(A) PGG was synthesized from tannic acid by a methanolysis reaction

(A) PGG was synthesized from tannic acid by a methanolysis reaction. suggesting possibilities in the therapy and prevention of several diseases. In the present study, we synthesized PGG chemically and found that it exhibited UVB radiation-induced skin protective activity. To assess this, we performed studies in human dermal fibroblasts and an study in a hairless mouse model with UVB radiation. PGG LY500307 alleviated UVB radiation-induced skin damage in the hairless mouse model, and this activity was associated LY500307 with its antioxidant and anti-inflammatory properties. MATERIALS AND METHODS Reagents and antibodies Tannic acid and 2,7-dichlorofluorescin diacetate (DCF-DA) were obtained from Sigma-Aldrich (USA). Antibodies specific for phospho-IB (Ser32/36), phospho-NF-B p65 (Ser536), phospho-IKK/ (Ser176/180), IKK, phospho-p38 (Thr180/Tyr 182), p38, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-JNK (Thr183/Tyr185), JNK, COX-2, ICAM-1, and GAPDH were obtained from Cell Signaling Technology (USA). Anti-IB and anti-NF-B p65 antibodies LY500307 were purchased from Santa Cruz Biotechnology (USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit antibody was obtained from Life Technologies (USA). All of the other chemicals used were analytical grade and purchased from Sigma-Aldrich unless normally noted. Synthesis and purification of PGG PGG was synthesized from tannic acid by a modification of Chen and Hagermans method (Chen and Hagerman, 2004). Briefly, tannic acid (10.0 g) was dissolved in methanolysis solution (70% methanol 140 ml and 0.1 M acetate buffer 60 ml). The combination was heated at 65C for 16 h, and then adjusted to pH 6.0 by addition of 0.25 M NaOH. After removal of methanol, the reactant was resuspended in distilled water and partitioned into ethyl ether and aqueous layers following ethyl acetate. PGG was purified from your ethyl acetate portion by high-performance liquid chromatography (HPLC) using a separation technology column (Jsphere ODS, 4 m, 250 4.6 mm), and an acetonitrile-water gradient with 0.1% trifluoroacetic acid (TFA) to afford 300 mg of PGG. This was recognized by reversed-phase HPLC and fast atom bombardment bass spectrometry (FAB-MS). PGG with a purity 97% was dissolved in dimethyl sulfoxide (DMSO) as a 10 mM stock solution, and kept at ?20C in aliquots. Cell culture and UVB radiation Normal human dermal fibroblasts were obtained from Modern Cell & Tissue Technologies (Korea), and managed in Dulbeccos Modified Eagles Medium (DMEM) made up of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Life Technologies) in a humidified atmosphere of 5% CO2 and 95% air flow at 37C. Cells pre-incubated with PGG for 1 h were washed with PBS and exposed to a 100 mJ/cm2 UVB light with a 312 nm UVB light source (VL-6.LM, Vilber Lourmat, France). The UVB intensity was measured with a Vav1 Waldmann UV meter (model 585100, Germany). After UVB LY500307 radiation, the cells were washed with PBS, and replaced with PGG made up of media for appropriate time periods. Cell viability assay Cell viability was decided using EZ-CyTox Enhanced Cell Viability Assay Kit (Daeil Lab Support, Korea) made up of the WST-1 reagent. Cells were treated with numerous concentrations of PGG or irradiated with numerous doses of UVB. After incubation for 24 h, assay reagent was added and absorbance of the soluble formazan was measured at 450 nm using a microplate reader (Molecular Devices, USA) after a reaction at 37C, as previously explained (Kim et al., 2013a; 2015). Measurement of ROS and superoxide LY500307 production UVB-irradiated cells were incubated for 6 h following incubation with PGG for 1 h in serum-free medium. ROS production was measured by staining with DCF-DA (10 M), as previously explained (Kim et al., 2013b). To examine superoxide production, cells pre-incubated with PGG for 30 min in the presence of lucigenin (25 M) were irradiated with UVB, and superoxide production was immediately measured by lucigenin-dependent chemiluminescence, as previously explained (Kim et al., 2013c). Measurement of superoxide- and peroxynitrite-scavenging activities Superoxide radicals were produced by the non-enzymatic NADH/phenazine methosulfate (PMS) system and the radical scavenging activity was measured by reduction of nitroblue tetrazolium (NBT), as previously explained (Kim et al., 2013c). The reaction mixtures made up of PGG, NBT (100 M), PMS (30 M), and NADH (150 mM) in 50 mM phosphate buffer (pH 7.4) were incubated at 25C for 5 min, and the absorbance was measured at 560.