) is a marine microalga belonging to Haptophyta, originally isolated from tropical seawater (Tahiti, French Polynesia), and currently used in aquaculture [1
]. The presence of n-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), vitamins, proteins, and xanthophylls such as fucoxanthin [3
], makes this microalga an interesting source of compounds with anti-inflammatory and hypolipidemic activities [5
]. Among the marine microalgae, Tisochrysis
contains a high amount of the pigment fucoxanthin (FX) (1.8% w/w) [8
]. In several in vitro and in vivo models, FX exerts anti-inflammatory effects by inhibiting pro-inflammatory cytokines and enzymes [9
]. FX also attenuates alcohol-induced oxidative lesions and inflammatory responses [13
]. However, Tisochrysis
is also a source of phenolic compounds [14
] which possess a high spectrum of biological activities including antioxidant, anti-aging, and anti-inflammatory effects [15
]. Despite the anti-inflammatory and antioxidant effects of T. lutea
that have been mostly attributed to FX, positive pharmacodynamic synergisms among various components, acting on different targets, cannot be excluded. Indeed, superior bioactivity of either the single component or the mixture was reported in studies on natural products [21
The aim of this study was to perform a direct comparison between the anti-inflammatory activity of a methanolic extract of T. lutea F&M-M36 and FX at equivalent concentrations in order to explore potential interactions among the components and pharmacological mechanisms involved. Lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse macrophages were used as an in vitro model of inflammation.
Inflammation is a key component of several chronic human diseases such as inflammatory bowel diseases, diabetes, cardiovascular diseases, neurodegeneration, and cancer [23
]. The identification of new anti-inflammatory compounds is a great challenge for the scientific community, and in this context, the microalga T. lutea
may represent an interesting source for the discovery of novel strategies for the prevention, and even control, of inflammation.
Overall, our results demonstrate that T. lutea
F&M-M36 methanolic extract and FX, at equivalent concentrations, exert anti-inflammatory activities by regulating a number of pro-inflammatory mediators. It is interesting to highlight that the effects on the COX-2/PGE2 axis are concentration-dependent and therefore suggestive of a pharmacological mechanism of action of T. lutea
F&M-M36 methanolic extract and FX; the prominent reduction of COX-2/PGE2 exerted by T. lutea
F&M-M36 methanolic extract also suggests that compounds other than FX may exert additive or synergistic effects. This is also consistent with previous reports documenting the superior activities of botanical extracts compared to single components [24
]. T. lutea
F&M-M36 methanolic extract contains polyphenols equivalent to 6.22 mg of gallic acid/g dry weight, exhibiting a much lower content of total polyphenols compared to that reported for other species, as a polyphenolic content of 515 mg GAE per 100 g DW and of 13.4 mg GAE/g EW measured in an ethanolic extract from the closely related species I. galbana
]. These differences may be ascribed to the extraction solvents used (methanol instead of ethanol), although differences in the analyzed species and in cultivation conditions may also have contributed [25
Despite the presence of phenolic compounds in T. lutea
being previously described, scarce information is available on their composition; our HPLC characterization showed that T. lutea
F&M-M36 methanolic extract contains a number of simple phenolic acids which have characteristic UV spectra (maximum absorption in the 200–290 nm range [26
Simple phenolic acids derivatives of hydroxybenzoic and gallic acids have been previously proved to exert anti-inflammatory activities; gallic acid exerted inhibitory effects on LPS-stimulated PGE2 and IL-6 production and COX-2 expression in RAW 264.7 cells [27
], and inhibited several NLRP3 inflammasome markers in an in vitro model of intestinal inflammation [28
]. Moreover, we previously demonstrated that hydroxytyrosol, p-coumaric acid, or foods rich in simple phenols exhibited anti-inflammatory properties in in vitro and in vivo models of colon inflammation [18
]. On the other hand, we cannot exclude the contribution of other, not characterized components of our methanolic extract. In particular, our T. lutea
F&M-M36 biomass contains 4.1% of dry-weight polyunsaturated fatty acids (PUFAs) and 2.61% of total ω-3 [7
] that are known to exert immunomodulatory and anti-inflammatory activities [30
