6.2  Molecular targets of PUFAs
There are several pharmacological studies suggesting molecular targets for the anti-inflammatory effects of n-3 PUFAs and their metabolites: PPAR-γ, GPR120, CMKLR1 (known as ChemR23), BLT1(leukotriene B4 receptor 1), GPR32 and ALX/FPR2 (Im, 2012). Thus, resolvins E1 and D1 exhibited a higher affinity for these receptors compared to EPA or DHA. Chem R23 and BLT1 are receptors of resolvin E1, while GPR32 and ALX/FPR2, bind to lipoxin A4 and resolvin D1 with high affinity. GPR120 was reported to be a receptor of EPA and DHA (EC50 ~ 1–10 μM), while ALX/FPR2 to annexin I and lipoxin A4 (Serhan and Petasis, 2011). Furthermore, some studies on GPR120 KO mice suggest that n-3 PUFAs that activate GPR120, interact with β-arrestin 2, and suppress NF-κB activation and macrophage-mediated inflammatory responses (Oh et al., 2010). However, it is important to highlight that the in vivo anti-inflammatory effects of n-3 PUFAs in humans are minor and might only occur at high n-3 PUFA levels, it was demonstrated in vitro that BSA-conjugated n-3 PUFA are incapable of activating GPR120 (Im, 2012).
DHA and EPA are weaker agonists of PPAR-γ (EC50 ~ 10–100 μM), while their oxidized metabolites (such as protectin D1) are much more potent (Yamamoto et al., 2005). Also, ALA or ARA has a similar potency to DHA or EPA for on PPAR-γ, and higher for PPAR-α (Calder, 2015). As PPAR-γ activation reduces inflammatory responses, via the NF-κB pathway, this mechanism could partially explain the anti-inflammatory effects of n-3 PUFAs. Furthermore, n-3 PUFAs were reported to suppress NF-κB activation in a PPAR-γ-independent manner by binding to TLR-4 under certain conditions (Im, 2012). Taking into account all these reports, it looks like three mechanisms are employed by n-3 PUFAs to suppress inflammatory signalling via NF-κB: (1) preventing NF-κB nuclear translocation via PPAR-γ activation, (2) interfering with membrane activation of NF-κB via TLR4 and (3) interaction with GPR120 initiating an anti-inflammatory signalling cascade (Calder, 2015).
Resolvin D1 is a potent agonist to GPR32 and ALX/FPR2 (EC50 = 8.8 pM and 1.2 pM), while Resolvin E1 strongly binds to Chem R23 (Kd = 4.5 nM), reducing IL-12 production (Krishnamoorthy et al., 2010) and is a partial agonist to BLT1, so it induces NF-κB activation via BLT1, inhibiting neutrophil migration (Arita et al., 2007). For the other resolvins, protectins or maresins, the molecular targets are not yet identified.
Isolated n-3 PUFAs and their bioactive mediators were extensively examined in animal models of colitis or arthritis or using specific transgenic models. n-3 PUFAs and RvD1, RvD5, PD1 and MaR1 administration proved effective in animal models of colitis, decreasing inflammation and chemically induced colonic damage. The beneficial effects are, in all cases, correlated with the reduction of ARA-derived mediators in the colonic mucosa (Bosco et al., 2013; Charpentier et al., 2018; Gobbetti et al., 2017; Marcon et al., 2013). Surprisingly, aspirin-triggered resolvin D1 (AT-RvD1) displayed a stronger anti-inflammatory effect than RvD2 in experimental colitis, through lipoxin A4 receptor (ALX) activation (Bento et al., 2011). Furthermore, n-3 PUFAs as fish oil has shown not just anti-inflammatory effects in peripheral tissues, but several beneficial effects in obesity-induced animal models, such as improved lipid profile, decreased hepatic steatosis and insulin resistance (Bargut et al., 2015; Pimentel et al., 2013). Indeed, beneficial effects of DHA and EPA in adipose tissue were reported in mice fed a high-fructose diet, including modulating pro- and anti-inflammatory markers and ameliorating adipocyte abnormalities. The effects were significantly higher for DHA compared to EPA (Bargut et al., 2017).
Additionally to anti-inflammatory effects, correlated with down-regulation of IL-6 and TNF-α expression in liver, n-3 PUFAs also exhibited triglyceridemia lowering effects in diabetic rats via modulation of PPAR-α (Devarshi et al., 2013; Ghadge et al., 2016). Additionally, Lee et al. demonstrated that a diet with a high n-6/n-3 PUFAs ratio (~9) induced dysbacteriosis of the gut microbiota in obesity-induced T2DM or high-fat-diet treated rats, while a low ratio (~3) enhanced blood glucose homeostasis (Lee et al., 2019). The outcomes of the most recent animal studies are summarized in Table 5 .
Table 5 Mittigating inflammation in animal model studies – effects of PUFAs.
