The green microalga Tetraselmis suecica reduces oxidative stress and induces repairing mechanisms in human cells

Green microalgae contain many active pigments such as carotenoids having antioxidant and protective activity on human cells. Here we investigate the biological activity of an ethanol/water extract of the marine green microalga Tetraselmis suecica containing high levels of carotenoids such as the xanthophylls lutein, violaxanthin, neoxanthin, antheraxanthin and loroxanthin esters. This extract has a strong antioxidant and repairing activity in the human lung cancer cell line (A549) as shown by the increased expression of dehydrocholesterol reductase-24 (DHCR24) and prostaglandin reductase 1 (PTGR1) genes and proteins. The extract also reduces prostaglandin E2 (PGE2) levels in cells damaged by H2O2 and has tissue repairing effects on reconstructed human epidermal tissue cells (EpiDermTM) indicating a potential cosmeceutical activity of this microalgal species.

and 400 μ g ml −1 ) with respect to the negative control. Within the experimental error, there was a plateau effect already at 2 μ g ml −1 of the extract and a further increase of this concentration did not affect cell viability. The expression of genes involved in oxidative stress and repairing pathways (Supplementary information, Table S2) were analyzed in A549 cells treated with 100, 200 and 400 μ g ml −1 of extract alone (Fig. 4D) and 100, 200 and 400 μ g ml −1 of extract after 1 h of exposure to 30 mM of H 2 O 2 (Fig. 4A,B and C respectively) since lower extract concentrations did not induce changes in gene and protein expression. Gene expression results are reported after 2 h of treatment since several factors implicated in oxidative damage repairing pathways were already expressed and activated after this time interval. Control genes for real-time qPCR were actin-beta (ACTB), beta-2-microglobulin (B2M), hypoxanthine phosphoribosyltransferase (HPRT1) and ribosomal protein large P 0 (RPLP 0 ), the expression of which remained constant. For gene expression studies, we chose the three highest concentrations (100, 200 and 400 μ g ml −1 ) that showed strong repairing activities even if we observed a slight cytotoxicity (about 20%) at the two highest concentrations (200 and 400 μ g ml −1 ). Notwithstanding the toxicity, there was a significant dose-dependent activation of specific oxidative stress response mechanisms without activating genes involved in cell death programs with all three concentrations. This suggests that the slight cytotoxicity did not inhibit the repairing activity of the extract.
Scientific RepoRts | 7:41215 | DOI: 10.1038/srep41215 treatment after 1 h of exposure to H 2 O 2 (from − 1.1 to 3.1, 6.5 and 11.2, and from 1.9 to 2.0, 2.8 and 6.7-fold change, respectively). Since DHCR24, GPX4 and PTGR1 genes play crucial roles in antioxidant/anti-inflammatory cell signaling pathways, A549 cells were treated with 200 and 400 μ g ml −1 extract for 24 h in the presence and absence of 30 mM H 2 O 2 and protein levels were analyzed by immunoblot. We chose the two most active concentrations used for PCR array analysis in order to compare the upregulation between gene and protein expression. Exposure time was 24 h since protein expression levels were too low before this time. Immunoblot analysis revealed a significant increase only in the expression of DHCR24 at the two concentrations tested (Fig. 5A), whereas the expression of GPX4 and PTGR1 significantly increased only after H 2 O 2 pretreatment at 400 μ g ml −1 (Fig. 5B,C). In light of these results, we hypothesized that the extract was able to repair peroxidative cell damage by reducing the quantity of prostaglandins. Quantitative ELISA test was used to determine prostaglandin E 2 (PGE 2 ) levels secreted by A549 cells in cell culture medium before and after extract treatment. As shown in Fig. 6A, A549 cells treated with extracts (100, 200 and 400 μ g ml −1 concentrations) had the same levels of prostaglandin E 2 (29, 30 and 28 pg μ l −1 , respectively) as the negative control (30 pg μ l −1 ). On the contrary, there was a significant dose-dependent decrease (90, 29 and 28 pg μ l −1 ) in prostaglandin E 2 levels with respect to the positive control (140 pg μ l −1 ) in A549 cells treated with extract (100, 200 and 400 μ g ml −1 ) after pretreatment with 30 mM of H 2 O 2 (Fig. 6B). Our data show that extract treatment results in a significant decrease in PGE 2 levels in cells damaged by H 2 O 2 .
