丹酚酸B激活自噬改善高磷诱导血管平滑肌细胞钙化的机制研究

Mechanism of salvianolic acid B attenuated phosphate induced vascular smooth muscle cells calcification through activating autophagy

目的:探讨丹酚酸B(Salvianolic acid B,SAB)改善高磷诱导的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)钙化的作用及机制。方法:采用大鼠血管平滑肌细胞(A7r5)钙化体外模型,用细胞增殖与活性检测试剂盒(cell counting kit-8,CCK-8)检测SAB对细胞活力的影响;2.6μmol·g^(-1)高磷诱导VSMCs钙化,加入丹参多酚酸类化合物丹酚酸B、丹酚酸C(Salvianolic acid C,SAC)、迷迭香酸(Rosmarinic acid,RA)及细胞自噬特异性抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA,5 nmol·g^(-1)),采用邻甲酚酞络合铜法及茜素红S染色检测细胞钙化程度;Western blot检测VSMCs钙化指标Runx2、OPN,自噬相关蛋白Beclin-1和LC3II/LC3I以及平滑肌收缩表型蛋白Calponin、SM22α的表达。结果:(1)钙离子浓度定量实验及茜素红染色实验显示SAB显著减轻高磷诱导的VSMCs钙化;(2)SAB浓度小于100 nmol·g^(-1)以下时对VSMCs生存活力无明显影响,选择3,10,30 nmol·g^(-1)剂量进行后续实验;Western blot显示SAB明显降低VSMCs成骨样分化的分子标志物Runx2、OPN蛋白表达(P<0.05),上调自噬标志物Beclin-1、LC3-I/II及VSMCs收缩表型标志物Calponin、SM22蛋白表达(P<0.05)。(3)SAB减轻高磷诱导VSMCs钙化的作用被自噬抑制剂3-MA阻断。结论:SAB可通过激活自噬抑制VSMCs钙化及向成骨样表型转化。

OBJECTIVE This study aims to investigate the effect and mechanism of salvianolic acid B(SAB)on the calcification of vascular smooth muscle cells(VSMCs)induced by high phosphorus.METHODS In vitro model of rat VSMCs(A7 r5)was used in this study.Cytotoxicity assay(1 mg·mLCCK8)was used to determined VSMCs viability at a different concentration of SAB.VSMCs calcification was induced by high phosphate calcifying medium(Pi)at a final concentration of 2.6μmol·g^(-1)for 3-7 days,SAB,SAC,RA and 3-Methyladenine,an autophagy inhibitor were added.Calcification degree was detected by calcium deposition and alizarin red staining.The protein expression Runx2,OPN and CNN,SM22 were measured by Western blot.In addition,we examined the effect of SAB on Beclin-1 and LC3 II/LC3 I expression on VSMCs and the effect of autophagy inhibitor on VSMC calcification.RESULTS Alizarin red staining and Calcium content assay showed that SAB at different concentrations significantly reduced calcification of VSMCs induced by high phosphorus(P<0.05).SAB concentrations less than 100 nmol·g^(-1)had no significant effect on the viability of VSMCs.Usage of SAB decreased the expression of runx2,OPN and increased the expression of CNN,SM22 compared with Pi model group,suggesting that SAB inhibited osteogenic differentiation of VSMCs.In addition,SAB can further upregulate the expression of autophagy marker after high phosphorus stimulation.Inhibition of autophagy by 3-MA blocked the inhibitory effect of SAB on VSMCs calcification.CONCLUSIONS SAB could inhibit the calcification and osteogenic phenotype transformation of VSMCs by activating autophagy.