异钩藤碱抑制PDGF-BB诱导肺动脉平滑肌细胞增殖的实验研究

目的 探讨异钩藤碱对血小板源性生长因子BB(PDGF-BB)诱导的肺动脉平滑肌细胞(PASMCs)增殖的影响及其机制。方法 建立PDGF-BB诱导的PASMCs增殖模型,通过MTT比色法和台盼蓝拒染法检测异钩藤碱对PASMCs增殖的影响及细胞毒性,流式细胞术检测细胞周期变化,Western blotting检测细胞周期相关蛋白的表达及血小板源性生长因子受体β(PDGF-Rβ)信号通路蛋白表达及磷酸化的影响。结果 与正常对照相比,加入PDGF-BB(20 ng/mL)培养24 h,可促进PASMCs增殖约2.5倍;异钩藤碱剂量依赖性地抑制PDGF-BB诱导的PASMCs增殖;异钩藤碱(25 μmol/L)明显抑制PDGF-BB诱导的S期细胞比例升高;Western blotting结果显示,异钩藤碱可抑制PDGF-BB诱导的cyclin D1、CDK6的表达和p27^kip1的降解,下调PDGF-Rβ及其下游信号分子AKT、糖原合成激酶(GSK)3β的磷酸化。结论 异钩藤碱通过将细胞周期阻滞于G₀/G₁期来抑制PDGF-BB诱导的PASMCs增殖,其机制可能为通过抑制PDGF-Rβ及其下游信号通路的磷酸化、进而调节细胞周期相关蛋白的合成和降解来实现。

Effect and mechanism of isorhynchophylline on platelet-derived growth factor(PDGF)-BB-induced pulmonary artery smooth muscle cell proliferation

Objective To investigate the effect of isorhynchophylline on platelet-derived growth factor (PDGF)-BB-induced pulmonary artery smooth muscle cells (PASMCs) proliferation and the related mechanism. Methods In PDGF-BB-induced PASMCs proliferation model, the effect and cytotoxicity of isorhynchophylline on PDGF-BB-induced PASMCs proliferation were measured by MTT assay and trypan blue exclusion, and the cell cycle progression was measured by flowcytometry. The expression of cell cycle-related protein and PDGF receptor β (PDGF-Rβ) signaling pathway in PDGF-BB-stimulated PASMCs were measured by Western blotting. Results Compared with the controls, the number of PASMCs increased approximately 2.5 fold after cultured with 20 ng/mL PDGF-BB for 24 h. Isorhynchophylline inhibited PDGF-BB-induced PASMCs proliferation in a concentration-dependent manner. 25 μmol/L isorhynchophylline inhibited the increase of PDGF-BB-induced PASMCs S phase proportion. Western blotting results indicated that isorhynchophylline decreased PDGF-BB-induced expressions of cyclin D1 and CDK6, and inhibited p27^kip1 degradation. Furthermore, isorhynchophylline negatively regulated PDGF-BB-induced phosphorylation of PDGF-Rβ and its downstream signaling molecules, including AKT and glycogen synthase kinase (GSK) 3β. Conclusion Isorhynchophyl-line inhibited PDGF-BB-induced PASMCs proliferation via inducing G₀/G₁ phase arrest, the possible mechanism of which is that isorhynchophylline modulate synthesization and degradation of cell cycle-related protein by inhibiting PDGF-Rβ and its downstream signaling pathway.