E. coli lipopolysaccharide (LPS) stimulated murine microglial BV-2 cells and incubated with PNLA at a concentration of 50 µM, led to a reduction in the synthesis of nitric oxide (NO), IL-6 and TNF-α by 27–74% ^[1][17]. PNLA also significantly reduced NO and PGE2 levels by 35% in rat primary peritoneal macrophages following LPS stimulation ^[2][29]. In another study in HepG2 cells, PNLA (25 μM) decreased NO produced by 50% ^[3][25]. Chen et al. assessed the effects of PNLA in LPS stimulated THP-1 macrophages and found that the levels of IL-6, TNF-α, and PGE2 were reduced by 46%, 18%, and 87%, respectively ^[4][19]. Previously, PNLA has been shown to reduce PGE2 synthesis in LPS-stimulated murine RAW264.7 macrophages ^[5][16]. In both studies, the effects of PNLA were dose dependent. PNLA also reduced PGE1 or PGE2 generation in RAW264.7 macrophages stimulated with DGLA or AA, by 53% and 22%, respectively ^[5][16].

When RAW264.7 macrophages are stimulated with LPS, iNOS and COX-2 mRNA and protein expression are increased, which then increases the production of NO and PGE2. The NF-κB pathway is normally activated during the up-regulation of the iNOS and COX-2 genes. Chen et al. (2015) demonstrated that PNLA reduced the expression of iNOS and COX-2 caused by LPS by 55% and 10%, respectively ^[1][17]. This shows that, as has been demonstrated for n-3 PUFAs, PNLA may block the activation of NF-κB ^[6][7][1,28]. Huang et al. reported similar results using 50 μM PNLA treatment which reduced COX-2 and PGE2 release from LPS-stimulated RAW264.7 and rat primary peritoneal macrophages ^[2][29]. However, Chuang et al. ^[5][16] found an increase in COX-2 protein expression (12%) in murine macrophages, despite lowered PGE2 production. They suggested that the decrease in PGE2 production may be due to the competition of PNLA or its metabolites with AA as a substrate for COX-2 ^[5][16]. Consistently with findings in LPS stimulated RAW264.7 cells, PNLA prevented NF-κB activation ^[2][29], and Baker et al., demonstrated that PNLA (50 μM) decreased TNF-α stimulated NF-κB activity in EA.hy296 cells ^[8][20]. Additionally, ICAM-1, MCP-1, regulated on activation, normal T cell expressed and secreted (RANTES) production by TNF-stimulated EA.hy296 cells were all lowered by PNLA treatment, as well as adhesion to human THP-1 monocytes ^[8][9][20,27].

One study reported that 50 and 100 μM PNLA reduced PGE2 generation by 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated human MDA-MB-231 breast cancer cells with lower COX-2 mRNA and protein levels ^[10][22]. Further assays in these cells showed that n-3 (DHA) or PNLA reduced cell invasion by 30% and 25%, respectively, and both PNLA and DHA inhibited cell motility ^[10][22]. The experiments demonstrated that PNLA was the most potent of the FAs (PNLA, DHA or EPA) in reducing PGE2 production. PNLA reduction of TPA induced PGE2 production was dose-dependent ^[10][22]. Reduction of PGE2 by PNLA, DHA, or EPA appeared to be mediated in part by decreased COX-2 expression ^[10][22].

Several research on animals has also highlighted PNLA’s anti-inflammatory properties. PNLA reduced the release of IL-1β, IL-6, TNF-α, and PGE2 in the TPA-stimulated dorsal skin of a mouse model where PNLA or vehicle was applied topically to the shaved back skin. This was linked to a decrease in phosphorylation of p38- and c-Jun N-terminal kinase (JNK)-mitogen-activated protein kinase (MAPK), but not of extracellular signal-regulated kinase-MAPK ^[4][19]. Supernatants from dorsal skin tissue homogenates were also assessed ^[4][19]. Leukocyte, neutrophil, and macrophage infiltration were reported to be decreased after a single injection of PNLA (3 g) in mice with TPA-induced ear swelling ^[4][19]. The authors suggested that these effects may result from the direct regulation of cell signalling rather than the uptake of PNLA into the cells.

In rats, PNLA administered orally prior to carrageenan injection into the right foot reduced oedema ^[11][24]. PNLA applied topically to the feet also reduced fever. Additionally, after PNO was injected into the right hind paw, the reaction time to a hot plate increased by 1.4-fold ^[11][24]. This shows that COX-2 activity and PGE2 release may play a role in PNLA’s analgesic effects.

The effects of dietary PNO on immune function were examined in other animal studies. Rats were given P. koraiensis oil by Matsuo et al. and then intraperitoneal ovalbumin as an immunization ^[12][30]. Rats given safflower oil (a source of LA; n-6) or evening primrose oil (EPO; a source of GLA; n-6) had reduced CD4 T-cell counts, as well as spleen cells’ synthesis of Leukotriene B4 (LTB4) and immunoglobulins (Ig)-E and-G, compared to those given PNO ^[12][30] suggesting that PNLA may modulate the immune response. Park et al. found that PNO feeding led to an increase in concanavalin A-stimulated splenic lymphocyte proliferation and IL-1β production by splenocytes activated with LPS ^[13][31]. Lin et al. (2017) also reported that a low dose of P. koraiensis could enhance the immune function in vivo, with elevated quantities of IL-2, IL-4, IL-10, and interferon (IFN)-γ in mice ^[14][32]. These observations contradicted the reported anti-inflammatory effects of PNLA ^[1][5][16,17]. However, these immune-enhancing effects may be due to non-PNLA components in PNO or inherent differences in the models.

More recently, Takala et al. confirmed some of the anti-inflammatory actions of PNLA in healthy volunteers and RA patients ^[15][5]. In PBMCs from RA patients stimulated with LPS, PNLA decreased IL-6 and TNF-α release by 60%, whereas in HCs, it did so by 50% and 35%, respectively. LPS-induced PGE2 levels in such PBMCs from RA patients and HCs were also reduced substantially by PNLA. Regarding IL-1β, levels were reduced in supernatants of activated PBMCs of HCs while unaffected in RA patients after treatment with PNLA.

When intracellular levels of IL-6, TNF-α, IL-1β and IL-8 in CD14 monocytes isolated from active patients with RA were assessed, there was a reduction in TNF-α, IL-6 and IL-1β, all approximately by 25%, and reduced IL-8 level by 20% without an effect on MCP-1 expression ^[16][6]. PNLA also significantly reduced the proportion of LPS-activated CD14 monocytes in RA patients from 66.8% to 58.4% and 56.3% for 25 and 50 μM PNLA, respectively ^[16][6]. There was no correlation between the reduction in different cytokines expressing CD14 monocytes by PNLA and the clinical and laboratory features of RA or disease activity scoring.

Takala et al. also used whole genome transcriptomics and investigated both inflammatory models of PBMCs from RA patients and healthy volunteers, and purified CD14 monocytes from active RA patients that were activated with 100 ng/mL LPS following pre-incubation with 25 μM PNLA. The bioinformatic analysis showed that NF-κB, STATs and chemokine receptor 2 (CCR2) were inhibited relative to the same model which was vehicle-treated and LPS stimulated, while up-regulated expression of PPARs was observed ^[15][16][5,6]. PNLA also regulated the expression of metabolic genes, including pyruvate dehydrogenase kinase-4 (PDK4) and serpin family E member 1 (SERPINE1) that codes for plasminogen activator inhibitor-1 (PAI-1) from HCs, and fructose-bisphosphatase 1(FBP1), PDK4 and N-Myc downstream regulator 2 (NDRG2) from RA patients.