改构黄藤素对钙化细胞的作用机制研究

The underlying mechanism of modified palmatine for calcified cells

摘要:

通过建立β半胱氨酸磷酸盐诱导大鼠血管平滑肌细胞（RASMCs）钙化的模型，研究改构黄藤素在血管钙化中的作用.通过 Von Kossa 染色技术检测发现改构黄藤素可以显著降低钙化细胞内的钙盐沉积；利用实时定量 PCR（Realtime Quantitative PCR）技术检测发现改构黄藤素可以将钙化细胞内的 Nrf-2 mRNA 水平由原来的 0.09±0.01 显著升高到 2.54±0.35；采用 Western Blot 技术检测发现改构黄藤素可以显著增强钙化细胞内的蛋白表达量；试剂盒检测活性氧（ROS）的含量发现改构黄藤素可以将钙化细胞内的荧光强度由原来的1.04±0.03 显著降低到 0.82±0.01；试剂盒检测一氧化氮（NO）的含量，发现改构黄藤素可以将钙化细胞内的 NO 含量由原来的 5.73 ± 0.05 μmol/L 显著降低到3.73 ± 0.15 μmol/L.

Abstract:

To explore the role of palmatine in vascular calcification，we employed a calcific cell model using β-glyceophosphate to stimulate the rat aortic smooth muscle cells (RASMCs). We performed a Von Kossa staining assay to detect the effect of palmatine on calcific RASMCs upon calcium deposition, we found that the modified palmatine could significantly inhibit intracellular calcium deposition of RASMCs after calcification. The mRNA level of the nuclear transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf-2) was determined by Real-time Quantitative PCR, we found that the modified palmatine could significantly increase the mRNA level of Nrf-2 from (0.09±0.01) fold to (2.54±0.35) fold in RASMCs following calcification. Further，we measured the protein level of Nrf-2 by Western Blot, we found that the modified palmatine could significantly activate the expression of Nrf-2 in RASMCs following calcification. We used a Reactive Oxygen Species (ROS) kit to detect the fluorescence intensity of ROS, we observed that the modified palmatine could respectively decrease the fluorescence intensity indicating ROS from (1.04±0.03) to (0.82±0.01) in RASMCs after calcification. We used a Nitric Oxide (NO) kit to measure the production of NO, we observed that the modified palmatine could decrease the production of NO from (5.73±0.05) μmol/L to (3.73±0.15) μmol/L in RASMCs after calcification.