Eicosapentaenoic acid and docosahexaenoic acid moderate inflammation in porcine cartilage explants

Objective: To characterize the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and arachidonic acid (ARA) in various combinations on indices of inflammation in recombinant porcine IL-1 beta stimulated porcine articular cartilage explants. Methods: Cartilage was obtained from the humeral-ulnar joints of Yorkshire x Landrace market sized gilts. Explants were harvested from the humeral-ulnar joints within 8 h of slaughter. Explants were allocated to culture plates and cultured in 1 mL of Dulbecco’s Modified Eagle serum free medium for 24 h with 10% fetal bovine serum. At 48 and 72 h, 1 mL of treatment media containing fatty acids and 15 ng/mL of recombinant porcine IL-1 was added to each well. At 48, 72, and 96 h after cartilage was allocated to wells, media were removed from each well and reserved for analysis. Media were analyzed for proteoglycan, nitric oxide (NO), interleukin-6 and prostaglandin E2 concentrations. Results: In general, when EPA and/or DHA are supplemented to explants in combination with linoleic acid (LA) NO and prostaglandin E2 release is decreased. Explants treated with 25 μg/mL DHA released 53% less NO into the media than explants treated with the same level of EPA and 60% less than explants treated with LA alone. Conclusions: These data demonstrate that EPA and DHA are capable of modulating the inflammatory response on porcine articular cartilage in vitro.


Introduction
Lameness is a common clinical condition in livestock usually resulting in culling of the affected animal. In livestock of all breeds this can be a cause of serious financial loss, including replacement cost, and is a source of major welfare concerns. Joint lesions have been identified as one of the main causes for culling sows in Danish herds, affecting 24% of sows [1], while in the U.S. 15.2% of sows are culled for locomotor problems [2]. Over a 6-month period, production sows culled for lameness were analyzed postmortem. It was found that 31 of the 45 sows were diagnosed with either osteochondrosis or athrosis [3].
The pathogenesis of osteoarthritis (OA) is strongly mediated by the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α [4]. The inflammatory response in cartilage is regulated through cell signaling pathways that control gene expression of proteins responsible for the production of nitric oxide (NO), prostaglandin E 2 (PGE 2 ), and interleukin-6 (IL-6). Nitric oxide production plays a significant role in the development of cartilage degradation through inhibition of proteoglycan and collagen synthesis [5]. When cartilage is stimulated with IL-1β and/or tumor necrosis factor-α, increased NO production occurs. Mastbergen et al., [6] demonstrated that increased proteoglycan release from human articular cartilage is positively correlated with an increase in both NO and PGE 2 production.
Omega-3 fatty acids have the potential to be potent modulators of osteoarthritic factors due to their ability to reduce prostaglandin and NO production. Eicosanoids formed from eicosapentaenoic acid (EPA) are 10-to 100-fold less potent than those produced from arachidonic acid (ARA) and therefore are associated with a decreased inflammatory response [7]. When mouse stromal cells were incubated with either 40 µM ARA or doi: 10.7243/2054-3425-3-4 EPA, ARA increased PGE 2 production, while EPA treated cells did not differ from the control. Additionally, Razzak et al., [8] demonstrated that when murine macrophages were incubated with lipopolysaccharide and a cyclooxygenase-2 (COX-2) inhibitor, NO production increased; however, when the same cells were incubated with EPA or a COX-2 inhibitor and EPA, NO production was minimal.
Both EPA and docosahexaenoic acid (DHA) can reduce the inflammatory response and the production of NO and PGE 2 in other tissues. The objective of this study was to determine the concentration of EPA and DHA, both alone and in combination, necessary to reduce inflammatory mediators in porcine articular cartilage explants ex vivo relative to either linoleic acid (LA) or ARA.

