Figure 15 Western blot analysis demonstrating the time course on degradation on immunoreactive IκB-α in THP-1 cells (1×106 cells/ml).
THP-1 cell were dispensed on 6-well plate till 70-80% confluent condition and treated with LPS 10 ng/ml (lane 2, 30 min; lane 3, 60 min; lane 4, 90 min; lane 5, 120 min; lane 6, 180 min) or vehicle (lane 1, control 30 min) as indicated. The data are representative example of three experiments.
1 2 3 4
IκB-α (36-kD)
α-tubulin (57-kD)
Vehicle
Cinnamophilin (µM)
LPS (10 ng/ml) 10
*
+
Resting
201 2 3 4
Figure 16 Effect of cinnamophilin on degradation of immunoreactive IκB-α in THP-1 cells.
THP-1 cells (1×106 cells/ml) were dispensed on 6-well plate till 70-80% confluent condition and treated with LPS (10 ng/ml) for 90 min as indicated. Cells were treated with cinnamophilin (lane 3, 10 µM; lane 4, 20 µM) or vehicle (lane 2) for 15 min before treatment with LPS 10 ng/ml. Then cell lysates were obtained and analysed for IκB-α protein expression by Western blot (lane 1, control). The data are representative example of three experiments. ∗: P<0.05 as compared with vehicle. ＋: P<0.01 as compared with resting.
1 2 3 4 IκB-α
(36-kD)
α-tubulin (57-kD)
Vehicle
HDI II (µM)
LPS (10 ng/ml) 2
+
Resting
51 2 3 4
Figure 17 Effect of HDI II on degradation of immunoreactive IκB-α in THP-1 cells. THP-1 cells (1×106 cells/ml) were dispensed on 6-well plate till 70-80% confluent condition and treated with LPS (10 ng/ml) for 90 min as indicated. Cells were treated with HDI II (lane 3, 2 µM; lane 4, 5 µM) or vehicle (lane 2) for 15 min before treatment with LPS 10 ng/ml.
Then cell lysates were obtained and analysed for IκB-α protein expression by Western blot (lane 1, control). The data are representative example of three experiments. ＋: P<0.001 as compared with resting.
1 2 3 4 5
P65
Vehicle Cinnamphilin HDI II PMC (20 µM) (2 µM) (10 µM) Resting
LPS (10 ng/ml)
Figure 18 Effects of cinnamophilin, HDI II and PMC on p65 expression in THP-1 cells.
THP-1 cells (1×107 cells/well) were dispensed on 6-well plate and treated with LPS (10 ng/ml) for 120 min as indicated. Cells were treated with cinnamophilin (lane 3, 20 µM), HDI II (lane 4, 2 µM), PMC (lane 5, 10 µM) or vehicle (lane 2) for 15 min before treatment with LPS 10 ng/ml. The cell nuclear extraction were obtained and analysed for p65 protein expression by Western blot (lane 1, control). The data are representative example of four experiments.
1 2 3 4 Phospho-JNK
Total-JNK
Resting
HDI II
20 2
Cinnamophilin
Vehicle
LPS (10 ng/ml)
+
*
*
*
1 2 3 4
Figure 19 Effect of cinnamophilin and HDI II on expression of JNK in THP-1 cells. THP-1 cells (1×106) were dispensed on 6-well plate till 70-80 % confluent condition and treated with LPS (10 ng/ml) for 15 min as indicated. Cells were then treated with cinnamophilin (lane 3, 20 μM) and HDI II (lane 4, 2μM) or vehicle (lane 2) for 15 min before treatment with LPS. Then cell lysates were obtained and analysed for JNK protein expression by Western blot (Lane 1, control) The data are representative example of three experiments. ∗: P<0.05, ∗∗: P<0.01 as compared with vehicle.＋: P<0.01 as compared with resting.
Resting Vehicle
HDI
20 2
Cinnamophilin
1 2 3 4
Total-ER Phospho-ERK
LPS (10 ng/ml)
Figure 20 Effect of cinnamophilin and HDI II on expression of ERK in THP-1 cells.
THP-1 cells (1×106) were dispensed on 6-well plate till 70-80 % confluent condition and treated with LPS (10 ng/ml) for 15 min as indicated. Cells were then treated with cinnamophilin (lane 3, 20μM) and HDI II (lane 4, 2μM) or vehicle (lane 2) for 15 min before treatment with LPS. Then cell lysates were obtained and analysed for ERK protein expression by Western blot (Lane 1, control) The data are representative example of three experiments.
*
(µM)
Figure 21 Effect of cinnamophilin on MCP-1-induced THP-1 cell migration. Cells (5×104) cultured for 6 hours in presence of different concentration of cinnamophilin (5, 20 µΜ) or vehicle were used for cell migration assay as described in Methods. Migrated cell numbers are presented as mean ± S.E.M. of three to four independent experiments. ∗: P<0.05, as compared with resting
*
(µM)
Figure 22 Effect of HDI II on MCP-1-induced THP-1 cell migration. Cells (5×104) cultured for 6 hours in presence of different concentration of HDI II (0.5, 2 µΜ) or vehicle were used for cell migration assay as described in Methods. Migrated cell numbers are presented as mean ± S.E.M. of three to four independent experiments. ∗:
P<0.05, as compared with resting
*
Resting Vehicle
Cinnamophilin HDI II
20 2
(µM)
Figure 23 Effect of cinnamophilin and HDI II on LPS-induced production of CD11b from conditioned medium of THP-1 cells. THP-1 cells (0.5×106 cells/well) were dispensed on 24-well plates and treated with cinnamophilin (lane 3, 20 µM) or HDI II (lane 4, 2 µM) or vehicle (lane 2) for 15 min before treatment with LPS (10 ng/ml) for 18.5 hrs. Then surpernatant were obtained and detected in flow cytometry using FITC-labeled mouse monoclonal antibody to human CD11b. Depicted are mean fluorescence (folds) and standard error of two to four independent experiments. ∗: P<0.05, as compared with resting
LPS (10 ng/ml)
*
*
*
*
*
* ＋
9
Resting Vehicle 1 5 20 LPS 100 (ng/ml)
Figure 24 Effect of cinnamophilin on LPS-induced production of tumor necrosis factor-α (TNF-α) from conditioned medium of THP-1 cells. THP-1 cells (1×106 cell/ml) were dispensed on 24-well plates till 70-80 % confluence and treated with various concentrations of cinnamophilin (1, 5, 20 µM) for 15 min before treatment with LPS (10 ng/ml) for 24 hrs.
