本實驗結果顯示，在體外試驗上，鼠尾草酸與熊果酸的總抗氧化力 分別是維生素 E 的 6.6 倍與 1.3 倍，且鼠尾草酸具有清除自由基 (DPPH) 的能力。在細胞試驗上，鼠尾草酸與熊果酸能抑制瘦體素所誘發大鼠血 管平滑肌細胞的增生，這可能是經由啟動了 ERK1/2、NF-κB、MMP-2 等相關的訊息基因調控蛋白的表現，因此透過西方點墨法可以觀察到鼠 尾草酸與熊果酸能抑制瘦體素所誘發大鼠血管平滑肌細胞 ERK1/2、NF-κB、MMP-2 蛋白的表現，利用明膠蛋白酵素電泳法可以觀察到鼠尾草 酸與熊果酸能抑制瘦體素所誘發大鼠血管平滑肌細胞 MMP-2 蛋白的分 泌，而免疫螢光染色法上，則可以觀察到鼠尾草酸與熊果酸能抑制瘦體 素所誘發大鼠血管平滑肌細胞 NF-κB 蛋白活化進入細胞核內的表現。
由以上的結果推論，瘦體素在肥胖病人動脈粥狀硬化發展過程中，可能 扮演重要角色之一，且馬鬱蘭中的鼠尾草酸與熊果酸，可能在抑制對瘦 體素誘發的血管平滑肌細胞增生上，具有重要的關鍵性，因此馬鬱蘭也 許具有預防心血管疾病發生的作用。
表 3. 鼠尾草酸與熊果酸的抗氧化能力
Table 1. Antioxidative abilities of carnosic acid and ursolic acid in Vitro. Carnosic acid Ursolic acid
DPPH radicals scavenging ability
(IC501 , μmol/L)
30.14±1.35 ND a
TEAC 6.6±0.02 1.3±0.14
1IC50 value meant the concentration (μmol/L) of sample required to scarenge 50% o DPPH radical.
aND, not detectable
Similar results were obtained in 3 separate experiments. All values are means ± SD for 3 separate experiments.
0 30 60 90 120
0 10 20 30 40 50
Carnosic acid (μmol/L) cell number (relative to control)
＊
＊
＊
圖 10. 鼠尾草酸對血管平滑肌細胞之影響-細胞毒性傷害
Cytotoxic effect of carnosic acid (CA) on vascular smooth muscle cells (VSMCs) with trypan blue exclusion test. VSMCs were treated with increasing concentrations (0~50μmol/L) of carnosic acid for 24 h in the 10% FBS-DMEM.
Values are means ± SD for triplicate determinations (
n
=3) repeated in 3 separate experiments.＊p
＜0.05 compared with control.0 30 60 90 120
0 10 20 30 40 50
Ursolic acid (μmol/L)
cell number (relative to control) ＊
＊
＊
圖 11. 熊果酸對血管平滑肌細胞之影響-細胞毒性傷害
Cytotoxic effect of ursolic acid (UA) on vascular smooth muscle cells (VSMCs) with trypan blue exclusion test. VSMCs were treated with increasing concentrations (0~50μmol/L) of carnosic acid for 24 h in the 10% FBS-DMEM.
Values are means ± SD for triplicate determinations (
n
=3) repeated in 3 separate experiments.＊p
＜0.05 compared with control.0 30 60 90 120 150
C M 100 200 300 500
Leptin concentration (ng/ml) Cell number (relative to control)
＊
＊ ＊
＊
圖 12. 評估不同濃度瘦體素對血管平滑肌細胞增生之影響
Leptin-induced proliferation of vascular smooth muscle cells (VSMCs). The numbers of VSMCs were counted after 24 h of incubation with leptin at the indicated concentrations. Values are means ± SD for triplicate determinations (
n
=3) repeated in 3 separate experiments.＊
p
＜0.05 compared with control.Abbreviation: C: control, M : 10% FBS medium.
