南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (11): 1357-1363.doi: 10.12122/j.issn.1673-4254.2019.11.14

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二甲双胍通过调控血管平滑肌肌动蛋白骨架系统抑制小鼠动脉粥样硬化形成

李 挺，何铭垣，李忠豪，王德奖，徐颖怡，吴 炜，燕 翼   

  • 出版日期:2019-12-05 发布日期:2019-11-20
  • 基金资助:

Metformin inhibits aortic atherosclerosis in mice by regulating actin skeleton in vascular smooth muscle cells

  

  • Online:2019-12-05 Published:2019-11-20

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摘要： 目的 探讨二甲双胍（Met）对代谢综合征小鼠动脉粥样硬化形成的影响与其作用机制。方法 用Met刺激转染了EGFPCLIP170或EGFP-Pdlim5质粒的小鼠主动脉平滑肌细胞，观察pAMPK、pCLIP-170、pPdlim5等蛋白的表达，免疫荧光染色观察细胞的微管系统及肌动蛋白骨架系统的变化。对小鼠主动脉平滑肌细胞行划痕实验，用Met梯度浓度（0、0.5、1 mmol/L）刺激细胞8 h，观察划痕愈合情况，评价各组细胞迁移能力。对ApoE-/-小鼠注射链脲佐菌素诱导实验性糖尿病，之后予以高脂饲料喂养建立代谢综合征兼动脉粥样硬化模型。治疗组（Met组，n=12）给予Met灌胃干预，对照组（Control组，n=10）予生理盐水。造模8周后提取动物主动脉全长及根部分别行油红O染色和α-平滑肌肌动蛋白染色，评价小鼠主动脉斑块内平滑肌细胞的异位迁移和脂质沉积情况。结果 小鼠主动脉平滑肌在Met处理后肌动蛋白骨架系统形态发生改变，应力纤维增粗，黏附斑增强，细胞的迁移运动受到明显抑制（P<0.05），但Met对微管骨架系统无明显影响；动物实验显示：在造模8周后，主动脉根部α-平滑肌肌动蛋白染色示Met组平滑肌细胞迁移运动受到抑制，异位扩散程度较Control组明显降低，油红O染色示Met组脂质沉积较Control组明显减少（P<0.05）。结论 二甲双胍通过调控细胞肌动蛋白骨架系统，抑制平滑肌细胞的迁移运动，从而抑制小鼠动脉粥样硬化的形成。

Abstract: Objective Investigate the effect and mechanism of metformin on the development of metabolic syndrome related atherosclerosis. Method Transfecting EGFP-CLIP170 or EGFP-Pdlim5 plasmid to the mouse aortic smooth muscle cell line, to test the expression of p-AMPK, pCLIP-170 and pPdlim5, and observe the microtubule or the actin skeleton system by immunofluorescence staining. Scratch the cells to perform wound healing experiment, stimulating the cells with gradient metformin (0, 0.5, 1 mmol/L) for 8 h, and observe the change of the scratch size and the dynamic change of cell skeleton and migration in vitro. ApoE-/- mice were injected with streptozotocin and followed by 8 weeks of high fat diet to induce metabolic syndrome model. In the therapeutic group, mice were treated metformin (Met) instead of saline in control group (Control, CTL group). In the end, the whole aorta and its root were isolated and performed oil red O staining and immol/Lunostaining of α-SMA to evaluate the migration of smooth muscle cells and he accumulation of lipids in the aorta. Results Mouse aortic smooth muscle cells showed an enhanced stress fiber and focal adhesion which representing the dynamic change of actin skeleton after Met stimulation, while the tubulin system rarely showed any change to Met. In animal model, The staining of α-SMA showed smooth muscle cells migrated to the intima or even to the lipid area from the media of aorta in CTL group compared to the Met group. Oil red O staining showed a reduced accumulation of lipids in the Met group than the controls (P<0.05). Conclusion Metformin reduces the formation of atherosclerosis by inhibiting the migration of smooth muscle cells through modulating cellular actin skeleton system in mice.