorylation obviously (P < 0.01). Next we examined the effect of fasudil on nuclear translocation of ERK1/2 induced by 5-HT. Result suggested that 5-HT increased nuclear translocation of ERK1/2 as determined by western blot analysis of nuclear-rich cellular fractions, with a peak at 15 min, consistent with its phosphorylation, which was reduced (P < 0.01) by pretreatment of cells with fasudil 25, 50 and 100μmol/L.
5 Effect of fasudil on mRNA expression of c-fos and c-jun
Treatment of cells with 1μmol/L 5-HT for 30 min increased mRNA expression of c-fos and c-jun (P< 0.01), which was decreased in different degree by fasudil 25, 50, 100μmo/L pretreatment (P < 0.05 or P<0.01).
6 Effect of m-Nis on 5-HT-mediated RhoA/ROCK signal pathway
5-HT induced a significant upregulation of mRNA expression of RhoA and ROCK1 (P < 0.01), which was reduced significantly (P < 0.05 or P < 0.01) by pretreatment with m-Nis 10-5 mol/L, m-Nis 10-6 mol/L also reduced ROCK1 mRNA expression (P < 0.05), but had no effect on RhoA mRNA expression. Phosphorylation of MYPT1 was induced by 5-HT with a peak at 10 min (P < 0.01), which was inhibited by m-Nis 10-5,10-6 and 10-7 mol/L in different degree (P < 0.05 or P < 0.01). However, m-Nis 10-8 mol/L had no effect on p-MYPT1 expression.
Conclusion: (1) 5-HT mediated ROCK activation, fasudil not only inhibited 5-HT-induced proliferation of PASMCs and cell-cycle progression, but also reduced JNK phosphorylation, nuclear translocation of ERK1/2 and c-fos, c-jun mRNA expression, which suggested that RhoA/ROCK signal pathway played an important role in 5-HT-induced proliferation of PASMCs. (2) M-Nis significantly inhibited 5-HT-induced RhoA, ROCK1 mRNA expression and the phosphorylation of MYPT1. So we conclude that the inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of RhoA/ROCK signal pathway.
Part 4 Effect of ROS in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathway
Objective: To study the effect of ROS in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathway.
Methods: PASMCs were cultured with the explant technique. ROS was measured by confocal microscopy with DCFH-DA. The MDA production and SOD activity were measured using kits supplied by Nanjing Jiancheng Biotechnological Company. The proliferation of PASMCs was evaluated by MTT assay. The expression of PCNA, the phosphorylation of ERK1/2 and JNK were detected by western blot.
Results:
1 Effect of m-Nis on the production of ROS in rat PASMCs
The fluorescence intensity of ROS was significantly increased and reached the peak at 15 min after the cells were treated with 5-HT (P<0.01). After that, the fluorescence intensity decreased gradually and reached to a stable state at 120 min and 240 min after 5-HT treatment, but still stronger than that in control group (P<0.01). Compared with control group, the fluorescence intensity of ROS increased by 288% 15 min after 5-HT treatment (P<0.01). Pretreatment with m-Nis 10-5, 10-6, 10-7 and 10-8 mol/L reduced the fluorescence intensity of ROS from 288±54% to 157±29%, 173±38%, 178±43% and 215±45%, respectively (P<0.01).
2 Effect of m-Nis on the activity of SOD and the content of MDA
Compared with the control group, the activity of SOD decreased obviously (P<0.01), and the content of MDA increased significantly (P<0.01) after 5-HT treatment. Compared with the 5-HT group, a significant reduction in MDA production was observed in m-Nis 10-5,10-6,10-7 mol/L groups (P<0.05 or P<0.01), which was associated with an increase in the SOD activity (P<0.05 or P<0.01). Meanwhile, m-Nis 10-8 mol/L also reduced the content of MDA (P<0. 05), but had no effect on the SOD activity.
3 Effect of m-Nis on H2O2-induced proliferation of PASMCs
The results of MTT assay suggested that 100μmol/L H2O2 significantly increased the A value (P<0.01), which was inhibited significantly (P <0.05 or P <0.01) by m-Nis 10-5,10-6,10-7 and 10-8 mol/L. Similarly, western blot analysis of PCNA indicated that the expression of PCNA was higher in H2O2 group than that in control group (P <0.01), which was also inhibited obviously by pretreatment of PASMCs with m-Nis (P<0.05 or P<0.01).
4 Effect of m-Nis on H2O2-induced phosphorylation of ERK1/2 and JNK
H2O2 caused obvious phosphorylation of ERK1/2 and JNK (P < 0.01), which was inhibited in different degree by m-Nis 10-5, 10-6 and 10-7 mol/L pretreatment (P < 0.05 or P < 0.01). Meanwhile, m-Nis 10-8 mol/L also inhibited the phosphorylation of ERK1/2 (P < 0.05), but had no effect on JNK activation.
Conclusion: (1) 5-HT induced the production of ROS obviously. H2O2 not only mediated proliferation of PASMCs, but also induced ERK1/2 and JNK phosphorylation, which suggested that ROS played an important role in 5-HT-induced proliferation of PASMCs. (2) M-Nis not only inhibited 5-HT-induced production of ROS, but also reduced the proliferation of PASMCs and phosphorylation of ERK1/2 and JNK induced by H2O2. So we conclude that the inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of ROS production and the downstream ERK1/2,JNK MAPK signal pathway.
CONCLUSIONS
1 M-Nis protects against MCT-induced pulmonary artery hypertension, which may be related to the antioxidation effect, the reduction of 5-HT exression and the suppression of the ERK/MAPK signal pathway.
2 M-Nis inhibited 5-HT-induced proliferation and migration of PASMCs, which may be related to the blockage of ERK1/2, JNK MAPK signal pathway through inhibiting the elevation of [Ca2+]i.
3 Fasudil inhibited 5-HT-induced proliferation of PASMCs obviously, which suggested that RhoA/ROCK signal pathway played an important role in 5-HT-induced proliferation of PASMCs.
4 M-Nis inhibited 5-HT-induced RhoA, ROCK1 mRNA expression and the phosphorylation of MYPT1 in different degree. So we conclude that the inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of RhoA/ROCK signal pathway.
5 ROS played an important role in 5-HT-induced proliferation of PASMCs. The inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of ROS production and the downstream ERK1/2,JNK MAPK signal pathway.
