Hot-Air-Dried Eruca sativa Mill. Extracts Prevent Retinal Inflammation and Unfolded Protein Responses in ARPE-19 Cells

Abstract

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) constitute leading causes of irreversible visual impairment; both are pathologically linked to chronic inflammation and endoplasmic reticulum (ER) stress in retinal pigment epithelial (RPE) cells. This study aimed to investigate the protective effects of hot-air-dried Eruca sativa Mill. extract (ESH) on lipopolysaccharide (LPS)- and thapsigargin (Tg)-induced inflammatory and ER stress responses, respectively, in ARPE-19 cells. ESH pretreatment significantly suppressed LPS-induced phosphorylation of nuclear factor kappa B (NF-κB), inhibitor of kappa B alpha, and c-Jun N-terminal kinase, indicating effective inhibition of inflammatory signaling cascades. At the transcriptional level, ESH markedly attenuated the expression of tumor necrosis factor-α mRNA, suggesting downstream prevention of NF-κB–mitogen-activated protein kinase-mediated inflammatory gene activation. Under ER stress conditions, ESH significantly attenuated the upregulation of CCAAT/enhancer-binding protein (C/EBP) homologous protein and X-box binding protein-1, along with reductions in the expressions of cleaved caspase-3 and −9, indicating mitigation of ER stress-associated retinal apoptosis. Additionally, ESH prevented Tg-inducible vascular endothelial growth factor (VEGF) mRNA expression, VEGF protein secretion, and intracellular calcium level. Strong positive correlations were observed between intracellular calcium and VEGF secretion (r = 0.888), and between VEGF mRNA and protein levels (r = 0.843), supporting a potential mechanistic link. Collectively, these findings demonstrate that ESH modulates inflammatory, ER stress, apoptotic, and angiogenic pathways, suggesting its potential as a functional dietary supplement to mitigate RPE dysfunction in AMD and DR.

Keywords

ARPE-19 cells endoplasmic reticulum stress hot-air drying inflammation

INTRODUCTION

Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are leading causes of irreversible vision loss worldwide. In 2020, DR affected more than 100 million adults and caused over 1 million cases of blindness, while AMD affected nearly 200 million individuals, with prevalence projected to rise to 288 million by 2040.^1,2 In the United States, DR affects approximately one-quarter of adults with diabetes, while advanced AMD occurs in up to 4% of individuals aged ≥ 50 years.^3,4 Across Europe and Asia, DR is observed in roughly 15%–30% of patients with diabetes, while AMD occurs in 8%–12% of elderly populations.^5–9 Despite the distinct etiologies—microvascular complications of hyperglycemia in DR and oxidative stress-related degeneration in AMD—both disorders share a central pathological hallmark: dysfunction of the retinal pigment epithelium (RPE).¹⁰ The RPE, a monolayer of pigmented cells essential for photoreceptor phagocytosis, trophic factor secretion, and blood–retina barrier regulation, is indispensable for retinal homeostasis.^11–13 Its disruption compromises photoreceptor viability and accelerates disease progression through neovascularization in DR and drusen accumulation in AMD.^14,15

Recent evidence highlights that both DR and AMD involve endoplasmic reticulum (ER) stress and chronic inflammation within RPE cells. The unfolded protein responses (UPR) pathways—PERK–eIF2α–C/EBP homologous protein (CHOP) and IRE1α–X-box binding protein-1 (XBP-1)—along with inflammatory mediators such as mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB), have been implicated in retinal degeneration.^16,17 UPR activation promotes secretion of pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin (IL)−1β, and IL-6), enhances vascular endothelial growth factor (VEGF) expression, disrupts intracellular calcium homeostasis, and induces apoptosis through B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 imbalance and caspase-3/9 activation.¹⁸ In addition, inflammation-induced post-translational modifications impair tight junction proteins, compromising blood–retina barrier integrity.¹⁹ Although pharmacological therapies such as anti-VEGF agents and corticosteroids can delay disease progression, they remain limited by adverse effects, therapeutic resistance, and high costs.^20,21 These limitations demonstrate the need for safer and more sustainable approaches to preserve or restore RPE integrity.

