Oleuropein Protects Human Retinal Pigment Epithelium Cells from IL-1β–Induced Inflammation by Blocking MAPK/NF-κB Signaling Pathways

Proinflammatory mediators such as interleukin (IL)-1β cause retinal pigment epithelium (RPE) inflammation, which is related to visual deterioration, including age-related macular degeneration and diabetic retinopathy. Oleuropein is a polyphenol compound that shows potent anti-inflammatory, antioxidant, and anti-cancer activities, but its effects on IL-1β–induced inflammation have not been examined in the adult RPE cell line ARPE-19. Here, we assessed the ability of oleuropein to attenuate this inflammation in ARPE-19 cells. IL-1β induced secretion of the inflammatory cytokines IL-6, monocyte chemoattractant protein-1 (MCP)-1, and soluble intercellular adhesion molecule (sICAM)-1. As measured by enzyme-linked immunosorbent assay, oleuropein significantly inhibited levels of all three proteins and led to decreased monocyte adhesiveness to ARPE-19 cells. To clarify the underlying anti-inflammatory mechanisms, we used western blots to evaluate the effect of oleuropein on inactivation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. The results showed that oleuropein significantly decreased levels of the inflammatory mediator cyclooxygenase-2 and increased anti-inflammatory protein HO-1 expression. We next examined if the anti-inflammatory activity of oleuropein arises via inactivated NF-κB. We found that suppressing phosphorylation of the JNK1/2 and p38 MAPK signaling pathways inhibited IL-6, MCP-1, and sICAM-1 secretion, implicating these pathways and NF-κB suppression in the effects of oleuropein. These results indicate that oleuropein shows potential for the prevention and treatment of inflammatory diseases of the retina.

the anti-inflammatory activity of oleuropein arises via inactivated NF-κB. We found that suppressing phosphorylation of the JNK1/2 and p38 MAPK signaling pathways inhibited IL-6, MCP-1, and sICAM-1 secretion, implicating these pathways and NF-κB suppression in the effects of oleuropein. These results indicate that oleuropein shows potential for the prevention and treatment of inflammatory diseases of the retina.

INTRODUCTION
Inflammation-induced damage of the retinal pigment epithelium (RPE) has been suggested as a major influence in retinal degenerative diseases (RDs), including age-related macular degeneration (AMD) and diabetic retinopathy (DR). Retinal pericytes are the cell layer surrounding endothelial cells. Inflammatory mediators can stimulate endothelial proliferative and induce formation of aberrant capillaries. Furthermore, activated RPE can release more inflammatory mediators to induce cell damage. In addition, the RPE is a main source of proinflammatory mediators [1][2][3]. AMD, which is classified into dry (non-neovascular) or wet (neovascular), involves irreversible visual disability [4,5]. RPE, which lies between the neural retina and the choroid, is a monolayer of pigmented cells and mainly functions as a retinal blood barrier and in photoreception [6]. Major risk factors for AMD are aging, oxidative stress, obesity, and chronic inflammation [7].
Interleukin (IL)-1β is a proinflammatory cytokine that initiates and propagates sterile inflammation, which is associated with retinal degenerative diseases [8]. In addition, IL-1β modulates expression of inflammatory mediators of RDs, including the proinflammatory cytokine IL-6, monocyte chemoattractant protein (MCP)-1, and soluble (s) intercellular adhesion molecule (ICAM)-1 [9]. IL-6 controls T cell proliferation, and this activation has been associated with inflammatory diseases such as DR [10]. MCP-1 draws monocytes and basophils to the site of inflammation, where macrophages or dendritic cells infiltrated in the tissues, activating the inflammatory reaction and development of related lesions [11]. During inflammation, leukocytes secrete the associated marker and regulator sICAM-1 [12]. Many studies have suggested that these activities are related to the regulation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways [13]. Results suggest that inflammatory mediators and leukocyte attractants would increase retinal inflammation and risk of AMD [14]. Therefore, anti-inflammatory therapies that inhibit these mediators could have potential in preventing progression in AMD [15].
Oleuropein (Fig. 1A) is a major phenolic compound of Olea europaea L. or olive leaf extract, and has antioxidant, anti-inflammatory, hepatoprotective, and anti-cancer effects [16,17]. Because of the potential link between the inflammatory state of the RPE and AMD development [18,19], here, we investigated the anti-inflammatory action of oleuropein in IL-1β-induced adult RPE (ARPE)-19 cells.

Cell Viability Assay
We used the CCK-8 kits to assess the effect of oleuropein on cell viability, as described previously [20]. Cells were seeded into 96-well plates (10 5 cells/well) and treated with oleuropein (3-100 μM) for 24 h, followed by addition of CCK-8 solution and incubation at 37 °C for 2 h. We used a 450-nm microplate reader for counting (Multiskan FC; Thermo, Waltham, MA, USA), and each concentration was evaluated in triplicate. Cell viability was given as percentage cell count relative to count without oleuropein treatment.