Finally, we used the reconstructed human epidermal tissue model EpiDerm EPI-200 (size 0.63 cm 2 ) as in vitro model to confirm the potential application of this extract as cosmeceutical. In particular, we chose this tissue because in vivo oxidative stress frequently occurs in the epidermidis causing aging and other oxidative stress-related diseases. EPI-200 was treated with 30 mM H 2 O 2 and with 200 μ g ml −1 of the ethanol/water extract for 1 h, after injury with H 2 O 2 . The epidermal tissue model was treated with 200 μ g ml −1 of the extract since this was the lowest concentration at which gene and protein expression data revealed a complete activation of all key factors in the antioxidant pathway. Treated medium was removed and replaced with extract, but without H 2 O 2 , to assess if the irritant effect persisted after 24 h of recovery (referred to as recovery time). Treatment with 200 μ g ml −1 extract for 1 h significantly affected tissue viability after 24 h recovery time (85% viability) compared to the irritant effect of 1 h treatment with H 2 O 2 (18% viability) (Fig. 7). The repairing effect by the extract was even more evident 1 h later after treatment with 30 mM H 2 O 2 and 200 μ g ml −1 of the extract tissue, with viability increasing from about 20% to 108.5% with respect to the negative control.

Discussion
Previous studies have shown that total extracts of Tetraselmis sp. have potential cosmetic and pharmaceutical applications for the human hair growth and pigmentation of the skin, and also for stimulating the increased production of skin structural proteins such as filaggrin and involucrin involved in dermal diseases as Psoriasis 8 . In a previous study, an ethanol/water extract of Tetraselmis suecica showed a strong scavenging activity against 2,2-difenyl-1-picrylhydrazyl (DPPH) and peroxyl radicals rather than against the superoxide anions from the xanthine/xanthine oxidase system 9 . Here we show that a similar type of extract from T. suecica also stimulates a strong response to cell damage and activates a repairing mechanism in human epidermal cells. Our extract contains high levels of xanthophylls (lutein, violaxanthin, neoxanthin, antheraxanthin and loroxanthin esters), pigments that are well known for their biological activities as antioxidants and which are precursors of other pigments or vitamins 10 . Lutein, which was particularly abundant in this extract, possesses pronounced free radical scavenging ability due to its polarity and number of conjugated double bonds 11 , and has been shown to significantly decrease neurogenic inflammatory response in the mouse skin 12 . The antioxidant activity of violaxanthin and neoxanthin is also well documented 13 , whereas the biological activity of antheraxanthin and loroxanthin esters recently characterized in T. suecica 14 have not yet been investigated.
In our study, the ethanol/water extract, containing high levels of carotenoids, from T. suecica showed marked radical scavenging ability when tested with the DPPH assay. The addition of extract led to 98% reduction of the radical DPPH (purple) into its reduced (yellow) form at the highest concentrations. As reported in Table 2, the radical scavenging activity of the extract was dose-dependent and its strength was 70% greater than α -Tocopherol at the highest concentrations. Moreover, the inhibition of DPPH free radicals induced by the extract was comparable to other well-known antioxidant molecules such as ascorbic acid where the percentage reduction of the free radical is about 95% 15 .