In addition, although FX is the main carotenoid found in T. lutea
, other compounds such as diadinoxanthin, diatoxanthin, and β-carotene were found in an ethyl acetate extract from T. lutea
containing a total amount of 132.8 mg of carotenoids/g of extract [31
]. The anti-inflammatory activities of carotenoids such as β-carotene at relatively high concentrations (50–100 µM) have been reported in LPS-induced RAW264.7, showing effects on IL-1β, IL-6, and TNF-α; [32
]. In the same model, other authors found significant effects of β-carotene 5 µM on IL-12, p40, and IL-1β expression [33
].MiRNAs are endogenous non-coding RNA molecules that silence target mRNA by binding to the 3′UTR of mRNA [34
]. Several miRNAs are regulated during the inflammatory process [35
]; mir-223 is emerging as an important regulator of the innate immune system, and its deficiency enhances pro-inflammatory macrophage activation [36
]. mir-223 targets NLRP3 result in reduced inflammation [38
]. Our results pointed out a peculiar superior effect of the T. lutea
F&M-M36 methanolic extract toward the NLRP3/mir223 axis. For the first time, we showed that T. lutea
F&M-M36 methanolic extract has the ability to enhance the secretion of mir-223 by LPS-stimulated RAW 264.7, although to a lesser extent than the selective COX-2 inhibitor Celecoxib, and that this effect may be attributable to the phenolic content of the extract, considering the negligible effects of FX alone.
The activity of T. lutea F&M-M36 methanolic extract was prominent over that of FX on the COX-2/PGE2 pathway and NLRP3/mir-223 axis, whereas similar effects were observed when other inflammatory mediators were investigated. The ability to simultaneously target different biological inflammatory networks certainly represents an added value of both the extract and FX.
Macrophages polarization between M1 and M2 phenotypes is an important regulatory mechanism for inflammation. M1 macrophages are classically activated by LPS and sustain inflammation, whereas M2 or M2-like phenotypes are associated with the resolution of inflammation [40
]. M1 macrophages express pro-inflammatory cytokines such as TNF-α, COX-2, and IL-6, while M2 macrophages express IL-10 and Arg1, thus exhibiting anti-inflammatory properties [41
T. lutea F&M-M36 methanolic extract and FX promoted some morphological and molecular characteristics of the M2 anti-inflammatory phenotype in RAW macrophages, such as increased expression of IL-10 and Arg1 and decreased expression of IL-6. The extent of these effects is almost completely attributable to the FX content.
Previous findings indicate that FX (100 µg/mL) inhibited the secretion of IL-1β and TNF-α and promoted that of IL-10 and IFN-γ in Caco-2 cells stimulated with LPS [8
]. In LPS-induced RAW 264.7, FX 15-60 µM (corresponding to about 10–40 µg/mL) significantly inhibited NO, TNF-α, and IL-6 production but slightly reduced PGE2 production [10
] and inhibited NF-κB activation and MAPK phosphorylation at 12–50 µM [11
]. In the same model, the half-maximal inhibitory concentration (IC50) for IL-6 production was 2.19 μM [12
]. In a recent report, Kim et al. (2021) [42
] found that the pretreatment of RAW 264.7 with FX 5 µM also significantly decreased LPS-induced expression of IL-6, IL-1β, and TNF-α by activating the NRF2/PI3K/AKT pathway. It is worth highlighting that all these studies were conducted with FX concentrations largely greater than ours (470 ng/mL). From a pharmacological point of view, the smaller is the concentration at which the molecule is active, the greater is its potential application. Recently, in a model of metabolic syndrome, a high-fat diet, supplemented with 12% (w/w) of freeze-dried T. lutea,
significantly reduced plasma TNF-α levels and increased IL-10 in abdominal adipose tissue [43
In addition, for the first time, we reported the ability of T. lutea
F&M-M36 methanolic extract to reduce the secretion of mir-146b, and this effect was almost completely attributable to FX [44
]. Increased levels of mir-146b are associated with inflammatory disease: in particular, mir-146b is increased in the serum of patients with inflammatory bowel disease and decreases after treatment with infliximab [45
]; moreover, circulating mir-146b correlates with endoscopic disease activity in patients with inflammatory bowel disease [46
is not approved for human consumption, and its safety has been evaluated only in short-term studies in animal models [2
]. However, T. lutea
is currently used in aquaculture [1
], and our data suggest that it could be added to animal feed not only for its high nutritional value, but also as an anti-inflammatory additive.