Given these limitations, dietary interventions using food-derived bioactive compounds have emerged as a promising strategy for RPE protection. Carotenoids such as lutein and zeaxanthin attenuate oxidative stress and inflammation in RPE cells, thereby supporting photoreceptor survival.^22,23 Edible wild plants, such as Aruncus dioicus var. kamtschaticus, exhibit anti-inflammatory and antioxidative activities in lipopolysaccharide (LPS)-induced ARPE-19 cells by downregulating NF-κB signaling.²⁴ Similarly, Gelidium amansii, a red seaweed rich in dietary fiber and polyphenols, reduces ER stress and oxidative damage in retinal cells through modulation of UPR markers and calcium homeostasis.²⁵ Additional studies have shown that diverse food-derived compounds protect ARPE-19 cells by modulating UPR markers and inflammatory cytokines, including CHOP, XBP-1, TNF-α, and IL-1β.^26–28 Beyond plant-derived phytochemicals, amino acid derivatives such as taurine, abundant in seafood,²⁹ also alleviate ER stress-induced apoptosis in ARPE-19 cells.³⁰ Similarly, resveratrol mitigates oxidative stress and inflammatory signaling in ARPE-19 cells by limiting ROS generation and regulating cytokine responses.³¹ Collectively, these findings support the potential of dietary bioactive compounds to preserve RPE function and mitigate pathogenic processes associated with DR and AMD.

Eruca sativa Mill. (commonly referred to as arugula, garden rocket, roquette, or rucola) is a leafy vegetable from the Brassicaceae family.³² In addition to its culinary value, E. sativa is rich in bioactive compounds, including phenolic compounds, glucosinolates, vitamin C, vitamin E, and unsaturated fatty acids.^33–36 Flavonoids, including kaempferol and quercetin, contribute to its robust antioxidant properties, protecting cellular components from oxidative damage.^37,38 Bioactive constituents of E. sativa have been associated with antioxidant,³⁹ anti-inflammatory,⁴⁰ anticancer,⁴¹ hepatoprotective,⁴² and anti-ulcer activities.⁴³ Furthermore, research highlights its potential in supporting cardiovascular health by inhibiting platelet aggregation and improving lipid profiles,^44,45 and promoting bone strength due to its high calcium, magnesium, and vitamin K content.⁴⁶ Despite these diverse pharmacological properties, research on the protective effects of E. sativa in retinal tissues—particularly against ER stress and inflammation—remains scarce. Given the involvement of these stressors in RPE dysfunction, the phytochemicals in E. sativa may confer retinal protection in degenerative conditions.

The functional efficacy of E. sativa depends on the preservation of bioactive components during processing.⁴⁷ Drying methods, in particular, markedly influence phytochemical stability and concentration.⁴⁸ Hot-air drying can enhance bioactivity by generating specific metabolites or increasing free amino acid content through heat-induced protein degradation.⁴⁹ Compounds produced or enriched during hot-air drying, including Maillard reaction products and free amino acids⁵⁰ which exhibit antioxidative and anti-inflammatory properties that may directly modulate ER stress and inflammation. Previous studies have reported that hot-air-dried cruciferous vegetables, including E. sativa, retain or even augment biological activity compared with freeze-dried counterparts.^51,52 This suggests that heat treatment may optimize functional potential rather than merely diminish bioactivity. Besides these biochemical advantages, hot-air drying is cost-effective, time-efficient, and scalable, making it suitable for both laboratory and industrial applications.⁵³

Taken together, the use of hot-air-dried materials not only ensures production feasibility but also harnesses unique biochemical changes that may enhance anti-inflammatory and ER stress-modulating effects. Therefore, this study aimed to investigate whether hot-air-dried E. sativa extract (ESH) can suppress ER stress and inflammatory signaling in ARPE-19 cells under conditions simulating retinal degeneration. The findings are expected to clarify the molecular mechanisms underlying the protective effects of E. sativa and support its potential application as a functional dietary supplement for preventing DR and AMD.

