Protective Effects of Violaxanthin on Cellular Oxidative Stress via NRF2 Activation in H₂O₂-Stimulated Human Keratinocytes

Abstract

Excessive accumulation of reactive oxygen species from exogenous and endogenous stressors can cause cellular damage. Chlorella contains diverse bioactive compounds, and violaxanthin, a major carotenoid pigment found in Chlorella sp. HS-V, has been reported to possess anti-inflammatory, anticancer, and antioxidant properties. We investigated the effect of violaxanthin on hydrogen peroxide (H₂O₂)-induced oxidative stress in human keratinocytes. Chlorella sp. HS-V extract significantly restored the H₂O₂-induced decrease in cell viability. Similarly, violaxanthin reduced H₂O₂-induced cytotoxicity and intracellular reactive oxygen species levels, which was associated with the upregulation of antioxidant enzyme expression. Under H₂O₂-induced oxidative stress conditions, violaxanthin may enhance cellular antioxidant defense by promoting nuclear factor erythroid 2-related factor 2 (NRF2) translocation through the phosphoinositide 3-kinase/protein kinase B/glycogen synthase kinase 3β (PI3K/AKT/GSK3β) signaling pathway. Additionally, violaxanthin improved H₂O₂-impaired wound healing in HaCaT human keratinocyte cells and reduced senescence-associated beta-galactosidase-positive normal human epidermal keratinocytes. Overall, these findings suggest that violaxanthin may serve as a potential therapeutic agent for mitigating oxidative stress-induced cellular dysfunction.

1. Introduction

The skin is the outermost organ of the body and acts as the first line of defense against bacteria and other organisms [1,2]. Keratinocytes are the primary cellular constituents of the epidermis and play a crucial role in maintaining skin barrier function [3]. Skin can be stimulated by reactive oxygen species (ROS) resulting from various factors, such as exposure to ultraviolet rays and the intrinsic aging process [4]. ROS are inherently unstable and react strongly with surrounding substances, resulting in various types of damage [5,6]. Many studies have revealed that the excessive accumulation of ROS induces oxidative stress, which can damage DNA and other biomolecules, thereby contributing to inflammation, aging, and tumorigenesis [7,8,9,10]. ROS can also lead to dysfunction and damage in keratinocytes, which play a key role in skin barrier function. O’Toole et al. showed that the migration of human keratinocytes, which is an essential process in wound healing, was inhibited by hydrogen peroxide (H₂O₂) [11]. Other studies have suggested that increased intracellular ROS production and deficiencies in antioxidant defense systems within keratinocytes contribute to the pathogenesis of keratinocyte-related inflammatory diseases, such as psoriasis [12,13]. Cellular defense against oxidative stress is regulated by multiple signaling pathways that coordinate ROS detoxification, antioxidant enzyme expression, inflammatory responses, and cell survival. Among these, the NRF2/antioxidant response element (ARE) pathway is a central defense system that induces cytoprotective and antioxidant genes, including HO-1, NQO1, SOD, CAT, and GPx [14,15]. In addition, upstream pathways such as PI3K/AKT/GSK3β, mitogen-activated protein kinases (MAPKs; ERK, JNK, and p38), and AMP-activated protein kinase (AMPK) have been reported to modulate oxidative stress responses by regulating NRF2 stabilization, nuclear translocation, antioxidant enzyme induction, and stress-adaptive cell survival [16,17,18]. Therefore, elucidating and activating these antioxidant defense mechanisms may be an important strategy for protecting keratinocytes against H₂O₂-induced oxidative stress.

Microalgae are photosynthetic microorganisms that possess diverse defense systems to survive under a variety of environmental conditions, enabling the synthesis of numerous bioactive metabolites [19]. Numerous studies have reported that microalgae contain a wide range of bioactive compounds, including proteins, fatty acids, phenolic compounds, vitamins, and photosynthetic pigments [20,21]. These microalgae-derived metabolites have attracted considerable attention for their potential applications in the cosmeceutical [22,23,24,25], pharmaceutical [24,25,26,27,28], and food industries [25,27,28,29]. Among various microalgae, Chlorella is a freshwater unicellular green alga [30]. Chlorella has been reported to contain vitamins, lipids, and carotenoids, including zeaxanthin, violaxanthin, and astaxanthin [19,31,32]. Due to its rich bioactive composition, Chlorella has been reported to exhibit diverse physiological activities [33,34,35]. Recent studies have demonstrated that Chlorella extract exhibits anticancer activity, including the suppression of apoptosis-related pathways, in various cancer cell lines [36,37,38]. Additionally, Chlorella extract has been shown to possess anti-inflammatory and immunomodulatory properties through the downregulation of several inflammatory cytokines [39,40,41,42]. Among the carotenoids found in Chlorella, violaxanthin is a major carotenoid that can be isolated from this microalga [38,43,44,45,46,47]. Previous studies have reported that violaxanthin exhibits higher antioxidant capacity compared to β-carotene and lutein [45]. Previous studies have indicated that violaxanthin exhibits antiproliferative activity in cancer cells and triggers biochemical changes indicative of early apoptosis [38,48]. Violaxanthin has also been shown to exert anti-inflammatory effects by inhibiting the nuclear factor kappa B (NF-κB) pathway in LPS-stimulated RAW 264.7 mouse macrophages [44]. Furthermore, violaxanthin derived from Nannochloropsis oceanica was found to protect human dermal fibroblasts against UVB-induced damage, notably restoring decreased collagen expression in UVB-mediated cellular senescence [49]. These studies suggest that violaxanthin has potential as a therapeutic agent in cosmetic and pharmaceutical applications. However, the protective effects and underlying mechanisms of violaxanthin against oxidative stress in keratinocytes remain unexplored. In this study, we investigated the protective effects of violaxanthin against H₂O₂-induced oxidative stress in human keratinocytes and explored the underlying molecular mechanisms.

