Antioxidant and Antimelanogenic Activities of Lactobacillus kunkeei NCHBL-003 Isolated from Honeybees

Excessive reactive oxygen species production can detrimentally impact skin cell physiology, resulting in cell growth arrest, melanogenesis, and aging. Recent clinical studies have found that lactic acid bacteria have a special effect directly or indirectly on skin organs, but the exact mechanism has not been elucidated. In this study, we investigated the mechanisms underlying the antioxidant protective effect and the inhibitory effect on melanin synthesis of Lactobacillus kunkeei culture supernatant (CSK), isolated from Apis mellifera Linnaeus (the Western honeybee). CSK exhibited notable efficacy in promoting cell migration and wound healing under oxidative stress, surpassing the performance of other strains. CSK pretreatment significantly upregulated the expression of Nrf2/HO-1 (nuclear factor erythroid 2-related factor 2/heme oxygenase-1), a key player in cellular defenses against oxidative stress, relative to the control H2O2-treated cells. The DCF-DA (dichloro-dihydro-fluorescein diacetate) assay results confirmed that CSK’s ability to enhance Nrf2 and HO-1 expression aligns with its robust ability to remove H2O2-induced reactive oxygen species. Furthermore, CSK upregulated MAPK (mitogen-activated protein kinase) phosphorylation, an upstream signal for HO-1 expression, and MAPK inhibitors compromised the wound-healing effect of CSK. Additionally, CSK exhibited inhibitory effects on melanin synthesis, downregulating melanogenesis-related genes in B16F10 cells. Thus, the present study demonstrated that CSK exhibited antioxidant effects by activating the Nrf2/HO-1 pathway through MAPK phosphorylation, thereby restoring cell migration and demonstrating inhibitory effects on melanin production. These findings emphasize the antioxidant and antimelanogenic potential of CSK, suggesting its potential use as a therapeutic agent, promoting wound healing, and as an active ingredient in skin-lightening cosmetics.


Introduction
The skin functions as a primary barrier, protecting the human body against microbes, ultraviolet radiation, and environmental pollutants.Exposure to these stimuli causes damage to keratinocytes, the outermost skin cells, triggering the production of reactive oxygen species (ROS) [1,2].Low-level ROS production is pivotal in numerous physiological activities, serving as an essential component of intercellular signaling pathways, including oxygen metabolism, inflammation, and immune responses [3,4].However, heightened oxidative stress due to excessive ROS production halts the cell cycle.This interruption prevents the migration or proliferation of cells and eventually leads to mechanisms for programmed cell death (i.e., apoptosis) [5][6][7][8][9].In melanocytes, oxidative stress promotes melanin biosynthesis, which is regulated by tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 (TYRP1), and TYRP2 [10,11].These intricate processes collectively contribute to skin aging.
Mitogen-activated protein kinases (MAPKs) are proteins that mediate signal transduction in response to external stimuli, participating in cellular responses, such as cell proliferation, growth, movement, and survival [21].In higher organisms, MAPKs encompass extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 kinase, which are phosphorylated in response to growth factors, oxidative stress, and inflammatory cytokines [22].These phosphorylated MAPKs then phosphorylate target transcription factors.Several studies have demonstrated the involvement of MAPKs in Nrf2/HO-1 signaling [23].Activated MAPKs induce the translocation of Nrf2 into the nucleus, enhancing oxidative stress mitigation [24,25].
Lactic acid bacteria (LAB), Gram-positive bacteria that convert sugar to lactic acid, generate numerous beneficial components [26,27] with anti-inflammatory and antibacterial properties, suppressing the proliferation of harmful bacteria [28].Researchers are increasingly interested in the treatment of skin wounds using LAB as a treatment, with fewer side effects on the skin.Recent studies have shown that Lactobacillus rhamnosus GG and Lactobacillus gasseri, which have been proven to increase gastric barrier repair, accelerate wound healing in primary human keratinocytes [29].Lactobacillus plantarum supernatant (CSP) has been shown to provide 60-100% protection against H 2 O 2 -induced stress in A549 cells; it has also been shown to promote wound healing more quickly than a positive control group in mice [30].For centuries, bee products, such as honey, propolis, royal jelly, and bee pollen, have been employed to enhance human health and address skin issues [31][32][33][34][35] owing to their anti-inflammatory, anticancer, and antioxidant properties.These effects are related to the microbiota in bee habitats and digestive systems.In particular, several studies have highlighted the benefits of honeybee-associated LAB [36][37][38][39][40].While most of these studies have demonstrated the effectiveness of probiotics in protecting hosts [41,42], their ability to safeguard the skin or inhibit melanin synthesis remains unexplored.This study explored the antioxidative and antimelanogenic properties of Lactobacillus kunkeei culture product on skin cells, shedding light on the skin protection potential of honeybee-associated LAB.

