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Z-Ligustilide protects vascular endothelial cells from oxidative stress and rescues high fat diet-induced atherosclerosis by activating multiple NRF2 downstream genes

  • Author Footnotes
    1 These authors contributed equally to this work.
    Yao Zhu
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Department of Cardiology, The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China

    First clinical medicine college of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Yajie Zhang
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Department of Central Laboratory, The Affiliated Nanjing Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China

    Department of Clinical Biobank, The Affiliated Nanjing Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China
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  • Xia Huang
    Affiliations
    Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China

    Department of Cardiology, The Affiliated Nanjing Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China
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  • Yong Xie
    Affiliations
    Department of Cardiology, The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China

    First clinical medicine college of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China
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  • Yuan Qu
    Affiliations
    Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China

    Department of Cardiology, The Affiliated Nanjing Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China
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  • Hongyan Long
    Affiliations
    Department of Central Laboratory, The Affiliated Nanjing Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China

    Department of Clinical Biobank, The Affiliated Nanjing Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China
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  • Ning Gu
    Correspondence
    Corresponding author. Department of Cardiology, The Affiliated Nanjing Hospital of Nanjing University of Chinese Medicine, Naning, Jiangsu, PR China.
    Affiliations
    Department of Cardiology, The Affiliated Nanjing Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China
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  • Weimin Jiang
    Correspondence
    Corresponding author. Department of Cardiology, The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China.
    Affiliations
    Department of Cardiology, The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China

    First clinical medicine college of Nanjing University of Chinese Medicine, Nanjing, Jiangsu, PR China
    Search for articles by this author
  • Author Footnotes
    1 These authors contributed equally to this work.

      Highlights

      • Nrf2 pathway protects vascular endothelium from oxidative stress in vivo and in vitro.
      • Z-Ligustilide ameliorates high fat induced-atherosclerosis in LDLR−/− mice.
      • Z-Ligustilide activates Nrf2 and its downstream genes containing the ARE element, in vascular endothelial cells.

      Abstract

      Background and aims

      Oxidative stress-induced endothelial dysfunction is considered to exert a vital role in the development of atherosclerotic coronary heart disease (CHD). NRF2 is a key transcriptional factor against oxidative stress through activation of multiple ARE-mediated genes. Z-Lig is derived from the Ligusticum species with antitumor, anti-inflammation and neuroprotection activities. However, the antioxidant potentials of Z-Lig on endothelial dysfunction and atherosclerosis have not been well elucidated. Therefore, in the present work, we appraise the cytoprotective property and anti-atherosclerosis effect of Z-Lig.

      Methods

      Potential NRF2 activators were screened and verified by luciferase reporter gene assay. The protein and mRNA levels of NRF2 and ARE-mediated genes, and GSH/GSSG level in EA.hy926 cells treated with Z-Lig were detected. The cytoprotective property of Z-Lig was assessed in the tert-butyl hydroperoxide (t-BHP)-evoked oxidative stress model. Cell viability and reactive oxygen species (ROS) levels in EA.hy926 cells were determined. An atherosclerosis model induced by HFD was used to determine the anti-atherosclerosis effect of Z-Lig in HFD-fed Ldlr-deficient mice.

      Results

      In vitro, 100 μM Z-Lig upregulated expressions of NRF2 and ARE-driven genes, promoted accumulation of nuclear NRF2 and unbound NRF2- KEAP1 complex in EA.hy926 cells. Furthermore, Z-Lig alleviated oxidative stress and cell injury caused by t-BHP via stimulation of the NRF2/ARE pathway. In vivo, intervention with 20 mg/kg Z-Lig markedly restrained atherosclerosis progression, including attenuation of HFD-induced atherosclerotic plaque formation, alleviation of lipid peroxidation and increase in antioxidant enzyme activity in aortas of HFD-fed Ldlr −/− mice. The chemopreventive effects of Z-Lig might be associated with the activation of NRF2 and ARE-driven genes.

      Conclusions

      The present study suggested that Z-Lig is an effective NRF2 activator, which can protect vascular endothelial cells from oxidative stress and rescue HFD-induced atherosclerosis.

      Graphical abstract

      Keywords

      Abbreviations:

      Z-Lig (Z-Ligustilide), ROS (reactive oxygen species), Nrf2 (nuclear factor-erythroid 2-related factor 2), Keap1 (Kelch like-ECH-associated protein 1), CVD (cardiovascular diseases), CHD (coronary heart disease), ARE (antioxidant response element), GSH (reduced glutathione), GSSG (glutathione disulfide), GCLM (glutamate-cysteine ligase modifier subunit), GCLC (glutamate-cysteine ligase catalytic subunit), GS (glutathione synthetase), GR (glutathione reductase), GPX (glutathione peroxidase), GRX (glutaredoxin), TR (thioredoxin reductase), TRX (thioredoxin), PRX (peroxiredoxin), SOD (superoxide dismutase), CAT (catalase), t-BHP (tert-butyl hydroperoxide), MDA (malondialdehyde), HFD (high fat diet), HO-1 (heme oxygenase-1), GST (glutathione S-transferase), NQQ1 (NAD(P)H quinone oxidoreductase 1), DMSO (dimethyl sulfoxide), DCFH-DA (2', 7'-dichlorodihydrofluorescein diacetate), DMEM (Dulbecco’s Modified Eagle Medium), TG (triglyceride), TC (total cholesterol), LDL-C (low density lipoprotein cholesterol), HDL-C (high density lipoprotein cholesterol), UBM (high resolution ultrasound biomicroscopy), ORO (Oil Red O), OCT (optimum cutting temperature)
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