Cell culture and primary astrocyte isolation
Dulbecco’s modified eagle medium (DMEM; C11995500BT) and DMEM/F-12 (C11330500BT) were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS; VS500T, Australia origin) was from Ausbian (Shanghai, China). BV2 microglia cells (provided by Dr. Zhu CQ, Fudan University) and SH-SY5Y cells (Cell Bank of Chinese Academy of Sciences, Shanghai, China) were grown in DMEM supplemented with 10% FBS and penicillin/streptomycin (100 U/mL and 100 μg/mL, respectively; P1400, Solarbio, Beijing, China). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2.
Primary astrocytes were prepared as previously described with slight modification [37, 38]. In brief, cerebral cortices were dissected from brains of new-born pups of Institute of Cancer Research (ICR) mice (postnatal 1-2 days; purchased from the Experimental Animal Center of Wenzhou Medical University) in cold Hank’s buffered saline. The animals used in this study were treated in accordance with protocols approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University. Cortices were gently triturated and digested with 0.25% Trypsin-EDTA solution (25200056, Gibco, Grand Island, NY, USA) for 15 min at 37°C. Thereafter, equal volume of culture medium, that is, DMEM/F-12 supplemented with 10% FBS and penicillin/streptomycin, was added and centrifuged at 200 g for 5 min. The pellets were gently resuspended in culture medium and filtered through a 100 μm pore mesh. The cells were then seeded in 75 cm2 flasks coated with poly-L-lysine (1 mg/mL) and cultured at 37 °C with medium changed every 4 days. When cells reached a confluency of 90-95%, the flasks were placed on an orbital shaker at 250 rpm and 37°C for 16 h. The remaining attached cells were then collected and re-cultured. Purity of astrocytes was evaluated by immunostaining of glial fibrillary acidic protein (GFAP). GFAP positive cells were counted in 3-random fields each time. Cultures were used for subsequent experiments when GFAP positivity was over 95% (Figure 1A).
Immunofluorescence
Cells were fixed in 4% paraformaldehyde for 30 min, followed by permeabilization in 0.2% Triton X-100 for 15 min and then blocked with 5% bovine serum albumin (Beyotime, Shanghai, China) for 1 h at room temperature. The washing buffer is phosphate-buffered saline. Primary antibodies against GFAP and p65 were from Millipore (MAB360, Billerica, MA, USA) and Cell Signaling (8242S, Boston, MA, USA), respectively. Alexa Fluor 488-conjugated anti-mouse (A11001) and 555-conjugated anti-rabbit (A21428) IgG antibodies and Hoechst 33258 (H3569) were from Thermo Fisher (Rockford, IL, USA).
Treatments and preparation of conditioned media
Microglia conditioned media were prepared as previously [19]. BV2 cells were seeded at a density of 2-3 × 106 in a 10-cm culture plate and cultured for 24 h followed by serum starvation overnight. Cells were then treated with or without LPS (100 ng/mL; L4391, Sigma, St. Louis, MO, USA) for 2 h, and then refreshed with serum-free medium with a continued incubation for 24 h. Following centrifugation at 20,000 g for 5 min, the media were collected as microglia conditioned medium without LPS stimulation (M/C) or microglia conditioned medium pre-activated with LPS (M/Lps), and stored at -80°C till use (Figure 1B).
Primary astrocytes of passage 3-5 were seeded at a density of 2-3 × 106 per well in a 6-well plate and cultured for 24h. Following starvation overnight, cells were pretreated with paroxetine (P9623, Sigma, St. Louis, MO, USA) at indicated concentrations for 30 min, followed by treatments of LPS (100 ng/mL) or the above M/C and M/Lps for indicated time. The resulted media of astrocytes treated with M/C, M/Lps or LPS for 24 h were centrifuged at 20,000 g for 5 min and designated as A/MC (conditioned medium of astrocytes cultured with M/C), A/MLps (conditioned medium of astrocytes cultured with M/Lps) and A/Lps (conditioned medium of astrocytes cultured with LPS), respectively (Figure 1B). Media were stored at -80°C till use.
