Biological impacts of resveratrol, quercetin, and N-acetylcysteine on oxidative stress in human gingival fibroblasts

Abstract

In periodontitis, production of reactive oxygen species (ROS) by neutrophils induces oxidative stress and deteriorates surrounding tissues. Antioxidants reduce damage caused by ROS and are used to treat diseases involving oxidative stress. This study summarizes the different effects of resveratrol, quercetin, and N-acetylcysteine (NAC) on human gingival fibroblasts (HGFs) under oxidative stress induced by hydrogen peroxide. Real-time cytotoxicity analyses reveals that resveratrol and quercetin enhanced cell proliferation even under oxidative stress. Of the antioxidants tested, resveratrol is the most effective at inhibiting ROS production. HGFs incubated with resveratrol and quercetin up-regulate the transcription of type I collagen gene after 3 h, but only resveratrol sustained this up-regulation for 24 h. A measurement of the oxygen consumption rate (OCR, mitochondrial respiration) shows that resveratrol generates the highest maximal respiratory capacity, followed by quercetin and NAC. Simultaneous measurement of OCR and the extracellular acidification rate (non-mitochondrial respiration) reveals that resveratrol and quercetin induce an increase in mitochondrial respiration when compared with untreated cells. NAC treatment consumes less oxygen and enhances more non-mitochondrial respiration. In conclusion, resveratrol is the most effective antioxidant in terms of real-time cytotoxicity analysis, reduction of ROS production, and enhancement of type I collagen synthesis and mitochondrial respiration in HGFs.

Keywords: N-acetylcysteine; human gingival fibroblasts; periodontitis; quercetin; resveratrol.

Fig. 1

Dynamic monitoring of cell viability and proliferation expressed as a cell index (CI) by xCELLigence system. HGFs (10,000 cells/well) were exposed to H₂O₂ (0.23 mM) to induce oxidative stress and treated with different concentrations of (A) resveratrol, (B) quercetin, and (C) NAC for 48 h. The percentage of variation of CI represents the changes recorded in CI values compared to the Base value. The graph shown is the result of the mean of three independent experiments (n = 6).

Fig. 2

Intracellular ROS scavenging activity of antioxidants in HGFs on oxidative stress. Representative double-stained, confocal laser microscopic images depicting ROS production induced by 0.23 mM H₂O₂ on HGFs (40×) with or without antioxidants after 30 min of incubation. Green fluorescence intensity corresponds to intracellular ROS production, and DAPI (blue) staining to viable cells. H₂O₂ affected the morphology of HGFs, which is typically large, flat, and elongated (spindle-shaped).

Fig. 3

Effect of resveratrol, quercetin, and NAC on type I collagen gene expression in HGFs under oxidative stress induced by H₂O₂ after different incubation periods. Relative mRNA of Type I collagen was detected by qRT-PCR. Bars represent mRNA expression normalized to GAPDH and relative to the control group. Data are expressed as mean ± SEM based on four independent experiments (n = 9). *p<0.05, **p<0.01: significantly different from H₂O₂ group.

Fig. 4

Effect of antioxidants on mitochondrial respiratory profiles in HGFs under oxidative stress. (A) Changes in HGFs’ cellular respiration under oxidative stress. OCR was measured in HGFs cells after 30 min of treatment with resveratrol (50 µM), quercetin (15 µM), and NAC (1.5 mM). Seahorse OCR protocol (mitochondrial stress test) included (1) H₂O₂ (1 mM) and three mitochondrial ETC complex inhibitors: (2) oligomycin, (3) FCCP, and (4) antimycin A with rotenone. OCR measured before (1) represents basal respiration, between (3) and (4) represents maximal respiratory capacity. Plots shown are means ± SEM (n = 6). (B) Simultaneous comparison of OCR versus ECAR in HGFs stressed by H₂O₂ (1 mM) and treated with resveratrol (50 µM), quercetin (15 µM), and NAC (1.5 mM). A higher OCR value indicates predominance of mitochondrial respiration. Similarly, a high ECAR value indicates predominance of non-mitochondrial respiration. Plots indicated the SEM (n = 6). (C) Antioxidant treatment enhances OCR rate in oxidative stress conditions. The bars show the comparison of the total amount of oxygen consumed by mitochondrial respiration in HGFs stressed by H₂O₂ (1 mM) following treatment with resveratrol (50 µM), quercetin (15 µM), and NAC (1.5 mM). Data are expressed as mean ± SEM (n = 6). *p<0.05: significantly different from the H₂O₂ group.