DHMEQ was synthesized by Umezawa and Chaicharoenpong (16), and for the present study it was dissolved in 100% DMSO at a concentration of 10 mg/ml and stored at −30°C (14,16). Before use in cell cultures, DHMEQ was diluted with the culture medium (DMEM/F-12; Invitrogen; Thermo Fisher Scientific, Inc.) to a final concentration of ≤0.1%. Dexamethasone was purchased from Sigma-Aldrich (Merck KGaA).

ARPE-19 cells were purchased from the American Type Culture Collection and maintained in DMEM/F-12 supplemented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all from Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂ in air. After reaching confluency, ARPE-19 cells were detached with a trypsin-EDTA solution (0.05%) (Thermo Fisher Scientific, Inc.) and plated for subcultures. Cells were passaged every 4–6 days, and those used in each experiment were confluent and exhibited no visible pigmentation. Moreover, cells were maintained for 3 weeks before the experimental procedures and were used at passages 4–6.

The effects of various concentrations of DHMEQ on ARPE-19 cells were evaluated by MTT. Cells were grown at 37°C in 96-well plates at a density of 2×10⁴ cells/well for 24 h. Upon confluency, the medium was replaced with a serum-free medium, and ARPE-19 cells were cultured at 37°C for 24 h with 0.1, 1.0, 5.0, 10.0, 50.0 or 100.0 µg/ml DHMEQ or without DHMEQ at 37°C for 24 h. After 24 h, the assay was performed by adding 10 µl MTT solution to the wells (Biotium, Inc.). After incubation for 4 h at 37°C, 200 µl DMSO was added to the cells. After incubation at room temperature for 5 min, the optical density (OD) at 570 nm (signal absorbance) and 630 nm (background absorbance) was measured using a microplate reader, and the normalized absorbance values (OD at 570 nm and OD at 630) were determined.

ARPE-19 cells were seeded in 6-well plates at 2×10⁵ cells/well and cultured for 24 h. Upon confluency, the medium was replaced with serum-free medium, and cells were or were not exposed to 20 ng/ml TNF-α (R&D Systems, Inc.) with DHMEQ (1.0, 10.0 and 100.0 µg/ml) or without DHMEQ at 37°C for 24 h. After exposure, cells were washed with phosphate-buffered saline (PBS, pH 7.4), detached by trypsin-EDTA (0.05%) and suspended in PBS. For staining of prepared cells, Annexin V-FITC solution (5 µl; Nacalai Tesque, Inc.) was added to 100 µl of cell suspension (1×10⁶ cells), then <1% propidium iodide (PI) solution (5 µl; Nacalai Tesque, Inc.) was added to the cell suspension, according to the manufacturer's instructions (cat. no. 15342; Nacalai Tesque, Inc.). The cells were incubated at room temperature for 15 min and analyzed by flow cytometry (FACSCalibur) using CellQuest Pro software version 6.0 (both BD Biosciences).

ARPE-19 cells were seeded in 6-well plates at 2×10⁵ cells/well and cultured for 24 h. Upon confluency, the medium was replaced with serum-free medium, and cells were or were not exposed to 20 ng/ml TNF-α (R&D Systems, Inc.) with DMSO (0.1%) or DHMEQ (1.0 or 10.0 µg/ml) at 37°C for 24 h. After exposure, cells were washed with PBS (pH 7.4), detached by trypsin-EDTA (0.05%) and suspended in PBS. The prepared ARPE-19 cells were incubated with phycoerythrin-conjugated monoclonal antibody to ICAM-1 (1:100; cat. no. 555511; BD Biosciences) at 4°C for 20 min and analyzed by flow cytometry (FACSCalibur) using CellQuest Pro software version 6.0 (both BD Biosciences).

ARPE-19 cells were seeded in 6-well plates at a density of 2×10⁵ cells/well and cultured for 24 h. Upon confluency (80-90% confluency), the medium was replaced with serum-free medium and cells were exposed to 20 ng/ml TNF-α and DMSO (0.1%), DHMEQ (1.0 µg/ml=4.0 µM, 10 µg/ml=40 µM) or dexamethasone (40 µM) at 37°C for 24 h. After exposure, the supernatant was collected from each well, and the levels of IL-8 and MCP-1 in the supernatant were determined by Quantikine^® Colorimetric Sandwich ELISA kits (R&D Systems, Inc.; IL-8. cat. no. D8000C; MCP-1, cat. no. DCP00).

In another experiment, ARPE-19 cells were exposed to 20 ng/ml TNF-α and DMSO (0.1%) or DHMEQ (10 µg/ml) at 37°C for 6, 12 and 24 h. After exposure, the supernatant was collected from each well and the levels of IL-8 and MCP-1 in the supernatant was determined by the Quantikine^® ELISA kits (R&D Systems, Inc.) as described above.