钙信号转导在肺动脉高压发生发展过程中起着非常重要的作用,细胞内钙离子浓度([Ca2+]i)的变化是触发细胞增殖相关信号转导的始动因素之一。在PAH发生过程中,5-HT等细胞因子激动相应的细胞表面受体后,都要通过胞质内游离钙浓度增高这一信号转导途径发挥作用,因此,[Ca2+]i的变化在PAH的形成过程中具有重要作用。
钙拮抗剂是近年来临床常用的抗肺动脉高压药物,但关于其逆转肺动脉高压的确切机制尚不甚明了。间尼索地平(m-Nis)是我校药学院合成的二氢吡啶类钙拮抗剂,前期研究表明其具有良好的稳定性以及抑制血管收缩的作用。本实验以野百合碱(MCT)诱导的大鼠肺动脉高压为模型,观察m-Nis对肺动脉高压的防治作用及肺血管重构的改善作用。还通过细胞免疫化学、流式细胞术、激光共聚焦及细胞分子生物学等技术研究m-Nis对5-HT诱导的肺动脉平滑肌细胞增殖、迁移的影响,深入探讨其作用机制。
第一部分m-Nis对野百合碱诱导的大鼠肺动脉高压的防治作用
目的:研究m-Nis对野百合碱诱导的大鼠肺动脉高压的防治作用及其作用机制。
方法:大鼠单剂量皮下注射MCT(60mg/kg)制备肺动脉高压模型。导管法测定肺血流动力学指标。光镜观察肺小动脉结构的改变。按试剂盒步骤测定血清中MDA含量及SOD活性。免疫印迹法测定PCNA、ERK1/2和p-ERK1/2的表达水平。免疫组化法观察5-HT和PCNA的表达。
结果:
1 m-Nis对野百合碱诱导的肺动脉高压大鼠平均肺动脉压(PAP)及其它血流动力学指标的影响
模型组PAP(17.4±1.6 mmHg)比对照组(10.5±1.6 mmHg)显著升高(P<0.01);与模型组相比,m-Nis 0.5, 1.0和2.0 mg/kg组的PAP分别降至14.4±1.1,14.0±1.3及13.6±0.9mmHg(P<0.01)。而平均颈动脉压(SAP)及心率在各组之间并无显著差异。
2 m-Nis对肺动脉高压大鼠右室肥厚的影响
与对照组相比,模型组大鼠体重显著降低(P<0.05),右心室重量/体重(RV/BW)明显升高(从0.79±0.09升至1.05±0.09,P<0.01),m-Nis 0.5, 1.0和2.0mg/kg对大鼠体重并无改善作用,却使RV/BW分别降至0.90±0.04,0.83±0.07,0.85±0.09(P<0.05或P<0.01,与模型组相比)。RV/(LV+SP)测定结果表明:与对照组相比,模型组大鼠RV/(LV+SP)明显升高(从0.307±0.007升至0.408±0.015,P<0.01),而m-Nis 0.5, 1.0, 2.0mg/kg组的RV/(LV+SP)分别降至0.365±0.013, 0.341±0.023和0.353±0.021(P<0.01,与模型组相比)。
3 m-Nis对肺动脉高压大鼠肺小动脉构型改变的影响
光镜下观察HE染色切片,发现模型组肺小动脉中膜厚度增加,血管管腔狭窄甚至闭塞。与对照组相比,MT%显著增加(P<0.01),VA%明显减少(P<0.01),而m-Nis各组均不同程度地减轻肺组织损伤程度,肺小动脉中膜增厚程度也有明显减轻,与模型组相比, MT%明显降低(P < 0.05或P < 0.01),而VA%明显增加(P < 0.01)。
4 m-Nis对肺动脉高压大鼠血清中SOD活性及MDA含量的影响
与对照组相比,模型组大鼠血清中MDA含量显著升高(从6.32±0.27升至7.62±0.63nmol/ml,P<0.01),SOD活性明显降低(从163±10降至122±11 U/ml,P < 0.01)。而m-Nis 0.5,1.0,2.0mg/kg组均不同程度地逆转了上述反应,MDA含量分别降至6.49±0.82,6.76±0.38及6.40±0.60(P< 0.05),SOD活性相应升至142±12(P<0.05),149±8和146±11(P<0.01)。
5 m-Nis对肺动脉高压大鼠肺组织中5-HT表达的影响
与对照组相比,模型组大鼠肺组织中5-HT阳性细胞百分率显著增加(从4.2±2.1%增至14.2±3.0%,P<0.01)。m-Nis 0.5,1.0,2.0mg/kg组5-HT阳性细胞百分率分别降至8.6±1.3%,7.1±1.9%和7.2±1.0%(P<0.01,与模型组相比)。
6 m-Nis对细胞增殖核抗原(PCNA)表达的影响
PCNA免疫组化染色结果表明,与对照组相比,模型组大鼠肺组织中PCNA阳性细胞百分数显著增加(P<0.01),胞核染色较深。而m-Nis各组PCNA阳性细胞百分数均明显减少,与模型组相比有明显差异(P<0.01)。western blot结果分析表明,与对照组相比,模型组大鼠肺动脉中PCNA蛋白表达显著增加(P<0.01),而m-Nis 0.5,1.0和2.0mg/kg组PCNA蛋白表达水平明显降低(P <0.05,与模型组相比)。
7 m-Nis对ERK1/2磷酸化水平的影响
western blot结果表明,与对照组相比,模型组大鼠肺动脉中p-ERK1/2蛋白表达显著增加(P<0.01),各浓度m-Nis均能明显抑制其表达(P<0.01)。而总ERK1/2的表达水平在各组之间并无显著性差异。各组条带灰度值计算结果表明,模型组p-ERK1/2/ ERK1/2的比值明显增加(P<0.01,与对照组相比),而m-Nis可明显降低此比值(P<0.05,与模型组相比)。
结论:m-Nis能明显降低肺动脉高压大鼠的平均肺动脉压,改善右室肥厚程度及肺小动脉构型改变,对野百合碱诱导的大鼠肺动脉高压有一定的防治作用,可能与其抗氧化作用,降低5-HT的表达及抑制ERK1/2 MAPK信号通路有关。
第二部分m-Nis对5-HT诱导大鼠肺动脉平滑肌细胞增殖、迁移及细胞内游离钙浓度变化的影响
目的:研究m-Nis对5-HT诱导大鼠肺动脉平滑肌细胞增殖、迁移及细胞内游离钙浓度变化的影响
方法:组织块种植法原代培养大鼠PASMCs。MTT法测定PASMCs增殖。Transwell迁移小室(8μm孔径)测定PASMCs的迁移能力。流式细胞仪测定细胞周期。免疫细胞化学染色及western blot法测定PCNA蛋白表达。激光共聚焦显微镜检测平滑肌细胞内Ca2+的变化。定磷法测定CaN活性。western blot法测定ERK1/2及JNK的磷酸化水平。RT-PCR法检测c-fos、c-jun、CaM及CaN mRNA的表达。
结果:
1 m-Nis平对5-HT诱导的PASMCs增殖的影响
MTT结果表明,与对照组比,5-HT组A值明显升高(从0.48±0.04升至0.74±0.09,P <0.01)。与5-HT组比,m-Nis 10-5,10-6,10-7,10-8 mol/L组A值分别下降至0.54±0.05,0.57±0.07,0.59±0.06(P <0.01),0.62±0.07 (P <0.05)。western blot结果显示,与对照组相比,5-HT组PASMCs的PCNA表达明显增加(P<0.01)。不同浓度m-Nis预处理,均明显降低5-HT诱导的PCNA的表达(P<0.05或P<0.01)。PCNA免疫细胞化学染色结果表明,5-HT处理组PCNA阳性细胞百分率明显高于对照组(P<0.01)。不同浓度m-Nis预处理后,均明显降低5-HT诱导的PCNA的阳性表达(P<0.01)。
