為實驗干擾因素。以 MTT 測定細胞存活率。分別測定 enterolactone, sesamin, genistein 及各種萃取物對於 PMA-differentiated THP-1 macrophages 之存活率 (圖 4-1)。另外也測定各種測試物單獨處理 HAEC 細胞後其細胞存活率 (圖 4-2)。
一、對於 PMA-differentiated THP-1 macrophages 的影響：
1. 在 chemical 測試物中， enterolactone、sesamin 及 genistein 分別在 1 µM，50 µM 與 20 µM 的單獨處理下，對 THP-1 macrophages 無顯著的 細胞毒性，故選用上述濃度作為後續實驗之條件。
2. 在萃物測試物中，山藥乙酸乙酯萃物、苜蓿芽乙酸乙酯萃物、茉莉甲醇
萃物及菊花甲醇萃物分別在 100 µg/ml 的單獨處理下，對 THP-1
macrophages 無顯著的細胞毒性，故選用上述濃度作為後續實驗之條件。
二、對於 Human Aortic Endothelial Cells (HAEC)的影響：
1. 在 chemical 測試物中， enterolactone、sesamin 及 genistein 分別在 1 µM，50 µM 與 25 µM 的單獨處理下，對 HAEC 無顯著的細胞毒性，故
0
yam EA extract (μg/ml) perc Fig 4-1. Effects of test compounds on viability of PMA-differentiated THP-1.
THP-1macrophages were treated with PMA at 75 ng/ml for 48hrs and test compounds for 48 hrs. Values are means ± SD. *Significantly different from control group (0)
0
alfalfa EA extract (μg/ml) perc
圖 4-2 不同處理對 HAEC 之細胞存活率影響
Fig 4-2. Effects of test compounds on viability of HAECs. HAECs were treated with test compounds for 24 hrs. Values are means ± SD
*Significantly different from control group(0) analyzed by Mann Whitney U t test, p
＜.05
yam EA extract (μg/ml) perc
alfalfa EA extract (μg/ml) perc
第二節 第二節 第二節
第二節 測試物對已分化 測試物對已分化 測試物對已分化 測試物對已分化 THP-1 細胞膽固醇堆積之影響 細胞膽固醇堆積之影響 細胞膽固醇堆積之影響 細胞膽固醇堆積之影響
一、氧化型低密度脂蛋白增加細胞內總膽固醇 (圖 4-3)
先以 PMA 處理 monocytic THP-1 細胞 72 小時形成 macrophage-like cells 為 PMA-differentiated THP-1 macrophages。再以氧化型低密度脂蛋白（oxLDL）25，
50 及 100 µg/ml 共同培養 48 小時，觀察細胞內膽固醇堆積情形。
結果發現分別投與 25, 50, 100µg/ml oxLDL 與 control 組比較均顯著升高細胞 內的總膽固醇量。Control 組的細胞內總膽固醇平均量為 33.73 ± 5.70 (µg
cholesterol/mg protein)，投與 25, 50, 100 µg/ml oxLDL 時，總膽固醇平均量分別為 43.33 ± 1.25, 49.72 ± 3.95, 49.72 ± 3.95 (µg cholesterol/mg protein)；分別提高總膽固 醇百分比分別為 28.5%、47.4%、43.9%。
由於添加 50 及 100 µg/ml oxLDL 對於總膽固醇量之提升無顯著差異，所以我 們選用 50 µg/ml oxLDL 為後續實驗所用之濃度。
圖 4-3 不同 oxLDL 濃度之處理對 THP-1 細胞內之總膽固醇/蛋白質比例之影響 Fig 4-3. Effects of oxidized LDL treatment on the content of cellular cholesterol in THP-1 cells stimulated with PMA. THP-1 cells (106/ml) were incubated alone or with the indicated concentrations of oxidized LDL for 48 hours. Levels of cellular
cholesterol are expressed relative to cellular protein. Values are means ± SD.