Overall, our results demonstrate that T. lutea F&M-M36 methanolic extract exerts promising anti-inflammatory activity, even more pronounced than that of FX alone, thus providing the background for conducting studies on its long-term safety and efficacy in inflammatory disease models.
4. Materials and Methods
4.1. Microalgal Biomass
The biomass of T. lutea
F&M-M36 strain belonging to the Fotosintetica & Microbiologica (F&M) S.r.l. Culture Collection (Florence, Italy) was produced at Archimede Ricerche S.r.l. (Camporosso, Imperia, Italy). T. lutea
F&M-M36 was cultivated in F medium [49
] in GWP®
-II photobioreactors [50
] in a semi-batch mode. The lyophilized biomass was stored at −20 °C until extraction.
4.2. Microalgal Extract Preparation
An aliquot of 250 mg of lyophilized T. lutea F&M-M36 biomass was extracted in 30 mL of methanol, overnight, at room temperature (RT). The mixture was then sonicated twice for 3 min at the maximum power. The solvent was separated from the biomass by filtration on paper. The residual biomass was extracted again with 15 mL of methanol at 37 °C for 4 h; then, the exhausted biomass was removed by filtration on paper, and the extract (30 + 15 = 45 mL) was evaporated under vacuum. The dry residue was solubilized in DMSO to obtain a final concentration of the extract of 65 mg/mL. Fucoxanthin (purity ≥ 95%) was purchased by Sigma Aldrich (Milan, Italy).
4.3. Sample Preparation and HPLC-DAD Analysis for Phenols Quantification and Characterization
The extract was dried under vacuum and resuspended in 9 mL of ethanol:water solution (75:25 v/v adjusted at pH 2 by formic acid addition) and partitioned with 5 mL of n-hexane in order to remove chlorophylls and other pigments, which could interfere in the analysis of phenolic compounds. The procedure was repeated three times. The last partition was carried out with chloroform instead of n-hexane. The polar phase was reduced to dryness, and the residue resuspended in 0.5 mL of methanol:water solution (50:50 v/v adjusted at pH 2.5 by formic acid addition).
Aliquots of the samples (15 μL) were injected into the Perkin® Elmer Flexar liquid chromatograph equipped with a quaternary 200Q/410 pump and an LC 200 diode array detector (DAD) (all from Perkin Elmer®, Bradford, CT, USA). The stationary phase was composed by a reverse-phase Agilent® Zorbax® SB-18 column (250 × 4.6 mm, 5 µm) (Agilent Technologies Inc., Santa Clara, CA, USA) kept at 30 °C. A gradient solvent system of solvent A (acidified water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid), over a 59 min run in a flow rate of 0.6 mL/ min was applied: 0–5 min (0% B), 5–8 min (0–3% B), 8–53 min (3–40% B), 53–58 min (40% B), 58–59 min (0% B).
The chromatograms were acquired at 280 and 350 nm, the most common wavelengths for the analysis of phenolic compounds. The putative identification of the phenolics detected was carried out based on the retention time, UV spectral characteristics, and comparison with standards, as well as based on literature data. A calibration curve of gallic acid (R2 = 0.99) was used to quantify the compounds and the result of the total phenolic content was given in mg GAE/g dry weight. The analysis was conducted in triplicate.