2 m-Nis对5-HT诱导的PASMCs细胞迁移的影响
与对照组比,5-HT(1μmol/L)能明显促进PASMCs迁移,平均每个视野下的迁移细胞数从对照组的34±7增加至95±13(P<0.01)。与5-HT组比,m-Nis 10-5,10-6,10-7,10-8 mol/L组平均每个视野下的迁移细胞数分别下降至43±9,44±10,58±9,62±9(P <0.01)。
3 m-Nis对PASMCs细胞周期的影响
与对照组比,5-HT组G0/G1期PASMCs百分率明显减少(从84%降至55%,P < 0.01),S期PASMCs百分率明显增加(从4%增至21%,P < 0.01),PI值也相应增加(从16%增至45%,P <0.01),提示5-HT能诱导PASMCs增殖。与5-HT组比,m-Nis 10-5,10-6,10-7 mol/L组G0/ G1期PASMCs百分率分别增至78%(P < 0.01),71%和64%(P < 0.05),S期PASMCs百分率也分别降至6%,10%和11%(P < 0.05或P < 0.01),G2/M期细胞无显著变化,说明m-Nis作用后使细胞停滞于G0/G1期。PI值分别降至22%,29%和36%(P <0.05或P <0.01)。而m-Nis 10-8 mol/L对细胞周期及PI值均无明显影响。
4 5-HT对培养的大鼠PASMCs内游离钙离子浓度的影响
Flou-3/AM负载的肺动脉平滑肌细胞图象清晰,可较好地显示细胞内游离钙离子浓度的变化过程,在加入1μmol/L 5-HT后,荧光强度增强,表明细胞内钙离子浓度迅速升高,在90s左右达到峰值,然后减弱,在200s左右荧光强度达到稳定的状态,而后有一个较长的平台期。
5 m-Nis对5-HT诱导大鼠PASMCs内[Ca2+]i升高的影响
在5-HT组,达峰值时其相对荧光强度(FI-F0)/F0为132.4±4.3%,与对照组相比明显升高(P<0.01)。与5-HT组相比,m-Nis 10-5,10-6,10-7,10-8 mol/L均能明显抑制5-HT诱导的细胞内钙离子浓度的升高,达峰值时相对荧光强度(FI-F0)/F0分别为20.1±4.1%,33.6±9.0%,41.5±2.3%和54.7±2.2%(P<0.01)。
6 m-Nis对5-HT诱导大鼠肺动脉平滑肌细胞CaN活性的影响
与对照组相比,5-HT组CaN活性明显升高(从1.7±0.5升至3.7±0.8μmol Pi/mg pro/h,P < 0. 01),m-Nis 10-5, 10-6,10-7,10-8mol/L均不同程度地降低了5-HT诱导的PASMCs中CaN活性的升高(分别降至2.4±0.6,2.9±0.6,3.0±0.7和3.1±0.5,P< 0. 05或P<0.01)。
7 m-Nis对5-HT诱导大鼠PASMCs内CaM、CaN mRNA表达的影响
与对照组相比,1μmol/L 5-HT作用24小时后明显诱导大鼠PASMCs内CaM及CaN mRNA表达水平升高(P < 0.01),加入m-Nis 10-5,10-6,10-7 mol/L预处理均明显抑制了5-HT诱导的CaM mRNA表达水平的升高(P < 0.05或P < 0.01),而m-Nis 10-8 mol/L抑制作用不明显。m-Nis 10-5,10-6,10-7,10-8 mol/L均明显抑制了5-HT诱导的CaN mRNA表达水平的升高(P < 0.05或P < 0.01)。
8 m-Nis对5-HT诱导的大鼠PASMCs中ERK1/2及JNK磷酸化的影响
western blot结果显示,1μmol/L 5-HT在5至30分钟内诱导大鼠动脉平滑肌细胞ERK1/2及JNK磷酸化水平明显升高,在15分钟时磷酸化水平最高。而m-Nis可浓度依赖性地拮抗5-HT的作用。m-Nis 10-5,10-6,10-7 mol/L可明显抑制5-HT诱导的ERK1/2磷酸化水平的升高(P < 0.05或P < 0.01),而m-Nis 10-8 mol/L的抑制作用并不明显。10-8 ~10-5 mol/L m-Nis均可抑制5-HT诱导的JNK磷酸化水平的升高(P < 0.05或P < 0.01)。
9 m-Nis对5-HT诱导的大鼠PASMCs中c-fos及c-jun mRNA表达的影响
1μmol/L 5-HT在5至60分钟内能明显诱导大鼠肺动脉平滑肌细胞c-fos及c-jun mRNA表达水平升高,在30分钟时表达水平达最高。而不同浓度m-Nis均明显抑制了5-HT诱导的c-fos及c-jun mRNA表达水平的升高(P < 0.05或P < 0.01)。
结论:(1)m-Nis对5-HT诱导的肺动脉平滑肌细胞增殖和迁移有明显的抑制作用,可能与其抑制增殖细胞核抗原(PCNA)表达及细胞周期进程有关。(2)m-Nis明显抑制了5-HT引起的[Ca2+]i的升高、CaM和CaNmRNA的表达以及CaN活性的升高;对5-HT诱导的ERK1/2, JNK磷酸化水平也有不同程度的抑制,还明显抑制了增殖相关基因c-fos, c-jun mRNA的表达水平。由此,我们推测m-Nis抗5-HT诱导的PASMCs增殖作用可能还与抑制细胞内[Ca2+]i的增高从而阻断了5-HT所介导的ERK1/2, JNK MAPK信号通路有关。
第三部分RhoA/Rho激酶信号通路在5-HT刺激PASMCs增殖中的作用及m-Nis对此信号通路的影响
目的:研究RhoA/Rho激酶信号通路在5-HT刺激PASMCs增殖中的作用及m-Nis对此信号通路的抑制作用。
方法:组织块种植法原代培养大鼠PASMCs。MTT法测定PASMCs增殖。流式细胞仪测定细胞周期。western blot法测定PCNA蛋白表达及ERK1/2、JNK、MYPT1磷酸化水平。RT-PCR法检测RhoA、ROCK1、c-fos及c-jun mRNA的表达。结果:
1法舒地尔对5-HT诱导的PASMCs增殖的影响
MTT实验结果表明,与对照组比,5-HT组A值明显升高(从0.37±0.05升至0.61±0.03,P <0.01)。法舒地尔25、50、100μmol/L各组A值分别降至0.48±0.05,0.46±0.03和0.43±0.04(P <0.01)。western blot结果显示,与对照组比,5-HT组PCNA蛋白表达明显增多(P <0.01),而25、50、100μmol/L法舒地尔不同程度地降低了PCNA的蛋白表达(P<0.05或P<0.01)。
2法舒地尔对5-HT作用下PASMCs细胞周期的影响
与对照组比,5-HT组G0/G1期PASMCs百分率明显减少(从83%降至55%,P < 0.01), S期PASMCs百分率明显增加(从4%增至21%,P < 0.01),PI值也相应增加(从17%增至45%,P <0.01),提示5-HT能诱导PASMCs增殖。与5-HT组比,法舒地尔25,50,100μmol/L组G0/ G1期PASMCs百分率分别增至65%,70%和78%(P<0.05或P<0.01),S期PASMCs百分率也分别降至12%,11%和9%(P < 0.05或P < 0.01),PI值分别降至35%,30%和22%(P <0.05或P <0.01)。
