Heat-Treated Haliotis discus hannai Shell Extract Attenuates Oxidative Stress and Modulates Cutaneous Homeostasis Through Calcium-Associated Keratinocyte Crosstalk: An In Vitro and Exploratory Human Skin Application Study

1. Introduction

Sebum is an essential component of the skin surface lipid barrier and contributes to epidermal hydration, antimicrobial defense, and cutaneous homeostasis. However, excessive or dysregulated sebum production is closely associated with sebum-related skin conditions and altered skin parameters, including oily skin, acne-prone skin, and seborrheic inflammatory responses [1,2]. Sebaceous glands are not merely lipid-producing structures but also actively participate in inflammatory and immune responses through interactions with keratinocytes, immune cells, microbial factors, and environmental stressors [1,3]. Therefore, strategies that simultaneously regulate epidermal barrier stress, sebocyte lipid accumulation, and cutaneous inflammation may provide a rational approach for improving sebum-related skin conditions.

Keratinocytes play a central role in maintaining epidermal barrier integrity and responding to inflammatory or oxidative stress. Barrier-associated proteins such as filaggrin and claudin-1 are important for epidermal differentiation, stratum corneum organization, and tight junction-associated barrier function [4,5]. Under inflammatory stress, impaired keratinocyte barrier function is often accompanied by increased oxidative stress and altered antioxidant defense responses [6,7,8]. The nuclear factor erythroid 2-related factor 2/heme oxygenase-1 axis is one of the major cytoprotective pathways involved in epidermal responses to oxidative stress [6]. Thus, modulation of oxidative stress and antioxidant defense in keratinocytes may be important for maintaining epidermal barrier function and cutaneous homeostasis under sebum-related inflammatory conditions.

Calcium signaling is a key regulator of epidermal homeostasis. The epidermal calcium gradient contributes to keratinocyte differentiation, barrier formation, cell-to-cell adhesion, and permeability barrier recovery [9,10]. Both extracellular calcium influx and intracellular calcium release have been implicated in the regulation of keratinocyte differentiation and epidermal barrier function [11,12]. Calcium-containing biomineral materials may therefore provide a biologically relevant platform for modulating epidermal responses if they can release calcium in a functionally meaningful manner. However, the relationship between calcium release from marine-derived mineral materials and keratinocyte barrier protection has not been fully clarified.

Abalone shell is a marine biomineral mainly composed of calcium carbonate polymorphs, including aragonite and calcite, together with trace minerals and organic matrix components. Thermal treatment can alter mineral phase composition, crystallinity, morphology, and surface-related properties of calcium carbonate-based materials [13,14]. These physicochemical changes may influence calcium release behavior and biological interaction with skin cells. Nevertheless, the potential of heat-treated abalone shell-derived materials as topical bioactive materials capable of modulating oxidative stress, barrier-associated responses, sebocyte lipid metabolism, and inflammatory skin microenvironments remains insufficiently investigated.

Sebocyte lipid production is regulated by multiple metabolic and transcriptional pathways, including sterol regulatory element-binding protein-1 and peroxisome proliferator-activated receptor-γ signaling [15,16]. In addition, lipid droplet-associated proteins and lipolysis-related pathways contribute to the balance between lipid storage and lipid mobilization in sebaceous cells [17]. Keratinocyte-derived soluble factors may also influence sebocyte behavior and macrophage-mediated inflammatory responses within the cutaneous microenvironment. Macrophage polarization is involved in the progression and resolution of inflammatory skin responses, with CD80 and CD86 commonly associated with pro-inflammatory activation and CD206 and interleukin-10 associated with anti-inflammatory phenotypes [18,19]. Therefore, an integrated evaluation of keratinocytes, sebocytes, macrophages, and human skin parameters is required to understand the functional potential of new sebum-regulating materials.

Based on these considerations, we hypothesized that heat-treated abalone shell extract at 300°C (H300) may improve sebum-related skin parameters by attenuating keratinocyte oxidative stress, enhancing antioxidant and barrier-associated responses, and subsequently modulating sebocyte lipid metabolism and macrophage inflammatory responses. The aim of this study was to characterize the physicochemical properties and calcium release profile of H300 and to evaluate its effects on oxidative stress, antioxidant defense, barrier-associated responses, EGTA-sensitive calcium-associated effects, sebocyte lipid metabolism, macrophage inflammatory mediators, and human sebum-related skin parameters.

