1. Introduction
Skin is considered as the outmost protective barrier in the body, protecting from detrimental substances, mechanical damage, pathological invasion and radiation that could cause perturbations to the skin structure. In this sense, skin is a well-known essential piece of the immune system. Several factors can contribute to the initiation and development of cutaneous alterations. In this line, the excessive exposure to UV radiation remains the main risk factor for the skin cancer [
1]. Solar UV radiation consists of three broad ranges of wavelength: UVC (100–280 nm), UVB (280–315 nm) and UVA (315–400 nm). UVB causes dermal changes, affects epidermal function and is the main factor responsible for the development of melanoma and non-melanoma skin cancer [
2]. In this regard, a high dose of UVB exposure promotes cutaneous inflammation, which is traduced in sunburn, photo-aging, DNA damage, immunosuppression and induction of skin cancer [
3].
It has been extensively studied that UVB-induced ROS production leads to activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB), among others, which further promote inflammation and apoptosis in cells and increase skin aging [
4]. Furthermore, this type of inflammation results in the production of cytokines as tumour necrosis factor alpha (TNF-α), IL-6 and IL-1β, which are released by keratinocytes after UVB irradiation [
5]. In this sense, it has been reported the relation between IL-1β secretion and activation of protein complexes called inflammasome [
6]. Inflammasome is a wide cytosolic multiprotein complex that acts as mediator of the innate immune system, which is activated by multiple types of tissue damages. The nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) is the most studied inflammasome. NLRP3 gen induction results in activation of caspase-1, which by cleavage catalyses the processing of pro-IL-1β in cytosol to mature form, leading to IL-1β production in the extracellular medium [
7]. In the last years, some authors have demonstrated the implication of NLRP3 inflammasome in tumorigenesis and cancer development, specifically in basal cell carcinomas [
8,
9].
Nowadays, non-melanoma skin cancer (NMSC) remains the most frequently diagnosed cancer in Caucasian people worldwide and strategies for its prevention are being developed [
10]. At this regards, photo-chemoprevention by natural products is one of the most studied alternatives for skin protection, due to their anti-oxidative actions [
1]. Although the anti-inflammatory properties of natural compounds have not been extensively investigated yet, in recent years, different studies have evaluated the beneficial effects of these compounds in photoprotection through their anti-inflammatory activity [
1].
In last years, microalgae have been employed as a vast source of bioactive molecules with potential activity in inflammation and cancer [
11]. Specifically, carotenoids have shown antioxidant, anti-inflammatory or anti-carcinogenic properties in several skin inflammatory models [
12,
13]. The orange carotenoid fucoxanthin (FX) is not exclusive from marine environmental [
14] but it is an extensive compound produced by microalgae and brown seaweeds, as previously published [
15] and it is known for its potent antioxidant properties. Nevertheless, its effect on NRLP3-inflammasome regulation has been little explored.
Polyphenols are the most popular antioxidant molecules in our diet, and exhibit other properties as anti-inflammatory or anti-neoplastic agents. For this reason, these compounds are also proposed as an important key for the management of skin protection [
16]. In this regard, rosmarinic acid (RA) is a phenolic ester that has been traditionally isolated from some terrestrial plants as
Rosmarinus officinalis L. or
Melissa officinalis L. [
17]. Moreover, this polyphenol has been abundantly found in
Zostera marina seagrass beds [
18,
19]. This aspect is very interesting since
Zostera has a rapid generation time and similar requirements to microalgae; thus it could be a great source to obtain a traditional phenolic compound like this. RA has been widely studied due to its remarkable biological and pharmacological activities, including anti-microbial, antioxidant and anti-inflammatory properties [
20]. Until now, only a limited number of studies have dealt with the effect of RA on NLRP3-inflammasome [
21,
22,
23], and none of them on human immortalized HaCaT keratinocytes.
These antecedents led us to evaluate the effects of a combination of FX and RA on ROS production, apoptosis prevention, cell cycle alterations, inflammasome regulation and Nrf2 pathway activation, in UVB-exposed human keratinocytes.
