Data_Sheet_1_Docosahexaenoic Acid Inhibits Inflammation-Induced Osteoclast Formation and Bone Resorption in vivo Through GPR120 by Inhibiting TNF-α Production in Macrophages and Directly Inhibiting Osteoclast Formation.PDF

Docosahexaenoic acid (DHA) is an n-3 fatty acid that is an important structural component of the cell membrane. DHA exerts potent anti-inflammatory effects through G protein-coupled receptor 120 (GPR120), which is a functional receptor for n-3 fatty acids. DHA also regulates osteoclast formation and function. However, no studies have investigated the effect of DHA on inflammation-induced osteoclast formation in vivo. In the present study, we investigated whether DHA influences osteoclast formation, bone resorption and the expression of osteoclast-associated cytokines during lipopolysaccharide (LPS)-induced inflammation in vivo, and then we elucidated the underlying mechanisms by using in vitro experiments. In vitro experiments revealed both receptor activator of NF-kB ligand (RANKL)- and tumor necrosis factor-α (TNF-α)-induced osteoclast formation was inhibited by DHA. Supracalvarial administration of LPS with or without DHA was carried out for 5 days and then the number of osteoclasts, ratio of bone resorption pits and the level of type I collagen C-terminal cross-linked telopeptide were measured. All measurements were significantly lower in LPS+DHA-co-administered mice than LPS-administered mice. However, this DHA-induced inhibition was not observed in LPS-, DHA-, and selective GPR120 antagonist AH7614-co-administered mice. Furthermore, the expression of RANKL and TNF-α mRNAs was lower in the LPS+DHA-co-administered group than in the LPS-administered group in vivo. TNF-α mRNA levels were decreased in macrophages co-treated with LPS+DHA compared with cells treated with LPS in vitro. In contrast, RANKL mRNA expression levels from osteoblasts co-treated with DHA and LPS in vitro were equal to that in cells treated with LPS alone. Finally, the inhibitory effects of DHA on osteoclast formation in vitro were not observed by using osteoclast precursors from GPR120-deficient mice, and inhibition of LPS-induced osteoclast formation and bone resorption by DHA in vivo was not observed in GPR120-deficient mice. These results suggest that DHA inhibits LPS-induced osteoclast formation and bone resorption in vivo via GPR120 by inhibiting LPS-induced TNF-α production in macrophages along with direct inhibition of osteoclast formation.

二十二碳六烯酸（Docosahexaenoic Acid，DHA）是一种n-3脂肪酸（n-3 fatty acid），是细胞膜（cell membrane）的重要结构组成成分。DHA可通过G蛋白偶联受体120（G protein-coupled receptor 120，GPR120）发挥强效抗炎作用，而GPR120是n-3脂肪酸的功能性受体。DHA还可调控破骨细胞的生成与功能。然而，目前尚无研究探讨DHA在体内对炎症诱导的破骨细胞生成的影响。本研究旨在探究DHA在体内脂多糖（lipopolysaccharide，LPS）诱导的炎症过程中，是否会影响破骨细胞生成、骨吸收以及破骨细胞相关细胞因子的表达，并通过体外实验阐明其潜在作用机制。体外实验结果显示，DHA可抑制核因子κB受体活化因子配体（receptor activator of NF-kB ligand，RANKL）及肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）诱导的破骨细胞生成。我们对小鼠进行为期5天的颅顶给药（supracalvarial administration），分别给予LPS单独处理或LPS联合DHA处理，随后检测破骨细胞数量、骨吸收陷窝比例以及I型胶原C端交联肽（type I collagen C-terminal cross-linked telopeptide）的水平。与仅接受LPS处理的小鼠相比，LPS联合DHA共处理的小鼠上述所有检测指标均显著降低。然而，在同时给予LPS、DHA及选择性GPR120拮抗剂AH7614的小鼠中，并未观察到DHA介导的抑制作用。进一步研究发现，体内LPS联合DHA共处理组的RANKL和TNF-α mRNA表达水平，均低于仅接受LPS处理的组别。体外实验中，与仅接受LPS处理的巨噬细胞相比，经LPS联合DHA共处理的巨噬细胞的TNF-α mRNA水平有所降低。与之相反，经DHA与LPS共处理的成骨细胞，其RANKL mRNA表达水平与仅接受LPS处理的成骨细胞并无差异。最后，在取自GPR120基因敲除（GPR120-deficient）小鼠的破骨细胞前体（osteoclast precursors）实验中，并未观察到DHA对破骨细胞生成的体外抑制作用；而在GPR120基因敲除小鼠体内，也未观察到DHA对LPS诱导的破骨细胞生成及骨吸收的抑制效果。上述研究结果表明，DHA可通过GPR120通路，一方面抑制巨噬细胞中LPS诱导的TNF-α生成，另一方面直接抑制破骨细胞生成，从而在体内抑制LPS诱导的破骨细胞生成与骨吸收。