3法舒地尔对5-HT诱导的ROCK激活的影响
与对照组比,1μmol/L 5-HT明显诱导了ROCK1 mRNA的表达(P < 0.01),法舒地尔25、50、100μmol/L预处理不同程度地降低了5-HT诱导的ROCK1 mRNA的表达(P < 0.05或P < 0.01)。western blot结果显示,1μmol/L 5-HT作用后明显升高了MYPT1的磷酸化水平,10分钟左右磷酸化水平最高(P < 0.01),法舒地尔25、50、100μmol/L不同程度地降低了5-HT诱导的MYPT1的磷酸化水平(P < 0.05或P < 0.01)。
4法舒地尔对5-HT作用下ERK1/2及JNK磷酸化水平的影响
与对照组比,5-HT作用15分钟明显诱导了ERK1/2的磷酸化(P<0.01),法舒地尔25、50、100μmol/L预处理对ERK1/2的磷酸化水平没有明显影响。而对于JNK的磷酸化水平,5-HT作用15分钟能明显诱导其升高(P < 0.01),法舒地尔25、50、100μmol/L预处理明显降低了JNK的磷酸化水平(P < 0.01)。另外,5-HT明显增加了核蛋白中p-ERK1/2的表达,15分钟左右表达水平最高(P <0.01),法舒地尔25、50、100μmol/L预处理明显降低了核蛋白中p-ERK1/2的表达(P < 0.01)。
5法舒地尔对5-HT作用下c-fos和c-jun mRNA表达的影响
与对照组比,5-HT作用30分钟明显诱导了c-fos及c-jun mRNA表达(P < 0.01)。法舒地尔25、50、100μmol/L预处理均明显抑制了5-HT诱导的c-fos (P < 0.01)及c-jun mRNA的表达(P < 0.05或P < 0.01)。
6 m-Nis对5-HT作用下PASMCs中RhoA/ROCK通路的影响
与对照组比,5-HT明显诱导了RhoA、ROCK1 mRNA的表达(P < 0.01),m-Nis10-5 mol/L明显降低5-HT诱导的RhoA mRNA的表达(P < 0.05)。m-Nis 10-5,10-6 mol/L还不同程度地降低了5-HT诱导的ROCK1 mRNA的表达(P < 0.05或P < 0.01)。western blot结果显示,5-HT作用10分钟明显升高了MYPT1的磷酸化水平(P < 0.01),m-Nis 10-5,10-6,10-7 mol/L预处理不同程度地降低了5-HT诱导的MYPT1的磷酸化水平(P < 0.05或P < 0.01),而m-Nis 10-8 mol/L对MYPT1的磷酸化水平无明显影响。
结论:(1)5-HT明显诱导了大鼠PASMCs中Rho激酶的表达及激活;Rho激酶抑制剂法舒地尔对5-HT诱导的PASMCs的增殖,细胞周期进展有明显的抑制作用,还不同程度地抑制了JNK的磷酸化,ERK1/2的核转位以及增殖相关基因c-fos、c-jun mRNA的表达。说明RhoA/ROCK通路在5-HT诱导的PASMCs增殖中起着重要作用。(2)m-Nis不同程度地抑制了5-HT诱导的RhoA, ROCK1 mRNA的表达以及Rho激酶的活化标志p-MYPT1的蛋白表达。说明m-Nis抗5-HT诱导的PASMCs的增殖作用可能与抑制RhoA/ROCK信号通路有关。
第四部分活性氧在5-HT刺激PASMCs增殖中的作用及m-Nis对此通路的影响
目的:研究活性氧(ROS)在5-HT刺激PASMCs增殖中的作用和m-Nis对ROS的产生及其下游信号通路的影响。
方法:组织块种植法原代培养大鼠PASMCs。激光共聚焦检测ROS生成量的变化。TAB法测定MDA含量。黄嘌呤氧化酶法测定SOD活性。MTT法测定PASMCs增殖。western blot法测定PCNA蛋白表达及ERK1/2、JNK磷酸化水平。
结果:
1 m-Nis对5-HT诱导的大鼠PASMCs内ROS生成的影响
1μmol/L的5-HT处理不同时间组,荧光强度均增强,5-HT处理15分钟组荧光强度最强,达对照组的288±54%(P<0.01),然后减弱,在5-HT处理120和240分钟组荧光强度达到稳定的状态,但仍明显高于对照组(P<0.01)。m-Nis 10-5,10-6,10-7,10-8mol/L均能明显抑制5-HT诱导的ROS的生成,在5-HT处理15分钟时荧光强度分别降为对照组的157±29%,173±38%,178±43%和215±45%(P<0.01)。
2 m-Nis对5-HT作用下大鼠PASMCs内SOD活性和MDA含量的影响
与对照组相比,5-HT处理组SOD活性明显降低(P < 0. 01),MDA含量明显升高(P < 0. 01)。m-Nis 10-5,10-6,10-7mol/L预处理均不同程度地逆转了5-HT诱导的PASMCs SOD活性的降低(P < 0. 05或P < 0. 01),同时也降低了MDA的含量(P < 0. 05或P < 0. 01),而m-Nis 10-8mol/L对SOD的活性没有明显影响,但可降低MDA的含量(P < 0.05)。
3 m-Nis平对H2O2诱导的大鼠PASMCs增殖的影响
MTT实验结果表明,100μmol/L H2O2能明显促进大鼠PASMCs增殖,与对照组比,H2O2处理组A值明显提高(P <0.01)。与H2O2处理组比,m-Nis各组A值均不同程度降低(P <0.05或P <0.01)。western blot结果显示,与对照组相比,H2O2处理组PASMCs的PCNA表达明显增加(P<0.01)。不同浓度m-Nis预处理后,均明显降低H2O2诱导的PCNA的表达(P<0.05或P<0.01)。
4 m-Nis对H2O2诱导的PASMCs中ERK1/2及JNK磷酸化水平的影响
与对照组相比,100μmol/L H2O2作用15分钟时能明显升高大鼠脉平滑肌细胞ERK1/2及JNK磷酸化水平(P<0.01)。m-Nis 10-5,10-6,10-7 mol/L可明显抑制H2O2诱导的ERK1/2及JNK磷酸化水平的升高(P < 0.05或P < 0.01),而m-Nis 10-8mol/L仅明显抑制了ERK1/2磷酸化水平的升高(P < 0.05),对JNK磷酸化水平无明显影响。
结论:(1)5-HT明显诱导了大鼠PASMCs中ROS的生成,H2O2不仅诱导了PASMCs增殖,还提高了ERK1/2及JNK的磷酸化水平。说明ROS在5-HT诱导的PASMCs增殖中起重要作用。(2)m-Nis不仅抑制了ROS的生成,还明显降低了H2O2诱导的PASMCs增殖及ERK1/2、JNK的磷酸化水平。说明m-Nis抗5-HT诱导的PASMCs增殖作用不仅与其抑制ROS的产生有关,还与其抑制ROS所介导的ERK1/2,JNK MAPK信号通路有关。
总结
1 m-Nis对野百合碱诱导的大鼠肺动脉高压有一定的防治作用,可能与其抗氧化作用,降低5-HT的表达及抑制ERK1/2 MAPK信号通路有关。
2 m-Nis对5-HT诱导的肺动脉平滑肌细胞增殖和迁移有明显的抑制作用,可能与其抑制细胞内[Ca2+]i的增高从而阻断了5-HT所介导的ERK1/2,JNK MAPK信号通路有关。
