Poly- And Oligosaccharide Ulva Sp. Fractions From Enzyme-Assisted Extraction Modulate The Metabolism 2

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3. Discussion

Our main scope is to evaluate the potential biological activities of poly-and oligosaccharide fractions from Ulon sp. obtained after an innovative Enzyme-Assisted Extraction (EAE) process, which is known to improve thee yield of extraction [5] and allow a significant fraction enrichment in ulvans when compared to maceration [36], on the metabolism of human skin dermal fibroblasts in culture.

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A focus on the expression of skin extracellular matrix components (ECM) involved in the anabolic pathway (particularly type I and Ⅲ collagens, GAGs and TIMP-1) and in the catabolic pathway (MMP-1) was done at proteomic and transcriptomic levels. In this work, we assumed that due to the high composition in carbohydrates and uronic acids of the fractions, the activity is related to ulvan (poly- and oligosaccharide) composition. Nevertheless, the authors should not neglect that polysaccharide ulvans from the Uloa sp. cell wall are closely linked to proteins, which are also known to promote in vitro total collagen and hyaluronic acid production in human dermal fibroblasts [39]. Thus, the authors suggest that these activities could be related to a synergy between ulvans and proteins, even though the focus is made on ulvans in this manuscript. 3.1.Action of Poly-and Oligosaccharide Uloa Fractions from EAE on ECM Fibroblasts Metabolism

Previous studies have demonstrated that fractions from Uloa sp. Cistanche Extract Anti Radiation (crude ulvans and low molecular weight ulvans) have the ability to modulate normal human dermal fibroblasts proliferation [36-38]. Our results indicated that both poly-and oligosaccharide fractions from EAE induced a significant increase of fibroblast metabolic activity (up to +68%). These results confirmed previous preliminary published data of the authors [36] and previous work on hydrolyzed Uloa pertusa extract at 250 ug/mL which induced significant pre- and senescent fibroblast proliferation [38]. Metabolic activity increase was not accompanied with the cytotoxicity effect of the fractions (evaluated by LDH assay), which means that the bioactivity results of poly- and oligosaccharide Uloa fractions from EAE are not biased by a cytotoxic effect in the range of tested concentrations. Our result is in accordance with previous studies highlighting ulvans as non-cytotoxic compounds on different cell types (macrophage cell lines, gut cells, fibroblasts, cells from mouse, and Vero cells)[13,14,4]

cistanche can anti-aging

As mentioned in the introduction, type Iand III collagens, and GAGs interact together to establish a molecular network for ECM assembly in the dermis. Thus, in this study, protein synthesis and mRNA level expression of major components of dermis ECM (type I and Ⅲ collagens, and GAGs either sulfated and non-sulfated) have been investigated in the presence of poly- and oligosaccharide Ullon sp. fractions from EAE. Furthermore,MMP-1,a key enzyme involved in type I collagen degradation (catabolism pathway of ECM) and its tissue inhibitor TIMP-1 involved in its regulation were also assed at protein and mRNA levels.

The results revealed that both poly-and oligosaccharide Ulva fractions from EAE enhanced total collagen synthesis at 1000 ug/mL (up to +2% evaluated with Red sirius assay and significative results except for DEP-AD PP-UE). These results differ from a previous study where crude vans and low molecular weight ulvans have no significant effect on total collagen synthesis [37]. However, our results are in accordance with previous work, which showed that L-rhamnose-rich polysaccharide preparations (ROPs) from Klebsiella pneumonia strains stimulate collagen synthesis[45]. Indeed, human dermal fibroblasts contain a lectin site that is able to recognize α-L-rhamnose [46]. In this way, the rhamnose moiety of ulvan fraction can be recognized by the human dermal fibroblast and then promote collagen synthesis. Particularly, type I collagen synthesis was increased for all fractions in ELISA (significant for UE and DS-UE) and in Western blot assays(NS). This result is in accordance with previous literature on hydrolyzed Ulva pertusa extracts [38] and depolymerized galactofucans(<10kDa)from Saccharina longicruris[47] which respectively increased the synthesis of type I pro and mature collagen. In our study, steady-state levels of COL1Al and COL1A2 were reduced after exposure to poly-and oligosaccharide Ulva fractions(significant for COL1A1 at 1000 ug/nnL for all fractions).Expression in coordinated manner of COL1Al and COL1A2 genes complies with the fact they are both subunits of type I collagen protein [48]. Regulation between type I collagen protein production and mRNA are different at the same observation time of 48 h, which suggests a different potential time response regulation between protein and mRNA[49]. Nevertheless, protein is the functional unit in ECM and exhibits an important increase after treatment with ulvans-derived EAE fractions. COL3A1 mRNA level expression was increased (significantly for UE and DS-UE at 1000 ug/mL) and correlated to an NS increase of type III collagen synthesis evaluated by WB. Moreover, the results showed that polysaccharide Uloa fractions from EAE (UE and DS-UE) boosted significantly GAGs(sulfated and non-sulfated) synthesis at 1000 ug/mL, while both oligosaccharide fractions had no effect. Our observations are in partial accordance with the work of Adrien etal. (2017) in which the increase in hyaluronic acid production in fibroblasts occurred with both ulvans extracts (crude and low molecular weight ulvans) [37]. cistanche herba Indeed, in our study, the low molecular weight ulvans had no effect on GAGs production (including non-sulfated GAGs hyaluronic acid).