Explant cultures
Yorkshire x Landrace cross gilts were slaughtered at market weight following the standard practices of the MSU Meat Laboratory. For experiments 1 and 2, front legs were collected from 8 gilts, and for experiments 3 and 4, front legs were collected from 6 gilts. Front legs were removed within 30 min of slaughter. Each experiment was conducted separately. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
The left and right humeral-ulnar joints were opened aseptically under sterile conditions and 50 cartilage disks were harvested with a 6 mm biopsy punch (Miltex, York, PA, USA) from the weight-bearing region of the articular surface of each gilt within 6 h of slaughter. Only visually normal cartilage was selected for biopsy.
After 24 h of conditioning media explants were washed twice with 1 mL of sterile phosphate buffered saline to remove FBS from the wells. Saline was completely removed from each well after each wash. Then, 1 mL of treatment media consisting of FBS free base media plus 1 μL/mL insulintransferrin-sodium selenite supplement (Roche Applied Science, Mannheim, Germany), 0.02 μg/mL thyroxine, and long chain polyunsaturated fatty acids (LCPUFA; Camen Chemical, Ann Arbor, MI, USA) were added to each well ( Table 1).
Each of the four experiments used a different combination of LCPUFA. Experiment 1 examined concentrations of EPA  (Figure 1). Between 24 and 48 h the explants were not exposed to IL-1β to serve as the unstimulated control. At 48, 72, and 96 h media were removed from each well and separated into two tubes for analysis. The first tube contained 10 μg/mL indomethacin to prevent further metabolism of PGE 2 and was stored at -20°C until analysis. The second tube did not contain any additives and was stored at 4°C for NO and proteoglycan analysis. Media were analyzed for proteoglycans (PG), NO, interleukin-6 (IL-6), and PGE 2 concentrations.

Proteoglycan analysis
Proteoglycan release into media was measured using the dimethylmethylene blue assay [10]. Proteoglycan content was determined by measuring sulfated glycosaminoglycan content using a chondroitin sulfate standard and expressed as µg PG/well. Absorbance at 530 nm with a correction at 590 nm was determined using a Spectramax 300 plate reader (Molecular Devices, Sunnyvale, CA, USA).

Nitric oxide analysis
Nitric oxide was measured indirectly by quantifying nitrite, a stable end-product of nitric oxide metabolism, in the media by using the Greiss reaction and a sodium nitrite standard [11]. Absorbance at 540 nm was determined using a Spectramax 300 plate reader (Molecular Devices, Sunnyvale, CA, USA). Results are expressed as µM of NO/well.

Prostaglandin E 2 analysis
Prostaglandin E 2 was measured in the media using a commercially available ELISA kit(EHPGE2; Thermo-Fisher Scientific, Pittsburgh, PA, USA) following the manufacturer's instructions. Media samples were diluted as needed in the provided assay buffer and analyzed. Absorbance at 405 nm with a correction at 580 nm was determined using a Spectramax 300 plate reader (Molecular Devices, Sunnyvale, CA, USA).

Interleukin-6 analysis
Interleukin-6 was measured in the media using a porcinespecific, commercially available ELISA kit (Porcine IL-6 Duoset; DY686, R & D Systems, Minneapolis, MN, USA) following the manufacturer's instructions. Samples were diluted as needed in the reagent diluent and analyzed in duplicate. Absorbance at 450 nm with a correction at 570 nm was determined using a Spectramax 300 plate reader (Molecular Devices, Sunnyvale, CA, USA).

Statistical analysis
Data were analyzed as a cumulative response following stimulation with IL-1, such that the response from media collected at 72 h was added to the response from media collected at 96 h. Data were analyzed using the mixed procedure of SAS (version 9.2; SAS Inst. Inc., Cary, NC, USA) with animal, treatment and time in the model. A random statement of animal nested in treatment was used. Differences between means were examined using the pdiff function. All data will be presented as lsmeans±SEM. P-values <0.05 will be discussed as significant while P-values <0.10 will be discussed as trends.

