Eupatin was obtained from the National Cancer Institute (NCI No. 122412). We purchased primary antibodies against p-GSK3b (Tye216) and p-p65 (Ser536) from Cell Signaling Technology (Danvers, MA, USA). We obtained antibodies against p-tau (Ser396) from Abcam (Cambridge, MA, USA) and those against p-tau (Ser202/Thr205), p-tau (Ser262), and tau w from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against GSK3b and a-tubulin were obtained from GeneTex Inc. (Hsinchu, Taiwan), and antibody against iNOS was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Jackson ImmunoResearch Inc. (West Grove, PA, USA) was our source for horseradish peroxidase (HRP)-conjugated antimouse and antirabbit immunoglobulin G secondary antibodies, and OA was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Unless otherwise stated, we obtained all chemicals not mentioned here from Sigma-Aldrich (St. Louis, MO, USA).
Murine RAW264.7 cells, a mouse macrophage cell line, were obtained from the Bioresource Collection and Research Center (Hsinchu city, Taiwan). Mouse BV-2 microglia were kindly provided by Prof. Shiow-Lin Pan (Graduate Institute of Cancer Biology and Drug Discovery, Taipei Medical University, Taipei, Taiwan). Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Life Technologies, Carlsbad, CA, USA) with supplementation of 100 μg/mL streptomycin (Biological Industries, Kibbutz Beit Haemek, Israel), 10% (vol/vol) fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA, USA), and 100 U/mL penicillin was used for cell culturing. We obtained the murine neuroblastoma cell line (Neuro-2a) from the Bioresource Collection and Research Center. After culturing the cells in minimum essential media consisting of streptomycin (100 μg/mL), penicillin (100 units/mL), and 10% fetal bovine serum, we incubated them at a temperature of 37°C in a humidified 5% CO2 air atmosphere.
Cell cytotoxicity assay
Colorimetric MTT assay was employed for measuring cell cytotoxicity. For 48 h, 1 × 104 cells were incubated in 96-well plates in 1 mL of medium with the control vehicle or vehicle containing the experimental compound. The next step after the application of different treatments was the addition of MTT (1 mg/mL) and incubation of the plates at 37°C for another 2 h. We then pelleted and lysed the cells in 10% sodium dodecyl sulfate (SDS) with 0.01 M HCl and used a microplate reader to measure the absorbance at 570 nm.
Nitrite production was measured in RAW264.7 macrophage and BV-2 microglia cell supernatants. In brief, after culturing 1 × 106 cells in 6-well plates and stimulating them using 100 ng/mL LPSs for 24 h, we mixed a Griess reagent with the cell supernatant (100 mL each) and measured the optical density at 550 nm. A standard curve based on obtained concentrations of sodium nitrite dissolved in DMEM was generated for calculating the nitrite concentration.
IL-6 enzyme-linked immunosorbent assay
Thirty-minute incubation of the cells with the experimental compounds was applied prior to and during 24-h incubation with LPSs (100 ng/mL). After collection of the medium, an enzyme-linked immunosorbent assay kit (PeproTech Inc., Cranbury, NJ, USA) was used to perform an assay for IL-6.
Cells were seeded 1 day before transfection. After 20 min of mixing 1 mL of TurboFect transfection reagent and pRK5-EGFP-Tau P301L plasmids (1 mg) at room temperature and addition of the mixture to the cells, the suspensions underwent 24-h incubation at 37°C in a humidified 5% CO2 atmosphere.
For 10 min at 4°C, 1 × 106 cells were incubated in lysis buffer (20 mM HEPES, pH 7.4; 2 mM EGTA, 0.1% Triton X-100; 50 mM β-glycerophosphate; 1 mM DTT; 10% glycerol; 1 μg/mL leupeptin; 1 mM sodium orthovanadate; 1 mM phenylmethylsulfonyl fluoride, and 5 μg/mL aprotinin). Next, the cells were removed, placed on ice for 10 min, and subjected to 30-min centrifugation (17,000 g) at 4°C. Next, we electrophoresed 20-μg protein samples on SDS polyacrylamide gels before transferring them onto a nitrocellulose membrane. The nitrocellulose membrane was subsequently blocked through 30-min incubation with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 (TBST) at room temperature. Immunoblots were obtained through incubation overnight at 4°C with primary antibodies in TBST and subsequent 1-h incubation at room temperature with secondary antibodies conjugated with HRP. Measurement of antibody binding was performed through photographic film exposure and application of an enhanced chemiluminescence reagent (GE Healthcare Corp., Buckinghamshire, UK).
Bioinformatics and protein modeling
Molecular docking analysis was performed using the Maestro docking module (Schrödinger). The GSK3b crystal structure was obtained from the Protein Data Bank (PDB ID: 1UV5) . The cocrystal ligand (HET ID: BRW) was used to identify the active site and define the center of the receptor grid. The compound (eupatin) was prepared using LigPrep. The ionization state was generated at a pH of 7.0 ± 2.0 by using the Epik module in LigPrep. The compound was docked to the active site using the Glide module of Schrödinger Maestro. A three-dimensional image of the docked compound and the GSK3b active site was generated using PyMOL (PyMOL Molecular Graphics System, Schrödinger LLC, New York, NY, USA), and a two-dimensional interaction diagram was generated using Maestro.
Data analysis and statistics
One-way analysis of variance was used for analyses, and data are presented as mean ± standard error of the mean. When significant differences were detected between groups, Tukey’s post hoc test was applied for identifying the significantly different group pairs. The significance level for parameters was set at p < 0.05.