In order to evaluate this effect at the cellular level we challenged cells with H 2 O 2 because a previous study showed that A549 lung cells have a multifaceted response when exposed to hydrogen peroxide 16 . D' Andrea et al. 16 have shown that H 2 O 2 induces damage to lipids, proteins, and nucleic acids due to the generation of reactive oxygen species (ROS). In our case, H 2 O 2 caused a reduction in A549 cell viability to 36% after 48 h, but addition of the extract induced a strong recovery in cell viability, with up to 100% recovery in some cases. In order to clarify this effect at the molecular level, we studied the difference in oxidative stress gene expression patterns between cells treated only with 30 mM H 2 O 2 and cells recovered with 100, 200 and 400 μ g ml −1 extract. The genes involved in ROS metabolism such as oxidative stress responsive genes ATOX1, CCL5 (RANTES), DHCR24, FOXM1, GPX1, GPX4, PDLIM1, PRDX5, SIRT2, SOD2 were all significantly up-regulated in a dose dependent manner after extract recovery treatment for the highest concentrations tested. In particular, antioxidant genes such as GPX1 and GPX4 showed a completely reversed expression after extract-induced recovery with respect to H 2 O 2 injury for all concentrations tested. On the contrary, PRDX5 showed an up-regulation with H 2 O 2 , because this gene codes for a mitochondrial peroxiredoxin 17 that decomposes hydrogen peroxide. However, after recovery treatment with extract, PRDX5 gene expression levels decreased in a dose dependent manner and were significantly down-regulated at 400 μ g ml −1 . This interesting finding is probably due to the ability of the extract to scavenge the effect induced by H 2 O 2 thereby reducing PRDX5 gene expression levels.
The genes involved in ROS metabolism such as SOD2 were up-regulated with H 2 O 2 . Recovery treatment with the extract caused an enhanced up-regulation of this gene in a dose dependent manner. This is an important  finding because, as demonstrated in previous studies, mitochondrial SOD2 plays a crucial role in protecting cells against oxidative stress 18 . Another important result regards the effect of the extract on up-regulation of DHCR24 because this is a multifunctional enzyme, which exerts resistance against oxidative stress and prevents apoptotic cell death when it is expressed at high levels 19 . The increase in expression levels of the inflammatory pathway gene PTGR1 suggests a potential anti-inflammatory activity because this enzyme is responsible for the biological inactivation of prostaglandins and related eicosanoids 20 .
The down-regulation of the GSTP1 gene after H 2 O 2 treatment indicates that cells were unable to defend themselves against injury. Surprisingly, after 200 and 400 μ g ml −1 of the recovery treatment with the extract, GSTP1 was significantly up-regulated indicating the restoration of the antioxidant defense mechanisms.
Peroxide metabolism genes such as AKR1C2, HSP90AA1 and SLC7A11 showed different expression patterns. In particular, the AKR1C2 gene, which catalyzes the conversion of aldehydes and ketones to their corresponding alcohols 21 , was down-regulated with H 2 O 2 treatment and up-regulated in a dose-dependent manner by the recovery treatment with the extract (only for 200 and 400 μ g ml −1 ). The activation of this gene indicates a specific cell response to hydrogen peroxide metabolites through the induction of detoxification mechanisms. Heat shock protein 90 kDa alpha (cytosolic) class A member 1 (HSP90AA1) was up-regulated by H 2 O 2 treatment because it is a pro-apoptotic factor which induces cell death in response to stress. The extract was able to down-regulate HSP90 expression leading to the induction of cytoprotective pathways through the inhibition of pro-apoptotic pathways 22 . Surprisingly, the extract was able to down-regulated the SLC7A11 gene which is up-regulated by the H 2 O 2 treatment. SLC7A11 encodes a subunit of the xCT cystine/glutamate aminoacid transport system, which is involved in the generation of glutathione and the protection of cells from oxidative stress. However, in a recent study the expression of SLC7A11 was shown to promote tumorigenesis and chemotherapy resistance 23 . The down-regulation of SLC7A11 by the extract may therefore be considered as a potential chemo-preventive agent.
In order to demonstrate the activation of an oxidative stress response pathway after T. suecica extract recovery treatment, we also analyzed the expression of key proteins involved in antioxidant mechanisms (GPX4 and DHCR24) and PTGR1. Immunoblot data confirmed gene expression results on the induction of an antioxidant pathway in A549 cells damaged with H 2 O 2 and then treated with extract (Schematic representation of the oxidative stress response pathway in Supplementary Information, Figure S3). We found that the physiological increase of the active form of GPX4 protein after 30 mM H 2 O 2 treatment was enhanced by recovery treatment with 200 and 400 μ g ml −1 extract. Another important finding was the increased expression of DHCR24 and PTGR1 proteins induced by extract treatment. The high up-regulation of PTGR1 could be linked to a reduction in prostaglandin release by cells. ELISA experiments showed a significant dose-dependent decrease in prostaglandin PGE 2 levels in the culture medium after recovery treatment confirming this hypothesis. To our knowledge, this is the first report that an ethanol/water extract from a marine green microalga acts as an inhibitor of prostaglandin release in an inflammatory response.