*Significantly different from control group analyzed by Mann Whitney U test at p
＜.05
＊
＊ ＊
二、 含植物性雌激素測試物對氧化型低密度脂蛋白增加 THP-1 macrophages 細 胞內之總膽固醇堆積之影響
先以 PMA 處理 THP-1 細胞 72 小時，再以氧化型低密度脂蛋白（oxLDL）50 µg/ml 培養 THP-1 macrophages 48 小時後，測量細胞內總膽固醇含量，並將數值 除以蛋白質濃度校正。
Control 組最後得到之膽固醇含量總平均為 33.73±5.70 (µg cholesterol/mg protein)，oxLDL treatment 組之總平均則為 49.01±4.02 (µg cholesterol/mg protein)，
因此得知以 oxLDL 處理後，細胞內總膽固醇量提升了 45.3％。以各次實驗 control 組之膽固醇含量為 100%，計算以測試物處理細胞後之相對增加或抑制百分比 (圖 4-4)。
結果發現 enterolactone 1 µM、sesamin 50 µM、茉莉甲醇萃物 100 µg/ml 及菊 花甲醇萃物 100 µg/ml，與單獨處理 oxLDL 的組別 (oxLDL treatment) 相比，可顯 著下降細胞內總膽固醇量。enterolactone 可下降 29.6%，sesamin 可下降 25.4%，
茉莉甲醇萃物可下降 42.8%，及菊花甲醇萃物可下降 38.6%之膽固醇堆積。
但是山藥乙酸乙酯萃物 100 µg/ml 顯著上升細胞內總膽固醇堆積達 93.8%。而 苜蓿芽乙酸乙酯萃物組及 genistein 組，與 oxLDL treatment 比較則無顯著差異。
圖 4-4 含植物性雌激素測試物對 oxLDL 處理細胞後之總膽固醇/蛋白質比例之影 響
Fig 4-4. The effects of chemicals and extracts on cellular cholesterol in
PMA-differentiated THP-1 macrophages incubated with oxLDL. THP-1 macrophages were treated with enterolactone (En), sesamin (Sn), yam EA extract (Y), alfalfa EA extract (A), genistein (G), jasmine methanol extract (J), and Chrysanthemum methanol extract (Ch) for 2 hrs and then co-cultured with oxLDL at 50 µg/ml for 48 hrs. Levels of cellular total cholesterol relative to protein were determined and the mean of control was set as 100%. Values are means ± SD.
*Significantly different from oxLDL analyzed by Mann Whitney U test at p＜.05 concentration
三、含植物性雌激素測試物對氧化型低密度脂蛋白刺激 THP-1 細胞內脂質堆積之 影響
THP-1 macrophages 培養 48 小時後，以 Oil-Red-O 染色，依此確定膽固醇酯 之生成 (圖 4.5)。之後以 isopropanol 溶解 Oil-Red-O 後，以 500 nm 讀取吸光值，
結果如圖 4.6。跟 control 組相比發現，oxLDL treatment 吸光值可增加 1.92 倍，顯 著高於 control 組。苜蓿芽乙酸乙酯萃物可增加 4.9 倍，顯著高於 oxLDL treatment；
而茉莉甲醇萃物之吸光值為 1.15 倍，顯著低於 oxLDL treatment。另外，sesamin、
genistein 之吸光值皆較 oxLDL treatment 低，但未達顯著。
圖 4-5 含植物性雌激素測試物經 oxLDL 處理對泡沫細胞形成之影響 Fig 4-5. Effects of chemicals and extracts on foam cell formation in
PMA-differentiated THP-1 macrophages incubated with oxLDL (50 µg/ml) for 48hrs.
THP-1 macrophages were pretreated with the test compounds for 2 hrs followed by oxLDL for 48 hrs. Cells were then stained with Oil-Red-O and then observed by phase-contrast microscopy (200X).
圖 4-6 含植物性雌激素測試物對 oxLDL 處理後對泡沫細胞生成之影響 Fig 4-6. Effects of chemicals and extracts on foam cell formation in
PMA-differentiated THP-1 macrophages incubated with oxLDL (50 µg/ml) for 48hrs.