0 30 60 90 120 150
C 24 48 72
Time (h) Cell number (relative to control)
＊
＊ ＊
圖 13. 評估瘦體素誘導血管平滑肌細胞增生在不同時間下之影響
Leptin-induced proliferation of vascular smooth muscle cells (VSMCs). When incubated with 500 ng/ml leptin, the number of VSMCs decreased time-dependently. Values are means ± SD for triplicate determinations (
n
=3) repeated in 3 separate experiments.＊p
＜0.05 compared with control.0
C L CA10 CA20 UA10 UA20
(μmol/L) Cell number ( relative to control )
cd Carnosic acid (CA) and Ursolic acid (UA) inhibited leptin-induced proliferation in vascular smooth muscle cells (VSMCs).VSMCs were pretreated or not
圖 15. 鼠尾草酸與熊果酸對瘦體素誘發之血管平滑肌細胞自由基生成 之影響-螢光拍照圖
Effect of carnosic acid (CA) and ursolic acid (UA) on Leptin-induced ROS production in vascular smooth muscle cells (VSMCs). VSMCs were pretreated or not pretreated for 1 h with CA (10、20 μmol/L), or UA (10、20 μmol/L) and then incubated for 24 h with 500 ng/ml leptin (L).Microphotograph of ROS production in VSMCs . Similar results were obtained in 4 separate experiments. Abbreviation: C:control, L:Leptin, CA:Carnosic acid, UA:Ursolic acid.
C L L+CA10
L+CA20 L+UA10 L+UA20
0
C L CA10 CA20 UA10 UA20
(μmol/L) ROS (% of control)
d production in vascular smooth muscle cells (VSMCs). VSMCs were pretreated or not pretreated for 1 h with CA (10、20 μmol/L), or UA (10、20 μmol/L) and then incubated for 24 h with 500 ng/ml leptin (L). Values are means ± SD for quadruplicate determinations (
n
=4) repeated in 3 separate experiments. a-dMeans with different letters are significantly different compared with control atp
＜0.05.Abbreviation: C:control, L:Leptin, CA:Carnosic acid, UA:Ursolic acid.
(A)
Density ratio of phospho-ERK to ERK% of control
＊ serum-deprived for 24 h and incubated with 500 ng/ml leptin for various times (0 min to 60 min). (A) Western blotting analysis of ERK1/2 phosphorylation. (B) Western blotting analysis of ERK1/2. (C) Density ratio of phospho-ERK1/2 to ERK1/2 data. Similar results were obtained in 4 separate experiments. Values are means ± SD for 4 separate experiments.＊
p
＜0.05 compared to control of time at 0 min.β actin ERK 1/2
Phospho-ERK 1/2
(A)
C L CA10 CA20 UA10 UA20
(μmol/L) Density ratio of phospho-ERK to ERK% of control
d c
phosphorylation stimulated by leptin. Vascular smooth muscle cells (VSMCs) were pretreated or not pretreated for 1 h with CA (10、20 μmol/L), or UA (10、20 μmol/L) and then incubated for 20 min with 500 ng/ml leptin (L). (A) Western blotting analysis of ERK1/2 phosphorylation. (B) Western blotting analysis of ERK1/2. (C) Density ratio of phosphor-ERK1/2 to ERK1/2 data.
Similar results were obtained in 4 separate experiments. Values are means ± SD for 4 separate experiments.a-e Different letters are significantly different at
P
＜ 0.05. Abbreviation: C:control, L:Leptin, CA:Carnosic acid, UA:Ursolic acid.β actin
Phospho-ERK 1/2
ERK 1/2
(A) NF-κB p50 (% of control)
c b
Effect of Carnosic acid (CA) and Ursolic acid (UA) on leptin induced activation of NF-κB p50 in vascular smooth muscle cells (VSMCs). VSMCs were pretreated or not pretreated for 1 h with CA (10、20 μmol/L), or UA (10、20 μmol/L) and then incubated for 24 h with 500 ng/ml leptin (L). After incubation, the cell lysates were subjected to 9% SDS-PAGE and transferred to nitrocellulose membrane. (A) Western blotting analysis of NF-κB p50. (B) Densitometric analysis of NF-κB p50 data. Similar results were obtained in 4 separate experiments. Values are means ± SD for 4 separate experiments.