2. Materials and Methods

2.1. Preparation of Abalone Shell-Derived Samples

Abalone shells were obtained from Wando, Jeollanam-do, Republic of Korea. The shells were washed three times with distilled water to remove residual organic matter and surface impurities and dried at 60°C for 24 h. The dried shells were mechanically ground and sieved to obtain a homogeneous powder. A portion of the powder was retained as the non-heated control material (NH300). For heat-treatment screening, aliquots were processed under air at the indicated temperatures (50°C, 100°C, 200°C, 300°C, and 400°C). The material used for subsequent biological and topical experiments was heated at 300°C for 30 min using an electric oven (KO-A21HL; Kitchenart, Seoul, Republic of Korea) and was designated H300.

2.2. Preparation of Abalone Shell Extracts

NH300 and H300 powders (10 g each) were extracted with 50% ethanol to a final volume of 1 L. The suspensions were sonicated at 40 kHz for 2 h using an SD-D200H sonicator (Seoul, Republic of Korea). The extracts were first filtered through filter paper and concentrated by rotary evaporation, followed by centrifugation at 4,500 rpm. The supernatants were collected and passed through a 0.22 μm filter (Sartorius, Göttingen, Germany) before use in subsequent physicochemical, cellular, and topical experiments.

2.3. X-Ray Diffraction Analysis

X-ray diffraction analysis was performed to evaluate heat-induced changes in the crystalline structure of abalone shell-derived samples. Powdered samples were placed on a glass sample holder and analyzed using an X-ray diffractometer (D8 Advance, Bruker, Billerica, MA, USA) with Cu Kα radiation. Diffraction patterns were recorded over a 2θ range of 20°–60° at a scanning rate of 2°/min. The diffraction peaks corresponding to calcite were analyzed, and the peak intensities at 29.4°, 39.5°, and 43.3° were quantitatively compared among samples treated at different temperatures.

2.4. ICP-OES Analysis

The elemental composition of NH300 and H300 was analyzed using inductively coupled plasma optical emission spectroscopy. Briefly, each powdered sample was digested in nitric acid (HNO3; Sigma-Aldrich, St. Louis, MO, USA) using a microwave digestion system (ETHOS UP, Milestone, Sorisole, Italy). After complete digestion, the solution was diluted with deionized water and filtered through a 0.22 μm syringe filter (Sartorius, Göttingen, Germany). Elemental analysis was performed using an ICP-OES system (Avio 200, PerkinElmer, Waltham, MA, USA). Calcium, magnesium, phosphorus, iron, silicon, strontium, sodium, and potassium were quantified based on standard calibration curves prepared using multi-element standard solutions (Merck, Darmstadt, Germany). The relative elemental composition was expressed as weight percentage.

2.5. UV Diffuse Reflectance Analysis

The UV attenuation properties of NH300 and H300 were evaluated by measuring diffuse reflectance in the UV region. Powdered samples were evenly placed in a quartz sample holder and analyzed using a UV–visible spectrophotometer equipped with an integrating sphere accessory (UV-2600i, Shimadzu, Kyoto, Japan). Diffuse reflectance values were measured at 290, 310, 330, and 360 nm, corresponding to the UVB–UVA wavelength range. The reflectance values of NH300 and H300 were compared to assess changes in UV attenuation capacity after heat treatment.

2.6. Determination of Sun Protection Factor Value

The sun protection factor (SPF) value of NH300 and H300 was determined using an in vitro spectrophotometric method. Each sample was dispersed in ethanol (Daejung Chemicals & Metals, Siheung, Republic of Korea) and sonicated for 10 min using a sonicator (SD-D200H, Seoul, Republic of Korea). The absorbance of each sample suspension was measured from 290 to 320 nm at 5-nm intervals using a UV-visible spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). SPF was calculated using the Mansur equation: SPF = CF × Σ[EE(λ) × I(λ) × Abs(λ)], where CF is the correction factor, EE(λ) is the erythemal effect spectrum, I(λ) is the solar intensity spectrum, and Abs(λ) is the absorbance of the sample at wavelength λ.

2.7. Bright-Field Microscopy

Morphological changes in abalone shell-derived materials before and after heat treatment were observed using bright-field microscopy. NH300 and H300 samples were dispersed in distilled water and briefly sonicated to obtain uniform suspensions. A small aliquot of each suspension was placed on a glass slide, covered with a coverslip, and observed using an optical microscope (Nikon Eclipse TS, Nikon, Tokyo, Japan). Representative images were used to compare particle morphology and crystal-like structures between NH300 and H300.