3. Discussion
Skin represents the first barrier that protects us from the deleterious effects of solar UV radiation, which is the main cause for skin cancer. Nevertheless, human skin is normally exposed to UV irradiation in a non-controlled way. Whereas a short-term UV exposure might suppress immune function or trigger an inflammatory response, chronic UV exposition conduces to photo-aging, DNA damage, or carcinoma [
29]. Skin has an antioxidant system, composed by enzymatic and non-enzymatic antioxidants, to prevent against toxic exogenous/endogenous ROS levels. Nowadays, it is well known the importance of natural substances that support the endogenous antioxidant systems of the skin via diet or dermatological preparations [
30]. To develop new skin photo-protective agents it is necessary to understand the molecular mechanism of UV-induced cellular responses and determine how these products may act in the skin cells. In this sense, many treatments with a unique compound have demonstrated successful results against photo-damage, but a more hopeful impact was obtained when a combination of several compounds was used [
31]. The efficacy of combinations could be due to the synergistic effect of their components. In this regard, natural products may be good candidates for skin photo-protection due to their low toxicity and high antioxidant capacity [
32]. Thus, the present study aimed to evaluate the preventive effect of a combination of RA and FX in UVB-exposed human HaCaT keratinocytes.
It is well reported that UVB radiation increases ROS production and DNA damage as well as promotes a strong inflammatory response characterized by pro-inflammatory mediator production [
33]. In this condition, apoptosis activation can eliminate the irreversibly damaged cells that could harbour oncogenic mutation [
25]. In this line, FX has demonstrated antioxidant activity offering protection against DNA damage and preventing cellular apoptosis in HaCaT human keratinocytes [
34,
35]. In a similar way, several studies have reported that RA reduces ROS production, protects against DNA damage and regulates apoptotic markers in vitro [
31,
36]. According to these findings, our results showed that the pre-treatment with FX or RA at 5 µM significantly attenuated UVB-induced damage by increasing cell viability and inhibiting ROS production in human HaCaT keratinocytes, but did not modify the number of apoptotic cells after UVB exposure. Nevertheless, the selected combination of RA and FX at 5 µM at equal dose (M2) not only improved cell viability and ROS levels in comparison with FX and RA administered separately, but also enhanced the cellular protection, reducing the number of cells in apoptosis after UVB irradiation. In addition to cellular responses previously detailed, UVB exposure also affected cell cycle dynamics. Previous studies have reported that UV exposure activates cell cycle arrest in both G1 and G2 phases [
37]. At this respect, our results reported that UVB irradiation significantly reduced cells at G0/G1 phase when compared with non-exposed cells. Furthermore, a S-phase delay was observed in UVB-exposed cells although it did not show to be highly affected by the treatments. The pre-treatment with FX, RA and concomitant administration of RA and FX (M2) significantly increased the percentage of cells at G0/G1 and reduced the percentage of cells at sub-G1. In this sense, our observations are in accordance with the results obtained in apoptosis assay. M2 prevented cell damage, reducing the percentage of cells under apoptosis 24 h after UVB exposure. In consequence, this combination promoted a higher percentage of viable cells at 48 h in UVB-exposed cells.
