In the present study, we found that peroxidized products of EPA/DHA, but not of unoxidized EPA/DHA, inhibited iNOS induction, followed by the reduction of NO production in proinflammatory cytokine-stimulated hepatocytes (Figures and , and ). MDA itself did not inhibit NO production. The inhibitory effects on NO production by peroxidized EPA/DHA were dependent on their peroxidation levels (expressed as MDA equivalent) (). Thus, it seems likely that peroxidized products of EPA/DHA, but not MDA, are involved in the inhibition of NO production. Peroxidized EPA/DHA markedly inhibited iNOS protein and its mRNA expression at earlier time periods, but less effectively thereafter (Figures and ). This observation is probably in part because of the reduction of peroxidized products by oxidoreductases such as super oxide dismutase in hepatocytes.
It is known that the induction of iNOS gene expression is regulated by the iNOS promoter transactivation and post-transcriptional modifications [32
]. EMSA revealed that peroxidized EPA/DHA inhibited NF-κ
B activation (). NF-κ
B typically exists in the form of p50/65 heterodimers attached to its inhibitory proteins (Iκ
, and Iκ
) in the cytoplasm of cells. The activation of NF-κ
B involves (i) proteolytic degradation of Iκ
Bs in proteasome after the phosphorylation by Iκ
B kinase, (ii) the translocation of NF-κ
B to the nucleus, and (iii) its binding to the promoter κ
B site [16
]. However, peroxidized EPA/DHA did not affect the phosphorylation of Iκ
(), and the degradation and recovery of Iκ
We previously found that the upregulation of IL-1RI through the activation of PI3K/Akt is essential for iNOS induction in addition to Iκ
B pathway [31
]. However, peroxidized EPA/DHA had no effect on the upregulation of IL-1RI protein, although they tended to reduce the levels of IL-1RI mRNA expression (data not shown). Thus, the inhibitory effect of peroxidized EPA/DHA is presumably NF-κ
B-dependent but IL-1RI-independent mechanism. The inhibitory effect of DHA on iNOS induction seems to be superior than that of EPA due to a higher susceptibility of DHA to peroxidation.
Regarding the posttranscriptional modifications, the 3′-UTR of the iNOS mRNA in rats has six AREs (AUUU(U)A), which are associated with ARE-binding proteins such as HuR and heterogeneous nuclear ribonucleoproteins L/I (PTB), thus contributing to the stabilization of the mRNA [34
]. Recently, we found that the antisense strand corresponding to the 3′-UTR of iNOS mRNA is transcribed from the iNOS gene, and that the iNOS mRNA antisense-transcript plays a key role in stabilizing the iNOS mRNA by interacting with the 3′-UTR- and ARE-binding proteins [33
]. Peroxidized EPA/DHA reduced the levels of the antisense-transcript expression (Figures and ). Taken together, peroxidized n-3 PUFAs including EPA/DHA inhibit NF-κ
B activation and iNOS antisense transcript expression, leading to the blockades of iNOS mRNA synthesis and its stabilization, resulting in the suppression of iNOS gene induction.
When we orally take n-3 PUFA-containing foods, such as grilled or boiled fish, peroxidized products of n-3 PUFAs are significant levels in human serum [8
] as similar as those used in this study. Although we agree with the common view that lipid peroxidized products are considered harmful to living body, it cannot negate the possibility that peroxidized products as well as n-3 PUFAs themselves have a variety of biological activities, such as anti-inflammatory and anticancer effects.
As mentioned before, Sethi et al. reported that anti-inflammatory effects of n-3 PUFAs were due to their peroxidized products [6
]. Peroxidized EPA inhibited NF-κ
B activation through PPARα
activation using PPARα
knockout mice [28
] and also inhibited the expression of chemokines in cytokine-stimulated endothelial cells through the inhibition of NF-κ
B activation via PPARα
-dependent pathway but not via the phosphorylation and degradation of Iκ
]. Similar with these observations, peroxidized EPA/DHA inhibited NF-κ
B activation not via phosphorylation and degradation of Iκ
in our IL-1β
-stimulated hepatocytes, while a participation of PPARα
remains unclear in the present study.
Accumulating evidence indicates that NO and iNOS are concerned with cancer development. There are many reports that NO can promote cancer development through induction of DNA damage, increased angiogenesis and blood flow, prevention of apoptotic cell death, and suppression of the immune system, although there are reports that NO can suppress cancer development [35
]. The anticancer effect of PUFAs, including our previous reports, may be in part due to NO suppression by peroxidized products of the PUFAs. Further investigation is needed to examine whether NO suppression by peroxidized n-3 PUFAs contributes to anticancer effect.
In conclusion, peroxidized products of n-3 PUFAs suppress iNOS induction and NO production in peroxidation-dependent manners. Results suggest that peroxidized products of n-3 PUFAs are in part involved in the anti-inflammatory effects. Further, the equation from the correlation between peroxidation level and NO suppression suggests that our in vitro NO production model in hepatocytes may be a candidate for biological quantification of lipid peroxidation.