Table 1.

Biological effects of n-3 PUFAs in in vitro models of OA. Up arrows refer to increase and down arrows to decrease.

Molecule	In Vitro Model	Treatment	Main Effects	Specific Outcomes	Ref.
DHA vs. palmitic acid (PA, SFA), oleic acid (OLA, MUFA) and linoleic acid (LA, n-6 PUFAs)	SW1353 chondrosarcoma cells ± LPS (1000 ng/mL)	6.25, 25, 100 μM	Reduced pyroptosis-dependent inflammatory response due to inhibition of TLR4/NF-κB and NLRP3/caspase-1/GSDMD signaling.	DHA, OLA ▪↓ TLR4; ▪↓ pNF-κB; ▪↓ NLRP3, caspase 1, GSDMD; ▪↓ IL-1β; ▪↑ Coll-II. PA, LA ▪↑ TLR4; ▪↑ pNF-κB.	[60]
DHA	SW1353 chondrosarcoma ± IL-1β (5–100 ng/mL)	3.125, 6.25, 12.5, 25, 50 µg/mL	Reduced inflammatory-dependent catabolic response by inhibition of p38 MAPK-dependent signaling.	▪↓ MMP-13; ▪↓ p38 phosphorylation.	[106]
DHA	Human chondrocytes ± TNF-α (50 ng/mL)	25 μM for 48 h	Reduced inflammatory and catabolic response through downregulating Wnt/β-catenin and NF-κB signaling pathways.	▪↓ CCL2, COX2, IL-1β; ▪↓ MMP-13; ▪↓ NF-κB p65 and β-catenin nucleus translocation.	[65]
DHA	Murine chondrocytes ± IL-1β (10 ng/mL)	5, 10, 25 μM, pre-treatment for 24 h	Reduced inflammatory and catabolic response by inhibition of NF-κB p65 and β-catenin by Malat-1.	▪↓ nitrites, iNOS, COX2; ▪↓ MMP-13; ▪↑ Coll II; ▪↓ NF-κB p65 subunit nucleus translocation and IκBα phosphorylation; ▪↓ Malat 1 (lncRNA).	[111]
EPA/DHA/ALA	Bovine chondrocytes ± IL-1α (10 ng/mL)	2.5, 5, 10, 20, 30 μg/mL for 8 h	Reduced inflammatory and catabolic response, efficiency EPA > DHA > ALA (modulation of more targets, modulation at low concentration).	EPA: ▪↓ ADAMTS-4/-5, MMP-3/-13; ▪↓ COX-2, IL-1α, IL-1 β, TNF-α; ▪↓ COX-1 not affected. DHA: ▪↓ ADAMTS-4; ▪↓ COX-2, IL-1α, IL-1 β, TNF-α; ▪↓ COX-1 not affected. ALA: ▪↓ ADAMTS-4/-5; ▪↓ COX-2, IL-1α, TNF-α; ▪↓ COX-1 not affected.	[112]
EPA/DHA vs. AA	Canine chondrocytes ± IL-1β (10 ng/mL)	10 μM for 8 days	Reduced inflammatory and catabolic response, efficiency EPA > DHA AA positively modulates some markers of inflammation.	EPA: ▪↓ iNOS, NO (EPA); ▪no modulations of MMP-3, 13, ADAMTS4,-5, TIMP-2, COX-2, PGE2. DHA: ▪ No modulations of all targets. AA: ▪↓ iNOS, NO; ▪↑ PGE2, ADAMTS5; ▪↓ MMP-3; ▪no modulations of MMP-13, ADAMTS4, TIMP-2, COX-2.	[120]
EPA/DHA	Bovine cartilage explants ± IL-1β (10 ng/mL)	0.1, 1, 10 μM EPA and/or DHA for 5 days	Reduce cytokine-induced articular cartilage degradation. Efficiency EPA > DHA at long term.	▪↓ GAG loss; ▪↓ ADAMTS 4-/-5; MMP-3/-13, COX-2.	[113]
Green-lipped mussel (GLM) (abundant in DHA)	Human OA chondrocytes ± IL-1β (20 ng/mL)	10, 100, 250 μg/mL	Reduced inflammatory response and necroptosis.	▪↓ RIPK1, RIPK3 and MLKL (necroptotic markers); ▪↓ IL-1β, IL-6; ▪↓ iNOS; ▪↓ MMP-3, -13; ▪↓ NF-KB;	[115]
EPA	Normal human knee chondrocytes ± SNP (1 mM)	10, 30, 50 µg/mL for 8 h	Reduced inflammatory-dependent catabolic response and apoptosis by inhibition of MAPK signaling.	▪↓phosphorylation of p38 MAPK and p53; ▪↓caspase 3 and poly (ADP-ribose) polymerase cleavage; ▪↓apoptosis; ▪↓ MMP-3, -13.	[114]
RvD1 (DHA metabolite)	Human OA chondrocytes ± IL-1β (1 ng/mL)	0–10 μM	Reduced inflammation by inactivation of NF-κB/p65, p38/MAPK and JNK1/2.	▪↓ COX-2, PGE2; ▪↓ iNOs, NO; ▪↓ MMP-13; ▪↓ apoptosis; ▪↓ NF-κB/p65; ▪↓ p38/MAPK; ▪↓ JNK1/2.	[117]
LA (n-6 PUFA) vs. ALA (n-3 PUFA)	Chondrocytes at high density ± IL-1β (100 pg/mL)	LA/ALA (1:1, 2:1, 4:1, 6:1, 8:1, 10:1) total amount 50 μg/mL for 1 h	Anti-catabolic effect, most effective ratio was 1:1, and 10:1 was not effective.	▪↓ MMP13.	[121]
