## ##
15% 0.50% PBS SDF-1α SDF-1α with Sal. B
SDF-1α with Mag
% of cell viability compared to 15% FBSS
##：p＜0.01 as compared with 15% FBS; † † ：p＜0.01 as compared with PBS; **：p＜0.01; as compared with SDF-1a, n=3.
圖 16、觀察 salvianolic acid B、magnolol 抑制由SDF-1a 刺激平滑肌 細胞增生 24 小時後之影響
Fig.16 Effect of salvianolic acid B and Magnolol on morphologic changes of VSMCs treated with 10ng/ml SDF-1a . VSMCs were incubated with either 0.075mg/ml of salvianolic acid B or 0.075mg/ml of
15% FBS 0.5% FBS 0.5% FBS in
PBS
SDF-1a SDF-1a+ Salivanolic acid B
SDF-1a+ magnolol
magnolol in 12 well culture plates in DMEM supplemented with 0.5%
FBS at 37℃ for 24 hours.
control Ballon injury
圖17、以二維電泳觀察氣球擴張後老鼠血液中蛋白質改變情形 Fig.17 Using two dimension electrophoress analysis rat plasma protein after Ballon injury.
(A)
(B)
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50
15% 0.50% PBS SDF-1α SDF-1α
+sal.B SDF-1α +Mag.
fold of PBS
圖 18、以西方墨點轉漬法觀察 salvianolic acid B 和 Magnolol 抑制由 SDF-1a 刺激血管平滑肌細胞 CXCR4 之影響
Fig.18 Effect of salvianolic acid B and magnolol on protein expression level of CXCR4 stimulated by SDF-1a. VSMCs were incubated with SDF-1a and 0.075mg/ml salvianolic acid B or Magnolol at 37℃ for 30
CXCR4 40 KDa
ß-actin 42 KDa
minutes. (A)Westeren blot analysis of CXCR4 by salvianolic acid B or Magnolol with or without SDF-1a(B) Quantification analysis of CXCR4 protein expression level effected by salvianolic acid B and Magnolol to PBS group.
15% 0.50% PBS SDF-1α SDF-1α
+Sal.B
SDF-1α +Mag.
( fold of PBS)
圖 18、以西方墨點轉漬法觀察 salvianolic acid B 和 Magnolol 抑制由 SDF-1a 刺激血管平滑肌細胞 Raf-1 之影響
Fig.19 Effect of salvianolic acid B and magnolol on protein expression level of Raf stimulated by SDF-1 a. VSMCs were incubated with
Raf-1 76 KDa
ß-actin
42 KDa
* *
SDF-1a and 0.075mg/ml salvianolic acid B or Magnolol at 37℃ for 30
minutes. (A)Westeren blot analysis of Raf by salvianolic acid B or Magnolol with or without SDF-1a(B) Quantification analysis of Raf protein expression level effected by salvianolic acid B and Magnolol to PBS group.
(A)
(B)
圖 20、以西方墨點轉漬法觀察 salvianolic acid B 和 Magnolol 抑制由 SDF-1a 刺激血管平滑肌細胞 MEK之影響
Fig.19 Effect of salvianolic acid B and magnolol on protein expression level of MEK stimulated by SDF-1a. VSMCs were incubated with
MEK 45 KDa
fold of PBS
#
* *
SDF-1a and 0.075mg/ml salvianolic acid B or Magnolol at 37℃ for 30
minutes. (A)Westeren blot analysis of MEK by salvianolic acid B or Magnolol with or without SDF-1a(B) Quantification analysis of MEK protein expression level effected by salvianolic acid B and Magnolol to PBS group.
