Topical Application of Glycolipids from Isochrysis galbana Prevents Epidermal Hyperplasia in Mice

marine drugs Article Topical Application of Glycolipids from Isochrysis galbana Prevents Epidermal Hyperplasia in Mice Azahara Rodríguez-Luna 1, Elena Talero 1, María del Carmen Terencio 2,3, María Luisa González-Rodríguez 4, Antonio M. Rabasco 4, Carolina de los Reyes 5, Virginia Motilva 1and Javier Ávila-Román1,*ID 1Department of Pharmacology, Faculty of Pharmacy, Universidad de Sevilla, 41012 Sevilla, Spain; [email protected] (A.R.-L.); etaler[email protected] (E.T.); [email protected] (V.M.) 2Department of Pharmacology, Faculty of Pharmacy, University of Valencia, 46010 Valencia, Spain; [email protected] 3Institute of Molecular Recognition and Technological Development (IDM), 46100 Valencia, Spain 4Department of Pharmaceutical Technology, Faculty of Pharmacy, Universidad de Sevilla, 41012 Sevilla, Spain; [email protected] (M.L.G.-R.); [email protected] (A.M.R.) 5Department of Organic Chemistry, Faculty of Marine and Environmental Sciences, University of Cadiz, 11510 Puerto Real, Cádiz, Spain; carolina.der[email protected] *Correspondence: [email protected]; Fax: +34-954-556-074 Received: 25 November 2017; Accepted: 7 December 2017; Published: 25 December 2017 Abstract: Chronic inflammatory skin diseases such as psoriasis have a significant impact on society. Currently, the major topical treatments have many side effects, making their continued use in patients difficult. Microalgae have emerged as a source of bio-active molecules such as glycolipids with potent anti-inflammatory properties. We aimed to investigate the effects of a glycolipid ( MGMG-A ) and a glycolipid fraction ( MGDG ) obtained from the microalga Isochrysis galbana on a TPA-induced epidermal hyperplasia murine model. In a first set of experiments, we examined the preventive effects of MGMG-A and MGDG dissolved in acetone on TPA-induced hyperplasia model in mice. In a second step, we performed an in vivo permeability study by using rhodamine-containing cream, ointment, or gel to determinate the formulation that preserves the skin architecture and reaches deeper. The selected formulation was assayed to ensure the stability and enhanced permeation properties of the samples in an ex vivo experiment. Finally, MGDG -containing cream was assessed in the hyperplasia murine model. The results showed that pre-treatment with acetone-dissolved glycolipids reduced skin edema, epidermal thickness, and pro-inflammatory cytokine production (TNFα , IL-1 β , IL-6, IL-17) in epidermal tissue. The in vivo and ex vivo permeation studies showed that the cream formulation had the best permeability profile. In the same way, MGDG -cream formulation showed better permeation than acetone-dissolved preparation. MGDG -cream application attenuated TPA-induced skin edema, improved histopathological features, and showed a reduction of the inflammatory cell infiltrate. In addition, this formulation inhibited epidermal expression of COX-2 in a similar way to dexamethasone. Our results suggest that an MGDG -containing cream could be an emerging therapeutic strategy for the treatment of inflammatory skin pathologies such as psoriasis. Keywords: glycolipids; MGDG ; skin; inflammation; epidermal hyperplasia; microalgae; Isochrysis galbana 1. Introduction Inflammatory skin diseases have a significant impact on the quality of life of patients; one of them is psoriasis, considered a common immune-mediated inflammatory skin disorder. It is estimated that 2–4% of the population suffers from psoriasis [ 1 ]. Although the exact mechanism of this pathology is Mar. Drugs 2018,16, 2; doi:10.3390/md16010002 www.mdpi.com/journal/marinedrugs Mar. Drugs 2018,16, 2 2 of 19 not completely understood, it is known that both genetic predisposition and environmental factors such as stress, infection, trauma, and use of some drugs play an important role in its etiology [ 2 ]. This disease is associated with several comorbidities as cardiovascular diseases, metabolic syndrome, and psychiatric disorders. Accumulating evidence has demonstrated that exposure of skin to the protein kinase Cactivator 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a pleiotropic tissue response and promotes macroscopic lesions, peeling, and erythema, mimicking an apparent psoriasis phenotype. Furthermore, an increase in epidermal thickness has been observed due to the hyperproliferation and aberrant differentiation of keratinocytes as well as the infiltration of inflammatory leukocytes into the epidermis and dermis [ 3 ]. Activated leukocytes cause uncontrolled production of reactive oxygen species (ROS), leading to peroxidative damage to skin membranes and contributing to the exacerbation of lesions. Moreover, these immune cells release growth factors, chemokines, and pro-inflammatory cytokines such as tumor necrosis factor (TNF)- α , interleukin (IL)-6, IL-1 β and IL-17, which interact as a network in the pathogenesis of psoriasis [ 4 ]. The inducible enzyme cyclooxygenase-2 (COX-2) has also been demonstrated to play a pivotal role in skin proliferative disorders through overproduction of pro-inflammatory prostaglandins such as PGE 2 [ 5 ]. Currently, the treatment of psoriasis includes topical agents (corticoids, vitamin Dderivatives, retinoids, and calcineurin inhibitors), photo-chemo-therapy, and systemic treatments (immunosuppressants and biological drugs) [ 6 ]. However, many patients, especially those with moderate to severe generalized psoriasis, are not adequately treated with effective or long-term therapies and most of them have various degrees of side effects. Thus, the development of well-tolerated immune-modulatory topical agents can offer an alternative option for the treatment of psoriatic patients. Microalgae have emerged as a source of bioactive compounds, including lipids, proteins, polysaccharides, and carotenoids, which have attracted the interest of the pharmaceutical industry based on their anti-oxidant, anti-inflammatory, or anti-carcinogenic activity in different skin inflammatory models [ 7 ]. Recently, the anti-inflammatory activity of galactosylglycerides isolated from the marine microalga Isochrysis galbana (I. galbana), including a monogalactosyldiacylglycerol ( MGDG ) fraction [ 8 ] and the pure compound