Results
Prior to IL-1β stimulation, release of proteoglycans, and production of nitric oxide and IL-6 did not exhibit a treatment effect for any of the 4 experiments. Additionally, experiments 1 and 3 did not exhibit a treatment effect for PGE 2 production prior to IL-1β stimulation. In experiment 2, explants treated with 12.5 and 25 µg/mL of DHA produced more PGE 2 prior to stimulation than those treated with 100 µg/mL of LNA, 6.25 µg/mL of DHA and 18.75 µg/mL of DHA (P<0.01; Table 3). In experiment 4, where ARA was used as the control fatty acid, prior to IL-1β stimulation the control produced 1.7 times more PGE 2 than the next highest treatment while the 25 µg/mL of DHA produced the least amount of PGE 2 (P<0.01; Table 5).
Cartilage explants treated with 0 to 25 µg/mL of EPA and stimulated with IL-1β exhibited a trend for an overall effect of  (Tables 3 and 4). Post IL-1β stimulation, explants with ARA as the control fatty acid and treated with 12.5 or 25 µg/mL of EPA released more proteoglycans than those treated with 12.5 and 25 µg/mL of DHA (Table 5). There was no difference in nitric oxide release from explants treated with varying concentrations of EPA and stimulated with IL-1β (P<0.05). Explants treated with DHA displayed an overall effect of treatment (P<0.01) and at 72 h the 12.5, 18.75 and 25 DHA treatments released less NO than the 100 LA or the 6.25 DHA ( Table 3). In explants treated with EPA and DHA alone or in combination NO release exhibited an overall effect of treatment (P<0.01) and all fatty acid treatments released less NO than the 100 LA treatment (P<0.05; Table 4 and Figure 2). Additionally, the 25 DHA treatment in experiment 3 released less NO than any other treatment (P<0.01). Explants with ARA as the control fatty acid exhibited a treatment effect for NO release with the 25 µg/mL of EPA treatment releasing the most NO (P<0.01; Table 5) while there was no difference between the control, 12.5 DHA and 25 DHA treatments.
Prostaglandin E 2 release was not affected by treatment with EPA and stimulation with IL-1β in experiment 1 (  Table 3. Cumulative proteoglycan (PG), nitric oxide (NO), prostaglandin E 2 (PGE 2 ), and interleukin-6 (IL-6) release from cartilage into media for experiment 2 when explants were treated with 0 to 25 µg/mL of docosahexaenoic acid (DHA) (  Table 3 and Figure 3) with no difference between the fatty acid treatments. Cumulative release of PGE 2 in experiment 4 was also affected by treatment (P<0.01) with the 25 EPA treatment producing the least amount of PGE 2 and the ARA control treatment producing the most (P<0.01; Table 5). Experiments 1, 2, and 3 did not have any effect on IL-6 release into the media regardless of fatty acid or concentration. However, experiment 4 demonstrated that cumulative release of IL-6 was affected by treatment (P<0.01) such that the 25 µg/mL of EPA treatment produced 50% more than the 12.5 EPA and DHA treatments and 86% more than the 100 ARA or 25 DHA treatments (P<0.01; Table 5).

Discussion and conclusion
This work investigated the anti-inflammatory effects of EPA and DHA by measuring inflammatory mediators and tissue degradation in a porcine explant model of inflammatory joint disease. The in vitro system of cartilage explants does mimic physiological conditions. The experiments were designed to determine the potential for omega-3 fatty acids to influence inflammatory molecules in porcine cartilage. Supplementation of EPA and/or DHA into porcine explant cultures consistently caused a reduction in NO and PGE 2 concentrations in the media; however, only EPA alone was able to alter PG release. In previous studies, Il-1β has been successfully used to induce cartilage degradation [12][13][14]. DHA was successful at altering chondrocyte metabolism to inhibit NO and PGE 2 production.   Table 5. Cumulative proteoglycan (PG), nitric oxide (NO), prostaglandin E 2 (PGE 2 ), and interleukin-6 (IL-6) release from cartilage into media for experiment 4 when explants were treated with 12.5 or 25 µg/mL of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ( Table 1) Table 1). NO exhibited a treatment effect (P<0.01). Bars with different letters differ at P<0.05.

