Cannabis sativa (Hemp) seed-derived peptides WVYY and PSLPA modulate the Nrf2 signaling pathway in human keratinocytes

2.2. Culture of human keratinocyte cells and HSDP treatment

Human HaCaT keratinocytes, sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA), underwent culture in dermal cell basal medium. This medium was enriched with 10% heat-inactivated fetal bovine serum (FBS) from Gibco (Carlsbad, CA, USA) and a penicillin/streptomycin mixture (100 U/mL) from Lonza (Walkersville, MD, USA). Cultivation occurred at 37°C in a humidified atmosphere infused with 5% CO2. Upon achieving 80–90% confluence, the cells were maintained in the same medium. Subsequently, the HaCaT cells were exposed to HSDP at concentrations of 25, 50, 75, and 100 μM for 24 hours. The experimental setup involved a control group treated with distilled water.

2.3. CCK-8 Assay

The study aimed to evaluate how HSDP affected the growth, propagation, and survival of human skin cells (HaCaT) using a CCK-8 assay. HaCaT cells were initially seeded at a density of 5 × 10⁴ cells per well in a 96-well plate and left to incubate for 24 hours. Following this, the cells were exposed to varying concentrations (25, 50, 75, and 100 μM) of HSCE for 24 hours, with sterile water utilized as the control. Post HSCE treatment, 1X CCK-8 solution (Cat. No. CCK-3000, Donginbio, Seoul, Korea) was introduced to each well, and the cells were further incubated for 3 hours. Measurement of wavelength absorbance at 450 nm was carried out using a Thermo Scientific Multiskan GO Microplate Spectrophotometer (Fisher Scientific Ltd., Vantaa, Finland). The percentage of cell viability was computed using the formula: (absorbance of treated cells / Average absorbance of control cells) x 100.

2.4. Wound healing assay

Culture inserts for wound healing assays (CBA-120, Cell Biolabs, USA) were added to each well of a 24-well plate. HaCaT cells were seeded inside the insert at a density of 1×10⁶ cells/well, ensuring 90% confluence, and cultured for 24 hours. Subsequently, the inserts were removed, capturing microscopic images of the cell morphology at 0 hours, followed by treatment with 100 ng/mL epidermal growth factor for both samples and positive control groups. After an additional 18-hour incubation, the media was removed, and 4% paraformaldehyde (PFA) was added, allowing it to react for 15 minutes at room temperature for fixation. Post-fixation, the cells were washed three times with PBS and microscopic images of the cell morphology were captured at 18 hours. The extent of wound healing was quantified by measuring the cell migration area between the images taken at the time of post-insert removal after additional incubation and those taken at 0 hours using the ImageJ software.

2.5. Assessment of UV protection

HaCaT cells are seeded at a density of 5×10⁴ cells/well in a 96-well plate and cultured. After 24 hours, the medium is replaced with serum-free medium for a 4-hour starvation period. Following this, the medium is removed, and the cells are exposed to UVB using the UVP® CL-1000® Ultraviolet Crosslinker equipment at a dose of 5–15 mJ/cm². Subsequently, the cells are treated with HSDP and further incubated for 24 hours. A CCK-8 assay is then performed to assess changes in cell viability.

2.6. 1,1-Diphenyl-2-picryhydrazyl (DPPH) assay

A 0.1 mM DPPH solution of 0.5 mL is mixed with 0.4 mL of ethanol, and the required volume of HCDP is added to achieve the necessary concentration, then supplemented with distilled water to reach a total volume of 1 mL. Positive control groups are also included. After vigorous vortexing for 10 seconds, the mixture is left to react for 30 minutes in the dark at room temperature. The absorbance is measured at 517 nm using a spectrophotometer. The DPPH radical scavenging activity is calculated using the following formula: DPPH Free Radical Inhibition (%) = [1 –(Absorbance of the test sample) / (Average absorbance of the control sample)] × 100