PDX	Rat chondrocytes ± IL-1β (10 ng/mL)	Pretreatment, 0.5, 1, 2, 4 μM	Inhibited inflammatory responses through the activation of AMPK and inhibition of NF-κB signaling pathway.	▪↑ Coll II; ▪↓ MMP-3, -13, ADAMTS4; ▪↓ iNOS, COX-2, NO, PGE2; ▪↓ NF-κB p65, IκBα phosphorylation; ▪↑ IκBα. ▪↑ AMPK; ▪Phosphorylation; ▪↓ Nuclear translocation of NF-κB p65.	[118]
Conjugated linoleic acids + AA or EPA linoleic acid (LA) + AA or EPA	Human OA chondrocytes	10 μM	Anti-inflammatory.	▪↓ PGE2, NO; ▪LA/EPA: lowest PGE2 production; ▪LA/AA: lowest NO production.	[131]
DHA	Murine bone marrow mesenchymal stromal cells during chondrogenesis ± IL-1β (10 ng/mL)	25 μM, pre-treatment for 21 days	Rescued IL-1β-impaired chondrogenesis by NF-κB signaling inhibition by Malat-1.	▪↑ chondrogenesis; ▪↑ COLL2, Acan, GAG, Sox-9; ▪↓ MMP-13, COLLX; ▪↓ NF-κB p65 subunit nucleus translocation and IκBα; ▪↓ Malat 1 (lncRNA).	[111]
DHA	293 T cells ± TNFα (10 ng/mL)	5, 10, 25 μM, for 24 h	Downregulated Wnt/β-catenin and NF-κB signaling.	Luciferase activities of reporter vector harbouring Wnt/β-catenin (TOPFlash) and NF-κB response element (NF-κB RE) showed a declining gradient.	[111]
13- and 16-H- DHEA and DHEA (endocannabinoid from DHA)	RAW264.7 macrophage ± LPS (1 µg/mL)	2.5–5 μM	Anti-inflammatory effects less pronounced compared to DHEA.	DHEA ▪↓ NO and IL-6; ▪↓ TNF-α, IL-1β; ▪↓ PGE2, PGD2. 13-H-DHEA ▪↓ TNF-α, IL-1β; ▪↓ InhbA, Ifit1; ▪↑ PPbp, Serpinb2; ▪↓ DHA. 16-H-DHEA ▪↓ IL-1 Ra; ▪↓ InhbA, Ifit1 (downstream to TLR4 activation); ▪↑ PPbp, Serpinb2; ▪↓ DHA.	[127]
13S,14S-epoxy- DHA (precursor of MaR-1)	Human macrophages (M1 and M2 subsets)	10 nM	Reduced inflammation and switching from M1 to M2 phenotype.	▪Higher conversion to MaR-1 in M2; ▪↓ LTB4 biosynthesis by LTA4; ▪↓ arachidonic acid conversion by hm12-LOX; ▪↓ CD54, CD80 (M1); ▪↑ CD163, CD206 (M2).	[128]
DHA/EPA	Equine synoviocytes ± IL-1β (5 ng/mL)	25–50 μM for 24 h	Reduced inflammatory and catabolic response due to increased integration within cell membranes and production of oxylipids (specialized pro-resolving mediators).	DHA ▪↑ Content in the cell membrane; ▪↑ RvD1, RvD2, Mar-1, Mar-2, 19,20DiHDPE, 19,20 EpDPE, 17HDoHE, 10,17 DiHDoHE; ▪↓ ADAMTS4, MMP-1, -13; IL-1β, IL-6, COX-2; ▪no significant reduction in TNF-α, MMP-3. EPA ▪↑ Content in cell membrane; ▪↓ ADAMTS4, MMP-1, -13; IL-1β, IL-6, COX-2.	[108]
RvD-1, -2, MaR-1, PDX	Equine synovial fibroblasts ± IL-1β (5 ng/mL)	Pre-treatment with 25 μM and 50 μM EPA and DHA	Reduced inflammatory and catabolic response due to increased integration within cells membrane.	▪↓ ADAMTS4, MMP-1, -13; IL-1β, IL-6, COX-2.	[108]
RvD1	Human OA fibroblast-like synoviocytes (FLs)	20, 50, 100, 200 nM	Reduced inflammatory and catabolic response due to Hippo-YAP signaling pathway activation.	▪↓ MMP13; ▪↓ IL-1β; ▪↑ YAP phosphorylation; ▪↓ YAP; ▪↓ Proliferation; ▪(G2 cell cycle arrest; ▪↓Cyclin D1, cyclin B1, PCNA; ▪↑ p53.	[123]
MaR-1 (DHA metabolite)	Rat FLSs ± IL-1β (10 ng/mL)	Pretreatment, 10, 100, 1000 nM, 1 h	Anti-inflammatory and anti-catabolic effect by stimulation of PI3k/Akt pathway and inhibition of NF-κB p65 pathway.	▪↓ MMP13; ▪↑ PI3k, Akt phosphorylation; ▪↓ pNF-κB p65.	[124]
RvD1/RvE1/MaR1	OA synovial fibroblast ± TNF-α (10 ng/mL)	Pretreatment, 100 nM	No anti-inflammatory effect.	▪No inhibition of COX-2, mPGES-1, IL-6, MMP-3.	[122]