Fucoxanthin-Containing Cream Prevents Epidermal Hyperplasia and UVB-Induced Skin Erythema in Mice

2.1. Effect of FX on Cell Viability

The effect of FX on THP-1 macrophages and HaCaT cells viability was measured by using the sulforhodamine B (SRB) assay. Results from cytotoxicity study showed that none of the tested concentrations affected cell viability. The inhibitory concentration 50 (IC₅₀) (half maximal inhibitory concentration) was above 100 µM at 24 h after treatment (Table S1).

2.2. Effects of FX on TNF-α Production in LPS-Stimulated THP-1 Macrophages and IL-6 and IL-8 Production in TNF-α-Stimulated HaCaT Human Keratinocytes

Non-cytotoxic concentrations of FX were used to determinate its effect on pro-inflammatory cytokines in both THP-1 macrophages and human keratinocytes. As shown in Figure 1, all concentrations tested significantly reduced the production of inflammatory cytokines in both cell models. Interestingly, the pre-treatment with the carotenoid at 50 µM showed a significant reduction of tumor necrosis factor alpha (TNF-α) production in lipopolysaccharide (LPS)-stimulated THP-1 macrophages, reaching similar values to dexamethasone (Dex) (p < 0.001) (Figure 1A). In relation to interleukin (IL)-6 and IL-8 production in HaCaT keratinocytes, similar results were exhibited in cells pretreated with the highest dose of FX (p < 0.001) (Figure 1B,C).

2.3. In Vitro Permeation Studies of BC from Different Topical Formulations

The objective of this study was to evaluate the in vitro permeation through artificial membranes of β-carotene (BC) from different topical formulations. BC was selected because of its structural similarity with FX. The formulations tested were: ethanolic solution as control, hydrogel, cream and ointment. BC was detected in the receiver medium in a time-dependent manner and profiles of cumulative amount of drug permeated have been obtained (Figure 2).

The permeation profiles showed the cream as the most favourable vehicle as penetration enhancer. When compared with the ethanolic control solution, a high percentage of BC permeated was obtained at a flux very similar to the control solution, as reported in Table 1. However, the ointment and hydrogel preparations made it difficult the pass through the membrane, probably due to their lipophilic and hydrophilic nature, respectively. This was visualized in terms of lower permeation percentages and flux rates. Hydrophilic cream provided the highest release of BC in comparison with lipophilic ointment or hydrogels, as previously reported for flavonoids [21]. Therefore, the hydrophilic cream (oil-in-water (OW) emulsion base) was chosen for incorporating FX and Dex for further studies. As these emulsions have hydrophilic external phase, they are miscible with water and skin secretions, thus they are not occlusive and are easily removed from skin.

2.4. Ex Vivo Permeation Studies of FX-Containing Cream

Once the cream was selected as the vehicle for drug formulation, FX-loaded cream was prepared following the same methodology that for BC. In addition, Dex-loaded cream was formulated as positive control for the in vivo assay. The permeation profiles of FX contained in the hydrophilic cream were evaluated with the aim of analyzing the permeation behavior in the mice skin compared to an ethanolic control solution and Dex-cream.

Results showed a clear improvement of FX permeation with time (Figure 2). As evidenced from the area under the drug permeation curve (AUC) calculated (48,610, 19,914 and 8671%/h for FX-cream, FX control and Dex-cream, respectively), the cream vehicle offered a higher amount of FX permeated with time, whereas the control solution of this molecule showed a lower percentage. Although the ethanol acts as permeation enhancer, the cream composition, rich in surfactants, made the formulation improve the amount of FX to cross the membrane. Surprisingly, the permeation profile of Dex-cream showed 15%, approximately, of drug permeation. This lack of diffusion across the membrane could be attributed to a lower partition coefficient than FX (1.83 vs. 14.76). Dex has a lower molecular weight than FX, but the lipophilic nature of the carotenoid makes it a better candidate to interact with the surfactant components of the cream, which aids the solubilized molecule to reach the receiver medium.