monogalactosylmonoacylglyceride (2S)-1-O-[(6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoyl]-3-Oβ -D-galactopyranosylglycerol ( MGMG-A ) (data not shown), has been reported through the inhibition of TNFα production in LPS-stimulated THP-1 human macrophages. However, data on these compounds’ effects on skin inflammatory pathologies have not yet been collected. Given the interesting anti-inflammatory properties and their high yield of these products in this microalga, we evaluated the preventive effects of the galactosylglycerides MGMG-A and MGDG from I. galbana in a murine model of TPA-induced epidermal hyperplasia by using a topical application of acetone-dissolved glycolipids. However, acetone application onto the skin has been reported to exhibit several drawbacks such as the amount of this organic solvent remaining in contact with the skin, spreading of the formulation and loss of sample, heterogeneity of the dose contacting with the skin, and difficulty of applying the sample [ 9 ]. It is well known that the topical application of bio-compounds requires their incorporation into a carrier that offers stability, good permeation, and sufficient time in contact with the skin. Currently, microalgae products are being used as cosmeceuticals through their incorporation in face and skin care products [ 10 ]. Therefore, our next objective was to use a pharmaceutical carrier to solve the above limitations of glycolipid solutions. The formulation of these substances involves the selection of appropriate combinations of formula ingredients with the aim of exerting a desirable local or systemic effect. Among them, topical formulations of different natures are used, including ointments, creams, and hydrophilic gels, in which the active compound is suspended or dissolved. In the present study, once a topical formulation was selected, we finally aimed to study its effect on a TPA-induced hyperplasia model and determinate its benefit to epidermal skin. Mar. Drugs 2018,16, 2 3 of 19 2. Results 2.1. Effects of Glycolipids on IL-6 and IL-8 Production in TNF-A-Stimulated HaCaT Human Keratinocytes Non-cytotoxic concentrations of the monogalactosylmonoacylglyceride (2S)-1-O-[(6Z,9Z,12Z,15Z) -octadeca-6,9,12,15-tetraenoyl]-3-Oβ -D-galactopyranosylglycerol ( MGMG-A ) and the monogalactosyldiacylglycerol fraction ( MGDG ) were selected to evaluate their effects on pro-inflammatory cytokines IL-6 and IL-8 production in HaCaT cells. The cytotoxic effect of MGMG-A and MGDG fraction was studied using the SRB method, resulting in 100% viability at the tested concentrations (Table S1). TNFα -stimulated HaCaT cells manifested high IL-6 and IL-8 levels in comparison with unstimulated control cells (p< 0.001) (Figure 1). Pre-treatment with the reference compound dexamethasone (Dex) as well as MGMG-A (10, 30, and 50 µ M) and MGDG fraction (10, 30, and 50 µ g/mL) significantly inhibited IL-6 and IL-8 production, with no significant differences between the different tested concentrations. Mar. Drugs 2017, 16, 2 3 of 19 2. Results 2.1. Effects of Glycolipids on IL-6 and IL-8 Production in TNF-Α-Stimulated HaCaT Human Keratinocytes Non-cytotoxic concentrations of the monogalactosylmonoacylglyceride (2S)-1-O-[(6Z,9Z,12Z, 15Z)-octadeca-6,9,12,15-tetraenoyl]-3-O-β-D-galactopyranosylglycerol (MGMG-A) and the monogalactosyldiacylglycerol fraction (MGDG) were selected to evaluate their effects on proinflammatory cytokines IL-6 and IL-8 production in HaCaT cells. The cytotoxic effect of MGMG-A and MGDG fraction was studied using the SRB method, resulting in 100% viability at the tested concentrations (Table S1). TNF-α-stimulated HaCaT cells manifested high IL-6 and IL-8 levels in comparison with unstimulated control cells (p < 0.001) (Figure 1). Pre-treatment with the reference compound dexamethasone (Dex) as well as MGMG-A (10, 30, and 50 µM) and MGDG fraction (10, 30, and 50 µg/mL) significantly inhibited IL-6 and IL-8 production, with no significant differences between the different tested concentrations. Figure 1. Effects of glycolipids from I. galbana on IL-6 and IL-8 production in TNF-α-stimulated HaCaT human keratinocytes. (a) IL-6 levels and (b) IL-8 levels in TNF-α-stimulated HaCaT human keratinocytes. Cells were pre-incubated with the glycolipid MGMG-A (10, 30, 50 µM) and the fraction MGDG (10, 30, 50 µg/mL) for 1 h, and then stimulated with TNF-α (10 ng/mL) for 24 h. Dexamethasone (Dex) was used as a positive reference compound at 1 µM. After 24 h, the production of cytokines in the supernatants was measured by ELISA assay. Results are representative of six independent experiments (n = 6). Values are means with standard errors represented by vertical bars. Mean value was significantly different compared with the control group (*** p < 0.001; Student t test). Mean value was significantly different compared with the TNF-α group (+ p < 0.05, ++ p < 0.01, +++ p < 0.001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test). 2.2. Topical Application of Acetone-Dissolved Glycolipids Inhibits Skin Inflammation and Hyperplasia in the Murine TPA-Induced Model We studied the effect of MGMG-A and MGDG on the murine TPA-induced epidermal hyperplasia model, which reproduces certain biochemical and histopathological parameters typical of human psoriasis [11]. TPA administration to mouse skin resulted in the development of macroscopic lesions (Figure 2a) and skin edema, confirmed by a higher weight of the 1 cm2 punch CTNF-αDex 10 30 50 10 30 50 0 50 100 150 *** + ++ +++++ MGMG-A MGDG TNF-α IL-6 production (pg/mL) CTNF-αDex 10 30 50 10 30 50 0 20000 40000 60000 MGMG-A MGDG TNF-α *** +++ ++ ++ ++ ++ ++ IL-8 production (pg/mL) (a) (b) Figure 1. Effects of glycolipids from I. galbana on IL-6 and IL-8 production in TNFα -stimulated HaCaT human keratinocytes. ( a ) IL-6 levels and ( b ) IL-8 levels in TNFα -stimulated HaCaT human keratinocytes. Cells were pre-incubated with the glycolipid MGMG-A (10, 30, 50 µ M) and the fraction MGDG (10, 30, 50 µ g/mL) for 1 h, and then stimulated with TNFα (10 ng/mL) for 24 h. Dexamethasone (Dex) was used as a positive reference compound at 1 µ M. After 24 h, the production of cytokines in the supernatants was measured by ELISA assay. Results are representative of six independent experiments (n= 6). Values are means with standard errors represented by vertical bars. Mean value was significantly different compared with the control group (*** p< 0.001; Student ttest) . Mean value was significantly different compared with the TNFα group (+ p< 0.05, ++ p< 0.01, +++ p< 0.001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test). 