(A)
(B)
圖 21、以西方墨點轉漬法觀察 salvianolic acid B 和 Magnolol 抑制由 SDF-1a 刺激血管平滑肌細胞 Erk 1/2 之影響
Fig.20 Effect of salvianolic acid B and magnolol on protein expression level of Erk stimulated by SDF-1 a. VSMCs were incubated with SDF-1a and 0.075mg/ml salvianolic acid B or Magnolol at 37℃ for 30
minutes. (A)Westeren blot analysis of Erk by salvianolic acid B or Magnolol with or without SDF-1a(B) Quantification analysis of Erk protein expression level effected by salvianolic acid B and Magnolol to PBS group.
fold of PBS
圖 22、以西方墨點轉漬法觀察 salvianolic acid B 和 Magnolol 抑制由
p-Erk 44 KDa
42 KDa ß-actin
42 KDa
SDF-1a 刺激血管平滑肌細胞立即性早期磷酸化 Erk 1/2 之影響 Fig.21 Effect of salvianolic acid B and magnolol on protein expression level of p-Erk stimulated by SDF-1a. VSMCs were incubated with SDF-1a and 0.075mg/ml salvianolic acid B or Magnolol at 37℃ for 30
minutes. (A)Westeren blot analysis of p-Erk by salvianolic acid B or Magnolol with or without SDF-1a(B) Quantification analysis of p-Erk protein expression level effected by salvianolic acid B and Magnolol to PBS group.
(A)
(B)
圖 23、以西方墨點轉漬法觀察 salvianolic acid B 和 Magnolol 抑制由
FAK 125 KDa
ß-actin
SDF-1α+Sal. B SDF-1α+Mag.
fold of PBS
##
** **
SDF-1a 刺激血管平滑肌細胞 FAK 之影響
Fig.22 Effect of salvianolic acid B and magnolol on protein expression level of FAK stimulated by SDF-1a. VSMCs were incubated with SDF-1a and 0.075mg/ml salvianolic acid B or Magnolol at 37℃ for 30
minutes. (A)Westeren blot analysis of FAK by salvianolic acid B or Magnolol with or without SDF-1a(B) Quantification analysis of FAK protein expression level effected by salvianolic acid B and Magnolol to PBS group.
salivanolic acid B magnolol
PBS SDF-1a 0.075 mg/ml 0.075 mg/ml 0.01 mg/ml 0.005 mg/ml 0.001 mg/ml
Day0
Day1
Day2
圖 24、觀察在不同時間點 salvianolic acid B、magnolol 抑制由 SDF-1a 刺激平滑肌細胞遷移之影響
Fig.23 Inhibtory effects of salvianolic acid B and magnolol on VSMCs migration stimulated by SDF-1a. VSMCs were incubated with 0.075mg/
ml magnolol in 24 well culture plates in DMEM supplemented with 0.5%
FBS at 37℃ at different time point.
(A)
(B)
圖 25、以西方墨點轉漬法觀察 salvianolic acid B 和 Magnolol 抑制由
JNk 54 KDa
ß-actin 42 KDa
0
SDF-1a 刺激血管平滑肌細胞 JNK 之影響
Fig.24 Effect of salvianolic acid B and magnolol on protein expression level of JNK stimulated by SDF-1a. VSMCs were incubated with SDF-1a and 0.075mg/ml salvianolic acid B or Magnolol at 37℃ for 30
minutes. (A)Westeren blot analysis of JNK by salvianolic acid B or Magnolol with or without SDF-1a(B) Quantification analysis of JNK protein expression level effected by salvianolic acid B and Magnolol to PBS group.
15% 0.50% PBS SDF-1α SDF-1α
+Sal.B
SDF-1α +Mag.
( fold of PBS)
pJNk 55 KDa
ß-actin
46 KDa 42 KDa
圖 26、以西方墨點轉漬法觀察 salvianolic acid B 和 Magnolol 抑制由 SDF-1a 刺激血管平滑肌細胞磷酸化 JNK 之影響
Fig.25 Effect of salvianolic acid B and magnolol on protein expression level of p-JNK stimulated by SDF-1a. VSMCs were incubated with SDF-1a and 0.075mg/ml salvianolic acid B or Magnolol at 37℃ for 30
minutes. (A)Westeren blot analysis of p-JNK by salvianolic acid B or Magnolol with or without SDF-1a(B) Quantification analysis of p-JNK protein expression level effected by salvianolic acid B and Magnolol to PBS group.
