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A5.1 Culture OF human chondrocytes in high glucose induces inflammatory markers and impairs autophagy
  1. A T Rufino1,2,
  2. M Ribeiro1,2,
  3. F Judas3,4,
  4. M C Lopes1,2,
  5. A F Mendes1,2
  1. 1Centre for Neuroscience and Cell Biology, University of Coimbra, Portugal
  2. 2Faculty of Pharmacy, University of Coimbra, Portugal
  3. 3Orthopaedics Department, University and Hospital Center of Coimbra, Portugal
  4. 4Faculty of Medicine, University of Coimbra, Portugal

Abstract

Background and Objectives Accumulating evidence indicates that Diabetes Mellitus is an independent risk factor for severe osteoarthritis (OA). Understanding the mechanisms involved is essential for designing preventive strategies and targeted therapies that can halt OA progression. Our previous studiesshowed that culture of human chondrocytes under excess glucose favours catabolic responses and oxidative stress. This study aims at elucidating whether exposure of chondrocytes to high glucose favours inflammatory responses and impairs autophagy, a crucial mechanism for the elimination of damaged molecules and organelles.

Materials and Methods Human chondrocytes isolated from knee cartilage obtained from multi-organ donors at the Orthopaedics Department of the University and Hospital Center of Coimbra and the human chondrocytic cell line, C28/I2, were cultured in medium containing regular (10 mM) or excess (30 mM) glucose for various periods. The expression of pro-inflammatory and pro-catabolic markers (iNOS, IL-1β and TNF-α) was evaluated by qRT-PCR. iNOS protein levels and the nuclear translocation of p65, a marker of NF-κB activation, were evaluated by western blot. NO production was assessed by the Griess reaction. Autophagy was assessed by determining the protein levels of LC3-I and II in the presence and absence of the lysosome inhibitor, chloroquine. To rule out possible osmotic effects, parallel experiments were performed in the presence of the cell-impermeable polyol, mannitol.

Results Culture of human chondrocytes in high glucose (30 mM) for 24h significantly increased iNOS mRNA and protein levels, as well as NO production relative to cells maintained in regular glucose. IL-1β and TNF-α mRNA levels were also significantly increased, while nuclear levels of NF-κB p65 increased in a time-dependent manner, peaking after 1h treatment. On the other hand, LC3-II levels were significantly reduced by culture of C28/I2 cells in high glucose for 24h, either in the presence or absence of chloroquine, indicating that its synthesis was decreased relative to cells maintained in regular glucose medium.

Conclusions Hyperglycemia-like glucose concentrations are sufficient to induce the inflammatory process and to impair autophagy in human chondrocytes which can contribute to the development and progression of OA.

Work supported by grants PEst-C/SAU/LA0001/2011 and PTDC/EME-TME/113039/2009 from FEDER through COMPETE and FCT.

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