Phloroglucinol Attenuates DNA Damage and Apoptosis Induced by Oxidative Stress in Human Retinal Pigment Epithelium ARPE-19 Cells by Blocking the Production of Mitochondrial ROS

Abstract

Phloroglucinol, a phenolic compound, is known to possess a potent antioxidant ability. However, its role in retinal cells susceptible to oxidative stress has not been well elucidated yet. Thus, the objective of this study was to evaluate whether phloroglucinol could protect against oxidative damage in cultured human retinal pigment epithelium ARPE-19 cells. For this purpose, ARPE-19 cells were stimula ted with hydrogen peroxide (H₂O₂) to mimic oxidative stress. Cell viability, cytotoxicity, apoptosis, reactive oxygen species (ROS) generation, mitochondrial function, DNA damage, and autophagy were then assessed. Our results revealed that phloroglucinol ameliorated cell viability, cytotoxicity, and DNA damage in H₂O₂-exposued ARPE-19 cells and blocked production of ROS. Phloroglucinol also counteracted H₂O₂-induced apoptosis by reducing Bax/Bcl-2 ratio, blocking activation of caspase-3, and inhibiting degradation of poly (ADP-ribose) polymerase. H₂O₂ caused mitochondrial impairment and increased expression levels of mitophagy markers such as PINK1and PARKIN known to be associated with mitochondrial ROS (mtROS) generation and cytosolic release of cytochrome c. However, these changes were significantly attenuated by phloroglucinol. Mito-TEMPO, a selective mitochondrial antioxidant, further enhanced the protective effect of phloroglucinol against dysfunctional mitochondria. Furthermore, H₂O₂ induced autophagy, but not when ARPE-19 cells were pretreated with phloroglucinol, meaning that autophagy by H₂O₂ contributed to the pro-survival mechanism and that phloroglucinol protected ARPE-19 cells from apoptosis by blocking autophagy. Taken together, these results suggest that phloroglucinol can inhibit oxidative stress-induced ARPE-19 cell damage and dysfunction by protecting DNA damage, autophagy, and subsequent apoptosis through mitigation of mtROS generation. Thus, phloroglucinol might have therapeutic potential to prevent oxidative stress-mediated damage in RPE cells.

Keywords: DNA damage; apoptosis; autophagy; mitochondrial ROS; phloroglucinol.

Figure 1

Phloroglucinol reverses H₂O₂-induced viability reduction and cytotoxicity of ARPE-19 cells. Cells were treated with different concentrations of phloroglucinol (PHG) or H₂O₂ alone for 24 h (A,B), or pretreated with or without phloroglucinol and/or N-acetyl-L-cysteine (NAC) for 1 h followed by treatment with phloroglucinol for 24 h (C,D). (A–C) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay was performed to determine cell viability. (D) Cytotoxicity was measured by lactate dehydrogenase (LDH) assay. * p < 0.05 and *** p < 0.001 vs. unstimulated control; ^# p < 0.05 and ^### p < 0.001 vs. H₂O₂ alone treatment.

Figure 2

Phloroglucinol inhibits apoptosis in H₂O₂-treated ARPE-19 cells. Cells were pretreated with phloroglucinol for 1 h and then treated with or without phloroglucinol for 24 h. (A,B) To quantitatively measure the frequency of apoptosis induction, flow cytometry was performed after double staining with annexin V and propidium iodide (PI). Representative histograms of (A) and quantitative analysis (B) are shown. (C) DNA isolated from cells was stained with ethidium bromide (EtBr) and then observed under UV light. (D) After extracting cell lysate of each treatment group, expression levels of presented proteins were investigated through immunoblotting. (E) Bar diagram showing the relative protein density after normalization with actin based on Western blot analysis. (F) Activity of caspase-3 was measured by DEVDase activity assay using cytoplasmic extracts and presented as relative values compared to control. *** p < 0.001 vs. unstimulated control; ^## p < 0.01 and ^### p < 0.001 vs. H₂O₂ alone treatment.

