Single Cell Confocal Raman Spectroscopy of Human Osteoarthritic Chondrocytes: A Preliminary Study

A great deal of effort has been focused on exploring the underlying molecular mechanism of osteoarthritis (OA) especially at the cellular level. We report a confocal Raman spectroscopic investigation on human osteoarthritic chondrocytes. The objective of this investigation is to identify molecular features and the stage of OA based on the spectral signatures corresponding to bio-molecular changes at the cellular level in chondrocytes. In this study, we isolated chondrocytes from human osteoarthritic cartilage and acquired Raman spectra from single cells. Major spectral differences between the cells obtained from different International Cartilage Repair Society (ICRS) grades of osteoarthritic cartilage were identified. During progression of OA, a decrease in protein content and an increase in cell death were observed from the vibrational spectra. Principal component analysis and subsequent cross-validation was able to associate osteoarthritic chondrocytes to ICRS Grade I, II and III with specificity 100.0%, 98.1%, and 90.7% respectively, while, sensitivity was 98.6%, 82.8%, and 97.5% respectively. The overall predictive efficiency was 92.2%. Our pilot study encourages further use of Raman spectroscopy as a noninvasive and label free technique for revealing molecular features associated with osteoarthritic chondrocytes.     

The Regulatory Role of Signaling Crosstalk in Hypertrophy of MSCs and Human Articular Chondrocytes

Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived in vitro differentiated chondrocytes and chondrocytes, with an emphasis on the crosstalk between these pathways. Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP)/Transforming growth factor-β (TGFβ), Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (IHH), Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF). This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC) repair, and improves understanding of the disease stages and cellular responses within an OA articular joint.     

Gremlin-1 and BMP-4 Overexpressed in Osteoarthritis Drive an Osteochondral-Remodeling Program in Osteoblasts and Hypertrophic Chondrocytes

Osteoarthritis (OA) is a whole joint disease characterized by an important remodeling of the osteochondral junction. It includes cartilage mineralization due to chondrocyte hypertrophic differentiation and bone sclerosis. Here, we investigated whether gremlin-1 (Grem-1) and its BMP partners could be involved in the remodeling events of the osteochondral junction in OA. We found that Grem-1, BMP-2, and BMP-4 immunostaining was detected in chondrocytes from the deep layer of cartilage and in subchondral bone of knee OA patients, and was positively correlated with cartilage damage. ELISA assays showed that bone released more Grem-1 and BMP-4 than cartilage, which released more BMP-2. In vitro experiments evidenced that compression stimulated the expression and the release of Grem-1 and BMP-4 by osteoblasts. Grem-1 was also overexpressed during the prehypertrophic to hypertrophic differentiation of murine articular chondrocytes. Recombinant Grem-1 stimulated Mmp-3 and Mmp-13 expression in murine chondrocytes and osteoblasts, whereas recombinant BMP-4 stimulated the expression of genes associated with angiogenesis (Angptl4 and osteoclastogenesis (Rankl and Ccl2). In conclusion, Grem-1 and BMP-4, whose expression at the osteochondral junction increased with OA progression, may favor the pathological remodeling of the osteochondral junction by inducing a catabolic and tissue remodeling program in hypertrophic chondrocytes and osteoblasts.     

Synovial Fluid Regulates the Gene Expression of a Pattern of microRNA via the NF-κB Pathway: An In Vitro Study on Human Osteoarthritic Chondrocytes

Synovial fluid (SF) represents the primary source of nutrients of articular cartilage and is implicated in maintaining cartilage metabolism. We investigated the effects of SF, from patients with osteoarthritis (OA), rheumatoid arthritis (RA), and controls, on a pattern of microRNA (miRNA) in human OA chondrocytes. Cells were stimulated with 50% or 100% SF for 24 h and 48 h. Apoptosis and superoxide anion production were detected by cytometry; miRNA (34a, 146a, 155, 181a), cytokines, metalloproteinases (MMPs), type II collagen (Col2a1), antioxidant enzymes, B-cell lymphoma (BCL)2, and nuclear factor (NF)-κB by real-time PCR. The implication of the NF-κB pathway was assessed by the use of NF-κB inhibitor (BAY-11-7082). RA and OA SF up-regulated miR-34a, -146a, -155, -181a, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, MMP-1, MMP-13, and ADAMTs-5 gene expression, while it down-regulated Col2a1. Pathological SF also induced apoptosis, reduced viability, and decreased BCL2 mRNA, whereas it increased superoxide anions, the expression of antioxidant enzymes, p65 and p50 NF-κB. Opposite and positive results were obtained with 100% control SF. Pre-incubation with BAY-11-7082 counteracted SF effects on miRNA. We highlight the role of the SF microenvironment in regulating some miRNA involved in inflammation and cartilage degradation during OA and RA, via the NF-κB pathway.     

Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1β-Induced Mitochondrial Dysfunction in Human Chondrocytes

Interleukin (IL)-1β is an important pro-inflammatory cytokine in the progression of osteoarthritis (OA), which impairs mitochondrial function and induces the production of nitric oxide (NO) in chondrocytes. The aim was to investigate if blockade of NO production prevents IL-1β-induced mitochondrial dysfunction in chondrocytes and whether cAMP and AMP-activated protein kinase (AMPK) affects NO production and mitochondrial function. Isolated human OA chondrocytes were stimulated with IL-1β in combination with/without forskolin, L-NIL, AMPK activator or inhibitor. The release of NO, IL-6, PGE₂, MMP3, and the expression of iNOS were measured by ELISA or Western blot. Parameters of mitochondrial respiration were measured using a seahorse analyzer. IL-1β significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1β-induced NO release and mitochondrial dysfunction but not IL-1β-induced release of IL-6, PGE₂, and MMP3. Enhancement of cAMP by forskolin reduced IL-1β-induced NO release and prevented IL-1β-induced mitochondrial impairment. Activation of AMPK increased IL-1β-induced NO production and the negative impact of IL-1β on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1β-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1β-induced NO release and mitochondrial dysfunction.     

Absolute Quantification of Selected Proteins in the Human Osteoarthritic Secretome

Osteoarthritis (OA) is characterized by a loss of extracellular matrix which is driven by catabolic cytokines. Proteomic analysis of the OA cartilage secretome enables the global study of secreted proteins. These are an important class of molecules with roles in numerous pathological mechanisms. Although cartilage studies have identified profiles of secreted proteins, quantitative proteomics techniques have been implemented that would enable further biological questions to be addressed. To overcome this limitation, we used the secretome from human OA cartilage explants stimulated with IL-1β and compared proteins released into the media using a label-free LC-MS/MS-based strategy. We employed QconCAT technology to quantify specific proteins using selected reaction monitoring. A total of 252 proteins were identified, nine were differentially expressed by IL-1 β stimulation. Selected protein candidates were quantified in absolute amounts using QconCAT. These findings confirmed a significant reduction in TIMP-1 in the secretome following IL-1β stimulation. Label-free and QconCAT analysis produced equivocal results indicating no effect of cytokine stimulation on aggrecan, cartilage oligomeric matrix protein, fibromodulin, matrix metalloproteinases 1 and 3 or plasminogen release. This study enabled comparative protein profiling and absolute quantification of proteins involved in molecular pathways pertinent to understanding the pathogenesis of OA.     

Shikonin Derivatives Inhibit Inflammation Processes and Modulate MAPK Signaling in Human Healthy and Osteoarthritis Chondrocytes

Osteoarthritis (OA) is the most common joint disorder and is characterized by the degeneration of articular cartilage. To develop new therapeutic approaches, we investigated the effect of shikonin derivatives on inflammation, MMP expression, and the regulation of MAPK signaling in human healthy (HC) and OA chondrocytes (pCH-OA). Viability was analyzed using the CellTiter-Glo^® Assay. Inflammatory processes were investigated using a proteome profiler™ assay. Furthermore, we analyzed the effects of the shikonin derivatives by protein expression analysis of the phosphorylation pattern and the corresponding downstream gene regulation using RT-qPCR. Both HC and pCH-OA showed a dose-dependent decrease in viability after treatment. The strongest effects were found for shikonin with IC₅₀ values of 1.2 ± 0.1 µM. Shikonin counteracts the inflammatory response by massively reducing the expression of the pro-inflammatory mediators. The phosphorylation level of ERK changed slightly. pJNK and pp38 showed a significant increase, and the downstream targets c/EBPs and MEF2c may play a role in the cartilage homeostasis. STAT3 phosphorylation decreased significantly and has a chondroprotective function through the regulation of cyclin D1 and Sox9. Our results demonstrate for the first time that shikonin derivatives have extensive effects on the inflammatory processes, MAPKs, and IL6/STAT3 downstream regulation in healthy and OA chondrocytes.     