ELISA Assay
ARPE-19 cells were pretreated or not with oleuropein (3-100 μM) for 1 h in 24-well plates, followed by addition of IL-1β (1 ng/ml) and culture for 24 h. Cell supernatants were assayed using ELISA kits to measure IL-6, MCP-1, and sICAM-1 levels, following the manufacturers' instructions. We used a microplate reader (Multiskan FC; Thermo) at 450 nm to determine optical density values.

Cell Adhesion Assay
ARPE-19 cells were pretreated with oleuropein for 1 h and added with 1 ng/ml IL-1β for 24 h. The control groups were incubated with IL-1β alone. ARPE-19 cells were co-cultured with THP-1 cells (10 6 cells/ ml), which labeled with Calcein-AM (Sigma-Aldrich) for 1 h in a humidified incubator containing 5% CO 2 at 37 °C. Attached THP-1 cells were assayed with fluorescence microscopy (3 per view; magnification, × 200; Olympus, Tokyo, Japan) with excitation and emission wavelengths of 490 and 515 nm, respectively. All experiments were repeated three times.

Preparation of Total Proteins
ARPE-19 cells were seeded in 6-well plates pretreated or not with oleuropein (3-100 μM) for 1 h, followed by addition of IL-1β (1 ng/ml) for 24 h to evaluate total protein content or for 30 min to detect protein phosphorylation. A protein lysis buffer (Sigma-Aldrich) containing protease inhibitor cocktail and phosphatase inhibitors was used to harvest cells. Total protein and phosphorylated proteins were quantified using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA), as previously described [19].

Immunofluorescence Staining
ARPE-19 cells were seeded onto 6-well plates and reached 50-60% confluence. Cells were pretreated or not for 1 h with oleuropein (3, 10, 30, or 100 μM), followed by addition of IL-1β for 15 min. Then, cells were suctioned from the medium, washed in phosphate-buffered saline (PBS), fixed in paraformaldehyde 4% (w/v), and incubated overnight at 4 °C with anti-NF-κB p65 antibody. Next, the medium was removed, and the cells were washed again in PBS, The presented data are mean ± SD. ### P < 0.001 compared to control, *P < 0.05, and **P < 0.01, comparisons to cells treated with IL-1β only. followed by incubation with secondary antibodies at room temperature for 1 h. After 2-3 more washes in PBS, the nuclei were stained with DAPI (Sigma). Images were captured using a fluorescence microscope (Olympus).

Statistical Analysis
One-way analysis of variance and post hoc analysis with Dunnett's test were used to analyze data. Data are presented as the mean ± standard deviation (SD) of at least three independent experiments. Differences were considered statistically significant at P < 0.05.

Effects of Oleuropein on Cell Viability in ARPE-19 Cells
Using the CCK-8 assay to detect cytotoxicity in ARPE-19 cells, we tested oleuropein concentrations from 3 to 200 μM. Oleuropein did not significantly affect cell viability compared with the DMSO negative control, suggesting that these concentrations were safe to use (cell viability ≥ 90% at oleuropein ≤ 200 μM) (Fig. 1B).

Oleuropein Suppression of IL-1β-Induced NF-κB Activation in ARPE-19 Cells
We wanted to explore whether the effects of oleuropein on HO-1, COX-2, and the cytokines sICAM-1, IL-6, and MCP-1 arise in part via suppression of IL-1β-induced NF-κB activation. We found that IL-1β caused a significant increase in NF-κB p65 translocation into the nucleus, which oleuropein ≥ 30 μM suppressed. Under oleuropein treatment, the p65 subunit was retained in the cytoplasm in IL-β-activated ARPE-19 cells (Fig. 5A). Oleuropein ≥ 30 μM significantly suppressed pP65 expression compared to IL-1β alone C A fluorescence plate reader was used to quantify calcein AM fluorescent intensity. Data are presented as mean ± SD. ### P < 0.001 compared to control, *P < 0.05, and **P < 0.01 for comparisons to cells treated with IL-1β only.