Since skin aging is characterized with an imbalance of ECM components (decreased levels of type I and II collagen, and GAGs), poly- and oligosaccharide fractions obtained from Uloa sp. after EAE treatment are of interest in anti-aging strategies, since they stimulate type I and III collagens, and GAGs synthesis.

However, our results also highlighted an increase in MP-1 synthesis, activity, and gene expression in the presence of poly-and oligosaccharide Uloa EAE-derived fractions. The rise in MMP-1 mRNA levels was correlated to the increase of MMP-1 synthesis in the presence of Ulon-derived EAE fractions and significatively for polysaccharide fractions (UE and DS-UE).The rise of MMP-1 enzyme synthesis is also related to a non-significant increase in MMP-1 activity in presence of the fractions. Our result differs from previous literature since hydrolyzed Uloa pertusa extracts inhibited the expression and secretion of MMP-1 in senescent fibroblasts[38], or fucose-containing polysaccharides (named fucoidans) inhibit UVB-induced MMP-1 expression in human skin fibroblasts [50]. However, our result is in accordance with the study of FRioux et al. (2013), since crude galactofucans (638-1529 kDa) increased the total catalytic activity MMPs(including MMP-1) of fibroblasts treated for 6 days[47]. Our data on MMP level increase is also in agreement with the work of Andres et al. (2006), since RROPs (rhamnose-rich oligo- and polysaccharide preparations from Klebsiella pneumonia and K. planticola strains) upregulate MMP-9 expression [45]Enhanced MMP-1 synthesis normally results in a decrease of ECM components amount such as type I collagen due to MMP-1 degradative activity, but this was not pointed out in our study, since an increase in typeI collagen occurred. Furthermore, Uloa EAE-derived fractions had no impact (NS increase or decrease) on the mRNA expression level and protein production of TIMP-1,a tissue inhibitor of MP-1 (except for DEP-HD PP-UE with a significant protein increase，p <0.05，at 1000 μg/mL）.It appears that the anabolic/catabolic balance of ECM could be in favor of collagen synthesis despite MMP-1 synthesis stimulation and activity. In this study, the authors thus showed that both poly- and oligosaccharide fractions derived from Ulloa sp. after EAE treatment are promoting fibroblast metabolism activity, ECM protein synthesis, and skin matrix renewal and remodeling, which is of interest for dermo-cosmetic applications in anti-aging strategies.

3.2.Role of the Ulvan Abundance, Sulfate Composition and Molecular Weight in ECM Fibroblast Modulation

The structural feature of ulvans that encompasses its degree of sulfation, sulfation pattern, monosaccharide composition, glycosidic linkages, degree of branching, as well as its molecular weight is known to impact its biological activity [10,1,37,40,44,51].

