Targeting PAR2-mediated inflammation in osteoarthritis: a comprehensive in vitro evaluation of oleocanthal's potential as a functional food intervention for chondrocyte protection and anti-inflammatory effects.

BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by chronic inflammation and progressive cartilage degradation, ultimately leading to joint dysfunction and disability. Oleocanthal (OC), a bioactive phenolic compound derived from extra virgin olive oil, has garnered significant attention due to its potent anti-inflammatory properties, which are comparable to those of non-steroidal anti-inflammatory drugs (NSAIDs). This study pioneers the investigation into the effects of OC on the Protease-Activated Receptor-2 (PAR-2) mediated inflammatory pathway in OA, aiming to validate its efficacy as a functional food-based therapeutic intervention. METHODS: To simulate cartilage tissue in vitro, human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes. An inflammatory OA-like environment was induced in these chondrocytes using lipopolysaccharide (LPS) to mimic the pathological conditions of OA. The therapeutic effects of OC were evaluated by treating these inflamed chondrocytes with various concentrations of OC. The study focused on assessing key inflammatory markers, catabolic enzymes, and mitochondrial function to elucidate the protective mechanisms of OC. Mitochondrial function, specifically mitochondrial membrane potential (ΔΨm), was assessed using Rhodamine 123 staining, a fluorescent dye that selectively accumulates in active mitochondria. The integrity of ΔΨm serves as an indicator of mitochondrial and bioenergetic function. Additionally, Western blotting was employed to analyze protein expression levels, while real-time polymerase chain reaction (RT-PCR) was used to quantify gene expression of inflammatory cytokines and catabolic enzymes. Flow cytometry was utilized to measure cell viability and apoptosis, providing a comprehensive evaluation of OC's therapeutic effects on chondrocytes. RESULTS: The results demonstrated that OC significantly downregulated PAR-2 expression in a dose-dependent manner, leading to a substantial reduction in pro-inflammatory cytokines, including TNF-α, IL-1ß, and MCP-1. Furthermore, OC attenuated the expression of catabolic markers such as SOX4 and ADAMTS5, which are critically involved in cartilage matrix degradation. Importantly, OC was found to preserve mitochondrial membrane potential (ΔΨm) in chondrocytes subjected to inflammatory stress, as evidenced by Rhodamine 123 staining, indicating a protective effect on cellular bioenergetics. Additionally, OC modulated the Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL)/Receptor Activator of Nuclear Factor Kappa-Β (RANK) pathway, suggesting a broader therapeutic action against the multifactorial pathogenesis of OA. CONCLUSIONS: This study is the first to elucidate the modulatory effects of OC on the PAR-2 mediated inflammatory pathway in OA, revealing its potential as a multifaceted therapeutic agent that not only mitigates inflammation but also protects cartilage integrity. The preservation of mitochondrial function and modulation of the RANKL/RANK pathway further underscores OC's comprehensive therapeutic potential in counteracting the complex pathogenesis of OA. These findings position OC as a promising candidate for integration into nutritional interventions aimed at managing OA. However, further research is warranted to fully explore OC's therapeutic potential across different stages of OA and its long-term effects in musculoskeletal disorders.

Anti-inflammatory effects of polydeoxyribonucleotide and adipose tissue-derived mesenchymal stem cells in a canine cell model of osteoarthritis.

IMPORTANCE: A relatively new therapeutic agent for osteoarthritis (OA), polydeoxyribonucleotide (PDRN), shows potential in treating human OA due to its regenerative and anti-inflammatory effects. However, studies on PDRN for canine OA are limited, and no study has investigated their use with mesenchymal stem cells (MSCs) conventionally used for OA treatment. OBJECTIVE: This study aimed to evaluate the potential of PDRN and explore its combined effect with adipose tissue-derived MSCs (AdMSCs) in treating canine OA. METHODS: To study the impact of PDRN, canine chondrocytes, synoviocytes, and AdMSCs were exposed to various PDRN concentrations, and viability was assessed using cell counting kit-8. The OA model was created by treating chondrocytes and synoviocytes with lipopolysaccharide, followed by treatment under three different conditions: PDRN alone, AdMSCs alone, and a combination of PDRN and AdMSCs. Using real-time quantitative polymerase chain reaction, the anti-inflammatory effects and mechanisms were investigated by quantitatively assessing pro-inflammatory cytokines, collagen degradation markers, adenosine A2a receptor (ADORA2A), and nuclear factor-kappa B. RESULTS: PDRN alone and combined with AdMSCs significantly reduced the expression of pro-inflammatory cytokines and collagen degradation markers in an OA model. PDRN promoted AdMSC proliferation and upregulated ADORA2A expression. AdMSCs exhibited comprehensive anti-inflammatory effects through paracrine effects, and both substances reduced inflammatory gene expression through different mechanisms, potentially enhancing therapeutic effects. CONCLUSIONS AND RELEVANCE: The results indicate that PDRN is a safe and effective anti-inflammatory material that can be used independently or as an adjuvant for AdMSCs. Although additional research is necessary, this study is significant because it provides a foundation for future research at the cellular level.