2.7. Recovery assessment against H₂O₂

During cell culturing, experiments were conducted using two sets: one for measuring cell viability and the other for staining with Methylene Blue solution. HaCaT cells were cultured in a 24-well plate, reaching approximately 90% confluence, and left to incubate for 24 hours. Subsequently, the cells were treated with samples and 1mM H₂O₂ for 12–16 hours. A positive control group with 2mM NAC was also included in the treatment. Three replicates were utilized to the CCK-8 assay for assessing cell viability, while the remaining three replicates were used for staining. After removing the medium from the 24-well plate, 500μL of 4% PFA solution per well was added for fixation at room temperature for 20 minutes. Following removal of the 4% PFA solution and PBS washing, the cells were stained using a 0.2% Methylene Blue solution. Post staining, images were captured using a microscope (Motic Asia, Hong Kong).

2.8. Minimum inhibitory concentration test (MIC assay)

For the MIC assay, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli strains were utilized. Bacterial stocks were thawed from -80°C and activated on a liquid medium. The bacterial suspension was adjusted to an OD600 of 0.1, and a maximum of 1% volume was inoculated into 5mL of liquid medium to prevent overgrowth. Two kinds of HCDP, WVYY, and PSLPA were treated respectively, with a positive control using 0.5 mg/mL Ampicillin. Following inoculation, bacterial cultures were incubated for 16 hours. Post-incubation, bacteria were serially diluted (10¹ to 10⁷ dilutions) and mixed in dilution solutions of the microbial medium. Thorough vortexing ensured homogeneous mixing before aliquoting 100μL of appropriately diluted bacterial suspension onto solid agar plates for culturing. Colonies on completed solid agar plates were counted. Colony-forming units (CFUs) were determined by multiplying the colony count with the dilution factor. Colony counts were measured using the Scan 1200 (Interscience, France) equipment, and CFU calculations were performed using the formula: Bacterial count (%) = (Number of colonies in the test sample / Number of colonies in the control sample) x 100.

2.9. Melanin content assay

B16F1 cells, a mouse melanoma cell line, were plated at a density of 1×10⁵ cells per well in 6-well plates and cultured for 24 hours under standard cell culture conditions. Subsequently, the cells were treated with HCDP and continued to be cultured for an additional 72 hours. A positive control containing 100 ppm Kojic acid was included, and each sample received an additional 1 μM α-MSH to stimulate melanin production. Following the incubation period, the upper layer of the medium was removed, and cells were harvested using 1X Trypsin-EDTA. To dissolve melanin, a solution of 1 N NaOH (300 μL) was added to the cell pellet, and the mixture was boiled at 100°C for 30 minutes. The resulting dissolved melanin solution was partially transferred to a 96-well plate for absorbance measurement at 405 nm. The absorbance value for each sample was quantified relative to the protein content, and the melanin production inhibition rate was expressed as a percentage. The calculation for the melanin production inhibition rate is given by the formula: Melanin production inhibition rate (%) = [1 – (test group melanin amount / α-MSH treated control melanin amount)] * 100.

2.10. Measurement of intracellular glutathione (GSH) and reactive oxygen species (ROS) levels

Following HCDP treatment, the cells underwent thorough PBS washing. Next, intracellular ROS and GSH levels in keratinocytes were measured using H2DCFDA (2’, 7’-dichlorodihydrofluorescein diacetate; Invitrogen) and CellTracker Blue (4-chloromethyl- 6.8- difluoro- 7- hydroxycoumarin; CMF2HC; Invitrogen), respectively. For staining, cells were exposed to either 10 μM of CellTracker Blue or 10 μM of H2DCFDA in PBS and left to incubate for 30 minutes at room temperature while avoiding light. After staining, cells were washed again in PBS multiple times. Fluorescence intensities were measured using a fluorescence microscope (BX-53; Olympus, Japan) with UV filters (460 nm for ROS and 370 nm for GSH), followed by image capture. Image J software was utilized for analysis, standardizing the intensities of the control group to 1.