2.5. Topical Application of FX-Containing Cream Decreases Skin Inflammation and Hyperplasia on the Murine TPA-Induced Model

To analyze whether topical pre-treatment with FX could reduce the in vivo inflammation, we studied this carotenoid on the murine TPA-induced epidermal hyperplasia model. Topical pre-treatment with FX-cream (100 mg per site containing 200 µg of FX) and Dex (100 mg per site containing 200 µg of Dex) was administered from 2 days before TPA-induced hyperplasia and 1 h after each TPA application (2 nmol per zone for three consecutive days). Then, 24 h after the last application of TPA, mice were sacrificed and skin biopsies were removed and weighted. Mice treated with TPA or TPA-cream resulted in the development of macroscopic lesions as peeling (Figure 3A), confirmed by an increase of weight of the 1 cm² punch biopsies of dorsal skin, in comparison with sham group (p < 0.001), with no significant differences between both groups (Figure 3B). This comparison led us to confirm that cream did not interfere in TPA action in our experiment. As expected, pre-treatment with the reference compound Dex significantly reduced the skin punch weight (p < 0.001). Similarly, mice treated with FX-cream showed a significant attenuation of skin edema when compared with TPA-cream group (p < 0.001) (Figure 3B). We next analyzed the skin homogenates to evaluate myeloperoxidase (MPO) activity as a neutrophil infiltration parameter with important relevance in hyperplasia model (Figure 3E). Our results confirm that this marker significantly increased after TPA application in comparison with sham group (p < 0.01). Dex application markedly diminished MPO activity in relation to TPA-cream group (p < 0.01). In a similar way, this parameter was reduced by FX-cream (p < 0.05) (Figure 3E). These results were confirmed with histological analysis of hematoxylin and eosin (H&E)-stained skin lesions in mice (Figure 3C), which showed that TPA administration produced a massive neutrophilic infiltration and an epidermal hyperplasia, because of uncontrolled and abnormal keratinocyte production. This effect was evidenced by a marked increase of epidermal thickness in this group (p < 0.001). Animals pre-treated with Dex and FX-cream evidenced a significant improvement in epidermal hyperplasia in relation to TPA-cream group (p < 0.001, and p < 0.01, respectively) (Figure 3C,D).

To support the beneficial effects of FX on skin inflammation and explore its possible mechanism of action, we measured cyclooxygenase-2 (COX-2) expression in skin samples (Figure 4). This enzyme has been shown to have an important role in pathogenesis of skin diseases. Immunohistochemical analysis of this enzyme exhibited that TPA-induced hyperplasia significantly enhanced COX-2-positive cell numbers (p < 0.001), principally located in epidermal layer, in comparison with sham (Figure 4B). Figure 4 shows that the results of mice pre-treated with Dex confirmed the anti-inflammatory effect of this corticoid (p < 0.001). Interestingly, pre-treatment with FX-cream resulted in a marked decrease in the number of epidermal COX-2-positive stained cells related with TPA-cream group, reaching expression levels lower than those in Dex group (p < 0.001).

2.6. FX Protects Human HaCaT Keratinocytes against UVB-Caused Damage

To examine the protective effect of FX in irradiated HaCaT cells, we evaluated cell viability by lactate dehydrogenase (LDH) enzyme activity, ROS levels and IL-6 production after UVB exposure. As expected, UVB irradiation significantly increased LDH activity, ROS content and IL-6 production in HaCaT keratinocytes compared to unirradiated control (p < 0.001) (Figure 5A–C). Pre-treatment of cells with FX 1 h prior to UVB exposure significantly decreased UVB-induced mortality, preserving cell membrane integrity in a dose-dependent manner at all concentrations used (10, 30 and 50 μM, p < 0.05, p < 0.01, p < 0.001, respectively) (Figure 5A). We next evaluated intracellular ROS levels in UVB-irradiated cells based on the dichlorofluorescein diacetate (DCF-DA) assay (Figure 5B). FX markedly reduced ROS levels by 23, 31 and 32% at the concentration of 10, 30 and 50 μM, respectively (p < 0.01, p < 0.001, p < 0.001) (Figure 5B).