Yiqi Yangxue formula inhibits cartilage degeneration in knee osteoarthritis by regulating LncRNA-UFC1/miR-34a/MMP-13 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Knee osteoarthritis (KOA) is a prevalent and disabling clinical condition affecting joint structures worldwide. The Yiqi Yangxue formula (YQYXF) is frequently prescribed in clinical settings for the treatment of KOA. Existing research has primarily focused on alterations in drug metabolism, with limited investigation into the epigenetic effects of YQYXF, particularly in relation to non-coding RNA. AIM OF THE STUDY: Exploring the effects of YQYXF on critical factors of long chain non-coding RNA UFC1/miR-34a/matrix metalloproteinase-13 (MMP-13) axis and their interrelationships. METHODS: UHPLC-QE-MS technology was used to identify the YQYXF ingredients in rat serum. KEGG and GO analysis were performed on the targets of blood components acting on KOA using a database. Simultaneously, a protein interaction network was constructed using target proteins and metabolites to identify the core components and key pathways of YQYXF. The KOA rat model was established using an improved Hulth method. SPF SD rats were randomly divided into normal group, sham surgery group, model group, celecoxib capsules group (18 mg/kg), YQYXF low, medium and high dose groups (4.6g/kg, 9.2g/kg, 18.4g/kg). Observe the synovial and cartilage tissues of rats using pathological methods. RT-PCR was used to detect the levels of UFC1, miR-34a, and MMP-13 in cartilage. Immunohistochemistry was used to detect the levels of MMP-13 and ADAMTS-5 in cartilage. ELISA method was used to detect the levels of MMP-13 and ADAMTS-5 in serum. In addition, we further validated the regulation of crucial factor expression levels of UFC1/miR-34a/MMP-13 axis in rat chondrocytes and degenerative chondrocytes of KOA patients by YQYXF, providing a basis for its treatment of KOA. RESULTS: The compounds that YQYXF enters the bloodstream mainly contain flavonoids and phenylpropanoid compounds. The core components that act on OA include quercetin, fisetin, and demethylweldelolactone. The main target pathways are the IL-17 signaling pathway, lipid and atherosclerosis, cellular sensitivity, inflammatory mediator regulation of TRP channels, TNF signaling pathway, relaxin signaling pathway and C-type lectin receptor signaling pathway. YQYXF inhibited the expression of miR-34a and MMP-13 mRNA, and reduced the protein levels of MMP-13 and ADAMTS-5. In vitro studies have confirmed that 20% YQYXF serum promoted UFC1 and reduce miR-34a levels. In addition, miR-34a in sh-UFC1+10% YQYXF serum and sh-UFC1+20% YQYXF serum groups significantly decreased compared to the sh-UFC1 group. CONCLUSION: The anti-KOA cartilage degeneration effect of YQYXF might be related to inhibiting cell apoptosis and promoting cell proliferation, which regulated the lncRNA-UFC1/miR-34a/MMP-13 axis.

Nuciferine ameliorates osteoarthritis: An in vitro and in vivo study.

Osteoarthritis (OA) is the most common musculoskeletal disease and a leading cause of pain and disability. A key hallmark of OA is cartilage degradation, which occurs due to an imbalance between the synthesis and degradation of the extracellular matrix (ECM). Interleukin-1ß(IL-1ß) has been reported to regulate ECM metabolism. Nuciferine (Nuc), a natural peptide extracted from the lotus leaf, possesses several significant pharmacological properties. However, the anti-inflammation of Nuc in OA has not been reported. In this study, ELISA and Western blot analyses were used to measure the production of inflammatory mediators in IL-1ß-Induced mouse chondrocytes. Additionally, mice with or without surgical destabilization of the medial meniscus (DMM) were treated with intra-articular injection of Nuc. We found that Nuc significantly reduces the level of iNOS, PEG2, and IL-6 in IL-1ß-induced chondrocytes. Furthermore, Nuc can ameliorate the development of OA in mice. Mechanistically, we found that the chondrocyte-protective effects of Nuc occur via the PTEN/NF-κB pathway. These findings suggest that Nuc could be a potential therapeutic agent for improving OA development.

