Oxya chinensis sinuosa (OC) Extracts Protects ARPE-19 Cells against Oxidative Stress via Activation of the Mitogen-Activated Protein Kinases (MAPKs)/ Nuclear Factor-κB (NF-κB) Pathway

Abstract

Oxya chinensis sinuosa (OC) is a well-known edible insect. Several researches on the health benefits of OC consumption have been performed to date; however, their effect on eye health remains largely unknown. This study aimed to assess the protective effects of OC extracts on the oxidative stress on the retinal pigment epithelium (RPE) cells. Oxidative damage has been identified as one of the key regulatory factors in age-related macular degeneration. H₂O₂-induced reactive oxygen species (ROS) production, a well-known oxidative stress factor, can cause cell death in retinal pigment epithelia cells. In this study, we found that three OC extracts effectively prevented H₂O₂-induced ROS production and subsequent death of ARPE-19 cells in a dose-dependent manner. In addition, the OC extracts inhibited the phosphorylation of mitogen-activated protein kinases including p38, JNK, and ERK. The OC extracts restored IκBα degradation induced by H₂O₂, indicating that OC extracts suppressed the activation of nuclear factor-κB. Furthermore, the three OC extracts were shown to have antioxidant effects by up-regulating the intracellular expression of key antioxidant proteins such as SOD, NQO, and HO-1. Here we demonstrated the antioxidant and anti-apoptotic effects of the OC extracts on ARPE-19, indicating their potential role in improving eye health. These results suggest that three OC extracts plays a critical role in oxidative stress-induced cell death protects in ARPE-19 cells.

Keywords: Oxya chinensis sinuosa; age-related macular degeneration; antioxidant; edible insect; eye health functional food.

Fig. 1.. Cytotoxicity of three OC extracts to ARPE-19 cells.

ARPE-19 cells (1×10⁴ cells/well) were plated in the complete DMEM/F12 medium. The cells were treated with (A) OCH, (B) OCE, or (C) OCM at 0.1, 0.5, 1, or 2 mg/mL for 24 h. After treatment, the cell viability was measured using a MTS assay. Data shown are mean±SD values of triplicate samples. ^*** Indicates a significant difference compared with non-treated cells (p<0.005). N.S indicates a non-significant difference. OC, Oxya chinensis sinuosa; OCH, hot water extract of OC; OCE, 70% ethanol extract of OC; OCM, 70% methanol extract of OC.

Fig. 2.. Three OC extracts protect human adult retina pigment epithelial cells from H₂O₂-induced cell death.

ARPE-19 cells (1×10⁴ cells/well) were plated in the complete DMEM/F12 medium. The cells were pre-treated with (A) OCH, (B) OCE, or (C) OCM for 1 h. Then, the cells were treated H₂O₂ (300 μM) for 24 h. After treatment, cell viability was measured using an MTS assay. The results are presented as mean±SD from triplicate samples (water, 70% ethanol, or 70% methanol). ^{*, **, ***} Indicate a significant difference compared with non-treated cells (p<0.05, 0.01, and 0.005, respectively). N.S indicates a non-significant difference. OC, Oxya chinensis sinuosa; OCH, hot water extract of OC; OCE, 70% ethanol extract of OC; OCM, 70% methanol extract of OC.

Fig. 3.. Protective effects of three OC extracts on H₂O₂-induced membrane damage.

ARPE-19 cells (1×10⁴ cells/well) were plated in the complete DMEM/F12 medium. The cells were pre-treated with (A) OCH, (B) OCE, or (C) OCM for 1 h. Then, the cells were treated with H₂O₂ (300 μM) for 24 h, followed by an LDH assay to determine the cell viability. The results are presented as mean±SD from triplicate samples (water, 70% ethanol, or 70% methanol). ^*** Indicates a significant difference compared with non-treated cells (p<0.005). LDH, lactate dehydrogenase; OC, Oxya chinensis sinuosa; OCH, hot water extract of OC; OCE, 70% ethanol extract of OC; OCM, 70% methanol extract of OC.

Fig. 4.. The OC extracts inhibit H₂O₂–induced ROS production in ARPE-19 cells.

ARPE-19 cells (1×10⁴ cells/well) were plated in the complete DMEM/F12 medium. The cells were pre-treated with OC extracts for 1 h. Then, the cells were treated with H₂O₂ (300 μM) for 24 h. After treatment, ROS production was determined using a ROS kit. The results are presented as mean±SD from triplicate samples (water, 70% ethanol, or 70% methanol). ^*** Indicates a significant difference compared with non-treated cells (p<0.005). ROS, reactive oxygen species; OC, Oxya chinensis sinuosa; OCH, hot water extract of OC; OCE, 70% ethanol extract of OC; OCM, 70% methanol extract of OC.

Fig. 5.. Inhibition of MAPK phosphorylation by OC extracts in ARPE-19 cells stimulated with H₂O₂.

ARPE-19 cells (1×10⁴ cells/well) were pre-treated with OCH, OCE, or OCM (2 mg/mL) for 1 h and then stimulated with H₂O₂ (300 μM) for 30 min. The phosphorylation of ERK 1/2, p38, and JNK were assessed using western blotting. The Fig. 5B is presented as mean±SD from triplicate samples (water, 70% ethanol, or 70% methanol). ^*** Indicates a significant difference compared with non-treated cells (p<0.005). OC, Oxya chinensis sinuosa; OCH, hot water extract of OC; OCE, 70% ethanol extract of OC; OCM, 70% methanol extract of OC; MAPK, mitogen-activated protein kinases.

Fig. 6.. Inhibition of NF-κB activation by the OC extracts in ARPE-19 cells stimulated with H₂O₂.

ARPE-19 cells (1×10⁴ cells/well) were pre-treated with OCH, OCE, or OCM (2 mg/mL) for 1 h and then stimulated with H₂O₂ (300 μM) for 30 min. The degradation of IκBα was assessed using western blot analysis. The Fig. 6B is presented as mean±SD from triplicate samples (water, 70% ethanol, or 70% methanol). ^*** Indicates a significant difference compared with non-treated cells (p<0.005). OC, Oxya chinensis sinuosa; OCH, hot water extract of OC; OCE, 70% ethanol extract of OC; OCM, 70% methanol extract of OC; NF-κB, nuclear factor-κB.

Fig. 7.. OC extracts alleviates oxidative stress in ARPE-19 cells.

ARPE-19 cells (1×10⁴ cells/well) were pre-treated with OCH, OCE, or OCM (2 mg/mL) for 1 h and then stimulated with H₂O₂ (300 μM) for 30 min. (A) The western blot analysis of (B) NQO1, (C) SOD1, and (D) HO-1. The Fig. 7 is presented as mean±SD from triplicate samples (water, 70% ethanol, or 70% methanol). ^*** Indicates a significant difference compared with non-treated cells (p<0.005). N.S indicates a non-significant difference. OC, Oxya chinensis sinuosa; OCH, hot water extract of OC; OCE, 70% ethanol extract of OC; OCM, 70% methanol extract of OC.