3 Rho激酶抑制剂法舒地尔明显抑制了5-HT诱导的PASMCs增殖,说明RhoA/ROCK通路在5-HT诱导的PASMCs增殖中起重要作用。
4 m-Nis不同程度地抑制了5-HT诱导的RhoA, ROCK1 mRNA的表达以及Rho激酶的活化标志p-MYPT1的蛋白表达。说明m-Nis抗5-HT诱导的PASMCs的增殖作用可能与抑制RhoA/ROCK信号通路有关。
5 ROS在5-HT诱导的PASMCs增殖中起重要作用。m-Nis抗5-HT诱导的PASMCs增殖作用不仅与其抑制ROS的产生有关,还与其抑制ROS所介导的ERK1/2,JNK MAPK信号通路有关。
Pulmonary artery hypertension (PAH) is a progressive and fatal disease characterized by an increase of vascular resistance, abnormal sustained pulmonary vasoconstriction, and progressive structural remodeling of pulmonary arteries, for which no clear pathogenesy has been developed. Recent studies suggest that constriction and remodeling of pulmonary arteries are the key point of PAH formation. The disequilibrium of proliferation of PASMCs and apoptosis results in pulmonary arteries remodeling. Therefore, the key points of PAH therapy include inhibiting the pulmonary artery constriction, improving the pulmonary circulation and preventing the proliferation of PASMCs.
Calcium signals play an important role in the formation of PAH. Studies suggest that the elevation of [Ca2+]i is one of the most important initiation agents to trigger proliferation of PASMCs. In the progress of PAH, many cell factors including 5-HT can elevate the [Ca2+]i after they bind to their receptors. Therefore, the elevation of [Ca2+]i is important to PAH.
Recently, calcium antagonists are frequently used in the treatment of PAH in clinic, but the clear mechanisms have not been elucidated. M-nisoldipine (m-Nis), a Ca2+ channel blocker of dihydropyridines (DHPs), is developed by Hebei Medical University. Early studies suggested that m-Nis had favourable stability and could inhibit vasoconstriction. Therefore, in the present study, monocrotaline (MCT) was use to induce PAH in rats, then the effect of m-Nis on PAH and pulmonary artery remodeling were studied. The methods such as immunhistochemistry, flow cytometry, confocal laser scanning microscope and molecular biology techniques were applied to explore the effect of m-Nis on 5-HT induced proliferation and migration of PASMCs and the mechanisms. Part 1 The effect of m-Nis on MCT-induced PAH in Rats
Objective: To study the effect of m-Nis on MCT-induced PAH in rats.
Methods: Rats were injected with a single dose (60mg/kg) of MCT subcutaneously to induce PAH. Pulmonary haemodynamic measurement and lung tissue morphological investigations were undertaken. The MDA production and SOD activity in the serum were tested. Expression of PCNA, ERK1 and p-ERK1/2 expression in pulmonary blood vessels was analyzed by western blot. The expression of 5-HT and PCNA in pulmonary tissues was observed by immunohistochemistry.
Results:
1 Effect of m-Nis on PAP and hemodynamic variables in MCT-induced PAH rats
Compared with the control group, mean PAP in the MCT group was significantly elevated (from 10.5±1.6 to 17.4±1.6mmHg, P<0.01). Compared with the MCT group, mean PAP in m-Nis 0.5, 1.0 and 2.0 mg/kg groups was decreased to 14.4±1.1,14.0±1.3 and 13.6±0.9 mmHg(P<0.01), respectively。Yet, SAP and heart rate did not differ significantly among the five groups.