Mesenchymal Stem Cell-Derived Exosomes as a Treatment Option for Osteoarthritis.

Osteoarthritis (OA) is a leading cause of pain and disability worldwide in elderly people. There is a critical need to develop novel therapeutic strategies that can effectively manage pain and disability to improve the quality of life for older people. Mesenchymal stem cells (MSCs) have emerged as a promising cell-based therapy for age-related disorders due to their multilineage differentiation and strong paracrine effects. Notably, MSC-derived exosomes (MSC-Exos) have gained significant attention because they can recapitulate MSCs into therapeutic benefits without causing any associated risks compared with direct cell transplantation. These exosomes help in the transport of bioactive molecules such as proteins, lipids, and nucleic acids, which can influence various cellular processes related to tissue repair, regeneration, and immune regulation. In this review, we have provided an overview of MSC-Exos as a considerable treatment option for osteoarthritis. This review will go over the underlying mechanisms by which MSC-Exos may alleviate the pathological hallmarks of OA, such as cartilage degradation, synovial inflammation, and subchondral bone changes. Furthermore, we have summarized the current preclinical evidence and highlighted promising results from in vitro and in vivo studies, as well as progress in clinical trials using MSC-Exos to treat OA.

Nanoparticle-delivered quercetin exhibits enhanced efficacy in eliminating iron-overloaded senescent chondrocytes.

Aim: The therapeutic potential of senolytic drugs in osteoarthritis (OA) is poorly known. Quercetin, a senolytic agent exhibits promising potential to treat OA, having limited bioavailability. We investigated the effects of Quercetin-loaded nanoparticles (Q-NP) with enhanced bioavailability in human chondrocytes mimicking OA phenotype.Materials & methods: The C-20/A4 chondrocytes were exposed to ferric ammonium citrate to induce OA phenotype, followed by treatment with free Quercetin/Q-NP for 24 and 48-h. Q-NP were synthesized by nanoprecipitation method. Following treatment chondrocytes were assessed for drug cellular bioavailability, viability, cell cycle, apoptosis, oxidative stress and expression of key senescence markers.Results: Q-NP exhibited 120.1 ± 1.2 nm particle size, 81 ± 2.4% encapsulation efficiency, increased cellular bioavailability and selective apoptosis of senescent chondrocytes compared with free Quercetin. Q-NP treatment also induced oxidative stress and reduced the expressions of senescence markers, including TRB3, p16, p62 and p21 suggesting their ability to eliminate senescent cells. Last, Q-NP arrested the cell cycle in the sub-G0 phase, potentially creating a beneficial environment for tissue repair.Conclusion: Q-NP propose a promising delivery system for treating OA by eliminating senescent chondrocytes through apoptosis. Furthermore, their enhanced cellular bioavailability and capacity to modify cell cycle and senescent pathways warrant further investigations.

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Isoquercetin Ameliorates Osteoarthritis via Nrf2/NF-κB Axis: An In Vitro and In Vivo Study.

Osteoarthritis (OA) is a progressive joint disease characterized by extracellular matrix (ECM) degradation and inflammation, which is involved with pathological microenvironmental alterations induced by damaged chondrocytes. However, current therapies are not effective in alleviating the progression of OA. Isoquercetin is a natural flavonoid glycoside compound that has various pharmacological effects including anticancer, anti-diabetes and blood lipid regulation. Previous evidence suggests that isoquercetin has anti-inflammatory properties in various diseases, but its effect on OA has not been investigated yet. In this study, through western bolt, qRT-PCR and ELISA, it was found that isoquercetin could reduce the increase of ADAMTS5, MMP13, COX-2, iNOS and IL-6 induced by IL-1ß, suggesting that isoquercetin could inhibit the inflammation and ECM degradation of chondrocytes. Through nuclear-plasma separation technique, western blot and immunocytochemistry, it can be found that Nrf2 and NF-κB pathways are activated in this process, and isoquercetin may rely on this process to play its protective role. In vivo, the results of X-ray and SO staining show that intra-articular injection of isoquercetin reduces the degradation of cartilage in the mouse OA model. In conclusion, the present work suggests that isoquercetin may benefit chondrocytes by regulating the Nrf2/NF-κB signaling axis, which supports isoquercetin as a potential drug for the treatment of OA.