2.8. Calcium Release Concentration Assay

To evaluate calcium release from coated abalone shell extracts, NH300 and H300 were adjusted to low (1 mg/mL), medium (5 mg/mL), and high (10 mg/mL) sample-equivalent concentrations. Each preparation was added to 96-well plates and dried under sterile conditions to form a uniform coating layer. Distilled water was then added, and the plates were incubated at room temperature for the indicated time points. Released Ca²⁺ was quantified using a Calcium Colorimetric Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Absorbance was measured using an AMR-100 microplate reader (Allsheng Instruments, Hangzhou, China), and Ca²⁺ concentration was calculated from a standard curve.

2.9. Cell Culture

Human HaCaT keratinocytes (CLS Cell Lines Service GmbH, Eppelheim, Germany) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific). Human SZ95 sebocytes (CLS Cell Lines Service GmbH, Eppelheim, Germany) were cultured in DMEM/F12 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin/streptomycin. THP-1 human monocytic cells (TIB-202, ATCC, Manassas, VA, USA) were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ using a HERAcell 150 CO₂ incubator (Thermo Fisher Scientific, Waltham, MA, USA). Cell culture procedures were performed under aseptic conditions. For macrophage differentiation, THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) at 100 nM for 24 h and then rested in PMA-free RPMI-1640 medium for 24 h before subsequent experiments.

2.10. Keratinocyte Stress Model and H300 Treatment

To establish a stress-induced keratinocyte damage model, keratinocytes were exposed to a 1:1 mixture of lipopolysaccharide (LPS; Escherichia coli O111:B4; Sigma-Aldrich, St. Louis, MO, USA) and recombinant human tumor necrosis factor-α (TNF-α; PeproTech, Cranbury, NJ, USA) at a final concentration of 3 ng/mL for 24 h. NH300 or H300 was applied at 1 mg/mL for 24 h, a concentration selected on the basis of preliminary cytotoxicity testing. Cells were treated under the following conditions: control, LPS+TNF-α stress, NH300, H300, LPS+TNF-α plus NH300, and LPS+TNF-α plus H300. After treatment, cells were harvested for flow cytometric analysis of cell survival-, apoptosis-, oxidative stress-, barrier-, and antioxidant defense-associated markers. BCL-2 was used as an anti-apoptotic cell-survival marker, caspase-3 as an apoptotic execution marker, DCF-DA staining as an intracellular reactive oxygen species indicator, monoamine oxidase B as a mitochondrial oxidative stress-associated marker, filaggrin as an epidermal barrier-associated marker, claudin-1 as a tight junction-associated barrier marker, and heme oxygenase-1 as an antioxidant defense-associated marker.

2.11. Flow Cytometric Analysis

Cells used for intracellular protein analysis were washed with phosphate-buffered saline (PBS; Gibco, Thermo Fisher Scientific), fixed, and permeabilized using a fixation/permeabilization solution (BD Cytofix/Cytoperm, BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Cells were then incubated with antibodies against BCL-2 (Cell Signaling Technology, Danvers, MA, USA), caspase-3 (Cell Signaling Technology), monoamine oxidase B (Abcam, Cambridge, UK), filaggrin (Abcam), claudin-1 (Abcam), heme oxygenase-1 (Cell Signaling Technology), protein kinase A (Sigma-Aldrich), peroxisome proliferator-activated receptor-γ (Cell Signaling Technology), and sterol regulatory element-binding protein-1 (Abcam), depending on the experiment. For macrophage polarization analysis, APC-conjugated anti-CD80 antibody (BioLegend, San Diego, CA, USA), FITC-conjugated anti-CD86 antibody (Thermo Fisher Scientific), and PE-conjugated anti-CD206 antibody (Thermo Fisher Scientific) were used. Intracellular reactive oxygen species were measured separately in viable, unfixed cells using 2′,7′-dichlorofluorescin diacetate (DCF-DA; Sigma-Aldrich), and DCF fluorescence was detected in the FITC channel by flow cytometry. Flow cytometric acquisition was performed using a FACSCalibur flow cytometer (BD Biosciences).

2.12. EGTA-Based Calcium Chelation Assay

To evaluate whether the keratinocyte-protective effects of H300 were associated with calcium release, an EGTA-based calcium chelation assay was performed. HaCaT keratinocytes were pretreated with ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA; Sigma-Aldrich) at 1 mM for 30 min and then treated with H300 in the continued presence of EGTA for 24 h. After treatment, cells were harvested and analyzed by flow cytometry to assess apoptosis-, oxidative stress-, barrier-, and antioxidant defense-associated markers, including caspase-3, reactive oxygen species, filaggrin, claudin-1, and heme oxygenase-1.