Adipose-Derived Extract Suppresses IL-1β-Induced Inflammatory Signaling Pathways in Human Chondrocytes and Ameliorates the Cartilage Destruction of Experimental Osteoarthritis in Rats

We investigated the effects of adipose-derived extract (AE) on cultured chondrocytes and in vivo cartilage destruction. AE was prepared from human adipose tissues using a nonenzymatic approach. Cultured human chondrocytes were stimulated with interleukin-1 beta (IL-1β) with or without different concentrations of AE. The effects of co-treatment with AE on intracellular signaling pathways and their downstream gene and protein expressions were examined using real-time PCR, Western blotting, and immunofluorescence staining. Rat AE prepared from inguinal adipose tissues was intra-articularly delivered to the knee joints of rats with experimental osteoarthritis (OA), and the effect of AE on cartilage destruction was evaluated histologically. In vitro, co-treatment with IL-1β combined with AE reduced activation of the p38 and ERK mitogen-activated protein kinase (MAPK) pathway and nuclear translocation of the p65 subunit of nuclear factor-kappa B (NF-κB), and subsequently downregulated the expressions of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, IL-6, and IL-8, whereas it markedly upregulated the expression of IL-1 receptor type 2 (IL-1R2) in chondrocytes. Intra-articular injection of homologous AE significantly ameliorated cartilage destruction six weeks postoperatively in the rat OA model. These results suggested that AE may exert a chondroprotective effect, at least in part, through modulation of the IL-1β-induced inflammatory signaling pathway by upregulation of IL-1R2 expression.     

Interleukin-1 Induces the Release of Lubricating Phospholipids from Human Osteoarthritic Fibroblast-Like Synoviocytes

(1) Background: Synovial fluid (SF) from knee joints with osteoarthritis (OA) has increased levels of phospholipids (PL). We have reported earlier that TGF-ß and IGF-1 stimulate fibroblast-like synoviocytes (FLS) to synthesize increased amounts of PLs. The current study examined whether IL-1ß induces the release of PLs in FLS and the underlying mechanism. (2) Methods: Cultured human OA FLS were treated with IL-1ß alone and with pathway inhibitors or with synthetic liver X receptor (LXR) agonists. Cholesterol hydroxylases, ABC transporters, apolipoproteins (APO), LXR, sterol regulatory binding proteins (SREBPs), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) were analyzed by RT-PCR, Western blot, and ELISA. The release of radiolabeled PLs from FLS was determined, and statistical analysis was performed using R (N = 5–9). (3) Results: Like synthetic LXR agonists, IL-1ß induced a 1.4-fold greater release of PLs from FLS. Simultaneously, IL-1ß upregulated the level of the PL transporter ABCA1 and of cholesterol hydroxylases CH25H and CYP7B1. IL-1ß and T0901317 stimulated the expression of SREBP1c, whereas only T0901317 enhanced SREBP2, HMGCR, APOE, LXRα, and ABCG1 additionally. (4) Conclusions: IL-1ß partially controls PL levels in OA-SF by affecting the release of PLs from FLS. Our data show that IL-1ß upregulates cholesterol hydroxylases and thus the formation of oxysterols, which, as natural agonists of LXR, increase the level of active ABCA1, in turn enhancing the release of PLs.     

