PPHs were purchased from Codebio Inc. (Cheonan, Republic of Korea). Hydrogen peroxide and thapsigargin were obtained from Sigma Chemical Company (St. Louis, MO, USA). Caspase-3 and -12 activity kits were obtained from BioVision (Mountain View, CA, USA). All other reagents used were of analytical grade and were generally obtained from Sigma.
Extraction and quantitation of PPHs using HPLC-DAD
PPHs were purchased from Codebio Inc. (Cheonan, Republic of Korea). Placentas were thawed using a defroster and then washed with saline at 16 °C to remove blood and cords. Placentas were hydrolyzed using papain, bromelain, pronase, and Alcalase at 70 ± 0.1 °C for 2 days (pH 4.5–6.0). Next, hydrolytic enzymes were inactivated at 100 ± 0.1 °C for 30 min and the resultant hydrolysates were filtered. The lipids were then removed by mixing with calcium and phosphate salts and by filtering the insoluble particulate matter. Finally, the hydrolysates were adjusted to pH 7.0 ± 0.2 using calcium and phosphate salts. Hydrolysates were mixed with AccQ-Fluor buffer and Acc-Fluor reagent (Waters Corporation, Milford, MA, USA), after which the mixture was heated at 80 °C. An aliquot of the sample was filtered and injected into an AccQ-Tag column (150 × 2.1 mm, 3 μm particle size; Waters Corporation, Milford, MA, USA) in an HPLC instrument for chromatographic separation. A gradient mixture of AccQ-Tag Eluent (A) and acetonitrile (B) was used as the mobile phase at a flow rate of 1 mL/min at 30 °C. The initial eluent was 100 % A, which linearly decreased to 67 % A over 33 min. The eluent was switched to 100 % B for 3 min and then switched back to 100 % A for 26 min. Amino acids were detected using a fluorescence detector at excitation and emission wavelengths of 250 and 395 nm, respectively.
Cell culture and viability analysis
The murine calvaria-derived MC3T3-E1 osteoblast-like cell line (4 to 10 passages) was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were seeded at 1 × 105 cells/mL and maintained in minimum essential medium (α-MEM) supplemented with 10 % fetal bovine serum (FBS) and penicillin-streptomycin (Invitrogen). Cells were incubated at 37 °C in a 95 % air/5 % CO2 atm. Cell viability was detected using a trypan blue exclusion assay. Briefly, MC3T3-E1 cells (5 × 104 cells/well) were incubated in 12-well plates overnight and treated for 24 h with drugs in medium containing 10 % serum. Cells were washed with sterile phosphate-buffered saline (PBS), treated with 0.25 % trypsin-EDTA (GIBCO BRL), and harvested. Cells were diluted in 0.1 % trypan blue (GIBCO BRL) and then counted under a light microscope.
For immunoblotting, MC3T3-E1 osteoblastic cells were lysed with extraction buffer. Proteins in the resultant lysates (40 μg) were resolved on a polyacrylamide gel and transferred to a nitrocellulose membrane. The blots were probed overnight at 4 °C with primary antibodies, washed, and probed again with species-specific secondary antibodies coupled to horseradish peroxidase (GE Healthcare, Piscataway, NJ, USA). Chemiluminescence reagents (GE Healthcare) were used for detection. Primary antibodies consisted of rabbit anti-GADD153/C/EBP homologous protein (CHOP), rabbit-anti-PERK, rat anti-GRP78, rabbit anti-ATF6α, mouse anti-eIF2α, mouse anti-β-actin (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA), rabbit anti-IRE1α, and rabbit anti-p-eIF2 (Cell Signaling Technologies, Inc., Danvers, MA, USA).
Measurement of caspase-3 activity
To analyze caspase-3 activity, pellets were resuspended in extraction buffer [25 mM HEPES (pH 7.4), 0.1 % Triton X-l00, 10 % glycerol, 5 mM DTT] and spun by centrifugation at 13,000 rpm at 4 °C for 30 min. Soluble protein (40 μg) was mixed with 100 μM caspase-3-specific substrate Ac-DEVD-AFC (Sigma-Aldrich) and incubated at 37 °C. Caspase-3 activity was analyzed by monitoring fluorogenic AFC release at 37 °C. Substrate cleavage was monitored at 405 nm using a SPECTRAmax 340 microplate reader and analyzed using SOFTmax PRO software (Molecular Devices, Sunnyvale, CA, USA).