Then supernatants were obtained and analysed for TNF-α protein expression by ELISA.
TNF-α protein level is presented as mean ± S.E.M. pg/106 cells of three independent experiments. ∗: P<0.05, ∗∗: P<0.01, ∗∗∗: P<0.001 as compared with vehicle. ＋: P<0.05 as compared with resting.
LPS 10 (ng/ml)
Cinnamophilin (µM)
＋
9
Resting Vehicle 0.5 1 2 LPS 100 (ng/ml)
Figure 25 Effect of HDI II on LPS-induced production of tumor necrosis factor-α (TNF-α) from conditioned medium of THP-1 cells. THP-1 cells (1×106 cell/ml) were dispensed on 24-well plates till 70-80 % confluence and treated with various concentrations of HDI II (0.5, 1, 2 µM) for 15 min before treatment with LPS (10 ng/ml) for 24 hrs. Then supernatants were obtained and analysed for TNF-α protein expression by ELISA. TNF-α protein level is presented as mean ± S.E.M. pg/106 cells of three independent experiments. ＋: P<0.05 as compared with resting.
LPS 10 (ng/ml) HDI II (µM)
9
Resting Vehicle 1
Cinnamophilin (µM) 20
Figure 26 Effect of cinnamophilin on MCP-1-induced production of tumor necrosis factor-α (TNF-α) from conditioned medium of THP-1 cells. THP-1 cells (1×106cell/ml) were dispensed on 24-well plates till 70-80 % confluence and treated with various concentrations of cinnamophilin (1, 20 µM) for 15 min before treatment with MCP-1 (240 ng/ml) for 24 hrs.
Then supernatants were obtained and analysed for TNF-α protein expression by ELISA.
TNF-α protein level is presented as mean ± S.E.M. pg/106 cells of three independent experiments.
MCP-1 (240 ng/ml)
9
Resting Vehicle 0.5
HDI II (µM) 2
Figure 27 Effect of HDI II on MCP-1-induced production of tumor necrosis factor-α (TNF-α) from conditioned medium of THP-1 cells. THP-1 cells (1×106 cell/ml) were dispensed on 24-well plates till 70-80 % confluence and treated with various concentrations of HDI II (0.5, 2 µM) for 15 min before treatment with MCP-1 (240 ng/ml) for 24 hrs. Then supernatants were obtained and analysed for TNF-α protein expression by ELISA.
TNF-α protein level is presented as mean ± S.E.M. pg/106 cells of three independent experiments.
MCP-1 (240 ng/ml)
A B(30 mins) C1hr D2hr E3hr F4hr
B lood pr essur e ( M A P , m m H g)
20 40 60 80 100 120 140 160
Figure 28 Hemodynamic effects in rats subjected to hemorrhagic shock were treated with either vehicle (○), YC-1 (10 mg/kg, ▼), or valproic acid (150 mg/kg, ▽). The surgical procedure without causing a hemorrage and treated with vehicle as sham-operated condition (●). The results are presented as mean ± S.E.M. (n = 2-8).
A B C1hr D2hr E3hr F4hr 5
10 15 20 25 30 35 40 45
BUN (mg/dL)
Figure 29 Serum levels of BUN in rats subjected to hemorrhagic shock were treated with either vehicle (○), YC-1 (10 mg/kg, ▼), or valproic acid (150 mg/kg, ▽). The surgical procedure without causing a hemorrage and treated with vehicle as sham-operated condition (●). The results are presented as mean ± S.E.M. (n = 2-8).
A B C1hr D2hr E3hr F4hr 0.2
0.4 0.6 0.8 1.0 1.2 1.4
Creati nin e (m g/ dL )
Figure 30 Serum levels of creatinine in rats subjected to hemorrhagic shock were treated with either vehicle (○), YC-1 (10 mg/kg, ▼), or valproic acid (150 mg/kg, ▽). The surgical procedure without causing a hemorrage and treated with vehicle as sham-operated condition (●). The results are presented as mean ± S.E.M. (n = 2-8).
A B C1hr D2hr E3hr F4hr 0
100 200 300 400 500 600
GOT (U/ L )
Figure 31 Serum levels of GOT in rats subjected to hemorrhagic shock were treated with either vehicle (○), YC-1 (10 mg/kg, ▼), or valproic acid (150 mg/kg, ▽). The surgical procedure without causing a hemorrage and treated with vehicle as sham-operated condition (●). The results are presented as mean ± S.E.M. (n = 2-8).
A B C1hr D2hr E3hr F4hr 0
50 100 150 200 250
GPT (U/ L )
Figure 32 Serum levels of GPT in rats subjected to hemorrhagic shock were treated with either vehicle (○), YC-1 (10 mg/kg, ▼), or valproic acid (150 mg/kg, ▽). The surgical procedure without causing a hemorrage and treated with vehicle as sham-operated condition (●). The results are presented as mean ± S.E.M. (n = 2-8).