Isolation and Identification of Gut Bacteria in Honeybees
The honeybees (Apis mellifera Linnaeus) were collected from three sites in Gwangju Metropolitan City, Republic of Korea.The sampling sites were located on the south side of the city.Twenty-five workers (free-flying bees) were collected from each colony at three different sites.All collections were carried out in June 2022, and samples were stored on ice before bacterial isolation.Fresh honeybee samples were surface-sterilized three times with 7% sodium hypochlorite in sterile plates.The bee midguts were removed with a sterile blade and homogenized in 1 mL of phosphate-buffered saline.The gut homogenate (100 µL) was spread, using the standard dilution method, onto MRS (de Man, Rogosa, and Sharpe) agar medium (Oxoid, Hampshire, UK).The isolates were incubated for 3-4 days, at 37 • C, under anaerobic conditions, using anaerobic jars (BD, Franklin Lakes, NJ, USA).For the acquisition of pure bacterial isolates, 100 colonies with different morphologies were collected and subcultured.Gram staining and biochemical characterizations (catalase, citrate, H 2 S, indole test, methyl red test, oxidase, urease, and Voges-Proskauer test) were conducted as initial screening for Lactobacillus.Gram-positive and catalase-negative bacilli were selected.The isolates were maintained at −80 • C. A DNA extraction kit (QIAGEN, Hilden, Germany) was used to perform a modified DNA extraction protocol.Genomic DNA was prepared from all thirty bacterial isolates.The 16S rDNA gene (1500 bp) was amplified using 27F and 1492R primers (5 ′ -AGAGTTTGATCCTGGCTCAG-3 ′ and 5 ′ -GGTTACCTTGTTACGACTT-3 ′ , respectively), targeting lactobacilli at the genus level.The polymerase chain reaction (PCR) amplifications consisted of initial denaturation at 95 • C for 60 s, followed by 35 cycles of 95 • C for 60 s, 55 • C for 60 s, and 72 • C for 60 s.The PCR fragment was sequenced according to the manufacturer's instructions for the BigDye Terminator Cycle Sequencing Kit, using a DNA sequencer (ABI 3730; Applied Biosystems, Foster City, CA, USA).DNA sequences obtained from the study were submitted to the DDBJ (DNA Data Bank of Japan) database.Species identification was conducted through a homology search of the 16S rRNA gene sequences, using the BLAST database on the NCBI website.Subsequently, the strains were designated as Lactobacillus kunkeei NCHBL-003, Lactobacillus plantarum NCHBL-004, and Lactobacillus reuteri NCHBL-005.

Preparation of the Culture Supernatants of Lactobacillus spp.
Probiotic strains L. kunkeei NCHBL-003, L. plantarum NCHBL-004, and L. reuteri NCHBL-005 were individually isolated from the digestive tracts of honeybees.In our experiments, each strain was used independently, without any combination treatments.For bacterial culture preparations, single colonies were inoculated into 10 mL of MRS broth and grown overnight at 37 • C, with shaking.A 1:10 dilution of the bacterial suspension was cultured, with shaking, until it reached an optical density of 0.6 at 600 nm, corresponding to 1 × 10 9 colony-forming units (CFU) mL −1 .In the preparation of cell-free culture supernatants for the in vitro assays, these Lactobacillus strains were grown in DMEM at 37 • C, with shaking, for 24 h.The culture supernatants were collected via centrifugation at 10,000× g for 10 min, adjusted to pH 7.4, and then sterilized using a syringe filter (0.22 µm).DMEM without bacterial culture served as a negative control.

Wound Healing Assay
HaCaT cells (3 × 10 5 cells/mL/well) were seeded into a 12-well plate (with 2-well silicone inserts) containing DMEM supplemented with 10% (v/v) FBS and 1% (v/v) P/S and incubated at 37 • C for 16 h.Subsequently, the medium was replaced with FBS-free DMEM, and the silicone inserts were removed to create a cell-free gap.Then, the medium was replaced with DMEM containing 1% (v/v) FBS and supplemented with 2.5%, 5%, or 10% (v/v) Lactobacillus spp.culture supernatant.After 1 h, the cells were treated with 500 tµM of H 2 O 2 , and images of the cells were captured after 24 h.The area of the cell-free gap was quantified using ImageJ software.