Measurement of caspase-12 activity
To analyze caspase-12 activity, pellets were measured by detecting free AFC cleavage by caspase-12-specific substrates. These experiments were performed with a caspase-12 Assay Kit (Biovision, San Francisco, CA, USA). After the lysates were incubated with ATAD-AFC for 2 h at 37 °C, the absorbance of each sample was analyzed at 505 nm.
Measurement of alkaline phosphatase activity
To analyze ALP activity, cells were treated with Krebs (control), PPHs (100 μg/mL), H2O2 (400 μM), and in the presence or absence of 100 μg/mL PPHs. After treatment for 4 h, cells were lysed. The amount of ALP activity and the protein concentration were measured in each supernatant using an ALP activity assay kit (Cell Biolabs, San Diego, CA, USA).
The mRNA levels of ALP, osteocalcin, Runx2, osterix, and GAPDH were determined using a PrimeScript™ RT reagent Kit (TaKaRa Bioscience, Kyoto, Japan). The sequences of the primers used for RT-PCR were as follows: ALP, forward primer: 5′-CCATGGTAGATTACGCTCACA-3′, reverse primer: 5′-ATGGAGGATTCCAGATACAGG-3′; osteocalcin, forward primer: 5′-AGCTATCAGACCAGTATGGCT-3′, reverse primer: 5′-TTTTGGAGCTGCTGTGACATC-3′; Runx2, forward primer: 5′-CTCAGTGATTTAGGGCGCATT-3′, reverse primer: 5′-AGGGGTAAGACTGGTCATAGG-3′; Osterix, forward primer: 5′- CGGGTCAGGTACAGTG-3′, reverse primer: 5′- ACCATGACGACAAGGG-3′; and GAPDH, forward primer: 5′-ATCACCATCTTCCAGGAG-3′, reverse primer: 5′-ATGGACTGTGGTCATGAG-3′. Reverse transcription was performed by incubating the reactions at 37 °C for 15 min and then at 85 °C for 5 s. For polymerase chain reaction amplification, an initial denaturation step was performed at 94 °C for 3 min, followed by annealing at 55 °C for 20 s and elongation at 72 °C for 45 s. In total, 35 cycles were performed.
NADPH oxidase activity assay
Cells were seeded in six-well plates and cultured for 48 h. Next, the cells were treated with 100 μM H2O2 for 6 h in the presence or absence of 100 μg/mL PPHs. NADPH oxidase activity was determined based on superoxide-induced lucigenin photoemission as described by Rao and Maddala et al. . Enzymatic assays were performed in a final volume of 0.2 ml containing 50 mM phosphate buffer (pH 7.0), 1 mM EGTA, 150 mM sucrose, 0.5 mM lucigenin, 0.1 mM NADPH, and cell lysis solution. Enzymatic reactions were initiated by the addition of lucigenin. Photoemission, expressed as relative light units, was measured every minute for 10 min using a luminometer. Assays were performed in the dark at room temperature with all appropriate controls.
Superoxide dismutase (SOD) activity assay
Cells were seeded in six-well plates and cultured for 48 h. The cells were then treated with 100 μM H2O2 for 6 h in the presence or absence of 100 μg/mL PPHs. Next, cells were harvested and the level of SOD activity was determined using a SOD assay kit (k335-100, Biovision) according to the manufacturer’s instructions.
DCFDA assay (ROS production)
The cellular ROS level was measured by following the protocol described by Badham et al. . Briefly, cells were treated with 100 μM H2O2 at 37 °C in the presence or absence of 100 μg/mL PPHs for 6 h. Next, cells were incubated with 10 μM 2′, 7′-dichlorofluorescein diacetate (DCFDA) at 37 °C for an additional 30 min. The fluorescence intensity of 2′,7′-dichlorofluorescein, a product of the reaction between DCFDA and cellular ROS, was analyzed using a fluorescence reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA). Data were normalized to the H2O2 absorbance values.
Results are presented as means ± standard errors of the mean (SEMs) for multiple wells tested in at least three separate experiments. MicroCal Origin software (Northampton, MA, USA) was used for statistical calculations. Differences were tested for significance using one-way analysis of variance (ANOVA) with Duncan’s multiple range test.