2 Effect of m-Nis on right ventricle hypertrophy in MCT-induced PAH rats
Body weight was significantly decreased by MCT injection (P<0.05). The ratio of right ventricular weight to body weight (RV/BW) was increased by MCT from 0.79±0.09 to 1.05±0.09 (P<0.01 vs control) and reduced by m-Nis 0.5, 1.0, 2.0mg/kg to 0.90±0.04 (P<0.05 vs MCT), 0.83±0.07 and 0.85±0.09 (P<0.01 vs MCT) respectively. Meanwhile, the right ventricular index RV/(LV+SP) was increased by MCT from 0.307±0.007 to 0.408±0.015 (P<0.01 vs control) and reduced obviously by m-Nis 0.5, 1.0, 2.0mg/kg to 0.365±0.013, 0.341±0.023 and 0.353±0.021 respectively (P<0.01 vs MCT).
3 Effect of m-Nis on the morphological alterations of small pulmonary arteries
Small pulmonary arteries in MCT group exhibited obvious medial wall thickening and lumen transverse area reducing, the MT% markedly increased, whereas the VA% decreased compared with those in control group (P<0.01). In m-Nis-treated groups, the pulmonary artery wall thickening was attenuated in different degrees, the MT% was decreased in m-Nis 0.5 mg/kg group (P<0.05) and m-Nis 1.0, 2.0 mg/kg groups (P<0.01) compared with that in MCT group. Meanwhile, the VA% was significantly increased in all the m-Nis groups compared with that in MCT group (P<0.01).
4 Effect of m-Nis on the activity of SOD and the content of MDA
Compared with control group, the content of MDA in MCT group was significantly elevated from 6.32±0.27 to 7.62±0.63 (P<0.01), and the activity of SOD was decreased from 163±10 to 122±11 (P<0.01). Compared with the MCT group, a significant reduction in MDA production was observed in all three m-Nis 0.5, 1.0 and 2.0mg/kg groups (from 7.62±0.63 to 6.49±0.82, 6.76±0.38 and 6.40±0.60 respectively) (P<0.05), which was associated with the increase of SOD activity (from 122±11 to 142±12, 149±8 and 146±11 respectively) (P<0.05 or P<0.01).
5 5-HT immunohistochemistry assessment
In MCT group, the percentage of 5-HT-positive cells (14.2±3.0%) was higher than that in control group (4.2±2.1%) (P<0.01), which was reduced to 8.6±1.3%,7.1±1.9% and 7.2±1.0% by m-Nis 0.5, 1.0 and 2.0mg/kg treatment (P<0.01 vs MCT group).
6 Effect of m-Nis on the expression of PCNA
From the result of immunohistological staining, we found that in MCT group the percentage of PCNA-positive cells was remarkably increased compared with the control group (P<0.01), which was decreased by pretreatment with m-Nis (P<0.01). Western blot analysis indicated that the expression of PCNA was significantly higher in MCT group than that in control group (P<0.01), which was markedly decreased (P<0.05 vs MCT group) by m-Nis 0.5, 1.0 and 2.0mg/kg pretreatment.
7 Effect of m-Nis on the activation of ERK1/2
Western blot analysis showed that p-ERK1/2 expression in MCT group exhibited a significant increase compared with that in control group (P<0.01), which was obviously decreased by m-Nis 0.5, 1.0 and 2.0mg/kg (P<0.01). About the expression of ERK1/2, no statistical difference was found among the five groups. So, the ratio of p-ERK1/2/ERK1/2 increased significantly in MCT group compared with that in control group (P<0.01), which was decreased significantly by m-Nis (P<0.05).
Conclusion: M-Nis protects against MCT-induced pulmonary artery hypertension, which may be related to the antioxidation effect, the reduction of 5-HT exression and the suppression of the ERK/MAPK signal pathway. Part 2 The effect of m-Nis on 5-HT-induced proliferation, migration of PASMCs and change of [Ca2+]i
Objective: To study the effect of m-Nis on 5-HT-induced proliferation, migration of PASMCs and change of [Ca2+]i.
Methods: PASMCs were cultured with the explant technique. The proliferation of PASMCs was evaluated by MTT assay. Transwell chambers were used to detect the migration. Cell-cycle distribution was determined by flow cytometry assay. The expression of PCNA was evaluated by immunocytochemical stain and western blot. The change of [Ca2+]i was measured by confocal microscopy. The activity of CaN was measured using kits. The phosphorylation of ERK1/2 and JNK was detected by western blot. The mRNA expression of c-fos, c-jun, CaM and CaN was evaluated by RT-PCR.
Results:
1 Effect of m-Nis on 5-HT-induced proliferation of PASMCs
The results of MTT assay suggested that 5-HT significantly increased the A value from 0.48±0.04 to 0.74±0.09 (P<0.01), which was decreased to 0.54±0.05,0.57±0.07,0.59±0.06 (P<0.01) and 0.62±0.07 (P<0.05) by m-Nis 10-5,10-6,10-7,10-8 mol/L, respectively. Similarly, western blot analysis of PCNA indicated that the expression of PCNA was significantly higher in 5-HT group than that in control group (P <0.01). Whereas, in four m-Nis treated groups, the level of PCNA was markedly decreased (P<0.05 or P<0.01). Next, the results of immunocytochemical stain suggested that in 5-HT group the percentage of PCNA-positive cells was remarkably increased compared with that in control group (P < 0.01), which was decreased by pretreatment with m-Nis 10-5,10-6,10-7 and 10-8 mol/L (P < 0.01).
2 Effect of m-Nis on 5-HT-induced PASMCs migration
Compared with control group, the number of migration cells increased from 34±7 to 95±13 (P<0.01) after 5-HT treatment, which was decreased to 43±9,44±10,58±9 and 62±9 (P <0.01) by m-Nis 10-5,10-6,10-7 and 10-8 mol/L pretreatment, respectively.
