Ganoderma lucidum Polysaccharides Reduce Lipopolysaccharide-Induced Interleukin-1β Expression in Cultured Smooth Muscle Cells and in Thoracic Aortas in Mice
Figure 2
EORP-mediated reduction in LPS-induced IL-1β expression is partly dependent on inhibition of phosphorylation of ERK. (a)–(c) Western blot analysis showing the effect of EORP pretreatment on the phosphorylation of (a) ERK1/2, (b) p38, (c) JNK, or (d) Akt in LPS-treated HASMCs. HASMCs were incubated for 24 h with or without 10 μg/mL of EORP, and then the cells were incubated with 10 μg/mL of LPS for 30 min and aliquots of cell lysate containing equal amounts of protein subjected to immunoblotting with the indicated antibodies. (e) Effect of inhibitors of MAPK phosphorylation on IL-1β expression in control and LPS-treated HASMCs. HASMCs were incubated for 1 h with 30 μM PD98059 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor), or SP600125 (a JNK inhibitor) and then for 24 h with or without 10 μg/mL of LPS in the continued presence of the inhibitor and then IL-1β expression was measured by Western blotting. (f) ERK-specific siRNA caused a 50% reduction in ERK protein expression by Western blotting. (g) The LPS-induced increase in IL-1β expression is inhibited by transfection of HASMCs with ERK1/2-specific siRNA (1 μM). HASMCs were transfected with either control siRNA or ERK1/2-specific siRNA (1 μM) for 48 h then were incubated with 10 μg/mL of EORP for 24 h and then with 10 μg/mL of LPS for 24 h in the continued presence of the same concentration of EORP, and IL-1β expression was measured in cell lysates by Western blotting. (h) ERK gene expression under different treatments was analyzed by the RT-PCR assay as described under Section 2. In panels (a)–(e) and (g), the data are expressed as a fold of the control value and are the means ± SEMs (). Total ERK and GAPDH, total p38 and GAPDH, total JNK and GAPDH, total Akt and GAPDH, β-actin, and α-tubulin were used as the loading control for panels (a), (b), (c), (d), (e), (f), and (g), respectively. * as compared to the untreated (control) cells. † as compared to the LPS-treated cells.
