Background: Disease modification is not yet possible for osteoarthritis (OA). Mitochondrial ROS and pro-inflammatory cytokines are involved in the pathogenesis of OA and are potential therapeutic targets. APPA, a combination of apocynin (AP) and paeonol (PA), has the potential capacity to modulate synthesis of pro-inflammatory stimuli.
Objectives: To investigate the anti-inflammatory effect of APPA in human articular chondrocytes and cartilage.
Methods: Tissue and chondrocytes from human OA cartilage were isolated. The effect of APPA on chondrocyte viability was analyzed using MTT. IL-1β 10 ng/mL and LPS 10 ng/mL were used as pro-inflammatory stimuli. ROS production was evaluated by flow cytometry using DCFH-DA and MitoSoxRed. The percentage of senescent cells was evaluated through the quantification of Fluorescein di-β-D-galactopyranoside (FDG) by flow cytometry. The effect of APPA on gene expression of pro-inflammatory cytokines (IL-8 and TNF-α) and enzymes degrading cartilage (MMP-13 and MMP-3) were analyzed in chondrocyte and cartilage by RT-PCR. Quantification of Toluidine Blue (TB) staining in cartilage was performed to evaluate proteoglycans content using software ImageJ/Fiji. Release of Glycosaminoglycan (GAGs) into the supernatant was quantified using BlyscanTM Glycosaminoglycan assay. Statistical analyses were performed with GraphPad Prism v6.
Results: Chondrocytes, incubated in presence of APPA 10 µg/mL for 24 h had viability >85%, reduced cytoplasmic ROS (p=0.028) and mitochondrial anion superoxide production induced by LPS 10 ng/mL (p=0.057). Chondrocytes incubated in presence of APPA 10 µg/mL for 2 hours contained significantly fewer senescent cells (p=0.0079). APPA significantly reduced the gene expression induced by IL-1β 10 ng/mL in chondrocytes of IL-8, TNF-α, MMP-13 and MMP-3. Cartilage incubated with APPA 60 and 100 µg/mL for 48 h showed decreased the MMP-3 gene expression induced by IL-1β (p=0.021 and p<0.0001 respectively). Quantification of TB showed that APPA 60 and 100 µg/mL during 48h increased the proteoglycans in intermedial layer, which had been decreased through the incubation with IL-1β (p=0.0018 and p=0.018 respectively). Quantification of release GAGs into the supernatant decreased significantly when the cartilage explants were incubated for 48h in presence of APPA 100 µg/mL (p=0.028).
Conclusion: APPA has a clear anti-inflammatory effect on human articular chondrocytes, and could reduce extracellular matrix degradation of cartilage. This could be mediated by the capacity to modulate ROS production and reduce senescence.
Disclosure of Interests: Mercedes Fernandez-Moreno: None declared, Nicholas Larkins Shareholder of: I am a shareholder in AKL Research and Development Ltd, Alan Reynolds Shareholder of: I have share options in AKL Research and Development Ltd, Speakers bureau: I have not been a paid speaker for a pharma company - at least not since 2008 whichI think is outside the scope of this, Consultant of: The last time I was a paid consultant was in 2017 when I acted as a consultant for Avillion and Norgine, Employee of: I am also an employee of AKL Research and Development Ltd, Tamara Hermida Gómez: None declared, Francisco J. Blanco Speakers bureau: Lilly
Galapagos, Consultant of: Lilly
Galapagos, Grant/research support from: Lilly
AKL Research and Development Ltd
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