It is known that UVB exposure induces abnormally augmented cytokine production from keratinocytes, leading to inflammatory skin disorders. To evaluate the effect of FX on IL-6 production, HaCaT cells were pre-treated for 1 h with this carotenoid, and then were exposed to UVB. Pre-treatment with FX at the concentrations tested of 10, 30 and 50 μM substantially inhibited this cytokine levels by 23, 31 and 59%, respectively (p < 0.001) (Figure 5C).

2.7. Topical Application of FX-Containing Cream Protects against UVB-Induced Skin Erythema in SKH-1 Hairless Mice

The possible photoprotective effect of the topical pre-treatment with FX-cream in hairless mice was assayed using an acute proinflammatory UVB dose (360 mJ/cm²) [22]. BC-cream was employed as reference compound due to its antioxidant and photoprotective activity [23]. Animals were examined with a dermatoscope for five days, evaluating UVB-induced skin alterations. As shown in Figure 6A, a single acute UVB exposure accelerated skin damage, showing typical symptoms such as peeling, loss of moisture, reduction of elasticity and increase of melanin production in comparison with sham controls (Figure 6A–D). The progressive evaluation of mice showed that pre-treatment with FX-cream increased the skin moisture (Figure 6B) and elasticity (Figure 6C) and decreased the production of melanin (Figure 6D), with similar values to BC-cream. In order to confirm the protective profile of FX, 48 h after UVB irradiation, the mice were sacrificed and dorsal skin was examined. Our data reflected significant increases of skin edema and MPO activity in UVB-irradiated group when compared with sham (p < 0.001). The reference group, BC-cream, showed pronounced lower levels of these parameters when compared to UVB-irradiated group (p < 0.001, p < 0.05, respectively) (Figure 6E,F). Similar results were found after pre-treatment with FX-cream (Figure 6E,F). These findings were confirmed by histological study by using H&E staining. Histologically, a significant increase in superficial skin layer thickness due to unregulated keratinocytes proliferation was detected in UVB-irradiated animals in comparison to sham (p < 0.001) (Figure 6G,H). FX-cream treatment was as effective as topical application of BC-cream; our data revealed a decrease in keratinocytes proliferation when compared to UVB-irradiated group, evidenced by a significant reduction of epidermal thickness (p < 0.001) (Figure 6G,H).

To further explore the possible mechanisms of action of FX, we examined the expression levels of different inflammatory and antioxidant proteins in dorsal skin samples. Exposure of skin to UVB induced a significant increase in the pro-inflammatory enzymes iNOS (p < 0.001) and COX-2 (p < 0.001) expression (Figure 7B,C). Neither of these proteins changed significantly in animals treated with the reference compound BC-cream. However, pre-treatment with FX-cream resulted in a significant downregulation of COX-2 expression levels in irradiated mice (p < 0.05) in comparison with UVB-irradiated animals. As regards the iNOS protein, although its expression tended to decrease in FX-cream-treated animals, no significant differences were observed in relation to UVB-treated mice. UVB exposure significantly down-regulated the expression of Nrf-2 protein (p < 0.05). BC-cream application induced a marked increase in Nrf2 levels (p < 0.001) accompanied with a rise of its target gene heme oxygenase-1 (HO-1) (p < 0.05). In a similar way, FX-cream significantly increased the expression of these antioxidant proteins in UVB-exposed skin (p < 0.01 and p < 0.05, respectively) (Figure 7D,E).

4.1. Cell Culture

The THP-1 human monocytic leukemia cell line and HaCaT human keratinocytes were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). THP-1 cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, New York, NY, USA) containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. HaCaT human keratinocytes were maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, Grand Island, New York, NY, USA). Both cell lines were grown in a humidified atmosphere containing 5% CO₂ at 37 °C [20].