3 Effect of m-Nis on cell-cycle progression
The percentage of cells in S phase increased dramatically from 4% to 21% after 5-HT stimulation (P<0.01) and was accompanied by a reduction in the percentage of cells in G1 phase from 84% to 55% (P<0.01). M-Nis 10-5,10-6,10-7 mol/L markedly blocked 5-HT-induced cell-cycle progression, with the percentages of PASMCs entering the S phase decreasing to 6%, 10% and 11% (P<0.05 or P<0.01), and those arrested in the G0/G1 phase increasing to 78%, 71% and 64% respectively (P<0.05 or P<0.01). Meanwhile, the proliferation index increased significantly from 16% to 45% after 5-HT stimulation (P<0.01) and was decreased to 22%, 29% and 36% respectively by preincubation with m-Nis 10-5,10-6,10-7 mol/L (P<0.05 or P<0.01).
4 Effect of 5-HT on fluorescence intensity of [Ca2+]i in rat PASMCs
The fluorescence intensity that represented the [Ca2+]i of PASMCs was significantly increased and reached the peak at 90 s after treated with 5-HT. After that, the [Ca2+]i decreased gradually and reached to a stable state at 200 s after 5-HT treatment.
5 Effect of m-Nis on [Ca2+]i in rat PASMCs
The relative fluorescence intensity of intracellular Ca2+ was significantly increased from 3.0±1.0% to 132.4±4.3% after perfusion with 5-HT (P <0.01 vs control), which was decreased to 20.1±4.1%,33.6±9.0%,41.5±2.3% and 54.7±2.2% (P<0.01) respectively by preincubation with m-Nis 10-5,10-6,10-7 and 10-8 mol/L.
6 Effect of m-Nis on the activity of CaN in rat PASMCs
The activity of CaN was significantly increased from 1.7±0.5 to 3.7±0.8 by 5-HT treatment (P <0.01 vs control), which was decreased to 2.4±0.6, 2.9±0.6 (P<0.01), 3.0±0.7 and 3.1±0.5 (P<0.05) respectively by preincubation with m-Nis 10-5, 10-6, 10-7 and 10-8 mol/L.
7 Effect of m-Nis on mRNA expression of CaM and CaN in rat PASMCs
The mRNA expression of CaM and CaN increased obviously after 1μmol/L 5 -HT treatment for 24h (P<0.01), which was significantly reduced (P<0.05 or P<0.01) by pretreatment with m-Nis 10-5, 10-6 and 10-7 mol/L. M-Nis 10-8 mol/L also significantly reduced the CaN mRNA expression, but have no effect on CaM mRNA expression.
8 Effect of m-Nis on the phosphorylation of ERK1/2 and JNK in rat PASMCs
Treatment of cells with 1μmol/L 5-HT caused phosphorylation of ERK1/2 and JNK with a peak at 15 min. M-Nis 10-5, 10-6 and 10-7 mol/L pretreatment reduced 5-HT-induced phosphorylation of ERK1/2 and JNK obviously (P<0.05 or P<0.01), and m-Nis 10-8 mol/L also significantly reduced the JNK phosphorylation, but have no significant effect on ERK1/2 phosphorylation.
9 Effect of m-Nis on mRNA expression of c-fos and c-jun in rat PASMCs
Treatment of cells with 1μmol/L 5-HT increased mRNA expression of c-fos and c-jun with a peak at 30 min, which was decreased in different degree by m-Nis 10-5, 10-6, 10-7 and 10-8 mol/L pretreatment (P<0.05 or P<0.01).
Conclusion: (1) M-Nis inhibited 5-HT-induced proliferation and migration of PASMCs, which may be related to the inhibition of PCNA expression and cell-cycle progression. (2) M-Nis inhibited 5-HT induced elevation of [Ca2+]i, mRNA expression of CaM, CaN, c-fos and c-jun, phosphorylation of ERK1/2 and JNK. So, the inhibition of m-Nis on proliferation of PASMCs may be related to the blockage of ERK1/2, JNK MAPK signal pathway through inhibiting the elevation of [Ca2+]i.
Part 3 The activation of RhoA/ROCK signal pathway in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathway
Objective: To study the the activation of RhoA/ROCK signal pathway in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathway.
Methods: PASMCs were cultured with the explant technique. The proliferation of PASMCs was evaluated by MTT assay. Cell-cycle distribution was determined by flow cytometry assay. The expression of PCNA, the phosphorylation of ERK1/2, JNK and MYPT1 was detected by western blot. The mRNA expression of Rho A, ROCK1, c-fos and c-jun was evaluated by RT-PCR.
Results:
1 Effect of fasudil on 5-HT-induced proliferation of PASMCs
The results of MTT assay suggested that 1μmol/L 5-HT significantly increased the A value from 0.37±0.05 to 0.61±0.031 (P<0.01), which was decreased to 0.48±0.05 , 0.46±0.03 and 0.43±0.04 by fasudil 25, 50, 100μmol/L pretreatment respectively (P<0.01). Similarly, western blot analysis of PCNA indicated that the expression of PCNA was higher in 5-HT group than that in control group (P <0.01), which was inhibited in different degree (P<0.05 or P<0.01) by pretreatment of PASMCs with fasudil.
2 Effect of fasudil on cell-cycle progression
The percentage of cells in S phase increased dramatically from 4% to 21% after 5-HT stimulation (P<0.01) and was accompanied by a reduction in the percentage of cells in G1 phase from 83% to 55% (P<0.01). Fasudil 25, 50, 100μmol/L markedly blocked 5-HT-induced cell-cycle progression, with the percentages of PASMCs entering the S phase decreasing to 12%, 11% and 9% (P<0.05 or P<0.01), and those arrested in the G0/G1 phase increasing to 65%, 70% and 78% respectively (P<0.05 or P<0.01). Meanwhile, the proliferation index increased dramatically from 17% to 45% after 5-HT stimulation (P < 0.01) and was decreased to 35%, 30% and 22% (P<0.05 or P<0.01) respectively by preincubation with fasudil 25, 50 and 100μmol/L.
3 Fasudil inhibited 5-HT- mediated ROCK activation
5-HT induced a significant upregulation of ROCK1 mRNA expression compared with that in quiescent PASMCs (P<0.01), and pretreatment with fasudil 25, 50, 100μmol/L resulted in a significant reduction in ROCK1 mRNA expression (P<0.05 or P<0.01). 5-HT caused phosphorylation of MYPT1, with a peak at 10 min (P<0.01), which was inhibited by fasudil in a concentration-dependent manner (P<0.05 or P<0.01).
