Effects of various dietary supplements on inflammatory processes in primary canine chondrocytes as a model of osteoarthritis

Abstract

The use of dietary supplements as an alternative treatment for joint-related pathologies such as osteoarthritis (OA) is increasing. However, there is little scientific evidence to support the intended use. The aim of this study was to evaluate the anti-inflammatory effects of creatine- and amino acid-based supplements in primary cultured canine chondrocytes (CnCs) as an in-vitro model of OA and compare the effects to more commonly used agents, such as the non-steroidal anti-inflammatory drug (NSAID), carprofen, and the joint supplement, glucosamine (GS). CnCs were stimulated with interleukin-1β (IL-1β) and the subsequent release of prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNFα) was measured using an enzyme-linked immunosorbent assay (ELISA). Changes in oxylipins were also assessed using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). All compounds examined were able to significantly reduce the release of PGE2 and TNFα and were associated with reductions in cyclooxygenase-2 (COX-2) expression and nuclear factor-kappaB (NF-κB) phosphorylation. The creatine- and amino acids-based supplements also altered the profile of oxylipins produced. All compounds examined were less effective at reducing the release of PGE2 than carprofen. Carprofen significantly increased release of TNFα from CnCs, however, while the other agents reduced TNFα release. This study suggests that creatine- and amino acid-based supplements may have a beneficial role in preventing inflammation within the joint and that further studies are warranted.

Figure 1

Effects of various treatments on cell viability. MTT assay to assess cell viability of primary cultured canine chondrocytes (CnCs) after 72-hour exposure to the inflammatory stimulant (IL-1β) and carprofen (A), CM (B), AlphaGEE (C), CRN (D), CHCL (E), or glucosamine (GS) (F). Values represent the mean ± SEM of 8 CnC monolayers.

Figure 2

Effects of carprofen and glucosamine (GS) on the release of inflammatory mediators from primary cultured canine chondrocytes (CnCs). Release of PGE2 (A and B) or TNFα (C and D) under control conditions and after stimulation with IL-1β with and without carprofen (A and C) or GS (B and D). Values represent the mean ± SEM of 4 monolayers per treatment group. *P < 0.05, **P < 0.01, ***P < 0.001, compared to IL-1β treatment group (2-way ANOVA), ****P < 0.0001.

Figure 3

Release of PGE2 from primary cultured canine chondrocytes (CnCs) under control conditions and after stimulation with IL-1β with and without CM (A), CHCL (B), CRN (C), or AlphaGEE (D). Values represent the mean ± SEM of 4 monolayers per treatment group. *P < 0.05, **P < 0.01, ***P < 0.001, compared to IL-1β treatment group (2-way ANOVA), ****P < 0.0001.

Figure 4

Western blot analysis of COX-2 (A) and NF-κB (B) in stimulated cells after 48 h for COX-2 and 1 h for NF-κB under the various treatment conditions. Values were expressed as mean ± SEM of 3 samples per treatment group. *P < 0.05, **P < 0.01, ***P < 0.001, compared to IL-1β treatment group (1-way ANOVA).

Figure 5

Release of TNFα from primary cultured canine chondrocytes (CnCs) under control conditions and after stimulation with IL-1β with and without CM (A), CHCL (B), CRN (C), or AlphaGEE (D). Values represent the mean ± SEM of 4 monolayers per treatment group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared to IL-1β treatment group (2-way ANOVA).

Figure 6

Levels of 12-HETE (A), 18-HEPE (B), dihomo PGF2α (C), and 20-HDoHE (D) in stimulated cells after 24 h under the various treatment conditions. Values were expressed as mean ± SEM of 4 samples per treatment group. Asterisks above the bars indicate significance compared to IL-1β treatment group; square brackets indicate significance compared to control (untreated group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns — nonsignificant (1-way ANOVA).