In terms of inflammation, it has been reported that pro-IL-1β is cleaved and IL-1β is released from cells as a pro-inflammatory cytokine after NLRP3 inflammasome activation [
38]. Thus, it could be concluded that IL-1β production in keratinocytes is inflammasome-dependent [
6]. Moreover, NLRP3 inflammasome activation by UVB increases not only IL-1β but also other pro-inflammatory cytokines such as IL-1α, IL-6, TNF-α or the mediator PGE2 [
7]. In this line, NLRP3 inflammasome plays an important role in UVB-induced skin inflammatory responses and has a critical function in skin pathologies initiation such as cancer [
8]. In addition, the inflammasome adaptor protein ASC has shown anti-tumour activity by activation of tumour suppressor protein p53 in UVB-irradiated keratinocytes [
39]. Due to the high connection between inflammasome, inflammation and cancer, the therapeutic strategies targeting inflammasome-related products are currently in development and suppose a pivotal tool for skin pathologies and cancer prevention [
40]. In previous studies, FX has shown anti-inflammatory activity through NF-κB and MAPKs signalling pathways regulation [
41], and consequently the down-regulation of IL-1β, TNF-α, inducible nitric oxide synthase, and cyclooxygenase-2 expression [
42,
43,
44]. On the other hand, the polyphenol RA has been previously reported to inhibit pro-inflammatory cytokines production, including IL-1β, and to down-regulate NF-κB signalling pathway in skin cells [
45]. In addition, this polyphenol has shown to modulate inflammasome activation, through down-regulation of caspase-1, NLRP3 and ASC expression in a poly(I:C)-induced inflammation in an infectious model by using epidermal keratinocytes [
21]. Moreover, due to its anti-inflammatory properties, RA has been proposed to ameliorate allergic reactions in allergic rhinitis and rhinoconjunctivitis [
46]. Furthermore, this compound has shown to prevent cisplatin-induced apoptosis in chemotherapy by down-regulating the caspase-1 expression [
47]. However, the role of FX or RA on NLRP3 inflammasome regulation in human keratinocytes has not been studied yet. In this line, our findings reported for the first time that the combination of RA and FX notably reduced inflammasome-related proteins such as NLRP3, ASC and caspase-1 in UVB-irradiated HaCaT keratinocytes. Interestingly, this effect was not detected when both compounds were administered separately, suggesting the beneficial effect of concomitant treatment with RA and FX.
The antioxidant role of FX and RA has been well-described. FX has shown to promote AKT/Nrf2/GSH-dependent antioxidant response in keratinocytes [
48,
49], as well as reduce wrinkles formation and epidermal hypertrophy in mice [
50] and supress melanogenesis and prostaglandin synthesis [
51]. In this line, due to its antioxidant activity, this carotenoid has been proposed as a photo-protective compound by stimulating skin barrier restoration in UVA-induced sunburn [
52]. Furthermore, a previous study by our group evidenced that FX increased Nrf2 and HO-1 expression in UVB-induced acute erythema in SKH-1 hairless mice [
44]. These results are in line with other authors that suggest that this compound could be an interesting strategy to counteract UVB-induced oxidative damage in skin [
53]. At this regard, similar properties have been demonstrated with RA applications, confirming the antioxidant activity of this polyphenol via activation of Nrf2-antioxidant system [
54,
55]. In accordance with these findings, the combination between FX and RA (M2) used in this study showed an up-regulation of Nrf2 transcriptional factor and its main target gene HO-1. These data indicate that the protective activity of M2 may be due to the reduction of oxidative stress through modulation of Nrf2 signalling pathway.
4. Materials and Methods
4.1. Compounds
RA and FX were obtained from Sigma-Aldrich (St. Louis, MO, USA). RA and FX stocks were prepared in DMSO at 10 mM and diluted to the desired concentration with culture medium. Controls were incubated with the corresponding quantity of DMSO, which was always below 1% and did not affect cell viability
4.2. Cell Line
Human immortalized keratinocytes HaCaT were obtained from the American Type Culture Collection (ATCC) and maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, Grand Island, NY, USA) containing 2 mM l-glutamine. The culture media was supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin (PAA Laboratories, Pasching, Austria). Cultures were incubated in an atmosphere of 5% CO2 at 37 °C.
4.3. UVB-Irradiation
The CL-1000M UV Crosslinker system (UVP, Upland, CA, USA), formed by 5 UVB tubes (8 W), was used to submit an energy spectrum of UVB radiation (peak intensity 302 nm, inside of the spectrum of UVB light 280–315 nm). To prevent UVB light absorption by the cell culture medium the medium was replaced by a thin layer of phosphate buffer solution (PBS) to cover the cells during irradiation. Three UVB intensities were evaluated (100, 150 and 225 mJ/cm
2) to determine the most suitable to evaluate cell cycle alterations and apoptosis [
22]. In this sense, 100 mJ/cm
2 was selected for the study.
