Introduction: Aging is one of the major risk factors for development of osteoarthritis (OA), and progressive articular cartilage degeneration is the central feature of the disease. It is known that the levels of nicotinamide adenine dinucleotide (NAD+), a key intermediate metabolite that is not only a cofactor for numerous enzymes involved in cellular energy metabolism but also a critical regulator of multiple cell signaling pathways, steadily decline during aging and in age-related diseases, associated with low-grade inflammation. We recently discovered that primary human knee OA chondrocytes and chondrocytes stimulated with pro-inflammatory cytokine IL-1betaexhibited reduced NAD+ levels, largely due to increased expression and activity of NADase CD38. Since the majority of NAD+ is synthesized through salvage pathways from NAD+ precursors such as nicotinmide riboside (NR) that is a pyridine nucleoside form of vitamin B3, we investigated the effect of supplementation of NR on chondrocytes and cartilage matrix metabolism in vitro and ex vivo. Methods: Primary human knee chondrocytes or cartilage explants were treated with IL-1beta in the presence or absence of NR (up to 100 M). The intracellular levels of NAD+ in chondrocytes were determined using the NAD/NADH quantification assay. Mitochondrial superoxide generation was determined in chondrocytes stimulated with menadione (an oxidative stress inducer) by MitoSox Red staining, followed by flow cytometry analysis. Gene expression of aggrecan (ACAN), type II collagen (col2a1), iNOS, MMP3 and MMP13 was examined by quantitative RT-PCR. Release of nitric oxide (NO), glycosaminoglycan (GAG), MMP3 and MMP13 from cartilage explants was assessed from conditioned media by Griess reaction, dimethylmethylene blue (DMMB) assay and ELISA, respectively. Results: Primary human knee chondrocytes from OA donors exhibited reduced levels of NAD+, compared to normal donors. Supplementation of NR to OA chondrocytes dramatically increased cellular NAD+ levels, and in addition, prevented IL-1beta-induced reduction of NAD+ content. Moreover, NR supplementation reversed the inhibitory effect of IL-1betaon ACAN and col2a1 mRNA expression, and attenuated IL-1beta-induced iNOS, MMP3 and MMP13 mRNA expression in a dose-dependent manner. Mitochondrial superoxide generation induced by menadione (evidenced by MitoSox Red staining) was significantly inhibited by NR. Furthermore, NR supplementation to cartilage explants mitigated IL-1beta-induced release of NO, GAG, MMP3 and MMP13. Discussion: Decline of NAD+ levels in articular chondrocytes is associated with OA. Restoration of chondrocyte NAD+ levels by NR supplementation may help maintain cartilage matrix homeostasis by preventing oxidative stress and excessive catabolic responses. Significance and Clinical Relevance: There are not yet effective disease-modifying therapies for OA. Given that oral NR supplementation is recently shown to safely boost human NAD+ metabolism in a dose-dependent manner, NR supplementation could be potentially used as a novel approach to inhibit cartilage degradation, therefore limiting OA progression.
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