Babcock Tricia A, Novak Todd, Ong Evan, Jho David H, Helton W Scott, Espat N Joseph
Laboratories of Surgical Metabolism, University of Illinois at Chicago, 60612, USA.
J Surg Res. 2002 Sep;107(1):135-9. doi: 10.1006/jsre.2002.6498.
The role of omega-3 fatty acids (FA) as anti-inflammatory agents involves the inhibition of macrophage (Mphi) cytokine production, but the mechanisms involved are not well defined. The effects of omega-3 FA on the transcription and translation of cyclooxygenase-2 (COX-2), the production of prostaglandin E(2) (PGE(2)), and the production of interleukin-10 (IL-10) were investigated as potential mechanisms for the down-regulation of lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha production.
RAW 264.7 Mphi were incubated with Omegaven (10 mg% omega-3 FA), Lipovenos (10 mg% omega-6 FA), or DMEM for 4 h of pretreatment. The cells were then exposed to LPS (1 microg/ml) or medium alone for 3 h. COX-2 mRNA levels were determined by semi-quantitative reverse transcriptase polymerase chain reaction, and COX-2 protein levels were determined by Western blotting. The levels of PGE(2) and IL-10 proteins secreted into the medium were quantified using enzyme-linked immunosorbent assays.
Pretreatment with omega-3 FA increased Mphi COX-2 protein expression levels without altering the levels of COX-2 mRNA in response to LPS stimulation. In addition, pretreatment with omega-3 FA dramatically decreased the PGE(2) and IL-10 production induced by LPS, whereas pretreatment with an equivalent dose of omega-6 FA only resulted in a modest increase in PGE(2) and a slight decrease in IL-10 production compared to controls.
As COX-2 protein levels were increased without a change in COX-2 mRNA levels with omega-3 FA pretreatment, this suggested that omega-3 FA did not upregulate COX-2 at the transcriptional level. The omega-3 FA may instead posttranscriptionally stabilize existing COX-2 mRNA. The increased COX-2 expression may thus be explained by increased translation of COX-2 and/or decreased COX-2 degradation. The decreased PGE(2) production could be attributed to the replacement of Mphi membrane omega-6 FA substrates by omega-3 FA and the competitive inhibition of COX-2 enzyme by omega-3 FA. The reduction of active COX-2 product associated with an increase in COX-2 enzyme implies the existence of a negative feedback mechanism. Surprisingly, IL-10 production was decreased by omega-3 FA pretreatment, indicating that the reduced IL-10 inhibition of Mphi cytokine production was superceded by the other actions of omega-3 FA.
ω-3脂肪酸（FA）作为抗炎剂的作用涉及抑制巨噬细胞（Mphi）细胞因子的产生，但其相关机制尚未完全明确。研究了ω-3 FA对环氧合酶-2（COX-2）转录和翻译、前列腺素E2（PGE2）产生以及白细胞介素-10（IL-10）产生的影响，将其作为脂多糖（LPS）诱导的肿瘤坏死因子-α产生下调的潜在机制。
将RAW 264.7 Mphi与Omegaven（10 mg% ω-3 FA）、Lipovenos（10 mg% ω-6 FA）或DMEM孵育4小时进行预处理。然后将细胞暴露于LPS（1 μg/ml）或单独的培养基中3小时。通过半定量逆转录聚合酶链反应测定COX-2 mRNA水平，通过蛋白质印迹法测定COX-2蛋白水平。使用酶联免疫吸附测定法定量培养基中分泌的PGE2和IL-10蛋白水平。
用ω-3 FA预处理可增加Mphi COX-2蛋白表达水平，而不改变LPS刺激后COX-2 mRNA水平。此外，用ω-3 FA预处理可显著降低LPS诱导的PGE2和IL-10产生，而与对照相比，用等量的ω-6 FA预处理仅导致PGE2适度增加和IL-10产生略有减少。
由于用ω-3 FA预处理后COX-2蛋白水平增加而COX-2 mRNA水平未改变，这表明ω-3 FA在转录水平上并未上调COX-2。相反，ω-3 FA可能在转录后稳定现有的COX-2 mRNA。因此，COX-2表达增加可能是由于COX-2翻译增加和/或COX-2降解减少所致。PGE2产生减少可能归因于ω-3 FA取代了Mphi膜上的ω-6 FA底物以及ω-3 FA对COX-2酶的竞争性抑制。与COX-2酶增加相关的活性COX-2产物减少意味着存在负反馈机制。令人惊讶的是，用ω-3 FA预处理可降低IL-10产生，这表明ω-3 FA的其他作用取代了IL-10对Mphi细胞因子产生的抑制作用减弱。