Melanin Generation
B16F10 cells (1 × 10 5 cells/mL/well) were seeded into a 12-well plate containing DMEM supplemented with 10% (v/v) FBS and 1% (v/v) P/S and cultured at 37 • C for 24 h to allow for attachment and stabilization.Subsequently, the culture medium was replaced with phenol red-free culture medium (Hyclone, Cytiva, Marlborough, MA, USA).Then, the cells were treated with 2.5%, 5%, or 10% CSK or with 2% arbutin.After 1 h, the cells were stimulated with α-melanocyte-stimulating hormone (α-MSH).After 72 h, the melanin levels in the medium were measured at 472 nm, using a microplate reader.

Protein Extraction and Western Blotting
HaCaT cells (1 × 10 5 cells/mL/well) were seeded onto a 12-well plate containing DMEM supplemented with 10% (v/v) FBS and 1% (v/v) P/S and incubated at 37 • C for 16 h.After the treatment for each experiment, the cells were lysed at the indicated time points in a buffer containing 1% Nonidet P-40, 50 mM Tris (pH 7.4), 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na 3 VO 4 , and 0.02% NaN 3 , supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (100×) and 2 mM dithiothreitol.In a parallel experiment, nuclear and cytoplasmic proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, Rockford, IL, USA), following the manufacturer's instructions.Protein samples were separated via 10% SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) and transferred onto nitrocellulose membranes.The membranes were blocked with EveryBlot Blocking Buffer for 1 h at room temperature and probed with primary antibodies against phospho-JNK, phospho-p38, phospho-ERK, Nrf2, HO-1, and β-actin overnight at 4 • C. Subsequently, the membranes were incubated with relevant secondary antibodies for 2 h at room temperature, and the proteins were detected using the enhanced chemiluminescence substrate.

Immunofluorescence Analysis
HaCaT cells (3 × 10 5 cells/mL/well) were seeded into an eight-well chamber containing DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) P/S and incubated at 37 • C for 16 h.After treatment with CSK or H 2 O 2 , the cells were fixed using a 4% formaldehyde solution and permeabilized using 0.2% TritonX-100 (Sigma, St. Louis, MO, USA).Subsequently, the cells were blocked with 3% bovine serum albumin and then incubated with an anti-Nrf2 antibody (1:50 in 3% bovine serum albumin) for 2 h at room temperature.Then, fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (Sigma, St. Louis, MO, USA) was added, and the cells were incubated for 2 h at room temperature.The cells were mounted onto the cover glass, using ProLong Gold Antifade Mountant with DNA Stain DAPI (Thermo Fisher, Waltham, MA, USA), and observed and photographed using a confocal microscope (Leica, Wetzlar, Germany).The exposure time was consistent across samples in all experiments.

Quantitative Real-Time PCR
Total RNA from HaCaT and B16F10 cells was extracted using the TRIzol method.Briefly, cells were lysed using TRIzol reagent and then treated with chloroform to induce phase separation.The aqueous phase containing RNA was collected and precipitated using isopropanol.The RNA pellet was washed with ethanol, air-dried, and then dissolved in RNase-free water.The quality and quantity of the extracted RNA were assessed using a NanoDrop.The RNA was reverse transcribed to cDNA, using the ReverTra Ace qPCR RT Master Mix (TOYOBO BioTechnology, Osaka, Japan), following the manufacturer's instructions.Quantitative real-time (qPCR) was performed using QGreen 2X SYBR Green PCR Master Mix (CellSafe, Yongin, Republic of Korea) and gene-specific primers (Supplementary Tables S1 and S2).

Statistical Analysis
Data were statistically analyzed using Student's t-test.Calculations to determine statistically significant differences between the experimental and control groups were performed using GraphPad Prism, version 5.01 (GraphPad Software, San Diego, CA, USA).Statistical significance was acknowledged when p < 0.05.

CSK Promotes Cell Migration during Oxidative Stress in HaCaT Cells
We isolated L. kunkeei NCHBL-003, L. plantarum NCHBL-004, and L. reuteri NCHBL-005 from honeybee gut microbiota and investigated the potential of their culture supernatants (CSK, CSP, and CSR, respectively) in promoting cell migration during oxidative stress through an in vitro wound healing assay.HaCaT cells with a cell-free gap were pretreated with various concentrations (2.5%, 5%, or 10%) of each culture supernatant for 1 h, and then oxidative stress was induced using H 2 O 2 .While H 2 O 2 -induced oxidative stress hindered cell migration and proliferation, pretreatment with Lactobacillus spp.culture supernatants enhanced wound healing in the oxidative environment (Figure 1A,B).Notably, CSK was associated with the highest recovery of migration ability under oxidative stress.Moreover, cytotoxicity assessments using MTT assays revealed that the Lactobacillus spp.culture supernatants did not have adverse effects on HaCaT cells (Figure 1C).Therefore, subsequent experiments were focused on elucidating the antioxidant mechanism of CSK, which demonstrated the most effective recovery from oxidative stress-induced impaired cell migration among the studied Lactobacillus spp.
1A,B).Notably, CSK was associated with the highest recovery of migration ability under oxidative stress.Moreover, cytotoxicity assessments using MTT assays revealed that the Lactobacillus spp.culture supernatants did not have adverse effects on HaCaT cells (Figure 1C).Therefore, subsequent experiments were focused on elucidating the antioxidant mechanism of CSK, which demonstrated the most effective recovery from oxidative stress-induced impaired cell migration among the studied Lactobacillus spp.

