2.1 Drug Administration
Buyang Huanwu Decoction is a granule made up of Radix Astragali seu Hedysari, Radix Angelicae Sinensis, Radix Paeoniae Rubra, Lumbricus, Semen Persicae, Flos Carthami, Rhizoma Ligustici Chuanxiong, according to the ratio of 120 : 6 : 4.5 : 3 :3 : 3 : 3.The decoction was made by boiling the mixture in distilled water at 100°C for 30 min three times, then the drug solution was cooled and dried to give the drug powder, weigh 400mg powder and dissolve in 40ml DMEM medium to make 10mg/ml mother liquor.
2.2 Cell Culture
BV2 mouse microglia cell come from Hunan University of Traditional Chinese Medicine. Cells were maintained in DMEM medium with 10% FBS (Procell), 10000u/mL penicillin and 10000μg/mL streptomycin(Solarbio) at 5%CO2,37℃ incubator.
2.3 Drugs and Treatment
BYHWT was dissolved in DMEM medium to a concentration of 10mg/mL, LPS(Sigma) was dissolved in PBS(Procell) to a concentration of 1mg/mL, then pass 0.22um sterile filter. In all experiment, cells were pretreated with BYHWT(1-8mg/ml) for 2h before stimulated with LPS(400ng/mL) for 24h.
2.4 CCK-8 Assay
BV2 cells were seeded in 96-well plates at a density of 8*103 cells per well(100μL per well). The cells were processed as described above, and 6 parallel holes were set for each group. After 24 hours of stimulation, remove the cell supernatant and add medium containing 10% CCK-8(Biosharpe) and the culture was continued for 3h, The absorbance was measured with a microplate reader (5LX800,Biotek)at 450 nm.
2.5 NO Assay
BV2 cells were seeded in 96-well plates at a density of 8*103 cells per well(100μL per well). The cells were processed as described above, and 6 parallel holes were set for each group. After incubation for 24h, the supernatant of culture was collected and the concentration of NO was measured by commercial kit (Beyotime )according to the manufacturer's instruction.
2.6 ELISA Assay
BV2 cells were seeded in 6-well plates at a density of 1*105 cells per well(2mL per well). The cells were processed as described above, and 3 parallel holes were set for each group. After incubation for 24h, the supernatant of culture was collected, and analyzed by ELISA according to the manufacturer’s protocol(MULTI SCIENCES)
2.7 Western Blotting
BV2 cells were seeded in 6-well plates at a density of 1*105cells per well (2mL per well), incubated and treated as mentioned above. Cultured cells were lysed with RIPA buff(add PMSF at 100:1),After lysing the cells on ice for 30 minutes, The supernatants were collected and then quantitated for protein determination using a BCA Protein Assay kit and the cells protein(30μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2h at 80V,and transferred to polyvinylidene fluoride (PVDF) membranes(Immobilon, USA) for 120 min at 200 mA. After blocking with 5% non-fat milk or BSA for 1h at 37℃, the membrane was incubated overnight with primary antibodies.(GAPDH, Proteintech 1:5000;NF-κB p65,Abcam,1:1000;p- NF-κB p65,Abcam,1:2000;IκB-α, Cell Signaling Technology , 1:1000; p-IκB-α, Cell Signaling Technology , 1:1000; NLRP3, Abcam, 1:1000) in antibody dilution(Solarbio) at 4℃ overnight. Then the membrane was washed three times for 15 min each with TBST buffer, and incubated for 60 min at 37℃ with HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H+L) and HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L)(Proteintech),secondary antibodies diluted in TBST buffer (1:8000). Finally, the membrane was washed three times for 15 min with TBST buffer. The protein bands were detected with enhanced chemiluminescence (Millipore), The GAPDH protein level and β-actin protein level was used as protein loading control.
2.8 Immunofluorescence staining
BV2 cells were seeded in 24-well plates at a density of 3*104cells per well(500μL per well), incubated and treated as mentioned above. After fixed with 4% paraformaldehyde for 20 min at room temperature, cell were permeabilized with 0.1% Triton X-100 for 20 min. Then blocking with 3% BSA for 1h at 37℃, and were incubated overnight at 4℃ with primary antibodies. (iNOS, Cell Signaling Technology , 1:400;NLRP3,1:50,Boste). On the flowing day, After washed with PBS for three times, cells were incubated with secondary antibodies (Fluorescein(FITC)-conjugated Affinipure Goat Anti-Rabbit IgG(H+L),1:400;Alexa Flour 594- conjugated Affinipure Goat Anti-Rabbit IgG(H+L),1:400;Donkey Anti-Goat IgG H&L (Alexa Fluor® 555),1:200, Proteintech )in 1%BSA dilution for 1h at 37℃. The cells were treated with DAPI(Solabio) for 10 min. All images were captured with a fluorescence microscope (Olympus)