4.2. Cell Viability Assay

SRB assay was used for determining the viability of THP-1 macrophages and HaCaT cells upon exposure to FX (Sigma-Aldrich, St. Louis, MO, USA) [51]. Firstly, for differentiation into macrophages, THP-1 cells were seeded into 96-well plates at 10⁴ cells/well in presence of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) for a final concentration of 0.2 µM for 72 h in 96-well plates and HaCaT cells were seeded into 96-well plates in the growth medium at 10⁴ cells/well for 24 h to ensure the adherence. Both cellular types were incubated in a humidified atmosphere of 5% CO₂ at 37 °C. After that, cells were treated with FX at final concentrations range of 10–100 µM in DMSO 0.1% (v/v) and the cytotoxicity was measured after 24, 48 and 72 h of incubation. The absorbance was determined at 492 nm in a microplate spectrophotometer (Sinergy HT, Biotek^®, Bad Friedrichshall, Germany).

4.3. Determination of TNF-α Production

THP-1 monocytes were differentiated into macrophages in 96-well plates (10⁴ cells/well). Then, cells were incubated for 1 h with FX (10, 30 and 50 µM). The positive reference compound used was Dex (1 µM) (Sigma-Aldrich, St. Louis, MO, USA). Inflammatory response was induced by addition of LPS (1 μg/mL) except for control group [52]. After 24 h, commercial enzyme-linked immunosorbent assay (ELISA) kit (Diaclone GEN-PROBE, Besançon, France) was used to quantify TNF-α according to the manufacturer’s protocol.

4.4. Determination of IL-6 and IL-8 Production

HaCaT cells were seeded in 6-well plates (5 × 10⁵ cells/well). After 24 h, cells were washed twice (PBS, 4 °C) and medium containing FX (10, 30 and 50 μM) or Dex (1 μM) was added for 1 h, and then cells were stimulated with TNF-α (10 ng/mL) except for control group. After 24 h, supernatant fluids were collected and stored −80 °C until measurements [20]. Commercial enzyme-linked immunosorbent assay (ELISA) kit (Diaclone GEN-PROBE, Besançon, France) was used to quantify IL-6 and IL-8 according to the manufacturer’s protocol.

4.5. Preparation of Topical Formulations

Based on previous studies, topical formulations were performed following the methodology detailed in Rodríguez-Luna et al. [20]. Thus, three different preparations were developed: hydrogel, cream and ointment. Concerning the hydrogel, BC (Sigma-Aldrich, St. Louis, MO, USA) solution in ethanol absolute (10 mg/mL) was gradually added to the polymer dispersion (1% w/v) under magnetic stirring. The ointment was prepared using both the melt emulsification and stirring steps. BC dissolved in ethanol (10 mg/mL) was added to the lipid mixture. Regarding cream, three different preparations were made containing 2 mg/g of drug (BC, FX or Dex). In all of them, the drug was dissolved in absolute ethanol (10 mg/mL) and added to the excipient cold mixture.

4.6. In Vitro Permeation Studies from Topical Formulations

In vitro drug permeation studies from the topical formulations were performed using a Franz diffusion cell apparatus (SES-Gmgh Analyses system, Bechenheim, Germany), with 14.5 mL receptor volume and 3.14 cm² diffusion area. An appropriately conditioned cellulose nitrate membrane (Tuffryn^®; Pall Corporation, Port Washington, New York, NY, USA), impregnated with lauryl alcohol (membrane weight increase 90–110%), was employed as artificial lipophilic membrane simulating the epidermal barrier [53,54]. The membrane was placed in the diffusion cell sandwiching the donor and receptor compartments. The receiver solution contained absolute ethanol and the donor chamber was filled with 1 g of formulation at 2 mg/g of BC (cream, ointment or hydrogel). In this study, BC was employed as reference compound because of its structural similarity with FX. The whole diffusion cell was thermostated maintaining the temperature at 32 °C. Aliquots of 0.5 mL were withdrawn from the receptor chamber after 0.5, 1, 1.5, 2 and 3 h and replaced with fresh ethanol. The concentrations of BC were spectrometrically assayed at 454 nm (UV/vis 1601 Shimadzu, Duisburg, Germany). The cumulative amounts of permeated drug per unit area in the receiver chamber (µg/cm²) were plotted as a function of time (h). The slope of the linear portion of the plot was presented as steady state flux (Jss, µg/cm²/h). From the permeation profiles, the AUC was calculated using the trapezoidal rule as a quantitative parameter for studying the permeation magnitude [55].