Figure 3.
Cumulative PGE 2 release from cartilage into media for Experiment 3 when explants were treated with 0 to 25 µg/ mL of EPA and DHA alone or in combination ( Table 1). PGE 2 exhibited a treatment effect (P<0.01). Bars with different letters differ at P<0.05.
The reduction in NO is likely due to a concurrent reduction in PGE 2. Omega-3 fatty acids are known for their anti-inflammatory properties with a reduction in PGE 2 being a consistent finding regardless of tissue. Arachidonic acid is an omega-6 fatty acid, which generally initiates a pro-inflammatory response via its metabolism to PGE 2 in the cell. Arachidonic acid is the preferred substrate for COX-2; however, EPA can also be used. In vitro COX-2 has higher specificity for ARA than for EPA and preferentially oxygenates ARA when both fatty acids are present even if ARA is at a low substrate concentration [15]. In the current experiments, when only EPA or DHA and LA were present in the media, once all ARA was released from the cellular membrane any additional reactions would utilize EPA as a substrate for COX-2, resulting in the production of PGE 3 . Since the ELISA used in the current study to measure the concentration of PGE 2 in the media has minimal cross reactivity with PGE 3, the lower concentration of PGE 2 in the EPA-treated media may be due to the production of PGE 3 and not alterations in COX-2 expression. In experiment 4 when ARA was present in the media as the control fatty acid instead of LA the PGE 2 levels were 122 times higher after IL-1β stimulation than in the LA treatment of the other experiments. Addition of EPA or DHA at any level was sufficient to reduce PGE 2 production even with an unlimited supply of ARA; however the 25 EPA treatment was able to create the most substantial reduction of approximately 75%. In the experiments using LA as the control, the presence of either EPA or DHA at any concentration or combination reduced PGE 2 concentrations similarly. The data from experiment 4 suggests that EPA may be utilizing competitive inhibition with ARA while DHA may beregulating COX-2 at a molecular level to reduce the production of PGE 2 post-stimulation.
In addition to a reduction in PGE 2 , supplementation of EPA and/or DHA resulted in the reduction of NO production. Nitric oxide production plays a significant role in the development of cartilage degradation through inhibition of proteoglycan and collagen synthesis [5]. Mastbergen et al., [6] demonstrated that increased proteoglycan release from human articular cartilage is positively correlated with an increase in both NO and PGE 2 production. Cyclooxygenase-2, inducible nitric oxide synthase (iNOS), and fatty acids have a very complicated interrelationship that has been explored by many researchers in various cell types [8,[16][17][18]. For example, when ARA was added to osteoblast cell cultures an increase in iNOS gene expression occurred; however, when EPA was added to ARAtreated cells, EPA prevented an increase in iNOS expression [16]. This suggests that EPA may be a more potent regulator of iNOS expression than ARA. However, when ARA was used as the control fatty acid NO production increased when EPA was added to the wells. Little research has been conducted using chondrocytes and it is possible that other cell types respond differently under these conditions. Mouse macrophages treated with 60 μM of LA, LNA, ARA, EPA, or DHA and stimulated with lipopolysaccharide and interferon-γ to simulate a bacterial endotoxin demonstrated that both EPA and DHA inhibited NO production, while only DHA inhibited iNOS protein and mRNA expression [17]. This suggests that in vitro DHA is a more potent regulator of NO and PGE 2 release than EPA, which agrees with our results. This is likely due to the difference in the mechanisms by which each of these fatty acids regulate the inflammatory mediators at a molecular level. Razzak et al., [8] demonstrated that when murine macrophages were incubated with lipopolysaccharide (LPS) and a COX-2 inhibitor, NO production increased. However, when the same cells were incubated with EPA or a COX-2 inhibitor and EPA, NO production was minimal. The authors attributed this decrease in NO production to a series of omega-3 fatty acid related, multi-factorial events involving iNOS and COX-2.
When DHA was added in conjunction with LA similar results were found as when EPA was used. When either EPA or DHA was added in conjunction with ARA only the 25 DHA treatment was able to reduce NO production; all other treatments produced NO concentrations higher than the control. This suggests that EPA and DHA are not working through the same pathways. EPA, DHA, and LNA are potent inhibitors of COX-2 catalyzed prostaglandin biosynthesis in in vitro studies [18]. In activated macrophages the transcription factor NF-κB must be activated for iNOS gene expression. When macrophages were supplemented with DHA the amount of NF-κB binding decreased, thus inhibiting both iNOS expression and NO production [17]. In vascular endothelial cell cultures, when cells were exposed to 25 µmol/L DHA for 48 h before exposure to 10 ng/mL IL-1α, inhibition of COX-2 increased greater than 50% [19]. Following a series of experiments in which endothelial cells were incubated with 25 µmol/L DHA for 48 h the researchers concluded that DHA inhibits COX-2 expression through two mechanisms. First, DHA reducesprotein kinase C-εactivation thereby inhibiting COX-2 and iNOS gene expression. Secondly, DHA scavenges reactive oxygen species preventing the production of H 2 O 2 , which is necessary for nuclear factor kappa-light-chain-enhancer of B cells activation [19]. DHA should interface with IL-1 signaling pathways in a similar manner regardless of tissue type, therefore reduction in NF-κB activation can explain the decrease in both NO and PGE 2 production in DHA treated cell cultures since both iNOS and COX-2 require NF-κB activation.
The ability of EPA and DHA to alter inflammatory mediators depends highly on the incorporation of these fatty acids into the cell membrane. Dietary omega-3 fatty acids are preferentially incorporated into certain tissues. DHA content in plasma, liver, brain, and other organs is highly correlated to erythrocyte DHA levels [20]. A suitable biomarker that correlates with EPA and DHA status in articular tissue has not been identified. At the in vivo level the concern would be whether or not sufficient levels of EPA and/or DHA could be obtained in the cartilage to elicit the reduction in inflammatory mediators. Dietary supplementation of 1% protected fish oil increases the DHA concentration in the cartilage of sows 2.4 fold when compared to a control diet when fed for an average of 2 years [21]. Additionally, synovial fluid concentrations of EPA and DHA were higher in sows fed protected fish oil as 1% of their diet [21]. Although the aforementioned study measured increased concentrations of DHA in the cartilage following fish oil supplementation the changes may not have been substantial enough to be biologically significant.
These data provide evidence that both EPA and DHA are able to alter the production of NO and PGE 2 in porcine articular cartilage explants. Further research is necessary to more