Due to difficulties in obtaining sufficient human lung epithelial cells to perform this study, we chose the A549 cell line derived from a lung carcinoma due to its considerable use in the literature as a surrogate cell type 24-27 due to its high levels of glutathione 28 and high (non-induced) heme oxygenase 1 (HO-1) gene expression levels 29 . We are aware that results obtained from this transformed cell line may not be applicable and immediately transferrable to normal lung epithelial cells in the respiratory tract in vivo. Thus, in order to use this extract as a potential cosmetic agent for topical application, we used human epidermidis tissue (EPI-200) as an experimental model and showed that the extract exerted a strong repairing effect after injury caused by hydrogen peroxide.
To date, intervention trials with single antioxidants in pharmacological doses have not supported a repairing effect in humans 12 . However, if many antioxidants work in a network, 'total antioxidants' may be a better concept than individual antioxidants. Thus, the potential synergistic effects of bioactive components, such as carotenoids, with different chemical structures and anti-oxidizing activities may be a promising agent for cosmeceutical use. The identification of potent marine microalgal species as peroxide scavengers and repairing agents can lead to new alternative cosmeceutical products or nutritional supplements for prevention of disorders related to oxidative damage, such as cancer, aging and skin inflammation diseases. In order to develop new natural cosmeceutical products for human health applications from marine microalgae, further studies are required to clarify if this bioactivity is ascribable to a single compound, classes of molecules (e. g. carotenoids) or the synergistic effect of several molecules contained in the ethanol/water extract of Tetraselmis suecica.

Methods
Strain and culture conditions. The prasinophyte Tetraselmis suecica was purchased from the American marine phytoplankton collection (CCMP 906) and was grown in Guillard's f/2 30 medium without silicic acid in two-liter polycarbonate bottles, constantly bubbled with air filtered through 0.2 μ m membrane filters. Cultures were grown at 19 °C, a photon-flux density of about 150 μ mol photons m −2 s −1 , and a photoperiod of 12:12 h light:dark (12 L:12D) cycle. Initial cell concentrations were about 5 × 10 3 cells ml −1 . Microalgal biomass was collected by centrifugation after 9 days (~8 × 10 3 cells ml −1 ).

Preparation of ethanol/water extract from Tetraselmis suecica. Extraction was performed accord-
ing to Goiris et al. 31 . Extraction procedure was conducted under dark conditions, at room temperature and under nitrogen atmosphere, in order to avoid oxidation of the sample. Freeze-dried biomass (~100 mg) was extracted with 1 ml ethanol/water (3/1 v/v) mixture for 30 min. The mixture was separated by centrifugation at 4500 × g, for 10 min, at 20 °C, and the upper layer was transferred to a clean tube. The pellet was resuspended in 1 ml of the ethanol/water mixture and extracted for a second time. The ethanolic extract was dried in a rotary vacuum evaporator (Buchi rotavapor R-114). Dry extract was stored under nitrogen atmosphere at − 20 °C prior to analysis.
