Poulsen Raewyn C, Wolber Frances M, Moughan Paul J, Kruger Marlena C
Institute of Food, Nutrition and Human Health, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand.
Prostaglandins Other Lipid Mediat. 2008 Feb;85(1-2):42-8. doi: 10.1016/j.prostaglandins.2007.10.004. Epub 2007 Nov 5.
Inflammation triggers an increase in osteoclast (bone resorbing cell) number and activity. Osteoclastogenesis is largely controlled by a triad of proteins consisting of a receptor (RANK), a ligand (RANK-L) and a decoy receptor (osteoprotegerin, OPG). Whilst RANK is expressed by osteoclasts, RANK-L and OPG are expressed by osteoblasts. The long chain polyunsaturated fatty acid (LCPUFA) arachidonic acid (AA, 20:4n-6) and its metabolite prostaglandin E2 (PGE2), are pro-inflammatory and PGE2 is a potent stimulator of RANKL expression. Various LCPUFAs such as eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and gamma-linolenic acid (GLA, 18:3n-6) have anti-inflammatory activity. We aimed to determine if AA itself can stimulate RANKL expression and whether EPA, DHA and GLA inhibit RANKL expression in osteoblasts. MC3T3-E1/4 osteoblast-like cells were cultured under standard conditions with each of the LCPUFAs (5microg/ml) for 48h. Membrane-bound RANKL expression was measured by flow cytometry and OPG secretion measured by ELISA. In a second experiment, RANKL expression in MC3T3-E1/4 cells was stimulated by PGE2 treatment and the effect of EPA, DHA and GLA on membrane-bound RANKL expression and OPG secretion determined. The percentage of RANKL-positive cells was higher (p<0.05) than controls following treatment with AA or GLA but not after co-treatment with the cyclooxygenase inhibitor, indomethacin. DHA and EPA had no effect on membrane-bound RANKL expression under standard cell culture conditions. Secretion of OPG was lower (p<0.05) in AA-treated cells but not significantly different from controls in GLA, EPA or DHA treated cells. Treatment with prostaglandin E2 (PGE2) resulted in an increase (p<0.05) in the percentage of RANK-L positive cells and a decrease (p<0.05) in mean OPG secretion. The percentage of RANKL positive cells was significantly lower following co-treatment with PGE2 and either DHA or EPA compared to treatment with PGE2 alone. Mean OPG secretion remained lower than controls in cells treated with PGE2 regardless of co-treatment with EPA or DHA. Results from this study suggest COX products of GLA and AA induce membrane-bound RANKL expression in MC3T3-E1/4 cells. EPA and DHA have no effect on membrane-bound RANKL expression in cells cultured under standard conditions however both EPA and DHA inhibit the PGE2-induced increase in RANKL expression in MC3T3-E1/4 cells.
炎症会引发破骨细胞（骨吸收细胞）数量和活性的增加。破骨细胞生成主要受由一种受体（核因子κB受体活化因子，RANK）、一种配体（RANK配体，RANK-L）和一种诱饵受体（骨保护素，OPG）组成的三联蛋白控制。虽然RANK由破骨细胞表达，但RANK-L和OPG由成骨细胞表达。长链多不饱和脂肪酸（LCPUFA）花生四烯酸（AA，20:4n-6）及其代谢产物前列腺素E2（PGE2）具有促炎作用，且PGE2是RANKL表达的强效刺激剂。各种LCPUFA，如二十碳五烯酸（EPA，20:5n-3）、二十二碳六烯酸（DHA，22:6n-3）和γ-亚麻酸（GLA，18:3n-6）具有抗炎活性。我们旨在确定AA本身是否能刺激RANKL表达，以及EPA、DHA和GLA是否能抑制成骨细胞中RANKL的表达。MC3T3-E1/4成骨样细胞在标准条件下与每种LCPUFA（5μg/ml）培养48小时。通过流式细胞术测量膜结合型RANKL的表达，通过酶联免疫吸附测定法测量OPG的分泌。在第二个实验中，用PGE2处理刺激MC3T3-E1/4细胞中RANKL的表达，并确定EPA、DHA和GLA对膜结合型RANKL表达和OPG分泌的影响。用AA或GLA处理后，RANKL阳性细胞的百分比高于对照组（p<0.05），但与环氧化酶抑制剂吲哚美辛共同处理后则不然。在标准细胞培养条件下，DHA和EPA对膜结合型RANKL的表达没有影响。AA处理的细胞中OPG的分泌较低（p<0.05），但GLA、EPA或DHA处理的细胞与对照组相比无显著差异。用前列腺素E2（PGE2）处理导致RANK-L阳性细胞的百分比增加（p<0.05），平均OPG分泌减少（p<0.05）。与单独用PGE2处理相比，PGE2与DHA或EPA共同处理后，RANKL阳性细胞的百分比显著降低。无论是否与EPA或DHA共同处理，用PGE2处理的细胞中平均OPG分泌仍低于对照组。本研究结果表明，GLA和AA的环氧化酶产物诱导MC3T3-E1/4细胞中膜结合型RANKL的表达。在标准条件下培养的细胞中，EPA和DHA对膜结合型RANKL的表达没有影响，然而EPA和DHA均抑制PGE2诱导的MC3T3-E1/4细胞中RANKL表达的增加。