Previous studies have shown that the molecular weight of bioactive compounds such as polysaccharides or protein appears to be a key factor for its effect on fibroblast proliferation and ECM modulation [37,39,47]. In our study, polysaccharide fractions, UE and DS-UE, with high molecular weights (Mw > 670 kDa)increased fibroblast proliferation. Although oligosaccharide fractions (Mw<10 kDa) also raise fibroblast proliferation, a DEP-AD PP-UE fraction with lower molecular weight (Mw of 1.5 kDa) has lower activity than DEP-HD PP-UE (Mw of 8 kDa). Similar results were obtained on fibroblasts treated with RROPs, since lower RROPs exhibited similar or lower proliferation activity compared to higher Mw RROPs[45]. Polysaccharide fractions treatment also enhanced GAGs synthesis, total and especially type I and III collagen synthesis, MP-1 synthesis by human skin fibroblasts, whereas oligosaccharide fractions (low molecular weight,<10 kDa) and in particular DEP-AD PP-UE (1.5 kDa) have lower capacities. Only DEP-HDPP-UE stimulated significatively total collagen synthesis and MP-1 synthesis, while DEP-AD PP-UE with lower molecular weight had no significant boost effect. These results are in accordance with work of Kidgell et al. (2020) where higher molecular weight ulvan fractions elicited a greater biological activity (immunomodulatory response) compared to lower Mw van fractions[44]. These results are also in agreement with work of Bodin et al. (20), since enzymatic hydrolyzed proteins from Uloa intestinalis did not stimulate ECM material biosynthesis (collagen and hyaluronic acid)[39].

The authors pointed out that the better bioactivity on GAGs synthesis was attributed to sulfated polysaccharides' high molecular weight fractions enriched in crude ulvans. This could be related to ulvans intrinsic composition in sulfates and its molecular weight, since oligosaccharide Ulva sp. fractions, partially completely free of sulfates, have no ability to stimulate GAGs synthesis. cistanche penis growth Another study on Ulku sp. showed the importance of the ulvan sulfation degree on biological activity since chemically sulfated Alvan fraction, doubled sulfate content from native polysaccharide, strongly enhanced anticoagulant activity [10]Moreover, DS-UE increased more ECM modulation regarding GAGs, total and type I and Ⅲ collagen synthesis, compared to UE. This better bioactivity could be linked to the higher ulvans abundance and greater sulfate groups content in dialyzed fraction compared to the other fractions.

Our data showed that poly-and oligosaccharide Uloa sp. fractions exhibited different metabolic activities, which may be attributed to the abundance of ulvan, its sulfation degree, and chemical structure. This work also highlighted a better activity for high molecular weight vans displaying sulfate groups. The fact that the polysaccharide fractions rich in crude ulvans (i.e, non-depolymerized) had the greatest effect on ECM fibroblast metabolism is ideal for upcoming studies and its future potential applications in dermo-cosmetic care, as minimal processing decreases production costs and time.

4. Materials and Methods

4.1.Macroalgal Material

Green seaweed, Ulva sp. (Chlorophyta, Ulvales, Ullvaceae), was sourced from the intertidal beach Landrezac(47°30°17.9" N°2°42°37.1" O) in Sarzeau (Brittany, France)on 28 May 2018 during the afternoon at low tide. Material was washed with tap water, ground to a 3mm diameter, frozen at-25℃, freeze-dried (Alpha 1-4LSC, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany), and stored at room temperature in the dark.

4.2.Poly-and Oligosaccharide Fractions Production and Characterization

In a previous work by Fourniere et al. (2019), detailed poly- and oligosaccharide fractions production from Enzyme-Assisted Extraction (EAE) was described (see Figure 9 and Table 3)[36].

To briefly summarize, endo-protease Protamex[(Novozymes, Bagsverd, Denmark)was used at 6%(w/dw) on dried Uloa sp. seaweed. Enzymatic hydrolysis was performed for 3 h at 50 ℃. Then, aqueous extract was undergone ethanolic precipitation (1:5, v Fisher Scientific, Illkirch, France)at 4℃for 244 h in order to obtain the fraction rich in crude ulvans (UE).

Then, fraction DS-UE was produced after dialysis of a fraction rich in crude Evans (10 mg/mL) for7 days at 4℃(cut-off 12-14 kDa, Spectra/Por94 Dialysis Membrane, Spectrum Laboratories,Fisher Scientific,Illkirch,France).