Scientific RepoRts | 7:41215 | DOI: 10.1038/srep41215 HPLC analysis of bioactive pigments. Pigment measurements were conducted by High Performance Liquid Chromatography (HPLC) on an aliquot of the ethanolic extract (10 mg), according to methods described in 32 . Prior to injection into the HPLC, 250 μ L of an Ion Pairing Agent (ammonium acetate 1 mol L −1 , final concentration of 0.33 mol L −1 ) was added to 0.5 mL of the pigment extract and incubated for 5 minutes in the dark at 4 °C. This extract was then injected in the 50 μ L loop of the Hewlett Packard series 1100 HPLC (Hewlett Packard, Wilmington, NC, USA), equipped with a reversed-phase column (C8 Kinetex column; 50 mm × 4.6 mm; 2.6 μ m particle size, Phenomenex ® , USA). The temperature of the column was steadily maintained at 20 °C, and the flow rate of the mobile phase was set up at 1.7 mL min −1 . The mobile phase was composed of two solvent mixtures: A, methanol/aqueous ammonium acetate (70/30, v/v) and B, methanol. During the 12-minutes elution, the gradient between the solvents was programmed: 75% A (0 min), 50% A (1 min), 0% A (8 min) isocratic for 3 min. Chlorophylls and carotenoids were detected by diode-array spectroscopy (spectrum data collected in the range 350-750 nm) using a Hewlett Packard photodiode array detector, model DAD series 1100 and absorbance chromatogram was reported at 440 nm. Chlorophylls were also detected by fluorescence using a Hewlett Packard standard FLD cell series 1100 with excitation and emission wavelengths set at 407 nm and 665 nm, respectively. Identification and quantification of pigments were carried out using pigment standards from the D.H.I. Water & Environment (Horsholm, Denmark). Pigment standards derived primarily from phytoplankton. The standards are flushed with 100% N2 and supplied in sealed vials with 2.5 mL, together with a certificate of analysis. All information regarding the accuracy of the preparation of the standards and reference numbers of all standards are available at the following address: http://c14.dhigroup.com/productdescriptions/phytoplanktonpigmentstandards. Moreover spectral information was compared with a library of chlorophyll and carotenoid spectra of pigments prepared from standard phytoplankton cultures 33 . Cell Viability. The effect of ethanol/water extract on cell viability was determined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay (Applichem A2231) in according to 34 . A549 cells, seeded in 96-well plates (2 × 10 3 cells/well), after treatment times, were treated with 10 μ l (5 mg ml −1 ) of MTT and incubated for 3 h. The absorbance was recorded on a microplate reader at a wavelength of 570 nm (Multiskan FC, THERMO SCIENTIFIC). The effect of the extract on cell viability was evaluated as percent of cell viability calculated as the ratio between mean absorbance of each sample and mean absorbance of control. Cells were lysed in the Petri dish by adding 1 ml of Trisure Reagent (Bioline, cat. BIO-38033) per 100 mm dish diameter. RNA was isolated according to the manufacturer's protocol. RNA concentration and purity was assessed using the nanophotomer NanodroP (Euroclone).

RNA Extraction and
About 200 ng RNA was subjected to reverse transcription reaction using the RT 2 first strand kit (Qiagen, cat.330401) according to the manufacturer's instructions. The qRT-PCR analysis was performed in triplicate using the RT 2 Profiler PCR Array kit (Qiagen, cat.330231), in order to analyze the expression of cell oxidative stress genes on A549 cells. Plates were run on a ViiA7 (Applied Biosystems 384 well blocks), Standard Fast PCR Cycling protocol with 10 μ l reaction volumes. Cycling conditions used were: 1 cycle initiation at 95.0 °C for 10 min followed by amplification for 40 cycles at 95.0 °C for 15 s and 60.0 °C for 1 min. Amplification data were collected via ViiA 7 RUO Software (Applied Biosystems). The cycle threshold (Ct)-values were analyzed with PCR array data analysis online software (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php, Qiagen).