2.2. Topical Application of Acetone-Dissolved Glycolipids Inhibits Skin Inflammation and Hyperplasia in the Murine TPA-Induced Model We studied the effect of MGMG-A and MGDG on the murine TPA-induced epidermal hyperplasia model, which reproduces certain biochemical and histopathological parameters typical of human psoriasis [ 11 ]. TPA administration to mouse skin resulted in the development of macroscopic lesions (Figure 2a) and skin edema, confirmed by a higher weight of the 1 cm 2 punch biopsies compared Mar. Drugs 2018,16, 2 4 of 19 with the sham group (p< 0.001) (Figure 2b). Topical treatment with Dex (200 µ M), MGMG-A and MGDG (200 µ M or 200 µ g/mL, respectively) 30 min prior to TPA application inhibited macroscopic damage and the skin punch weight (p< 0.001 and p< 0.05, respectively), suggesting an inhibition of skin edema (Figure 2b). We next examined hematoxylinand eosin-stained sections of mouse skin (Figure 2c). Consistent with macroscopic changes, TPA-treated animals exhibited a clear evidence of edema, epidermal hyperplasia, and massive neutrophilic infiltration compared with the sham (Figure 2c). Moreover, a marked increase in epidermal thickness was evident in the TPA group (p< 0.001) (Figure 2d). These results correlated with increased MPO activity, an established marker for inflammatory cell infiltration into the skin (Figure 2e). Treatment with the pure compound and glycolipid fraction markedly prevented epidermal hyperplasia (p< 0.01 and p< 0.001, respectively) (Figure 2c,d), which was associated with a reduction in MPO activity, being significant for MGMG-A (p< 0.01) (Figure 2e). Mar. Drugs 2017, 16, 2 4 of 19 biopsies compared with the sham group (p < 0.001) (Figure 2b). Topical treatment with Dex (200 µM), MGMG-A and MGDG (200 µM or 200 µg/mL, respectively) 30 min prior to TPA application inhibited macroscopic damage and the skin punch weight (p < 0.001 and p < 0.05, respectively), suggesting an inhibition of skin edema (Figure 2b). We next examined hematoxylinand eosinstained sections of mouse skin (Figure 2c). Consistent with macroscopic changes, TPA-treated animals exhibited a clear evidence of edema, epidermal hyperplasia, and massive neutrophilic infiltration compared with the sham (Figure 2c). Moreover, a marked increase in epidermal thickness was evident in the TPA group (p < 0.001) (Figure 2d). These results correlated with increased MPO activity, an established marker for inflammatory cell infiltration into the skin (Figure 2e). Treatment with the pure compound and glycolipid fraction markedly prevented epidermal hyperplasia (p < 0.01 and p < 0.001, respectively) (Figure 2c,d), which was associated with a reduction in MPO activity, being significant for MGMG-A (p < 0.01) (Figure 2e). Figure 2. Topical application of acetone-dissolved glycolipids from I. galbana inhibits skin inflammation and hyperplasia on the murine 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced model. The glycolipid MGMG-A (200 µM per site) or the fraction MGDG (200 µg/mL per site) were topically administered 30 min before TPA application (2 nmol per zone) during three consecutive days. Dex was used as a positive reference compound (200 µg per site). (a) Representative images of macroscopic appearance of the dorsal skin; (b) skin edema as punch biopsy; weight of edema (mg/cm 2 ) was employed as marker of inflammatory skin process; (c) histological appearance of mouse dorsal skin after H&E-staining (n = 4); Bar = 100 µm. Original magnification 100×. (d) Epidermal thickness assessment in H&E-stained skin slides; (e) yeloperoxidase (MPO) activity in dorsal skin. Values are means with standard errors represented by vertical bars. Data are means ± SEM (n = 10 mice/group). Mean value was significantly different compared with the sham group (*** p < 0.001; Student t test). Mean value was significantly different compared with TPA group (+ p < 0.05, ++ p < 0.01, +++ p < 0.001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test). Figure 2. Topical application of acetone-dissolved glycolipids from I. galbana inhibits skin inflammation and hyperplasia on the murine 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced model. The glycolipid MGMG-A (200 µ M per site) or the fraction MGDG (200 µ g/mL per site) were topically administered 30 min before TPA application (2 nmol per zone) during three consecutive days. Dex was used as a positive reference compound (200 µ g per site). ( a ) Representative images of macroscopic appearance of the dorsal skin; ( b ) skin edema as punch biopsy; weight of edema (mg/cm 2 ) was employed as marker of inflammatory skin process; ( c ) histological appearance of mouse dorsal skin after H&E-staining (n= 4); Bar = 100 µ m. Original magnification 100 × . ( d ) Epidermal thickness assessment in H&E-stained skin slides; ( e ) yeloperoxidase (MPO) activity in dorsal skin. Values are means with standard errors represented by vertical bars. Data are means ± SEM (n= 10 mice/group). Mean value was significantly different compared with the sham group (*** p< 0.001; Student ttest). Mean value was significantly different compared with TPA group (+ p< 0.05, ++ p< 0.01, +++ p< 0.001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test). Mar. Drugs 2018,16, 2 5 of 19 To support the beneficial effects of glycolipids on skin inflammation, we analyzed the production of several pro-inflammatory cytokines that are highly involved in psoriasis as well as the anti-inflammatory cytokine IL-10. Immune cell infiltration detected in the histological examination of the skin from TPA-treated mice correlated with increased levels of the pro-inflammatory cytokines TNFα , IL-1 β , IL-6 and IL-17, in comparison with the sham group (p< 0.05, p< 0.01, p< 0.01 , and p< 0.001 , respectively) (Figure 3). In accordance with the reduction of the skin edema, the production of TNFα , IL-6, and IL-17 was significantly reduced in animals treated with the glycolipid MGMG-A (p< 0.05, p< 0.001 ,p< 0.05, respectively) (Figure 3a–d). Regarding