Two depolymerization procedures (radical depolymerization by H-OZ and ion-exchange resin depolymerization) were performed on ethanolic precipitate solution from a fraction rich in crude ulvans (25 mg/mL).For radical depolymerization, hydrogen peroxide (8%, v/v, Fisher Scientific, Illkirch, France) was mixed to the solution for 24 h at 50 ℃, and the product underwent 48 h dialysis at 4 ℃(cut-off of 500-1000 Da, Biotech CE Tubing, Spectra/Por@, Fisher Scientific, IIllkirch, France). For acidic depolymerization, resin Amberlite” FPC23H(10mL equivalent, Sigma-Aldrich, Saint Quentin Fallavier, France)was mixed to the solution for24 h at 80 ℃, and the product was neutralized with NaOH (0.1 and 1 M) before undergoing 48 h dialysis at 4℃(cut-off of 500-100 Da, Biotech CE Tubing, Spectra/Por", Fisher Scientific, Illkirch, France). cistanche salsa benefits These two procedures led to oligosaccharide fractions called DEP-HD PP-UE and DEP-AD PP-UE for H2OZ and the acidic resin procedure, respectively.

All fractions were freeze-dried (Alpha 1-4LSC, Martin Christ Gefriertrocknungsanla-gen GmbH, Osterode am Harz, Germany) and stored at 4℃ before analysis. Characterization of these fractions: polysaccharide molecular weight distribution by high-pressure size exclusion chromatography (HPSEC, UHPLC Ultimate 300, Thermo Fisher Scientific, Waltham, MA, USA), biochemical composition, monosaccharide composition by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD, Dionex" ICS-5000* DC, Thermo Fisher Scientific, Illkirch, France) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF, Bruker, Billerica, Waltham, MA, USA) were also conducted in a previous study [36].

4.3.Cell Culture

Human dermal fibroblast samples were provided by"Laboratoire Interactions Ep-itheliums Neurones"(LIEN,EA 4685), Brest, France. Human dermal samples were obtained from skin biopsies of healthy donors undergoing abdominoplasty surgery. The study was conducted in accordance with the Declaration of Helsinki, and all patients signed an informed consent agreement form. Sample collections adhered to the local agreement comity ("Comite de protection des personnes" Ouest VI) and referenced under DC 2016-2833.

Normal human dermal fibroblasts (NHDF) were cultured in Dulbecco's modified eagle medium (DMEM,Lonza, Basel, Switzerland) with 10%(v/v) fetal bovine serum (FBS, Gibco, Fisher Scientific, Illkirch, France), a 1% antibiotic solution (v/v)(Penicillin, Streptomycin 10,000 U/mL, Gibco, Fisher Scientific, Illkirch, France),and 1% antifungal (v/v)(Fungizone, amphotericin B, Gibco, Fisher Scientific, Illkirch, France), in a temperature-controlled incubator with 5%CO2 at 37°C(Forma Steri-Cyclei160, Thermo Fisher Scientific Langenselbold, Germany). Cells were sub-cultured by trypsinization (0.05%) and EDTA (ethylenediaminetetraacetic acid)solution((Gibco, Fisher Scientific, Illkirch, France) after reaching confluence. All experiments were performed between the 3rd and 8th passages.

Cells were seeded onto 96-well microplates at a density of 400 cells/well for WST-1 and LDH (lactate dehydrogenase) assays onto 12-well plates, at a density of 5000 cells/well for Red sirius and Blue Alcian staining and RT-qPCR assay, and onto 6-well plates at a density of 200,000 cells/well for ELISA and Western blot assays.

In all experiments, after reaching 80% confluency, cells were incubated in DMEM with 2% FBS in the absence or presence of the poly- and oligosaccharide fractions for 48 h at 50 and 1000 μg/mL.

4.4.WST-1 Cell Proliferation Assay

The WST-1, in vitro, the assay is based on the conversion of tetrazolium salt WST-1 (4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) into formazan (orange dye) by cellular mitochondrial dehydrogenases. The color change is directly proportional to the viability and proliferation of cells in the culture.

After 48 h incubation, the medium was removed, and 100uL of WST-1 reagent (WST-1 cell proliferation kit, Roche Diagnostics, Meylan, France; dilution 1:40 in DMEM with 2%FBS) were added into each well. After 45 min incubation at 37℃ with 5% CO2, absorbance was measured at 450 against 630 nm with a microplate reader (Varioskan Lux, Thermo Fisher Scientific, Vantaa, Finland). 4.5.LDH Cytotoxicity Assay

The LDH cytotoxicity assay is based on the measurement of lactate dehydrogenase (LDH), which is a stable cytosolic enzyme that is released upon cell lysis. Released LDH in culture supernatants is measured via the conversion of a tetrazolium salt (iodonitrotetrazolium violet) into red formazan product. cistanche tubulosa dosage reddit The amount of red formazan measured is proportional to the number of lysed cells.