4.7. Animals

Female Swiss CD-1 mice (25–30 g) were purchased from Janvier-Labs (Le Genest St Isle, France) and female SKH-1 hairless mice (18–20 g) from Charles River Laboratories (Écully, France). Mice were maintained under standard conditions (temperature of 24–25 °C, humidity of 70–75% and 12 h light-12 h dark cycle) and were allowed free access to a standard diet (Panlab) and water ad libitum. All studies were performed in accordance with the recommendations of the European Union regarding animal experimentation (Directive of the European Council 2010/63/EU). The experiments followed protocols approved by the Animal Ethics Committee of the University of Seville (Protocol 06/04/2018/042).

4.8. Ex Vivo Permeation Studies from Creams

Once the cream carrier was chosen from the previous study, ex vivo diffusion studies were performed using excised mice skin as a membrane for evaluating the permeation behaviour of FX-loaded cream. Moreover, Dex-cream was included because it was used as positive control in the in vivo hyperplasia study. Swiss CD-1 mice were sacrificed by cervical dislocation and full dorsal skin was excised. A specific portion of the skin was washed with distilled water and cut for the permeation assay. The study was carried out following the methodology previously reported [56]. Animal skin was inserted between the donor and receiving compartments and adjusted by means of a pinch clamp. The receiving chamber was filled with 14.5 mL of degassed ethanol absolute and was thermostated by means of a water bath circulator and a jacket surrounding the cell, maintaining 32 °C in the skin surface. The receiving medium was continuously stirred to avoid diffusion layer effect.

FX-cream (0.2% w/w), FX control solution in ethanol (0.2% w/w) and Dex-cream (0.2% w/w) were accurately measured and placed on stratum corneum in the donor compartment and sealed with parafilm. The same procedure as previously described was followed [56]. Absolute ethanol was used as solvent in the receiver compartment. The concentrations of Dex were spectrometrically assayed at 254 nm (UV/vis 1601 Shimadzu). The FX content in the ethanol solutions was analyzed by using HPLC method. The analysis was performed on a Hitachi Elite Lachron HPLC system equipped with a L-2130 isocratic pump, a diode array detector L-2455 and a L-2200 autosampler. Separation was carried out within a chromatographic C18 column (Merck, RP-18 LichroCART^® 150 mm × 4 mm, 5 µm). For drug analysis, the injection volume was 10 µL, and the flow rate and column temperature were set at 1.0 mL/min and 25 °C, respectively. The mobile phase consisted of A (formic acid 0.1%) and B (acetonitrile). The elution program was: 6 min isocratic at 90% B, followed by a gradient to 100% B at 10 min. Afterwards, the column was re-equilibrated during 5 min at 90% B. The cumulative amount of drug in receptor chamber for the three formulations (FX-cream, FX control solution and Dex-cream) was measured as a function of time (t, h). The cumulative amount (%) of drug permeated through the skin (P%) was determined as per the following equation [57]:

where C_n is the drug concentration of the nth sampling point (mg/mL), C_i is the drug concentration of the ith sample point (mg/mL), V is the total volume (14.5 mL) of liquid in receiving pool, V_i is the volume (0.5 mL) of the ith sampling points and M is the mass of drug (FX or Dex) [20].