THP-1 macrophages were pretreated with the test compounds for 2 hrs followed by oxLDL for 48 hrs. Cells were then stained with Oil-Red-O and lysed with isopropanol.
Values are means ± SD.
＊Significantly different from oxLDL treatment analyzed by student Mann Whitney U test at p＜.05
concentration
四、 含植物性雌激素測試物對 THP-1 macrophages 之 CD36 mRNA 表現的影響 經過共同培養 16 小時後，發現 genistein、茉莉甲醇萃物與菊花甲醇萃物皆能 顯著抑制 CD36 mRNA 表現，分別可抑制 30.4% (p=.05)，35.9% (p=.05)及 27.4%
(p=.05)。
經過共同培養 48 小時後，發現 enterolactone、sesamin 與 genistein 皆能顯著 抑制 CD36 mRNA 表現。Enterolactone 可抑制 29.2% (p=.05)，sesamin 可抑制 38.3%
(p=.05)，genistein 可抑制 52.2% (p=.05)。
圖 4-7 含植物性雌激素測試物對氧化型低密度脂蛋白刺激細胞後 CD36 mRNA 表 現之影響
Fig 4-7. Effects of chemicals and extracts on CD36 mRNA expression in
PMA-differentiated THP-1 macrophages incubated with oxLDL (50 µg/ml) for 16 and 48hrs. THP-1 macrophages were pretreated with the test compounds for 2 hrs followed by oxLDL for 16 and 48 hrs. Expression levels were adjusted by GAPDH mRNA levels and are represented relative to control, which is set as 100%. Values are means ± SD.
*Significantly different from oxLDL treatment analyzed by Mann Whitney U test at p <
0.05.
concentration
C D 3 6 e x p re s s io n ( % o f C )
三、含植物性雌激素測試物對 THP-1 macrophages 之 ABCA1 mRNA 表現的影響 在測試物與 oxLDL 共同培養 16 小時後，發現與 oxLDL 刺激組相較之下，
enterolactone、sesamin、genistein 與茉莉甲醇萃物會降低 ABC A1 mRNA (p=.05)。
但是培養 48 小時後，所有測試物對於 ABCA1 mRNA 表現量，與 oxLDL treatment 比較皆無顯著差異。
圖 4-8 含植物性雌激素測試物對氧化型低密度脂蛋白刺激細胞後 ABCA1 mRNA 表現之影響
Fig 4-8. Effects of chemicals and extracts on ABC A1 mRNA expression in
PMA-differentiated THP-1 macrophages incubated with oxLDL (50 µg/ml) for 16 and 48hrs. THP-1 macrophages were pretreated with the test compounds for 2 hrs followed by oxLDL for 16 and 48 hrs. Expression levels were adjusted by GAPDH mRNA levels and are represented relative to control, which is set as 100%. Values are means ± SD.
*Significantly different from oxLDL treatment analyzed by Mann Whitney U test at p <
0.05.
concentration
A B C A 1 e x p re s s io n ( % o f C )
第三節 第三節
第三節 第三節 植物性雌激素測試物質對 植物性雌激素測試物質對 植物性雌激素測試物質對 植物性雌激素測試物質對 THP-1 macrophages 表現 表現 表現 表現 促發炎因子之影響
促發炎因子之影響 促發炎因子之影響 促發炎因子之影響
一、含植物性雌激素測試物對 THP-1 macrophages 生成 IL-1β 之影響
oxLDL treatment 組測得之 IL-1β 之濃度為 337.7 ± 22.8 pg/ml，而 control 組之 濃度為 2.0 ± 3.8 pg/ml。以 oxLDL treatment 組各次實驗的濃度設定為 100%，將實 際濃度換算成百分比後，表示如圖 4-9。
本次研究發現，所有測試物皆可顯著下降 THP-1 macrophages 之 IL-1β 生合 成量。苜蓿乙酸乙酯萃物組可下降 42.6% (p<.001)，山藥乙酸乙酯萃物組可下降 42.1% (p<.001)，菊花甲醇萃物組可下降 41.1% (p<.001)，genistein 組可下降 35.5%
(p=.003)，sesamin 組可下降 35.4% (p<.001)，enterolactone 組可下降 25.3%
(p<.001)，茉莉甲醇萃物組可下降 24.0% (p<.001)。
其中以山藥、苜蓿乙酸乙酯萃物及菊花甲醇萃物處理的組別下降幅度最高。
圖 4-9 含植物性雌激素測試物對氧化型低密度脂蛋白刺激 THP-1 細胞生成 IL-1β 之影響
Fig 4-9. Effects of chemicals and extracts on IL-1β concentration in PMA-differentiated THP-1 macrophages incubated with oxLDL 50 µg/ml for 24hrs. THP-1 macrophages were pretreated with the test compounds for 2 hrs followed by oxLDL for 24 hrs.