2. Materials and Methods

2.1. Cell Culture and Reagents

For this study, the immortalized human keratinocyte cell line (HaCaT) was selected, since it is commonly applied to evaluate protective mechanisms against skin oxidative stress [17,50]. HaCaT cells were obtained from Cell Line Service (DKFZ, Eppelheim, Germany). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, WelGene, Inc., Daegu, Republic of Korea), supplemented with 10% fetal bovine serum (FBS, Corning Inc., Corning, NY, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). nHEK cells, primary normal human epidermal keratinocytes, were obtained from CELLnTEC (Bern, Switzerland). nHEK cells were cultured in Keratinocyte Basal Medium-2 (Lonza, Basel, Switzerland), supplemented with bovine pituitary extract, epinephrine, gentamicin sulfate-amphotericin, hydrocortisone, human epidermal growth factor, insulin, and transferrin, as recommended by the manufacturer. All cell lines were maintained in a humidified incubator at 37 °C with 5% CO₂. Dimethyl sulfoxide (DMSO), violaxanthin, ascorbic acid, N-acetylcysteine (NAC), and hydrogen peroxide (H₂O₂) were purchased from Sigma-Aldrich (St. Louis, MO, USA). LY294002 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Violaxanthin was dissolved in DMSO, and the final concentration of DMSO in the culture medium did not exceed 0.1% (v/v).

2.2. Microalgal Strain and Culture Conditions

Chlorella sp. HS-V (KCTC 13850BP; Korean Collection for Type Cultures, Jeongeup, Republic of Korea) was cultivated in 2 L Erlenmeyer flasks with a working volume of 500 mL. The culture medium consisted of 1 g/L S-feed fertilizer (Farmhannong, Seongnam, Republic of Korea), 3 g/L yeast extract (BD Biosciences, Franklin Lakes, NJ, USA), and 15 g/L glucose, with the initial pH adjusted to 7.0. Cultures were incubated at 30 °C with continuous shaking at 120 rpm for 72 h under continuous illumination at an intensity of 150 μmol photons m⁻² s⁻¹. After cultivation, the biomass was harvested by centrifugation and subsequently lyophilized for further analysis.

2.3. Preparation of Chlorella sp. HS-V Extract

Bioactive compounds were extracted from the lyophilized microalgal biomass via saponification followed by solvent fractionation. Briefly, 2 g of lyophilized biomass was combined with 8 mL of 60% (w/v) KOH aqueous solution and incubated at 40 °C for 1 h. The saponified mixture was then extracted with 60 mL of hexane/ethyl acetate (1:1, v/v) at 40 °C for 1 h with continuous stirring. Phase separation was induced by the addition of 30 mL of distilled water followed by vigorous agitation. The upper organic phase was collected and concentrated under reduced pressure using a rotary evaporator to yield the solid crude extract. The resulting extract was dissolved in medium-chain triglyceride (MCT) oil and filtered to remove insoluble particulates, yielding the final oil-based formulation.

2.4. Cell Viability Assay

HaCaT cells (4.0 × 10⁴) were seeded in 24-well plates and incubated for 24 h, followed by treatment with the indicated concentrations of Chlorella sp. HS-V extract, violaxanthin or H₂O₂ for 24 h. Before each subsequent treatment, the cells were washed with DPBS to remove residual compounds. Subsequently, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution (0.5 mg/mL) was added to each well, and cells were incubated for an additional 2 h. The culture medium was removed, and DMSO was added to each well to dissolve the formazan crystals. Cell viability was determined by measuring the absorbance at 520 nm using a Synergy™ HTX Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA), and was calculated using the following formula: cell viability (%) = [(absorbance of treated cells − absorbance of blank)/(absorbance of control cells − absorbance of blank)] × 100, with the untreated control set at 100%.

2.5. High-Performance Liquid Chromatography (HPLC) Analysis

The chemical profile of the ethanol-dissolved Chlorella sp. HS-V extract was analyzed by HPLC using an Agilent 1260 Infinity Binary LC system coupled with a diode array detector (DAD; Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was performed on a Waters Spherisorb^® S5 ODS1 column (4.6 mm × 250 mm, 5 μm; Waters Corporation, Milford, MA, USA) maintained at 40 °C. The injection volume was 20 μL, and the flow rate was set at 1.2 mL/min. Solvent A consisted of acetonitrile/methanol/0.1 M Tris-HCl buffer at 84:2:14 (v/v/v), while solvent B consisted of methanol/ethyl acetate at 68:32 (v/v). The gradient program was set as follows: 100% A from 0 to 15 min, a linear change to 100% B from 15 to 19 min, to 100% B from 19 to 25 min, and a return to 100% A for re-equilibration from 25 to 30 min. The detection wavelength was 445.4 nm.

2.6. ABTS Radical Scavenging Assay

The ABTS radical cation solution was prepared using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; MedChemExpress, Monmouth Junction, NJ, USA). Briefly, ABTS was reacted with potassium peroxydisulfate (Supelco, Bellefonte, PA, USA) to obtain a final potassium peroxydisulfate concentration of 2.45 mM. The mixture was then allowed to stand for 16 h at room temperature under light-protected conditions to generate ABTS radical cations. Before use, the resulting ABTS•+ solution was adjusted with distilled water to an absorbance of 0.70 ± 0.02 at 734 nm. This diluted solution was then mixed with ascorbic acid (28.4 µM) and violaxanthin (2, 5, and 10 µM), followed by shaking for 5 min in a dark room. The ABTS radical scavenging activity was analyzed by measuring the absorbance at 734 nm using a Synergy^TM HTX Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA).

2.7. Intracellular Reactive Oxygen Species (ROS) Measurement Assay

HaCaT cells (8.0 × 10³) were seeded in 96-well plates and incubated for 24 h. Following 24 h of pre-treatment with violaxanthin (2, 5, and 10 µM) or NAC (5 mM), cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS, WelGene) and incubated with 10 µM 2′,7′-dichlorofluorescein diacetate (DCF-DA; Sigma-Aldrich) for 30 min in the dark. The DCF-DA solution was aspirated, and cells were treated with H₂O₂ (1 mM) for 20 min to induce a ROS response. Fluorescence intensity was quantified at an excitation wavelength of 485 nm and an emission wavelength of 520 nm using a Synergy™ HTX Multi-Mode Microplate Reader.