the fraction MGDG , its application resulted in a strong significant suppression of TNFα and IL-6 levels (p< 0.01, and p< 0.001) comparable to Dex (Figure 3a,c). IL-10 production analysis revealed increased levels in the TPA group when compared with the sham (p< 0.05). Nevertheless, pre-treatments showed lower IL-10 levels when compared to the TPA group, reflecting similar values to the sham (Figure 3e). Mar. Drugs 2017, 16, 2 5 of 19 To support the beneficial effects of glycolipids on skin inflammation, we analyzed the production of several pro-inflammatory cytokines that are highly involved in psoriasis as well as the anti-inflammatory cytokine IL-10. Immune cell infiltration detected in the histological examination of the skin from TPA-treated mice correlated with increased levels of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-17, in comparison with the sham group (p < 0.05, p < 0.01, p < 0.01, and p < 0.001, respectively) (Figure 3). In accordance with the reduction of the skin edema, the production of TNF-α, IL-6, and IL-17 was significantly reduced in animals treated with the glycolipid MGMG-A (p < 0.05, p < 0.001, p < 0.05, respectively) (Figure 3a–d). Regarding the fraction MGDG, its application resulted in a strong significant suppression of TNF-α and IL-6 levels (p < 0.01, and p < 0.001) comparable to Dex (Figure 3a,c). IL-10 production analysis revealed increased levels in the TPA group when compared with the sham (p < 0.05). Nevertheless, pre-treatments showed lower IL-10 levels when compared to the TPA group, reflecting similar values to the sham (Figure 3e). Figure 3. Effect of the glycolipid MGMG-A and the fraction MGDG from I. galbana on the production of cytokines in skin homogenates in the murine 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced hyperplasia model. (a) TNF-α (pg/mg tissue); (b) IL-1β (pg/mg tissue); (c) IL-6 (pg/mg tissue); (d) IL17 (pg/mg tissue); and (e) IL-10 (pg/mg tissue). Values are means with standard errors represented by vertical bars. Data are means ± SEM (n = 10). Mean value was significantly different compared with the sham group (* p < 0.05, ** p < 0.01, *** p < 0.001; Student t test). Mean value was significantly different compared with TPA group (+ p < 0.05, ++ p < 0.01, +++ p < 0.001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test). 2.3. Effect of the Formulation The development of topical formulations implies the selection of excipients leading to improvement in the drug skin delivery. In order to evaluate the skin accumulation and penetration properties of the examined formulations, sections of the mice skin were analyzed by confocal laser scanning microscopy (CLSM) at the end of permeation experiments. For these studies, rhodamine Sham TPA Dex MGMGA MGDG 0 10 20 30 ** ++ +++ +++ TPA IL-6 (pg/mg tissue) Sham TPA Dex MGMG-A MGDG 0 5 10 15 TPA ++ ++ * TNF-α (pg/mg tissue) Sham TPA Dex MGMG-A MGDG 0.0 0.5 1.0 1.5 2.0 TPA * * IL-10 (pg/mg tissue) Sham TPA Dex MGMGA MGDG 0.0 0.5 1.0 1.5 ++ TPA *** + IL-17 (pg/mg tissue) Sham TPA Dex MGMG-A MGDG 0 5 10 15 20 ++ TPA ** + IL-1β (pg/mg tissue) (a) (b) (c) (d) (e) Figure 3. Effect of the glycolipid MGMG-A and the fraction MGDG from I. galbana on the production of cytokines in skin homogenates in the murine 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced hyperplasia model. ( a ) TNFα (pg/mg tissue); ( b ) IL-1 β (pg/mg tissue); ( c ) IL-6 (pg/mg tissue); ( d ) IL-17 (pg/mg tissue); and ( e ) IL-10 (pg/mg tissue). Values are means with standard errors represented by vertical bars. Data are means ± SEM (n= 10). Mean value was significantly different compared with the sham group (* p< 0.05, ** p< 0.01, *** p< 0.001; Student ttest). Mean value was significantly different compared with TPA group (+ p< 0.05, ++ p< 0.01, +++ p< 0.001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test). 2.3. Effect of the Formulation The development of topical formulations implies the selection of excipients leading to improvement in the drug skin delivery. In order to evaluate the skin accumulation and penetration Mar. Drugs 2018,16, 2 6 of 19 properties of the examined formulations, sections of the mice skin were analyzed by confocal laser scanning microscopy (CLSM) at the end of permeation experiments. For these studies, rhodamine 6G, a fluorescent hydrophobic probe, was added as a model drug [ 12 ]. The penetration depth of the fluorescent probe and the relative intensity of fluorescence in the skin layers were compared in three types of semisolid formulations (gel, cream, and ointment described in Section 4.10). Confocal images revealed that all the examined formulations penetrated deeply into the stratum corneum (SC) and diffused into the whole skin thickness, except for the ointment (Figure 4a). Cream showed the higher probe permeation in 24 h, following the control formulation containing only ethanol and incorporated into Carbopol®gels. However, the rhodamine 6G incorporated into the lipid ointment was observed to show a low penetration capacity. In addition to the effect of the carrier nature, deeper skin layers were more easily visualized when ethanol was present in the composition, as occurred in all the formulations except for the ointment, where the labeling probe was dissolved in propylene glycol. The quantitative parameters of histogram distribution revealed a higher fluorescent intensity and accumulation of rhodamine 6G in the presence of ethanol (Figure 4b). Among all the samples, the cream system offered the higher fluorescence intensity and adequate symmetry of the normal distribution of histogram. Mar. Drugs 2017, 16, 2 6 of 19 6G, a fluorescent hydrophobic probe, was added as a model drug [12]. The penetration depth of the fluorescent probe and the relative intensity of fluorescence in the skin layers were compared in three types of semisolid formulations (gel, cream, and ointment described in Section 4.10). Confocal images revealed that all the examined formulations penetrated deeply into the stratum corneum (SC) and diffused into the whole skin thickness, except for the ointment (Figure 4a). Cream showed the higher probe permeation in 24 h, following the control formulation containing only ethanol and incorporated into Carbopol ® gels. However, the rhodamine 6G incorporated into the lipid ointment