LDH assay (CytoTox96@, Promega, Madison, WI, USA) was performed according to supplier instructions. Positive control(LDH maximum release) was performed by adding 10 uL of lysis solution 10×(Triton X-100 at 0.8%)for 45 minutes before adding the reagent CytoTox969. After incubation， 50 μL of cell culture supernatant was transferred onto a new 96-well microplate （non-sterile）. Then， 50 μL of reagent CytoTox96° were added. The microplate was incubated for 30 minutes at room temperature in the dark. Then, the reaction was stopped by the addition of 50 μL of "Stop solution".LDH release was measured by absorbance quantification with a microplate reader at 490 nm (Varioskan Lux, Thermo Fisher Scientific, Vantaa, Finland).

4.6.Alcian Blue Staining for Glycosaminoglycans Quantification

Alcian blue staining is a coloration method that allows quantifying two types of glycosaminoglycans (GAGs): sulfated (heparan sulfate, keratan sulfate, chondroitin sulfate, and dermatan sulfate) and non-sulfated (hyaluronic acid). Alcian blue solution at pH 1 colors sulfated GAGs, while solution at pH2.5 colors non-sulfated GAGs.

At the end of the incubation, the culture medium was removed, and cells were rinsed twice with PBS 1X (Fisher Scientific, Illkirch, Irance). Then, cells were rinsed for 5 min with 0.1N HCl(pH1, Fisher Scientific, Illkirch, France) or with 3M acetic acid (pH2.5, Fisher Scientific, Illkirch, France) in order to decrease pH and reveal sulfated and non-sulfated glycosaminoglycans, respectively. Then, cells were stained with 1% Alcian Blue(8GX, Sigma-Aldrich, Saint Quentin Fallavier, France) in HC10.1N solution (pH1) or in 3% acetic acid solution (pH2.5)for 30 min. Cells were rinsed twice with tap water for 5 min, dried under the hood,and rinsed with distilled water for 5 min. Then, bound dye was solubilized with Guanidine hydrochloride solution 6M (Sigma-Aldrich, Saint Quentin Fallavier, France)for 5 min under agitation at room temperature. The colored solution was transferred to a new 96-well microplate, and absorbance reading was performed at 600 nm (Varioskan Lux, Thermo Fisher Scientific, Vantaa, Finland).

4.7.Red Sirius Staining for Total Collagen Quantification

The Red Sirius staining procedure allows quantifying total collagen. Picrosirius red, a strong linear anionic dye comprising six sulfonate groups, associates along cationic collagen fibers.

After incubation, the culture medium was removed, and cells were rinsed twice with PBS 1X(Fisher Scientific, Illkirch, France). Then,1 mL of Bouin solution Gigma-Aldrich, Saint Quentin Fallavier, France) was added in each well, and cells were stained for 1 h at room temperature. Then, cells were rinsed twice with distilled water and dried under the hood. Cells were stained with 1 mL of Red Sirius solution (Sirius Red 80 in aqueous saturated picric acid solution, Sigma-Aldrich,Saint Quentin Fallavier, France)for 1 h. After staining, the solution was removed, and cells were rinsed successively with distilled water and HC10.01N to remove unbound dye. Bound dye was solubilized with NaOH0.1N for 30 min. Colored solution was transferred to a new 96-well microplate, and an absorbance read was performed at 550 nm (Varioskan Lux, Thermo Fisher Scientific, Vantaa, Finland). 4.8.Type I Collagen and MMP-1 ELISAs

After incubation, cells were washed twice with PBS 1X (Fisher Scientific, Illkirch, France). Fibroblasts were lysed in RIPA(radioimmunoprecipitation assay)buffer (Sigma-Aldrich, Saint Quentin Fallavier, France) supplemented with protease inhibitor cocktail (Sigma-Aldrich, Saint Quentin Fallavier, France) at 4℃ for 1 h. Cells were scraped, and samples were vortexed for 30 min and centrifuged (12,00x g for 30 min at 4℃). Supernatants containing intracellular proteins were collected and then stored at-80 ℃until analysis for protein determination and Western blot analysis. Protein concentration was determined by Pierce BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA) using Bovine Serum Albumin (BSA) as standard.

