In the previous study, we fermented microalgae Pavlova lutheri by H. polymorpha as below (7). The cellulose-degraded microalga P. lutheri was autoclaved at 121°C for 30 min in a buffer (pH 7) solution at a ratio of 1:15 (w/v). Subsequently, H. polymorpha was inoculated with the autoclaved solution at a concentration of 1% (v/v) and incubated at 37°C for 12 days. Fermented microalgae was lyophilized and stored at 80°C until used. Human osteoblastic (MG-63) cells were obtained from American Type Culture Collection (Manassas, VA, USA). Dulbecco's Modified Eagle's Medium (DMEM), Trypsin-ethylenediaminetetraacetic acid (EDTA), penicillin/streptomycin, fetal bovine serum (FBS) and other materials required for culturing cells were purchased from Gibco BRL, Life Technologies (Grand Island, NY, USA). The p38 inhibitor (SB203580), NF-κB inhibitor [pyrrolidine dithiocarbamate (PDTC)] and 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents used in this study were of analytical grade.

Human osteoblastic MG-63 cells were cultured in DMEM containing 5% FBS in a humidified atmosphere with 5% CO₂ at 37°C. Cells were sub-cultured at 3-day intervals using trypsin-EDTA and were seeded in 96-well plates at a density of 5×10³ cells per well. 24 h post plating, media was replaced with DMEM without serum and cells were subjected to sample treatment for another 3 days. Subsequently, 100 µl of 1 mg/ml MTT reagent was added to each well, and incubated for 3 h. The supernatant was removed and formazan was dissolved in DMSO and its formation was observed by monitoring the signal at 540 nm using a microplate reader.

Cells were seeded into 12-well plates at a density of 5×10⁵ cells per well for 24 h and then treated with samples in DMEM media without serum for 3 days. Cells were then washed three times with PBS, and were lysed with a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl and 1% Triton X-100. Briefly, the lysate was mixed with p-NPP substrate solution which consists of 1.5 mM MgCl₂ and 15 mM p-nitrophenyl phosphate (p-NPP). The activity of phosphatases to catalyze the hydrolysis of p-NPP to p-nitrophenol was evaluated by measuring the absorbance at 405 nm (19). ALP activity was normalized according to the cellular total protein content of cell lysate determined by BCA protein assay (Sigma-Aldrich).

The lyophilized fermented microalgae was dissolved in distilled water and passed through disposable Silica-based bonded-phase cartridges (Sep-Pak Vac C₁₈ 20 cc/5 g; Waters, Milford, MA, USA). The cartridges were activated with 100% methanol in 0.1% trifluoroacetic acid (TFA) and subsequently the cleaned product was eluted with 10% methanol in 0.1% TFA. The eluent was lyophilized then further purified as described below.

The cleaned eluent from previous step was loaded onto HiPrep 16/10 DEAE ion exchange column equilibrated with 20 mM sodium acetate buffer (pH 4.0) and eluted with a linear gradient of NaCl (0–2 M) at a flow rate of 1.0 ml/min by 280 nm absorbance using FPLC (Amersham Bioscience Co., Uppsala, Sweden). Separated fractions were pooled and desalted by an electrodialyzer (Micro Acilyzer model G3; Asahi Chemical Industry Co., Tokyo, Japan) with a 100 Da molecular mass cutoff membrane (Asahi Chemical Industry Co.). The desalted fractions were lyophilized and investigated for their ALP activity. The fraction which had the highest ALP activity was further purified on a Primesphere 10 C₁₈ (20×250 mm) column at reversed-phase HPLC (Dionex Corp., Sunnyvale, CA, USA) with a linear gradient of acetonitrile (0–30% in 30 min) containing 0.1% TFA at a flow rate 1.0 ml/min by 215 nm absorbance. Elution peaks were collected and tested for ALP activity. The highest active fraction was re-applied to a Acclaim 120 C₁₈ analytical column with 15% acetonitrile (20% v/v, in 15 min) containing 0.1% TFA at flow rate of 1 ml/min. The purified peptide was subjected to amino acid sequence analysis.

The accurate molecular mass and amino acid sequence of the purified peptide was determined by quadrupole time-of-flight mass spectroscopy (Q-TOF MS; Micromass UK Ltd., Altrincham, UK) coupled to electrospray ionization (ESI) source. The molecular weight of the purified peptide dissolved in methanol/water (1:1, vol/vol) was detected by a doubly charged (M+2H)⁺² state in the mass spectrum and the amino acid sequence was identified by tandem MS analysis. Spray voltage was 4,000 V and nitrogen was maintained at 40 psi for nebulization. Mass spectra were acquired over the range from 50 to 1,500 m/z.

The levels of mineralization were determined in the 24-well plates using Alizarin Red staining (Sigma-Aldrich) after 7 days treatment with the peptide. Briefly, cells were fixed with 70% (v/v) ethanol for 1 h and then stained with 40 mM Alizarin Red S (pH 4.2) for 15 min at room temperature. After removing Alizarin Red S solution by aspiration, cells were incubated in PBS for 15 min at room temperature on an orbital rotator. Cells were then rinsed once with fresh PBS and subsequently distained for 15 min with 10% cetylpyridinium chloride in 10 mM sodium phosphate (pH 7.0). The extracted stain was then transferred to a 96-well plate and the absorbance at 562 nm was measured using a microplate reader (Tecan Austria GmbH, Grödje, Austria).

