Paeoniflorin Protects Retinal Pigment Epithelial Cells from High Glucose-Induced Oxidative Damage by Activating Nrf2-Mediated HO-1 Signaling

Abstract

Oxidative stress due to hyperglycemia damages the functions of retinal pigment epithelial (RPE) cells and is a major risk factor for diabetic retinopathy (DR). Paeoniflorin is a monoterpenoid glycoside found in the roots of Paeonia lactiflora Pall and has been reported to have a variety of health benefits. However, the mechanisms underlying its therapeutic effects on high glucose (HG)-induced oxidative damage in RPE cells are not fully understood. In this study, we investigated the protective effect of paeoniflorin against HG-induced oxidative damage in cultured human RPE ARPE-19 cells, an in vitro model of hyperglycemia. Pretreatment with paeoniflorin markedly reduced HG-induced cytotoxicity and DNA damage. Paeoniflorin inhibited HG-induced apoptosis by suppressing activation of the caspase cascade, and this suppression was associated with the blockade of cytochrome c release to cytoplasm by maintaining mitochondrial membrane stability. In addition, paeoniflorin suppressed the HG-induced production of reactive oxygen species (ROS), increased the phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), a key redox regulator, and the expression of its downstream factor heme oxygenase-1 (HO-1). On the other hand, zinc protoporphyrin (ZnPP), an inhibitor of HO-1, abolished the protective effect of paeoniflorin against ROS production in HG-treated cells. Furthermore, ZnPP reversed the protective effects of paeoniflorin against HG-induced cellular damage and induced mitochondrial damage, DNA injury, and apoptosis in paeoniflorin-treated cells. These results suggest that paeoniflorin protects RPE cells from HG-mediated oxidative stress-induced cytotoxicity by activating Nrf2/HO-1 signaling and highlight the potential therapeutic use of paeoniflorin to improve the symptoms of DR.

Keywords: High glucose; Nrf2/HO-1; Oxidative stress; Paeoniflorin; Retinal pigment epithelial cells.

Fig. 1

Suppression of HG-induced cytotoxicity by paeoniflorin in ARPE-19 cells. Cells were treated with different concentrations of glucose (0, 5, 10, 20, 30 and 40 mM, A) or paeoniflorin (0, 50, 100, 200, 300 and 400 μM, B) for 48 h, or with the indicated concentrations of paeoniflorin for 2 h, and then treated with HG (30 mM D-(+)-glucose) for 48 h (C). Cell viability was determined using an MTT assay. (D) Relative levels of LDH released into cell supernatant were determined using an LDH activity assay kit. Values are the means ± SD for at least three independent experiments, and analysis of variance followed by Tukey’s post hoc test showed significant differences (*p<0.05, **p<0.01 and ***p<0.001 vs. control cells; ^#p<0.05 and ^###p<0.001 vs. HG-treated cells).

Fig. 2

Amelioration of HG-induced apoptosis and DNA damage by paeoniflorin in ARPE-19 cells. Cells were pretreated with or without paeoniflorin (0, 50 and 100 μM) for 2 h and then stimulated with HG (30 mM D-(+)-glucose) for 48 h. (A) Apoptosis was analyzed by annexin V/PI staining. Each number represents the total frequency of cells in the early stage (annexin V positive) of apoptosis and cells in the late stage (annexin V and PI double positive) of apoptosis. (B, C). Morphological changes in nuclei were observed using a fluorescence microscope after DAPI staining (1 μg/mL, scale bar 75 µm). Representative images (B) and the proportion of apoptotic nuclei (C). (D) DNA was isolated from cells and separated by agarose gel electrophoresis to visualize DNA fragmentation. (E) Changes in caspase-3, PARP, and γH2AX expressions were determined using total protein levels. β-actin served as a loading control. (F) Caspase-3 activity was assessed using a commercially available kit. (G) Extent of DNA damage was determined using a Comet assay (scale bar 50 µm). (A, C) Values are the means ± SD for at least three independent experiments, and analysis of variance followed by Tukey’s post hoc test showed significant differences (***p<0.001 vs. control cells; ^##p<0.01 and ^###p<0.001 vs. HG-treated cells).