2.8. Reverse Transcription–Polymerase Chain Reaction (RT-PCR) and Reverse Transcription–Quantitative Polymerase Chain Reaction (RT-qPCR)

Total mRNA expression levels of antioxidant-related genes were assessed by RT-PCR and RT-qPCR. HaCaT cells (6.0 × 10⁵) were seeded in 100 mm dishes and treated with violaxanthin (2, 5, and 10 µM) and/or H₂O₂ (500 µM) for 24 h. Total RNA was extracted using RiboEx reagent (GeneAll Biotechnology, Seoul, Republic of Korea). Complementary DNA (cDNA) was synthesized using oligo-dT primers, 2.5 mM dNTP, and Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Thermo Fisher Scientific). The RT-PCR mixture contained 10X reaction buffer with MgCl₂, 2.5 mM dNTP, DMSO, and Taq DNA polymerase (Bioneer, Daejeon, Republic of Korea). For RT-qPCR, the EvaGreen qPCR master mix (Solis BioDyne, Tartu, Estonia) was used, along with the StepOnePlus™ Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). All primer sequences were designed using the NCBI Primer-BLAST online tool (National Center for Biotechnology Information, Bethesda, MD, USA) based on reference sequences deposited in the NCBI database. The primer sequences used in this study are listed in

Table 1. List of primers used for RT-PCR and RT-qPCR analysis.

Target mRNA	Primer Sequences
CAT	F_5′-GGCCAGTCCTGACAAAATGC-3′
	R_5′-TGGGGTAGTAATTTGGAGCA-3′
GPx1	F_5′-GCAGCTCGTTCATCTGGGTG-3′
	R_5′-ATGTGTGCTGCTCGGCTAGC-3′
GSS	F_5′-TAGATGCCCCACGTGCTTGT-3′
	R_5′-ATCCTCATGGAGAAGATCGAACC-3′
HO-1	F_5′-GCCCTTCAGCATCCTCAGTTCC-3′
	R_5′-AGTGGTCATGGCCGTGTCAAC-3′
NQO1	F_5′-GGGAGACAGCCTCTTACTTGCC-3′
	R_5′-AACACCCAGCCGTCAGCTATTG-3′
SOD1	F_5′-CCAGTGCAGGGCATCATCA-3′
	R_5′-TTGGCCCACCGTGTTTTCT-3′
NRF2	F_5′-ATAGCTGAGCCCAGTATC-3′
	R_5′-CATGCACGTGAGTGCTCT-3′
GAPDH	F_5′-TCCAAAATCAAGTGGGGCGATGC-3′
	R_5′-GCCAGTAGAGGCAGGGATGATGT-3′

.

2.9. Immunoblotting

HaCaT cells (6.0 × 10⁵) and nHEK cells (8.0 × 10⁵) were seeded in 100 mm dishes. For Western blot analysis, reagent-treated cells were harvested and subjected to protein extraction using a RIPA lysis buffer containing a PhosSTOP phosphatase inhibitor cocktail (Roche, Basel, Switzerland). The lysis buffer was composed of 150 mM NaCl, 25 mM Tris-HCl, 5 mM EDTA, 1% sodium deoxycholate, 1% NP-40, and 0.025% sodium dodecyl sulfate. The total protein in each lysate was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific).

Equivalent amounts of protein were loaded onto SDS-polyacrylamide gels, separated by electrophoresis, and transferred to 0.2 µm nitrocellulose membranes (Cytiva, Amersham, UK). Non-specific binding was minimized by blocking the membranes with 2% skim milk in TBST for 2 h at room temperature. The membranes were incubated with primary antibodies at 4 °C overnight and then reacted with HRP-conjugated secondary antibodies for 2 h at room temperature. The protein signals were developed using Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA).

The anti-actin antibody (sc-47778) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against nuclear factor erythroid 2-related factor 2 (NRF2; #12721), lamin A/C (#4777), β-tubulin (#2128), AKT (#9272), phosphorylated AKT at S473 (#9271), glycogen synthase kinase 3β (GSK3β; #9315), phosphorylated GSK3β at S9 (#9323), p16 (#18769), p21 (#2947), p53 (#9282), phosphorylated p53 at S15 (#9284), anti-rabbit IgG, and anti-mouse IgG were obtained from Cell Signaling Technology (Beverly, MA, USA).

2.10. Cell Fractionation Assay

To examine the subcellular localization of NRF2, nuclear and cytoplasmic extracts were isolated from HaCaT cells using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). All extraction steps were performed following the manufacturer’s protocol. In brief, after the designated treatments, cells were harvested, rinsed twice with ice-cold DPBS, and pelleted by centrifugation at 500× g for 5 min.

For cytoplasmic extraction, the cell pellet was first suspended in ice-cold Cytoplasmic Extraction Reagent I (CER I) via vigorous vortexing and then incubated for 10 min. CER II was subsequently added, and the samples were subjected to additional vortexing and brief incubation on ice. After centrifugation at 16,000× g for 5 min at 4 °C, the supernatant was recovered as the cytoplasmic extract. The residual pellet was further processed with ice-cold nuclear extraction reagent (NER) through repeated vortexing and ice-incubation cycles to extract nuclear proteins. The samples were then centrifuged at 16,000× g for 10 min, and the supernatant was collected as the nuclear extract.

The obtained fractions were analyzed by immunoblotting to assess NRF2 protein distribution. Lamin A/C and β-tubulin served as nuclear and cytoplasmic fractionation controls, respectively.