Engineering exosomes derived from TNF-α preconditioned IPFP-MSCs enhance both yield and therapeutic efficacy for osteoarthritis.

BACKGROUND: The pathogenesis of osteoarthritis (OA) involves the progressive degradation of articular cartilage. Exosomes derived from mesenchymal stem cells (MSC-EXOs) have been shown to mitigate joint pathological injury by attenuating cartilage destruction. Optimization the yield and therapeutic efficacy of exosomes derived from MSCs is crucial for promoting their clinical translation. The preconditioning of MSCs enhances the therapeutic potential of engineered exosomes, offering promising prospects for application by enabling controlled and quantifiable external stimulation. This study aims to address these issues by employing pro-inflammatory preconditioning of MSCs to enhance exosome production and augment their therapeutic efficacy for OA. METHODS: The exosomes were isolated from the supernatant of infrapatellar fat pad (IPFP)-MSCs preconditioned with a pro-inflammatory factor, TNF-α, and their production was subsequently quantified. The exosome secretion-related pathways in IPFP-MSCs were evaluated through high-throughput transcriptome sequencing analysis, q-PCR and western blot analysis before and after TNF-α preconditioning. Furthermore, exosomes derived from TNF-α preconditioned IPFP-MSCs (IPFP-MSC-EXOsTNF-α) were administered intra-articularly in an OA mouse model, and subsequent evaluations were conducted to assess joint pathology and gait alterations. The expression of proteins involved in the maintenance of cartilage homeostasis within the exosomes was determined through proteomic analysis. RESULTS: The preconditioning with TNF-α significantly enhanced the exosome secretion of IPFP-MSCs compared to unpreconditioned MSCs. The potential mechanism involved the activation of the PI3K/AKT signaling pathway in IPFP-MSCs by TNF-α precondition, leading to an up-regulation of autophagy-related protein 16 like 1(ATG16L1) levels, which subsequently facilitated exosome secretion. The intra-articular administration of IPFP-MSC-EXOsTNF-α demonstrated superior efficacy in ameliorating pathological changes in the joints of OA mice. The preconditioning of TNF-α enhanced the up-regulation of low-density lipoprotein receptor-related protein 1 (LRP1) levels in IPFP-MSC-EXOsTNF-α, thereby exerting chondroprotective effects. CONCLUSION: TNF-α preconditioning constitutes an effective and promising method for optimizing the therapeutic effects of IPFP-MSCs derived exosomes in the treatment of OA.

The novel HSP90 monoclonal antibody 9B8 ameliorates articular cartilage degeneration by inhibiting glycolysis via the HIF-1 signaling pathway.

Osteoarthritis (OA) is a prevalent chronic degenerative disease that affects the bones and joints, particularly in middle-aged and elderly individuals. It is characterized by progressive joint pain, swelling, stiffness, and deformity. Notably, treatment with a heat shock protein 90 (HSP90) inhibitor has significantly curtailed cartilage destruction in a rat model of OA. Although the monoclonal antibody 9B8 against HSP90 is recognized for its anti-tumor properties, its potential therapeutic impact on OA remains uncertain. This study investigated the effects of 9B8 on OA and its associated signaling pathways in interleukin-1ß (IL-1ß)-stimulated human chondrocytes and a rat anterior cruciate ligament transection (ACLT) model. A specific concentration of 9B8 preserved cell viability against IL-1ß-induced reduction. In vitro, 9B8 significantly reduced the expression of extracellular matrix-degrading enzyme such as disintegrin and metallopeptidase-4 (ADAMTS4) of thrombospondin motifs, matrix metalloproteinase-13 (MMP-13), as well as cellular inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which were upregulated by IL-1ß. In vivo, 9B8 effectively protected the articular cartilage and subchondral bone of the rat tibial plateau from ACLT-induced damage. Additionally, gene microarray analysis revealed that IL-1ß substantially increased the expression of SLC2A1, PFKP, and ENO2 within the HIF-1 signaling pathway, whereas 9B8 suppressed the expression of these genes. Thus, 9B8 effectively mitigates ACLT-induced osteoarthritis in rats by modulating the HIF-1 signaling pathway, thereby inhibiting overexpression involved in glycolysis. These results collectively indicate that 9B8 is a promising novel drug for the prevention and treatment of OA.