2.13. Preparation of Keratinocyte-Conditioned Media

Keratinocyte-conditioned media (KCM) were prepared from HaCaT keratinocytes exposed to each experimental condition. Briefly, HaCaT cells were treated for 24 h under the following conditions: control, LPS+TNF-α stress, NH300, H300, LPS+TNF-α plus NH300, and LPS+TNF-α plus H300. After treatment, culture supernatants were collected and centrifuged to remove cellular debris. The resulting conditioned media were designated C_CM, S_CM, NH300_CM, H300_CM, S+NH300_CM, and S+H300_CM, respectively. Based on preliminary viability testing, KCM was applied to sebocytes and THP-1-derived macrophages at a final sample-equivalent concentration of 300 μg/mL for 24 h.

2.14. Sebocyte Lipid Accumulation and Lipid Metabolism Analysis

Sebocytes were treated for 24 h with keratinocyte-conditioned media at a final sample-equivalent concentration of 300 μg/mL. Lipid accumulation was evaluated by Oil Red O staining. Cells were fixed with 4% paraformaldehyde, washed with PBS, and stained with Oil Red O solution (Sigma-Aldrich). Stained lipid droplets were observed under an optical microscope. For lipid metabolism-related signaling analysis, cells were fixed and subjected to immunofluorescence staining for hormone-sensitive lipase, phosphorylated perilipin, and AMP-activated protein kinase. Protein kinase A, peroxisome proliferator-activated receptor-γ, and sterol regulatory element-binding protein-1 expression levels were analyzed by flow cytometry.

2.15. Macrophage Polarization and Inflammatory Mediator Analysis

THP-1-derived macrophages were treated for 24 h with KCM at 300 μg/mL. Macrophage polarization was evaluated by flow cytometric analysis of CD80, CD86, and CD206. Macrophage population shifts based on CD80 and CD86 expression were further visualized using t-distributed stochastic neighbor embedding analysis. Nitric oxide production was measured using Griess reagent. Inducible nitric oxide synthase levels were quantified using a human inducible nitric oxide synthase ELISA kit (Abcam), and interleukin-10 levels were quantified using a human interleukin-10 ELISA kit (R&D Systems).

2.16. Exploratory Human Skin Application Study

A 14-day blinded exploratory human skin application study was conducted in 23 male participants in their twenties with oily or seborrheic skin tendencies. Participants were allocated to the H300 group (n=10), NH300 group (n=10), or placebo reference group (n=3). Participants were withdrawn from the study if they developed an adverse skin reaction after the initial application or used medication for inflammatory skin conditions during the study period. No treatment-related adverse skin reactions were observed during the 14-day application period. H300 and NH300 formulations contained 1 mg/mL of the corresponding abalone shell extract in a vehicle composed of 10% glycerin in PBS; the placebo contained the identical vehicle without abalone shell extract. Identical containers and coded labels were used, and allocation information was withheld from both participants and investigators during the application and assessment period. Participants applied the assigned formulation twice daily after cleansing for 14 days, with a total daily application amount of 1 mL. Skin measurements were obtained at baseline (Day 0), Day 7, and Day 14 after approximately 20 min of acclimatization under controlled room conditions (22 ± 2°C; relative humidity, 40–60%). Facial surface images were acquired from the same region of interest (ROI) using the APM PRO 100 system (Aram Huvis, Republic of Korea) at ×30 magnification and analyzed using ARM PRO v1.1.4 software. Redness-positive area was additionally quantified using ImageJ where indicated. Skin moisture and oil levels were measured using an RBX-916 skin diagnostic device (Ningbo, China); each ROI was measured three times at each visit, and the mean value was used for analysis. Subjective inflammation-related symptoms, sebum-related discomfort, and overall skin satisfaction were assessed using questionnaire items scored on a 5-point Likert scale. The placebo arm was included as an exploratory vehicle reference; because of its small sample size, inferential comparisons in the primary comparative analyses focused on H300 and NH300. The study was reviewed and approved by the Institutional Review Board of Hansung University, Seoul, Republic of Korea (approval number 70094841-202509-HR-001-01), and was conducted in accordance with the Declaration of Helsinki and relevant institutional guidelines. Written informed consent was obtained from all participants before enrollment. Participant anonymity was preserved in the manuscript and figures.

Clinical Trial Registration

The study protocol was reviewed and approved by the Institutional Review Board of Hansung University before participant enrollment, and the human skin application study was completed in 2025. At the time the study was designed and conducted, it was treated as an exploratory cosmetic-material skin application study, and prospective registration in a public clinical trial registry was not undertaken. To ensure transparency and compliance with current publication standards, the study was retrospectively registered with the Clinical Research Information Service (CRIS), Republic of Korea (registration number: KCT0012699; registration date: 30 September 2026).

