新育種苦瓜對3T3-L1細胞之細胞週期、脂質生合成及發炎反應之影響及其分子機制探討

Effects and mechanisms of Momordica charantia on cell cycle, adipogenesis, and inflammation in 3T3-L1 cells

邱天惠(Tien-Hui Chiu)

指導教授 ： 徐慶琳

中山醫學大學/健康管理學院/營養學研究所/碩士(2013年)

摘要

肥胖為世界盛行的疾病之一，亦會造成許多慢性疾病的發生，例如：心血管疾病、第二型糖尿病、高血壓與癌症等。山苦瓜已長期且廣泛的運用在治療第二型糖尿病與發炎反應上，然而目前新育種苦瓜對3T3-L1細胞在肥胖與肥胖發炎之影響尚未釐清，因此，本實驗利用三種新育種苦瓜，分別為蘋果苦瓜、苦瓜明道選72與苦瓜沖繩選33之果肉與種子，分別以水、甲醇、乙醇、丙酮與乙酸乙酯進行萃取，利用3T3-L1細胞為實驗模式，探討三種新育種苦瓜對3T3-L1細胞之細胞週期、脂質生合成與發炎反應之影響。在新育種苦瓜對3T3-L1前脂肪細胞之細胞週期影響上，蘋果苦瓜種子水萃取物可藉由活化p-p53、p27與p18之蛋白表現，以及抑制CDK2、CDK6、cyclin D1與p-Rb之蛋白表現，使細胞週期停滯在G1期，進而抑制3T3-L1前脂肪細胞增生。在新育種苦瓜對3T3-L1脂肪細胞脂質生合成影響上，以蘋果苦瓜果肉水萃取物對3T3-L1脂肪細胞在胞內三酸甘油酯、甘油-3-磷酸去氫酶活性與脂肪細胞數目 (油紅染色) 上具有最佳之抑制效果。其可透過調控MAPK路徑之蛋白質表現 (p-ERK與p-JNK)，抑制3T3-L1脂肪細胞分化相關基因 (PPARγ、SREBP-1c、C/EBPα與C/EBPβ)，以及抑制脂肪酸合成酶 (FAS) 之基因與蛋白表現量，並提升脂肪酸分解與氧化之基因表現 (ATGL、HSL、ACO與CPT-1)，進而抑制脂肪細胞油滴堆積之作用。此外，其亦可抑制leptin之基因表現，並顯著增加adiponectin之基因與蛋白表現。在新育種苦瓜對3T3-L1脂肪細胞發炎反應之影響上，苦瓜明道選72果肉甲醇萃取物具有抑制發炎細胞激素IL-6與MCP-1之分泌並提升adiponectin含量之能力，其是經由提升SIRT1與AMPK，並抑制NF-κB與CD40之基因表現，進而抑制發炎相關基因IL-6、MCP-1、PAI-1、CRP與leptin，並提升adiponectin之基因表現。綜合以上結果得知，新育種苦瓜對3T3-L1細胞具有使細胞週期停滯於G1期 (蘋果苦瓜種子水萃取物)、抑制脂質生合成 (蘋果苦瓜果肉水萃取物) 與降低發炎反應 (苦瓜明道選72果肉甲醇萃取物) 之作用。

關鍵字

3T3-L1細胞,新育種苦瓜 ； 細胞週期,脂質生合成 ； 發炎反應

並列摘要

The prevalence of obesity is increasing worldwide and is considered a risk factor associated with cardiovascular, type 2 diabetes, hypertension, and cancer. Bitter melon was widely used as herbal medicine for anti-diabetes and anti-hypertension activities. However, the inhibitory effects of obesity and obesity-related inflammation response in new variety selection of bitter melon remain unclear. Therefore, the aim of this study was investigate the effects of different solvents (water, methanol, ethanol, acetone, and ethyl acetate) extracts from the fruit and seed of Momordica charantia, Momordica charantia MDS72, and Momordica charantia ONS33 on cell cycle arrest, adipogenesis, and inflammation response in 3T3-L1 cells. In cell cycle arrest, the result indicated that the water extract from the seed of Momordica charantia (WESMC) caused cell cycle arrest in the G1 phase in 3T3-L1 preadipocytes through regulates cell cycle-related proteins, such as activate the protein expressions of p-p53, p27, and p18. Furthermore, WESMC also suppress the protein expressions of CDK2, CDK6, cyclin D1, and p-Rb in 3T3-L1 preadipocytes. In the adipogenesis, the data indicated that water extract from the fruit of Momordica charantia (WEFMC) significantly decreased the intracellular triglyceride, glycerol-3-phosphate dehydrogenase, and the number of adipocytes (oil red o staining) through regulate MAPK pathway protein expressions (p-ERK and p-JNK), inhibit adipocytes differentiation related gene (PPARγ, SREBP-1c, C/EBPα, and C/EBPβ), lipogenesis related gene and protein expressions of FAS, and increase the lipolysis and fatty acid oxidation related gene expressions of ATGL, HSL, CPT-1, and ACO. In addition, WEFMC also suppress the gene expressions of leptin and promote gene and protein expressions of adiponectin. In the inflammation response, the result indicated that methanol extract from the fruit of Momordica charantia MDS72 (MEFMCMDS72) suppress the secretion of inflammation cytokines, such as IL-6 and MCP-1, and stimulate the secretion of adiponectin in 3T3-L1 adipocytes through increase the gene expressions of SIRT1 and AMPK, suppress the gene expressions of NF-κB and CD40, thus decrease the gene expressions of IL-6, MCP-1, PAI-1, CRP, and leptin, and enhance the gene expressions of adiponectin. These results demonstrate that new variety selection of bitter melon can regulate the cell cycle arrest in the G1 phase (WESMC), inhibit adipogenesis (WEFMC), and suppress obesity-related inflammation response (MEFMCMDS72) in 3T3-L1 cells.

並列關鍵字

3T3-L1 cells ； bitter melon ； cell cycle ； adipogenesis ； inflammation

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