Medium was collected for IL-1β analysis using ELISA. The secretion of IL-1β by cells treated with oxLDL only was expressed as a value of 100. Values are means ± SD.
*Significantly different from oxLDL treatment analyzed by Mann Whitney U test at p <
0.05.
concentration
R el a ti v e le v el o f IL -1 ββββ (% )
二、含植物性雌激素測試物對 THP-1 macrophages 生成 TNF-α 之影響
oxLDL treatment 組測得 TNF-α 之濃度為 1218.5±113.8 pg/ml，而 control 組之 濃度為 8.07±9.09 pg/ml。以 oxLDL treatment 組各次實驗的濃度設定為 100%，將 實際濃度換算成百分比後，表示如圖 4-10。
將所有測試物與 oxLDL treatment 相比，除 enterolactone 外，皆可顯著下降 PMA-differentiated THP-1 macrophages 之 TNF-α 生合成量。菊花甲醇萃物組可下 降 67.3% (p<.001)，genistein 組可下降 62.6% (p<.001)，sesamin 組可下降 61.6%
(p=.001)，苜蓿乙酸乙酯萃物組可下降 52.6% (p<.001)，茉莉甲醇萃物組可下降 47.0% (p<.001)，山藥乙酸乙酯萃物組可下降 38.2% (p<.001)。
其中以 sesamin、genistein 及菊花甲醇萃物處理之組別，可以下降 TNF-α 分 泌量的幅度最多，皆可超過 60%。
圖 4-10 含植物性雌激素測試物對氧化型低密度脂蛋白刺激 THP-1 細胞生成 TNF-α 之影響
Fig 4-10. Effects of chemicals and extracts on TNF-α concentration in
PMA-differentiated THP-1 macrophages incubated with oxLDL 50 µg/ml for 24hrs THP-1 macrophages were pretreated with the test compounds for 2 hrs followed by oxLDL for 24 hrs. Medium was collected for TNF-α analysis using ELISA. The secretion of TNF-α by cells treated with oxLDL only was expressed as a value of 100.
Values are means ± SD.
*Significantly different from oxLDL treatment analyzed by Mann Whitney U test at p <
0.05.
concentration
R el a ti v e le v el o f T N F -αααα (% )
三、 含植物性雌激素測試物對 THP-1 macrophages 生成 MCP-1 之影響 oxLDL treatment 組測得 MCP-1 之濃度為 97.4 ± 41.5 pg/ml。以 oxLDL treatment 組各次實驗的濃度設定為 100%，將實際濃度換算成百分比後，表示如 圖 4-11。
將所有測試物質組別與 oxLDL treatment 相比，以 enterolactone、sesamin、
genistein、苜蓿芽乙酸乙酯萃物處理之組別，可以顯著下降 MCP-1 之生合成量。
山藥乙酸乙酯萃物則是會顯著上升。
Genistein 可下降 32.9% (p=.007)，苜蓿乙酸乙酯萃物組可下降 25.8%
(p=.002) ，Enterolactone 可下降 24.4% (p=.002)，sesamin 可下降 19.8% (p=.027)。
山藥乙酸乙酯萃物組則會上升 48.4% (p=.001)。
圖 4-11 含植物性雌激素測試物對 THP-1 細胞生成 MCP-1 之影響 Fig 4-11. Effects of chemicals and extracts on MCP-1 concentration in
PMA-differentiated THP-1 macrophages incubated with oxLDL 50 µg/ml for 48hrs.