Fig. 3

Attenuation of HG-induced mitochondrial dysfunction and cytosolic release of cytochrome c by paeoniflorin in ARPE-19 cells. (A) Cells were treated with HG (30 mM D-(+)-glucose) in the presence or absence of paeoniflorin (0, 50 and 100 μM) for 48 h, JC-1 stained, and subjected to followed by flow cytometry. Changes in JC-1 monomer ratio (indicating MMP loss) are shown. JC-1, a cationic carbocyanine dye, shows voltage-dependent accumulation in mitochondria and begins to form J aggregates in mitochondria. Since it remains as a monomer upon depolarization of the mitochondrial membrane, the high frequency of monomers in paeoniflorin-treated cells indicates the loss of MMP. Values are the means ± SD for at least three independent experiments, and analysis of variance followed by Tukey’s post hoc test showed significant differences (***p<0.001 vs. control cells; ^##p<0.01 and ^###p<0.001 vs. HG-treated cells). (B) Changes in cytochrome c expression were analyzed using mitochondrial (Mito) and cytosolic fractions (Cyto). COX IV and β-actin were used as loading controls for the mitochondrial and cytosolic fractions, respectively.

Fig. 4

Attenuation of ROS production and induction of Nrf2-mediated HO-1 by paeoniflorin in HG-treated ARPE-19 cells. Cells were pretreated with or without paeoniflorin (0, 50 and 100 μM) for 2 h and then stimulated with HG (30 mM D-(+)-glucose) for 1 h (A, B) or 48 h (C). (A) After DCF-DA staining, fluorescence intensities, representing ROS production, were measured under a fluorescence microscope (scale bar 50 µm). (B) Changes in intracellular ROS levels were investigated by flow cytometry after DCF-DA staining. Values are the means ± SD for at least three independent experiments, and analysis of variance followed by Tukey’s post hoc test showed significant differences (***p<0.001 vs. control cells; ^###p<0.001 vs. HG-treated cells). (C) Changes in the expressions of Nrf2, HO-1, and Keap1 were investigated using total proteins isolated from cells.

Fig. 5

Loss of the ROS scavenging and mitochondrial protective effects of paeoniflorin after inhibiting HO-1 activity in HG-treated ARPE-19 cells. Cells were pretreated with 100 μM paeoniflorin and 10 μM ZnPP for 2 h and then stimulated with HG (30 mM D-(+)-glucose) for 1 h (A, B) or 48 h (C-E). (A) Changes in intracellular ROS levels were measured by DCF-DA staining. (B) Flow cytometric analysis was performed on JC-1-stained. (A, B) Values are the means ± SD for at least three independent experiments, and analysis of variance followed by Tukey’s post hoc test showed significant differences (***p<0.001 vs. control cells; ^###p<0.001 vs. HG-treated cells; ^$$$p<0.001 vs. HG and paeoniflorin-treated cells). (C) Changes in cytochrome c expression were analyzed in mitochondrial and cytosolic fractions isolated from cells.

Fig. 6

Abrogation of the protective effect of paeoniflorin against HG-induced apoptosis and cytotoxicity by HO-1 inhibition in ARPE-19 cells. Cells were pretreated with 100 μM paeoniflorin and 10 μM ZnPP for 2 h and then stimulated with HG (30 mM D-(+)-glucose) for 48 h. (A) The average degree of apoptosis (annexin V-positive cells) determined by Annexin V/PI staining is shown. (B) DNA fragmentation was visualized by agarose gel electrophoresis. (C) Caspase-3, PARP, and γH2AX expressions were determined using total proteins. (D) Caspase-3 activity was assessed using a commercial assay kit. (E) Cell viabilities were determined using an MTT assay. (A, D, E) Values are the means ± SD for at least three independent experiments, and analysis of variance followed by Tukey’s post hoc test showed significant differences (***p<0.001 vs. control cells; ^###p<0.001 vs. HG-treated cells; ^$$$p<0.001 vs. HG and paeoniflorin-treated cells).

Fig. 7

Schematic diagram showing the protective effect of paeoniflorin against HG-mediated oxidative stress-induced cytotoxicity in RPE ARPE-19 cells. ARE, antioxidant response element; HO-1, heme oxygenase-1; Keap1, Kelch-like ECH associated protein 1; Nrf2, nuclear factor erythroid 2-related factor 2; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; ZnPP, zinc protoporphyrin; γH2AX, phosphorylated form of H2AX.