*4.1. In Vitro Results*

Using the BV-2 microglial cell line, Lu et al. have shown that DHA (30 μM) reduced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumor necrosis factor α (TNF-α) induced by interferon-γ (INF-γ), and antagonized IFN-γ induced NO production [21]. Inoue et al. investigated the implication of the sirtuin (SIRT) signaling in the anti-inflammatory response mediated by microglia, with BV-2 cells and with the MG6 line. They also found that DHA inhibited production of TNF-α and IL-6 induced by stimulation in two cellular models (BV-2 cells and MG6 microglia), but had no effect on IL-10 production induced by LPS. Results obtained with the MG6 microglia cells and a treatment with DHA (100 μM) + eicopentaenoic acid (EPA, 100 μM) suggest that the anti-inflammatory properties of DHA and/or EPA could be due to a SIRT1-mediated NF-κB (nuclear factor-kappa B) p65 deacetylation, through a positive feedback regulation of SIRT1 gene expression [22]. Others studies have shown that DHA (30 μM) decreased IL-1β [17,23] and IL-6 [23] expression in BV-2 cells stimulated with LPS. In primary cultures of mice microglial cells, treatment by DHA (20 μM to 80 μM) prior to LPS induction significantly attenuated LPS-induced NO and TNF-α release in a dose dependent manner. The inhibitory effect of DHA (20 μM) on TNF-α and NO release was also observed when cells were treated with myelin + IFNγ. In the same study, the authors observed that DHA probably modulates phenotypic polarization of microglia, with upregulation of M2-associated genes (including chemokine ligands (*CCL), CCL2, CCL17, Arg1*, and *IL-5*) and downregulation of M1-associated genes (including *IL-6, CCL5, TNF-α*, and *IL-1α*) [24]. With the human CHME3 microglial cells treated by DHA (0.1 to 1 μM) and exposed to amyloid-β42 (Aβ42), Hjorth et al. have shown a decrease in the levels of TNF-α and cluster of differentiation (CD) CD40 and CD86, as well as an increase in CD206 [25].

Furthermore, in rat glial primary cell cultures, DHA (100 μM) seems to have an active role in the regulation of the pro-inflammatory response. Indeed, pre-incubation of rat glial primary cell cultures with DHA before LPS/IFN-γ stimulation led to a decrease in the DNA binding activity of the activating protein-1 (AP1) and phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun. This pre-incubation also led to an increase of the expression of Nrf2 and HO-1. Using DHA before IFN-γ stimulation counteracted the elevation of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6, CCL2, and C-C chemokine receptor type 2 (CCR2) [26]. With macrophages, Cai et al. have demonstrated that 24 h of DHA (20 μM) treatment increased the expression of arginase-1 and TGF-β and suppressed production of CCL2, C-X-C motif chemokine ligand 10 (CXCL10), IL-1α, and TNF-α in primary macrophage cultures [43]. Reduction of the production of cytokines TNF-α and IL-6 could be induced through toll-like receptor-3 (TLR-3) and TLR-4 activation in EOC20 microglia cells treated by polyinosinic–polycytidylic acid (synthetic double-stranded RNA consisting of one strand of poly(inosinic acid) and one strand of poly(cytidyl acid) paired by wobble pairing, structurally similar to the double-stranded RNA of certain viruses, triggering an immune response) or 10 μg/mL of imiquimod (immune response modifier) [27].

Other forms of DHA have been used to decrease LPS-induced inflammation in BV-2 cells. Triglycerides forms of DHA (20 μM), or endogenous derivatives, have the capability to significantly reduce the production of IL-6 and TNF-α [29]. N-docosahexaenoyl dopamine (DHDA, 2 μM) decreases production of IL-6 and CCL-20 (macrophage-inflammatory protein-3α). Authors have also demonstrated that the level of prostaglandine E2 is reduced by using DHDA [30]. Synaptamide, an endogenous metabolite of DHA, leads to similar effects on inflammation. Using primary cultures of rat microglia and BV-2 cells, Park et al. found that synaptamide suppressed LPS-induced TNF-α and iNOS mRNA expression in a dose dependent manner. Furthermore, synaptamide decreased expression of IL-1β, IL-6, and CCL2. The authors suggest that the anti-inflammatory effects of synaptamide could be due to its fixation on the GPR110 receptor, as the synaptamide effects were suppressed by blocking synaptamide binding to it. This interaction could lead to an upregulation of cyclic adenosine 3 ,5 -monophosphate/protein kinase A (cAMP/PKA) signaling by inhibiting NF-κB p65 nuclear translocation [31,32]. Pro-inflammatory effects have also been demonstrated with resolvins (RvD) that are metabolites of DHA. RvD2 could counteract the mRNA pro-inflammatory upregulation induced by LPS (CD11b, ionized calcium binding adaptor molecule 1 (Iba-1), TNF-α, NF-κB p65, iNOS, IL-1, IL-18, IL-6, the nuclear factor of kappa light polypeptide gene enhancer in the B-cells inhibitor, alpha (IkBα), the the inhibitor of the nuclear factor kappa-B kinase subunit β (IKKβ), and IL-1β) and decrease the ROS production [44].

In their recent study, Chang et al. showed that the neuroprotective and antiinflammatory properties of DHA could attenuate effects of Japanese encephalitis virus (JEV). This virus, when it invades the central nervous system, causes a robust inflammatory response that leads to neuronal cell death. When infected with the JEV, primary neuron/glia rat primary cultures (i.e., neurons, astrocytes, and microglial cells), the authors measured an increase of neurotoxin cytokines production (NO, TNF-α, IL-1 β, prostaglandine E2 (PGE2), and ROS) that is counteracted by a DHA treatment (25 or 50 μM, 12 h) [28].

Taken together, these studies demonstrate the ability of DHA or its derivatives to limit the inflammatory effects, to be neuroprotective, and even to promote anti-viral effects in different types of cell cultures and in different models of inflammation.

Some data obtained in vitro are also observed in in vivo models.
