由前面實驗結果，山苦瓜乙酸乙酯萃取物有刺激脂肪細胞棕色脂肪相關基因 表現的潛力，乃進而探討其中可能的活性成分。先前實驗室經分離純化，鑑定出 苦瓜中9c,11t,13t-CLN 為可活化 PPARs 之化合物。CLN 由於具有共軛三烯的結構，
故在268 nm 有最大吸光值 (而 CLA 具有共軛雙烯結構，在 235 nm 有最大吸光) (Igarashi et al., 2000) 。根據紫外光吸光圖譜結果顯示，EAE 與 S、NS 相比，在 270 nm 吸光值最高，顯示 EAE 中 CLN 含量較高，S 其次，NS 最低 (圖 2-15) 。
(一) 共軛次亞麻油酸 CLN
脂肪細胞在CLN 處理下，棕色脂肪相關基因 PGC1α、UCP1、Cidea 及 Prdm16 mRNA 表現量有高於空白處理組的趨勢，皆在濃度 100 μM 有最大增加幅度 (幅度 分別為1.5 倍、2.4 倍、3.1 倍及 4.1 倍) 。其中 PGC1α 基因表現在濃度 25 μM 有 顯著增加 (1.2 倍) (p<0.005) (圖 2-16) 。粒線體生合成作用相關基因 Tfam 在濃度 50 μM 顯著高於空白組，但幅度並不大 (1.1 倍) (p<0.05) ，而 NrF1 和空白組並無 顯著差異 (p>0.05)。整體而言，Cox7a1 及 Cox8b 在各濃度下皆高於空白組，但只 有濃度100 μM 下，Cox7a1 基因表現顯著高於空白組 (p<0.05) (圖 2-17 )。白色脂 肪相關基因表現上，Retn 與 Chemerin 和空白組相比並無顯著差異 (p>0.05) (圖
(二) 植醇 phytol
脂肪細胞在 phytol 處理下，棕色脂肪相關基因 PGC1α 及 cidea 和空白處理組 相比，雖表現較高，但並無顯著差異 (p>0.05) 。而 UCP1 在各濃度下的表現量皆 顯著增加 (700、350 及 175 μM 分別增加 1.8 倍、1.5 倍及 1.5 倍) (p<0.05 ~ p<0.005) 。 在高濃度phytol 處理下 prdm16 表現較低，但隨著濃度減少有增加的趨勢，但並無 差異 (p>0.05) (圖 2-16) 。粒線體生合成作用相關基因 Tfam 在各濃度下皆顯著高 於空白組，但幅度並不大 (1.1 倍) (p<0.05) ，而 NrF1 mRNA 表現在濃度 350 及 175 μM 則顯著被抑制 (0.9 倍) (p<0.05) 。整體而言，Cox7a1 及 Cox8b 在各濃度 phytol 下皆高於空白組，唯因誤差較大，統計差異並不顯著 (p>0.05) (圖 2-17 ) 。白色脂 肪相關基因表現，Retn 和對照組相比並無顯著差異，Chemerin 則是在 350 μM 濃 度下，顯著較高 (p<0.05) (圖 2-18) 。PPARα、PPARγ 及 C/EBPβ 等轉錄調控因子 基因表現，phytol 處理組和空白組相比並無顯著差異，而 C/EBPα 在濃度 700 μM 處理下表現顯著升高 (1.2 倍) (p<0.05) (圖 2-19) 。
(三) 葉黃素 lutein
脂肪細胞在 lutein 低濃度 8.75 μM 處理下，UCP1 mRNA 表現顯著高於空白組 (p<0.05) 。而棕色脂肪相關基因 PGC1α、Cidea 及 prdm16 和空白組相比雖無顯著 差異，但隨著濃度降低有升高的趨勢 (p>0.05) ，在濃度 8.75 μM 皆有最大增加幅 度 (幅度分別為 1.2 倍、1.3 倍、2.4 倍及 1.5 倍) (圖 2-16) 。粒線體生合成作用相 關基因Tfam, NrF1 在各濃度處理下，和空白處理組並無顯著差異 (p>0.05) 。特別 的是，不同於其他棕色脂肪相關基因，在較高濃度 (35 μM) 處理下，Cox7a1 及 Cox8b 有最大增加幅度 (2.0 倍及 1.9 倍) ，其中除了 Cox7a1 基因表現在 8.75 μM 濃度處理下有顯著增加 (1.6 倍) ，其餘皆無顯著差異 (p>0.05) (圖 2-17 ) 。白色脂 肪相關基因表現上，Retn 與 Chemerin 和空白組相比並無顯著差異 (p>0.05) (圖 2-18) 。轉錄調控因子基因表現在 lutein 處理下，PPARα、C/EBPα 及 C/EBPβ mRNA 表現和空白組相比並無顯著差異，而濃度0.2 μg/mL 處理下，PPARγ 表現有顯著增 加 (1.3 倍) (圖 2-19) 。
PGC1a Figure 2-1 Troglitazone treatment during last 2 days of differentiation induced brown gene expression in 3T3-L1 adipocytes.