MATERIALS AND METHODS

Preparation of ESH

The extraction procedure for ESH was performed as described by Son et al.⁵⁰ Fresh E. sativa Mill. leaves were procured from Greemfarm (Seoul, Korea) in May 2018. After thoroughly washing under running water to remove debris, the samples underwent hot-air drying in a drying oven (Hanil, Jangseong, Korea) at 60°C for 40 h. The dried material was ground with a grinder (SMX-M41KP, Shinil, Cheonan, Korea) and passed through an 80-mesh sieve. The powdered samples were stored at –70°C prior to extraction. For ethanol extraction, 100 g of powder was mixed with 1.5 L of 80% ethanol and subjected to reflux extraction at 65°C for 3 h; the process was repeated three times. The extract was filtered using Whatman No. 2 filter paper (GE Healthcare, Chicago, IL, USA), concentrated under reduced pressure with a rotary vacuum evaporator (N-1110S-W, EYELA, Tokyo, Japan), and freeze-dried. The resulting extract was stored at –70°C until further use.

ARPE-19 cell culture and treatment

The ARPE-19 cell line, derived from human RPE, was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 100 µg/mL gentamicin (Thermo Scientific, Waltham, MA, USA), and maintained at 37°C in a humidified incubator (VS-2180CW, Vision Scientific, Korea) under 5% CO₂. ESH was administered at concentrations of 100, 500, and 1,000 µg/mL for 24 h. Inflammation was induced with 1 µg/mL LPS (E. coli O55:B5, Sigma-Aldrich, St. Louis, MO, USA), while ER stress was induced with 5 µM thapsigargin (Tg) for 24 h. For quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis, ARPE-19 cells (5.0 × 10⁵ cells/well) were seeded into 6-well plates and incubated for 48 h prior to treatment.

Cell viability assay

Cell viability was evaluated using the EZ-CYTOX assay kit (DoGenBio, Seoul, Korea) according to the manufacturer’s instructions.⁵⁴ ARPE-19 cells were seeded at 2.0 & 10⁴ cells/well in 96-well plates and incubated for 24 h. Absorbance was measured using a microplate reader (SpectraMax M2, Molecular Devices, San Jose, CA, USA).

qRT-PCR

Total RNA was isolated using NucleoZOL reagent (Macherey-Nagel, Germany). ARPE-19 cells were pretreated with ESH, followed by treatment with LPS or Tg. Residual genomic DNA was eliminated using the DNA-free™ Kit (Thermo Fisher, Waltham, MA, USA). RNA quantity was measured using a SpectraDrop™ Microplate (SpectraMax iD3).⁵⁵ cDNA was synthesized from 1 µg RNA using the iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) under the following conditions: priming at 25°C for 5 min, reverse transcription at 46°C for 20 min, and enzyme inactivation at 95°C for 1 min. qRT-PCR was performed using the CFX Connect Real-time PCR System (Bio-Rad, Hercules, CA, USA) with SsoAdvanced SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Each 20 µL reaction contained 2 µL cDNA, 1 µL forward and reverse primers (Table 1), 11 µL SYBR mix, and 5 µL nuclease-free water (Thermo Fisher Scientific, Waltham, MA, USA). Cycling conditions were as follows: 95°C for 30 s, followed by 39 cycles at 95°C for 10 s and 55°C for 30 s, and melt curve analysis. Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and analyzed using the 2^–ΔΔCt method.²⁵

Table 1.

Sequences of Primers Used for Quantitative Real-Time Polymerase Chain Reaction

Gene	Priimer sequence (5′→3′)
IL-1β
Foward	CTC GCC AGT GAA ATG ATG GCT
Reverse	GTC GGA GAT TCG TAG CTG GAT
IL-6
Foward	CTT CTC CAC AAG CGC CTT C
Reverse	CAG GCA ACA CCA GGA GCA
TNF-α
Foward	GGC AGT CAG ATC ATC TTC TCG
Reverse	GGT TTG CTA CAA CAT GGG CTA
VEGF
Foward	CTA CCT CCA CCA TGC CAA GT
Reverse	GCA GTA GCT GCG CTG ATA GA
GAPDH
Foward	GGC CTC CAA GGA GTA AGA CC
Reverse	AGG GGA GAT TCA GTG TGG TG

GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL-1β, interleukin 1 beta; IL-6, interleukin 6; TNF-α, tumor necrosis factor alpha; VEGF, vascular endothelial growth factor.