4.4. Fucoxanthin Determination in the Methanolic Extract
FX content of T. lutea
F&M-M36 extract was carried out by chromatographic analysis according to a modification of the method by Kim et al. [8
]. FX separation was achieved with an HPLC 1050 (Hewlett Packard, Palo Alto, CA, USA) equipped with a C30 reverse-phase column (YCM Carotenoid, 4.6 mm × 250 mm, 5 μm particle size) (Waters, MA, USA), and a UV photodiode array detector 1050 (Hewlett Packard, Palo Alto, CA, USA). A gradient method with two eluents were used; eluent A: 81% Methyl Tert-Butyl Ether (MTBE), 10% methanol, and 9% deionized water, and eluent B: 93% MTBE and 7% methanol. The injection volume was 20 μL with a constant flow rate of 1 mL/min, at 25 °C temperature. The detection was performed at 450 nm. The quantification was performed by internal standard calibration. Commercial FX (Sigma-Aldrich, Milan, Italy) standard solutions (20, 40, 60, 80, 100, 120 μg/mL in methanol/MTBE 4:1), with β-apo-carotenal (50 μg/mL) and Sudan Red (90 μg/mL) were prepared. The rate between the area under the peaks of FX standard solutions and the area under the internal standard peak was plotted against FX standard solution concentrations (μg/mL) to obtain a calibration curve adopted to quantify the concentration of FX in the T. lutea
4.5. In Vitro Model of Inflammation and Anti-Inflammatory Assay
RAW 264.7 macrophages were purchased from the American Tissue Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Milan, Italy) with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 100 U/mL penicillin-streptomycin (Thermo Fisher Scientific), in 5% CO2
at 37 °C. The cytotoxicity of the extracts was first evaluated by MTS assay as previously described [18
]. FX was dissolved in DMSO and diluted in a complete cell-culture medium in order to obtain the appropriate concentrations to be tested. The final concentrations of DMSO were below 0.1%, and the control cells were exposed only to DMSO 0.1%. The cultured cells were treated with lipopolysaccharide (LPS, 1 μg/mL Sigma-Aldrich, Milan, Italy) and with T. lutea
F&M-M36 methanolic extract (1–100 µg/mL) or FX (4.7–470 ng/mL) (Sigma-Aldrich, Milan, Italy). After incubating for 18 h at 37 °C, the cells were harvested for RNA and protein extraction, and the cell medium was collected and stored at −20 °C for PGE2 determination [18
4.6. Morphological Analysis: Hematoxylin and Eosin (H&E) Staining
RAW 264.7 were seeded in Poly-D-lysine-coated glass dishes for 24 h then treated with LPS and T. lutea
F&M-M36 extract, FX, or Celecoxib as described above. After 18 h, cells were fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature. Next, cells were washed in H2
O and then stained with hematoxylin for 2 min, differentiated in saturated lithium carbonate solution for 30 s, stained with eosin for 2 min, and dehydrated with ethanol series (50, 75, 96, and 100%), and finally xylene. Subsequently, glass dishes were mounted on microscope slides with a mounting medium and allowed to dry. Microscopic analysis was performed with ACT-2U software program (Nikon, Instruments Europe, Badhoevedorp, The Netherlands) connected via a camera to the microscope (Optiphot-2; Nikon). Five photomicrographs were randomly taken for each sample to evaluate cell morphology. The percentage of cells with dendritic changes (number of cells with clear morphological changes/total number of cells in the field × 100) were counted using ImageJ 1.33 image analysis software (http://rsb.info.nih.gov/ij
(accessed on 22 April 2021)).
4.7. PGE2 Determination
PGE2 levels were measured in the RAW 264.7 cell media using an ELISA kit (Cayman Chemical, MI, USA) according to the manufacturer’s specifications, and expressed as pg/mL. Celecoxib (Sigma-Aldrich, Milan, Italy) 3 µM (1.14 µg/mL), was used as a positive control.
