Induction of Oxidative Stress and Inflammation in a Canine Chondrocyte Culture Model
ACVIM 2008
D.L. Dycus1; A.Y. Au2,3,4; M.W. Grzanna2; C.G. Frondoza1,2,3
1Mississippi State University, Mississippi State, MS, USA; 2Nutramax Laboratories, Inc., Edgewood, MD, USA; 3Johns Hopkins University, Baltimore, MD, USA; 4Syracuse University, Syracuse, NY, USA

Osteoarthritis (OA) is a chronic progressive disease affecting diarthrodial joints. It is characterized by cartilage breakdown resulting in pain and joint dysfunction. The etiology of OA is still unclear but evidence points to the critical role of pro-inflammatory mediators, particularly cytokines and prostaglandins. Cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) are suspected to induce reactive oxygen species (ROS). Overproduction of these molecules may be a cause of oxidative stress resulting in chondrocyte apoptosis. ROS generation may thus contribute to the pathogenesis of OA. However, little is known about the effect of oxidative stress on canine chondrocytes and its role in the development of OA. To address this problem, we determined whether oxidative stress can be induced by cytokines in a canine chondrocyte culture model. We evaluated whether the chondrocytes continue to proliferate and maintain the cartilage phenotype. In addition, we subjected chondrocytes to oxidative stress by activation with cytokines. Oxidative stress was assessed by measurement of reduced glutathione (GSH) levels and superoxide dismutase (SOD) enzyme activity. As positive control for inhibiting oxidative stress, we used silybin phosphatidylcholine complex (SPC). This agent has anti-oxidant, as well as anti-inflammatory properties, and is used for hepatoprotection (Marin®).

The phenotype expression was verified by immunostaining and Western blot analyses of aggrecan, as well as collagen types I and II. Chondrocytes (5x105 cells/well) were incubated with control media alone or SPC (298 ng/ml) for 24 hrs. The cultures were re-incubated with control media alone or the combination of IL-1β (10 ng/ml) and TNF-α (1 ng/ml) for 24 hrs or 48 hrs. The spent culture supernatant was measured for PGE-2 by ELISA. Cell lysates were assayed for GSH and SOD. Statistical significance was set at p <0.05 using one-way ANOVA, Tukey post-hoc test. Cultured canine chondrocytes continued to produce type II collagen and aggrecan. There was no detectable production of type I collagen. Activation significantly increased GSH levels by 37% and decreased SOD levels by 50%. Activation also significantly increased production of PGE-2, a pain producing substance. Pretreatment with SPC significantly reduced GSH levels, increased SOD activity, and decreased PGE-2 production. The present study demonstrates that the canine chondrocyte model maintains the cartilage phenotype and respond to cytokines. Here we demonstrate that oxidative stress and inflammation can be induced by cytokines and suppressed by SPC. The chondrocyte culture model helps clarify cellular mechanisms involved in oxidative stress and inflammation. Moreover, this model may be used to screen potential agents that minimize induction of oxidative stress and inflammation in cartilage.

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David Dycus

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