a-d Different letters are significantly different at
P
＜0.05. Abbreviation: C:control, L:Leptin, CA:Carnosic acid, UA:Ursolic acid.Histone P50
(A)
Effect of Carnosic acid (CA) and Ursolic acid (UA) on leptin induced activation of NF-κB p65 in vascular smooth muscle cells (VSMCs). VSMCs were pretreated or not pretreated for 1 h with CA (10、20 μmol/L), or UA (10、20 μmol/L) and then incubated for 24 h with 500 ng/ml leptin (L). After incubation, the cell lysates were subjected to 9% SDS-PAGE and transferred to nitrocellulose membrane. (A) Western blotting analysis of NF-κB p65. (B) Densitometric analysis of NF-κB p65 data. Similar results were obtained in 4 separate experiments. Values are means ± SD for 4 separate experiments.
a-d Different letters are significantly different at
P
＜0.05. Abbreviation: C:control, L:Leptin, CA:Carnosic acid, UA:Ursolic acid.Histone P65
圖 21. 鼠尾草酸與熊果酸對瘦體素誘發之血管平滑肌細胞核轉錄因子 NF-κB 之影響 (ICC)
Effect of Carnosic acid (CA) and Ursolic acid (UA) on leptin induced NF-κB nuclear translocation in vascular smooth muscle cells (VSMCs). VSMCs were pretreated or not pretreated for 1 h with CA (10、20 μmol/L), or UA (10、20 μmol/L) and then incubated for 24 h with 500 ng/ml leptin (L). After incubation, the cell were fixed in 100% ice-cold acetone and immunofluorescence staining with rabbit anti-NF-κB/p50 followed by Alexa-488 secondary antibody. The cell nuclei were stained with PI. The green signal represents NF-κB/p50 expression and the red signal corresponds to PI. Similar results were obtained in 3 separate experiments.Abbreviation: C-B(control-blank; The rat VSMCs labeled in the absence of primary antibody to NF-κB/p50), C:control, L:Leptin, CA:Carnosic acid, UA:Ursolic acid.
p50
PI
Merge
C L L+CA 10 L+CA 20 L+UA 10 L+UA 20
C-B
(A)
C L CA10 CA20 UA10 UA20
(μmol/L) MMP-2 (% of control)
a
then incubated for 24 h with 500 ng/ml leptin (L). (A) Western blotting analysis of MMP-2. (B) Densitometric analysis of MMP-2 data. Similar results were obtained in 5 separate experiments. Values are means ± SD for 5 separate experiments. a-eDifferent letters are significantly different atP
＜0.05.Abbreviation: C:control, L:Leptin, CA:Carnosic acid, UA:Ursolic acid..
MMP-2 β actin
(A)
C L CA10 CA20 UA10 UA20
(μmol/L) MMP-2 (% of control)
bc
pretreated or not pretreated for 1 h with CA (10、20 μmol/L), or UA (10、20 μmol/L) and then incubated for 24 h with 500 ng/ml leptin (L). (A) Gelatin zymography of MMP-2. (B) Densitometric analysis of gelatin zymography data.Similar results were obtained in 5 separate experiments. Values are means ± SD for 5 separate experiments. a-d Different letters are significantly different at
P
＜ 0.05. Abbreviation: C:control, L:Leptin, CA:Carnosic acid, UA:Ursolic acid.MMP-2
圖 24.鼠尾草酸與熊果酸對動脈粥狀硬化形成機制之影響
Carnosic acid Ursolic acid
Origanum majorana L.
ROS
ERK 1/2 IKK
NF-κB
MMP-2
Proliferation
Atheroslcerosis
Leptin