β-Asarone Alleviates High-Glucose-Induced Oxidative Damage via Inhibition of ROS Generation and Inactivation of the NF-κB/NLRP3 Inflammasome Pathway in Human Retinal Pigment Epithelial Cells

Abstract

Diabetic retinopathy (DR) is the leading cause of vision loss and a major complication of diabetes. Hyperglycemia-induced accumulation of reactive oxygen species (ROS) is an important risk factor for DR. β-asarone, a major component of volatile oil extracted from Acori graminei Rhizoma, exerts antioxidant effects; however, its efficacy in DR remains unknown. In this study, we investigated whether β-asarone inhibits high-glucose (HG)-induced oxidative damage in human retinal pigment epithelial (RPE) ARPE-19 cells. We found that β-asarone significantly alleviated cytotoxicity, apoptosis, and DNA damage in HG-treated ARPE-19 cells via scavenging of ROS generation. β-Asarone also significantly attenuated the excessive accumulation of lactate dehydrogenase and mitochondrial ROS by increasing the manganese superoxide dismutase and glutathione activities. HG conditions markedly increased the release of interleukin (IL)-1β and IL-18 and upregulated their protein expression and activation of the nuclear factor-kappa B (NF-κB) signaling pathway, whereas β-asarone reversed these effects. Moreover, expression levels of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome multiprotein complex molecules, including thioredoxin-interacting protein, NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain, and cysteinyl aspartate-specific proteinase-1, were increased in ARPE-19 cells under HG conditions. However, their expression levels remained similar to those in the control group in the presence of β-asarone. Therefore, β-asarone protects RPE cells from HG-induced injury by blocking ROS generation and NF-κB/NLRP3 inflammasome activation, indicating its potential as a therapeutic agent for DR treatment.

Keywords: NF-κB; NLRP3 inflammasome; ROS; high glucose; β-asarone.

Figure 1

Inhibitory effects of β-asarone on high-glucose (HG)-induced cell viability reduction and cytotoxicity in ARPE-19 cells. Cells were grown for 48 h in a medium with various doses of β-asarone (0, 10, 25, 50, and 100 μM) (A) and glucose (0, 5, 10, 20, and 40 μM) (B) or pre-treated with β-asarone (50 μM) or N-acetyl-cysteine (NAC; 10 mM) for 1 h and cultured for an additional 48 h under HG (20 mM glucose) conditions in presence of β-asarone or NAC (C,D). Cell viability and cytotoxicity were determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) (A–C) and lactate dehydrogenase (LDH) (D) assays, respectively. Numerical values are represented as the mean ± standard deviation of three independent experiments (SD; * p < 0.05 and *** p < 0.001 vs. control group; ### p < 0.001 vs. HG-treated group).

Figure 2

Inhibition of HG-induced apoptosis and DNA damage by β-asarone in ARPE-19 cells. Cells were treated with β-asarone (50 μM) for 1 h and cultured for an additional 48 h under HG (20 mM glucose) conditions. (A) Representative images of cell morphology changes as observed by using inverted microscope. (B,C) 4′,6-diamidino-2-phenylindole (DAPI) staining was performed to observe morphological changes in the nuclei by using fluorescence microscopy. Representative images of DAPI-stained nuclei (B) and quantification results of apoptotic nuclei (C). (D,E) Apoptosis was measured using flow cytometry after Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Representative images of flow cytometry (D) and quantification results (E). (F,G) After the lysis of collected cells, equal amounts of protein from each cell lysate were loaded and blotted with poly(ADP ribose) polymerase (PARP) and β-actin antibodies. (H,I) DNA damage was assessed using the 8-hydro’y-2′-deoxyguanosine (8-OHdG) (H) and comet (I) assays. (C,E,G) Numerical values are represented as the mean ± SD of three independent experiments (*** p < 0.001 vs. control group; ### p < 0.001 vs. HG-treated group).

Figure 3

Suppression of HG-induced cytosolic reactive oxygen species (ROS) accumulation by β-asarone in ARPE-19 cells. (A–D) Cells pre-treated with β-asarone for 1 h were cultured in an HG medium for 1 h and stained with 5,6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). (A,B) Representative images of flow cytometry (A) and quantification results (B). (C) Representative images (green) of cytosolic ROS levels visualized using fluorescence microscopy (200×). (E) Reduced glutathione/oxidized glutathione (GSH/GSSG) ratio was measured using a commercially available kit. (B,D,E) Numerical values are represented as the mean ± SD of three independent experiments (*** p < 0.001 vs. control group; ### p < 0.001 vs. HG-treated group).

Figure 4

Blockade of HG-induced mitochondrial ROS accumulation by β-asarone in ARPE-19 cells. Cells pre-treated with β-asarone for 1 h were cultured in an HG medium for 1 h and stained with MitoSOX. (A,B) Representative images of flow cytometry (A) and quantification results (B). (C,D) Representative images of mitochondrial ROS levels (red) visualized using fluorescence microscopy (400×). Nuclei (blue) were stained with DAPI. (E,F) After the lysis of collected cells, equal amounts of protein from each cell lysate were loaded and blotted with manganese superoxide dismutase (MnSOD) and β-actin antibodies. (G) Activity of Mn-SOD was measured using a commercially available kit. (B,D,F,G) Numerical values are represented as the mean ± SD of three independent experiments (*** p < 0.001 vs. control group; ### p < 0.001 vs. HG-treated group).

Figure 5

Attenuation of HG-induced cytokine production and nuclear factor-kappa B (NF-κB) activation by β-asarone in ARPE-19 cells. (A,B) Concentrations of interleukin (IL)-1β (A) and IL-18 (B) released in the supernatant of cells exposed to HG for 48 h in the presence or absence of β-asarone were measured using enzyme-linked immunosorbent assay (ELISA) kits. (C,D) Expression levels of IL-1β and IL-18 proteins were determined via Western blot analysis using cells cultured under the same conditions. (E–G) Cells pre-treated with β-asarone for 1 h were further cultured in an HG medium for another 1 h. (E,F) Nuclear and cytosolic fractions were used to detect the indicated proteins. (G) Cellular localization of p-NF-κB p65 (red) in cells cultured under the same experimental setting was observed using the immunofluorescence assay. Nuclei (blue) were counterstained with DAPI. (A,B,D,F) Numerical values are represented as the mean ± SD of three independent experiments (* p < 0.05, *** p < 0.001 vs. control group; ### p < 0.001 vs. HG-treated group).

Figure 6

Suppression of HG-induced NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation by β-asarone in ARPE-19 cells. Cells pre-treated with β-asarone for 1 h were further cultured in an HG medium for 48 h, and Western blot analysis was conducted to determine the expression levels of the indicated proteins (A,B). (C–F) Cellular localization of NLRP3 (green) and apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) (red) in cells cultured under same experimental setting was observed using the immunofluorescence assay. Nuclei (blue) were counterstained with DAPI. (G) Activity of caspase-1 was measured using a fluorescence assay kit. (B,D,F,G) Numerical values are represented as the mean ± SD of three independent experiments (*** p < 0.001 vs. control group; ### p < 0.001 vs. HG-treated group).

Figure 7

Schematic diagram of the inhibitory effects of β-asarone on mitochondrial ROS-mediated activation of NF-κB/NLRP3 signaling, resulting in the alleviation of inflammation in hyperglycemic RPE cells.