Antioxidant activity of glucosamine and its effects on ROS production, Nrf2, and O-GlcNAc expression in HMEC-1 cells

Abstract

Glucosamine (GlcN) is the most used supplement for osteoarthritis treatment. In vitro studies have related GlcN to beneficial and detrimental effects on health. The aim of this study was to evaluate the effects of O-linked-N-acetylglucosaminylation (O-GlcNAc) on GlcN-induced ROS production and Nrf2 expression in human dermal microvascular endothelial cells-1 (HMEC-1) and to evaluate the antioxidant capacity of GlcN compared to well-known antioxidants. For this, we evaluate the antioxidant capacity by in vitro assays. Besides, the GlcN (5-20 mM) effects on cell viability, reactive oxygen species (ROS) production, O-GlcNAc, and nuclear factor erythroid-2-related factor 2 (Nrf2) expression with and without the O-GlcNAc inhibitor OSMI-1 (10 μM) in HMEC-1 were evaluated. GlcN showed high inhibitory concentration (low scavenging activity) against superoxide (O₂^•─, IC₂₀ = 47.67 mM), 2,2-diphenyl-1-picrylhydrazyl (DPPH^•, IC₅₀ = 21.32 mM), and hydroxyl (HO^•, IC₅₀ = 14.04 mM) radicals without scavenging activity against hydrogen peroxide (H₂O₂) and low antioxidant capacity determined by oxygen radical absorbance capacity (ORAC, 0.001 mM Trolox equivalent) and ferric reducing antioxidant power (FRAP, 0.046 mM Trolox equivalent). In cell culture, GlcN (20 mM) reduced cell viability up to 26 % and induced an increase in ROS production (up to 70 %), O-GlcNAc (4-fold-higher vs. control), and Nrf2 expression (56 %), which were prevented by OSMI-1. These data suggest an association between O-GlcNAc, ROS production, and Nrf2 expression in HMEC-1 cells stimulated with GlcN.

Keywords: Endothelium; Glucosamine; Nrf2; O-GlcNAc; ROS production; Scavenging activity.

Graphical abstract

Fig. 1

Scavenging activity of glucosamine (GlcN) against A: hydroxyl (HO^•), B: 2,2-Diphenyl-1-picrylhydrazyl (DPPH^•) and C: superoxide (O₂^•-). Data are shown as mean ± SEM, n ≥ 3.

Fig. 2

GlcN decreases cell viability in human dermal microvascular endothelial cells-1 (HMEC-1). GlcN (5–20 mM) treatment for 24 h reduced cell viability by the MTT assay. Data are shown as mean ± SD, * p ≤ 0.05 vs. control group (C), n = 5.

Fig. 3

Time-course of O-linked-N-acetylglucosaminylation (O-GlcNAcylation) induced by 5, 10, and 20 mM GlcN in HMEC-1 cells. A: Representative micrographs (10X) illustrating O-GlcNAcylation expression. B: Quantification of O-GlcNAcylation expression. Data are mean ± SEM, n = 3, *p ≤ 0.05 vs. control.

Fig. 4

GlcN-induced O-GlcNAc and nuclear factor erythroid-2-related factor 2 (Nrf2) expression in HMEC-1 is related to O-GlcNAcylation. A: Representative images (20X) of HMEC-1 cells treated with 20 mM GlcN (6 h), 10 μM OSMI-1 (24 h, O-GlcNAc transferase inhibitor), OSMI-1 + GlcN or 100 μM Q (6 h; as a Nrf2 inducer). The red, green, and blue signals represent the O-GlcNAcylation and Nrf2 expression, and nuclear stain, respectively. B: Quantification of O-GlcNAcylation expression. C: Quantification of Nrf2 expression. Data are mean ± SEM, n = 3 (independent experiments), *p ≤ 0.05 vs. control, #p ≤ 0.05 vs. GlcN.

Fig. 5

GlcN induced reactive oxygen species (ROS) in a concentration-dependent manner in HMEC-1 cells. A: Representative images of intracellular ROS production (20X) by 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) fluorochrome is observed in green, while the dihydroethidium (DHE) fluorochrome is shown in red. B: Quantification using H₂DCFDA and C: DHE. Data are shown as mean ± SEM, n = 3. *p ≤ 0.05 vs. control. Treatment of 1 mM H₂O₂ for 2 h was used as the positive control.

Fig. 6

Inhibition of O-GlcNAcylation by OSMI-1 reduced GlcN-induced ROS production in HMEC-1 cells. A: representative images of intracellular ROS production (20X). Quantitative analysis of fluorescence intensity of B: H₂DCFDA (green signal) and C: DHE (red signal). Each bar represents the mean ± SEM, n = 3. *p ≤ 0.05 vs. control.

Fig. 7

Summary of the main findings on HMEC-1. GlcN is incorporated into the cell by glucose transporters (purple line), then is incorporated into the hexosamine biosynthetic pathway (HBP), where the uridine diphosphate (UDP)-GlcNAc substrate is used by the OGT enzyme for O-GlcNAc (blue box) several proteins, while the enzyme OGA removes it. GlcN treatment increases O-GlcNAcylation (blue lines), reactive oxygen species (ROS) production, and nuclear factor erythroid-2-related factor 2 (Nrf2) expression in human dermal microvascular endothelial cells-1 (HMEC-1). The inhibition of O-GlcNAcylation, with OSMI-1, prevents (orange lines) ROS production and Nrf2 expression, suggesting that these processes are related. In addition, GlcN also could scavenge some ROS. Biorender.com GPx = glutathione peroxidase, CAT = catalase, SOD = superoxide dismutase, GSH = glutathione, NQO1 = NAD(P)H quinone oxidoreductase 1, HOCl = hypochlorous acid, ONOO^– = peroxynitrite anion, O₂^•- = superoxide anion, ROO^• = peroxyl radicals, H₂O₂ = hydrogen peroxide, OH^• = hydroxyl radicals, HMEC-1 = human dermal microvascular endothelial cells-1. Created with BioRender.com.