Background/Purpose:

Background: Effective treatments for Osteoarthritis (OA) are not available. In aging-related diseases, including OA, failure of cellular homeostasis mechanisms, such as autophagy can cause extracellular matrix destruction and cell death. Chondrocytes are essential for the maintenance of cartilage integrity. With aging, chondrocyte function is diminished, contributing to a cellular senescence phenotype often observed in OA chondrocytes. In addition, a defect in autophagy is observed in both aging and cartilage degeneration. The objective of this study was to identify anti-senescence and pro-autophagy molecules by a cell-based high-throughput screening (HTS) in human chondrocytes.

Methods:

Methods: To induce cellular senescence or reduced autophagy, immortalized human chondrocytes, TC28a2 were seeded in 384 well plates, and treated with IL-6 (20ng/ml) for 72 or 18 hours, respectively. Then, chondrocytes were incubated with Prestwick Chemical Library (1120 approved drugs) at 10μM for 72 hours. To identify anti-senescence hits, nuclei was stained with Hoechst 33342 (2,5µg/ml), while β–galactosidase subcellular structures was stained by using ImaGene Green C₁₂FDG substrate (30μM). To evaluate autophagic flux, a reporter cell line was generated by lentiviral transfection of pBABE-mCherry-EGFP-LC3 plasmid in TC28a2 chondrocytes. Plates were imaged by using Operetta® High Content Screening (HCS) System. Relative intensity of C₁₂FDG in cytoplasm and relative spot intensity of cytoplasm were determined to quantitate β–galactosidase activity and autophagic flux respectively. For compound validation, senescence markers, autophagic flux, inflammation and apoptosis were evaluated in human chondrocytes. Moreover, cartilage degradation and autophagy were evaluated by safranin O staining and immunohistochemistry in human cartilage explants.

Results:

Results: A primary screening was performed to identify anti-senescence compounds by measurement of senescence-associated β-galactosidase activity. 252 compounds with anti-senescence effects were identified. The anti-senescence hits were analyzed by monitoring autophagic flux. 26 compounds with both anti-senescence and pro-autophagy effects were selected. Then, one compound was selected for further validation. The compound reduced senescence (p <0.001) and increased autophagic flux (p <0.0001) in response to IL-6. Western Blot analysis showed protective effect against defective autophagy, senescence and inflammation. Interestingly, this protective effect was partially mediated by mTOR inhibition, a proposed mechanism to prevent cartilage aging. Moreover, the selected compound conferred protection against cell death by apoptosis and reduced nitric oxide (NO) production into supernatants (p < 0.05) and cartilage degradation in response to IL-1β.

Conclusion:

Conclusions: These observations provide a unique opportunity to study cartilage aging with the objective to explore the therapeutic potential of pharmacological prevention of chondrocyte senescence and autophagy as a strategy to slow or reverse aging-associated changes, prevent the onset of OA and provide benefits for its clinical management.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeting-cartilage-aging-as-osteoarthritis-therapeutics-by-drug-repurposing/