法舒地尔通过 ERK 信号通路上调 p27kip¹ 抑制血小板衍生生长因子诱导的人肺动脉平滑肌细胞增殖。
Fasudil inhibits platelet-derived growth factor-induced human pulmonary artery smooth muscle cell proliferation by up-regulation of p27kip¹ via the ERK signal pathway.
机构信息
Department of Pediatric Cardiac Surgery Center, Fuwai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100037, China.
出版信息
Chin Med J (Engl). 2011 Oct;124(19):3098-104.
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), p27(Kip1).
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 p27(Kip1) 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 G(0)/G(1) 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 p27(Kip1) 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 p27(Kip1), which may be associated with inhibition of ERK activity.
背景
RhoA/Rho 激酶（ROCK）途径参与肺动脉高压（PAH）和肺动脉平滑肌细胞（PASMC）增殖。抑制 ROCK 已被提议作为 PAH 的治疗方法。但是，RhoA/ROCK 途径及其下游信号在人 PASMC 增殖中的机制尚不清楚。我们研究了 ROCK 抑制剂法舒地尔对血小板衍生生长因子（PDGF）诱导的人 PASMC 增殖的影响，以及 RhoA/ROCK 与细胞外信号调节激酶（ERK）、p27（Kip1）之间的可能关联。
方法
用人 PASMC 培养物，用 10ng/ml PDGF 刺激，在加入有丝分裂原之前加入不同浓度的法舒地尔。分别用 MTT 和流式细胞术测定细胞活力和细胞周期。用免疫印迹法测定 ROCK 活性、ERK 活性以及增殖细胞核抗原（PCNA）和 p27（Kip1）的蛋白表达。
结果
通过 MTT 测定，PDGF 显著增加了代表人 PASMC 增殖的 OD 值，法舒地尔预处理呈剂量依赖性显著逆转了这种作用。在 PDGF 刺激后，细胞周期 S 期的百分比从 15.6%显著增加到 24.3%，而 G0/G1 期的百分比从 80.6%降低到 59%。法舒地尔预处理呈剂量依赖性显著逆转了 PDGF 的细胞周期作用。PDGF 显著诱导 ROCK 活性和 ERK 活性，在 15 分钟时达到高峰，法舒地尔明显抑制了这些活性。此外，法舒地尔显著抑制 PDGF 诱导的 PCNA 表达，并上调人 PASMCs 中的 p27（Kip1）表达，在 PDGF 刺激后下降。
结论
RhoA/ROCK 对 PDGF 诱导的人 PASMC 增殖至关重要，法舒地尔通过上调 p27（Kip1）有效抑制 PDGF 诱导的人 PASMC 增殖，这可能与抑制 ERK 活性有关。
Zotero 插件