Hop Extract Anti-Inflammatory Effect on Human Chondrocytes Is Potentiated When Encapsulated in Rapeseed Lecithin Nanoliposomes

Hop (Humulus lupulus L.) is a plant used as an ingredient in beer or employed for its anti-inflammatory properties. The cultivation of hops is currently dedicated to the brewing industry, where mainly female flowers are used, whereas aerial parts, such as leaves, are considered coproducts. Osteoarthritis is the most common musculoskeletal disease associated with low-grade cartilage inflammation. Liposomes have been shown to be promising systems for drug delivery to cartilage cells, called chondrocytes. The aim of our work was to vectorize hop extract valorized from coproducts as a therapeutic agent to alleviate inflammation in human chondrocytes in vitro. Liquid chromatography allowed the identification of oxidized bitter acids in a methanolic extract obtained from the leaves of Cascade hops. The extract was encapsulated in rapeseed lecithin nanoliposomes, and the physicochemical properties of empty or loaded nanoliposomes exhibited no difference. Increasing concentrations of the hop extract alone, empty nanoliposomes, and loaded nanoliposomes were tested on human chondrocytes to assess biocompatibility. The appropriate conditions were applied to chondrocytes stimulated with interleukin-1β to evaluate their effect on inflammation. The results reveal that encapsulation potentiates the hop extract anti-inflammatory effect and that it might be able to improve joint inflammation in osteoarthritis. Furthermore, these results also show that a “zero waste” chain is something that can be achieved in hop cultivation.     

Effects of Visfatin on Intracellular Mechanics and Catabolism in Human Primary Chondrocytes through Glycogen Synthase Kinase 3β Inactivation

Osteoarthritis (OA) is still a recalcitrant musculoskeletal disease on account of its complex biochemistry and mechanical stimulations. Apart from stimulation by external mechanical forces, the regulation of intracellular mechanics in chondrocytes has also been linked to OA development. Recently, visfatin has received significant attention because of the clinical finding of the positive correlation between its serum/synovial level and OA progression. However, the precise mechanism involved is still unclear. This study determined the effect of visfatin on intracellular mechanics and catabolism in human primary chondrocytes isolated from patients. The intracellular stiffness of chondrocytes was analyzed by the particle-tracking microrheology method. It was shown that visfatin damages the microtubule and microfilament networks to influence intracellular mechanics to decrease the intracellular elasticity and viscosity via glycogen synthase kinase 3β (GSK3β) inactivation induced by p38 signaling. Further, microtubule network destruction in human primary chondrocytes is predominantly responsible for the catabolic effect of visfatin on the cyclooxygenase 2 upregulation. The present study shows a more comprehensive interpretation of OA development induced by visfatin through biochemical and biophysical perspectives. Finally, the role of GSK3β inactivation, and subsequent regulation of intracellular mechanics, might be considered as theranostic targets for future drug development for OA.     

Histone Extraction from Human Articular Cartilage for the Study of Epigenetic Regulation in Osteoarthritis

Osteoarthritis (OA) is a chronic disease that affects articular cartilage, causing its degeneration. Although OA is one of the most prevalent pathologies globally, there are no definitive treatments available. Recently, research has focused on elucidating the complex interplay that takes place between inflammatory processes and epigenetic regulation, showing that histone post-translational modifications (PTMs) can exert a pronounced effect on the expression of OA-related genes. OA chondrocytes enhance the production of interleukin 1β (IL-1β) and interleukin 8 (IL-8), which are epigenetically regulated. These cytokines upregulate the synthesis of matrix metalloproteinases (MMPs) and aggrecanases, which promote the extracellular matrix (ECM) destruction. This motivates the study of histone PTMs to investigate the epigenetic regulation of proinflammatory molecules, but the absence of specific protocols to extract histones from human articular cartilage has complicated this task. The lack of effective methods can be explained by the structural complexity and low cellularity of this tissue, which are responsible for the biomechanical properties that allow the movement of the joint but also complicate histone isolation. Here, we provide a histone extraction procedure specifically adapted for cryopreserved human articular cartilage that can be useful to understand epigenetic regulation in OA and accelerate the search for novel strategies.     