The cereblon-AMPK (AMP-activated protein kinase) axis in chondrocytes regulates the pathogenesis of osteoarthritis.

OBJECTIVE: AMP-activated protein kinase (AMPK) dysregulation is implicated in osteoarthritis (OA), but the mechanisms underlying this dysregulation remain unclear. We investigated the role of cereblon, a substrate-recognition protein within the E3-ligase ubiquitin complex, in AMPK dysregulation and OA pathogenesis. METHODS: Cereblon expression was examined in human (n = 5) and mouse (n = 10) OA cartilage. The role of cereblon was investigated through its adenoviral overexpression (n = 10) or knockout (KO, n = 15) in the destabilization of the medial meniscus (DMM)-operated mice. The therapeutic potentials of the chemical cereblon degrader, TD-165, and the AMPK activator, metformin, were assessed through intra-articular (IA) injection to mice (n = 15). RESULTS: Immunostaining revealed that cereblon is upregulated in human and mouse OA cartilage. In DMM model mice, cartilage destruction was exacerbated by overexpression of cereblon in mouse joint tissues (OARSI grade; 1.11 [95% CI: 0.50 to 2.75]), but inhibited in global (-2.50 [95% CI: -3.00 to -1.17]) and chondrocyte-specific (-2.17 [95% CI: -3.14 to -1.06]) cereblon KO mice. The inhibitory effects were more pronounced in mice fed a high-fat diet compared to a regular diet. The degradation of cereblon through IA injection of TD-165 inhibited OA cartilage destruction (-2.47 [95% CI: -3.22 to -1.56]). Mechanistically, cereblon exerts its catabolic effects by negatively modulating AMPK activity within chondrocytes. Consistently, activation of AMPK by IA injection of metformin inhibited posttraumatic OA cartilage destruction (-1.20 ([95% CI: -1.89 to -0.45]). CONCLUSIONS: The cereblon-AMPK axis acts as a catabolic regulator of OA pathogenesis and seems to be a promising therapeutic target in animal models of OA.

METTL14 promotes chondrocyte ferroptosis in osteoarthritis via m6A modification of GPX4.

BACKGROUND: Ferroptosis is caused by iron-dependent peroxidation of membrane phospholipids and chondrocyte ferroptosis contributes to osteoarthritis (OA) progression. Glutathione peroxidase 4 (GPX4) plays a master role in blocking ferroptosis. N6-methyladenosine (m6A) is an epigenetic modification among mRNA post-transcriptional modifications. This study investigated the effect of methyltransferase-like 14 (METTL14), the key component of the m6A methyltransferase, on chondrocyte ferroptosis via m6A modification. METHODS: An OA rat model was established through an intra-articular injection of monosodium iodoacetate in the right knee. OA cartilages in rat models were used for gene expression analysis. Primary mouse chondrocytes or ADTC5 cells were stimulated with IL-1ß or erastin. The m6A RNA methylation quantification kit was used to measure m6A level. The effect of METTL14 and GPX4 on ECM degradation and ferroptosis was investigated through western blotting, fluorescence immunostaining, propidium iodide staining, and commercially available kits. The mechanism of METTL14 action was explored through MeRIP-qPCR assays. RESULTS: METTL14 and m6A expression was upregulated in osteoarthritic cartilages and IL-1ß-induced chondrocytes. METTL14 depletion repressed the IL-1ß or erastin-stimulated ECM degradation and ferroptosis in mouse chondrocytes. METTL14 inhibited GPX4 gene through m6A methylation modification. GPX4 knockdown reversed the si-METTL14-mediated protection in IL-1ß-induced chondrocytes. CONCLUSION: METTL14 depletion inhibits ferroptosis and ECM degradation by suppressing GPX4 mRNA m6A modification in injured chondrocytes.

Fibronectin isoforms promote postnatal skeletal development.