Total RNA was extracted from cell lysates using the Nucleo Spin®
RNA kit (Macherey-Nagel, Bethlehem, PA, USA) according to the manufacturer’s instructions. For first-strand cDNA synthesis, 1 µg of total RNA from each sample was reverse-transcribed. Primers were designed based on the mouse GenBank sequences for HO-1, IL-10, IL-6, IL1-β, COX-2, iNOS, TNF-α, SOD2, NLRP3, and Arg1, and are reported in Table 2
. Ribosomal protein large P1 (RPLP-1) was co-amplified as the reference [18
]. For each target gene, the relative amount of mRNA in the samples was calculated as the ratio to RPLP-1 mRNA [19
4.9. Real-Time PCR for mir-146b and mir-223 Expression Analysis
For miRNA expression analysis, the total RNA was extracted from cell culture media by using TRIzol (Invitrogen, Carlsbad, CA, USA). Reverse-transcription of RNA was performed using the miRCURY LNA RT Kit according to the manufacturer’s instructions (Qiagen). qRT-PCR assays were carried out in a Rotor-Gene®Q PCR System (Qiagen) using a miRCURY LNA SYBR® Green PCR Kit and miRCURY LNA miRNA PCR Assay according to the manufacturer’s instructions (Qiagen). Briefly, each reaction was performed in a final volume of 10 μL containing two μL of the cDNA, a master mix containing 5 μL of 2× miRCURY SYBR Green PCR Master Mix, 1 μL of miRCURY LNA miRNA PCR Assay, and RNase-free water. The amplification profile was: PCR initial heat activation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 10 s and combined annealing/extension at 56 °C for 60 s. The expression of mir-146b and mir-223 was normalized to RNU6B and calculated as 2-ΔΔCt.
4.10. Dot-Blotting for COX-2 Protein Expression
Cells were lysed in a 300 µL radioimmunoprecipitation assay buffer (RIPA) (Sigma-Aldrich, Milan, Italy). Total protein content was measured by using the Bio-Rad DC protein assay kit (Bio-Rad, Milan, Italy). Equal aliquots (30 μg) of proteins were applied to a nitrocellulose membrane (Millipore, Burlington, VT, USA) and allowed to dry for 30 min at RT. After blocking with 6% nonfat dry milk for 1 h at RT, the membranes were incubated overnight at RT with the Rabbit anti-COX-2 polyclonal antibody (1:200) (Cayman Chemical, MI, USA, catalog number 160126) followed by incubation with anti-rabbit IgG horseradish peroxidase-linked antibody (Cell Signaling, Danvers, MA, USA), 1:4000 for 1 h at RT. Chemiluminescence was developed by using the Immobilon Horseradish Peroxidase Substrate (Merck Millipore, Darmstadt, Germany), and immunoreactive spots were quantified using Quantity-One software (Bio-Rad Laboratories S.r.l., Milan, Italy).
4.11. Immunocytochemistry for COX-2 Protein Expression
RAW 264.7 cells were grown in Poly-D-lysine-coated glass dishes for 24 h then treated with LPS and compounds and extracts tested as described above. After treatment, cells were fixed with cold 4% (w/v) paraformaldehyde for 20 min, washed in PBS, and then incubated for 15 min with 0.1% (w/v) TritonX-100 and 3% Bovine Serum Albumin (BSA). Thereafter, the cells were incubated with Rabbit anti-COX-2 polyclonal antibody (1:200) (Cayman, Ann Arbor, MI, USA, catalog number 160126) at 4 °C overnight, followed by the fluorescent secondary antibody: AlexaFluor 586 goat anti-rabbit (1:333) (Invitrogen, Carlsbad, CA, USA). Nuclei were also counterstained with DAPI dye (Sigma-Aldrich, Milan, Italy). Microscopic analysis was performed with an Olympus BX63 microscope equipped with a Metal Halide Lamp (Prior Scientific Instruments Ltd., Cambridge, UK) and a digital camera, Olympus XM 10 (Olympus, Milan, Italy).
4.12. Statistical Analysis
Data were analyzed by ANOVA test and Dunnett’s Multiple Comparison test and expressed as the means ± standard error (SEM) of four independent experiments. All analyses were carried out using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). p values less than 0.05 were considered significant.