4.9. TPA-Induced Epidermal Hyperplasia Model and Treatments

Female Swiss CD-1 mice (n = 10 per group) were used to study the effect of FX on TPA-induced hyperplasia in mice dorsal skin. Briefly, the dorsal hair of animals was removed with an electric clipper and treated with depilatory cream (Deliplus, Barcelona, Spain) [35]. After 24 h, the animals were assigned to the different groups: Sham (vehicle: 100 mg of cream with 0.2% of ethanol); TPA control group; TPA-Cream (vehicle: 100 mg of cream with 0.2% of ethanol); Dex-cream (100 mg of cream per site, containing 200 µg of Dex dissolved in ethanol at 10 µg/mL); FX-cream formulation (100 mg of cream per site, containing 200 µg of FX dissolved in ethanol at 10 µg/mL). The creams were applied to the dorsal skin in an area of 1 cm² using a syringe, during 2 days before the hyperplasia induction. Mice were anesthetized with ketamine (100 mg/kg of animal) and diazepam (5 mg/kg of animal) during treatments and TPA challenge. On day 4, TPA (2 nmol of TPA, dissolved in 20 µL of ethanol) was topically applied to the same areas on all groups except in sham group. After 1 h, FX-cream, the Dex-cream or vehicle were administered following the mentioned protocol. This procedure was replicated during two consecutive days. After 24 h of the last TPA dorsal application (day 7), mice were sacrificed by cervical dislocation and punch biopsies from the treated dorsal skin were weighed to evaluate edema, before further processing for histology and biochemical parameters.

4.10. MPO Activity

The measurement of MPO activity was used as a marker of neutrophil infiltration [58]. The tissue was thawed, weighed and homogenized in 10 volumes of 50 mM-PBS (pH 7.4). Then, the homogenate was centrifuged at 20,000× g for 20 min at 4 °C and the pellet was again homogenized in 10 volumes of 50 mM-PBS (pH 6) containing hexadecyl trimethylammonium bromide (0.5%) and 10 mM-EDTA. This homogenate was subjected to one cycle of freezing/thawing and sonicated. The homogenate samples (50 µL) were added to 96-well microplate and incubated at 37 °C for 3 min with a measurement mix (o-dianisidine dihydrochloride (0.067%), hexadecyl trimethyl-ammonium bromide (0.5%) and 0.3 mM-H₂O₂). The changes in absorbance were monitorized at 450 nm with a microplate reader (Labysystem Multiskan EX, Thermo Scientific, New York, NY, USA). MPO activity unit was defined as the amount of enzyme present that produced a change in absorbance of 1.0 unit/min at 37 °C in the final reaction volume containing acetate. Results are expressed as units/mg tissue [20].

4.11. Histological Study

The samples were fixed in paraformaldehyde, dehydrated by increasing concentrations of ethanol and embedded in paraffin. For H&E stains, tissue sections were cut to 7 μm on a rotary microtome (Leica Microsystems, Wetzlar, Germany), mounted on slides, deparaffinized with xylene, rehydrated through graded alcohols and stained according to standard protocols. All tissue sections were examined under an Olympus BH-2 microscope (GMI Inc., Ramsey, MN, USA) for determination of histopathological changes. Epidermal thickness was measured by using Scientific Imaging Systems (Biophotonics ImageJ Analysis Software; National Institutes of Health, Rockville, MD, USA).

4.12. Immunohistochemical Analysis

COX-2 expression was measured by immunohistochemical analysis by using a streptavidin-biotin-peroxidase method [59]. Paraffin-embedded dorsal skin sections (7 µm) were mounted on slides, deparaffinized with xylene and rehydrated through graded alcohols. These sections were boiled (10 mM citrate buffer (pH 6.0) for 3 min) for antigen retrieval, followed by cooling at room temperature for 20 min. Endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide for 20 min and the sections were washed (PBS, 10 min). To minimize the non-specific adsorption the sections were incubated in normal horse serum (Vectastain Kit; Vector Laboratories, Burlingame, CA, USA) for 20 min. Afterwards, slides were incubated with rabbit polyclonal anti-COX-2 antibody (Cayman Chemical, Ann Arbor, MI, USA) (1:300) overnight at 4 °C. Then, the samples were treated with anti-mouse IgG antibody. After 30 min, the cells were incubated with the streptavidin–peroxidase complex for 30 min, at room temperature (Vectastain Kit; Vector Laboratories, Burlingame, CA, USA). The enzymatic activities were developed with 3,3′-diaminobenzidine (DAB), and the sections were counterstained with hematoxylin. Negative control sections were treated in the same way, omitting the primary antibody. To examine COX-2 immunoreactivity, the microscope Olympus BX61 was used (Olympus Optical Co. Ltd., Tokyo, Japan). The quantification of immunohistochemical data was done by counting the number of immunostained cells as percent of total epidermal cells from 10 microscopic fields of immunostained tissues per animal.

