Oxidative and carbonyl stress induced AMD and Codonopsis lanceolata ameliorates AMD via controlling oxidative and carbonyl stress

Abstract

Age-related macular degeneration (AMD) is one of the leading causes of blindness. AMD is currently incurable; the best solution is to prevent its occurrence. To develop drugs for AMD, it is crucial to have a model system that mimics the symptoms and mechanisms in patients. It is most important to develop safer and more effective anti-AMD drug. In this study, the dose of A2E and the intensity of blue light were evaluated to establish an appropriate atrophic in vitro model of AMD and anti-AMD effect and therapeutic mechanism of Codonopsis lanceolata. The experimental groups included a control group an AMD group treated with A2E and blue light, a lutein group treated with 25 μM lutein after AMD induction, and three groups treated with different doses of C. lanceolata (10, 20, and 50 μg/mL) after AMD induction. Intrinsic apoptotic pathway (Bcl-2 family), anti-oxidative system (Keap1/Nrf2/HO-1 antioxidant response element), and anti-carbonyl effect (4-hydroxynonenal [4-HNE]) were evaluated using immunofluorescence, MTT, TUNEL, FACS, and western blotting analyses. A2E accumulation in the cytoplasm of ARPE-19 cells depending on the dose of A2E. Cell viability of ARPE-19 cells according to the dose of A2E and/or blue light intensity. The population of apoptotic or necrotic cells increased based on the A2E dose and blue light intensity. Codonopsis lanceolata dose-dependently prevented cell death which was induced by A2E and blue light. The antiapoptotic effect of that was caused by activating Keap1/Nrf2/HO-1 pathway, suppressing 4-HNE, and modulating Bcl-2 family proteins like increase of antiapoptotic proteins such as Bcl-2 and Bcl-XL and decrease of proapoptotic protein such as Bim. Based on these findings, 30 μM A2E and 20 mW/cm² blue light on adult retinal pigment epithelium-19 cells was an appropriate condition for AMD model and C. lanceolata shows promise as an anti-AMD agent.

Keywords: Codonopsis lanceolata; 4-HNE; Age-related macular degeneration (AMD); Apoptosis; Keap1/Nrf2/HO-1 pathway; Oxidative/carbonyl stress.

Figure 1

N-retinylidene-N-retinyl-ethanolamine (A2E) synthesis and the light intensity of the manufactured blue light transilluminator. (A) The scheme of A2E synthesis. (B) The result of A2E synthesis. Synthetic A2Es consisted of one A2E and two A2E isotypes. (C) The intensity of the blue light transilluminator. The peak wavelength of the irradiated light was 454.6 nm, with the majority of light falling within the range of blue light.

Figure 2

Cell culture protocols depending on the analysis and sample harvest time.

Figure 3

Cytotoxicity and A2E accumulation in adult retinal pigment epithelial (ARPE)-19 cells, depending on the dose of A2E and the intensity of blue light. (A) A2E accumulation in the cytoplasm of ARPE-19 cells depending on the dose of A2E. A clear presence of A2E in the cytoplasm was observed, particularly at a dose of 20 μM A2E. Scale bar, 50 μm. Magnification, 1000 × . (B) Cell viability of ARPE-19 cells according to the dose of A2E and/or blue light intensity. N = 6. The results are presented as the mean ± standard deviation (SD). ^*, p < 0.05; ^**, p < 0.001 compared to 0 μM A2E. ^$, p < 0.05; ^$$ p < 0.01 compared to 10 μM A2E. ^#, p < 0.05; ^##, p < 0.001 compared to 20 μM A2E.

Figure 4

Apoptosis, oxidative stress, and carbonyl stress in ARPE-19 cells depending on the level of A2E and the intensity of blue light. (A) The results of the TUNEL assay for ARPE-19 cell death at various doses of A2E and/or blue light intensities. Scale bar, 50 μm. Magnification, 1000 × . (B) The results of apoptosis-related pathway analysis. N = 3.

Figure 5

Codonopsis lanceolata prevented cell death induced by A2E and blue light. (A) C. lanceolata dose-dependently ameliorated cell death induced by A2E and blue light (N = 3). ^*, p < 0.05; ^**, p < 0.001 compared to CON; ^$, p < 0.05 compared to the A2E and blue light treatment group; ^#, p < 0.05 compared to the lutein treatment group. (B) C. lanceolata dose-dependently suppressed RPE-19 cell apoptosis induced by A2E and blue light (green spot). Scale bar, 50 μm. Magnification, 1000 × .

Figure 6

Codonopsis lanceolata regulated the apoptosis of ARPE-19 cells through the activation of the Keap1/Nrf2/HO-1 pathway and the suppression of 4-HNE. (A) C. lanceolata modulated the Bcl-2 family, increasing the levels of the antiapoptotic proteins Bcl-2 and Bcl-XL, while decreasing the level of the proapoptotic protein Bim (N = 3). (B) C. lanceolata treatment activated the anti-oxidative Keap1/Nrf2/HO-1 pathway but decreased the expression of 4-HNE in a dose-dependent manner (N = 3). (C) C. lanceolata treatment stimulated the expression of nuclear Nrf2, which functions as a transcription factor for HO-1 (green spots). (D) C. lanceolata treatment dose-dependently induced the expressions of Keap1 and HO-1. Scale bar, 50 μm. Magnification, 1000 × .