Dexamethasone Attenuates the Expression of MMP-13 in Chondrocytes through MKP-1

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is upregulated in inflammation and reduces the activity of proinflammatory mitogen-activated protein kinases (MAP kinases) by dephosphorylation. MAP kinases are intracellular signaling pathways that mediate the cellular effects of proinflammatory cytokines. In the present study, we investigated the effects of the glucocorticoid dexamethasone on the expression of catabolic enzymes in chondrocytes and tested the hypothesis that these effects are mediated through MKP-1. Dexamethasone was found to significantly attenuate the expression of matrix metalloproteinase (MMP)-13 in human OA chondrocytes as well as in chondrocytes from MKP-1 WT mice, but not in chondrocytes from MKP-1 KO mice. Dexamethasone also increased the expression of MKP-1 in murine and human OA chondrocytes. Furthermore, p38 MAP kinase inhibitors significantly attenuated MMP-13 expression in human OA chondrocytes, while JNK MAP kinase inhibitors had no effect. The results indicate that the effect of dexamethasone on MMP-13 expression in chondrocytes was mediated by an MKP-1 and p38 MAP kinase-dependent manner. These findings, together with previous results, support the concept of MKP-1 as a protective factor in articular chondrocytes in inflammatory conditions and as a potential drug target to treat OA.     

Identification and Characterization of the Intra-Articular Microbiome in the Osteoarthritic Knee

Osteoarthritis (OA) is the most common joint disorder in the United States, and the gut microbiome has recently emerged as a potential etiologic factor in OA development. Recent studies have shown that a microbiome is present at joint synovia. Therefore, we aimed to characterize the intra-articular microbiome within osteoarthritic synovia and to illustrate its role in OA disease progression. RNA-sequencing data from OA patient synovial tissue was aligned to a library of microbial reference genomes to identify microbial reads indicative of microbial abundance. Microbial abundance data of OA and normal samples was compared to identify differentially abundant microbes. We computationally explored the correlation of differentially abundant microbes to immunological gene signatures, immune signaling pathways, and immune cell infiltration. We found that microbes correlated to OA are related to dysregulation of two main functional pathways: increased inflammation-induced extracellular matrix remodeling and decreased cell signaling pathways crucial for joint and immune function. We also confirmed that the differentially abundant and biologically relevant microbes we had identified were not contaminants. Collectively, our findings contribute to the understanding of the human microbiome, well-known OA risk factors, and the role microbes play in OA pathogenesis. In conclusion, we present previously undiscovered microbes implicated in the OA disease progression that may be useful for future treatment purposes.     

Curcuminoids Inhibit Angiogenic Behaviors of Human Umbilical Vein Endothelial Cells via Endoglin/Smad1 Signaling

Background: Angiogenesis is primarily attributed to the excessive proliferation and migration of endothelial cells. Targeting the vascular endothelial growth factor (VEGF) is therefore significant in anti-angiogenic therapy. Although these treatments have not reached clinical expectations, the upregulation of alternative angiogenic pathways (endoglin/Smad1) may play a critical role in drug (VEGF-neutralizing agents) resistance. Enhanced endoglin expression following a VEGF-neutralizing therapy (semaxanib^®) was noted in patients. Treatment with an endoglin-targeting antibody augmented VEGF expression in human umbilical vein endothelial cells (HUVECs). Therefore, approaches that inhibit both the androgen and VEGF pathways enhance the HUVECs cytotoxicity and reverse semaxanib resistance. The purpose of this study was to find natural-occurring compounds that inhibited the endoglin-targeting pathway. Methods: Curcuminoids targeting endoglin were recognized from two thousand compounds in the Traditional Chinese Medicine Database@Taiwan (TCM Database@Taiwan) using Discovery Studio 4.5. Results: Our results, obtained using cytotoxicity, migration/invasion, and flow cytometry assays, showed that curcumin (Cur) and demethoxycurcumin (DMC) reduced angiogenesis. In addition, Cur and DMC downregulated endoglin/pSmad1 phosphorylation. Conclusions: The study first showed that Cur and DMC demonstrated antiangiogenic activity via the inhibition of endoglin/Smad1 signaling. Synergistic effects of curcuminoids (i.e., curcumin and DMC) and semaxanib on HUVECs were found. This might be attributed to endoglin/pSmad1 downregulation in HUVECs. Combination treatment with curcuminoids and a semaxanib is therefore expected to reverse semaxanib resistance.     