CSK Inhibits ROS Generation by Modulating the Nrf2/HO-1 Pathway
Given that CSK mitigated the deleterious effects of H2O2 on wound healing, we investigated whether CSK is involved in inhibiting H2O2-induced ROS production.To de-

CSK Inhibits ROS Generation by Modulating the Nrf2/HO-1 Pathway
Given that CSK mitigated the deleterious effects of H 2 O 2 on wound healing, we investigated whether CSK is involved in inhibiting H 2 O 2 -induced ROS production.To determine the protective effects of CSK against H 2 O 2 -induced oxidative stress, we measured intracellular ROS generation in HaCaT cells, using the DCF-DA assay.The green fluorescence signals of DCF were stronger in H 2 O 2 -treated cells than in untreated cells, indicating that H 2 O 2 induced abundant ROS production in HaCaT cells.However, pretreatment with 2.5%, 5%, and 10% CSK was associated with significant attenuation of the increase in DCF fluorescence intensity (Figure 2A,B), demonstrating CSK's antioxidant capacity.Generally, Nrf2/HO-1 activity increases under H 2 O 2 -induced oxidative stress due to its role as an antioxidant [43].To confirm that CSK protected cells from oxidative stress through Nrf2/HO-1 activity, we induced oxidative stress in CSK-treated HaCaT cells, using H 2 O 2 , and assessed the expression of Nrf2 and HO-1, using Western blot analyses and qPCR.H 2 O 2 treatment increased the mRNA and protein levels of Nrf2 and HO-1 in HaCaT cells, indicating that oxidative stress activated the Nrf2/HO-1 pathway (Figure 2C,D).The expression of Nrf2 and HO-1 was significantly higher in the presence of CSK than in the sole presence of H 2 O 2 .Furthermore, CSK treatment augmented Nrf2 expression within the nucleus (Figure 2C,E).Thus, CSK amplifies the expression of Nrf2 and HO-1 during oxidative stress, leading to the activation and translocation of Nrf2 from the cytoplasm to the nucleus.These results indicate that CSK exerts its antioxidative effects, at least in part, by activating the Nrf2/HO-1 signaling pathway, amplifying the cellular defense mechanisms against oxidative damage.crease in DCF fluorescence intensity (Figure 2A,B), demonstrating CSK's antioxidant capacity.Generally, Nrf2/HO-1 activity increases under H2O2-induced oxidative stress due to its role as an antioxidant [43].To confirm that CSK protected cells from oxidative stress through Nrf2/HO-1 activity, we induced oxidative stress in CSK-treated HaCaT cells, using H2O2, and assessed the expression of Nrf2 and HO-1, using Western blot analyses and qPCR.H2O2 treatment increased the mRNA and protein levels of Nrf2 and HO-1 in HaCaT cells, indicating that oxidative stress activated the Nrf2/HO-1 pathway (Figure 2C,D).The expression of Nrf2 and HO-1 was significantly higher in the presence of CSK than in the sole presence of H2O2.Furthermore, CSK treatment augmented Nrf2 expression within the nucleus (Figure 2C,E).Thus, CSK amplifies the expression of Nrf2 and HO-1 during oxidative stress, leading to the activation and translocation of Nrf2 from the cytoplasm to the nucleus.These results indicate that CSK exerts its antioxidative effects, at least in part, by activating the Nrf2/HO-1 signaling pathway, amplifying the cellular defense mechanisms against oxidative damage.