2.11. Wound-Healing Assay

HaCaT cells (4.0 × 10⁵) were seeded in 60 mm dishes and grown to approximately 90% confluence. Cells were pretreated with 500 µM H₂O₂ for 24 h, after which a linear scratch wound was created across the cell monolayer using a sterile 1000 µL pipette tip. Cells were then washed with DPBS to remove debris and treated with the indicated concentrations of violaxanthin. Wound closure was quantified using ImageJ software (ImageJ 1.52a, National Institutes of Health, Bethesda, MD, USA) and is expressed as the percentage of wound closure relative to the initial wound area at 0 h.

2.12. Senescence-Associated Beta (SA-β)-Galactosidase Staining Assay

Cellular senescence was assessed by SA-β-gal staining based on a modified protocol from Debacq-Chainiaux et al. [51]. nHEK cells (8.0 × 10⁵) were seeded and incubated at 37 °C for 24 h. Cells were pretreated with violaxanthin (5 and 10 µM) or NAC (5 mM) for 24 h, followed by exposure to 200 µM H₂O₂ for 4 h to induce oxidative stress-mediated cellular senescence. Subsequently, cells were fixed for 5 min and incubated in a staining solution for 8 h at 37 °C without CO₂. SA-β-gal-positive cells were visualized under a bright-field microscope and quantified by counting the cells.

2.13. Statistical Analysis

All quantitative data are presented as the mean ± standard deviation (SD) of at least three independent experiments. Statistical analyses and graphical representations were performed using GraphPad Prism software (version 8.0.1, GraphPad Software, San Diego, CA, USA). The statistical significance of differences between multiple groups was evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s test. A p-value of less than 0.05 was considered to indicate statistical significance, and specific significance levels are denoted in the respective figure legends.

3. Results

3.1. Chlorella Extract Restores H₂O₂-Induced Cytotoxicity in HaCaT Cells

Previous studies have reported that Chlorella exerts protective effects against H₂O₂-induced cellular damage through its antioxidant capacity [52,53]. We therefore investigated whether Chlorella extract exerts a similar protective effect against H₂O₂-induced cytotoxicity in HaCaT cells. As shown in Figure 1A, cells were treated with Chlorella sp. HS-V extract at 12.5, 25, 50, 100, 150, 200, and 250 µg/mL for 24 h. Cell viability decreased to approximately 66% at 200 µg/mL, whereas no significant cytotoxicity was observed at concentrations up to 150 µg/mL. To determine the optimal H₂O₂ concentration for inducing cytotoxicity, cells were treated with H₂O₂ at 100, 200, 300, 400, 500, 750, and 1000 µM for 24 h (Figure 1B). Treatment with 500 µM H₂O₂ reduced cell viability to approximately 80%. A minimal viability reduction may be insufficient to assess the recovery effect of protective compounds, whereas excessive H₂O₂-induced cytotoxicity may shift the experimental readout toward irreversible cell death, such as apoptosis or necrosis, rather than a recoverable oxidative stress response [54,55,56]. Consistent with this approach, previous HaCaT oxidative stress studies have selected H₂O₂ concentrations that reduced cell viability to approximately 70–80% for evaluating antioxidant or cytoprotective effects [57,58]. Therefore, the Chlorella sp. HS-V extract at 50, 100, and 150 µg/mL and 500 µM H₂O₂ were selected for subsequent experiments. To evaluate the protective effect of the Chlorella sp. HS-V extract against H₂O₂-induced cytotoxicity, cells were pretreated with the extract (50, 100, and 150 µg/mL) for 24 h, followed by treatment with 500 µM H₂O₂ for 24 h. As shown in Figure 1C, the Chlorella sp. HS-V extract restored the H₂O₂-induced decrease in cell viability in a dose-dependent manner. These results suggest that the Chlorella sp. HS-V extract attenuates H₂O₂-induced cytotoxicity in HaCaT cells.

3.2. Violaxanthin, a Major Carotenoid in Chlorella Extract, Protects HaCaT Cells from H₂O₂-Induced Cytotoxicity

Given that the Chlorella sp. HS-V extract exhibited a protective effect against H₂O₂-induced cytotoxicity (Figure 1), we performed HPLC analysis to identify the major active component responsible for this activity (Figure 2A). HPLC analysis showed a distinct peak corresponding to violaxanthin in the Chlorella sp. HS-V extract, and the violaxanthin content was 12,798.8 µg/g of evaporated extract. These results indicate that violaxanthin is a major carotenoid present in the Chlorella sp. HS-V extract among the detected pigments. To evaluate whether violaxanthin itself exerts cytotoxic effects, HaCaT cells were treated with violaxanthin at concentrations ranging from 1 to 100 µM for 24 h, which revealed no significant cytotoxicity at concentrations up to 10 µM (Figure 2C). Therefore, subsequent experiments were conducted using 2, 5, and 10 µM violaxanthin to evaluate its protective effects within a non-cytotoxic concentration range. Based on these findings, we next investigated whether violaxanthin exerts a similar protective effect against H₂O₂-induced cytotoxicity, as observed with the Chlorella sp. HS-V extract (Figure 1C). Cells were pretreated with violaxanthin (2, 5, and 10 µM) for 24 h, and then treated with 500 µM of H₂O₂. As a result, violaxanthin restored the H₂O₂-induced decrease in cell viability in a dose-dependent manner. Notably, pretreatment with 10 µM violaxanthin restored cell viability to levels comparable to those of the untreated control (Figure 2D). These data show that violaxanthin, the major carotenoid in the Chlorella sp. HS-V extract, protects HaCaT cells against H₂O₂-induced cytotoxicity.

