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: LillyPfizerSanofiGalapagos, Consultant of: LillyPfizerSanofiGalapagos, Grant/research support from: LillyMSDMerck SeronoPfizerPierre-FabraRocheSanofiServierUCBAbbvieAmgenBioibericaBristol MayerCelgeneCelltrionCellerixGrunenthalGebro PharmaAKL Research and Development Ltd
Interleukin-26 Has Synergistic Catabolic Effects with Palmitate in Human Articular Chondrocytes via the TLR4-ERK1/2-c-Jun Signaling Pathway