Tested compound(s) Animal model Main anti-inflammatory findings References
n-3 PUFA (fish oil or mix fish and olive oil or flaxseed oil) TNBS colitis ↓IL-1β; IL-12p70; ↓IL-6; ↓TNFα; ↑PGE3,↑ TXB3; ↑ LTB5 Bosco et al. (2013)
TNBS colitis ↓colon iNOS, ↓COX-2 expression, ↓IL-6, ↓LTB4, ↓TNFα production Charpentier et al. (2018)
DSS colitis ↓TNF-α; ↓ COX-2; ↑anti-inflammatory PG; Sharma et al. (2019)
Carrageenan induced inflammation ↓TNF-α; ↓ IL-6 Zadeh-Ardabili and Rad (2019)
STZ- diabetic rats ↑ gene expression PPRγ; ↓ NF-κB activity Ghadge et al. (2016)
STZ- diabetic rats ↓TNF-α; ↓ IL-6 Lee et al. (2019)
STZ-NIC diabetic rats ↑ PPAR-α only by flaxseed oil; both (flaxseed oil and fish oil):↑ D5 and D6 desaturases; ↓TNF-α; ↓ IL-6; Devarshi et al. (2013)
STZ-NIC diabetic rats ↑ renal SOD-1; ↑ GPx-1 expression; ↑ CAT; ↓ renal AGEs formation ↓AGE protein expression; ↓ IL-6; ↓ NF-κB expression Jangale et al. (2016)
STZ-NIC diabetic rats ↓ IL-1β; ↓TNFα; ↓IL-6; ↓IL-17 A; ↓MDA Zhu et al. (2020)
Wistar rats ↓ IL-6; ↓ TNF-α; ↓IL- 10 receptor Pimentel et al. (2013)
C57BL/6 mice ↓ NF-κB expression; ↓ IL-6; ↓ TNF-α Bargut et al. (2015)
EPA monogliceride DSS colitis ↓PMN infiltration; ↓ NF-κB activity; ↓IL-1β; ↓TNF-α; ↓ IL-6; ↓expression of COX2 in colon Morin et al. (2016)
ALA TNBS colitis ↓ IP-8, ↓ LTB4, ↓ colon NF-κB DNA binding activity Hassan et al. (2010)
EPA vs. DHA high-fructose fed C57BL/6J mice ↓ TNF-alpha and IL-6 gene expressions; ↓MCP-1 pERK and NFkB protein expressions Bargut et al. (2017)
EPA free fatty acid APCMin/+ FAP model ↓ COX-2 expression; ↑ EPA tissue uptake; ↓ lipid peroxidation Fini et al. (2010)
CAC model C57BL/6J mouse ↓PGE2; ↑ EPA tissue uptake Piazzi et al. (2014)
Endogenous conversion n-6 into n-3 PUFA CAC modelFat-1 mouse ↓ COX-2 expression; ↓ NF-κB activity; ↓PGE2 Han et al. (2016)
Chronic arthritisFat-1 mouse vs WT mouse ↓ IL-17; ↑mRNA expression of Foxp3 (in Fat-1 mouse) Kim et al. (2018)
AT-RvD1 DSS colitis/TNBS colitis ↓PMN infiltration; ↓ NF-κB activity and mRNA expression; ↓IL-1β; ↓MIP-2; ↓mRNA expression of VCAM-1, ICAM-1 Bento et al. (2011)
Adjuvant-induced arthritis ↓TNF-α; IL-1β Lima-Garcia et al. (2011)
RvD2 DSS colitis ↓IL-1β; ↓ murine KC (IL-8 human homolog) Campbell et al. (2010)
TNBS colitis ↓PMN infiltration; ↓ NF-κB activity and mRNA expression; ↓IL-1β; ↓MIP-2; ↓mRNA expression of VCAM-1, ICAM-1 Bento et al. (2011)
RvE1 DSS colitis ↓PMN infiltration; ↓TNF-α; ↓mRNA expression of IL-6, TNFα, IL-1β Ishida et al. (2010)
Collagen-induced arthritis No statistical significant effect on TNF-α de Molon et al. (2019)
RvD5 DSS colitis ↓PMN infiltration; ↓TNF-α; ↓ IL-6; ↓IL-1β; Gobbetti et al. (2017)
MaR1 DSS colitis/TNBS colitis ↓PMN infiltration; ↓ NF-κB activity; ↓IL-1β; ↓TNF-α; ↓ IL-6; ↓mRNA expression of ICAM-1 Marcon et al. (2013)
PD1 DSS colitis ↓PMN infiltration; ↓IL-1β only partially Gobbetti et al. (2017)
CAC – colitis associated cancer; CAT – catalase DSS – dextran sulfate sodium; GPx – glutathione peroxidase NIC – Nicotinamide; PD – protectin; SOD – superoxide dismutase; STZ – Streptozotocin; TNBS – trinitrobenzene sulphonic acid; EPA – Eicosapentaenoic Acid; DHA – Docosahexaenoic Acid; TNF – α-Tumour Necrosis Factor alpha; LTB – Leukotriene, PPAR – peroxisome proliferator-activated receptor; COX – cyclooxygenase; AGE – advanced glycation end products; TXB – Thromboxane; PG – prostaglandins; ICAM – Intercellular Adhesion Molecule; VCAM – Vascular Cell Adhesion molecule; IL – Interleukin; NF-κB, nuclear factor kappa B; PMN – polymorphonuclear leukocyte; MIP-2 –macrophage inflammatory protein 2; IP-8 – Isoprostane-8; Foxp3 – Forkhead box P3; pERK – protein kinase RNA-like endoplasmic reticulum kinase; MCP-1 – Monocyte chemoattractant protein-1; murine KC – murine chemokine.