Cell culture supernatant were collected and centrifuged to remove cell debris (200× g for 10min at 4 ℃)and stored at-80℃ until analysis.

Type I collagen and MMP-1 measurements were evaluated in culture media with Abcam kits (ab210966 Human Pro-Collagen I alpha 1 SimpleStep ELISA Kit and ab215083 Human MMP1 SimpleStep ELISA Kit, Cambridge, MA, USA) according to the manufacturer's instructions. Absorbance was measured at 450 nm with a microplate reader (Varioskan Lux, Thermo Fisher Scientific, Vantaa, Finland). Results were normalized with cell protein concentrations previously determined. 4.9.Western Blot Protein samples extraction was described in previous Section 4.8. Protein samples with Laemmli buffer (Bio-Rad, Marnes-La-Coquette,France) were boiled 5 min at 95℃and then centrifuged at 16,000×g for 1 min. Equal amounts of protein (10 ug) were loaded into wells of7.5% or 4-15% TGX Stain-Free gel (Bio-Rad, Marnes-La-Coquette, France), along with Precision Plus Protein "Unstained Protein standard (Bio-Rad, Marnes-La-Coquette, France). The gel was run at 200 V for 30 min (PowerPac"Basic with Mini-PROTEAN Tetra System, Bio-Rad, Marnes-La-Coquette, France). The transfer of protein from gel to a mini PVDF (polyvinylidene fluoride)membrane (Bio-Rad,Marnes-La-Coquette, France)was performed with a Trans-Blot Turbo Transfer System (Bio-Rad, Marnes-La-Coquette, France).The membrane was blocked with 3% BSA Gigma-Aldrich, Saint Quentin Fallavier, France) or 5% non-fat dry milk in TBST(Bio-Rad,Marnes-La-Coquette, France) for 1 h at room temperature and washed with TBST. Membrane was probed with type I collagen (Novotec, Reuver, The Netherlands), type III collagen (Novotec, Reuver, The Netherlands), MMP-1 (EnoGene, New York, NY, USA), or TIMP-1(RayBiotech, Peachtree Corners, GA, USA) antibodies; then, it was washed several times with TBST and incubated for1h at room temperature with secondary peroxidase-conjugated antibodies (Jackson ImmunoResearch, Ely, UK).Then, clarity Western ECL substrate (Bio-Rad, Marnes-La-Coquette, France) was applied for 5 min to membranes and revelation was performed using ChemiDoc XRS+Molecular Imager (Bio-Rad, Marnes-La-Coquette, France). Protein level was quantified using ImageLab v6.01.1 software.

4.10.Zymography

Zymography was performed to analyze the active form of collagenase MMP-1 accord-ing to Inanc et al. (2017) adapted method [52].

SDS-PAGE gels (10% polyacrylamide, Bio-Rad, Marnes-La-Coquette, France) were copolymerized with 1 mg/mL of type I collagen rat tail Gibco, Fisher Scientific, Illkirch, France).Protein samples(5 ug) were mixed with non-reducing sample buffer (62.5 mM Tris HCl pH 6.8,20% glycerol,4% SDS,0.01% bromophenol blue), centrifuged 1 min at 4℃,loaded into wells of the gel,along with Precision Plus Protein" All Blue Standards (Bio-Rad, Marnes-La-Coquette,France),and then electrophoresed at 110 V at 4℃ for2h The gel was washed twice in 2.5% Triton X-100 (v/v) for 15 min each time in order to remove SDS. Then, the gel was incubated in Zymogram Development Buffer (Bio-Rad, Marnes-La-Coquette,France) for 48 h at 37℃.After incubation, the gel was stained for 1h with 0.5% Coomassie Blue R-250 (Bio-Rad, Marnes-La-Coquette, France) solution (w/v)and then destained in 40% methanol and 10% glacial acetic acid solution (Sigma-Aldrich, Saint Quentin Fallavier, France).MMP-1 activity was detected as a clear band against a Coomassie Blue-stained gel background. Quantification was performed on performed ChemiDoc XRS+ Molecular Imager (Bio-Rad, Marnes-La-Coquette, France) with Image Lab version 6.01.1 software (Bio-Rad). 4.11.RNA Isolation and Real-Time RT-PCR