Cell viability measurement
Cell viability was determined by the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT; C0009, Beyotime, Shanghai, China) assay [39]. Primary astrocytes or SH-SY5Y cells were seeded in triplicates in 96-well plates at 5 × 104 and 1 × 104 per well, respectively, and cultured for 24 h. Astrocytes were treated with paroxetine at different concentrations for 24 h following overnight starvation. SH-SY5Y cells were incubated with A/MC, A/MLps, A/Lps or LPS directly (100 ng/mL) for 24 h. Thereafter, MTT (5 mg/mL prepared in phosphate-buffered saline) was added into each well and incubated at 37°C for 4 h. The resulting formazan crystals were dissolved in dimethyl sulfoxide. Optical density was measured at 490 nm.
RNA isolation and quantitative PCR
Total RNA was isolated using TRIzol Reagent (15596018, Invitrogen, Carlsbad, CA, USA), and reverse-transcribed to cDNA using PrimeScript RT kit (RR037A, TaKaRa, Dalian, China). Quantitative PCR was performed using SYBR Green Supermix (1725260, Bio-Rad, Hercules, CA, USA) following the manufacturer’s instruction. Primers were as follows: TNF-α, 5’-CGT CAG CCG ATT TGC TAT CT-3’ and 5’-CGG ACT CCG CAA AGT CTA AG-3’; IL-1β, 5’-GAA ATG CCA CCT TTT GAC AGT G-3’ and 5’-TGG ATG CTC TCA TCA GGA CAG-3’; BDNF, 5’-TCA TAC TTC GGT TGC ATG AAG G-3’ and 5’-AGA CCT CTC GAA CCT GCC C-3’; MANF, 5’-TCT GGG ACG ATT TTA CCA GGA-3’ and 5’-CTT GCT TCA CGG CAA AAC TTT-3’; β-actin, 5’-AGC CAT GTA CGT AGC CAT CC-3’ and 5’-CTC TCA GCT GTG GTG GTG AA-3’.
Determination of TNF-α and BDNF production
Medium TNF-α and BDNF were measured using ELISA kits respectively from R&D Systems (VAL609, Minneapolis, MN, USA) and Westang (F10200, Shanghai, China) according to the manufacturers’ instructions. Absorbance was read at 450 nm. The concentration of each sample was calculated from the standard curve prepared using the included standards.
NO production assay
Medium nitrite was measured as an indicator of NO production [40]. In brief, 50 μl of supernatant was mixed with an equal volume of Griess reagent I, followed by an addition of 50 μl of Griess reagent II (S0021, Beyotime, Shanghai, China) at room temperature. Absorbance was immediately measured at 540 nm. The concentration of each sample was calculated from a standard curve generated using sodium nitrite.
Western blot analysis
Cells were lysed in sample buffer containing 60 mM Tri-HCl, pH 6.8, 2% SDS and 5% glycerol, and boiled for 5 min. Total protein concentration was measured using a BCA kit (P0010, Beyotime, Shanghai, China). Equal amount of protein from each sample was loaded and analyzed by Western blot as previously described [41]. Pyrrolidine dithiocarbamic acid (PDTC; P8765) was purchased from Sigma (St. Louis, MO, USA). Primary antibodies against p-p65 (3031), p65 (3034), p-JNK1/2 (9251), JNK1/2 (9252), p-p38 (9211), p38 (9212), p-ERK1/2 (9101), ERK1/2 (9102), p-STAT3 (9145), STAT3 (12640), iNOS (2977) and β-actin (4970), anti-rabbit (7074) and anti-mouse (7076) secondary antibodies, and LumiGLO® Reagent and Peroxide chemiluminescence detection kit (7003) were all purchased from Cell Signaling (Boston, MA, USA).
Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test for multiple comparisons and two-way ANOVA for factorial design experiments using the SPSS 23.0 statistics software. Values were expressed as mean ± SE from at least three independent experiments. The p < 0.05 was considered statistically significant.