血管紧张素Ⅱ通过诱导自噬促进血管平滑肌细胞增殖和迁移

AngiotensinⅡPromotes Vascular Smooth Muscle Cells Proliferation and Migration through Inducing Autophagy

目的探讨血管紧张素Ⅱ(angiotensin II,AngⅡ)诱导血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖迁移过程中自噬的作用。方法体外培养小鼠原代主动脉平滑肌细胞,采用AngⅡ(10^(-7)mol/L)刺激VSMCs 24 h,免疫印迹法检测细胞自噬标志物LC3B、P62的表达。荧光显微镜下观察并统计对照组、AngⅡ组的自噬小体和自噬溶酶体的数量,探究AngⅡ对VSMCs自噬的影响。通过EdU增殖实验和划痕实验,检测AngⅡ刺激后VSMCs增殖和迁移的变化情况。使用自噬抑制剂3-MA(10 mmol/L)干预后,使用Western blot和mCherry-GFP-LC3双荧光指示系统观察检测LC3Ⅱ、P62表达变化和自噬体、自噬溶酶体的数量变化,确认自噬受到抑制。通过EdU增殖实验和划痕实验,检测3-MA对AngⅡ诱导的VSMCs增殖和迁移的影响。结果与对照组相比,AngⅡ显著增加LC3Ⅱ表达、LC3Ⅱ/LC3Ⅰ比值,显著减少P62表达,且显著增加自噬体和自噬溶酶体的数量,说明AngⅡ可诱导VSMCs自噬发生。AngⅡ还可显著增加EdU阳性细胞数及VSMCs的迁移距离,表明AngⅡ可促进VSMCs迁移和增殖。与AngⅡ组相比,3-MA可显著增加P62的表达,减少LC3Ⅱ表达、LC3Ⅱ/LC3Ⅰ比值以及自噬体和自噬溶酶体的数量,抑制自噬的发生;同时3-MA还可显著减少EdU阳性细胞数及VSMCs的迁移距离,抑制VSMCs的增殖和迁移。结论AngⅡ可通过诱导自噬发生,促进VSMCs的增殖和迁移。

Objective To explore the role of autophagy in the process of angiotensinⅡ(AngⅡ)induced proliferation and migration of vascular smooth muscle cells(VSMCs).Methods VSMCs from primary cultures of mice were stimulated with AngⅡ(10^(-7)mol/L)for 24 hours.The expression of autophagy markers LC3B and P62 were detected by immunoblotting.Under the fluorescence microscope,the number of autophagosomes and autolysosomes in the control group(Con group)and AngⅡgroup were observed and counted to investigate the effect of AngⅡon VSMCs autophagy.Changes in VSMCs proliferation and migration after AngⅡstimulation were evaluated through EdU proliferation assay and scratch assay.After intervention with the autophagy inhibitor 3-MA(10 mmol/L),Western blot and mCherry-GFP-LC3 dual fluorescence indicator system were used to observe and detect changes in LC3Ⅱand P62 expression,as well as changes in the number of autophagosomes and autolysosomes,confirming the inhibition of autophagy.Through EdU proliferation experiments and scratch tests,the influence of 3-MA on AngⅡ-induced VSMCs proliferation and migration was examined.Results Compared with the Con group,AngⅡsignificantly increased the expression of LC3Ⅱand the ratio of LC3Ⅱ/LC3Ⅰ,and significantly reduced the expression of P62.Moreover,there was a significant increase in the number of autophagosomes and autolysosomes,indicating that AngⅡcan induce VSMCs autophagy.AngⅡalso significantly increased the number of EdU positive cells and the migration distance of VSMCs,indicating that AngⅡcan promote VSMCs migration and proliferation.Compared with the AngⅡgroup,3-MA significantly increases the expression of P62,decreases the expression of LC3Ⅱand the ratio of LC3Ⅱ/LC3Ⅰ,as well as the number of autophagosomes and autolysosomes,inhibiting the occurrence of autophagy.At the same time,3-MA significantly reduces the number of EdU positive cells and the migration distance of VSMCs,inhibiting the proliferation and migration of VSMCs.Conclusion AngⅡpromotes VSMCs proliferation and migration by inducing autophagy.