2.17. Statistical Analysis

Unless otherwise indicated, in vitro experiments were performed with five replicates per condition. In vitro data are presented as mean ± standard deviation, whereas longitudinal human skin measurements are presented as mean ± standard error of the mean where indicated. Statistical analyses were performed using GraphPad Prism. Two-group comparisons were performed using two-sided Student's t-tests, and paired comparisons used paired t-tests. Experiments involving more than two independent groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple-comparisons test. Longitudinal human skin measurements across treatment groups and time points were analyzed using two-way repeated-measures ANOVA followed by Sidak's multiple-comparisons test. Other two-factor designs were analyzed using two-way ANOVA followed by Sidak's multiple-comparisons test. All tests were two-sided, and p < 0.05 was considered statistically significant.

3. Results

3.1. Heat Treatment at 300°C Alters Physicochemical Properties and Enhances Calcium Release from Abalone Shell-Derived Material

The physicochemical changes induced by heat treatment were evaluated using X-ray diffraction, inductively coupled plasma optical emission spectroscopy, ultraviolet diffuse reflectance analysis, sun protection factor measurement, bright-field microscopy, and calcium release concentration assays. X-ray diffraction analysis showed that the intensities of the characteristic calcite-associated peaks at 29.4°, 39.5°, and 43.3° progressively increased with increasing heat-treatment temperature from 50°C to 400°C. A marked increase was observed at 300°C, whereas no significant difference was detected between 300°C and 400°C, suggesting that heat-induced calcite-associated structural reorganization was prominent at 300°C (Figure 1a). Elemental analysis by ICP-OES showed that calcium and magnesium were the major elements detected in both NH300 and H300, together with minor elements such as phosphorus, iron, silicon, strontium, sodium, and potassium (Figure 1b). Quantitative elemental comparison indicated that heat treatment increased calcium and magnesium levels while reducing selected trace elements, suggesting mineral enrichment after thermal processing. H300 showed lower diffuse reflectance than NH300 across the UV region from 290 to 360 nm (Figure 1c). Consistent with this result, H300 exhibited a higher sun protection factor value than NH300, indicating enhanced UV attenuation potential after heat treatment (Figure 1d). Morphological observation further confirmed heat-induced structural changes. NH300 showed elongated needle-like structures and irregular debris, whereas H300 displayed smoother polyhedral crystalline particles (Figure 1e). Calcium release concentration assays showed higher released Ca²⁺ concentration from H300 than from NH300, particularly at 0.5 h (Figure 1f).

3.2. H300 Reduces Oxidative Stress-Associated ROS and MAOB Responses in Stressed Keratinocytes

To evaluate whether H300 protects keratinocytes from stress-induced damage, apoptosis-, survival-, and oxidative stress-associated markers were analyzed by flow cytometry. Under LPS+TNF-α-induced stress conditions, BCL-2 expression was decreased, whereas H300 and S+H300 treatment restored BCL-2 expression (Figure 2a, e). In contrast, caspase-3 expression was increased in the stress-treated group and decreased in the H300 and S+H300 groups (Figure 2b, e). Oxidative stress-associated changes were further examined using DCF-DA-based intracellular ROS analysis and MAOB expression. ROS generation was increased under stress conditions, whereas H300 and S+H300 treatment reduced intracellular ROS levels (Figure 2c, e). MAOB was also increased in the stress-treated group but decreased following H300 and S+H300 treatment (Figure 2d, e). Notably, the S+H300 group showed lower ROS- and MAOB-associated responses than the S+NH300 group. To assess barrier-associated responses, filaggrin expression was analyzed. Filaggrin expression was reduced under stress conditions but increased following H300 and S+H300 treatment (Figure 2f). Claudin-1 and HO-1 were also restored or increased by H300 treatment under stress conditions (Figure 2g).

3.3. Calcium Chelation Partially Attenuates H300-Associated Antioxidant and Barrier Responses

To determine whether the protective effects of H300 were associated with calcium-related responses, oxidative stress-, barrier-, and antioxidant defense-associated markers were evaluated under EGTA-based calcium chelation conditions. Intracellular ROS generation was increased in the S-treated group, whereas H300 and S+H300 treatment reduced ROS levels (Figure 3a). However, ROS generation increased again in the S+H300+EGTA group compared with the S+H300 group, indicating that EGTA-mediated calcium chelation partially attenuated the ROS-reducing effect of H300. Filaggrin expression decreased under stress conditions but increased following H300 and S+H300 treatment, while it was reduced in the S+H300+EGTA group compared with the S+H300 group (Figure 3b). A similar pattern was observed for claudin-1 (Figure 3c). HO-1 expression was also increased by H300 and reduced in the S+H300+EGTA group (Figure 3d). These results suggest that calcium-associated responses contribute, at least in part, to the keratinocyte-protective effects of H300.