After 48 h incubation, total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA) and chloroform (Acros Organics,Fisher Scientific, Illkirch, France), precipitated with isopropanol (Fisher Scientific, Illkirch, France), washed with ethanol 75%, dried under the hood before resuspension in DEPC (diethylpyrocarbonate)-treated water (Fisher Scientific, IIlkirch, France). RNA quantity and purity level (between 1.8 and 2.0) were estimated with absorbance measurement on a microplate reader (Varioskan Lux, Thermo Fisher Scientific, Vantaa, Finland) at 260 and 280 nm using drop TM Plate (Thermo Fisher Scientific, Waltham, MA, USA).

One microgram of total RNA was treated with DNaseI (iScript Dnase Master Mix, Bio-Rad, Marnes-La-Coquette, France) for 5 min at 25℃ to remove DNA contaminants. The enzyme was inactivated for 5 min at 75°C.

Reverse transcription was performed on 1 μg of total RNA， previously treated with DNase I, using iScript Reverse Transcription (RT) Supermix (Bio-rad, Marnes-La-Coquette, France).Steps followed for cDNa synthesis were 5 min at 25°C,20 min at 46°C,and 1min at95°C.

Controls of non-template were included in PCR experiments. qPCR was performed in 96-well plates for a total volume of 20 μL containing 1 uL of 1∶15 diluted cDNA samples from RT，1 μL of primer（see Table 4， Bio-Rad， Marnes-La-Coquette， France），10 μL of iTaq Universal SYBR Green Supermix （Bio-Rad， Marnes-La-Coquette， France）， and 8μL of nuclease-free water. The amplification conditions were 2 min at 95 ℃ followed by 40 cycles of 5s at 95℃and 30s at 60℃,and end with5s at 95℃and 5s at 65 ℃ before protocol for the melting curve: increase of 0.5 ℃ from 65℃ to 95 ℃. PCR experiments were performed on CFX 96 system (Bio-Rad, Marnes-La-Coquette, France), and results were collected from Bio-Rad CFX Manager software v3.1 (Marnes-La-Coquette, France). The mRNA amounts were normalized to RPL13A mRNA, and analysis of relative gene expression was calculated using the 2-AACt method.

4.12. Statistical Analysis

All values were expressed as the mean of n experiment ± standard error (SE).

Statistical analyses were performed using Addinsoft (2020). Evaluation of normal distribution of the data was assessed using Shapiro-Wilk's test. Significant differences between control and experimental samples were analyzed by Student's-t-test (independent, two-sided), when normal distribution occurred, and by Mann-Whitney's test, when normal distribution was rejected. Significant differences compared with control according to statistical test are indicated by asterisks(*p<0.05,**p<0.01 and **p<0.001). Differences that are not statistically significant are named "NS" in the text and are characterized by the absence of asterisks on the figures.

5. Conclusions

This work demonstrates that poly-and oligosaccharide fractions,enriched in ulvans, recovered after green novel technology EAE from the green seaweed Uloa sp., influence fibroblast proliferation, ECM protein synthesis, matrix renewal, and remodeling. Potential application in cosmetic anti-aging strategies could emerge since skin aging is characterized by a collagen synthesis decrease. Thus, skin matrix renewal is beneficial in this context. Thus, ulvans, from Ulva sp. fractions obtained after EAE, are biologically active on human dermal skin fibroblasts metabolism. The authors suggest that this activity is a function of the ulvan abundance, sulfate composition, and molecular weight of the fractions. The authors speculate that these sulfated rhamnose-rich molecules correspond to signaling molecules, which are recognized by lectin sites of the membrane fibroblasts and subsequently stimulate their metabolic activity at both anabolic and catabolic points of view. However, further investigations are necessary to assess deeply the molecular mechanism activated by poly-and oligosaccharide Ulou sp. fractions at the transcription factors level, the penetration capacity of high and low molecular weight ulvans in 3D skin or ex vivo skin explant models, and confirm these results of ECM modulation with Uloa EAE-derived fractions in these models for an anti-aging target.

This article is extracted from Mar. Drugs 2021, 19, 156. https://doi.org/10.3390/md19030156 https://www.mdpi.com/journal/marinedrugs