Resting Vehicle ____________________10 20 EK5 (µM)
_________________________________
MCP-1 (50 ng/ml)
##
**
Figure 15. The effect of EK5 on MCP-1-induced ERK phosphorylation in THP-1 cells. THP-1 cells (1×10
6cells/ml) were dispensed on 6-well plates and treated with MCP-1 (100 ng/ml) for 15 minutes as indicated. Cells were treated with the indicated concentration of EK5 (10 and 20
µM) or vehicle (DMSO 0.2 % v/v) for 15 minutesbefore treatment with MCP-1. Then the cell lysates were obtained and analyzed for ERK protein expression by Western blot. The data are representative example of three experiments.
##P<0.01 as compared with the resting; **P<0.01 as compared with the vehicle.
Resting Vehicle 10 20
Rel ati ve e x p re ssi on of p- ERK1/ 2 (f ol ds)
0.0 0.5 1.0 1.5 2.0
Resting Vehicle ____________________5 10 EK8 (µM)
_________________________________
MCP-1 (50 ng/ml)
#
Figure 16. The effect of EK8 on MCP-1-induced ERK phosphorylation in THP-1 cells. THP-1 cells (1×10
6cells/ml) were dispensed on 6-well plates and treated with MCP-1 (100 ng/ml) for 15 and 30 minutes as indicated. Cells were treated with the indicated concentration of EK8 (5 and 10
µM) or vehicle (DMSO 0.2 % v/v) for 15minutes before treatment with MCP-1. Then the cell lysates were obtained and analyzed for ERK protein expression by Western blot. The data are representative example of three experiments.
#P<0.05 as compared with the resting.
Resting Vehicle 5 10
0
Relative expression of p-p38 (folds)
0.0
Figure 17. Effects of MCP-1 on p38 phosphorylation in THP-1 cells.
THP-1 cells (1×10
6cells/ml) were dispensed on 6-well plates and treated with MCP-1 (50 and 100 ng/ml) for 3, 5, 10, 15, 30, 45, 60, and 120 minutes as indicated. Cells were treated with vehicle (DMSO 0.1 % v/v) for 15 minutes before treatment with MCP-1. Then the cell lysates were obtained and analyzed for p38 protein expression by Western blot. The data are representative example of one experiments.
MCP-1 (50 ng/ml)
0.0
0 _____________________________________5 15 30 60 (min) MCP-1 (100 ng/ml)
Relative expression of p-JNK (folds)
Figure 18. Effects of MCP-1 on JNK phosphorylation in THP-1 cells.
THP-1 cells (1×10
6cells/ml) were dispensed on 6-well plates and treated with MCP-1 (50 and 100 ng/ml) for 3, 5, 10, 15, 30, 45, 60, and 120 minutes as indicated. Cells were treated with vehicle (DMSO 0.1 % v/v) for 15 minutes before treatment with MCP-1. Then the cell lysates were obtained and analyzed for JNK protein expression by Western blot. The data are representative example of one experiments.
MCP-1 (100 ng/ml)
Resting
M igr ated ce ll num b er ( fo lds )
0
Figure 19. Effects of EK5 and EK8 on MCP-1-induced migration in
human primary monocytes. THP-1 cells (1×10
5cells) were dispensed
on the inserts and treated with the incubated concentration of EK5 (10
µM), EK8 (10 µM) or vehicle (DMSO 0.1% v/v) for 30 minutes beforetreatment with MCP-1 (100 ng/ml). After incubation for 1.5 hrs, the
migrated human primary monocytes number was counted by
phase-contrast microscopy as indicated in Methods. The data are shown
as the means ± S.E.M. of three independent experiments.
#P<0.05 as
compared with the resting.
Resting 50 100 250
Resting
Relative activation of MMP-9 (folds)
0
Figure 20. Effects of MCP-1 and TNF-
αααα on MMP-9 activation inhuman primary monocytes. Human primary monocytes (1×10
6cells/ml) were dispensed on 24-well plates and treated with MCP-1 (50, 100, 250 ng/ml) for 24 hours as indicated. Cell-free supernatants were then assayed for MMP-9 activity by gelatin zymography. Values represent means ± S.E.M. from three independent experiments.
#P<0.05;
###P<0.001 as compared with the resting.
MMP-9 →
Figure 21. The inhibitory mechanisms of aristolochic acid and
N-hydroxycinnamoylphenalkylamides analogues (EK5 and EK8) on
MCP-1-activated monocytic cells.
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