Poster AA5 - Oxidative Stress Induces Calcium signaling, NF-kB signaling, and Reactive Oxygen Species Production in a Human Chondrocyte Osteoarthritis Model

Introduction: Osteoarthritis, the most common form of arthritis, is a degenerative joint disease that affects 32.5 million adults. Osteoarthritis (OA) is characterized by the breakdown of the articular cartilage (AC). The AC is divided into four zones: superficial, middle, deep and the zone of calcified cartilage and is composed of specialized metabolic cells, chondrocytes. In the AC the oxygen levels range from 6% in the superficial zone to 1% in the deep zone. Under oxidative stress, the transcription factor NF-κB is upregulated due to elevated levels of reactive oxygen species (ROS). NF- κB is a family of transcription factors which modulate multiple biological processes, such as cell growth, apoptosis, and immune and inflammatory responses. The NF- κB pathway can be activated by ROS, pro-inflammatory cytokines, ECM degradation, and calcium [Ca2+] signaling, an essential universal secondary messenger that mediates cellular metabolic activity in chondrocytes. In OA, the dysregulation of [Ca2+] signaling and NF- κB can lead to an increase in ROS production and degradation of ECM protein. Since NF- κB is stimulated by ROS and [Ca2+], in this study we investigated the crosstalk between [Ca2+] and NF- κB in OA under different physiological oxygen (O2) levels. This work aims to ascertain further the mechanism that facilities the crosstalk between [Ca2+] and NF-κB, two therapeutic agents that can mitigate OA.

Materials and

Methods: C28/I2 human chondrocytes were cultured under hyperoxic (20% O2) and physioxic (5% O2) conditions. C28/I2 human chondrocytes were treated for 5 days at 37°C, 5%CO2 and varying O2 (5,20%). In this study NF- κB and Ca2+ were inhibited in chondrocytes to determine their role on ROS, protein concentration and cell viability. IL- 1β was used to induce the OA model. Chondrocytes were treated with 10ng/mL of IL-1β, 200μg/mL of Chondroitin sulfate (iNF- 𝝹B), and10μM of Verapamil (iCa2+) using the following treatment groups: A)Cells only (Negative control), B)IL-1β (Positive control), C)iNF- 𝝹B, D)iCa2+ , E)iCa2+ + iNF- 𝝹B, F)IL-1β + iNF- 𝝹B , G)IL-1β +iCa2+, H)iCa2++ iNF- 𝝹B + IL-1β. After the treatment, the following biochemical assays were performed to analyze the effect of inhibition of [Ca2+] and NF-κB on cell viability: MTT, Collagen. Western blotting was used to analyze protein content of NF-kB and upstream proteins: MMP-13 and IL-1β. In order to determine the effect of NF- κB and Ca2+ on ROS production, ROS concentration was evaluated via measuring oxidative stress using the Cell Rox Assay, which detects a panel of ROS: hydroxyl radical, superoxide anion, hydrogen peroxide, peroxynitrite, nitric oxide, tert-butyl hydroperoxide, hypochlorite anion.

Results, Conclusions, and Discussions: Results/Conclusion/Discussion
The results from this study shows promise in controlling ROS in OA by inhibiting [Ca2+] and NF- κB at different oxygen tensions. Under hyperoxic conditions, the Cell Rox assay confirmed that when NF- κB is inhibited, oxidative stress levels are evaluated, but when [Ca2+] is inhibited, oxidative stress activity is decreased. For cells treated with and without IL-1β and [Ca2+] and NF- κB inhibited, oxidative levels were increased. These preliminary results confirmed that both [Ca2+] and NF- κB signaling play a role in oxidative stress in OA (Figure 1 and Figure 2) and on cell viability. Under physioxic conditions similar results were observed. Collectively this study shows promise in controlling ROS in OA by inhibiting [Ca2+] and NF- κB. These preliminary results will help advance the understanding of oxidative stress when [Ca2+] and NF- κB is inhibited in OA. The knowledge gained from this study will contribute to potential therapeutic methods that can reduce the inflammatory response caused by Ca2+ and ROS in osteoarthritis.

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