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. 2020 Mar 2;9(3):205.
doi: 10.3390/antiox9030205.

Inhibitory Effect and Mechanism of Action of Quercetin and Quercetin Diels-Alder anti-Dimer on Erastin-Induced Ferroptosis in Bone Marrow-Derived Mesenchymal Stem Cells

Xican Li  1   2 , Jingyuan Zeng  1 , Yangping Liu  3 , Minshi Liang  1   2 , Qianru Liu  1   2 , Zhen Li  4 , Xiaojun Zhao  1   2 , Dongfeng Chen  4
Affiliations

Inhibitory Effect and Mechanism of Action of Quercetin and Quercetin Diels-Alder anti-Dimer on Erastin-Induced Ferroptosis in Bone Marrow-Derived Mesenchymal Stem Cells

Xican Li et al. Antioxidants (Basel). .

Abstract

In this study, the anti-ferroptosis effects of catecholic flavonol quercetin and its metabolite quercetin Diels-Alder anti-dimer (QDAD) were studied using an erastin-treated bone marrow-derived mesenchymal stem cell (bmMSCs) model. Quercetin exhibited higher anti-ferroptosis levels than QDAD, as indicated by 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY), 2',7'-dichlorodihydrofluoroscein diacetate (H2DCFDA), lactate dehydrogenase (LDH) release, cell counting kit-8 (CCK-8), and flow cytometric assays. To understand the possible pathways involved, the reaction product of quercetin with the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH●) was measured using ultra-performance liquid-chromatography coupled with electrospray-ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-Q-TOF-MS). Quercetin was found to produce the same clusters of molecular ion peaks and fragments as standard QDAD. Furthermore, the antioxidant effects of quercetin and QDAD were compared by determining their 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide radical-scavenging, Cu2+-reducing, Fe3+-reducing, lipid peroxidation-scavenging, and DPPH●-scavenging activities. Quercetin consistently showed lower IC50 values than QDAD. These findings indicate that quercetin and QDAD can protect bmMSCs from erastin-induced ferroptosis, possibly through the antioxidant pathway. The antioxidant pathway can convert quercetin into QDAD-an inferior ferroptosis-inhibitor and antioxidant. The weakening has highlighted a rule for predicting the relative anti-ferroptosis and antioxidant effects of catecholic flavonols and their Diels-Alder dimer metabolites.

Keywords: Diels-Alder dimer; QDAD; anti-ferroptosis; antioxidant; erastin; quercetin.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The structures and molecular models of quercetin and quercetin Diels-Alder anti-dimer (QDAD): (A) structure of quercetin; (B) structure of QDAD; (C) molecular model of quercetin; (D) molecular model of 2R,3R-QDAD. The molecular model was created based on the preferential conformation by using Chem3D Pro 14.0 (PerkinElmer, Waltham, MA, USA).
Figure 2
Figure 2
Fluorescence images of normal and ferroptotic bone marrow-derived mesenchymal stem cells (bmMSCs). (A) Control group (bmMSCs with no erastin treatment); (B) model group (erastin-treated bmMSCs); (C) positive control group (bmMSCs treated with erastin + ferrostatin-1); (D) quercetin sample group (bmMSCs treated with erastin + quercetin); (E) QDAD sample group (bmMSCs treated with erastin + QDAD). Lipid peroxidation (LPO) accumulation was probed using 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY) and is depicted in green; cell nuclei were stained using DAPI (4‘,6-diamidino-2-phenylindole) and are shown in blue; and mitochondria were stained using BBcell ProbeTM and are shown as red fluorescence.
Figure 3
Figure 3
Representative flow cytometry analysis of the 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY) fluorescence intensity (A–F) and the percentage of the relative mean fluorescence intensity of C11-BODIPY (F) of bmMSCs. (A) Control group; (B) model (erastin) group; (C) positive control (erastin + ferrostatin-1) group; (D) erastin + 0.03 μM quercetin; (E) erastin + 0.03 μM QDAD; (F) relative percentage of the mean fluorescence intensity of experimental groups. * The quercetin-treated samples (0.03 μM) have a significantly (p < 0.01) lower fluorescence than the erastin-treated group. Other groups do not show a significant decrease (p > 0.01). FL1-A (fluorochromes emitting A channel).
Figure 4
Figure 4
Representative flow cytometry analysis of the 2′,7′-dichlorodihydrofluoroscein diacetate (H2DCFDA) fluorescence intensity (A–E) and the percentage of the relative mean fluorescence intensity of H2DCFDA (F) of bmMSCs. (A) Control group; (B) model (erastin) group; (C) positive control (erastin + ferrostatin-1) group; (D) erastin + 0.03 μM quercetin; (E) erastin + 0.03 μM QDAD; (F) relative percentage of the mean fluorescence intensity of experimental groups. * The quercetin-treated sample group (0.03 μM) has a significantly (p < 0.01) lower fluorescence intensity than the erastin-treated samples. H2DCFDA: 2′,7′-dichlorodihydrofluoroscein diacetate; FL1-A: fluorochromes emitting A channel.
Figure 5
Figure 5
The lactate dehydrogenase (LDH) release assay (A) and cell counting kit-8 (CCK-8) assay (B) of erastin-induced ferroptotic bmMSCs. The erastin group is the model group, while erastin + ferrostatin-1 is the positive control group. * p < 0.05, compared with the erastin-treated group. Significant (p < 0.05) difference is shown between the 0.03 μM quercetin-treated group and the 0.09 μM quercetin-treated group in B.
Figure 6
Figure 6
Main results of ultra-performance liquid-chromatography coupled with electrospray-ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-Q-TOF-MS/MS) analysis. (A) Chromatogram of quercetin when the formula [C15H10O7−H]− was extracted. (B) Primary MS spectra of quercetin. (C) Secondary MS spectra of quercetin. (D) Chromatogram of possible dimeric products of quercetin-quercetin when the formula [C30H18O14−H]− was extracted. (E) Primary MS spectra of possible dimeric products. (F) Secondary MS spectra of the radical adduct formation (RAF) product. (G) Chromatogram of standard QDAD when the formula [C30H18O14−H]− was extracted. (H) Primary MS spectra of standard QDAD. (I) Secondary MS spectra of standard QDAD.
Figure 7
Figure 7
The proposed MS elucidation of QDAD (the accurate m/z values are simply expressed as integers; electron transfer has not been marked. Other reasonable cleavages should not be excluded in the MS elucidation).
Figure 8
Figure 8
Scheme for the cause of nomenclature of the “(±) quercetin Diels-Alder anti-dimer”. (A) quercetin oxidation by the DPPH● radical; (B) Diels-Alder dimerization reaction (the single-barbed curved arrow indicates one electron transfer in Figure A).
Figure 9
Figure 9
The structures of catecholic flavonols and the relevant Diels-Alder dimers in Dysosma versipellis.
Figure 10
Figure 10
The prediction of relative antioxidant levels for catecholic flavonols and the Diels-Alder dimers.
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