was observed to show a low penetration capacity. In addition to the effect of the carrier nature, deeper skin layers were more easily visualized when ethanol was present in the composition, as occurred in all the formulations except for the ointment, where the labeling probe was dissolved in propylene glycol. The quantitative parameters of histogram distribution revealed a higher fluorescent intensity and accumulation of rhodamine 6G in the presence of ethanol (Figure 4b). Among all the samples, the cream system offered the higher fluorescence intensity and adequate symmetry of the normal distribution of histogram. Figure 4. Effect of the vehicle composition and physicochemical properties of the drug on the permeation characteristics. (a) Confocal micrographs of mice skin cross sections corresponding to rhodamine-loaded ethanolic control solution, cream, hydrogel, and ointment. Bar = 200 µm. Original magnification 100×; (b) Numerical data corresponding to the intensity histogram for each sample. Mean: arithmetical mean value; mean energy: average image energy; RMS: root mean square value; skewness: skewness of the distribution; (c) Ex vivo permeability percentages of MGDG formulations in 24 h (ethanolic control solution, cream, and ointment). 2.4. Ex Vivo Permeation Studies Permeation profiles of MGDG from the ethanol solution and cream through mice skin membranes were obtained from the equation described in Section 4.11. Dex-loaded cream was used as the control formulation. Results showed that permeation of MGDG from the cream (100 ± 1.9% of the applied dose) was twice that observed from the ethanolic control solution (49.3 ± 3.5%). On the other hand, the permeated amount of Dex from cream was lower (15 ± 3.1%) in comparison with the other preparations (Figure 4c). This value can be attributed to the lower partition coefficient of this Figure 4. Effect of the vehicle composition and physicochemical properties of the drug on the permeation characteristics. ( a ) Confocal micrographs of mice skin cross sections corresponding to rhodamine-loaded ethanolic control solution, cream, hydrogel, and ointment. Bar = 200 µ m. Original magnification 100 × ; ( b ) Numerical data corresponding to the intensity histogram for each sample. Mean: arithmetical mean value; mean energy: average image energy; RMS: root mean square value; skewness: skewness of the distribution; ( c ) Ex vivo permeability percentages of MGDG formulations in 24 h (ethanolic control solution, cream, and ointment). 2.4. Ex Vivo Permeation Studies Permeation profiles of MGDG from the ethanol solution and cream through mice skin membranes were obtained from the equation described in Section 4.11. Dex-loaded cream was used as the control formulation. Results showed that permeation of MGDG from the cream (100 ± 1.9% of the applied Mar. Drugs 2018,16, 2 7 of 19 dose) was twice that observed from the ethanolic control solution (49.3 ± 3.5%). On the other hand, the permeated amount of Dex from cream was lower (15 ± 3.1%) in comparison with the other preparations (Figure 4c). This value can be attributed to the lower partition coefficient of this molecule (logP 1.83) compared to MGDG , whose lipophilicity resembled a reference diacylglycerol in terms of lipophilic acyl groups (logP 3.85) [ 13 ]. It is well known that the partition coefficient has been widely used as a measurement for defining the lipophilicity of a drug and the diffusion efficiency across the membranes [14]. 2.5. Topical Pre-Treatment with MGDG-Cream Decreases Skin Inflammation and Hyperplasia in the Murine TPA-Induced Model We evaluated the effect of the MGDG -cream formulation on the murine TPA-induced epidermal hyperplasia model. This cream formulation enabled lipid preservation and high permeation in comparison with the acetone vehicle. After treatment with TPA for three consecutive days, mice exhibited the expected psoriasis phenotype, including peeling, erythema, and thickening of the back skin, accompanied by a marked increase in dorsal skin thickness, weight, and substantial inflammatory cell infiltration in the dermis (p< 0.001) (Figure 5). Pre-treatment with MGDG -cream (100 mg per site containing 200 µ g of MGDG ) attenuated the macroscopic lesions formation (Figure 5a) and significantly reduced skin edema (p< 0.001) when compared with the cream-TPA group (Figure 5b). These results were accompanied by a clear inhibition of MPO activity following MGDG -cream administration (p< 0.001); interestingly, the glycolipid formulation was as effective as the reference topical treatment with Dex-cream, reaching similar levels to those in the healthy group (Figure 4e). Histological analysis of H&E-stained skin lesions confirmed an improvement in the microscopic features of hyperplasia in mice treated with MGDG -cream, evidenced by a reduction of epidermal thickness (p< 0.05) in relation to the cream-TPA group (Figure 5c,d). It is known that COX-2 plays an important role in skin pathologies. Immunohistochemical analysis of this enzyme showed that stimulation with TPA significantly increased COX-2-positive cell numbers (p< 0.001), predominantly localized in the epidermal layer (Figure 6a), when compared with the sham group. As shown in Figure 6b, skin from MGDG -cream-treated mice revealed a significant downregulation in the number of epidermal COX-2-positive stained cells in comparison with the cream-TPA group (p< 0.001). Mar. Drugs 2017, 16, 2 7 of 19 molecule (logP 1.83) compared to MGDG, whose lipophilicity resembled a reference diacylglycerol in terms of lipophilic acyl groups (logP 3.85) [13]. It is well known that the partition coefficient has been widely used as a measurement for defining the lipophilicity of a drug and the diffusion efficiency across the membranes [14]. 