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. 2021 Aug 31;12(9):1380.
doi: 10.3390/genes12091380.

Beta vulgaris rubra L. (Beetroot) Peel Methanol Extract Reduces Oxidative Stress and Stimulates Cell Proliferation via Increasing VEGF Expression in H2O2 Induced Oxidative Stressed Human Umbilical Vein Endothelial Cells

Laila Naif Al-Harbi  1 , Subash-Babu Pandurangan  1 , Alhanouf Mohammed Al-Dossari  1 , Ghalia Shamlan  1 , Ahmad Mohammad Salamatullah  1 , Ali A Alshatwi  1 , Amna Abdullah Alotiby  2
Affiliations

Beta vulgaris rubra L. (Beetroot) Peel Methanol Extract Reduces Oxidative Stress and Stimulates Cell Proliferation via Increasing VEGF Expression in H2O2 Induced Oxidative Stressed Human Umbilical Vein Endothelial Cells

Laila Naif Al-Harbi et al. Genes (Basel). .

Abstract

The antioxidant capacity of polyphenols and flavonoids present in dietary agents aids in arresting the development of reactive oxygen species (ROS) and protecting endothelial smooth muscle cells from oxidative stress/induced necrosis. Beetroot (Beta vulgaris var. rubra L.; BVr) is a commonly consumed vegetable representing a rich source of antioxidants. Beetroot peel's bioactive compounds and their role in human umbilical vein endothelial cells (HUVECs) are still under-researched. In the present study, beetroot peel methanol extract (BPME) was prepared, and its effect on the bio-efficacy, nuclear integrity, mitochondrial membrane potential and vascular cell growth, and immunoregulation-related gene expression levels in HUVECs with induced oxidative stress were analysed. Gas chromatography-mass spectroscopy (GC-MS) results confirmed that BPME contains 5-hydroxymethylfurfural (32.6%), methyl pyruvate (15.13%), furfural (9.98%), and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-Pyran-4-one (12.4%). BPME extract effectively enhanced cell proliferation and was confirmed by MTT assay; the nuclear integrity was confirmed by propidium iodide (PI) staining assay; the mitochondrial membrane potential (Δψm) was confirmed by JC-1 staining assay. Annexin V assay confirmed that BPME-treated HUVECs showed 99% viable cells, but only 39.8% viability was shown in HUVECs treated with H2O2 alone. In addition, BPME treatment of HUVECs for 48 h reduced mRNA expression of lipid peroxide (LPO) and increased NOS-3, Nrf-2, GSK-3β, GPX, endothelial nitric oxide synthase (eNOS) and vascular cell growth factor (VEGF) mRNA expression levels. We found that BPME treatment decreased proinflammatory (nuclear factor-κβ (F-κβ), tissue necrosis factor-α (TNF-α), toll-like receptor-4 (TLR-4), interleukin-1β (IL-1β)) and vascular inflammation (intracellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), EDN1, IL-1β)-related mRNA expressions. In conclusion, beetroot peel treatment effectively increased vascular smooth cell growth factors and microtubule development, whereas it decreased vascular inflammatory regulators. BPME may be beneficial for vascular smooth cell regeneration, tissue repair and anti-ageing potential.

Keywords: angiogenesis; beetroot; inflammation; mitochondria; oxidative stress.

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Conflict of interest statement

There is no conflict of interest for this study.

Figures

Figure 1
Figure 1
Diagrammatic representation of experimental design and timeline for the present study.
Figure 2
Figure 2
Results for GC-MS chromatogram of beetroot peel methanol extract (a), in vitro cell proliferation (b) and light microscopy images for cell morphology (c) in HUVECs treated with increasing concentration of beetroot peel methanol extract (BPME) (uniform shape of adherent and proliferating cells mentioned by red arrowhead). All values are means ± SD (n = 6). * p ≤ 0.05 by comparison with vehicle control.
Figure 3
Figure 3
Analysis of microtube formation based on florescence microscopic in vehicle control, 0.1 and 0.2 μg/mL dose of beetroot peel methanol extract (BPME) treated normal and H2O2-induced oxidative-stressed HUVECs after 48 h. After 48 h, H2O2-induced oxidative-stressed HUVECs showed altered morphology when compared to vehicle control. Volumes of 0.1 and 0.2 μg/mL of beetroot peel methanol extract (BPME)-treated HUVECs showed normal morphology with vascular microtubules with proliferating cells resembling the common morphology of smooth muscle cell behaviour.
Figure 4
Figure 4
Propidium iodide (PI) staining analysis for nuclear morphology in vehicle control, 0.1 and 0.2 μg/mL of beetroot peel methanol extract (BPME)-treated normal (3a) and H2O2-induced oxidative-stressed (3b) HUVECs after 48 h. In PI staining, vehicle control showing the nucleus appeared to be normal with no signs of shrunken, pyknosis or apoptotic nucleus. In H2O2 alone, treated cells showed polarized nuclear membrane after 48 h. Treatment with 0.2 μg/mL of BPME normalized the H2O2-induced nuclear membrane polarization when compared to 0.1 μg/mL BPME or Quercetin 10 μM.
Figure 5
Figure 5
Analysis of mitochondrial membrane potential using JC-1 staining for vehicle control, 0.1 and 0.2 μg/mL of beetroot peel methanol extract (BPME) treated normal (a) and H2O2-induced oxidative-stressed (b) HUVECs after 48 h. JC-1 fluorescence images showing merged images of the red and green signals of the dye, corresponding to JC-1 in J-aggregates vs. monomeric form. We found less J-aggregates H2O2 alone treated HUVECs. In 0.2 μg/mL BPME-treated HUVECs showing high j-aggregates directly representing (high MMP, Δψm) high mitochondrial membrane potential compared to 0.1 μg/mL of BPME or 10 μM of Quercetin.
Figure 6
Figure 6
Flow cytometry-based analysis of mitochondrial membrane capacity using JC-1 (Δψm, BD Mito Scan) in normal and H2O2-induced oxidative-stressed HUVECs treated with 0.2 μg/mL of BPME. Untreated cells were considered a negative control, whereas H2O2 (10 mM) was added for positive control.
Figure 7
Figure 7
Annexin-V/propidium iodide double staining-based apoptosis, necrosis or late apoptotic characteristic analysis of BPME (0.2 μg/mL)-treated normal and H2O2-induced oxidative-stressed HUVECs. Untreated cells were considered a negative control, whereas H2O2 (10 mM) was added for positive control. (Annexin-V/apoptosis quadrants: A3: viable cells; A4: early apoptosis; A2: late apoptosis; A1: necrosis).
Figure 8
Figure 8
Oxidative stress, antioxidant (a), pro-inflammation (b) and vascular cell inflammation (c)-related gene expression levels of vehicle control, 0.1 and 0.2 μg/mL of beetroot peel methanol extract (BPME)-treated normal and H2O2-induced oxidative-stressed HUVECs after 48 h. All values are means ± SD (n = 6). * p ≤ 0.05 and ** p ≤ 0.001 were compared with vehicle control or H2O2 alone treated HUVECs.
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