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槲皮素通过Nrf2/HO-1通路改善血管紧张素Ⅱ诱导的血管平滑肌细胞表型转化

Quercetin regulates phenotypic transformation of vascular smooth muscle cells induced by angiotensin Ⅱ through Nrf2/HO-1 signaling pathway

    摘要
  • 摘要:
    目的 探讨槲皮素（quercetin, QR）对血管紧张素Ⅱ（angiotensin Ⅱ, Ang Ⅱ）诱导的血管平滑肌细胞（vascular smooth muscle cells, VSMCs）损伤和表型转化的保护作用及机制。
    方法 用Ang Ⅱ处理VSMCs，并分为4组：空白对照组（Ctrl组）、QR对照组（QR组）、模型组（Ang Ⅱ组）、QR治疗组（Ang Ⅱ＋QR组）。检测细胞内活性氧（reactive oxygen species, ROS）水平和超氧化物歧化酶（superoxide dismutase, SOD）活性。采用蛋白质印迹法检测炎症、细胞凋亡、细胞外基质重塑、表型转化及核因子E2衍生相关因子2（nuclear factor erythroid 2-related factor 2, Nrf2）信号通路相关蛋白表达。采用实时荧光定量反转录聚合酶链反应检测炎症因子白介素（interleukin, IL）1β、IL-6和肿瘤坏死因子α（tumor necrosis factor-α, TNF-α）的转录水平。采用划痕实验检测细胞迁移能力。用Nrf2转录活性抑制剂ML385进行挽救实验，验证QR对VSMCs的保护机制。
    结果 与Ctrl组相比较，Ang Ⅱ组细胞的ROS水平更高，SOD活性被抑制，炎症相关蛋白表达上调，炎症因子mRNA水平增高，基质金属蛋白酶（matrix metalloproteinase, MMP）表达升高（P＜0.05），VSMCs的迁移能力增强，收缩表型标志物平滑肌相关蛋白22α（smooth muscle 22α, SM22α）和α平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）表达减少，分泌表型标志物骨桥蛋白（osteopontin, OPN）表达上调，细胞凋亡增加（P＜0.05）。QR干预后，细胞内ROS水平明显降低，SOD活性增高，血管细胞黏附分子1和磷酸化核因子κB的蛋白表达下降，炎症因子的转录水平下降，收缩表型标志物表达上调，细胞凋亡和迁移能力减弱（P＜0.05）。挽救试验证实，QR通过激活Nrf2，减轻VSMCs氧化应激和炎症相关损伤， 并抑制VSMCs向合成型转化。
    结论 QR通过促进Nrf2的表达，减轻Ang Ⅱ诱导的VSMCs氧化应激、炎症和凋亡，抑制细胞外基质重塑和VSMCs迁移，从而抑制VSMCs表型转化。

     

    Abstract:
    Objective To investigate the protective effect and mechanism of quercetin (QR) against angiotensin Ⅱ (Ang Ⅱ)-induced injury and phenotypic transformation in vascular smooth muscle cells (VSMCs).
    Methods VSMCs were treated with Ang Ⅱ and divided into four groups: control (Ctrl), QR control, model (Ang Ⅱ), and treatment (Ang Ⅱ＋QR) groups. Intracellular reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity were measured. Western blotting was used to assess protein expression related to inflammation, apoptosis, extracellular matrix (ECM) remodeling, phenotypic transformation, and the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify mRNA levels of inflammatory cytokines (interleukin IL-1β, IL-6, and tumor necrosis factor-α TNF-α). Cell migration was evaluated using a scratch assay. A rescue experiment employing the Nrf2 transcriptional activity inhibitor ML385 was conducted to further validate protective mechanism of QR for VSMCs.
    Results Compared to the Ctrl group, Ang Ⅱ-treated VSMCs exhibited significantly higher ROS levels, suppressed SOD activity, upregulated expression of inflammatory proteins and mRNA (IL-1β, IL-6, TNF-α), increased matrix metalloproteinase (MMP) protein expression (P＜0.05), enhanced migration capacity, reduced expression of contractile phenotype markers (smooth muscle 22 alpha SM22α and α-smooth muscle actin α-SMA), upregulated expression of the synthetic phenotype marker osteopontin (OPN), and significantly increased apoptosis (P＜0.05). QR intervention significantly reduced intracellular ROS level, increased SOD activity, decreased protein expression of vascular cell adhesion molecule-1 (VCAM-1) and phosphorylated nuclear factor-κB (NF-κB), downregulated inflammatory cytokine transcription, upregulated contractile phenotype marker expression, and attenuated apoptosis and migration capacity (P＜0.05). The rescue experiment confirmed that QR alleviated Ang Ⅱ-induced oxidative stress, inflammation-related injury, and suppressed the transition of VSMCs towards a synthetic phenotype via activation of Nrf2.
    Conclusions  QR mitigates Ang Ⅱ-induced oxidative stress, inflammation, and apoptosis in VSMCs, inhibits ECM remodeling and VSMCs migration, and consequently suppresses VSMCs phenotypic transformation, primarily through promoting Nrf2 expression.

     

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