0.50% 15% PBS SDF-1α SDF-1α
+Sal. B
SDF-1α +Mag.
( normalize with β-gal)
**
#
**
#
?
#：與 15% FBS-DMEM 比較有顯著差異 P≦0.05； †：與 PBS 比較有
顯 著 差 異
P≦0.05 ； *與 SDF-1a 比較有顯著差異 P≦0.05 ； ** 與 SDF-1a 比較有顯著差異 P≦0.01Figure 26. Inhibitory effects of salvianolic acid B and magnolol on kB promter activity stimulated by SDF-1ain VSMCs. Activation of NF-kB activity stimulated by SDF-1awas s ignificantly s uppressed b y salvianolic acid B and Magnolol. #： p＜ 0.05, † ：p＜0.05, **：p＜
0.01, #：as compared with 15% FBS, †：as compared with PBS, *：as compared with SDF-1a, n=3.
(A)
15% 0.50% PBS SDF-1α SDF-1α
+Sal.B
SDF-1α +Mag.
fold of PBS
**
圖 28、以西方墨點轉漬法觀察 salvianolic acid B 和 Magnolol 抑制由 SDF-1a 刺激血管平滑肌細胞磷酸化 caspase-3 之影響
Fig.27 Effect of salvianolic acid B and magnolol on protein expression level of Caspase 3 stimulated by SDF-1a. VSMCs were incubated with SDF-1a and 0.075mg/ml salvianolic acid B or Magnolol at 37℃ for 30
minutes. (A)Westeren blot analysis of caspase 3 by salvianolic acid B or Magnolol with or without SDF-1a(B) Quantification analysis of Caspase 3 protein expression level effected by salvianolic acid B and magnolol to PBS group.
(A)
(B)
ß-actin
42 KDa 23 KDa Bax
0
(fold with PBS)
圖 29、以西方墨點轉漬法觀察 salvianolic acid B 和 Magnolol 抑制由 SDF-1a 刺激血管平滑肌細胞磷酸化 Bax 之影響
Fig.28 Effect of salvianolic acid B and magnolol on protein expression level of Bax stimulated by SDF-1a. VSMCs were incubated with SDF-1a and 0.075mg/ml salvianolic acid B or Magnolol at 37℃ for 30 minutes.
(A)Westeren blot analysis of Bax by salvianolic acid B or Magnolol with or without SDF-1a(B) Quantification analysis of Bax protein expression level effected by salvianolic acid B and magnolol to PBS group.
(A)
(B)
圖 30、以西方墨點轉漬法觀察 salvianolic acid B 和 Magnolol 抑制由 SDF-1a 刺激血管平滑肌細胞磷酸化 Bcl-2 之影響
Fig.29 Effect of salvianolic acid B and magnolol on protein expression level of Bcl-2 stimulated by SDF-1a. VSMCs were incubated with SDF-1a and 0.075mg/ml salvianolic acid B or Magnolol at 37℃ for 30
minutes. (A)Westeren blot analysis of Bcl-2 by salvianolic acid B or Magnolol with or without SDF-1a(B) Quantification analysis of Bcl-2 protein expression level effected by salvianolic acid B and magnolol to PBS group.
15% 0.5% PBS SDF-1a 0.1 0.075 0.075 0.001 mg/ml
Salivanolic acid B Magnolol
圖 31、觀察 salvianolic acid B 和 magnolol 誘導平滑肌細胞 DNA 斷 裂的情形
Figure.30 DNA fragmentation of VSMCs induced by salvianolic acid B and magnolol at 48h. VSMCs stimulated by SDF-1a were incubated with 0.075mg/ml salvianolic acid B and Magnolol in 10cm culture dish at 37℃ for 48 hours. DNA fragmentation assay manifested are DNA laddering were clearly observed after 48h, treated of salvianolic acid B and Magnolol.