Fucoxanthin and Rosmarinic Acid Combination Has Anti-Inflammatory Effects through Regulation of NLRP3 Inflammasome in UVB-Exposed HaCaT Keratinocytes

Abstract

Excessive exposure to ultraviolet (UV) radiation is the main risk factor to develop skin pathologies or cancer because it encourages oxidative condition and skin inflammation. In this sense, strategies for its prevention are currently being evaluated. Natural products such as carotenoids or polyphenols, which are abundant in the marine environment, have been used in the prevention of oxidative stress due to their demonstrated antioxidant activities. Nevertheless, the anti-inflammatory activity and its implication in photo-prevention have not been extensively studied. Thus, we aimed to evaluate the combination of fucoxanthin (FX) and rosmarinic acid (RA) on cell viability, apoptosis induction, inflammasome regulation, and anti-oxidative response activation in UVB-irradiated HaCaT keratinocytes. We demonstrated for the first time that the combination of FX and RA (5 µM RA plus 5 μM FX, designated as M2) improved antioxidant and anti-inflammatory profiles in comparison to compounds assayed individually, by reducing UVB-induced apoptosis and the consequent ROS production. Furthermore, the M2 combination modulated the inflammatory response through down-regulation of inflammasome components such as NLRP3, ASC, and Caspase-1, and the interleukin (IL)-1β production. In addition, Nrf2 and HO-1 antioxidant genes expression increased in UVB-exposed HaCaT cells pre-treated with M2. These results suggest that this combination of natural products exerts photo-protective effects by down-regulating NRLP3-inflammasome and increasing Nrf2 signalling pathway.

Keywords: NRLP3; UVB; anti-inflammatory; anti-oxidative; fucoxanthin; inflammasome; photo-protection; rosmarinic acid.

Figure 1

Selection of UVB dose and effect of rosmarinic acid (RA), fucoxanthin (FX) and combinations on cell viability and ROS production in UVB-irradiated HaCaT cells. (A) Effect of different UVB doses on cell viability determined by MTT assay. Results are expressed as percentage respect to untreated control cells and bars represents mean ± SEM of four independent experiments (n = 4) in duplicate. (*** p < 0.001 vs. untreated cells; Student t test) (B) Effect of RA, FX and their combinations on cell viability in human HaCaT keratinocytes was measured by MTT assay after 24 h of UVB exposition. (C) Intracellular ROS generation was evaluated 30 min after UVB irradiation by relative fluorescence intensity using 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA) assay. For experiments B and C, cells were pre-treated with RA (2.5 and 5 µM), FX (5 µM) and two combinations M1 (2.5 RA plus 5 µM FX) and M2 (5 µM RA plus 5 μM FX), for 1 h. Then, cells were irradiated with selected UVB dose (100 mJ/cm²) and incubated for the required times. Results are expressed as percentage respect to untreated control cells (B) or UVB-exposed control cells (C), and bars represents mean ± SEM of at least six independent experiments (n = 6) in duplicate. The mean was significantly different compared to control cells (*** p < 0.001; Student t test). The mean was significantly different compared to UVB-irradiated cells (⁺ p < 0.05, ⁺⁺ p < 0.01 and ⁺⁺⁺ p < 0.001; one-way ANOVA followed by Bonferroni’s multiple comparison test).

Figure 2

Effect of rosmarinic acid (RA), fucoxanthin (FX) and combinations on apoptosis and cell cycle arrest by using flow cytometry. (A) Results of Annexin V-FITC assay as percentage of cells in different apoptotic phases; R1: necrotic cells (Annexin V-FITC negative, PI positive), R2: viable cells (Annexin V-FITC negative, PI negative), R3: early apoptotic cells (Annexin V-FITC positive, PI negative), R4: late apoptotic cells (Annexin V-FITC positive, PI positive). Cells were pre-treated with RA (2.5 and 5 µM), FX (5 µM) and two combinations M1 (2.5 RA plus 5 µM FX) and M2 (5 µM RA plus 5 μM FX), then were irradiated with selected UVB dose (100 mJ/cm²) and incubated for 24 h. (B) Cell cycle phase distribution of UVB-exposed HaCaT cells. Cells were incubated with treatments for 48 h. Data are expressed as percentage and bars represents mean ± SEM of four independent experiments (n = 4) in duplicate. Results are expressed as percentage and bars represents mean ± SEM of four independent experiments (n = 4) in duplicate. The mean value was significantly different compared with control group (*** p < 0.001, ** p < 0.01, * p < 0.05 vs. untreated cells; Mann–Whitney U-test). The mean value was significantly different compared with UVB-irradiated cells ⁺ p < 0.05, ⁺⁺ p < 0.01 vs. UVB-irradiated cells; Kruskal–Wallis test followed by Dunn’s multiple comparison test).

Figure 3

Effect of rosmarinic acid (RA), fucoxanthin (FX) and concomitant administration of RA and FX (M2) on inflammasome components expression and IL-1β levels in UVB-exposed HaCaT keratinocytes. Cells were pre-treated with RA (5 µM), FX (5 µM) and M2 (5 µM RA plus 5 μM FX) for 1h, then were UVB-irradiated (100 mJ/cm²) and incubated for 24 h. Densitometry analysis of (A) nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain- containing 3 (NLRP3), (B) inflammasome adaptor protein (ASC) and (C) caspase-1 positivity were performed following normalization to the control (β-actin housekeeping gene). (D) IL-1β production was evaluated by ELISA assay. Results are expressed as percentage respect to UVB-irradiated control and bars represents mean ± SEM of four independent experiments (n = 4). The mean value was significantly different compared with control group (* p < 0.05, ** p < 0.01 and *** p < 0.001; Mann–Whitney U-test). The mean value was significantly different compared with UVB-irradiated cells (⁺ p < 0.05 and ⁺⁺ p < 0.01; Kruskal–Wallis test followed by Dunn’s multiple comparison test).

Figure 4

Effect of rosmarinic acid (RA), fucoxanthin (FX) and concomitant administration of RA and FX (M2) on Nrf2 and HO-1 expression in UVB-exposed HaCaT keratinocytes. Cells were pre-treated with RA (5 µM), FX (5 µM) and M2 (5 µM RA plus 5 μM FX) for 1 h, then were UVB-irradiated (100 mJ/cm²) and incubated for 24 h. Densitometry analysis of (A) Nrf2 and (B) HO-1 were performed following normalization to the control (β-actin housekeeping gene). Data shown are expressed as percentage respect to UVB-irradiated control and bars represents mean ± SEM of four independent experiments (n = 4). The mean value was significantly different compared with control group (* p < 0.05; Mann–Whitney U-test). The mean value was significantly different compared with UVB-irradiated cells (⁺ p < 0.05 and ⁺⁺ p < 0.01; Kruskal–Wallis test followed by Dunn’s multiple comparison test.