4.4. Cell Proliferation Assay
Cell viability was evaluated by the colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) assay, which determines the formation of purple formazan in viable cells and allows estimate cellular viability [
56]. MTT assay was performed according to protocol described with slight modifications. Briefly, HaCaT cells were seeded at a density of 10
4 cells/well in a 96-well plate for 24 h. Cells were pre-treated with different doses either RA (2.5, 5, 7.5 and 10 µM) or FX (5, 10, 15 and 20 µM) and the sixteen possible combinations between RA and FX, for 1 h. Then, the culture medium was replaced with a thin layer of PBS and exposed to a single dose of UVB radiation at 100 mJ/cm
2. Cells were supplied with fresh culture medium and incubated for 24 h. Absorbance was measured at 570 nm using a Synergy HT Multi-mode Microplate Reader (BioTek Instruments, Winooski, VT, USA).
4.5. Intracellular ROS Scavenging Activity
The 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA) assay was used to detect intracellular ROS levels in HaCaT keratinocytes [
57]. Briefly, keratinocytes were seeded at 10
4 cells/well in 96-well plates and were treated with RA (2.5 and 5 µM), FX (5 µM) and the combinations M1 (2.5 RA plus 5 µM FX) and M2 (5 µM RA plus 5 μM FX) for 1 h. After UVB exposure (100 mJ/cm
2), cells were incubated with 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) solution (5 mg/mL) in PBS for 30 min at 37 °C. Then, the medium was discarded and the cells were washed with PBS. The fluorescence of the 2’,7’-dichlorofluorescein (DCF) product was determined by using a fluorescence plate reader (Sinergy HT, Biotek
®, Bad Friedrichshall, Germany) at 485 nm for excitation and 535 nm for emission.
4.6. Apoptosis Determination
Apoptosis was evaluated by flow cytometry using Annexin V-FITC Apoptosis detection kit from BD Pharmingen (Franklin Lakes, NJ, USA), as previously reported [
26]. Briefly, HaCaT cells were seeded at 5 × 10
5 cells/well in 6-well plates and incubated for complete adhesion. The day after seeding, cells were treated with RA (2.5 and 5 µM), FX (5 µM) and mixtures M1 (2.5 RA plus 5 µM FX) and M2 (5 µM RA plus 5 μM FX) for 1 h. After that, treatments were removed and cells were irradiated at 100 mJ/cm
2, and then incubated with fresh medium for 24 h. Cells were harvested and suspended in binding buffer at 10
5 cells/mL. Then, annexin V-FITC and propidium iodide (PI) were added as indicated in the manufacture’s protocol. Subsequently, samples were kept in darkness for 15 min and 400 µL of binding buffed were added. Fluorescence intensity of PI and FITC-Annexin-V-stained cells was determined on a Coulter Epics XL Flow Cytometer (Beckman Coulter, Hialeah, FL, USA). Data were obtained using the SYSTEM II (v.2.5.) software examining 10
4 events. The cytogram of FITC fluorescence in log scale versus PI fluorescence in log scales allows the identification of viable cells (Annexin V-FITC negative, PI negative), early apoptotic cells (Annexin V-FITC positive, PI negative), late apoptotic cells (Annexin V-FITC positive, PI positive) and necrotic cells (Annexin V-FITC negative, PI positive). Flow cytometry data were analysed by FlowJo software (Tree Star Inc., Ashland, OR, USA).
