Figure 5

BW-B 70C inhibits LPS-induced NF-κB activation in microglial BV2 cells and prevents nuclear translocation of p65 in rat primary microglia. (A) Microglial cells (BV2) were transiently co-transfected with 1.5 μg of NF-κB luciferase reporter construct. Cells were pre-treated with BW-B 70C (25–75 μM) for 30 min followed by LPS (1 μg/ml) stimulation for 4 h. Luciferase activity was normalized with respect to β-galactosidase activity. Data are means ± SD of three different experiments. Immunoblot was performed at 1 h post treatment with LPS (1 μg/ml) for p65 (B) and p50 (C) in nuclear extract from primary microglia. A non-specific band (NS) was taken as internal standard. Blots are representative of three different experiments. (D) Immunohistochemcal analysis of rat primary microglia showing nuclear translocation of p65 1 h post LPS treatment. Cells were pretreated with BW-B 70C (75 μM) for 30 min before stimulation with LPS (1 μg/ml). Red fluorescence shows positive reaction for p65 and blue fluorescence showed nuclear staining with DAPI. LPS treatment translocated p65 to the nucleus, and treatment with BW-B 70C reversed it. Figures are representative of 3 experiments. (Magnification 200 X). ^#p < 0.001 vs. untreated; *p < 0.001 vs. untreated; **p < 0.001 vs. LPS; ***p < 0.001 vs. LPS+BW-B 70C (50 μM).