3T3-L1 adipocytes were treated for the last 2 days before mature with 5 μM troglitazone (Trog) or vehicle (DMSO) for 2 days. 3T3-L1 cells were differentiated according to protocol 1 (A) or protocol 2 (B). Protocol 1：cells were differentiated in Differention Medium (DM) Ⅰ&Ⅱ containg 10 %
PGC1a Figure 2-2 Troglitazone treatment for 4 days (last 2 days of differentiation and 2 days post differentiation) induced brown gene expression in 3T3-L1 adipocytes. 3T3-L1 adipocytes were 4 days (last 2 days of differentiation and 2 days post differentiation) with 5 μM troglitazone (Trog) or vehicle (DMSO). 3T3-L1 cells were differentiated according to protocol 1 (A) or protocol 2 (B). Protocol 1：cells were differentiated in Differention Medium (DM) Ⅰ&Ⅱ containg 10 % BS/DMEM, and cells were differentiated for 8 days in DMⅡ. Protocol 2：cells were differentiated in DMⅠ&Ⅱ containg 10 % FBS/DMEM, and cells were differentiated for 4 days in DMⅡ. Each bar represents the mean value ± SD (n=3). Mean mRNA expression levels of vehicle treated cells were taken as 1 and those Trog treated cells were calculated as folds those of vehicle treated cells. * and *** denote significantly different from vehicle treated cells at p<0.05(*) or p<0.005 (***) analyzed by Student’s t-test.
PGC1a Figure 2-3 Troglitazone treatment during last 4 days of differentiation induced brown gene expression in 3T3-L1 adipocytes.
3T3-L1 adipocytes were treated for the last 4 days before mature with 5 μM troglitazone (Trog) or vehicle (DMSO) for 4 days. 3T3-L1 cells were differentiated according to protocol 1 (A) or protocol 2 (B). Protocol 1：cells were differentiated in Differention Medium (DM) Ⅰ&Ⅱ containg 10 %
PGC1a Figure 2-4 Comparison of the effects of differentiation protocol and treatment time on the mRNA expression of brown-fat selective and mitochondria biogensis gene in troglitazone treated 3T3-L1 adipocytes. 3T3-L1 cells were differentiated as protocol 1and protocol 2, and were treated (A) for the last 2 days before mature with 5 μM troglitazone (Trog) or vehicle (DMSO) for 2 days；(B) Since the last 2 days before mature with 5 μM Trog or DMSO for 4 days and (C) for the last 4 days before mature with 5 μM Trog or DMSO for 4 days (C). Each bar represents the mean value ± SD (n=3). Mean mRNA expression levels of vehicle treated cells were taken as 1 and those Trog treated cells were calculated as folds those of vehicle treated cells. *, ** and *** denote significantly different from DMSO treated cells at p<0.05(*), p<0.01 (**) or p<0.005 (***) analyzed by Student’s t-test. # and ## denote significantly different between protocol 1 and 2 at p<0.05(#) or p<0.01 (##) analyzed by Student’s t-test.
(A) (B)
Figure 2-5 mRNA expression of brown-fat-selective genes in 3T3-L1 adipocytes treated with ethyl acetate extract (EAE) of wild bitter gourd and its saponifiable (S) or non-saponifiable (NS) fractions .
