丹参酮ⅡA对血管紧张素Ⅱ诱导的血管平滑肌细胞增殖及迁移的影响

Effect of tanshinoneⅡA on angiotensinⅡ-induced proliferation and migration of vascular smooth muscle cells

目的探讨丹参酮ⅡA(TanⅡA)对血管紧张素Ⅱ(AngⅡ)诱导血管平滑肌细胞(VSMC)增殖和迁移的潜在影响及其与自噬调控的关系。方法以小鼠主动脉平滑肌细胞株为研究对象,加入AngⅡ建立细胞增殖模型后,使用不同浓度TanⅡA干预,分别采用CCK-8法、细胞划痕法检测TanⅡA对细胞增殖、迁移的影响,采用Western blotting法检测VSMC收缩表型蛋白α-平滑肌肌动蛋白(α-SMA)、合成表型蛋白骨桥蛋白(OPN),自噬相关蛋白p62、Beclin-1、LC3A/B的表达水平,观察TanⅡA对VSMC表型转化以及自噬的影响。再使用自噬抑制剂3-甲基腺嘌呤(3-MA)干预细胞,检测细胞增殖、迁移以及细胞表型蛋白与自噬相关蛋白的表达水平。结果AngⅡ处理后,VSMC的增殖和迁移能力显著增强,TanⅡA显著抑制AngⅡ诱导的VSMC的增殖和迁移。AngⅡ明显降低α-SMA的表达,增加OPN、p62、Beclin-1、LC3A/B的表达,随着TanⅡA浓度增加(4、8、12、16μg/mL),α-SMA的表达逐渐增加,OPN、p62、Beclin-1、LC3A/B的表达逐渐减少。加入3-MA处理细胞后,3-MA抑制AngⅡ诱导的VSMC增殖和迁移,同时增加α-SMA的表达,降低OPN、p62、Beclin-1、LC3A/B的表达,与TanⅡA作用一致。结论TanⅡA对AngⅡ诱导的VSMC的表型转化、增殖、迁移以及自噬具有抑制作用,其机制与抑制自噬有关。

Objective To determine whether TanshinoneⅡA(TanⅡA)has an effect on angiotensinⅡ(AngⅡ)-induced proliferation and migration of vascular smooth muscle cells(VSMCs),phenotypic switching,or autophagy.Methods In this study,murine aortic smooth muscle cell lines were treated with AngⅡto establish a model of cell proliferation.Different concentrations of TanⅡA were added and effects on cell proliferation and migration were determined by cell counting using a CCK-8 assay and a cell scratch assay,respectively.Alpha-smooth muscle actin(α-SMA),a marker of contractile VSMCs,was detected by Western blotting.Expression levels of osteopontin(OPN)and the autophagy-related proteins(p62,Beclin-1,LC3A/B)were assayed to determine the effect of TanⅡA on VSMC phenotypic transformation and autophagy.Cells were treated with the autophagy inhibitor 3-methyladenine(3-MA),combined with TanⅡA,and then cell proliferation,migration and expression levels of phenotypic markers and autophagy-related proteins were assessed.Results After AngⅡtreatment,the proliferation and migratory capacity of VSMCs was significantly enhanced,and phenotypic transformation was significantly inhibited by AngⅡ.Western blotting revealed that AngⅡreduced the expression ofα-SMA,increased the expression of OPN,p62,Beclin-1,and LC3A/B,and that these effects increased with higher TanⅡA concentrations.After the addition of 3-MA to the treated cells,the proliferation and migration of VSMCs induced by AngⅡwas inhibited,the expression ofα-SMA was increased,and the expression of OPN,p62,Beclin-1,and LC3A/B was decreased,which was consistent with the effect of TanⅡA.Conclusion TanⅡA may have inhibitory effects on phenotypic transformation,proliferation,migration,and autophagy of AngⅡ-treated VSMC,suggesting that the inhibition of the proliferation and migration may be regulated by autophagy.