4 Effect of fasudil on ERK1/2 and JNK phosphorylation
5-HT caused obvious phosphorylation of ERK1/2 and JNK with a peak at 15 min (P < 0.01). Pretreatment with fasudil 25, 50 and 100μmol/L did not block ERK1/2 phosphorylation, but reduced JNK phosphorylation obviously (P < 0.01). Next we examined the effect of fasudil on nuclear translocation of ERK1/2 induced by 5-HT. Result suggested that 5-HT increased nuclear translocation of ERK1/2 as determined by western blot analysis of nuclear-rich cellular fractions, with a peak at 15 min, consistent with its phosphorylation, which was reduced (P < 0.01) by pretreatment of cells with fasudil 25, 50 and 100μmol/L.
5 Effect of fasudil on mRNA expression of c-fos and c-jun
Treatment of cells with 1μmol/L 5-HT for 30 min increased mRNA expression of c-fos and c-jun (P< 0.01), which was decreased in different degree by fasudil 25, 50, 100μmo/L pretreatment (P < 0.05 or P<0.01).
6 Effect of m-Nis on 5-HT-mediated RhoA/ROCK signal pathway
5-HT induced a significant upregulation of mRNA expression of RhoA and ROCK1 (P < 0.01), which was reduced significantly (P < 0.05 or P < 0.01) by pretreatment with m-Nis 10-5 mol/L, m-Nis 10-6 mol/L also reduced ROCK1 mRNA expression (P < 0.05), but had no effect on RhoA mRNA expression. Phosphorylation of MYPT1 was induced by 5-HT with a peak at 10 min (P < 0.01), which was inhibited by m-Nis 10-5,10-6 and 10-7 mol/L in different degree (P < 0.05 or P < 0.01). However, m-Nis 10-8 mol/L had no effect on p-MYPT1 expression.
Conclusion: (1) 5-HT mediated ROCK activation, fasudil not only inhibited 5-HT-induced proliferation of PASMCs and cell-cycle progression, but also reduced JNK phosphorylation, nuclear translocation of ERK1/2 and c-fos, c-jun mRNA expression, which suggested that RhoA/ROCK signal pathway played an important role in 5-HT-induced proliferation of PASMCs. (2) M-Nis significantly inhibited 5-HT-induced RhoA, ROCK1 mRNA expression and the phosphorylation of MYPT1. So we conclude that the inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of RhoA/ROCK signal pathway.
Part 4 Effect of ROS in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathway
Objective: To study the effect of ROS in 5-HT-induced proliferation of PASMCs and the inhibitory effect of m-Nis on this pathway.
Methods: PASMCs were cultured with the explant technique. ROS was measured by confocal microscopy with DCFH-DA. The MDA production and SOD activity were measured using kits supplied by Nanjing Jiancheng Biotechnological Company. The proliferation of PASMCs was evaluated by MTT assay. The expression of PCNA, the phosphorylation of ERK1/2 and JNK were detected by western blot.
Results:
1 Effect of m-Nis on the production of ROS in rat PASMCs
The fluorescence intensity of ROS was significantly increased and reached the peak at 15 min after the cells were treated with 5-HT (P<0.01). After that, the fluorescence intensity decreased gradually and reached to a stable state at 120 min and 240 min after 5-HT treatment, but still stronger than that in control group (P<0.01). Compared with control group, the fluorescence intensity of ROS increased by 288% 15 min after 5-HT treatment (P<0.01). Pretreatment with m-Nis 10-5, 10-6, 10-7 and 10-8 mol/L reduced the fluorescence intensity of ROS from 288±54% to 157±29%, 173±38%, 178±43% and 215±45%, respectively (P<0.01).
2 Effect of m-Nis on the activity of SOD and the content of MDA
Compared with the control group, the activity of SOD decreased obviously (P<0.01), and the content of MDA increased significantly (P<0.01) after 5-HT treatment. Compared with the 5-HT group, a significant reduction in MDA production was observed in m-Nis 10-5,10-6,10-7 mol/L groups (P<0.05 or P<0.01), which was associated with an increase in the SOD activity (P<0.05 or P<0.01). Meanwhile, m-Nis 10-8 mol/L also reduced the content of MDA (P<0. 05), but had no effect on the SOD activity.
3 Effect of m-Nis on H2O2-induced proliferation of PASMCs
The results of MTT assay suggested that 100μmol/L H2O2 significantly increased the A value (P<0.01), which was inhibited significantly (P <0.05 or P <0.01) by m-Nis 10-5,10-6,10-7 and 10-8 mol/L. Similarly, western blot analysis of PCNA indicated that the expression of PCNA was higher in H2O2 group than that in control group (P <0.01), which was also inhibited obviously by pretreatment of PASMCs with m-Nis (P<0.05 or P<0.01).
4 Effect of m-Nis on H2O2-induced phosphorylation of ERK1/2 and JNK
H2O2 caused obvious phosphorylation of ERK1/2 and JNK (P < 0.01), which was inhibited in different degree by m-Nis 10-5, 10-6 and 10-7 mol/L pretreatment (P < 0.05 or P < 0.01). Meanwhile, m-Nis 10-8 mol/L also inhibited the phosphorylation of ERK1/2 (P < 0.05), but had no effect on JNK activation.
Conclusion: (1) 5-HT induced the production of ROS obviously. H2O2 not only mediated proliferation of PASMCs, but also induced ERK1/2 and JNK phosphorylation, which suggested that ROS played an important role in 5-HT-induced proliferation of PASMCs. (2) M-Nis not only inhibited 5-HT-induced production of ROS, but also reduced the proliferation of PASMCs and phosphorylation of ERK1/2 and JNK induced by H2O2. So we conclude that the inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of ROS production and the downstream ERK1/2,JNK MAPK signal pathway.
CONCLUSIONS
1 M-Nis protects against MCT-induced pulmonary artery hypertension, which may be related to the antioxidation effect, the reduction of 5-HT exression and the suppression of the ERK/MAPK signal pathway.
2 M-Nis inhibited 5-HT-induced proliferation and migration of PASMCs, which may be related to the blockage of ERK1/2, JNK MAPK signal pathway through inhibiting the elevation of [Ca2+]i.
3 Fasudil inhibited 5-HT-induced proliferation of PASMCs obviously, which suggested that RhoA/ROCK signal pathway played an important role in 5-HT-induced proliferation of PASMCs.
4 M-Nis inhibited 5-HT-induced RhoA, ROCK1 mRNA expression and the phosphorylation of MYPT1 in different degree. So we conclude that the inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of RhoA/ROCK signal pathway.
5 ROS played an important role in 5-HT-induced proliferation of PASMCs. The inhibition of m-Nis on 5-HT-induced proliferation of PASMCs may be related to the blockage of ROS production and the downstream ERK1/2,JNK MAPK signal pathway.
引文
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