(M-496) 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 O₂ levels. This work aims to ascertain the potential mechanism that facilities the role of oxidative stress on chondrocyte cells and [Ca²⁺] and NF-κB, two potential therapeutic agents that can mitigate OA.




Materials and Methods::
C28/I2 human chondrocytes were cultured under hyperoxic (20% O₂) conditions.C28/I2 human chondrocytes were treated up to 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 and Live/Dead Assay) and DNA (Hoechst Assay). 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::
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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