Western blot analysis

ARPE-19 cells were lysed with radioimmunoprecipitation buffer containing protease and phosphatase inhibitors (Thermo Fisher, 99:1). Lysates were ultrasonicated and centrifuged at 12,000×g for 20 min at 4°C. Protein concentrations were determined using the bicinchoninic acid assay (Thermo Fisher). Equal amounts of protein (30 µg) were separated on 10%–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene fluoride membranes. Membranes were blocked in 5% skim milk in Tris-buffered saline with Tween 20 and incubated with primary antibodies (Table 2) overnight at 4°C. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, and signals were detected using SuperSignal West Pico PLUS (Thermo Fisher) with an imaging system (Azure Biosystems, Dublin, CA, USA). Band intensities were quantified with ImageJ and normalized to α-tubulin.

Table 2.

Antibodies Used for Western Blot Analysis

Type of antibody	Antibody	Catalog number	Dilution factor	Corporation
Primary antibody	p-JNK	9251	1:500	Cell Signaling
	p-IkBɑ	2859	1:1000	Cell Signaling
	p-NF-ĸB	3033	1:1000	Cell Signaling
	CHOP	2895	1:2000	Cell Signaling
	BiP	3183	1:1000	Cell Signaling
	XBP-1	83418	1:1000	Cell Signaling
	Cleaved-caspase 3	9661	1:1000	Cell Signaling
	Cleaved-caspase 9	9507	1:1000	Cell Signaling
	Bax	2772	1:1000	Cell Signaling
	Bcl-2	sc-7382	1:1000	Santa Cruz
	β-actin	sc-47778	1:2000	Santa Cruz
	ɑ-tublin	sc-8035	1:1000	Santa Cruz
Secondary antibody	Anti-rabbit IgG	7074	1:3000	Cell Signaling
Secondary antibody	Anti-mouse IgG	7076	1:1000	Cell Signaling

α-tubulin, alpha-tubulin; β-actin, beta-actin; Bax, bcl-2-associated X protein; Bcl-2, b-cell lymphoma 2; BiP, binding immunoglobulin protein; CHOP, C/EBP homologous protein; Cleaved-caspase 3, cleaved caspase-3; Cleaved-caspase 9, cleaved caspase-9; IgG, immunoglobulin G; p-IκBα, phosphorylated inhibitor of nuclear factor kappa B alpha; p-JNK, phosphorylated c-Jun N-terminal kinase; p-NF-κB, phosphorylated nuclear factor kappa B; XBP-1, X-box binding protein 1.

VEGF secretion assay

VEGF levels in culture supernatants were measured using a human VEGF Enzyme-Linked Immunosorbent Assay (ELISA) kit (Abcam, UK), according to the manufacturer’s protocol.⁵⁶

Intracellular calcium measurement

Intracellular calcium levels were determined using the Fluo-4 NW Calcium Assay Kit (Thermo Fisher). ARPE-19 cells were seeded at a density of 5.0 × 10⁴ cells/well in black-walled 96-well plates (Corning, NY, USA) and cultured overnight, then treated with ESH or vehicle for 3 h. Fluo-4 NW dye (100 µL) was added to each well and incubated at 37°C in the dark for 30 min. Following dye loading, 5 µM Tg was added, and fluorescence was recorded using a microplate reader (Molecular Devices).

Statistical analysis

Data are presented as means ± standard deviations from three independent experiments. Statistical significance was assessed using one-way analysis of variance followed by Tukey’s post hoc test for multiple comparisons. Pearson’s correlation coefficients were calculated to assess the relationships between VEGF mRNA expression and secreted VEGF protein levels, as well as between intracellular calcium levels and secreted VEGF protein. Differences with P < .05 were considered statistically significant.