Figure 3

Phloroglucinol suppresses H₂O₂-induced reactive oxygen species (ROS) production in ARPE-19 cells. Cells treated with or without phloroglucinol or NAC for 1 h were stimulated with H₂O₂ for another 1 h. After 2′,7′-dichlorofluorescein diacetate (DCF-DA) staining, ROS generation level was investigated through flow cytometry (A,B) and fluorescence microscopy (C). Representative histograms (A) and quantitative analysis (B) are shown. (C) Representative images of DCF-DA fluorescence are presented. *** p < 0.001 vs. unstimulated control; ^### p < 0.001 vs. H₂O₂ alone treatment.3.4. Phloroglucinol Abolishes H₂O₂-Induced DNA Damage.

Figure 4

Phloroglucinol alleviates H₂O₂-induced DNA damage in ARPE-19 cells. Cells were treated with or without phloroglucinol or NAC for 1 h prior to treatment with H₂O₂ for 24 h. (A) Representative immunofluorescence images of comet assay are indicated. (B) Representative images of γH2AX immunofluorescence (red) observed with a fluorescence microscope are shown. The location of the nucleus was indicated by counterstaining with DAPI (blue). (C) After treatment, contents of 8-hydroxy-2′-deoxyguanosine (8-OHdG) were measured using an ELISA kit. *** p < 0.001 vs. unstimulated control; ^### p < 0.001 vs. H₂O₂ alone treatment.

Figure 5

Phloroglucinol eliminates H₂O₂-mediated mtROS generation in ARPE-19 cells. MitoSOX staining was performed to determine the abundance of mtROS in cells treated with H₂O₂ for 24 h after pretreatment for 1 h with or without phloroglucinol and/or Mito-TEMPO. (A) Representative images of cells stained for mitochondrial peroxide (MitoSOX, red) and nuclei (4’,6-diamidino-2-phenylindol, DAPI, blue) are shown. (B,C) For quantitative evaluation of mtROS production, flow cytometric analysis was performed. Representative histograms (B) and average values (C) are shown. (D) Isolated total cell lysates were immunoblotted with antibodies corresponding to indicated mitophagy-marker proteins. (*** p < 0.001 vs. unstimulated control; ^### p < 0.001 vs. H₂O₂ alone treatment; ^&&& p < 0.001 vs. phloroglucinol + Mito-TEMPO group.

Figure 6

Phloroglucinol protects H₂O₂-induced mitochondrial impairment and cytosolic release of cytochrome c in ARPE-19 cells. Cells were preincubated with or without phloroglucinol and/or Mito-TEMPO for 1 h, followed by treatment with H₂O₂ for another 24 h. (A,B) After with 5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethyl-imidacarbocyanune iodide (JC-1) staining, representative histograms (A) and average values of JC-1 monomer ratios (B) are presented. (C) After isolation of mitochondrial and cytoplasmic fractions, the expression of cytochrome c in each fraction was investigated by immunoblotting. (D) Bar diagram showing the relative protein density after normalization with actin based on Western blot analysis. *** p < 0.001 vs. unstimulated control; ^## p < 0.01 and ^### p < 0.001 vs. H₂O₂ alone treatment; ^& p < 0.05 vs. phloroglucinol + Mito-TEMPO group.

Figure 7

Phloroglucinol attenuates ARPE-19 cells against H₂O₂-induced autophagy. (A,B) Cells were incubated with phloroglucinol or 3-MA for 1 h and then treated with H₂O₂ for 24 h, stained with Cyto-ID, and subjected to flow cytometry. Representative histograms (A) and mean values of Cyto-ID-positive cells (B) are presented. (C) H₂O₂-teated Cells in the presence or absence of phloroglucinol were stained with Cyto-ID. Representative images are shown. (D) Isolated total proteins were immunoblotted with indicated antibodies corresponding to autophagy-marker proteins. (E) Bar diagram showing the relative protein density after normalization with actin based on Western blot analysis. ** p < 0.01 and *** p < 0.001 vs. unstimulated control; ^## p < 0.01 and ^### p < 0.001 vs. H₂O₂ alone treatment.

Figure 8

Schematic showing the protective effect of phloroglucinol on oxidative injury in human RPE ARPE-19 cells. As a scavenger of mtROS, phloroglucinol protects against oxidative stress-induced apoptosis by blocking mitochondrial and DNA damage and autophagy.