CSK Induces Nrf2/HO-1 through MAPK Phosphorylation in HaCaT Cells
Previous studies have reported that MAPKs mediate the activity of antioxidant-related factors, such as Nrf2 and HO-1, in various cell types [24,25].Therefore, we investigated the potential of CSK to activate MAPKs.HaCaT cells were subjected to treatment with CSK for specified time points (0, 0.5, 1, 3, and 6 h) to assess the dynamic changes in MAPK activation.CSK treatment induced the strong phosphorylation of JNK, p38, and ERK at 0.5 h after treatment (Figure 3A).The protein expression of Nrf2 and HO-1 increased strongly after 3 h (Figure 3A).Furthermore, the Nrf2 and HO-1 genes showed a strong increase after 1 h and 6 h of CSK treatment, respectively (Figure 3B).Considering that CSK induced the activity of MAPKs at an earlier time point than Nrf2/HO-1, CSK could be expected to regulate Nrf2/HO-1 by activating MAPKs in HaCaT cells.To further verify whether CSK-induced Nrf2/HO-1 expression is related to MAPKs, HaCaT cells were treated with SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor), followed by CSK treatment.The CSK-induced expression of Nrf2 and HO-1 was inhibited by MAPK inhibitors (Figure 3C).Moreover, the addition of inhibitors significantly reduced the expression of Nrf2 and HO-1 mRNA relative to that observed in groups without inhibitors (CSK alone) (Figure 3D).These results indicate that CSK upregulates Nrf2 and HO-1 expression by activating MAPKs.

CSK Promotes Cell Migration through MAPKs during Oxidative Stress in HaCaT Cells
CSK increased the expression of the antioxidant factors Nrf2 and HO-1 through MAPKs; therefore, we further investigated whether CSK promoted cell migration through MAPKs during oxidative stress.HaCaT cells with a cell-free gap were pretreated with SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), and PD98059 (ERK inhibitor) and then treated with CSK.Subsequently, the cells were treated with H 2 O 2 to induce oxidative stress for 24 h, and the cell-free gap was observed under a microscope.CSK restored cell migration impaired by oxidative stress, and MAPK inhibitors reversed the effect of CSK during oxidative stress (Figure 4A,B).These results indicate that MAPKs mediate the protective effect of CSK against H 2 O 2 -induced impairments in cell migration in HaCaT cells.

CSK Inhibits Melanin Synthesis by Modulating the Expression of Melanogenic Genes in B16F10 Cells
In the skin, oxidative stress leads to cellular damage and promotes melanin biosynthesis [44].The inhibitory effects of CSK on melanin synthesis were investigated via the treatment of B16F10 cells with CSK at concentrations of 2.5%, 5%, and 10%.First, no CSKinduced cytotoxicity was observed (Figure 5A).When B16F10 cells were stimulated with α-MSH to enhance melanin synthesis, CSK treatment resulted in a concentration-dependent reduction in melanin production (Figure 5B).In particular, the 10% CSK treatment exhibited stronger inhibition of melanin synthesis relative to the positive control group treated with arbutin.Additionally, CSK treatment decreased the mRNA expression levels of TYR, a protein involved in melanin precursor production, and melanogenesis-related genes (Mitf, Tyrp1, and Tyrp2) (Figure 5C).The suppression of mRNA expression by CSK was significant and comparable to the effect of arbutin treatment.These results suggest that CSK not only reduces oxidative stress in the skin but also prevents melanin production by suppressing the expression of melanogenesis-related genes.
oxidative stress for 24 h, and the cell-free gap was observed under a microscope.CSK restored cell migration impaired by oxidative stress, and MAPK inhibitors reversed the effect of CSK during oxidative stress (Figure 4A,B).These results indicate that MAPKs mediate the protective effect of CSK against H2O2-induced impairments in cell migration in HaCaT cells.The wound area was photographed at 0 h and 24 h after treatment; the cells were first pretreated with inhibitors for 1 h, then treated with CSK (10%) for 1 h, and finally treated with H2O2 (500 µM) for 24 h.Scale bar: 200 µm.(B) Wound areas were quantified using ImageJ and expressed as the percentage difference before and after H2O2 treatment.Data are presented as mean ± SD. ### p < 0.001, relative to untreated cells.*** p < 0.001, relative to untreated or H2O2-treated cells.

CSK Inhibits Melanin Synthesis by Modulating the Expression of Melanogenic Genes in B16F10 Cells
In the skin, oxidative stress leads to cellular damage and promotes melanin biosynthesis [44].The inhibitory effects of CSK on melanin synthesis were investigated via the treatment of B16F10 cells with CSK at concentrations of 2.5%, 5%, and 10%.First, no CSK-induced cytotoxicity was observed (Figure 5A).When B16F10 cells were stimulated with α-MSH to enhance melanin synthesis, CSK treatment resulted in a concentration-dependent reduction in melanin production (Figure 5B).In particular, the 10% CSK treatment exhibited stronger inhibition of melanin synthesis relative to the positive control group treated with arbutin.Additionally, CSK treatment decreased the mRNA expression levels of TYR, a protein involved in melanin precursor production, and melanogenesis-related genes (Mitf, Tyrp1, and Tyrp2) (Figure 5C).The suppression of mRNA expression by CSK was significant and comparable to the effect of arbutin treatment.These results suggest that CSK not only reduces oxidative stress in the skin but also prevents melanin production by suppressing the expression of melanogenesis-related genes.