Cells were treated with p38 inhibitor (SB203580, 20 µM) or NF-κB inhibitor (PDTC, 10 µM) for 1 h prior to treatment with the peptide (in the presence of inhibitor) for 72 h. Cells were then harvested in lysis buffer (RIPA; Sigma-Aldrich). Cleared total cell lysates (20 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech, Amersham, UK). Membrane was blocked with 5% skim milk and probed with the primary antibody (diluted 1:1,000) followed by incubation with a secondary antibody conjugated with horseradish peroxidase (diluted 1:5,000) at room temperature. The proteins of interest were detected using a chemiluminescent ECL assay kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Primary and secondary antibodies including ALP (sc-373737), OCN (sc-365797), β-actin (sc-47778), p-p38 (sc-7973), p-pERK (sc-7383), p-Jun (sc-822), p-p65 (sc-166748) and anti-mouse IgG-HRP (7076) purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Cell Signaling Technology, Inc. (Danvers, MA, USA). Western blots were visualized using an LAS3000^® Luminescent image analyzer and protein expression was quantified by Multi Gauge v3.0 software (Fujifilm Life Science, Tokyo, Japan).

All experiments were repeated at least three times and expressed as the means ± SD. Differences between the means of individual groups were assessed by two-way ANOVA with Tukey's multiple comparisons test using the statistical software package GraphPad Prism 6 software (San Diego, CA, USA).

We previously investigated the antioxidant activities of the fermented P. lutheri microalgae by H. polymorpha or C. rugopelliculosa and suggested the potential of fermented P. lutheri as protein source possessing antioxidant activity (7). In this study, we investigated the difference between the effect of fermented and non-fermented P. lutheri on osteoblastic differentiation in MG-63 cells. The lyophilized P. lutheri was treated with cellulose (1% dry powder weight of the raw substance) to break its cell wall and was subsequently fermented by inoculation with H. polymorpha for 12 days (7). Different concentrations (10, 50, 100 µg/ml) of fermented and non-fermented P. lutheri (with degraded cell-wall) were added to the cells for 3 days followed by measuring their toxicity and ALP activity. The cells treated with either fermented or non-fermented P. lutheri did not show any significant difference in viability compared to control which is non-treatment group (Fig. 1A), however a significant increase in the ALP activity of the cells were observed when treated with 100 µg/ml non-fermented P. lutheri with P<0.05 and 50 and 100 µg/ml fermented P. lutheri with P<0.01 (Fig. 1B).

We then further traced the purification profile of peptide from fermented P. lutheri and characterized the effect on osteoblastic differentiation in MG-63 cells.

The lyophilized fermented P. lutheri was dissolved in distilled water and passed through disposable Sep-Pak Vac C₁₈ cartridges to salt out. The cleaned product was then concentrated and further separated using a weak anion exchange chromatographic method on a FPLC at 280 nm. The monitored peaks were desalted and analyzed for their ALP activity (data not shown). The selected fraction with highest ALP activity was split to four portions through reversed-phase chromatographic method on a HPLC at 215 nm (Fig. 2A). Each portion was measured for its ALP activity at 100 µg/ml and Fraction 4 (Fr.4) showed the highest activity with of 1.642 fold compared to the control (P<0.01; Fig. 2B). The peptide PPLF was purified from the additional analysis using an Acclaim 120 C₁₈ analytical column and was determined as Glu-Pro-Gln-Trp-Phe-Leu (MW 908.9 Da) by ESI/MS spectroscopy (Fig. 2C and D).

The effect of PPLF on ALP activity and mineralization which are main makers for osteoblastic differentiation (8,18) was assessed in MG-63 cells. Cells treated with PPLF showed significantly increased levels of ALP release (Fig. 3A) and mineralization (P<0.05 and P<0.01) (Fig. 3B). In order to further assess the effects of PPLF, we analyzed protein levels of two of the known early markers of differentiation ALP and OCN in cells treated with PPLF (10, 20, 50 µM) for 3 days. Both protein levels showed to be up-regulated by PPLF treatment compared to the non-treated cells (P<0.05; Fig. 4). These results suggest that PPLF induces the expression of the early markers of osteoblastic differentiation in MG-63 cells.

To determine the mechanism of the PPLF effect on differentiation in MG-63 cells, the phosphorylation levels of MAPK pathway (p38, ERK1/2, Jun) and NF-κB (p65) which are known to control ALP and OCN expression and are involved in osteoblastic differentiation were examined (8,10,18). As shown in Fig. 5, p-p38 and p-p65 exhibited a significant increment by PPLF at 20 and 50 µM (P<0.05), however p-ERK1/2 and p-Jun did not show any change with PPLF treatment. The result suggests that PPLF treatment activates p38/p65 signaling pathway. To examine whether the p38/p65 phosphorylation by PPLF treatment was connected to the expression of ALP and OCN, we assessed the expression of ALP and OCN in the cells treated PPLF (50 µM) when pre-treated with p38 inhibitor SB203580 (20 µM) or NF-κB inhibitor PDTC (10 µM). Pre-treatment with SB203580 inhibited both p38 and p65 phosphorylation levels and resulted in a decrease in the expression of ALP and OCN (Fig. 6A). We also observed the same result on ALP and OCN expression by PDTC treatment, however p38 phosphorylation showed no-significant change compared to PPLF treatment suggesting that p38 is upstream of p65 (Fig. 6B). These data suggest that treatment with PPLF induces ALP and OCN expression via p38/p65 signaling pathway in MG-63 cells.