RESULTS

Effects of ESH on ARPE-19 cell viability

The EZ-CYTOX assay, which quantifies cell viability based on the reduction of a water-soluble tetrazolium salt by dehydrogenase enzymes in metabolically active cells to produce a colored formazan product,⁵⁰ was used to determine the optimal concentration of ESH extract for pretreatment. ARPE-19 cells were treated with ESH at concentrations of 0, 25, 100, 400, 600, 800, and 1,000 µg/mL (Fig. 1). No significant cytotoxicity was observed at concentrations up to 1,000 µg/mL, indicating that ESH did not adversely affect cell viability. Based on these results, concentrations of 0, 100, 500, and 1,000 µg/mL were selected for subsequent experiments.

Effects of ESH on NF-κB and MAPK signaling pathways and inflammatory gene expression in ARPE-19 cells

Figure 2 illustrates the effects of ESH pretreatment on post-translational modification of inflammation-related signaling proteins and transcriptional regulation of pro-inflammatory cytokines in LPS-stimulated ARPE-19 cells. LPS stimulation robustly activated inflammatory signaling, increasing the phosphorylation of NF-κB and IκBα by 140% and 458%, respectively, relative to the untreated control. At the post-translational level, ESH significantly inhibited the phosphorylation of NF-κB and IκBα, a key mediator of inflammatory signaling. Specifically, ESH at a concentration of 1,000 µg/mL of ESH significantly inhibited NF-κB phosphorylation by 53.53%. Moreover, phosphorylated IκBα levels were reduced in a concentration-dependent manner, with decreases of 34.92%, 51.38%, and 74.25% at 100, 500, and 1,000 µg/mL, respectively (Fig. 2A–C). Additionally, phosphorylation of the MAPK family protein c-Jun N-terminal kinase (JNK) was elevated by 109% following LPS treatment. ESH significantly suppressed this increase only at 1,000 µg/mL, with a 67.47% reduction relative to the LPS group (Fig. 2D). ESH may partially block IκBα phosphorylation in inflammation-related signaling, thereby limiting NF-κB activation; at high concentrations, the inhibitory effect extends to the MAPK/JNK pathway. These results suggest that ESH may have a complex mechanism of action that affects the transcriptional regulation and signaling process of the inflammatory response.

At the transcriptional level, ESH pretreatment significantly suppressed the gene expression of pro-inflammatory cytokine TNF-α in a concentration-dependent manner. Specifically, TNF-α mRNA expression, which was elevated by approximately 30% upon LPS stimulation relative to the control, was reduced by 20.15% and 25.59% in the 500 and 1,000 µg/mL ESH-treated groups, respectively. Modulation of inflammatory gene expression can be interpreted as a transcriptional repression effect due to the inhibition of the NF-κB and MAPK pathways. TNF-α is regulated by both pathways, and dual signal blockade can have a synergistic effect on the decrease in expression (Fig. 2E).^20,57 IL-1β and IL-6 expression were induced by LPS, increasing by approximately 1,300% and 140%, respectively, relative to the control. Conversely, IL-1β and IL-6 did not show a significant decrease in the presence of ESH (Fig. 2F–G). This discrepancy may be explained by the distinct induction kinetics of inflammatory cytokines, with IL-1β and IL-6 generally peaking later than TNF-α in response to LPS.⁵⁸ These findings suggest that ESH attenuates LPS-induced inflammatory responses in ARPE-19 cells by inhibiting the phosphorylation of NF-κB and MAPKs and transcriptionally downregulating TNF-α expression.