3.4. H300-Stimulated Keratinocyte-Conditioned Media Regulate Lipid Accumulation and Lipogenesis-Associated Signaling in Sebocytes

To evaluate whether H300-treated keratinocytes indirectly regulate sebocyte lipid metabolism, sebocytes were treated with KCM. Oil Red O staining showed that intracellular lipid accumulation increased in the S_CM-treated group, whereas H300_CM and S+H300_CM treatment reduced lipid accumulation (Figure 4a). In particular, the S+H300_CM group showed lower lipid accumulation than the S+NH300_CM group. Lipid metabolism-associated responses were further examined by immunofluorescence staining of HSL, p-perilipin, and AMPK. The S_CM-treated group showed reduced fluorescence signals for HSL, p-perilipin, and AMPK, whereas H300_CM and S+H300_CM treatment increased these signals (Figure 4b). PKA expression decreased in the S_CM-treated group, whereas H300_CM and S+H300_CM treatment increased PKA expression (Figure 4c). The expression of PPARγ and SREBP1 was increased in the S_CM-treated group, but reduced by H300_CM and S+H300_CM treatment (Figure 4d).

3.5. H300-Stimulated Keratinocyte-Conditioned Media Modulate Macrophage Inflammatory Responses

To evaluate the indirect regulatory effect of H300-treated keratinocytes on macrophage inflammatory responses, THP-1-derived macrophages were treated with KCM. CD80 and CD86 expression were analyzed as M1-associated polarization markers. The S_CM-treated group showed an increased proportion of CD80-positive cells, whereas H300_CM and S+H300_CM treatment reduced CD80 expression (Figure 5a, b). Macrophage population shifts were further examined by t-SNE analysis based on CD80 and CD86 expression (Figure 5c). CD206 expression was relatively low in the S_CM-treated group but increased following H300_CM and S+H300_CM treatment (Figure 5d). NO and iNOS levels were increased in the S_CM-treated group, whereas H300_CM and S+H300_CM treatment reduced NO and iNOS levels (Figure 5e). In contrast, IL-10 concentration increased following H300_CM and S+H300_CM treatment.

3.6. H300 Application was Associated with Favorable Changes in Redness, Hydration, Oiliness, and Subjective Skin Condition in an Exploratory Human Skin Application Study

A 14-day human skin application study was conducted in 23 participants allocated to H300 (n=10), NH300 (n=10), or placebo (n=3); the primary inferential comparisons shown in Figure 6 focus on the two active groups. Serial facial ROI images showed that the H300-treated group exhibited gradual attenuation of local redness and inflammatory-looking skin features over time (Figure 6a). Close-up skin images showed reduced roughness around pores, local redness, and color irregularity over 14 days in the H300 group (Figure 6b). ImageJ-based quantification showed no significant between-group difference in redness-positive area at baseline, whereas the H300-treated group had significantly lower redness-positive area than the NH300-treated group at Days 7 and 14 (Figure 6c). Skin moisture increased in the H300-treated group at Days 7 and 14 (Figure 6d), whereas skin oil levels decreased over the same period (Figure 6e). Subjective assessments of inflammation-related symptoms, sebum-related discomfort, and overall skin satisfaction also showed favorable changes after H300 application relative to NH300 (Figure 6f).

4. Discussion

This study showed that heat-treated abalone shell extract at 300°C (H300) was associated with favorable changes in sebum-related skin parameters and with calcium-associated keratinocyte protection, modulation of sebocyte lipid metabolism, and altered macrophage inflammatory responses. The major findings were as follows: first, heat treatment at 300°C promoted calcite-associated structural reorganization, mineral enrichment, UV attenuation, and calcium release from abalone shell-derived material. Second, H300 protected keratinocytes against LPS+TNF-α-induced stress by reducing apoptosis- and oxidative stress-associated markers while restoring barrier- and antioxidant defense-associated markers. Third, EGTA chelation partially attenuated the protective effects of H300, supporting the involvement of calcium-associated responses. Fourth, conditioned media from H300-treated keratinocytes reduced sebocyte lipid accumulation and lipogenic signaling while enhancing lipid metabolism-regulatory responses. Finally, H300-derived keratinocyte-conditioned media attenuated macrophage inflammatory responses and increased CD206- and IL-10-associated anti-inflammatory responses, while 14-day H300 application was associated with favorable changes in redness, hydration, oiliness, and subjective skin condition. These findings suggest that H300 may function as a mineral-derived topical material capable of modulating oxidative stress-associated and keratinocyte-centered cutaneous responses.