2.11. Detection of lipid peroxidation

Following the administration of HCDP, cells underwent a comprehensive PBS wash and were subsequently exposed to 5 μM of BODIPY™ 581/591 C¹¹ (A12410; Molecular Probes, USA) in PBS for 1 hour at 37°C in a light-avoided environment. Upon staining, cells were once again washed with PBS and mounted on glass slides, covered with coverslips. For image acquisition, a fluorescence microscope (BX-53; Olympus, Japan) equipped with fluorescent filters (591nm for non-peroxidation and 510nm for peroxidation) was utilized. The ImageJ software (version 1.46r; National Institutes of Health, USA) was employed to measure lipid peroxidation intensities. The intensities of the control group were standardized to 1.

2.12. Immunofluorescence staining

Following treatment with HCDP, the cells were thoroughly washed with PBS. Subsequently, the cells were fixed using 4% PFA (w/v) in PBS for 1 hour at room temperature. To permeabilize the cells, 1% Triton X-100 (v/v) in distilled water (DW) was applied for 1 hour at 37°C, followed by four washes in 1% PVA/PBS. To minimize nonspecific binding, the cells were then incubated in 2% BSA in 1% PVA/PBS for 2 hours. The cells were directly exposed to primary antibodies for NRF2 (1:200; PA5–34401; Thermo Fisher Scientific) andHO-1 (1:200; ab13248; Abcam), and incubated overnight at 4°C. After this incubation, the cells underwent multiple washes in 1% PVA/PBS and were then treated with a secondary fluorescein isothiocyanate-conjugated anti-rabbit polyclonal antibody (1:200; ab6717; Abcam, Cambridge, UK) at 37°C for 2 hours while avoiding light. Following the secondary antibody incubation and subsequent washes with 1% PVA/PBS, the cells were immediately counterstained with 5 μg/mL Hoechst-33342 for 8 minutes. Post thorough washing, they were mounted on glass slides, covered with cover slips, and examined under a fluorescence microscope. The evaluation of fluorescence was conducted using ImageJ software (version 1.46r; National Institute of Health, USA).

2.13. Analysis of gene expression by quantitative real-time PCR

The cells treated with HSDP were detached using trypsin and washed with PBS before storage at -80°C until RNA extraction. For this purpose, a minimum of 1 x10⁶ cells per experimental group were processed using the RNeasy Mini kit (#74104; QIAGEN). The mRNA levels were measured with the NanoDrop (DS-11, DeNovix), followed by cDNA synthesis using RT Master Mix (FSQ-201, TOYOBO, Japan) following the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) involved reaction mixtures containing 10 μL THUNDERBIRDTM Next SYBR® qPCR Mix (QPS-201, TOYOBO, Japan), 1 μL (10 pmol/μL) of forward primer, 1 μL (10 pmol/mL) of reverse primer, 7 μL of Nuclease-Free Water, and 1 μL of cDNA in a PCR plate. The amplification was carried out with the Rotor-Gene Q 6plex System (Rotor-Gene Q, Qiagen, Germany) through 40 cycles: denaturation at 95°C for 15 s, annealing at 62°C for 1 min, and extension at 72°C for 1 min. Each plate had at least three replications. The quantification of each target gene expression was standardized against the endogenous control gene (GAPDH) and a list of the primers is shown in S1 Table in S1 File. Relative expressions were calculated using the formula:

