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Background Chondrosenescence, chondroptosis and autophagy contribute to cell death and tissue damage in OA. The mitochondria are related with these three process implicated in the cartilage degeneration. Mitochondrial dysfunction is well documented in OA and has the capacity to promote abnormalities in chondrocyte viability contributing to cartilage degeneration. Cybrids are optimal cellular models to study the mitochondrial function, since they carry different mitochondrial variants with the same nuclear background, therefore, excluding the variations because of nuclear genome. Objectives The aim of this work is to study the role of human OA mitochondria in the cellular senescence, apoptosis and autophagy. Methods Cybrids were developed using 143B.TK- Rho-0 cell line (nuclear donor) and platelets (mitochondrial donors) from healthy (N) and knee OA donors. Senescence level was measured by real-time PCR method The percentage of mitochondrial depolarized was evaluated incubating cells with DilC1(5) in presence of FCCP 1μM using Flow Cytometer. The percentage of apoptotic cells was measured by Flow Cytometry using Annexin-V. Autophagy was evaluated through the developed of Microtubule-associated protein 1A/1B-light chain 3 (LC3) WB. Appropriate statistical analyses were performed with GraphPad Prism v6. Results The gene expression corresponding to senescence marker protein (SMP30) showed higher levels in OA cybrids than in N (4.535±1.63; 1.21±0.42 respectively, p≤0.0005). OA cybrids showed higher increment depolarized mitochondria under negative stimuli in comparison with basal condition than N (2.57±1.20; p≤0.0005; 1.76±0.99; p≤0.05 respectively). The analysis of apoptotic levels, when the cells were submitted for a positive stimuli with staurosporine (2 μM) and an inflammatory environment with IL-1β (10 ng/ml), OA cybrids reflected a statistically significant increase in positive cells for Anexine-V in comparison with N cybrids in both conditions (staurosporine 15.68±6.39; 6.41±4.88 respectively, p≤0.05. IL-1β 0.924±0.19; 0.47±0.24 respectively, p≤0.05). Autophagy was analyzed studying LC3 a marker for autophagosome formation and the results showed that LC3 activation was reduced in OA cybrids (1.19±0.24; 1.41±0.21 respectively, p≤0.05). Conclusion Mitochondria from OA patients is involved in cellular senescence, apoptosis and autophagy (three relevant processes involved in OA). Disclosure of Interests Andrea Dalmao-Fernandez: None declared, Tamara Hermida Gomez: None declared, Maria Eugenia Vazquez Mosquera: None declared, Sara Relano-Fernandez: None declared, Ignacio Rego-Perez: None declared, Francisco J. Blanco Consultant for: AbbVie, Bioiberica, BMS, GSK, Grunenthal, Janssen, Lilly, Pfizer, Regeneron, Roche, Sanofi, TRB Chemedica, and UCB, Mercedes Fernandez-Moreno: None declared |