3.3. Violaxanthin Decreases Intracellular Reactive Oxygen Species Levels in HaCaT Cells

Violaxanthin has previously been shown to exhibit antioxidant properties comparable to those of other carotenoids, including β-carotene and zeaxanthin [47]. Therefore, we next examined whether the protective effect of violaxanthin against H₂O₂-induced cellular damage was associated with its radical-scavenging activity using an ABTS assay. As shown in Figure 3A, violaxanthin showed concentration-dependent ABTS radical scavenging activity. Although its activity was weaker than that of ascorbic acid, which was used as a representative antioxidant control [59], these results indicate that violaxanthin possesses measurable antioxidant activity. To determine whether violaxanthin reduces intracellular ROS levels, cells were pretreated with violaxanthin (2, 5, and 10 µM) or NAC (5 mM) for 24 h, followed by exposure to 1 mM H₂O₂ for 20 min to induce intracellular ROS accumulation. As shown in Figure 3B, violaxanthin significantly reduced intracellular DCF fluorescence intensity in a dose-dependent manner. Notably, treatment with 10 µM violaxanthin reduced ROS levels to a degree comparable to those of NAC. Collectively, these results suggest that violaxanthin exerts a protective effect against H₂O₂-induced oxidative stress by reducing intracellular ROS levels in HaCaT cells.

3.4. Violaxanthin Increases the mRNA Expression of Antioxidant Enzymes in HaCaT Cells

To alleviate oxidative stress caused by ROS, cells regulate antioxidant defense systems through antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidases (GPxs), glutathione synthetase (GSS), heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase (NQO1). We therefore examined whether the ROS-scavenging effect of violaxanthin is associated with the upregulation of antioxidant enzyme expression. HaCaT cells were treated with violaxanthin (2, 5, and 10 µM) for 3 h, and the mRNA expression levels of CAT, GPx1, GSS, HO-1, NQO1, and SOD1 were assessed by RT-PCR and RT-qPCR. Violaxanthin increased the mRNA expression of CAT, GSS, HO-1, NQO1, and SOD1 relative to the untreated control (Figure 4A,B). To examine whether violaxanthin still upregulates antioxidant enzyme expression under oxidative stress conditions, RT-PCR and RT-qPCR were performed. Cells were pretreated with violaxanthin, followed by treatment with 500 µM H₂O₂. Even in the presence of H₂O₂, violaxanthin significantly upregulated the mRNA expression of CAT, HO-1, NQO1, and SOD1 (Figure 4C,D). Collectively, these results indicate that violaxanthin reduces intracellular ROS levels by upregulating the mRNA expression of antioxidant enzymes, not only under basal conditions but also under H₂O₂-induced oxidative stress.

3.5. Violaxanthin Promotes the Nuclear Translocation of NRF2 in HaCaT Cells

As shown in Figure 4, violaxanthin increased the mRNA expression of antioxidant enzymes. Previous studies have reported that nuclear factor erythroid 2-related factor 2 (NRF2) is a key transcription factor that regulates cellular antioxidant responses by upregulating the expression of antioxidant enzymes [60,61,62]. To determine whether violaxanthin affects the activation of NRF2, immunoblotting was performed. As shown in Figure 5C, the expression of NRF2 was increased by violaxanthin. This finding raised the possibility that NRF2 activation by violaxanthin may contribute to the observed upregulation of antioxidant enzymes. RT-PCR and RT-qPCR analyses were performed to determine whether the increase in NRF2 protein was attributable to enhanced transcription, but violaxanthin did not significantly alter NRF2 mRNA expression (Figure 5A,B). These results suggest that violaxanthin may regulate NRF2 at a post-translational level rather than enhancing its transcription. As NRF2 requires nuclear translocation for its transcriptional activity, and the expression of its downstream target genes, including CAT, SOD, and GPxs, is regulated by this process [63], we examined whether violaxanthin promotes the nuclear translocation of NRF2. To this end, cytoplasmic and nuclear fractions were separated, and the NRF2 protein levels in each fraction were assessed by immunoblotting (Figure 5D). Treatment with violaxanthin increased nuclear NRF2 levels while decreasing cytoplasmic NRF2 levels. Consequently, these results suggest that violaxanthin may promote antioxidant response by promoting nuclear translocation for the activation of NRF2 rather than by transcriptional expression.

3.6. Violaxanthin Activates NRF2 via the AKT/GSK3β Signaling Pathway in HaCaT Cells

Previous studies have reported that the PI3K/AKT/GSK3β signaling pathway acts as an upstream regulator of NRF2 nuclear translocation [64,65,66]. To examine whether violaxanthin itself affects AKT/GSK3β signaling under basal conditions, HaCaT cells were treated with violaxanthin in the absence of H₂O₂. Violaxanthin did not markedly alter the total protein levels of AKT or GSK3β, but it increased the phosphorylation levels of AKT (Ser473) and GSK3β (Ser9) (Supplementary Figure S1). These results suggest that violaxanthin may activate AKT/GSK3β signaling under basal conditions without changing the total expression levels of AKT and GSK3β. To further investigate whether this pathway is involved in violaxanthin-induced NRF2 nuclear translocation, immunoblotting was performed (Figure 6A). The treatment with H₂O₂ (500 µM), which was performed to induce oxidative stress, decreased the phosphorylation levels of AKT (S473) and GSK3β (S9), whereas pretreatment with violaxanthin restored these levels. To further verify that the AKT/GSK3β pathway mediates the effect of violaxanthin on NRF2 translocation, the cells were pretreated with the PI3K/AKT inhibitor LY294002 prior to violaxanthin and H₂O₂ treatment. The MTT analysis showed that the recovery effect of violaxanthin on H₂O₂-induced cytotoxicity was inhibited by LY294002 (Figure 6B). Furthermore, LY294002 suppressed violaxanthin-induced AKT (Ser473) phosphorylation and NRF2 protein expression (Figure 6C). These data suggest that the violaxanthin-induced translocation of NRF2 may be mediated by the AKT/GSK3β signaling pathway.