ARPE-19 cells were seeded in 6-well plates at a density of 2×10⁵ cells/well and cultured for 24 h. Upon confluency, the medium was replaced with serum-free medium and cells were exposed to 20 ng/ml TNF-α in the absence or presence of DHMEQ (10 µg/ml) at 37°C for 24 h. After exposure, cells were washed with PBS, detached by trypsin-EDTA (0.05%) and suspended in PBS. Total RNA from the prepared ARPE-19 cells was extracted with ISOGEN (Nippon Gene Co., Ltd.) according to the manufacturer's instructions. Briefly, 10 µg total RNA from each sample was reverse-transcribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems; Thermo Fisher Scientific, Inc.; reverse transcription conditions were as follows: Initial incubation at 37°C for 60 min and 95°C for 5 min), then loaded onto Human NFκB Pathway TaqMan^® Array plates (cat. no. 4414095; Applied Biosystems; Thermo Fisher Scientific, Inc.) for profiling of NF-κB-associated 92 genes expression levels. PCR was performed on a QuantStudio™ 12K Flex Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Raw cycle threshold (Cq) values were calculated with SDS software 1.2.3. (Applied Biosystems; Thermo Fisher Scientific, Inc.). The sequences of the forward and reverse primers are not commercially available. Data were analyzed according to the comparative Cq method, and the global median normalization method was used (22). The 2^−ΔΔCq method was performed to calculate the expression level of the fold change (23). The fold change in ARPE-19 cells exposed to 20 ng/ml TNF-α in the absence or presence of DHMEQ was calculated for each gene; genes with a 2-fold increase in this ratio were defined arbitrarily as upregulated in cells exposed to DHMEQ, whereas those with a 2-fold decrease were defined as downregulated genes.

In order to validate the expression levels of genes [lymphotoxin β receptor (LTBR), MCP-1, and Toll-like receptor 4 (TLR4)], which were either upregulated or downregulated in ARPE-19 cells treated with DHMEQ, quantitative PCR was carried out in duplicate using the TaqMan^® Universal PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) on a QuantStudio™ 12K Flex Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), following the manufacturer's protocol. ARPE-19 cells were seeded in 6-well plates at a density of 2×10⁵ cells/well and cultured for 24 h. Upon confluency, the medium was replaced with serum-free medium and cells were exposed to 20 ng/ml TNF-α exposed to DMSO (0.1%) or DHMEQ (10 µg/ml) at 37°C for 24 h. After exposure, cells were washed with PBS, detached by trypsin-EDTA (0.05%) and suspended in PBS. Total RNA from the prepared ARPE-19 cells was extracted with ISOGEN (Nippon Gene Co., Ltd.) as described above and reverse-transcribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) PCR conditions were as follows: Initial incubation at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles two-step cycling (denaturing at 95°C for 15 sec, annealing/extension at 60°C for 60 sec). The TaqMan primers/probes pairs were obtained from Applied Biosystems (Thermo Fisher Scientific, Inc.) using inventoried TaqMan gene expression assays [LTBR assay ID, Hs01101194_m1; MCP-1 assay ID, Hs00234140_m1; TLR4 assay ID, Hs00152939_m1]. For an endogenous control mRNA, the β-actin (assay ID, Hs99999903_m1, Thermo Fisher Scientific, Inc.) was used for data normalization of the mRNA expression levels.

Data are presented as the mean ± SD. Statistical significance was evaluated with unpaired t-tests. To compare data among ≥3 groups, one-way ANOVA using the Bonferroni's multiple comparison tests was performed. P<0.05 was considered to indicate a statistically significant difference.

Confluent ARPE-19 cells were exposed to various concentrations of DHMEQ, and it was revealed that DHMEQ at concentrations ≤10 µg/ml did not have toxic effects on cells. However, DHMEQ at concentrations of 50 and 100 µg/ml significantly inhibited the viability of ARPE-19 cells compared with the viability of cells cultured without DHMEQ (Fig. 1). Therefore, these results indicated that higher concentrations of DHMEQ, such as 50 and 100 µg/ml, reduce the viability of ARPE-19 cells.