3.2. HSDPs WVYY and PSLPA exhibit protective effects against ultraviolet radiation and free radicals

Next, we evaluated the protective abilities of the HSDPs, WVYY and PSLPA, against ultraviolet stimulation. To this end, we used a positive control (PC), i.e., cells that were not subjected to ultraviolet exposure, and a negative control (NC), i.e., cells that were subjected to ultraviolet exposure in the absence of peptide treatment. Interestingly, at 5 or 15 mJ/cm², only treatment with the combination group exhibited significant protection against ultraviolet radiation exposure in fibroblasts, even surpassing the PC (p < 0.0001). On the other hand, all experimental groups significantly protected keratinocytes against ultraviolet radiation exposure, as evident by increased viability compared to that in the NC (p < 0.05; Fig 1D). These findings suggest that the two HSDPs possess ultraviolet protective properties, although further investigation is warranted. Given these findings, the free radical scavenging activity of the two HSDPs was assessed using DPPH, and ascorbic acid (10 ppm) was used as the PC. A significant decrease in free radical activity was observed at several concentrations with both HSDPs compared to that in the control (p < 0.05; Fig 1E). In summary, the two peptides themselves possess defensive properties against ultraviolet radiation and free radicals. Therefore, the final working concentrations of WVYY and PSLPA for subsequent analysis were determined through multiple approaches.

3.3. Assessment of recovery and defense properties of WVYY and PSLPA

Given that we determined the optimal concentrations of the two peptides, we further examined various biological effects including effect on melanin content, antibacterial activity, effect on wound healing, and H₂O₂ defense capacity. No differences were observed in the melanin content in keratinocytes in any of the experimental groups, suggesting that the two peptides did not have any inhibitory effects on melanin synthesis (S1C Fig in S1 File). For the assessment of antibacterial effects, we used three bacterial strains: Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis. MIC assays were performed to assess the antibacterial effects and identify suitable concentrations for bacterial growth inhibition. Initially, liquid bacterial cultures were evaluated to measure the effect of the two HSDPs on colony-forming units by determining the OD 600. A significant reduction in the colony counts for E. coli was noted with 75 μM WVYY and 50 μM PSLPA (p < 0.0001 and p < 0.01, respectively; Fig 2A’). Similarly, a significant reduction was also noted in the colony counts for S. aureus with 75 μM WVYY and 75 μM PSLPA (both p < 0.001; Fig 2B’). However, reduction in S. epidermidis colony counts was not noted in any of the experimental groups (Fig 2C’). Thus, using the optimized concentrations, the antibacterial effect of the two HSDPs was reassessed. Consistently, we found that WVYY and PSLPA inhibited E. coli growth by 29.24% and 14.04% (Fig 2A and 2A’’), respectively. Similarly, WVYY and PSLPA inhibited S. aureus growth by 48.81% and 35.63%, respectively (Fig 2B and 2B’’). Interestingly, and in contrast to E. coli and S. aureus, stimulation of S. epidermidis growth was noted with both WVYY and PSLPA, with inhibition rates of −47.70% and −81.18%, respectively (Fig 2C and 2C’’). Therefore, it has been demonstrated that WVYY and PSLPA exhibit inhibitory functions against harmful bacteria and even contribute to the proliferation of beneficial bacteria, such as Staphylococcus epidermidis.

Next, the effect of the two HSDPs on wound healing was evaluated using keratinocytes. Significant wound healing was noted across all groups, WVYY, PSLPA, and their combination (p < 0.05; Fig 3B). Although a statistical comparison was not performed, when calculating the area at the pixel level, WVYY showed 38.10% compared to T0, PSLPA showed 43.63%, and the combination treatment showed 66.70%. Furthermore, when compared to T18, WVYY showed 20.52%, PSLPA showed 26.05%, and the combination treatment showed 49.12%. Lastly, antioxidant effects of the two HSDPs against H₂O₂ were assessed. A significant defense against free radicals in cell viability for the PSLPA alone (85.3%) and the combination of WVYY and PSLPA (85.85%) significantly increased the viability of H₂O₂-treated cells (p < 0.05; Fig 3C, 3C’ and 3D), suggesting antioxidant effects; however, the same was not observed with WVYY (73.72%). Collectively, these findings suggest that both HSDPs demonstrate antibacterial, wound-healing, and antioxidative effects.

We would like to thank Editage (www.editage.co.kr) for English language editing and Ye-Eun Kim for figure arrangement.