3.7. Violaxanthin Promotes Wound Healing in H₂O₂-Treated HaCaT Cells

Accumulating evidence has implied that excessive cellular ROS levels cause impaired function in fibroblasts and keratinocytes by modifying and degrading extracellular matrix proteins or increasing cell death [67,68]. It has been reported that one therapeutic approach to delayed wound-healing is the regulation of excessive ROS [69]. NAC has also been suggested as a potential wound-healing agent by promoting cell proliferation and migration through the antioxidant defense systems [70]. Since 10 μM of violaxanthin demonstrated a comparable ROS-scavenging ability to that of NAC, we hypothesized that violaxanthin would have an effect on H₂O₂-induced wound-healing delay. As shown in Figure 7, the results of the wound healing assay showed that violaxanthin induced faster scratch closure than in H₂O₂ treatment alone at 16 h. Consequently, these data indicated that violaxanthin might have a protective effect on wound healing delayed by ROS.

3.8. Violaxanthin Suppresses H₂O₂-Induced Premature Senescence in nHEK Cells

Extensive studies have reported that intracellular ROS, produced by endogenous and exogenous stressors, induce the senescence of keratinocytes and also delay wound healing [71]. It has been revealed that exposure to low doses of H₂O₂ in keratinocytes leads to premature senescence by decreasing population doublings and increasing SA-β-gal-positive cells [72]. Since HaCaT cells have been reported to not express SA-β-galactosidase [73,74], SA-β-gal staining was performed in nHEK cells to determine whether violaxanthin can inhibit the senescence of keratinocytes induced by H₂O₂. After pretreatment with violaxanthin (5 and 10 µM), SA-β-gal-positive cells were significantly decreased compared to the H₂O₂ treatment alone, suggesting that violaxanthin alleviated cellular senescence under oxidative stress conditions (Figure 8A,B). In addition, the expression of p16, p21, and p53, which were involved in the senescence signaling pathway [75], was analyzed by immunoblotting, as shown in Figure 8C. The expression of p21 and p53 and the phosphorylation of p53 (S15) were increased by H₂O₂, effectively inducing cellular senescence in nHEK cells. Interestingly, violaxanthin (2, 5, and 10 µM) significantly reduced the expression of p21 and p53, and the phosphorylation of p53 (S15) in a dose-dependent manner. These results suggest that violaxanthin can suppress H₂O₂-induced senescence.

4. Discussion

Microalgae are rich sources of physiologically active compounds with diverse biological activities, such as anticancer, antioxidant, and anti-inflammatory properties [32,76,77]. Chlorella, a unicellular green microalga of Chlorophyta [78], is one of the most widely cultivated microalgal species owing to its broad biological activities, rapid growth rate, and resilience to adverse environmental conditions [21]. It has been reported that peptides extracted from Chlorella have protective effects against oxidative stress caused by free radicals [79]. Bae MJ et al. indicated that a hydrothermal extract from Chlorella inhibited histamine release, suggesting its potential as an antiallergic agent [80]. In the present study, the Chlorella sp. HS-V extract significantly recovered the H₂O₂-induced cytotoxicity in a dose-dependent manner (Figure 1). Based on these findings, the Chlorella sp. HS-V extract is suggested to exert antioxidant activity in keratinocytes.

Previous studies have indicated that Chlorella contains numerous active ingredients such as vitamins, lipids, proteins, and carotenoids [31,32]. As shown in Figure 2A, HPLC analysis indicated that a major component of the Chlorella sp. HS-V extract was violaxanthin. Violaxanthin is a carotenoid that can be isolated from microalgae such as Chlorella ellipsoidea and Nannochloropsis oceanica, as well as orange-colored fruits and green vegetables [38,43,44,46]. Violaxanthin has been reported to possess antioxidant, anti-inflammatory, and antiproliferative properties [38,44,45]. Furthermore, violaxanthin has been demonstrated to inhibit UVB-induced cellular senescence in human dermal fibroblasts, highlighting its potential as an anti-photoaging agent [49]. However, the effects of violaxanthin on keratinocytes remain limited. In the present study, we demonstrated for the first time that violaxanthin exerts protective effects against H₂O₂-induced oxidative damage in keratinocytes.

The primary function of the skin is to form an effective barrier between the body and the external environment [81]. ROS are generated in the skin upon exposure to external stressors or as byproducts of intracellular metabolic processes [82]. Excessive ROS not only cause skin aging and inflammation but also inhibit the proliferation of keratinocytes, which act as a primary barrier against the penetration of exogenous chemicals and microorganisms [82]. Furthermore, ROS have been reported to affect various intracellular signaling pathways and molecular processes in keratinocytes [83]. Accumulating evidence suggests that H₂O₂-induced oxidative stress triggers autophagy and apoptosis in keratinocytes [84,85,86]. In order to maintain keratinocyte homeostasis and overall skin integrity, excessive ROS need to be controlled through cellular antioxidant defense systems [83]. Several studies have explored the use of exogenous antioxidants to activate intracellular antioxidant defense systems and protect keratinocytes from oxidative stress [87,88]. Zheng J et al. showed that americanin B protected HaCaT keratinocytes from H₂O₂ by reducing the level of ROS and alleviating DNA damage [87]. Consistent with these findings, both the Chlorella sp. HS-V extract and violaxanthin restored the H₂O₂-induced decrease in cell viability in a dose-dependent manner in the present study (Figure 1 and Figure 2). Moreover, as shown in Figure 3, treatment with 10 µM violaxanthin reduced intracellular ROS levels to a degree comparable to that of NAC. Collectively, these results suggest that violaxanthin reduces intracellular ROS levels and modulates cellular antioxidant defense systems, thereby attenuating H₂O₂-induced cytotoxicity. Additionally, while 10 μM of violaxanthin demonstrated near-complete recovery of cell viability without causing cytotoxicity, the absolute optimal concentration may lie between 10 and 20 μM. Further fine-tuning of the dosage within this narrow window will be necessary to establish the exact therapeutic index for future formulation and clinical applications.