Discussion
The numerous health benefits of probiotics are well recognized, with recent studies particularly emphasizing their positive effects on skin health, such as antioxidant, antipigmentation, and anti-wrinkle properties [45][46][47].However, the specific mechanisms underlying these beneficial effects on the skin remain poorly understood.The primary objective of our study was to explore the antioxidant and antimelanogenic properties of

Discussion
The numerous health benefits of probiotics are well recognized, with recent studies particularly emphasizing their positive effects on skin health, such as antioxidant, antipigmentation, and anti-wrinkle properties [45][46][47].However, the specific mechanisms underlying these beneficial effects on the skin remain poorly understood.The primary objective of our study was to explore the antioxidant and antimelanogenic properties of L. kunkeei cultures isolated from the honeybee gut.Our focus was on elucidating the mechanism of action of CSK, particularly its impact on the Nrf2/HO-1 activation pathway through MAPK, aiming to understand its role in wound healing via the inhibition of oxidative stress.Additionally, we examined CSK's effect on α-MSH-induced melanin synthesis inhibition in melanocytes.
Honeybee products have a long history of use in traditional remedies, and there is increasing scientific interest in the LAB they contain.LAB derived from honeybees exhibit diversity due to environmental factors and individual bee characteristics, with Lactobacillus spp.being a predominant genus in the honeybee gut microbiota [48,49].Consistent with previous reports, we isolated and identified three honeybee-derived LAB, namely L. kunkeei NCHBL-003, L. plantarum NCHBL-004, and L. reuteri NCHBL-005.In this study, Lactobacillus culture supernatants (CSK, CSP, and CSR) were utilized, representing a form of postbiotics derived from probiotic fermentation [50].Postbiotics have gained considerable interest due to their demonstrated efficacy, which is comparable to that of probiotic bacteria, making them increasingly relevant in academic research [51].Recent studies have explored how to enhance skin repair by using not only live probiotics but also postbiotics, including heat-killed bacteria and their metabolites.It was found that the fermented supernatant of L. kefiri can postpone cellular senescence by reducing oxidative stress and the expression of aging-related genes in human skin cells affected by H 2 O 2 [52].Furthermore, the culture supernatant of L. gasseri BNR17 has been shown to inhibit oxidative stress and hyperpigmentation in skin cells [53].Oxidative stress, induced by free radicals, is intricately linked to age-related phenomena, including skin wrinkles.Treatment with H 2 O 2 in cellular models leads to an excess of ROS, disrupting the oxidative balance [54], which is useful for establishing an oxidative stress model to investigate cellular responses in an environment that simulates oxidative stress [55].Such models are crucial for studying aging-related changes, particularly in skin aging and wrinkle formation.
In this study, we confirmed that pretreatment with CSR and CSK inhibited H 2 O 2induced cell migration impairment without causing cytotoxicity.Specifically, CSK reduced the wound area by more than 90%, even at low concentrations.Furthermore, DCF staining confirmed CSK's effective inhibition of ROS production, which facilitated the recovery of cell migration disrupted by H 2 O 2 .We then proceeded to examine the potential pathways through which CSK exerts its antioxidant action in HaCaT cells.
Numerous studies have established the role of Nrf2/HO-1 signaling in HaCaT cells for cellular protection against oxidative stress, suggesting its relevance in treating various diseases [56][57][58].To investigate the cytoprotective mechanism of CSK against oxidative stress, we examined its association with Nrf2/HO-1 activation.Remarkably, the H 2 O 2 -induced protein and gene expression of Nrf2 and HO-1 increased significantly following CSK pretreatment.Interestingly, HO-1 expression was more pronounced in the presence of H 2 O 2 than in the presence of CSK alone.This suggests that CSK further enhances HO-1 activity facilitated by Nrf2 under oxidative stress conditions.In various cell types, the expression of antioxidant enzymes, including Nrf2 and HO-1, is regulated by MAPKs [20,59,60].In the present study, CSK pretreatment rapidly increased JNK, p38, and ERK phosphorylation and was followed by an upregulation of Nrf2 and HO-1.This consistent pattern suggests that CSK activates MAPK pathways, contributing to enhanced Nrf2 and HO-1 expression in response to oxidative stress.Moreover, MAPK inhibitors reversed the CSK-induced upregulated phosphorylation of JNK, p38, and ERK, attenuating Nrf2/HO-1 expression.Additionally, our findings demonstrated that CSK treatment facilitated the recovery of cell migration impaired by oxidative stress.However, this beneficial effect was reversed when MAPK inhibitors were introduced.These results underscore the crucial role of MAPK signaling in the cytoprotective actions of CSK, emphasizing its potential as a therapeutic target to mitigate oxidative stress-induced cell damage.
Our study demonstrated that CSK effectively reduced α-MSH-induced melanogenesis in melanocytes without causing cytotoxicity, exhibiting efficacy comparable to arbutin-a recognized skin-lightening agent.Ultraviolet-stimulated ROS in skin cells stimulate α-MSH through MC1R, inducing melanin synthesis [61].Our results showed the increased melanin content of melanocytes following α-MSH treatment.Melanin synthesis in melanocytes primarily involves three key enzymes: TYR, TYRP1, and TYRP2.TYR initiates melanogenesis by converting tyrosine to dopaquinone, while TYRP1 contributes to the later stages, including DHICA (5,6-dihydroxyindole-2-carboxylic acid) formation.TYRP2 also influences melanogenesis.Thus, all three of these enzymes play central roles in regulating skin pigmentation [62].Additionally, MITF, a nuclear transcription factor, regulates both gene expression and pigment production in melanocytes [63], akin to TYR, TYRP1, and TYRP2.Melanin overproduction in melanocytes contributes to various skin conditions, necessitating the exploration of effective agents to regulate pigmentation [64].Arbutin, a cosmetic ingredient, lightens skin by inhibiting TYR via its conversion to hydroquinone, a compound known for its depigmenting effects [65].Consistent with previous studies, our findings show that arbutin treatment reduced melanin secretion and the mRNA expression of tyr, mitf, tyrp1, and tyrp2 in the presence of α-MSH, a major stimulator of melanin biosynthesis in melanocytes.Furthermore, CSK treatment yielded a similar effect to arbutin, reducing melanin secretion and the mRNA expression of tyr, mitf, tyrp1, and tyrp2 in a concentration-dependent manner.
While our study successfully validated the antioxidant and antimelanogenic effects of the investigated probiotic culture media, we did not conduct a comprehensive analysis of the individual components directly responsible for these observed effects.The primary focus of our investigation was assessing the overall efficacy of probiotic culture media rather than identifying specific bioactive compounds.Therefore, while our results provide robust evidence for the beneficial effects of the investigated culture media, further studies are warranted to elucidate the precise molecular constituents contributing to the observed antioxidant and antimelanogenic properties.To address these limitations in our future experiments, we will focus on unveiling the precise mechanisms underlying the CSK effect.This will involve employing techniques such as amino acid analysis and high-performance liquid chromatography analysis of culture media.