DISCUSSION
Inflammation is thought to cause RPE damage related to vision loss and RDs such as dry AMD and DR [20,21]. Therefore, ophthalmologic agents are needed to attenuate RPE inflammation or ameliorate RDs. Oleuropein is reported to have powerful anti-inflammatory Oleuropein inactivated the pJNK1/2 pathway. C, D Oleuropein inactivated the p38 pathway. E, F Oleuropein had no effect on the pERK1/2 pathway. ARPE-19 cells (10 6 cells/ml) were incubated in the absence or presence of oleuropein 3-100 μM for 1 h and then exposed to IL-1β (1 ng/ml) for 24 h (total proteins) or 30 min (phosphorylated proteins). Protein levels were determined by western blot. Results are mean ± SD. Statistical significance: ### P < 0.001 compared to control, *P < 0.05, and **P < 0.01 for comparisons to cells treated with IL-1β only.
properties in vivo and in vitro [16,22], but research into its effects in ARPE-19 cells has so far been limited [23]. In line with earlier work, our current results indicate that polyphenols such as oleuropein may be candidates for supplementation or focused dietary intake in the treatment or prevention RDs, especially AMD [24,25]. We found that oleuropein, which did not substantially affect ARPE-19 cell viability up to 200 μM (Fig. 1B), is effective in vitro in reducing IL-1β-induced inflammation in ARPE-19 cells.
from the cytoplasm to the nucleus, with the p65 subunit being retained in the cytoplasm in oleuropein-pretreated IL-β-activated cells (Fig. 5). Taken together, these results show a pattern of inactivated NF-κB and down-regulated phosphorylation of JNK1/2 and p38 MAPKs following oleuropein pretreatment in IL-1β-induced ARPE-19 cells.
Earlier reports have indicated that the proinflammatory cytokine IL-1β initiates and propagates inflammation in RDs, including AMD and DR [8,26]. Inflammation in ARPE-19 cells can induce expression of other chemokines and cytokines, including IL-6, MCP-1, and sICAM-1 [17,19,27]. MCP-1 is primarily produced by Müller cells in RDs, and it recruits macrophages to the site of damage [28,29]. In the presence of IL-1β, mesenchymal stem cells simultaneously secrete a significant amount of IL-6, leading to production of this cytokine in the inflammatory environment of the diseased retina [30]. Studies also have shown that ICAM-1 is involved in leukocyte-endothelial interactions and that cell migration may contribute to the development of choroidal neovascularization. In addition, sICAM-1 appears to be more closely related to neovascular AMD [31][32][33]. Our results demonstrated that oleuropein could reduce chemokine and proinflammatory cytokine expressions via blocking MAPK and NF-κB pathways in inflammatory APRE-19 cells.
When RPE cells were active by IL-1β will release cytokines and chemokines to trigger inflammatory responses in the inflammation-relative eye diseases progressive. We firstly observed that oleuropein has the ability to inhibit the inflammation in IL-1β-induced ARPE-19 cells. In ophthalmology, oleuropein has not been used to treat inflammation-relative eye diseases. However, quercetin is also a phenolic compound that has recently been used to treat dry eye, corneal inflammation, and corneal neovascularization [9]. These studies provide a basal theoretical for in vivo study, even clinical application of oleuropein in the prevention and treatment of retinal inflammatory diseases in the future. On this regard, it could be useful to develop a biodegradable deliver system to inject oleuropein, avoiding a multiple treatment [34].
In addition, oleuropein is a natural compound from Olea europaea L. Previous studies found that 400 μM oleuropein did not significantly affect cell viability in 3T3-L1 adipocytes and 300 μM oleuropein inhibited sterol regulatory element-binding transcription factor 1 and fatty acid synthase gene expressions for suppressed lipogenesis in 3T3-L1 adipocytes [35].Other studies indicated that 400 μM oleuropein could activate the phosphorylation AMPK and AKT for promoted insulin sensitivity in C2C12 muscle cells [36]. Colorectal cancer mice treated with 100 mg/kg oleuropein, the result demonstrated that could improve clinical symptoms of colonic tumors by ameliorating inflammatory responses [37]. Therefore, we suggest that the experimental treatment of ARPE-19 cells with a maximum dose of 100 μM oleuropein has anti-inflammatory effects that should have physiologically usable dosage significance in ARPE-19 cells. The results of this experiment, we hope to develop a novel eye drops to treat in diabetic mice-induced eye disease and can be used in clinical practice in the future.

CONCLUSIONS
These results indicate that oleuropein attenuates IL-1β-induced IL-6, MCP-1, and sICAM-1 secretion and leads to increased anti-inflammatory HO-1 expression by repressing the NF-κB and JNK1/2-pP38 MAPK signal pathways. Oleuropein thus shows potential for anti-inflammation in IL-1β-induced ARPE-19 cells. In further, we will confirm that oleuropein inhibits inflammatory effects in RDs in vivo study.

AUTHOR CONTRIBUTION
Designed and performed the experiments: SJW, WCH, and CHH. Analysis and interpretation of data: MLH and YRZ. Drafting the manuscript: SJW and SH. All authors have read and agreed to the published version of the manuscript.

FUNDING
The present study was supported by grants from the Chang Gung Memorial Hospital (grants CMRPF1L0011, CMRPF1K0081, and CMRPF1H0111) and the Ministry of Science and Technology in Taiwan (grant 109-2320-B-255-006-MY3).

AVAILABILITY OF DATA AND MATERIALS
The data that support the findings of this study are available from the corresponding author upon reasonable request.

DECLARATIONS
Ethics Approval and Consent to Participate Not applicable.