2.5. Topical Pre-Treatment with MGDG-Cream Decreases Skin Inflammation and Hyperplasia in the Murine TPA-Induced Model We evaluated the effect of the MGDG-cream formulation on the murine TPA-induced epidermal hyperplasia model. This cream formulation enabled lipid preservation and high permeation in comparison with the acetone vehicle. After treatment with TPA for three consecutive days, mice exhibited the expected psoriasis phenotype, including peeling, erythema, and thickening of the back skin, accompanied by a marked increase in dorsal skin thickness, weight, and substantial inflammatory cell infiltration in the dermis (p < 0.001) (Figure 5). Pre-treatment with MGDG-cream (100 mg per site containing 200 µg of MGDG) attenuated the macroscopic lesions formation (Figure 5a) and significantly reduced skin edema (p < 0.001) when compared with the cream-TPA group (Figure 5b). These results were accompanied by a clear inhibition of MPO activity following MGDGcream administration (p < 0.001); interestingly, the glycolipid formulation was as effective as the reference topical treatment with Dex-cream, reaching similar levels to those in the healthy group (Figure 4e). Histological analysis of H&E-stained skin lesions confirmed an improvement in the microscopic features of hyperplasia in mice treated with MGDG-cream, evidenced by a reduction of epidermal thickness (p < 0.05) in relation to the cream-TPA group (Figure 5c,d). It is known that COX2 plays an important role in skin pathologies. Immunohistochemical analysis of this enzyme showed that stimulation with TPA significantly increased COX-2-positive cell numbers (p < 0.001), predominantly localized in the epidermal layer (Figure 6a), when compared with the sham group. As shown in Figure 6b, skin from MGDG-cream-treated mice revealed a significant downregulation in the number of epidermal COX-2-positive stained cells in comparison with the cream-TPA group (p < 0.001). Figure 5. Cont. Mar. Drugs 2018,16, 2 8 of 19 Mar. Drugs 2017, 16, 2 8 of 19 Figure 5. Topical pre-treatment with cream containing the glycolipid fraction MGDG from I. galbana decreases skin inflammation and hyperplasia on the murine 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced model. Glycolipid cream formulation (100 mg per site containing 200 µg of MGDG), dexamethasone (Dex) (100 mg per site, equivalent at 200 µg of compound), or vehicle (cream with a comparable volume of ethanol) was topically administered from two days before hyperplasia induction and 30 min after each TPA application (2 nmol per zone for three consecutive days). Dex was used as the positive reference compound. (a) Representative images of macroscopic appearance of the dorsal skin; (b) determination of skin edema as punch biopsy weight; (c) histological appearance of mouse dorsal skin after H&E-staining (n = 4); Bar = 100 µm. Original magnification 100×. (d) Epidermal thickness assessment in H&E-stained skin slides; (e) myeloperoxidase (MPO) activity. Values are means with standard errors represented by vertical bars. Data are means ± SEM (n = 10 mice/group). Mean value was significantly different compared with the sham group (*** p < 0.001; Student t test). Mean value was significantly different compared with cream-TPA group (+ p < 0.05, +++ p < 0.001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test). Figure 5. Topical pre-treatment with cream containing the glycolipid fraction MGDG from I. galbana decreases skin inflammation and hyperplasia on the murine 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced model. Glycolipid cream formulation (100 mg per site containing 200 µ g of MGDG ), dexamethasone (Dex) (100 mg per site, equivalent at 200 µ g of compound), or vehicle (cream with a comparable volume of ethanol) was topically administered from two days before hyperplasia induction and 30 min after each TPA application (2 nmol per zone for three consecutive days). Dex was used as the positive reference compound. ( a ) Representative images of macroscopic appearance of the dorsal skin; ( b ) determination of skin edema as punch biopsy weight; ( c ) histological appearance of mouse dorsal skin after H&E-staining (n= 4); Bar = 100 µ m. Original magnification 100 × . ( d ) Epidermal thickness assessment in H&E-stained skin slides; ( e ) myeloperoxidase (MPO) activity. Values are means with standard errors represented by vertical bars. Data are means ± SEM (n= 10 mice/group). Mean value was significantly different compared with the sham group (*** p< 0.001; Student ttest). Mean value was significantly different compared with cream-TPA group (+ p< 0.05, +++ p< 0.001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test). Mar. Drugs 2017, 16, 2 8 of 19 Figure 5. Topical pre-treatment with cream containing the glycolipid fraction MGDG from I. galbana decreases skin inflammation and hyperplasia on the murine 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced model. Glycolipid cream formulation (100 mg per site containing 200 µg of MGDG), dexamethasone (Dex) (100 mg per site, equivalent at 200 µg of compound), or vehicle (cream with a comparable volume of ethanol) was topically administered from two days before hyperplasia induction and 30 min after each TPA application (2 nmol per zone for three consecutive days). Dex was used as the positive reference compound. (a) Representative images of macroscopic appearance of the dorsal skin; (b) determination of skin edema as punch biopsy weight; (c) histological appearance of mouse dorsal skin after H&E-staining (n = 4); Bar = 100 µm. Original magnification 100×. (d) Epidermal thickness assessment in H&E-stained skin slides; (e) myeloperoxidase (MPO) activity. Values are means with standard errors represented by vertical bars. Data are means ± SEM (n = 10 mice/group). Mean value was significantly different compared with the sham group (*** p < 0.001; Student t test). Mean value was significantly different compared with cream-TPA group (+ p < 0.05, +++ p < 0.001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test). Figure 6. Cont. Mar. Drugs 2018,16, 2 9 of 19 Mar. Drugs 2017, 16, 2 9 of 19 Figure 6. Topical pre-treatment with cream containing the glycolipid fraction MGDG from I. galbana attenuates 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression in mouse skin. (a) Representative photographs of epidermal COX-2 distribution by immunohistochemical detection; Bar = 200 µm. Original magnification 200×. (b) Positive COX-2 epidermal layer was assessed by counting the COX-2 positive cells versus total cells in different immunostained dorsal skin sections per animal. Representative photomicrographs showing positive epidermal COX-2 staining yielded a brown product. Values are means with standard errors represented by vertical bars. Data are means ± SEM (n = 4). Mean value was significantly different compared with the sham group (*** p < 0.001; Student’s t test). Mean value was significantly different to the cream-TPA group (+++ p < 0.001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test). 