3T3-L1 adipocytes were differentiated using protocol 1 and were treated with 5 μM Trog, wild bitter gourd extracts or vehicle (DMSO) during the last 4 days of differentiation. Each bar represents the mean value ± SD (n=3). Mean mRNAexpression levels of vehicle treated cells were taken as 1 and those Trog/extracts treated cells were calculated as folds those of vehicle treated cells. (A) PGC1α, (B) UCP1, (C) cidea, (D) prdm16. *, ** and *** denote significantly different from vehicle treated cells at p<0.05(*), p<0.01 (**) or p<0.005 (***) analyzed by Student’s t-test.
(A) (B)
Figure 2-6 mRNA expression of mitochondria biogensis genes in 3T3-L1 adipocytes treated with ethyl acetate extract (EAE) of wild bitter gourd and its saponifiable (S) or non-saponifiable (NS) fractions.
3T3-L1 adipocytes were differentiated using protocol 1 and were treated with 5 μM Trog, wild bitter gourd extracts or vehicle (DMSO) during the last 4 days of differentiation. Each bar represents the mean value ± SD (n=3). Mean mRNAexpression levels of vehicle treated cells were taken as 1 and those Trog/extracts treated cells were calculated as folds those of vehicle treated cells. (A) Tfam, (B) NrF1, (C) cox7a1, (D) cox8b. * and *** denote significantly different from vehicle treated cells at p<0.05(*) or p<0.005 (***) analyzed by Student’s t-test.
(A)
Figure 2-7 mRNA expression of white-fat-selective genes in 3T3-L1 adipocytes
treated with ethyl acetate extract (EAE) of wild bitter gourd and its saponifiable (S)
or non-saponifiable (NS) fractions.
3T3-L1 adipocytes were differentiated using protocol 1 and were treated with 5 μM Trog, wild bitter gourd extracts or vehicle (DMSO) during the last 4(A) (B)
Figure 2-8 mRNA expression of transcription factors genes in 3T3-L1 adipocytes treated with ethyl acetate extract (EAE) of wild bitter gourd and its saponifiable (S) or non-saponifiable (NS) fractions.
3T3-L1 adipocytes were differentiated using protocol 1 and were treated with 5 μM Trog, wild bitter gourd extracts or vehicle (DMSO) during the last 4 days of differentiation. Each bar represents the mean value ± SD (n=3). Mean mRNAexpression levels of vehicle treated cells were taken as 1 and those Trog/extracts treated cells were calculated as folds those of vehicle treated cells. (A) PPARα, (B) PPARγ, (C) C/EBPα, (D) C/EBPβ. *, ** and *** denote significantly different from vehicle treated cells at p<0.05(*), p<0.01(*) or p<0.005 (***) analyzed by Student’s t-test.
(A) (B) Figure 2-9 mRNA expression of (A) UCP1 and (B) C/EBPβ in 3T3-L1 adipocytes treated with wile bitter gourd extracts or its fraction in the presence of forskolin. 3T3-L1 adipocytes were differentiated using protocol 1 and were treated with 5 μM Trog, wild bitter gourd extracts or vehicle (DMSO) during the last 4 days of differentiation, with or without forskolin for 8 hr. Each bar represents the mean value ± SD (n=2).
(A) (B)
Figure 2-10 mRNA expression of brown-fat-selective genes in 3T3-L1 adipocytes
treated with water extract (WE) of wild bitter gourd or WE pretreated with
bacillus subtilis natto (WEn). 3T3-L1 adipocytes were differentiated using protocol 1 and were treated with 5 μM Trog, wild bitter gourd extracts (WE, WEn) or vehicle (DMSO) during the last 4 days of differentiation. Each bar represents the mean value ± SD (n=2). Mean mRNA expression levels of vehicle treated cells were taken as 1 and those Trog/extracts treated cells were calculated as folds those of vehicle treated cells. (A) PGC1α, (B) UCP1, (C) cidea, (D) prdm16. * and *** denote significantly different from vehicle treated cells at p<0.05(*)or p<0.005 (***) analyzed by Student’s t-test.(A) (B)