THP-1 macrophages were pretreated with the test compounds for 2 hrs followed by oxLDL for 48 hrs. Medium was collected for MCP-1 analysis using ELISA. The secretion of MCP-1 by cells treated with oxLDL only was expressed as a value of 100.
Values are means ± SD.
*Significantly different from oxLDL treatment analyzed by Mann Whitney U test at p <
0.05.
concentration
R el a ti v e le v el o f M C P -1 ( % )
四、 含植物性雌激素食材萃物對 THP-1 macrophages 生成 MMP-9 之影響 oxLDL treatment 組測得 MMP-9 之三次實驗平均濃度為 78.12±59.63 mg/ml，
control 組則為 35.51±17.86 mg/ml。以 oxLDL treatment 組各次實驗的濃度設定為 100%，將實際濃度換算成百分比後，表示如圖 4-12。
將所有測試物質組別與 oxLDL treatment 相比，以 enterolactone、genistein、
山藥乙酸乙酯萃物、苜蓿芽乙酸乙酯萃物處理之組別，可以顯著下降 MMP-9 之 生合成量。其中苜蓿乙酸乙酯萃物組可下降 43.1% (p=.016) ，genistein 可下降 34.2% (p=.007) ，Enterolactone 可下降 24.5% (p<.001) ，山藥乙酸乙酯萃物組可 下降 12.2% (p=.001)。
圖 4-12 含植物性雌激素測試物對 THP-1 細胞生成 MMP-9 之影響 Fig 4-12. Effects of chemicals and extracts on MMP-9 concentration in
PMA-differentiated THP-1 macrophages incubated with oxLDL 50 µg/ml for 48hrs.
THP-1 macrophages were pretreated with the test compounds for 2 hrs followed by oxLDL for 48 hrs. Medium was collected for MMP-9 analysis using ELISA. The secretion of MMP-9 by cells treated with oxLDL only was expressed as a value of 100.
Values are means ± SD.
*Significantly different from oxLDL treatment analyzed by Mann Whitney U test at p <
0.05.
concentration
R el a ti v e le v el o f M M P -9 ( % )
五、含植物性雌激素食材萃物對 THP-1 細胞 IL-1β mRNA 表現之影響
所有測試物無論於 16 小時或 48 小時測得的 IL-1β mRNA 表現量，與 oxLDL treatment 相比皆無顯著差異。
圖 4-13 含植物性雌激素測試物對氧化型低密度脂蛋白刺激細胞後 IL-1β mRNA 表現之影響
Fig 4-13. Effects of chemicals and extracts on IL-1β mRNA expression in
PMA-differentiated THP-1 macrophages incubated with oxLDL (50 µg/ml) for 16 and 48hrs. THP-1 macrophages were pretreated with the test compounds for 2 hrs followed by oxLDL for 16 and 48 hrs. Expression levels were adjusted by GAPDH mRNA levels and are represented relative to control, which is set as 100%. Values are means ± SD.
*Significantly different from oxLDL treatment analyzed by Mann Whitney U test at p <
0.05.
concentration
IL-1
β β β β
expression (% of C)
六、含植物性雌激素食材萃物對 THP-1 細胞 TNFα mRNA 表現之影響
在測試物與 oxLDL 共同培養 16 小時後，enterolactone、sesamin、山藥乙酸乙 酯萃物及苜蓿乙酸乙酯萃物，其 TNF-α mRNA 表現顯著低於 oxLDL treatment。而
在測試物與 oxLDL 共同培養 16 小時後，enterolactone、sesamin、山藥乙酸乙 酯萃物及苜蓿乙酸乙酯萃物，其 TNF-α mRNA 表現顯著低於 oxLDL treatment。而