Fibronectin (FN) is a ubiquitous extracellular matrix glycoprotein essential for the development of various tissues. Mutations in FN cause a unique form of spondylometaphyseal dysplasia, emphasizing its importance in cartilage and bone development. However, the relevance and functional role of FN during skeletal development has remained elusive. To address these aspects, we have generated conditional knockout mouse models targeting the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and both isoforms together in a double knockout (FNdKO). We used these mice to determine the relevance of the two principal FN isoforms in skeletal development from postnatal day one to the adult stage at two months. We identified a distinct topological FN deposition pattern in the mouse limb during different gestational and postnatal skeletal development phases, with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Cartilage-specific cFN emerged as the predominant isoform in the growth plate, whereas circulating pFN remained excluded from the growth plate and confined to the primary and secondary ossification centers. Deleting either isoform independently (cFNKO or pFNKO) yielded only relatively subtle changes in the analyzed skeletal parameters. However, the double knockout of cFN in the growth plate and pFN in the circulation of the FNdKO mice significantly reduced postnatal body weight, body length, and bone length. Micro-CT analysis of the adult bone microarchitecture in FNdKO mice exposed substantial reductions in trabecular bone parameters and bone mineral density. The mice also showed elevated bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice demonstrated changes in the resting, proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and X, decreased apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. Transforming growth factor-ß1 and downstream phospho-AKT levels were significantly lower in the FNdKO than in the control mice, revealing a crucial FN-mediated regulatory pathway in chondrogenesis and bone formation. In conclusion, the data demonstrate that FN is essential for chondrogenesis and bone development. Even though cFN and pFN act in different regions of the bone, both FN isoforms are required for the regulation of chondrogenesis, cartilage maturation, trabecular bone formation, and overall skeletal growth.

Eriodictyol attenuates osteoarthritis progression through inhibiting inflammation via the PI3K/AKT/NF-κB signaling pathway.

Eriodictyol, a flavonoid distributed in citrus fruits, has been known to exhibit anti-inflammatory activity. In this study, destabilized medial meniscus (DMM)-induced OA model was used to investigate the protective role of eriodictyol on OA. Meanwhile, we used an IL-1ß-stimulated human osteoarthritis chondrocytes model to investigate the anti-inflammatory mechanism of eriodictyol on OA. The production of nitric oxide was detected by Griess reaction. The productions of MMP1, MMP3, and PGE2 were detected by ELISA. The expression of LXRα, ABCA1, PI3K, AKT, and NF-κB were measured by western blot analysis. The results demonstrated that eriodictyol could alleviate DMM-induced OA in mice. In vitro, eriodictyol inhibited IL-1ß-induced NO, PGE2, MMP1, and MMP3 production in human osteoarthritis chondrocytes. Eriodictyol also suppressed the phosphorylation of PI3K, AKT, NF-κB p65, and IκBα induced by IL-1ß. Meanwhile, eriodictyol significantly increased the expression of LXRα and ABCA1. Furthermore, eriodictyol disrupted lipid rafts formation through reducing the cholesterol content. And cholesterol replenishment experiment showed that adding water-soluble cholesterol could reverse the anti-inflammatory effect of eriodictyol. In conclusion, the results indicated eriodictyol inhibited IL-1ß-induced inflammation in human osteoarthritis chondrocytes through suppressing lipid rafts formation, which subsequently inhibiting PI3K/AKT/NF-κB signaling pathway.

Pilot screening of potential matrikines resulting from collagen breakages through ionizing radiation.

Little is known regarding radiation-induced matrikines and the possible degradation of extracellular matrix following therapeutic irradiation. The goal of this study was to determine if irradiation can cut collagen proteins at specific sites, inducing potentially biologically active peptides against cartilage cells. Chondrocytes cultured as 3D models were evaluated for extracellular matrix production. Bystander molecules were analyzed in vitro in the conditioned medium of X-irradiated chondrocytes. Preferential breakage sites were analyzed in collagen polypeptide by mass spectrometry and resulting peptides were tested against chondrocytes. 3D models of chondrocytes displayed a light extracellular matrix able to maintain the structure. Irradiated and bystander chondrocytes showed a surprising radiation sensitivity at low doses, characteristic of the presence of bystander factors, particularly following 0.1 Gy. The glycine-proline peptidic bond was observed as a preferential cleavage site and a possible weakness of the collagen polypeptide after irradiation. From the 46 collagen peptides analyzed against chondrocytes culture, 20 peptides induced a reduction of viability and 5 peptides induced an increase of viability at the highest concentration between 0.1 and 1 µg/ml. We conclude that irradiation promoted a site-specific degradation of collagen. The potentially resulting peptides induce negative or positive regulations of chondrocyte growth. Taken together, these results suggest that ionizing radiation causes a degradation of cartilage proteins, leading to a functional unbalance of cartilage homeostasis after exposure, contributing to cartilage dysfunction.

