[PubMed] [CrossRef] [Google Scholar]Macmicking J, Xie QW, Nathan C. (Cimino ((occurring naphthoquinone compound. Both shikonin and its analogs are potential pharmaceutical brokers with anticancer activities via inhibiting topoisomerase-I, inducing apoptosis, regulating the activities of phosphorylated extracellular regulated protein kinase (pERK) and c-Jun N-terminal kinase (JNK), and suppressing the expression of tumor necrosis factor receptor-associated protein 1 (TRAP1) (Xuan and Hu, 2009; Wu in LPS-treated BV2 microglial cells by inhibiting crosstalk between ROS and NF-B. MATERIALS AND METHODS Chemicals CP 31398 2HCl The roots of were purchased from an herb market (Jecheon, Republic of Korea). A voucher specimen has been deposited in Division of Wood Chemistry & Microbiology, CP 31398 2HCl Department of Forest Products, Korea Forest Research Institute (Seoul, Republic of Korea). The roots (1 kg) were extracted with acetone (4 L) by Ultrasonic (JAC 4020P, Republic of Korea) for 4 h at room temperature and repeated three times. After filtration, the solution was evaporated to remove CHCl3. Purification was carried out on Sephadex LH-20 column (10 400 mm) eluting with CHCl3/EtOH (2:1, v/v) and separated into four fractions. Subfraction was separated by MPLC (EYERA system) with YMC-GEL ODS-A (S-75 m, AA12S75, 30100 mm, Kyoto, Japan) column chromatography and eluted with MeOH/H2O (7:3, v/v). UV detected at 280 nm to yield shikonin (15 mg). The chemical structures of shikonin (as shown in Fig. 1A) were determined by IH-NMR and 13C-NMR (Varian Unity-Inova 500 MHz, Palo Alto, CA, USA). Open in a separate window Fig. 1. Effects of shikonin around the viability of BV2 microglial cells. (A) Chemical structure of shikonin isolated from (forward 5-cct cct CP 31398 2HCl cca ccc tac caa gt-3 and reverse 5-cac cca aag tgc ttc agt ca-3), (forward 5-aag act tgc cag gct gaa ct-3 and reverse 5-ctt ctg cag tcc agg ttc aa-3), (forward 5-tgt gat ggt ggg aatggg tc-3 and reverse 5-ttt gat gtc acg cac gat tt-3). The following PCR conditions were applied: and 4C for 10 min to obtain the supernatants. The supernatants were collected and protein concentrations determined using Rftn2 a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). The samples were stored at ?80C or immediately used for western blot analysis. The proteins were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). Proteins were detected using an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL, USA). NO assay BV2 microglial cells (2105 cells/ml) were plated onto 24-well plates and pretreated with the indicated concentrations of shikonin 1 h prior to stimulation with 500 ng/ml LPS for 24 h. Supernatants were collected and assayed for NO production using Griess reagent. Briefly, the samples were mixed with equal volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride) and then incubated at room temperature for 10 min. The absorbance was measured at 540 nm on a microplate reader (Thermo Electron Corporation). Sodium nitrite dilution series were used as a standard to determine the nitrite concentration in the supernatants. Measurement of PGE2 and TNF- The expression levels of PGE2 and TNF- were measured by an enzyme immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturers instructions. Briefly, BV2 microglial cells (2105 cells/ml) were plated in 24-well plates and pretreated with the indicated concentrations of shikonin 2 h prior to stimulation with 500 ng/ml LPS for 24 h. One hundred microliters of culture medium supernatant was collected for determination of PGE2 CP 31398 2HCl and TNF- concentration by ELISA. ROS analysis BV2 microglial cells were seeded on 24-well plate at a density of 2105 cells/ml CP 31398 2HCl and preincubated with florescence dye 6-carboxy-2,7-dichlorofluorescein diacetate (H2DCFDA, Molecular Probes, Eugene, OR, USA) for 1 h and then treated the indicated concentrations of shikonin, NAC, and GSH 1 h before stimulation with LPS (500 ng/ml) for 24 h. The cells were lysed with triton and the sample was centrifuged and supernatant was analyzed for ROS production using GLOMAX luminometer (Promega). Electrophoretic mobility assay (EMSA) EMSA was performed with the nuclear extract. Synthetic complementary NF-B (5-agt tga ggg gac ttt ccc agg c-3) binding oligonucleotides (Santa Cruz Biotechnology) were 3-biotinylated using the biotin 3-end DNA labeling kit (Pierce) according to the manufacturers instructions, and annealed for 30 min at.