Effects of ESH on ER stress-related protein expression in ARPE-19 cells

Building on the observed anti-inflammatory effects of ESH, we further investigated its potential to modulate ER stress responses in ARPE-19 cells. Figure 3 presents the effects of ESH treatment on UPR- and apoptosis-related proteins in Tg-induced ER stress conditions. Western blot analysis revealed that Tg treatment markedly upregulated the expression of ER stress markers CHOP and XBP-1 by 2,478% and 2,899%, respectively, compared with the untreated control. These elevations were significantly suppressed by ESH treatment at 1,000 µg/mL by 51.91% and 40.59%, respectively. Binding immunoglobulin protein (BiP) expression increased by 374% after Tg stimulation, with no significant change following ESH treatment at any concentration. The observed downregulation of CHOP and XBP-1 suggests that ESH interferes with the UPR, mitigating ER stress signaling pathways (Fig. 3A–D).²⁴

Moreover, Tg stimulation also strongly activated apoptosis-related proteins, with cleaved caspase-3 and cleaved caspase-9 expression increasing by 1,464% and 2,150%, respectively. ESH markedly inhibited the activation of apoptosis-related proteins, including cleaved caspase-3 and cleaved caspase-9, at 1,000 µg/mL by 48.67% and 41.20%, respectively, demonstrating a protective effect against ER stress-induced apoptotic cell death (Fig. 3E and F). However, the expressions of Bax and Bcl-2 were not significantly affected by ESH treatment, suggesting that the anti-apoptotic effect of ESH may primarily involve caspase-dependent pathways rather than modulation of Bcl-2 family proteins.⁵³ Hence, the Bax/Bcl-2 ratio showed a slight increasing trend following ESH treatment, but the differences were not statistically significant (Fig. 3G–I).

Collectively, these results demonstrate that ESH significantly attenuates ER stress and ER stress-mediated apoptotic signaling in ARPE-19 cells, suggesting potential protective roles of ESH against cellular pathological responses in the retina.

Effects of ESH on Tg-induced increases in VEGF secretion and calcium

Given the established link between ER stress and VEGF expression, which is typically upregulated under ocular stress conditions,²⁰ the impact of ESH on VEGF levels was investigated. VEGF mRNA expression increased by approximately 497% in ARPE-19 cells following Tg treatment, while pretreatment with ESH at concentrations of 100, 500, and 1,000 µg/mL resulted in reductions of 29%, 37%, and 30%, respectively, relative to the Tg-only group (Fig. 4A). Intracellular calcium levels, which increased by 39.57% following Tg exposure relative to control, were significantly suppressed by ESH pretreatment only at 1,000 µg/mL, with an 18.49% decrease relative to the Tg-treated group (Fig. 4B). VEGF secretion, measured using ELISA, increased by 88.17% following Tg treatment relative to the control and was significantly inhibited by ESH at 500 and 1,000 µg/mL, showing reductions of approximately 19.32% and 30.50%, respectively, relative to Tg-treated cells (Fig. 4C). To further confirm the mechanistic involvement of the MAPK pathway, U0126—a selective MEK1/2 inhibitor—was employed as a pharmacological control.⁵⁹ While U0126 treatment did not significantly alter intracellular calcium levels, it markedly suppressed VEGF secretion to levels comparable to those in the control group. Our findings may suggest that MAPK signaling plays a key role in VEGF upregulation under ER stress conditions, independently of intracellular calcium regulation. In contrast, ESH reduced VEGF secretion partly through suppression of intracellular calcium and transcriptional downregulation, indicating that ESH may act through both MAPK-dependent and -independent mechanisms. Notably, VEGF mRNA levels showed a strong positive correlation with VEGF protein levels measured using ELISA (r = 0.843), and intracellular calcium levels were also strongly correlated with VEGF protein levels (r = 0.888), supporting the mechanistic link among calcium homeostasis, transcriptional regulation, and VEGF secretion.⁶⁰

VEGF expression is closely associated with ocular ER stress, as prolonged ER stress triggers the UPR, leading to hypoxia-related signaling cascades that include VEGF upregulation.^21,22 In pathological conditions, such as AMD and DR, chronic ER stress contributes to excessive VEGF secretion, promoting neovascularization and retinal damage.^20,60 Since ESH significantly attenuated VEGF induction, it may play a potential role in alleviating ocular ER stress and its downstream pathological consequences. Mitigation of UPR activation and VEGF expression, as mentioned earlier, supports the protective effects of ESH against ER stress-associated ocular diseases.

