Nutr Res Pract.  2021 Jun;15(3):319-328. 10.4162/nrp.2021.15.3.319.

Extract of Curcuma zedoaria R. prevents atherosclerosis in apolipoprotein E-deficient mice

Affiliations
  • 1Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Korea
  • 2Department of Korean Life Science and Technology, University of Science and Technology, Daejeon 34113, Korea
  • 3Department of Physiology, School of Medicine, Chungnam National University, Daejeon 34134, Korea
  • 4Department of Pharmacognosy, College of Pharmacy, Chungnam National University, Daejeon 34134, Korea
  • 5Department of Life Science and Biotechnology, Soonchunhyang University, Asan 31538, Korea

Abstract

BACKGROUND/OBJECTIVES
Curcuma zedoaria R. (Zingiberaceae) has been used to treat headache, fever, and hypertension-related symptoms in Asian countries, including Korea, China, and Japan. We investigated whether dietary intake of a C. zedoaria extract (CzE) affected atherosclerosis in vivo.
MATERIALS/METHODS
Apolipoprotein E-deficient (ApoEM−/− ) mice (n = 32) were fed a normal diet (ND), a high-cholesterol diet (HCD), an HCD containing CzE (100 mg/kg/day), or an HCD containing simvastatin (10 mg/kg/day) for 12 weeks. The anti-atherosclerotic effects were evaluated by observing changes in fatty streak lesions, immunohistochemical analysis, ex vivo fluorescence imaging, lipid profiles, and western blot analysis.
RESULTS
The CzE-fed group showed a 41.6% reduction of atherosclerosis. Furthermore, CzE significantly reduced the levels of serum triglyceride, high-density lipoprotein, the chemokine (C-X3-C-motif ) ligand 1, the adhesion molecules vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin; down-regulation of tumor necrosis factor-α, interleukin-6, high mobility group box-1, and cathepsin levels in the aortic sinuses and aortas of ApoE −/− mice were also observed.
CONCLUSIONS
The results suggest that the inclusion of a water extract of C. zedoaria in a HCD is closely correlated with reducing the risk of vascular inflammatory diseases in an ApoE mouse model.

Keyword

C. zedoaria; atherosclerosis; ApoE knockout mice; VCAM-1; chemokine CX3CL1

Figure

  • Fig. 1 Representative images of Oil Red O-stained gross atherosclerotic lesions in HCD-fed and control mice (A-C). Typical cross-sections of aortic valves (× 100) in hearts from control (ND) (A), HCD (B), and CzE-treated HCD (C) mice. Treatment with CzE (100 mg·kg−1·d−1) for 12 weeks affects atherosclerosis in ApoE−/− mice. (D) Mean atherosclerotic lesion area in the aortic sinus was determined; data are expressed as mean ± SD values (n = 6 for each group).ND, normal diet; HCD, high-cholesterol diet; CzE, Curcuma zedoaria extract; ApoE−/−, apolipoprotein E-deficient.**P < 0.01 vs. HCD group.

  • Fig. 2 Immunohistochemical identification of inflammatory molecules in ApoE−/− mouse aortas. ApoE−/− mice were fed an HCD for 12 weeks, after which, inflammatory molecules in plaques in the aortic sinuses were assessed histologically and immunohistochemically. In the control aorta, TNF-α, CX3CL1, and HMGB-1 showed negligible staining (brown staining). HCD-fed ApoE−/− mice showed over-expression of TNF-α, CX3CL1, and HMGB-1 not only in endothelial cells, including in some intimal areas, but also in smooth muscle cells (dark-brown staining: black arrows). However, in HCD–CzE-fed ApoE−/− mice, the expressions of these inflammatory molecules were significantly inhibited. Arrowheads indicate TNF-α-, CX3CL1-, or HMGB-1-positive cells. All results are shown as mean ± SD values.ND, normal diet; HCD, high-cholesterol diet; CzE, Curcuma zedoaria extract; ApoE−/−, apolipoprotein E-deficient; TNF-α, tumor necrosis factor-α; CX3CL1, chemokine (C-X3-C-motif) ligand 1; HMGB-1, high mobility group box-1.*P < 0.05 vs. HCD group; ##P < 0.01 vs. ND group.

  • Fig. 4 Effect of CzE on adhesion molecules and inflammatory mediators in the aortas of ApoE−/− mice. The data were collected from ND, HFD with CzE (100 mg/kg), and HFD with simvastatin (10 mg/kg) fed mice (treated orally for 12 weeks). Representative photomicrographs of western blot results and densitometric analysis show the relative protein expression levels of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) and inflammatory mediators (TNF-α, IL-6, CX3CL1, and HMGB-1), normalized to β-actin. Values are mean ± SD.ND, normal diet; HCD, high-cholesterol diet; CzE, Curcuma zedoaria extract; ApoE−/−, apolipoprotein E-deficient; ICAM-1, intracellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; CX3CL1, chemokine (C-X3-C-motif) ligand 1; HMGB-1, high mobility group box-1.*P < 0.05, **P < 0.01 vs. HCD group; ##P < 0.01 vs. ND group.

  • Fig. 3 Visualization of cathepsin activity in atherosclerotic mouse aortas. Visible light images show that the aortas of ApoE−/− mice appear normal, whereas those of ApoE−/− mice fed an HCD exhibit increased proteolytic fluorescence in the carotid arteries. The fluorescent density distributed from the ascending aorta to the infrarenal aorta in each group indicates the location of cathepsin activity. In the ApoE−/− mice fed the HCD with CzE diet (100 mg·kg−1·d−1), the inflammatory cathepsin expression level was significantly inhibited. The presence of internally quenched fluorogenic peptides containing Abz-Dnp, which are activatable by pan-cathepsin, confirmed the inhibition of inflammatory cardiovascular disease by CzE treatment in ApoE−/− mice. Values presented are mean ± SD.ND, normal diet; HCD, high-cholesterol diet; CzE, Curcuma zedoaria extract; ApoE−/−, apolipoprotein E-deficient.**P < 0.01 vs. HCD group; ##P < 0.01 vs. ND group.


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