GSP attenuated LPS or RANKL induced over-expression of MDR1 by inhibiting NF-κB and MAPK/ERK medicated YB-1 activation.

<p>(A) A2780/T cells were incubated with 0∼40 μM of GSP for 6h then stimulated with or without 1 μg/ml LPS for 30min. 10μM PDTC and U1026 were used as NF-κB and MAPK/ERK inhibitor respectively. The lysates were subjected to Western blot analysis. GSP inhibited LPS-induced NF-κB, phospho-AKT, phosphor-ERK1/2 and phospho-iκBα levels with activated LPS-reduced iκBα level. The GAPDH and phospho-protein relevant total protein band confirmed the integrity and equal loading of total protein and phospho-protein respectively. *Significantly different from LPS-treated control cells (P<0.05); **Significantly different from LPS-treated control cells (P<0.01). (B) A2780/T cells were pre-incubated with 40 μM GSP for 0∼6h then simulated with or without 1 μg/ml LPS for 30 min. The nuclear and cytosolic protein were extracted from cell lysates. Western blot analysis were performed using the antibodies against nuclear p65, YB-1 and laminB1 as well as cytosolic p65, YB-1 and GAPDH. The Lamin B1 and GAPDH band confirms the integrity and equal loading of nuclear and cytosolic protein respectively. **Significantly different from untreated cells (P<0.01). (C) A2780/T cells treated with 1 μg/ml LPS or 50 ng/ml RANKL followed by pre-treatment with or without 40 μM GSP for 24 h were fixed for immunofluorescence test with staining with anti-p65 (green) and DAPI (blue). The pre-treatment with GSP before LPS or RANKL stimulation prevented the nuclear translocation of p65.