DISCUSSION

AMD and DR are progressive retinal diseases characterized by the deterioration of the RPE, a monolayer of specialized cells essential for photoreceptor maintenance and visual function.¹⁴ RPE dysfunction is precipitated by various stressors, notably chronic inflammation, ER stress, and pathological angiogenesis.⁶¹ These pathological triggers are particularly detrimental due to their interconnected nature, where persistent inflammation exacerbates ER stress and promotes angiogenic signaling through VEGF upregulation.^20,24 Given the pivotal role of the RPE in retinal homeostasis, strategies that mitigate these pathological cascades may prevent or attenuate AMD and DR progression. In the present study, we investigated the cytoprotective effects of ESH in ARPE-19 cells exposed to LPS- and Tg-induced retinal pathologies.

Inflammation is a key initiating trigger in RPE dysfunction and retinal degeneration, with the NF-κB and MAPK signaling pathways central to cytokine transcription and immune activation.⁶² Our findings demonstrated that ESH pretreatment significantly inhibited the phosphorylation of NF-κB, IκBα, and JNK in LPS-stimulated ARPE-19 cells, indicating effective suppression of inflammatory signaling cascades at the post-translational level.⁶³ Inflammation inhibitory effects of ESH translated to reduced transcriptional activity, as evidenced by decreased TNF-α gene expression, a downstream target of both NF-κB and MAPK. IL-1β and IL-6 gene levels were not significantly altered, which may reflect differential temporal expression patterns or involvement of parallel signaling pathways less responsive to ESH.^58,64 Overall, the ability of ESH to modulate early-phase inflammatory responses highlights its potential in preventing inflammation-induced RPE damage and slowing the initiation of AMD and DR.

Beyond inflammation, unresolved ER stress contributes to RPE apoptosis and secretion of angiogenic mediators. In retinal disease, chronic ER stress activates the UPR, leading to upregulation of CHOP and XBP-1, which are implicated in pro-apoptotic signaling.⁶⁵ In this study, ESH significantly attenuated Tg-induced expression of CHOP and XBP-1, suggesting its efficacy in disrupting maladaptive UPR activation.⁶⁶ Concomitantly, cleaved caspase-3 and −9 levels were reduced, indicating suppression of ER stress-induced apoptotic signaling. However, the expressions of Bax and Bcl-2 as well as the Bax/Bcl-2 ratio remained unchanged, implying that ESH may primarily inhibit caspase-dependent apoptosis rather than modulate mitochondrial apoptotic regulators.⁶⁷ These findings underscore ESH’s capacity to buffer ER stress burden and preserve RPE cell viability by targeting key pro-apoptotic nodes within the UPR axis.⁶⁸

VEGF overexpression and intracellular calcium dysregulation are hallmark features of neovascularization in AMD and DR.²⁴ ER stress has been shown to promote VEGF production, partly via calcium leakage from the ER lumen and activation of downstream transcriptional machinery.²⁰ In this study, ESH pretreatment markedly suppressed VEGF mRNA expression, protein secretion, and intracellular calcium elevation in Tg-stimulated ARPE-19 cells. The strong correlations between intracellular calcium levels and VEGF secretion (r = 0.888) and between VEGF mRNA and protein levels (r = 0.843) support a mechanistic link involving both transcriptional and post-transcriptional regulation.⁶⁹ Notably, the MEK1/2 inhibitor U0126, which selectively blocks the MAPK/ERK kinase pathway upstream of ERK1/2 activation, reduced VEGF secretion independently of calcium, whereas ESH modulated both pathways, suggesting dual MAPK-dependent and calcium-mediated mechanisms of VEGF inhibition.^24,70 These results position ESH as a multi-target agent capable of counteracting both angiogenic signaling and calcium imbalance in the stressed RPE.