Sebum-related skin conditions are not limited to excessive lipid production but are closely associated with epidermal barrier impairment, oxidative stress, and inflammatory responses. Sebaceous glands actively participate in skin immune and inflammatory regulation through interactions with keratinocytes, immune cells, microbial factors, and environmental stressors [1,2,3]. Oxidative stress has also been recognized as an important contributor to acne-prone and sebum-related inflammatory skin conditions, linking reactive oxygen species production with inflammatory signaling, lipid oxidation, and sebaceous gland dysfunction [20]. Therefore, an approach for managing oily or seborrheic skin may need to address not only lipid accumulation but also keratinocyte barrier-associated responses and inflammatory microenvironments. In this context, the present study is meaningful because H300 was evaluated across multiple skin-relevant systems, including keratinocytes, sebocytes, macrophages, and human skin parameters, rather than focusing on sebum production alone.

Keratinocytes are central regulators of epidermal barrier integrity and inflammatory responses. Filaggrin and claudin-1 are key barrier-associated proteins involved in epidermal differentiation, stratum corneum organization, and tight junction-associated barrier function [4,5]. In the present study, LPS+TNF-α-induced stress reduced filaggrin and claudin-1 expression, whereas H300 increased the expression of these markers under stress conditions. These findings suggest that H300 may help maintain epidermal barrier-associated responses under inflammatory stress conditions. In addition, H300 reduced ROS and MAOB-associated oxidative stress while increasing HO-1 expression. Because oxidative stress and impaired antioxidant defense are closely related to epidermal barrier dysfunction [6,7,20], the increase in HO-1 expression and reduction in ROS levels by H300 may contribute to improved keratinocyte resilience under stress conditions.

A key finding of this study is the calcium-associated contribution of H300 to keratinocyte protection. Calcium signaling plays an essential role in epidermal homeostasis, including keratinocyte differentiation, cell-to-cell adhesion, and barrier recovery [10,11]. In particular, extracellular calcium and the calcium-sensing receptor are known to regulate keratinocyte differentiation and epidermal barrier function [9,10]. Heat treatment of abalone shell extract increased calcite-associated structural features and enhanced calcium release concentration, suggesting that physicochemical reorganization of the material may influence its biological activity. The EGTA experiment further supported this interpretation. EGTA partially attenuated the H300-mediated reduction in ROS levels and increases in filaggrin, claudin-1, and HO-1 expression, suggesting that calcium-associated responses are involved in H300-mediated keratinocyte protection. However, EGTA did not completely abolish the effects of H300, suggesting that additional factors, such as mineral composition, surface properties, or other ion-related responses, may also contribute to its biological effects.

The physicochemical changes observed after heat treatment may be important for understanding the functional characteristics of H300. Abalone shell is a calcium carbonate-based marine biomineral composed mainly of aragonite and calcite polymorphs, together with trace minerals and organic matrix components. Previous studies have shown that mollusk shell macromolecules can influence calcium carbonate polymorphism and that thermal treatment can alter calcite-related structural properties [13,14]. In this study, H300 showed increased calcite-associated peak intensities, changes in elemental composition, altered particle morphology, and increased calcium release concentration. These findings suggest that heat treatment at 300°C generates a structurally reorganized and calcium-releasing abalone shell-derived material.

Sebocyte lipid metabolism is regulated by multiple transcriptional, enzymatic, and lipid droplet-associated pathways. SREBP1 is a central transcriptional regulator of fatty acid synthesis, while PPARγ contributes to sebocyte differentiation and lipid production [15,16]. In addition, lipid droplet-associated proteins and lipolysis-related pathways contribute to sebaceous lipid accumulation and mobilization [16]. Recent evidence also supports that regulation of SREBP1-associated lipogenic activity can restrict lipid production in human sebocytes [21]. In the present study, S_CM increased lipid accumulation and upregulated PPARγ and SREBP1, whereas H300_CM and S+H300_CM reduced these lipogenic responses. In contrast, H300-derived conditioned media increased PKA, HSL, p-perilipin, and AMPK-associated lipid metabolism-regulatory signals. These results suggest that H300 may indirectly regulate sebocyte lipid accumulation through keratinocyte-mediated soluble factors and may shift sebocytes away from a predominantly lipogenic response.

