Preparation of LBP
LBP was prepared as described previously []. Dried L. barbarum was made into a powder and decocted with water (60 °C) by a traditional method used for Chinese medicinal herbs after degreasing. Then it was filtered by regenerated cellulose membranes of 300 kDa, 100 kDa, 80 kDa, 50 kDa and 30 kDa (0.2 MPa, 60 °C) after centrifuging. The resulting fraction was retained and vacuum-dried at 40 °C. Neutral sugars were determined by phenol-H2SO4, acidic sugars by carbazole and proteins by the Coomassie Brilliant Blue G-250 method .
LBP we prepared was a brown powder composed of neutral sugars (78.23%) and acidic sugars (14.83%). The protein content was < 6.92%.
Cell lines and reagents
The mouse macrophage RAW264.7 cells were purchased from China Center for Type Culture Collection (Shanghai, China).
Rabbit anti-β-Actin monoclonal antibody, rabbit monoyclonal antibody against NF-κB p65, IκB etc. were purchased from Abcam (Cambridge, MA). Secondary antibodies were obtained from Boster Co.(Wuhan, China). Goat anti rabbit IgG labeled fluorescent antibody was purchased from Santa cruz (Shanghai, China). DMEM medium, fetal bovine serum (FBS), Penicillin and Streptomycin Solution were purchased from HyClone (Logan, UT, USA). LPS were purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents used in this study were of analytical grade.
Cell culture
RAW 264.7 cells were cultured in DMEM medium, supplemented with 10% FBS, 100 U/mL Penicillin, 100 µg/mL Streptomycin Solution and maintained at 37 °C in a humidified atmosphere with 5% CO2.
Time dependent regulation of I κ B phosphorylation by LBP
RAW264.7 cells were grown in six-well plates (1 × 106 cells/well) for 24 h. In brief, 1 × 106 RAW264.7 cells were pre-treated with control, LPS(1 µg/mL), LBP༈25 µg/mL༉, LPS + LBP༈1 µg/mL + 25 µg/mL༉, respectively, for 5, 10, 20, 30 min. At the end of culture, the protein was extracted and the expression of I κ B and P-I κ B was detected by Western blot.
Dose effect regulation of I κ B phosphorylation by LBP
RAW264.7 cells were grown in six-well plates (1 × 106 cells/well) for 24 h. 1 × 106 RAW264.7 cells were pre-treated with control, LPS(1 µg/mL), LBP༈25, 50, 100 µg/mL༉, LPS༈1 µg/mL༉+LBP༈25、50、100 µg/mL༉ for 5 min. At the end of culture, the protein was extracted and the expression of I κ B and P-I κ B was detected by Western blot.
Total and nuclear protein extraction
For I κ B, P-I κ B, NF - κ B etc. analysis, protein expression by western blotting, total and nuclear protein extracts were prepared from pure macrophage using commercial kits (Biosynthesis Biotechnology Co., LTD, Beijing, China). The protein concentration was determined by bicinchoninic acid (BCA) assay and stored at − 80 °C until analyzed.
Western blotting
Sixty (60) µg of cell extract were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membrane was blocked for 1 h with 5% non fat milk in TBST and then incubated with a rabbit monoyclonal antibody against I κ B (AbcamCompany, UK) at 4 °C over night. After washing with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary anti-rabbit antibodies (1:5000; Boster Co., Wuhan, China) for 60 min at room temperature. After additional washing, bound conjugates were detected by ECL superSignalTM West Pico substrate (Pierce, Rockford, IL, USA). Proteins were visualized by exposing the blot to X-ray film, photographed with a digital camera, and then the net intensities of the individual bands were measured using Bandscan 5.0 software. Rabbit anti-β-Actin monoclonal antibody (AbcamCompany, UK) was used as the loading control, and I κ B protein expression was normalized to Actin.
The WB process of P-I κ B, NF - κ B are the same as that of I κ B.
Immunofluorescence staining
RAW264.7 cells on glass coverslips were incubated with control, LPS(1 µg/mL), LBP༈25 µg/mL༉, LPS + LBP༈1 µg/mL + 25 µg/mL༉, respectively, for 30 min. After 30 min fixation with 4% paraformaldehyde and 30 min permeabilization with 0.1% Triton X-100, cells were blocked for 1 h with 5% bovine serum albumin (BSA) at room temperature. Then, cells were first incubated with the primary rabbit anti-NF-κB p65 antibody at 4 °C overnight, followed by incubation with the secondary Alexa Fluor-conjugated secondary antibody for 1 h at room temperature. Nuclear of cells were stained with DAPI. A fluorescence microscope was employed for observation of the images.
RAW264.7 cells were grown in six-well plates (1 × 106 cells/well) for 24 h .1 × 106 RAW264.7 cells were pre-treated with control, LPS(1 µg/mL), LBP༈25, 50, 100 µg/mL༉, LPS༈1 µg/mL༉+LBP༈25、50、100 µg/mL༉ for 30 min. At the end of culture, cytoplasmic protein and nucleoprotein were extracted and the number of nuclear translocation of NF - κ B was detected by Western blot.
Statistical analysis
All data are expressed as means ± standard error (SEM) and represented at least three independent experiments. ANOVA and Student's t-test were used to perform multiple comparisons for statistical significance, and p < 0.05 was considered statistically significant.