Protein Extraction and Western
Blotting. A549 cells (2 × 10 6 ), used for protein extraction and analysis, were seeded in Petri dishes (100 mm diameter) to obtain four types of samples: negative control without any treatment, positive control with cells treated with 30 mM H 2 O 2 , cells treated with 100, 200 and 400 μ g ml −1 , and cells recovered with 100, 200 and 400 μ g m −1 of extract after pre-treatment for 1 h with 30 mM H 2 O 2 . A549 cell lysate was prepared after 24 h of treatment by scraping the cells of each Petri dish into 1 ml of Radio Immune Precipitation Assay buffer (RIPA, Cell Signaling, cat. 9806), supplemented with 1 μ M of protease inhibitor PMSF (Cell Signaling, cat. 8553). The lysate was incubated on ice for 15 min and then clarified by centrifugation at 14000 × g, for 20 min. Total protein concentration was determined according to the Bradford method using a Protein Assay Reagent (Applichem, cat. A6932) with bovine serum albumin (BSA, Sigma Aldrich, cat. A2058) as a standard. The protein extract was stored at − 20 °C until use. Before electrophoresis, protein samples were incubated at 100 °C for 5 min. Following 10% SDS-PAGE, gels were stained with Coomassie or blotted onto nitrocellulose membrane (Biorad, cat. 170-4159). Membranes were incubated for 1 h in blocking reagent (1X Tris Buffered Saline-TBS), with 0.1% Tween-20 with 5% w/v nonfat dry milk, and incubated overnight at 4 °C with the primary antibodies diluted in 1X TBS, 0.1% Tween-20 with 5% BSA.
After incubation, membranes were washed three times for 5 min each with 15 ml of TBS/Tween and then incubated with HRP-conjugated secondary antibody with gentle agitation for 1 h at room temperature. For β -actin and DHCR24 antibodies, we used HRP-conjugated secondary antibody anti-mouse (1:10000, Santa Cruz Biotechnology); for GPX4 and PTGR1 antibodies, we used HRP-conjugated secondary antibody anti-rabbit (1:10000, Jackson ImmunoResearch).
After incubation, membranes were washed three times for 5 min each with 15 ml of TBS/Tween. Blotted membranes were immunodetected using clarity Western ECL (Biorad, cat. 170-5060). Proteins were visualized with Fuji medical X-ray film (cat. 47410). Densitometric analysis of immunopositive bands was performed using Image J software.

Treatment of human epidermis. EpiDerm TM was purchased from the MatTek Corporation (EPI-200-SIT)
and maintained in culture medium provided by the manufacturer and according to a standardized protocol. The EpiDerm skin model (area of 0.6 cm 2 ) consisted of normal human derived epidermal keratinocytes (NHEK) cultured to form a multilayered, highly differentiated model of the human epidermis.
EpiDerm tissues were conditioned by incubation with culture medium in order to release transport-stress related compounds and debris overnight (in a 5% CO 2 atmosphere at 37 °C). About 200 μ g ml −1 carotenoid extract was dissolved in dimethyl sulfoxide (DMSO, final concentration 1% v/v) and used for the treatment of human epidermis; after 1 h of incubation with the extract, a complete recovery medium was used for 24 h of recovery time.
Tissue Viability. The effect of the extract on tissue viability was determined using the MTT assay. After 1 h treatment and 24 h of recovery time, tissues were transferred to 24-well plates containing MTT medium (1 mg ml −1 ). After 3 h incubation in MTT, the blue formazan salt was extracted with 2 ml/tissue of isopropanol and the optical density of the extracted formazan was determined using a spectrophotometer at a wavelength of 570 nm. Relative tissue viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin irritation potential of the test extract was predicted if the remaining relative tissue viability was below 50% (MatTek Corporation-Protocol).
Statistical Analysis. Statistical significance of the DPPH assay was determined by Students-t test (*p values ≤ 0.05). Statistical differences between treated and control cells for cell viability counts were determined by One-way ANOVA and significant differences between the treated groups by Students-t test (*) and ANOVA followed by Dunnett's test (#) (p values ≤ 0.001) using Microsoft Excel software (365 version, 2013). Gene expression data were analyzed by PCR array data analysis online software (http://pcrdataanalysis.sabiosciences.com/ pcr/arrayanalysis.php, Qiagen ® ). Only expression values greater than a 2.0-fold difference with respect to the controls were considered significant. Immunoblotting protein expression was calculated as the percentage of integral area of every single gel band with respect to total gel lane area, represented as pixels. Statistical differences between treated and controls were determined by Students-t test with significant p values ≤ 0.05. Data significantly different from controls, with p values < 0.001 are marked with two asterisks in the figures. Significant differences between treated groups, after epidermal human tissue experiments, were determined using Students-t test (*p ≤ 0.05) and ANOVA.