The Role of the Notch Signaling Pathway in Recovery of Cardiac Function after Myocardial Infarction

Myocardial infarction (MI) is a pathological process, evidencing as massive death of cardiomyocytes associated with hypoxic and oxidative stress. The formation of areas of fibrosis ultimately leads to heart failure. There are some mechanisms that contribute to the functional repair of the heart. In most mammals, including humans, the Notch signaling pathway has cardioprotective effects. It is involved in the formation of the heart in embryogenesis and in the restoration of cardiac function after MI due to: (1) reducing oxidative stress; (2) prevention of apoptosis; (3) regulation of inflammation; (4) containment of fibrosis and hypertrophy of cardiomyocytes; (5) tissue revascularization; and (6) regulation of proliferation and differentiation of cardiomyocytes. In addition, the Notch signaling pathway interacts with other signaling cascades involved in the pathogenesis of MI and subsequent cardiac repair. In this review, we consider the Notch signaling pathway as a potential target for therapeutic approaches aimed at improving cardiac recovery after MI.     

Dynamics of Connexin 43 Down Modulation in Human Articular Chondrocytes Stimulated by Tumor Necrosis Factor Alpha

Connexin 43 (Cx43) exerts pivotal functions in articular chondrocytes (CH). It is involved in the communication among cells and between cells and the extracellular environment, and it contributes to the maintenance of the correct cell phenotype. The pro-inflammatory cytokine TNFα induces a reduction in Cx43 expression in CH. Here, we studied the dynamics of this decrease in expression. We evaluated Cx43 protein and gene expression and the involvement of C-terminal domain (CTD) cleavage and proteasomal degradation. Treatments able to counteract TNFα action were also examined, together with Gap Junction (GJ) functionality and Cx43 localization. TNFα induced a significant reduction in Cx43 expression already at day 1, and the down modulation reached a peak at day 3 (−46%). The decrease was linked to neither gene expression modulation nor CTD cleavage. Differently, the proteasome inhibitor MG132 reverted TNFα effect, indicating the involvement of proteasomal degradation in Cx43 reduction. In addition, the co-treatment with the anabolic factor TGF-β1 restored Cx43 levels. Cx43 decrease occurred both at the membrane level, where it partially influenced GJ communication, and in the nucleus. In conclusion, TNFα induced a rapid and lasting reduction in Cx43 expression mostly via the proteasome. The down modulation could be reverted by cartilage-protective factors such as MG132 and TGF-β1. These findings suggest a possible involvement of Cx43 perturbation during joint inflammation.     

Nuclear Magnetic Resonance Therapy Modulates the miRNA Profile in Human Primary OA Chondrocytes and Antagonizes Inflammation in Tc28/2a Cells

Nuclear magnetic resonance therapy (NMRT) is discussed as a participant in repair processes regarding cartilage and as an influence in pain signaling. To substantiate the application of NMRT, the underlying mechanisms at the cellular level were studied. In this study microRNA (miR) was extracted from human primary healthy and osteoarthritis (OA) chondrocytes after NMR treatment and was sequenced by the Ion PI Hi-Q™ Sequencing 200 system. In addition, T/C-28a2 chondrocytes grown under hypoxic conditions were studied for IL-1β induced changes in expression on RNA and protein level. HDAC activity an NAD⁽⁺⁾/NADH was measured by luminescence detection. In OA chondrocytes miR-106a, miR-27a, miR-34b, miR-365a and miR-424 were downregulated. This downregulation was reversed by NMRT. miR-365a-5p is known to directly target HDAC and NF-ĸB, and a decrease in HDAC activity by NMRT was detected. NAD⁺/NADH was reduced by NMR treatment in OA chondrocytes. Under hypoxic conditions NMRT changed the expression profile of HIF1, HIF2, IGF2, MMP3, MMP13, and RUNX1. We conclude that NMRT changes the miR profile and modulates the HDAC and the NAD⁽⁺⁾/NADH signaling in human chondrocytes. These findings underline once more that NMRT counteracts IL-1β induced changes by reducing catabolic effects, thereby decreasing inflammatory mechanisms under OA by changing NF-ĸB signaling.