吴茱萸次碱通过Sirt1改善血管平滑肌细胞钙化

Rutecarpine Attenuates Vascular Smooth Muscle Cell Calcification via Sirt1

【目的】探讨吴茱萸次碱对血管平滑肌细胞钙化的作用及机制。【方法】本研究采用大鼠血管平滑肌细胞钙化体外模型,用不同浓度的吴茱萸次碱处理细胞,qRT-PCR检测成骨样分化基因Runx2、BMP2和Osterix的表达和茜素红染色测细胞钙化程度。此外,观察吴茱萸次碱对血管平滑肌细胞Sirt1表达水平的影响以及Sirt1抑制剂对细胞钙化的影响。【结果】茜素红素染色及钙离子浓度定量实验均显示不同浓度的吴茱萸次碱可明显地改善高磷高钙诱导的大鼠血管平滑肌细胞的钙化程度(136±10、75±6、52±6、31±5、P <0.05)。使用吴茱萸次碱明显降低血管平滑肌细胞成骨样分化的分子标志物ALP活性,下调Runx2(2.85±0.25、1.75±0.18、1.62±0.13、1.36±0.16、P <0.05),BMP2(3.2±0.32、1.85±0.17、1.65±0.15、1.43±0.12,P <0.05)和Osterix(2.60±0.27、1.82±0.16、1.55±0.15、1.36±0.17;P <0.05)的表达水平,说明吴茱萸次碱可抑制血管平滑肌细胞成骨样分化过程。另外,高磷高钙可下调血管平滑肌细胞Sirt1的表达水平,qRT-PCR和Western blot结果证实吴茱萸次碱可明显地上调Sirt1 mRNA(0.35±0.06、0.75±0.11;0.22±0.08、0.87±0.13;P <0.05)和Sirt1的蛋白(0.38±0.09、0.71±0.13;P <0.05)的表达水平。钙定量结果显示Sirt1抑制剂EX-527能阻断吴茱萸次碱改善血管平滑肌细胞钙化的作用(138±13、36±7、87±8,P <0.05)。【结论】吴茱萸次碱可通过调节Sirt1改善血管平滑肌细胞钙化。

【Objective】This study aims to investigate whether rutecarpine has an effect on calcification of VSMC and its underlying mechanism.【Methods】In vitro model of rat VSMC calcification was used in this study. Rutecarpine at different concentrations was used to treat cultured rat VSMC. The expression of Runx2,BMP2 and Osterix was analyzed by qRT-PCR and mineral deposition was detected by alizarin red staining. In addition,we examined the effect of rutecarpine on Sirtuin-1(Sirt1)expression in VSMC and the effect of Sirt1 inhibitor on VSMC calcification.【Results】Alizarin red staining and calcium content assay showed that rutecarpine at different concentrations significantly reduced calcification of rat VSMC induced by high phosphorus and high calcium(136±10,75±6,52±6,31±5.29,P<0.05). Usage of rutecarpine decreased the activity of ALP,an osteogenic differentiation molecular marker,and down-regulated the expression of Runx2(2.85±0.25,1.75±0.18,1.62±0.13,1.36±0.16,P<0.05),BMP2(3.2±0.32,1.85±0.17,1.65±0.15,1.43±0.12,P<0.05)and Osterix(2.60±0.27,1.82±0.16,1.55±0.15,1.36±0.17,P<0.05),suggesting that rutecarpine inhibited osteogenic differentiation of VSMC. In addition,high phosphorus and high calcium down-regulated the expression of Sirt1 in VSMC. qRT-PCR and western blot analysis confirmed that rutecarpine up-regulated the expression of Sirt1 at both mRNA(0.35±0.06,0.75±0.11;0.22±0.08,0.87±0.13,P<0.05)and protein levels(0.38±0.09,0.71± 0.13,P<0.05). Quantification of calcium content analysis showed inhibition of Sirt1 by EX-527 blocked the inhibitory effect of rutecarpine on VSMC calcification(138±13,36±7,87±8,P<0.05).【Conclusion】We demonstrate that rutecarpine attenuates VSMC calcification via up-regulation of Sirt1.