Antioxidant enzymes serve as key components of cellular antioxidant defense systems, playing a central role in maintaining redox homeostasis and protecting against oxidative damage [62]. These enzymes include SOD, CAT, and the GPx family [89]. However, both acute and chronic H₂O₂ exposure have been reported to reduce their activities, thereby inducing oxidative stress [90]. Accordingly, various studies have focused on alleviating oxidative stress by enhancing antioxidant enzyme activity [86,91,92]. As shown in Figure 4, violaxanthin alone increased the expression of CAT, GSS, HO-1, NQO1, and SOD1. Moreover, in H₂O₂-treated cells, violaxanthin significantly upregulated the expression of CAT, NQO1, and SOD1. Interestingly, the regulatory effects of violaxanthin on the mRNA levels of individual antioxidant enzymes showed subtle differences depending on the presence or absence of H₂O₂. Similar differential regulation has been reported in previous studies, and this pattern may reflect the gene-specific and condition-dependent nature of antioxidant defense systems. For example, in normal human dermal fibroblasts, avenanthramide C treatment alone dose-dependently increased HO-1 expression via NRF2 activation, whereas the expression of other antioxidant genes remained unchanged or even decreased. However, under H₂O₂-stimulated conditions, avenanthramide C reduced the H₂O₂-induced increase in antioxidant gene transcripts, including HO-1, GSS, SOD1, GPx1, and CAT [93]. In primary rat hepatocytes, antioxidant enzyme expression also changed differently depending on the culture condition and H₂O₂ exposure; catalase expression decreased during basal culture, whereas H₂O₂ exposure prevented this decrease and induced catalase mRNA expression, indicating that antioxidant enzyme regulation can be strongly influenced by oxidative conditions [94]. Pilat et al. showed that oxidative stress did not uniformly regulate all antioxidant enzymes in ARPE-19 cells, where HO-1 was induced by sublethal H₂O₂ treatment, whereas GPx and catalase showed distinct or limited responses depending on the stress condition [95]. These findings suggest that antioxidant enzymes do not necessarily respond uniformly to antioxidant compounds or oxidative stimuli, but may be selectively regulated according to cellular redox status, stress intensity, and enzyme-specific physiological roles. Thus, the differential responses observed in Figure 4 may represent adaptive fine-tuning of antioxidant defense pathways under basal versus H₂O₂-induced oxidative stress conditions. Further studies examining time-dependent changes in antioxidant enzyme activity, and NRF2 target gene activation would help clarify the gene-specific regulatory effects of violaxanthin.

Many studies have shown that NRF2 regulates ROS homeostasis by modulating the cellular antioxidant defense system through various mechanisms [62,96,97,98]. The NRF2–ARE signaling pathway is a major transcriptional regulatory pathway controlling the expression of antioxidant enzymes [99,100]. NRF2 is known to be regulated by Kelch-like ECH-associated protein 1 (Keap1), as well as by GSK3β-mediated beta-transducin repeat-containing protein (β-TrCP)-dependent degradation [101]. Under normal conditions, Keap1 forms homodimers that bind to NRF2, thereby promoting its cytoplasmic sequestration and ubiquitin-dependent proteasomal degradation [102,103]. In addition to Keap1, NRF2 is also subject to degradation through GSK3β-mediated phosphorylation and β-TrCP-dependent ubiquitin ligase activity, providing an additional layer of regulation [104]. Therefore, the inhibition of GSK3β activity may stabilize NRF2 protein by preventing its β-TrCP-dependent degradation. Under oxidative stress conditions, NRF2 is released from Keap1, translocates to the nucleus, and binds to ARE sequences to drive the transcription of antioxidant enzyme genes [105]. As shown in Figure 5, violaxanthin enhanced NRF2 protein stability and promoted its nuclear translocation under H₂O₂-induced oxidative stress conditions, suggesting that violaxanthin may activate the NRF2–ARE signaling axis to reinforce cellular antioxidant defense systems.

Previous studies have demonstrated that NRF2 can be upregulated by the activation of AKT, as well as by the inactivation of GSK3β [104,106]. It has been well established that PI3K-mediated phosphorylation of AKT at Ser473 leads to the inactivation of GSK3β [107]. Consistent with this, protective effects mediated by PI3K/AKT–NRF2 activation against oxidative stress have been reported in various cell types, including human trabecular meshwork cells, 661W cells, and keratinocytes [108,109,110,111,112]. For instance, fisetin has been shown to activate the PI3K/AKT/NRF2 signaling pathway, thereby inhibiting H₂O₂-induced apoptosis in HaCaT keratinocytes [113]. Based on the observed increase in NRF2 protein expression and nuclear accumulation by violaxanthin, we next investigated the upstream signaling mechanism underlying these effects. Immunoblotting analysis revealed that H₂O₂ treatment significantly reduced the phosphorylation of both AKT (Ser473) and GSK3β (Ser9), whereas pretreatment with violaxanthin dose-dependently restored these phosphorylation levels (Figure 6A). To confirm the involvement of AKT/GSK3β signaling in violaxanthin-mediated NRF2 activation, cells were treated with the PI3K/AKT inhibitor LY294002 prior to violaxanthin treatment. LY294002 reduced the cytoprotective effect of violaxanthin against H₂O₂-induced cytotoxicity (Figure 6B) and suppressed violaxanthin-induced AKT phosphorylation (Figure 6C); however, it only partially inhibited NRF2 protein expression. This partial suppression suggests that while the PI3K/AKT pathway is an important mediator, it may not be solely responsible for NRF2 regulation in this context. Previous studies have established that NRF2 activation is governed by a complex network of upstream kinases; in addition to the PI3K/AKT axis, mitogen-activated protein kinases (MAPKs), particularly p38 and ERK, are known to facilitate NRF2 nuclear translocation and the subsequent induction of antioxidant enzymes in response to various chemicals and oxidative stimuli [114,115]. Therefore, further studies are required to investigate the potential involvement of other upstream signaling pathways, including p38 MAPK or ERK, in violaxanthin-mediated NRF2 regulation. Taken together, these results suggest that violaxanthin activates NRF2 signaling, at least in part, through the AKT/GSK3β pathway, thereby reinforcing cellular antioxidant defense against H₂O₂-induced oxidative stress (Figure 9).