Conclusions
Our study highlighted the multifaceted benefits of CSK in promoting skin health through its antioxidant and melanogenesis regulation activities.The observed activation of the Nrf2/HO-1 pathway via MAPKs underscores the cytoprotective mechanisms of CSK against oxidative stress.Notably, CSK exhibits an antipigmentation effect by suppressing genes related to melanin production in melanocytes.These findings strongly suggest the potential of CSK as a novel therapeutic agent in skincare, offering a dual approach to managing oxidative stress and regulating melanin.In the future, our research will focus on identifying the active components of CSK and investigating its antioxidant, antimelanogenic, and anti-wrinkle and moisturizing effects.Such findings will contribute to solidifying the role of CSK in skincare formulations.

Figure 1 .
Figure 1.Wound−healing effects of Lactobacillus spp.culture supernatant in H2O2−treated HaCaT cells.(A) After a cell−free gap (wound area) was formed, cells were treated with Lactobacillus spp.culture supernatant at the indicated concentrations.After 1 h, cells were treated with H2O2 (500 µM).The wound margin was photographed 24 h after treatment.Scale bar: 200 µm.(B) Wound areas were quantified using ImageJ and expressed as the percentage difference between the H2O2−treated group and the supernatant-treated group.(C) HaCaT cells were treated with Lactobacillus spp.culture supernatant at the indicated concentrations for 24 h, and cell viability was determined using the MTT assay.Data are presented as the mean ± standard deviation (SD).*** p < 0.001, relative to H2O2-treated cells.ns, not significant; CSP, culture supernatant of Lactobacillus plantarum; CSR, culture supernatant of Lactobacillus reuteri; CSK, culture supernatant of Lactobacillus kunkeei.