4.7. Cell Cycle Determination
For cell cycle determination, a similar procedure to apoptosis evaluation was carried out, with the difference of that after irradiation, cells were incubated with fresh medium for 48 h. After incubation, keratinocytes were harvested by trypsinization as previously detailed [
26], fixed in 85% cold ethanol (5 × 10
5 cells/mL) and kept at −20 °C until further analysis. Ethanol was eliminated, cells were resuspended in PBS, and the cell suspension was filtered through a 35 µm nylon mesh to separate aggregated cells. Then, 50 μL RNase (1 mg/mL) and 50 μL PI (1 mg/mL) were added to each sample, which was then incubated for 20 min in darkness at room temperature until analysis. DNA content was determined on a Coulter Epics XL Flow Cytometer (Beckman Coulter, Hialeah, FL, USA). Data were acquired using the SYSTEM II (v. 2.5.) software examining 10
4 events. Percentages of cells in apoptotic-sub G1, G0/G1, S and G2/M were calculated using CXP software.
4.8. Determination of IL-1β Production
HaCaT cells were seeded at 5 × 105 cells/well in 6-well plates, incubated for 24 h and treated with either RA (5 µM), FX (5 µM) or M2 (5 µM RA plus 5 μM FX), for 1 h. Then, the cells were irradiated at 100 mJ/cm2, and incubated with fresh medium for 24 h. Supernatant fluids were collected and stored at −80 °C until use. Commercial enzyme-linked immunosorbent assay (ELISA) kits (Diaclone GEN-PROBE, Besançon cedex, France) was used to quantify IL-1β production according to the manufacturer’s protocol. The absorbance at 450 nm was read by a microplate reader (Sinergy HT, Biotek®, Bad Friedrichshall, Germany). To calculate the concentration of IL-1β (pg/mL), a standard curve was constructed using serial dilutions of cytokine standards provided with the kit.
4.9. Western Blot Analysis
The cell culture and treatments were similar to those carried out for cytokine production. Then, HaCaT cells were harvested by trypsinization, centrifuged and resuspended in lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton-X-100, 1 mM phenylmethylsulphonyl fluoride, protease inhibitor cocktail tablet, 0.5 mM sodium orthovanadate, 20 mM sodium pyrophosphate). The homogenates were centrifuged (12,000 g, 15 min, 4 °C), and the supernatants were collected and stored at −80 °C. Bradford colorimetric method was utilized to determine the protein concentration of the homogenates [
58]. Samples of the supernatants with equal amounts of protein (20 μg) were separated on 10% acrylamide gel by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Next, the proteins were electrophoretically transferred onto a nitrocellulose membrane and incubated with specific primary antibodies: rabbit anti-ASC (Bio-Rad, Hercules, CA, USA) (1:1000), rabbit anti-NLRP3 (1:1000), rabbit anti-Nrf2 (1:1000), rabbit anti-HO-1 (1:1000), rabbit anti-caspase-1 (1:1000) (Cell Signaling, Danvers, MA, USA) overnight at 4 °C. After rising, the membranes were incubated with the horseradish peroxidase-linked (HRP) secondary antibody anti-rabbit (Cayman Chemical
®, Ann Arbor, MI, USA) (1:1000) or anti-mouse (Dako
®, Atlanta, GA, USA) (1:1000) containing blocking solution for 1 h at room temperature. To prove equal loading, the blots were analysed for β-actin expression using an anti-β-actin antibody (Sigma Aldrich
®, St. Louis, MO, USA). Immunodetection was performed employing an enhanced chemiluminescence light-detecting kit (SuperSignal West Pico Chemiluminescent Substrate, Pierce, IL, USA). Then, the immunosignals were monitored by using an Amersham Imaging 600 (GE Healthcare Life Sciences, Barcelona, Spain) and densitometric data were analysed after normalization to the control (housekeeping gene). The signals were analysed and quantified with a Scientific Imaging Systems (Biophotonics ImageJ Analysis Software, National Institute of Mental Health, Bethesda, MD, USA) and expressed as total percentage respect to UVB-exposed control group.
4.10. Statistical Analysis
All values in the figures and text are expressed as arithmetic means ± standard error of the mean (S.E.M.). Data were evaluated with GraphPad Prism® Version 5.00 software (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 analysed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test. p values < 0.05 were considered statistically significant.