The use of keratinocyte-conditioned media provides an important conceptual link between epidermal stress responses and sebocyte behavior. Keratinocytes can influence adjacent skin cells through soluble mediators, and sebaceous gland activity is closely integrated with inflammatory and immune responses in the cutaneous microenvironment [1,2,3]. In this study, stressed keratinocyte-conditioned media promoted sebocyte lipid accumulation, whereas conditioned media from H300-treated keratinocytes attenuated this response. These results suggest that H300 may not directly suppress sebocytes only as a material stimulus, but may instead remodel keratinocyte-derived signaling environments that subsequently regulate sebocyte lipid metabolism.

Macrophage polarization is another important component of inflammatory skin responses. CD80 and CD86 are commonly associated with pro-inflammatory macrophage activation, whereas CD206 and IL-10 are associated with anti-inflammatory or tissue-resolving phenotypes [18,19]. Macrophage activation is now understood as a broad and dynamic spectrum rather than a simple binary M1/M2 process [22]. Therefore, marker-based interpretation should be made cautiously and within the context of the experimental stimuli and measured functional responses [23]. In this study, S_CM promoted CD80-associated M1-like activation and increased NO/iNOS production, indicating that stressed keratinocytes can generate a conditioned medium environment that promotes macrophage inflammatory responses. In contrast, H300_CM and S+H300_CM reduced CD80-associated inflammatory polarization and NO/iNOS production while increasing CD206 and IL-10 responses. These findings suggest that H300-treated keratinocytes may indirectly modulate macrophage inflammatory responses and support CD206- and IL-10-associated anti-inflammatory responses within the cutaneous microenvironment.

The exploratory human skin application study further suggests the potential relevance of the in vitro findings. After 14 days of application, H300 reduced redness-positive area and skin oiliness while increasing skin moisture and subjective improvement scores. These changes are consistent with the cellular findings showing keratinocyte protection, increased barrier-associated marker expression, reduced sebocyte lipid accumulation, and modulation of macrophage inflammatory responses. Although the human study was exploratory, the consistency between cellular responses and skin parameter changes supports further investigation of H300 as a topical material for sebum-related and seborrheic skin conditions.

This study has several limitations. First, although EGTA partially attenuated the effects of H300, the precise calcium-associated signaling events were not fully defined. Further studies should evaluate calcium-sensing receptors, calcium channels, and downstream calcium-responsive signaling molecules involved in keratinocyte differentiation and barrier-associated responses [9,10]. Second, the conditioned-media experiments suggest keratinocyte-mediated indirect regulation of sebocytes and macrophages, but the soluble factors responsible for these effects were not identified. Cytokine profiling, proteomic analysis, or metabolomic analysis of conditioned media may help clarify the keratinocyte-derived mediators involved. Third, although PKA–HSL–p-perilipin–AMPK-related changes and PPARγ/SREBP1 regulation were observed, direct validation of these pathways using pharmacological inhibitors or gene silencing is required. Fourth, the human skin application study was exploratory, short in duration, and used an intentionally small placebo reference arm; future studies should use larger, adequately powered, balanced placebo-controlled cohorts, longer follow-up, and standardized instrumental measurements. Finally, the safety, formulation stability, and skin penetration behavior of H300 should be further evaluated before broader topical application.

In conclusion, H300 was associated with favorable changes in sebum-related skin parameters, together with calcium-associated keratinocyte protection, keratinocyte-mediated regulation of sebocyte lipid metabolism, and modulation of macrophage inflammatory responses. The present findings suggest that heat treatment at 300°C produces a calcite-associated, calcium-releasing abalone shell-derived material that may contribute to keratinocyte-centered regulation of cutaneous homeostasis. Therefore, H300 warrants further investigation as a topical material for oily, seborrheic, and inflammation-associated skin conditions.

5. Conclusions

Taken together, H300 was associated with reduced oxidative stress-related responses and improved barrier-associated marker profiles in stressed keratinocytes. Calcium chelation partially attenuated these responses, supporting a calcium-associated component without establishing a specific calcium receptor pathway. Keratinocyte-conditioned medium further modulated sebocyte lipid-associated and macrophage inflammatory responses. The small exploratory human application study provides complementary skin-parameter observations but does not directly establish antioxidant efficacy in vivo. Further mechanistic studies and adequately powered prospective human studies are warranted to confirm these findings.