Figure 1 .
Figure 1.Wound-healing effects of Lactobacillus spp.culture supernatant in H 2 O 2 -treated HaCaT cells.(A) After a cell-free gap (wound area) was formed, cells were treated with Lactobacillus spp.culture supernatant at the indicated concentrations.After 1 h, cells were treated with H 2 O 2 (500 µM).The wound margin was photographed 24 h after treatment.Scale bar: 200 µm.(B) Wound areas were quantified using ImageJ and expressed as the percentage difference between the H 2 O 2 -treated group and the supernatant-treated group.(C) HaCaT cells were treated with Lactobacillus spp.culture supernatant at the indicated concentrations for 24 h, and cell viability was determined using the MTT assay.Data are presented as the mean ± standard deviation (SD).*** p < 0.001, relative to H 2 O 2 -treated cells.ns, not significant; CSP, culture supernatant of Lactobacillus plantarum; CSR, culture supernatant of Lactobacillus reuteri; CSK, culture supernatant of Lactobacillus kunkeei.

Microorganisms 2024 , 16 Figure 2 .Figure 2 .
Figure 2. Oxidative stress attenuation by the culture supernatant of Lactobacillus kunkeei (CSK) in H2O2−treated HaCaT cells.(A) HaCaT cells were pretreated with CSK for 1 h and then treated with H2O2 (500 µM) for 3 h.Subsequently, ROS generation was assessed using the dichloro−dihydro−fluorescein diacetate (DCF−DA) assay.Scale bar: 100 µm.(B) The fluorescence intensity of DCF was quantified using ImageJ, and data are presented as levels relative to the fluorescence of the control (cells without H2O2 treatment).(C-E) HaCaT cells were pretreated with CSK for 1 h and then treated with H2O2 (500 µM) for 6 h.(C) The cytoplasmic, nuclear, and total protein Nrf2 expression and HO-1 total protein expression were examined using Western blotting, and (D)Figure 2. Oxidative stress attenuation by the culture supernatant of Lactobacillus kunkeei (CSK) in H 2 O 2 -treated HaCaT cells.(A) HaCaT cells were pretreated with CSK for 1 h and then treated with

Figure 4 .
Figure 4. MAPK activation−based wound-healing effect of CSK in H2O2−treated HaCaT cells.(A)The wound area was photographed at 0 h and 24 h after treatment; the cells were first pretreated with inhibitors for 1 h, then treated with CSK (10%) for 1 h, and finally treated with H2O2 (500 µM) for 24 h.Scale bar: 200 µm.(B) Wound areas were quantified using ImageJ and expressed as the percentage difference before and after H2O2 treatment.Data are presented as mean ± SD. ### p < 0.001, relative to untreated cells.*** p < 0.001, relative to untreated or H2O2-treated cells.

Figure 4 . 16 Figure 5 .
Figure 4. MAPK activation-based wound-healing effect of CSK in H 2 O 2 -treated HaCaT cells.(A) The wound area was photographed at 0 h and 24 h after treatment; the cells were first pretreated with inhibitors for 1 h, then treated with CSK (10%) for 1 h, and finally treated with H 2 O 2 (500 µM) for 24 h.Scale bar: 200 µm.(B) Wound areas were quantified using ImageJ and expressed as the percentage difference before and after H 2 O 2 treatment.Data are presented as mean ± SD. ### p < 0.001, relative to untreated cells.*** p < 0.001, relative to untreated or H 2 O 2 -treated cells.Microorganisms 2024, 12, x FOR PEER REVIEW 11 of 16

Figure 5 .
Figure 5.The effects of CSK on melanogenesis in α-melanocyte stimulating hormone (α-MSH)stimulated B16F10 cells.(A) B16F10 cells were treated with arbutin and CSK at the indicated concentrations for 24 h, and cell viability was determined using the MTT assay.(B) Antimelanogenic activity was evaluated by quantifying the amount of melanin synthesized in the B16F10 cells.After CSK treatment for 1 h, α-MSH (100 nM) was added to stimulate melanin synthesis.After 72 h, melanin content was measured at 472 nm, using a spectrophotometer.Arbutin (2%) was used as a positive control.(C) Melanogenesis-related gene expression was measured after 1 h of CSK treatment, followed by 48 h of α-MSH (100 nM) treatment, using real-time PCR.Data are presented as the mean ± SD. # p < 0.05, ## p < 0.01, and ### p < 0.001, relative to untreated cells.* p < 0.05, ** p < 0.01, and *** p < 0.001, relative to α-MSH-treated cells.