2.1 Chemicals and reagent.
Antibodies (iNOS (2982), COX-2 (12282), phospho-Akt (9271), ERK (9102), phosphor-ERK (9101), JNK (9252), phosphor-JNK (9251), p38 (9212) and phosphor-p38 (9211) primary antibodies as well as an anti-rabbit immunoglobulin G (IgG) secondary antibody) were purchased from Cell Signaling Technology (Boston, MA, USA). Fetal bovine serum (FBS) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Penicillin/streptomycin (P/S) and Dulbecco’s modified Eagle’s medium (DMEM/F12) were purchased from Gibco (Grand Island, NY, USA). LPS originating from Escherichia coli O55:B5 and sinapic acid (purity > 98%) were obtained from Sigma–Aldrich (St. Louis, MO, USA).
2.2 Cell culture.
The BV-2 mouse microglial cell line was obtained from Prof. Lee Sung Jung’s research team at the School of Dentistry, Seoul National University and was cultured in Dulbecco's modified Eagle medium (DMEM; Gibco, Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen, Grand Island, NY, USA) and 1% penicillin/streptomycin (P/S; Gibco, Invitrogen, Grand Island, NY, USA) at 37°C under a humidified atmosphere containing 5% CO2.
2.3 Cell Viability Assay.
Cell viability was measured by a colorimetric assay to detect cell metabolic ability according to a previously reported protocol (Zhao et al., 2019). BV-2 microglial cells were cultured in DMEM/F12 medium (Gibco) in 96-well plates (1×104 cells/well) for 24 hours. Cells were then treated for 12 hours with various concentrations of sinapic acid and compounds in the presence or absence of 100 ng/ml LPS. After incubation for 12 hours, 10 µl of EZ-Cytox reagent (Daeil Lab Co., Ltd., Seoul, Republic of Korea) was added to each well followed by incubation for 30 min at 37°C. The absorbance of each reaction product was then measured using a microplate reader (Infinite 1000, Tecan Trading AG, Switzerland) at a wavelength of 450 nm. The results are presented as a percentage of the MTT absorbance of the control cells.
2.4 Measurement of Nitric Oxide.
Cells were grown in 6-well plates (5×105 cells/well) for 24 hours. Cells were then pretreated with 25, 50 and 100 µg/ml WIN-1001X for 1 hour and then treated with 100 ng/ml LPS for 12 hours. The medium was collected and centrifuged at 13,000 rpm to remove dead cells. The supernatant medium was collected and mixed with an equal volume (50 µl) of Griess reagents (1% sulfanilamide and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5% H3PO4) for 10 min followed by incubation at room temperature. The nitric oxide concentration was measured by a sodium nitrite standard curve at 540 nm using a microplate reader (Multiskan SkyHigh Plate Reader, Thermo Fisher Scientific).
2.5 Animal experiments.
Male ICR mice (aged 8 weeks and weighing 26 ± 2 g) were purchased from Orient Bio, Inc., Seongnam, Republic of Korea. Animals were kept in the housing facility for one week to adapt to the environment before the experiment and were maintained at 24 ± 1°C under a 16/8 h light/dark cycle with food and water provided ad libitum. Five groups of animals were maintained in different cages with 6 mice per cage. The normal control group and LPS-injected only group were administered PBS orally. The positive control group was administered dexamethasone (2.5 mg/kg/day) orally. The other two groups were administered sinapic acid (dissolved in PBS) orally (10 and 20 mg/kg/day). The oral administration continued for 8 days. Three days before sampling the LPS-injected group, the positive control group and the two sinapic acid-administered groups were i.p. (intraperitoneal) injected with LPS (2.5 mg/kg/day) after 30 min of oral gavage treatment. Animals were anesthetized by diethyl ether and sacrificed after the last LPS stimulation for 3 h. Brain tissue samples were collected and stored at -80°C until further analysis.
2.6 Total Protein Isolation from BV-2 Cells.
Total protein was isolated according to a previous study (Zhao et al., 2019). Briefly, cells were washed 3 times with ice-cold PBS and centrifuged at 13,000 rpm for 5 min to remove dead cells. After discarding the supernatant, the collected cells were lysed with lysis buffer (Cell Signaling Technology, Boston, MA, USA), containing protease inhibitor cocktail (PIC, Roche, Penzberg, Germany) and phenylmethylsulfonyl fluoride (PMSF), for 20 min on ice. The lysates were centrifuged (13,000 rpm) for 20 min. Protein quantification was conducted with the Bradford reagent (Bio–Rad, Hercules, CA, USA), and the BSA standard (0 − 20 mg/ml) was prepared as a standard curve. Western blot samples were prepared with lysate and an equal volume of 2× NuPAGE LDS sample buffer (Thermo Fisher Scientific, Inc., Lafayette, CA, USA) with 10% 2-mercaptoethanol. After denaturation at 99°C, the samples were stored at -80°C until further analysis.
2.7 Total Protein Isolation from the Mouse Brain.
For the preparation of mouse brain samples as described previously(Zhao et al., 2019), the mouse cerebral cortex was collected, stored at -80°C and then homogenized with PRO-PREP protein extraction buffer (iNtRON, Seongnam-si, Republic of Korea) supplemented with 1× PIC set III (Sigma–Aldrich, St. Louis, MO, USA). The homogenates were then centrifuged (13,000 rpm) for 20 min at 4°C. Protein quantification assays were performed as described for total protein samples.
2.8 Western blot Analysis.
Total protein samples (20 µg) were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS − PAGE) with 10% acrylamide/bis gels and electrotransferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membranes were then blocked with 5% BSA in Tris-buffered saline with Tween (TBST) and incubated overnight at 4°C with specific primary antibodies (1:1000). After washing with TBST, the membranes were incubated with secondary HRP-conjugated IgG (1:2000) at room temperature for 1 h and visualized using SuperSignal™ West Femto Chemiluminescent Substrate (Thermo Fisher Scientific). Band signal densitometry analysis was performed with the LAS4000 system (Fujifilm, Tokyo, Japan), and the intensities of the proteins were quantified by Multi Gauge V3.0 software (Fujifilm, Japan).
Data are expressed as the mean ± SEM of each independent replication. For comparison of three or more replications, data were analyzed by one-way analysis of variance (ANOVA) with Dunnett's post-hoc test. A value of P < 0.05 was considered statistically significant. Statistical tests were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).