4.13. UVB Irradiation of HaCaT Keratinocytes

Human keratinocytes were exposed to UVB radiation as previously described [60]. Briefly, the cells were grown to confluence and were treated with different concentrations of FX (10, 30 and 50 µM) for 1 h. Then, the medium was removed and a thin layer of PBS was added. Cells were exposed to a single dose of UVB radiation (50 mJ/cm²) for 1 min. The UVB source was a CL-1000M UV Crosslinker (UVP, Upland, CA, USA), which was used to deliver an energy spectrum of UVB light (280–315 nm; peak intensity, 302 nm). After UVB irradiation, the cells were supplied with fresh complete medium and incubated for 24 h.

4.14. Analysis of Intracellular LDH Activity

The measure of cytosolic enzyme LDH is one of the commonly used methods for assessing loss of cellular membrane integrity. The assay is based on the conversion of lactate to pyruvate in the presence of LDH with the consequence oxidation of NADH as previously described [61]. HaCaT cells were seeded in 6-well plates (5 × 10⁵ cells/well). After 24 h, cells were treated with FX (10, 30 and 50 μM) for 1 h and then, were irradiated with UVB. After 24 h, cell-free supernatants and cell lysates were mixed in a 96-well plate and the absorbance was read by using a microplate reader system (Sinergy HT, Biotek^®, Bad Friedrichshall, Germany). LDH leakage was estimated calculating the LDH activity in the cell-free medium and LDH activity in lysates ratio. Results were represented as the percentage (%) of change in activity compared with the control cells.

4.15. Intracellular ROS Scavenging Activity

For quantification of ROS in HaCaT cells, the DCF-DA assay was employed [62]. HaCaT cells were seeded in nin96-well plates (10⁴ cells/well). Non-irradiated cells were used as negative control. After 24 h, cells were treated with FX (10, 30 and 50 µM) for 1 h and then, were exposed to UVB. Post irradiation, cells were incubated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) solution (5 mg/mL) in PBS for 30 min [63]. The fluorescence of the 2′,7′-dichlorofluorescein (DCF) product was determined using a fluorescence plate reader (Sinergy HT, Biotek^®, Bad Friedrichshall, Germany) at 485 nm for excitation and 535 nm for emission.

4.16. Determination of IL-6 Production in UVB-Exposed HaCaT Keratinocytes

HaCaT cells (5 × 10⁵ cells/well) were seeded in 6-well plates. After 24 h, cells were treated with FX (10, 20 and 50 μM) for 1 h and then, were exposed to UVB. After 24 h, supernatant fluids were collected and ELISA kit (Diaclone GEN-PROBE, Besançon, France) was employed to quantify IL-6 according to the manufacturer’s protocol. The absorbance at 450 nm was read by a microplate reader (Labysystem Multiskan EX, Thermo Scientific, New York, NY, USA).