Further mechanistic insight was gained by evaluating specific components of the UPR. Although ESH significantly downregulated CHOP and XBP-1, BiP expression remained unchanged, suggesting selective modulation of downstream UPR mediators rather than complete blockade of stress detection.⁷¹ This partial modulation may allow RPE cells to retain adaptive capacity while preventing transition to apoptotic signaling.⁷² Such selective interference is particularly advantageous, as ESH treatment may preserve normal protein-folding homeostasis while minimizing pathological outcomes. These nuanced effects highlight ESH’s potential as a fine-tuned modulator of ER stress rather than as a non-specific suppressor.^16–18

The anti-inflammatory, anti-ER stress, VEGF-regulatory, and intracellular calcium-modulating effects observed in this study are likely attributable to specific bioactive constituents of E. sativa that become enriched or stabilized during hot-air drying. The protective effects of ESH observed in the present study may be attributed, at least in part, to well-characterized phytochemical constituents of E. sativa. Among these, glucosinolates such as glucoerucin and their isothiocyanate derivatives have been reported to suppress pro-inflammatory signaling by downregulating cyclooxygenase 2, Toll-like receptor 4, and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 expression, thereby attenuating NF-κB-mediated cytokine transcription.⁴⁰ Heat processing of E. sativa has also been shown to increase γ-aminobutyric acid, which supports intracellular calcium homeostasis, along with natural antioxidants and polyphenolic compounds that mitigate oxidative stress and indirectly alleviate ER stress.⁷³ Furthermore, glucosinolate-derived isothiocyanates, including sulforaphane and its structural analogs, modulate UPR markers (e.g., CHOP and BiP) and apoptotic markers (Bax and Bcl-2).⁷⁴ In addition, essential minerals such as magnesium and zinc act as cofactors that stabilize calcium homeostasis and ER function.^75,76 Collectively, these phytochemical and mineral attributes provide a plausible mechanistic basis for the anti-inflammatory, ER stress-modulating, VEGF-regulatory, and calcium-regulating activities observed in our experiments.

Despite these promising findings, several limitations should be acknowledged. The concentrations of ESH tested in vitro (100–1,000 µg/mL) may exceed physiologically achievable levels following oral consumption due to potential degradation, low solubility, or first-pass metabolism. Moreover, as a crude extract comprising a complex mixture of phytochemicals, it remains difficult to identify the primary bioactive components responsible for the observed effects. The absence of pharmacokinetic data—covering absorption, distribution, metabolism, and excretion—further limits prediction of systemic efficacy and retinal bioavailability. Additional studies employing targeted metabolomics, bioactivity-guided fractionation, and pharmacokinetic profiling, together with in vivo validation, will therefore be required to confirm and extend the present findings and to establish the potential of ESH as a functional dietary agent for preventing or delaying RPE dysfunction in AMD and DR.

CONCLUSION

This study demonstrated that ESH effectively attenuated key pathological processes involved in RPE dysfunction, including NF-κB-MAPK–mediated inflammation, UPR-driven ER stress, VEGF-mediated angiogenesis, and intracellular calcium dysregulation, in ARPE-19 cells under degenerative conditions. ESH significantly suppressed inflammatory signaling and TNF-α expression, reduced CHOP and XBP-1 levels without affecting BiP, and inhibited caspase-dependent apoptotic signaling while modulating VEGF expression via both MAPK–dependent and calcium-mediated pathways.

AUTHORS’ CONTRIBUTIONS

Conceptualization: M.C., J.L., and J.-H.H. Methodology: M.C., J.L., and Y.J.K. Writing—original draft preparation: M.C. and J.L. Writing—review and editing: M.C., J.L., Y.J.K., M.K., and J.-H.H. Supervision: M.K. and J.-H.H. Project administration: M.K. and J.-H.H. Funding acquisition: M.K. and J.-H.H. All the authors have read and agreed to the published version of the article.

Footnotes

AUTHOR DISCLOSURE STATEMENT

The authors have no conflicts of interest to declare.

FUNDING INFORMATION

There are no funders to report for this submission.

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