血管紧张素Ⅱ诱发自噬对血管平滑肌细胞表型转换的调控作用

Effect of Ang Ⅱ-stimulated autophagy on phenotype conversion of vascular smooth muscle cells

目的观察血管紧张素Ⅱ(AngⅡ)对小鼠主动脉血管平滑肌细胞(VSMC)自噬的影响以及自噬对细胞表型转换的调控作用。方法原代培养小鼠VSMC,用10^(-6)mol/L AngⅡ作用VSMC不同时间,采用Western blot检测微管相关蛋白轻链-3-Ⅱ(LC3-Ⅱ)的表达以观察AngⅡ对VSMC自噬的影响,透射电镜观察对照组及AngⅡ组的自噬小体。用Western blot检测自噬抑制剂3-MA和Baf-A1干预后对AngⅡ诱导自噬及细胞表型转换的影响。使用siRNA抑制自噬相关基因Atg7的表达,qRT-PCR检测转染后Atg7的表达变化,Western blot检测转染siRNA Atg7后对LC3-Ⅱ及细胞表型蛋白标志物的影响。结果 AngⅡ以时间依赖方式促进LC3-Ⅱ表达,自噬抑制剂3-MA抑制AngⅡ促LC3-Ⅱ的表达作用,而Baf-A1则增强AngⅡ促LC3-Ⅱ的表达作用,两种自噬抑制剂均可抑制AngⅡ促VSMC表型转换作用。转染siRNA Atg7后显著抑制AngⅡ促LC3-Ⅱ的表达作用,并可抑制AngⅡ促细胞表型转换作用。结论 AngⅡ促进VSMC从收缩表型转化为合成表型可能是自噬依赖性的,抑制自噬可以抑制AngⅡ诱导的VSMC表型转换。

Aim To observe the effect of angiotensinⅡ （AngⅡ） on the autophagy of mouse aortic smooth muscle cells and the regulation of autophagy on cell phenotype conversion. Methods Mouse vascular smooth muscle cells were obtained by primary culture, vascular smooth muscle cells were incubated with 10-6 mol/L AngⅡ at different time, Western blot was used to detect the expression of LC3-Ⅱ. Transmission electron microscope was used to observe the autophagic body of different groups. Vascular smooth muscle cells were pretreated with 3-MA, Baf-A1 and then incubated with 10-6 mol/L AngⅡ for 24 h, Western blot was used to detect the expression of LC3-Ⅱ and the contractile phenotype marker （SM22α and SM-α-actin） and synthetic phenotype marker （OPN） of vascular smooth muscle cells in different groups. qRT-PCR was performed to detect the Atg7 mRNA level after transfection with siRNA Atg7. Western blot was performed to detect the expression of LC3-Ⅱ, SM22α, SM-α-actin and OPN to observe the effect of siRNA Atg7 on AngⅡ-induced autophagy and VSMC phenotype conversion. Results AngⅡ stimulated LC3-Ⅱ expression in a time-dependent manner, 3-MA significantly attenuated the AngⅡ-enforced expression of LC3-Ⅱ while Baf-A1 further increased the expression of LC3-Ⅱ. Both 3-MA and Baf-A1 significantly suppressed AngⅡ-stimulated VSMC phenotype conversion. Transfection with siRNA Atg7 remarkably decreased the expression of LC3-Ⅱ stimulated by AngⅡ and suppressed AngⅡ-stimulated VSMC phenotype conversion. Conclusion AngⅡ-stimulated VSMC phenotype conversion may be autophagy-dependent, and inhibition of autophagy significantly suppressed AngⅡ-stimulated VSMC phenotype conversion.