多元不飽和脂肪酸對脂肪細胞和蝕骨細胞分化之影響

Effects of polyunsaturated fatty acids on adipogenesis of 3T3-L1 cells and RANKL-induced osteoclast differentiation of RAW 264.7 macrophages

葉佳珍(Chia-Chen Yeh)

指導教授 ： 劉凱莉

中山醫學大學/健康管理學院/營養學研究所/碩士(2010年)

https://doi.org/10.6834/CSMU.2010.00153

摘要

亞麻油酸(linoleic acid，LA ) 和次亞麻油酸 (α-linolenic acid，LNA) 為必需脂肪酸，人體無法合成需由飲食得到，LA可藉由δ6去飽和酵素 ( δ6 desaturase ) 形成γ-次亞麻油酸 ( Gamma Linolenic Acid ﹐GLA )，再經由延長酵素 ( elongase ) 和δ5去飽和酵素 ( δ5 desaturase ) 合成花生四烯酸 ( Arachidonic Acid ﹐AA )。本實驗室之前研究發現n-6多元不飽和脂肪酸LA及GLA 皆可降低細菌內毒素Lipopolysaccharide 誘發小鼠RAW264.7巨噬細胞發炎反應。研究證實身體長期處於慢性發炎狀態會增加肥胖和骨質疏鬆症發生，減少促發炎物質生成量可減少脂肪細胞及蝕骨細胞(osteoclast)生成。本研究以 Receptor Activator of Nuclear Factor-kB Ligand (RANKL)誘發小鼠RAW264.7巨噬細胞分化為蝕骨細胞為研究模式，探討LA、GLA、AA對RANKL誘發小鼠RAW264.7巨噬細胞分化為蝕骨細胞之影響。LA、GLA和AA與RANKL 10ng/ml共同處理4天不影響細胞存活率下。與小鼠RAW264.7巨噬細單獨處理RANKL 10ng/ml 4天控制組相較，RANKL 10ng/ml共同50 μM LA及50和100 μM ( GLA、AA )細胞中Tartrate-Resistant Acid Phosphatase、Matrix Metallopeptidase 9和Nuclear Factor of Activated T-cells, Cytoplasmic 1 mRNA的表現顯著被抑制，且以100 μM GLA抑制效果最佳。
此外，本研究亦探討LA、GLA、AA和LNA對3T3-L1小鼠前脂肪細胞分化為脂肪細胞之影響。LA、GLA、AA、LNA和DM1 [Dexamethasone (DEX) 0.25 μM、3-Isobutyl-1-Methylxanthine (IBMX) 0.25 mM、insulin 0.1 μg/ml]及DM2 ( insulin 0.1 μg/ml )共同處理7天不影響細胞存活率。與只處理DM1之 3T3-L1小鼠前脂肪細胞相較，處理DM1及50、100、200 μM GLA和200 μM ( LA、AA、LNA) 8小時可增加ERK1/2磷酸化和抑制CCAAT/enhancer Binding Protein β (C/EBP β) 蛋白質表現。與只處理DM1和DM2之3T3-L1小鼠脂肪細胞相較，脂肪酸、DM1及DM2共同處理八天，會減少脂肪細胞內油滴的累積及抑制Peroxisome-Proliferator Activated Receptor γ (PPAR γ)、C/EBP α mRNA和蛋白質的表現，降低Fatty Acid Synthase mRNA和蛋白質表現。此外於脂肪細胞分化過程中添加脂肪酸，會顯著增加脂肪細胞中Adenosine Monophosphate-Activated Arotein Kinase 磷酸化及抑制肪細胞激素 ( adipokine ) resistin mRNA的表現。以於脂肪細胞分化過程中添加200 μM GLA對抑制脂肪細胞分化及脂肪細胞中脂肪酸合成和脂肪細胞激素的效果最好。綜合以上結果得知，在RANKL誘發小鼠RAW264.7巨噬細胞分化為蝕骨細胞和3T3-L1小鼠前脂肪細胞分化為脂肪細胞過程中，多元不飽和n-3和n-6脂肪酸可抑制蝕骨細胞分化 (抑制功效GLA＞LA＞AA) 及脂肪細胞分化 (抑制功效GLA＞LA＞AA LNA GLA＞LA＞AA LNA)。

關鍵字

小鼠RAW264.7巨噬細胞 ； 小鼠3T3-L1前脂肪細胞 ； 亞麻油酸 ； γ-次亞麻油酸 ； α-次亞麻有酸 ； 花生四烯酸

並列摘要

Linoleic acid ( LA ) and α-linolenic acid ( LNA ) are the essential fatty acids which human is not able to synthesize and must obtain by the food. LA can convert to gamma linolenic acid ( GLA ) by δ6 desaturase. Then GLA can convert to arachidonic acid ( AA ) by elongase and δ5 desaturase. The data form our lab have shown that LA and GLA could reduced lipopolysaccharide induced inflammatory response in RAW 264.7 macrophages. Studies have demonstrated that chronic inflammation play an important role in obese and osteoporosis. Moreover, modulating pro-inflammatory mediator production could reduce adipogenesis and osteoclast differentiation. In this study, explore the effect of polyunsaturated fatty acids on Receptor Activator of Nuclear Factor-kB Ligand (RANKL) induced osteoclasts differentiation of RAW264.7 macrophage. Compared with cells treated with RANKL only, 50 μM LA, cells treated with RANKL and 50 and 100 μM (GLA, AA) significantly decreased Tartrate-Resistant Acid Phosphatase、Matrix Metallopeptidase 9和Nuclear Factor of Activated T-cells, Cytoplasmic 1 mRNA expression. Moreover the 100 μM GLA treatment have the highest inhibitory capacity. We also assessed the effects of polyunsaturated fatty acids on adipogenesis.3T3 preadipocytes treated with LA, GLA, AA, LNA and DM1 (Dexamethasone (DEX) 0.25 μM, 3-Isobutyl-1-Methylxanthine (IBMX) 0.25 mM, and insulin 0.1 μg/ml] 48 h and then DM2 (insulin 0.1 μg/ml) every 48 h for 6 days did not affect the cell viability by 3-( 4,5-Dimethylthiazol-2-yl ) -2,5-Diphenyltetrazolium Bromide assay. Compared cells treated with DM1 only, cells treated with DM1 and fatty acids 8h significantly increased the ERK1/2 phosphorylation and suppressed CCAAT/enhancer Binding Protein β (C/EBP β) protein expression. During differentiation of the 3T3 preadipocyte to adipocytes, exogenous fatty acid addition could decrease lipid accumulation and decreased Peroxisome-Proliferator Activated Receptor γ (PPAR γ)、C/EBP α and fatty acid synthase expression. Moreover, exogenous addition of fatty acid during adipogenesis increase phosphorylation of Adenosine Monophosphate-Activated Arotein Kinase expression and reduced resistin production. In conclusion, n-3 and n-6 polyunsaturated fatty acids could reduced RNAKL induced osteoclast differentiation of RAW 264.7 macrophages and 3T3 cell adipogenesis.

並列關鍵字

RAW264.7 macrophage ； 3T3-L1 preadipocyte ； linoleic acid ； Arachidonic Acid ； Gamma Linolenic Acid ； linolenic acid

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