Fasudil inhibits platelet-derived growth factor-induced human pulmonary artery smooth muscle cell proliferation by up-regulation of p27^kipl via the ERK signal pathway

背景 RhoA/ Rho kinase (岩石) 小径涉及肺的动脉的高血压(哼) 并且肺的动脉光滑的肌肉房间(PASMC ) 增长。岩石的抑制作为一个处理被建议了为哼。但是 RhoA/ROCK 小径并且它在人的 PASMC 的增长的下游的发信号的机制是不清楚的。我们调查了 fasudil 的效果，一个选择岩石禁止者，在导出血小板的生长上，因素( PDGF )导致了人的 PASMC 增长，和在 RhoA/ROCK 和细胞外的调整信号的 kinase (英皇家空军之阶级最低之兵)之间的可能的协会， p27KiP1.Methods 人 PASMC 与 10 ng/ml PDGF 的刺激是有教养的，并且 fasudil 的不同集中在 mitogen 的增加前被增加。房间生存能力和房间周期分别地与 MTT 和流动 cytometry 被决定。岩石活动，英皇家空军之阶级最低之兵活动和增殖的房间的蛋白质表示原子 angigen (PCNA ) 和 p27Kip1 被 MTT 试金被 immunoblotting.Results 测量， PDGF 显著地增加了代表了人的 PASMC 增长的 OD 值，并且有 fasudil 的预告的处理显著地颠倒了以一种剂量依赖者方式的这效果。在 PDGF 刺激以后，在 S 阶段的房间的百分比从 15.6 ～ 24.3 戏剧性地增加了，当在 G0/G1 阶段的百分比从 80.6 ～ 59 被减少时。并且有 fasudil 的预告的处理以一种剂量依赖者方式显著地颠倒了 PDGF 的房间周期效果。PDGF 显著地在 15 分钟与一座山峰导致了岩石活动和英皇家空军之阶级最低之兵活动，它被 fasudil 显著地禁止。另外， fasudil 显著地也禁止了导致 PDGF 的 PCNA 表示和 fasudil 在人的 PASMC 的 upregulated p27Kip1 表示，在 PDGF stimulation.Conclusion RhoA/ROCK 为导致 PDFG 的人的 PASMC 增长是重要的以后，它减少了，和 fasudil 有效地由 p27Kip1 的起来规定禁止了导致 PDGF 的人的 PASMC 增长，它可以与英皇家空军之阶级最低之兵的抑制被联系活动。

Background RhoA/ Rho kinase （ROCK） pathway is involved in pulmonary arterial hypertension （PAH） and pulmonary artery smooth muscle cell （PASMC） proliferation. Inhibition of ROCK has been proposed as a treatment for PAH. But the mechanism of RhoA/ROCK pathway and its downstream signaling in proliferation of human PASMCs is unclear. We investigated the effect of fasudil, a selective ROCK inhibitor, on platelet-derived growth factor （PDGF） induced human PASMC proliferation, and the possible association between RhoA/ROCK and extracellular signal-regulated kinase （ERK）,p27KiP1.Methods Human PASMCs were cultured with the stimulation of 10 ng/ml PDGF, and different concentrations of fasudil were added before the addition of mitogen. Cell viability and cell cycle were determined with MTT and flow cytometry respectively. ROCK activity, ERK activity and protein expression of proliferating cell nuclear angigen （PCNA） and p27Kip1 were measured by immunoblotting.Results By MTT assay, PDGF significantly increased the OD value that represented human PASMC proliferation, and pretreatment with fasudil significantly reversed this effect in a dose-dependent manner. After PDGF stimulation, the percentage of cells in S phase increased dramatically from 15.6% to 24.3%, while the percentage in G0/G1 phase was reduced from 80.6% to 59%. And pretreatment with fasudil reversed the cell cycle effect of PDGF significantly in a dose-dependent manner. PDGF markedly induced ROCK activity and ERK activity with a peak at 15 minutes, which were significantly inhibited by fasudil. In addition, fasudil significantly inhibited PDGF-induced PCNA expression and fasudil also upregulated p27Kip1 expression in human PASMCs, which decreased after PDGF stimulation.Conclusion RhoA/ROCK is vital for PDFG-induced human PASMC proliferation, and fasudil effectively inhibited PDGF-induced human PASMC proliferation by up-regulation of p27Kip1, which may be associated with inhibition of ERK activity.