Beyond their role in keratinocyte homeostasis, ROS also play an important role in wound healing [116]. A controlled ROS level is required for the transition from the inflammatory to the proliferation phase [117], but prolonged or excessive ROS exposure can result in tissue damage and delay the wound healing process [118]. Therefore, proper regulation of ROS levels through cellular antioxidant defense systems is essential for normal wound healing [116]. Resveratrol, for example, has been shown to accelerate wound healing by restoring oxidative-stress-impaired cell proliferation and migration [119]. Similarly, in the present study, wound-healing analysis revealed that violaxanthin restored H₂O₂-induced impairment of wound closure in HaCaT cells (Figure 7). This effect is likely attributable, at least in part, to the antioxidant capacity of violaxanthin. Furthermore, the observed changes in AKT/GSK3β and NRF2 signaling may also be associated with the wound-healing-promoting effects of violaxanthin. Collectively, these findings suggest that violaxanthin may serve as a potential candidate for alleviating the oxidative-stress-induced impairment of wound-healing in keratinocytes.

Accumulating evidence indicates that cellular senescence and skin aging are promoted by increased ROS production resulting from harmful environmental factors and chronic ultraviolet (UV) exposure [120,121]. Violaxanthin, which is abundantly found in the microalga Nannochloropsis oceanica, has been shown to inhibit UVB-induced senescence in normal human dermal fibroblasts by suppressing G1 phase arrest, ERK phosphorylation, and the upregulation of p16 and p21 [49]. Based on these findings, we hypothesized that violaxanthin may similarly inhibit H₂O₂-induced senescence in keratinocytes. Cellular senescence is characterized by increased senescence-associated β-galactosidase (SA-β-gal) activity, which is widely used as a marker of senescent cells [51]. Liu L et al. reported that although HaCaT cells may exhibit senescence-associated phenotypes upon H₂O₂ exposure, they do not express SA-β-galactosidase activity [73]. In the present study, nHEK cells were therefore used to evaluate H₂O₂-induced senescence rather than HaCaT cells. As shown in Figure 8, violaxanthin reduced the number of SA-β-gal-positive cells and downregulated the expression of p21 and p53, as well as the phosphorylation of p53 at Ser15, all of which are key mediators of the senescence signaling pathway. These results suggest that violaxanthin may inhibit H₂O₂-induced cellular senescence in nHEK cells primarily through suppression of the p53/p21 signaling pathway. This finding is partially consistent with a previous study showing that violaxanthin suppresses cellular senescence [49]; however, the present study extends these findings to epidermal keratinocytes and provides additional mechanistic and functional evidence under H₂O₂-induced oxidative stress conditions. Moreover, while the previous study mainly focused on ERK phosphorylation, G1 phase arrest, and ECM-related photoaging responses, the present study differs in that it suggests the possible involvement of the AKT/GSK3β–NRF2 axis as an upstream signaling pathway involved in violaxanthin-mediated antioxidant defense, as supported by NRF2 nuclear translocation and antioxidant enzyme induction. Another notable finding is that p16 expression was not significantly altered by violaxanthin treatment under the present experimental conditions. This result does not necessarily exclude the involvement of p16 in cellular senescence, but suggests that p16 may not be the major senescence-associated target regulated by violaxanthin in this model. Consistent with our findings, Ido et al. reported that H₂O₂ treatment in primary human keratinocytes induced p21 expression but not p16 expression, and suggested that low-dose H₂O₂-induced premature senescence was mainly associated with activation of the p53/p21 pathway rather than the p38MAPK/p16 pathway [122]. Similarly, in a stress-induced premature senescence model using human skin fibroblasts, low-dose H₂O₂ exposure did not significantly increase p16 expression compared with untreated control cells, whereas p16 upregulation became more evident under higher-dose or more sustained senescence conditions [123]. Mechanistically, the p53/p21 and p16/RB pathways are two major but partially distinct regulatory axes of senescence-associated cell cycle arrest. The p53/p21 pathway is generally associated with the early DNA damage and oxidative stress response, whereas the p16/RB pathway is more closely related to the maintenance and stabilization of senescence [124]. Taken together, these findings indicate that p16 expression is not always uniformly increased in H₂O₂-induced senescence models and may depend on cell type, stress intensity, and the stage of senescence. Further studies using other senescence induction conditions or additional p16/RB pathway markers, such as phosphorylated RB and CDK4/6 activity, would be needed to clarify whether the p16/RB pathway contributes to the anti-senescent effects of violaxanthin. In addition, we acknowledge that the present study was conducted using 2D monolayer cell culture models, which lack the complex structural organization and multi-cellular interactions of actual human skin. To better understand the physiological relevance and translational potential of violaxanthin as a cosmeceutical or pharmacological agent, future studies should evaluate its protective efficacy and tissue penetration using 3D human skin equivalents or in vivo animal models of oxidative skin damage.

5. Conclusions

In this study, we investigated the protective effects of violaxanthin against H₂O₂-induced oxidative damage in human keratinocytes. Violaxanthin, a major bioactive component in the Chlorella sp. HS-V extract, attenuated H₂O₂-induced cytotoxicity and reduced intracellular ROS levels. Furthermore, it upregulated the mRNA expression of key antioxidant enzymes, including CAT, NQO1 and SOD1, both in the presence and absence of H₂O₂, suggesting its capacity to reinforce cellular antioxidant defense systems. Mechanistically, violaxanthin promoted nuclear translocation of NRF2 via the AKT/GSK3β signaling pathway. In addition, violaxanthin restored H₂O₂-impaired wound healing in HaCaT cells and suppressed oxidative-stress-induced senescence in nHEK cells. In conclusion, violaxanthin may serve as a potential therapeutic agent for alleviating ROS-induced damage in keratinocytes, with its cytoprotective effects mediated, at least in part, through activation of the AKT/GSK3β–NRF2 antioxidant signaling axis.