To determine whether DHMEQ induces apoptosis or necrosis in ARPE-19 cells, Annexin-V and/or PI-positive cells were analyzed by flow cytometry. It was demonstrated that concentrations of 1.0 and 10 µg/ml DHMEQ did not alter the percentage of apoptotic cells (Annexin-V-positive and PI-negative; Fig. 2A and B). However, a dose of 100 µg/ml DHMEQ significantly increased the number of apoptotic cells compared with cells treated with TNF-α (20 ng/ml) without DHMEQ (Fig. 2A and B). In addition, concentrations of 100 µg/ml DHMEQ significantly increased the number of necrotic cells compared with cells treated with TNF-α (20 ng/ml) without DHMEQ (Annexin-V-positive and PI-positive; Fig. 2A and B). Collectively, the results indicated that a high concentration of DHMEQ, such as 100 µg/ml, promoted the induction of apoptosis and necrosis of ARPE-19 cells. Based on these findings, a concentration of DHMEQ <100 µg/ml was used in subsequent experiments.

Previous studies have revealed that TNF-α can enhance the protein expression of ICAM-1 synthesized by ARPE-19 cells (24). To assess these findings, the present study stimulated ARPE-19 cells with TNF-α (20 ng/ml) for 24 h and evaluated the protein expression of ICAM-1 by flow cytometry. The results revealed that the expression of ICAM-1 was increased 7-fold in cells stimulated by TNF-α compared with cells cultured without TNF-α (Fig. 3A and B).

The effects of DHMEQ on the protein expression level of ICAM-1 were also examined in ARPE-19 cells stimulated with TNF-α. It was revealed that DHMEQ (10 µg/ml) significantly reduced the expression of ICAM-1 in cells by ~50% compared with cells treated with DMSO (Fig. 3C-E). Thus, it was speculated that DHMEQ may be able to decrease TNF-α-induced ICAM-1 expression in ARPE-19 cells.

MCP-1 and IL-8 have been revealed to be the major chemokines produced by TNF-α-stimulated ARPE-19 cells (25), and dexamethasone has been reported to have anti-inflammatory effects on ARPE-19 cells (20). Therefore, the present study investigated whether DHMEQ is able to decrease the protein expression levels of MCP-1 and IL-8 in ARPE-19 cells stimulated with TNF-α. Moreover, the anti-inflammatory effect of DHMEQ was compared with that of dexamethasone in cells. It was demonstrated that the production of IL-8 and MCP-1 from cells treated with DHMEQ (40 µM=10 µg/ml) was significantly decreased compared with ARPE-19 cells treated with DMSO. In addition, there was a significant difference in the production of IL-8 and MCP-1 between cells treated with DHMEQ (40 µM) and those treated with dexamethasone (40 µM; Fig. 4A and B). These findings revealed that DHMEQ has strong suppressive effects on the production of MCP-1 and IL-8 by ARPE-19 cells compared with dexamethasone. Furthermore, the results indicated that DHMEQ significantly reduced the protein expression levels of MCP-1 and IL-8 at 6, 12 and 24 h (Fig. 4C and D). Therefore, DHMEQ may be able to decrease the TNF-α-induced chemokine production in ARPE-19 cells at several time-points after co-culturing.

To determine the alterations of the expression levels of NF-κB-associated inflammatory genes in ARPE-19 cells exposed to DHMEQ, the present study compared RNA isolated from TNF-α-stimulated cells in the absence or presence of DHMEQ, using the Human NF-κB Pathway TaqMan^® Array Plates that analyze 92 NF-κB-associated inflammatory genes. Moreover, summaries of the differentially expressed genes between the two cell populations are presented in Table I. A total of 19 genes were revealed to be upregulated and 25 genes were revealed to be downregulated in cells exposed to DHMEQ compared with those in the absence of DHMEQ. The differentially expressed genes are presented in Tables I and II. The gene expression levels of cytokines and chemokines, including MCP-1, ICAM-1, IL-6 and IL-8, and TLR2, TLR3 and TLR4, were downregulated in ARPE-19 cells treated with DHMEQ (Table I). In addition, DHMEQ suppressed TNF superfamily member 15 (TNFSF15) and TNF-α-induced protein 3 (TNFAIP3; Table I). However, it was revealed that DHMEQ increased the expression levels of numerous genes associated with the NF-κB signaling pathway, including prostaglandin E synthase (PTGES), mitogen-activated protein kinase 14 (MAP3K14), LTBR and TNFRSF1A associated via death domain (TRADD).

In addition, representative genes, LTBR, MCP-1 and TLR4, which were identified by NF-κB Pathway Array, were assessed by quantitative PCR analysis. Although the fold changes were not exactly the same between the two methods, the gene expression level of LTBR was significantly increased in the presence of DHMEQ compared to that in the presence of DMSO, whereas the gene expression levels of MCP-1 and TLR4 were significantly decreased in the presence of DHMEQ compared to that in the presence of DMSO (Fig. 5).