阿托伐他汀通过调控MMP-7/Gα13增强ApoE^-/-小鼠动脉粥样硬化斑块稳定性^*

Study on the Mechanism by Which Atorvastatin Enhances the Stability of Atherosclerotic Plaques in ApoE^-/- Mice Through Regulation of MMP-7/Gα13

摘要

目的 探究阿托伐他汀（atorvastatin, Ator）调控ApoE^-/-小鼠动脉粥样硬化（atherosclerosis, AS）的多效性分子机制。方法 30只8周龄雄性ApoE^-/-小鼠经高脂饮食诱导AS模型后，随机分为对照组（生理盐水）、低剂量Ator组〔5 mg/(kg·d)〕和高剂量Ator组〔10 mg/(kg·d)〕，干预10周。主要结局指标为油红O、HE染色检测动脉粥样硬化斑块面积，次要结局指标包括血液化学分析仪检测血清血脂指标，Masson、天狼星红染色结合偏振光检测斑块稳定性相关指标（胶原纤维含量、纤维帽厚度），免疫组化法检测α-平滑肌肌动蛋白（alpha smooth muscle Actin, α-SMA）与CD68表达，荧光酶标仪检测活性氧（reactive oxygen species, ROS）水平，Western blot法、RT-qPCR法分别检测炎症、氧化应激、凋亡相关分子及基质金属蛋白酶7（matrix metallopeptidase 7, MMP-7）、G蛋白α13亚基（guanine nucleotide-binding protein subunit α-13, Gα13）的蛋白与基因表达。结果 血脂与斑块：Ator降低血清低密度脂蛋白胆固醇（low-density lipoprotein cholesterol, LDL-C）、总胆固醇（total cholesterol, TC）、甘油三酯（triglycerides, TG）水平（P<0.05），并剂量依赖性减少主动脉脂质沉积面积和斑块面积〔对照组斑块面积（1.62±0.15） mm²，低剂量组（1.13±0.06） mm²，高剂量组（0.83±0.07） mm²，P<0.001〕。斑块稳定性：Ator提升胶原纤维含量（P<0.001）、增加纤维帽厚度，上调α-SMA表达、下调CD68表达（P<0.001）。氧化应激：Ator显著降低动脉组织ROS及血清丙二醛（malondialdehyde, MDA）水平（P<0.001）。分子机制：Ator下调促炎因子〔肿瘤坏死因子α（tumor necrosis factor-alpha, TNF-α）、白细胞介素（interleukin, IL）-6〕、NLRP3、促凋亡蛋白（caspase-3、p53）、诱导型一氧化氮合酶（inducible nitric oxide synthase, iNOS）及MMP-7；上调抗凋亡蛋白Bcl-2、抗氧化蛋白〔线粒体去乙酰化酶3（sirtuin 3, SIRT3）/超氧化物歧化酶2（superoxide dismutase 2, SOD2）〕及Gα13（P<0.001）。上述蛋白与基因表达趋势一致，高剂量组效应更显著。结论 阿托伐他汀通过调节血脂、抑制炎症反应、减轻氧化应激、调控细胞凋亡，同时影响MMP-7和Gα13的表达，发挥抗AS的多效作用。

Abstract

Objective To explore the pleiotropic molecular mechanisms by which atorvastatin (Ator) regulates atherosclerosis (AS) in ApoE^-/- mice. Methods Thirty 8-week-old male ApoE^-/- mice were induced to develop atherosclerosis by a high-fat diet. They were randomly assigned to a control group (normal saline), a low-dose Ator group (5 mg/[kg·d]), and a high-dose Ator group (10 mg/[kg·d]), and received interventions for 10 weeks. The primary outcome indicators were assessed by Oil Red O and HE staining to measure the area of atherosclerotic plaques. Secondary outcome indicators included serum lipid levels detected by a blood chemical analyzer, Masson and Sirius Red staining combined with polarized light to assess plaque stability indicators (collagen fiber content, fibrous cap thickness), immunohistochemistry to detect the expression of α-smooth muscle actin (α-SMA) and CD68, fluorescence microplate reader to measure reactive oxygen species (ROS) levels, Western blot and RT-qPCR to detect protein and gene expression of inflammation, oxidative stress, apoptosis-related molecules, matrix metallopeptidase 7 (MMP-7), and guanine nucleotide-binding protein subunit α-13 (Gα13). Results Blood lipids and plaques: Ator reduced serum low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TG) levels (P < 0.05), and dose-dependently decreased the area of aortic lipid deposition and plaque area (Control group: plaque area [1.62 ± 0.15] mm², Low-dose group: [1.13 ± 0.06] mm², High-dose group: [0.83 ± 0.07] mm², P < 0.001). Plaque stability: Ator increased collagen fiber content ( P < 0.001), increased fibrous cap thickness, upregulated α-SMA expression, and downregulated CD68 expression ( P < 0.001). Oxidative stress: Ator remarkably diminished the levels of ROS in arterial tissue and malondialdehyde (MDA) in serum ( P < 0.001). Molecular mechanism: Ator downregulated pro-inflammatory factors (tumor necrosis factor-alpha [TNF-α], interleukin-6 [IL-6]), NLRP3, pro-apoptotic proteins (caspase-3, p53), inducible nitric oxide synthase (iNOS), and MMP-7; and upregulated anti-apoptotic protein Bcl-2, antioxidant proteins [mitochondrial deacetylase 3 (sirtuin 3, SIRT3)/superoxide dismutase 2 (SOD2)], and Gα13 ( P < 0.001). The expression trends of these proteins and genes were consistent, with a more significant effect in the high-dose group. Conclusion Atorvastatin exerts its anti-atherosclerotic effects by regulating blood lipids, inhibiting inflammatory responses, reducing oxidative stress, and regulating apoptosis. It also affects the expression of MMP-7 and Gα13.

关键词

阿托伐他汀 / ApoE^-/-小鼠 / 动脉粥样硬化 / 基质金属蛋白酶7 / G蛋白α13亚基

Key words

Atorvastatin / ApoE^-/- mice / Atherosclerosis / Matrix metallopeptidase 7 / Guanine nucleotide-binding protein subunit α-13

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基金资助

*武汉市医学科研项目(WX21Q11)