3. Discussion Inflammatory skin diseases have a significant impact on society, with atopic dermatitis, acne, sunburn, and psoriasis being the most common manifestations. Psoriasis is a chronic, autoimmune, and multisystem inflammatory disease that affects 2–4% of the population [15]. Currently, conventional treatments for this disease are based on the degree of severity and range from topical therapy and systemic agents through to phototherapy or combinations of those. However, many of these therapies are not recommended for the vast majority of patients afflicted with mild forms of psoriasis due to their potential risk [16]. Therefore, other treatment approaches for mild psoriasis that require topical therapy only are still needed. In this regard, natural products provide some options for increasing the safety and efficacy in the management of this pathology [17]. Microalgae species are a promising source of a variety of bioactive molecules, including polar lipids such as glycolipids. Lipid-enriched extracts or pure glycolipids have previously demonstrated their in vitro antiinflammatory [18,19] and antitumor properties [20], which make them suitable candidates for further investigation. However, the use of galactosylglycerides to prevent skin pathologies such as psoriasis has not been previously evidenced. In this sense, we have recently observed that this kind of metabolite protects human HaCaT keratinocytes against UVB radiation through inhibition of ROS generation and a decrease in the production of the pro-inflammatory cytokine IL-6 (data not shown). These findings suggest that this type of molecule could play a main role not only in protecting the skin from UVB exposure but also in preventing the skin inflammatory process. In this context, we aimed to evaluate the anti-inflammatory effects of the glycolipid MGMG-A and MGDG fraction in an experimental TPA-induced hyperplasia model in mice. Moreover, we used different semisolid formulations in which the glycolipid was loaded in order to facilitate its topical application and to enhance the permeation mechanism compared to conventional liquid preparations. Firstly, we tried to demonstrate the anti-inflammatory potential of the compounds under study in the in vitro model of TNF-α-stimulated HaCaT keratinocytes. This cytokine plays a crucial role in the pathogenesis of skin inflammatory diseases such as psoriasis [21]. We observed that pretreatment with the compound MGMG-A or the fraction MGDG significantly reduced the production of the pro-inflammatory cytokines IL-6 and IL-8 in stimulated HaCaT keratinocytes. These results encouraged us to evaluate the preventive effects of these products on TPA-induced Figure 6. Topical pre-treatment with cream containing the glycolipid fraction MGDG from I. galbana attenuates 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression in mouse skin. ( a ) Representative photographs of epidermal COX-2 distribution by immunohistochemical detection; Bar = 200 µ m. Original magnification 200 × . ( b ) Positive COX-2 epidermal layer was assessed by counting the COX-2 positive cells versus total cells in different immunostained dorsal skin sections per animal. Representative photomicrographs showing positive epidermal COX-2 staining yielded a brown product. Values are means with standard errors represented by vertical bars. Data are means ± SEM (n= 4) . Mean value was significantly different compared with the sham group ( *** p< 0.001 ; Student’s ttest) . Mean value was significantly different to the cream-TPA group (+++ p< 0.001; one-way ANOVA followed by Bonferroni’s Multiple Comparison test). 3. Discussion Inflammatory skin diseases have a significant impact on society, with atopic dermatitis, acne, sunburn, and psoriasis being the most common manifestations. Psoriasis is a chronic, autoimmune, and multisystem inflammatory disease that affects 2–4% of the population [ 15 ]. Currently, conventional treatments for this disease are based on the degree of severity and range from topical therapy and systemic agents through to phototherapy or combinations of those. However, many of these therapies are not recommended for the vast majority of patients afflicted with mild forms of psoriasis due to their potential risk [ 16 ]. Therefore, other treatment approaches for mild psoriasis that require topical therapy only are still needed. In this regard, natural products provide some options for increasing the safety and efficacy in the management of this pathology [ 17 ]. Microalgae species are a promising source of a variety of bioactive molecules, including polar lipids such as glycolipids. Lipid-enriched extracts or pure glycolipids have previously demonstrated their in vitro anti-inflammatory [ 18 , 19 ] and antitumor properties [ 20 ], which make them suitable candidates for further investigation. However, the use of galactosylglycerides to prevent skin pathologies such as psoriasis has not been previously evidenced. In this sense, we have recently observed that this kind of metabolite protects human HaCaT keratinocytes against UVB radiation through inhibition of ROS generation and a decrease in the production of the pro-inflammatory cytokine IL-6 (data not shown). These findings suggest that this type of molecule could play a main role not only in protecting the skin from UVB exposure but also in preventing the skin inflammatory process. In this context, we aimed to evaluate the anti-inflammatory effects of the glycolipid MGMG-A and MGDG fraction in an experimental TPA-induced hyperplasia model in mice. Moreover, we used different semisolid formulations in which the glycolipid was loaded in order to facilitate its topical application and to enhance the permeation mechanism compared to conventional liquid preparations. Firstly, we tried to demonstrate the anti-inflammatory potential of the compounds under study in the in vitro model of TNFα -stimulated HaCaT keratinocytes. This cytokine plays a crucial role in the pathogenesis of skin inflammatory diseases such as psoriasis [ 21 ]. We observed that pre-treatment with the compound MGMG-A or the fraction MGDG