4.17. UVB-Induced Erythema in Hairless Mice

Female SKH-1 hairless mice (n = 8 per group) were used to study the effect of FX on UVB-induced erythema. Animals were distributed to the different groups: Sham (vehicle: 100 mg of cream with 0.2% of ethanol); UVB (100 mg of cream with 0.2% of ethanol); BC-cream (100 mg of cream per site, containing 200 µg of BC dissolved in ethanol at 10 µg/µL) as reference control; FX-cream (100 mg per site, containing 200 µg of FX dissolved in ethanol at 10 µg/µL). The formulations were applied on the dorsal skin (1 cm²/area) by using a syringe, and starting 2 days before irradiation. On day 3, 30 min after application of the substances, all groups except the sham control were exposed to an acute UVB dose (360 mJ/cm²) as previously described [22]. Then, 48 h after UVB exposure, mice were macroscopically evaluated and sacrificed. At this end-point time, a pronounced UVB-induced cutaneous inflammation was shown [4].

4.18. Dermatoscope Measurements

Dorsal skin was macroscopically examined every day by using a multi-dermatoscope (Dermatoscope MDS 800, Microcaya, Bilbao, Spain). Corneometer^® was used to determinate the hydration in the stratum corneum through electrical capacitance of the skin surface [64]. A Cutometer^® probe was used as suction method to analyse the real elasticity of skin [65]. In the last step, Mexameter^® was employed to analyse the redness skin by melanin index [66]. Every day, dorsal skin of mice was assessed twice and finally the results were expressed in arbitrary units (a. u.) or by a melanin index scale (1-36).

4.19. Western Blot Assay

Frozen dorsal skin tissues from UVB-induced erythema model were randomly selected (6 per group), weighed and homogenized in ice-cold buffer (50 mM Tris-HCl, pH 7.5, 8 mM MgCl₂, 5 mM ethylene glycol bis (2-aminoethyl ether)-N,N,N′,N′-tetra acetic acid, 0.5 mM EDTA, 0.01 mg/mL leupeptin, 0.01 mg/mL pepstatin, 0.01 mg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 250 mM NaCl). The homogenates were centrifuged (12,000× g, 15 min, 4 °C), and the supernatants were collected and stored at −80 °C. To determinate the protein concentration of the homogenates was used the Bradford colorimetric method [67]. Samples of the supernatants with equal amounts of protein (25 μg) were separated on 10% acrylamide gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then, the proteins were electrophoretically transferred onto a nitrocellulose membrane and incubated with specific primary antibodies: rabbit anti-inducible nitric oxide synthase (iNOS) (1:1000; Stressgen-Enzo Life Sciences, Farmingdale, NY, USA); rabbit anti-COX-2 (1:3000; Cayman Chemical^®, Ann Arbor, MI, USA); rabbit anti-Nrf2, rabbit anti-HO-1 (1:1000; Cell Signaling, Danvers, MA, USA) overnight at 4 °C. After rinsing, the membranes were incubated with the horseradish peroxidase-linked (HRP) secondary antibody anti-rabbit (1:1000; Cayman Chemical^®, Ann Arbor, MI, USA) or anti-mouse (1:1000; Dako^®, Atlanta, GA, USA) containing blocking solution for 1 h at room temperature. To prove equal loading, the blots were analyzed for β-actin expression using an anti-β-actin antibody (1:1000; Sigma Aldrich^®, St. Louis, MO, USA). Immunodetection was performed using an enhanced chemiluminescence light-detecting kit (SuperSignal West Pico Chemiluminescent Substrate, Pierce, IL, USA). Then, the immunosignals were monitorized by using LAS-3000 Imaging System (Fujifilm Image Reader, Valhalla, NY, USA) and densitometric data were studied after normalization to the control (housekeeping gene). The signals were analyzed and quantified with a Scientific Imaging Systems (Biophotonics ImageJ Analysis Software, National Institute of Mental Health, Bethesda, MD, USA) and expressed as percentage respect to sham control group [68].

4.20. Statistical Analysis

All values in the figures and text are expressed as arithmetic means ± SEM. Data were evaluated with GraphPad Prism version 5.00 software (GraphPad Software, Inc., San Diego, CA, USA). In all cases, the Shapiro-Wilk test was used to verify the normality of the data. The Mann-Whitney U-test was chosen for non-parametric values. The parametric values groups were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni’s Multiple Comparison Test. p values < 0.05 were considered statistically significant.