significantly reduced the production of the pro-inflammatory cytokines IL-6 and IL-8 in stimulated HaCaT keratinocytes. These results encouraged Mar. Drugs 2018,16, 2 16 of 19 4.12. TPA-Induced Epidermal Hyperplasia Model and MGDG-Cream Treatment Given acetone is considered moderately toxic and irritant and we aim to elaborate an optimal glycolipid-containing pharmacological formulation, we evaluated the effect of MGDG -cream on TPA-induced hyperplasia in murine skin. The dorsal skin of female Swiss mice was shaved as described above. In this second experimental model (n= 10 per group), a pre-treatment was carried out two days (day − 2 and day − 1) before the first TPA challenge (day 0). MGDG -cream formulation (100 mg per site, containing 200 µ g of MGDG dissolved in ethanol at 10 µ g/ µ L), Dex-cream (100 mg per site, equivalent at 200 µ g of compound dissolved in ethanol at 10 µ g/ µ L) or vehicle (cream with a comparable volume of ethanol) were applied to the shaved dorsal skin of the animals in an area of 1 cm 2 using a syringe. On day 0, TPA (2 nmol per site, dissolved in ethanol) was topically applied to the same areas using a micropipette (total volume of 20 µ L). After 1 h, MGDG -cream, Dex-cream, or vehicle was topically administered. Mice were anesthetized with ketamine (100 mg/kg of animal) and diazepam (5 mg/kg of animal) during treatments and TPA challenge. This protocol was repeated for two consecutive days. Mice were sacrificed on day 3 by cervical dislocation and punch biopsies from the treated dorsal skin were weighed to evaluate edema, before further processing for MPO activity and histology. 4.13. Immunohistochemical Analysis Staining of COX-2 was performed using a streptavidin-biotin-peroxidase method [ 46 ]. Paraffin-embedded dorsal skin sections (7 µ m) 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. Sections were rinsed with PBS for 10 min. Nonspecific adsorption was minimized by incubating sections in normal horse serum (Vectastain Kit; Vector Laboratories, Burlingame, CA, USA) for 20 min. Subsequently, slides were incubated with rabbit polyclonal anti-COX-2 antibody (Cayman Chemical, Ann Arbor, MI, USA) (1:300) overnight at 4 ◦ C. Then, slides were treated with anti-mouse IgG antibody for 30 min and incubated with the streptavidin–peroxidase complex for 30 min at room temperature (Vectastain Kit; Vector Laboratories, CA, USA). The enzymatic activities were developed with 3,3 0 -diaminobenzidine (DAB), and the sections were counterstained with hematoxylin. Negative control sections were treated in the same way, omitting the primary antibody [ 47 ]. COX-2 immunoreactivity was examined on all sections using a microscope Olympus BX61 (Olympus Optical Co. Ltd., Tokyo, Japan). The quantification of immunohistochemical data was done by counting the number of immunostained brown cells as the percent of total epidermal cells from 10 microscopic fields of immunostained tissues per animal. 4.14. 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. pvalues < 0.05 were considered statistically significant. In the histological experiment, results shown are representative of at least four independent experiments performed on different days. 5. Conclusions In conclusion, our study demonstrates for the first time the preventive effects of topical administration of the glycolipid MGMG-A or a fraction MGDG from I. galbana, in the inflammatory model of TPA-induced skin hyperplasia. These actions may be associated with a reduction of Mar. Drugs 2018,16, 2 17 of 19 edema, leukocyte infiltration, pro-inflammatory cytokines production and COX-2 expression in skin mouse. Topical application of MGDG -cream enhanced the sample permeability and consequently, increased the preventive effects of this product. Future studies are needed to expand the vision of the mechanisms by which these lipid products improve skin inflammation and will support their potential use in the development of effective therapeutic strategies for skin pathologies as psoriasis. Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/16/1/2/s1, Table S1: Viability of HaCaT human keratinocytes treated with different concentrations of MGMG-A and MGDG fraction isolated from the microalgae Isochrysis galbana. Acknowledgments: This study was supported by grants from Ministerio de Economía y Competitividad MICIIN INNPACTO-IPT-2012-1370-060000 and Consejería de Innovación, Ciencia y Empresa-Junta de Andalucía POLFANAT-P12-AGR-430. The authors thank “Centro de Investigación, Tecnología e Innovación” of the University of Seville for providing technical assistance. Author Contributions: María del Carmen Terencio, Elena Talero, Virginia Motilva, and Javier Ávila-Román designed the study protocol; Carolina de los Reyes performed chemical characterization of glycolipids from I. galbana; Azahara Rodríguez-Luna, Elena Talero, and Javier Ávila-Román conducted in vivo and histological experiments and analyzed the data; Azahara Rodríguez-Luna, María Luisa González-Rodríguez, and Antonio M. Rabasco executed technologic experiments; Azahara Rodríguez-Luna, Elena Talero, María Luisa González-Rodríguez, Virginia Motilva, and Javier Ávila-Román wrote the draft of the manuscript. All the authors critically reviewed and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Abbreviations CLSM confocal laser scanning microscopy Dex dexamethasone IL Interleukin I. galbana Isochrysis galbana MGDG Monogalactosyldiacylglycerol fraction MGMG-A Monogalactosylmonoacylglyceride (2S)-1-O-[(6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoyl]- 3-O-β-D-galactopyranosylglycerol MPO myeloperoxidase RMS root mean square SC stratum corneum TNF-αTumor Necrosis Factor alpha TPA 12-O-tetradecanoylphorbol-13-acetate References 1. An, J.; Li, Z.; Dong, Y.; Ren, J.; Huo, J. Amentoflavone protects against psoriasis-like skin lesion through suppression of NF-κB-mediated inflammation and keratinocyte proliferation. Mol. Cell. Biochem. 2016,413, 87–95. [CrossRef] [PubMed] 2. Arasa, J.; Martos, P.; Terencio, M.C.; Valcuende-Cavero, F.; Montesinos, M.C. Topical application of the adenosine A2A receptor agonist CGS-21680 prevents phorbol-induced epidermal hyperplasia and inflammation in mice. Exp. Dermatol. 2014,23, 555–560. [CrossRef] [PubMed] 3. 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