Myrislignan ameliorates the progression of osteoarthritis: An in vitro and in vivo study.

Osteoarthritis (OA) is a prevalent disease of the musculoskeletal system that causes functional deterioration and diminished quality of life. Myrislignan (MRL) has a wide range of pharmacological characteristics, including an anti-inflammatory ability. Although inflammation is a major cause of OA, the role of MRL in OA treatment is still not well-understood. In this study, we analyze the anti-inflammatory and anti-ECM degradation effects of MRL both in vivo and in vitro. Rat primary chondrocytes were treated with interleukin-1ß (IL-1ß) to simulate inflammatory environmental conditions and OA in vitro. The in vivo OA rat model was established by anterior cruciate ligament transection (ACLT) on rat. Our investigation discovered that MRL lowers the IL-1ß-activated tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX2) and inducible nitric-oxide synthase (iNOS) expression in chondrocytes. Moreover, MRL effectively alleviates IL-1ß-induced extracellular matrix (ECM) degradation and promotes ECM synthesis in chondrocytes by upregulating the mRNA level expression of collagen-II and aggrecan (ACAN), downregulating the expression of matrix metalloproteinases-3,-13 (MMP-3, MMP-13), and a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5). Gene expression profiles of different groups identified DEGs that were mainly enriched in functions associated with NF-κB signaling pathway, and other highly enriched in functions related to TNF, IL-17, Rheumatoid arthritis and cytokine-cytokine receptor signaling pathways. Venn interaction of DEGs from the abovementioned five pathways showed that Nfkbia, Il1b, Il6, Nfkb1, Ccl2, Mmp3 were highly enriched DEGs. In addition, our research revealed that MRL suppresses NF-κB and modulates the Nrf2/HO-1/JNK signaling pathway activated by IL-1ß in chondrocytes. In vivo research shows that MRL slows the progression of OA in rats. Our findings imply that MRL might be a viable OA therapeutic choice.

Synovial microenvironment in temporomandibular joint osteoarthritis: crosstalk with chondrocytes and potential therapeutic targets.

Temporomandibular joint osteoarthritis (TMJOA) is considered to be a low-grade inflammatory disease involving multiple joint tissues. The crosstalk between synovium and cartilage plays an important role in TMJOA. Synovial cells are a group of heterogeneous cells and synovial microenvironment is mainly composed of synovial fibroblasts (SF) and synovial macrophages. In TMJOA, SF and synovial macrophages release a large number of inflammatory cytokines and extracellular vesicles and promote cartilage destruction. Cartilage wear particles stimulate SF proliferation and macrophages activation and exacerbate synovitis. In TMJOA, chondrocytes and synovial cells exhibit increased glycolytic activity and lactate secretion, leading to impaired chondrocyte matrix synthesis. Additionally, the synovium contains mesenchymal stem cells, which are the seed cells for cartilage repair in TMJOA. Co-culture of chondrocytes and synovial mesenchymal stem cells enhances the chondrogenic differentiation of stem cells. This review discusses the pathological changes of synovium in TMJOA, the means of crosstalk between synovium and cartilage, and their influence on each other. Based on the crosstalk between synovium and cartilage in TMJOA, we illustrate the treatment strategies for improving synovial microenvironment, including reducing cell adhesion, utilizing extracellular vesicles to deliver biomolecules, regulating cellular metabolism and targeting inflammatory cytokines.

SerpinA3N limits cartilage destruction in osteoarthritis by inhibiting macrophage-derived leucocyte elastase.

OBJECTIVES: Inflammatory mediators such as interleukin 6 (IL-6) are known to activate catabolic responses in chondrocytes during osteoarthritis (OA). This study aimed to investigate the role of a downstream target gene of IL-6, the serine protease inhibitor SerpinA3N, in the development of cartilage damage in OA. METHODS: RNA sequencing was performed in murine primary chondrocytes treated with IL-6, and identified target genes were confirmed in human and murine OA cartilage samples. Male cartilage-specific Serpina3n-deficient mice and control mice underwent meniscectomy (MNX) or sham surgery at 10 weeks of age. Intra-articular injections of SerpinA3N or sivelestat (an inhibitor of leucocyte elastase (LE), a substrate for SerpinA3N) were performed in wild-type mice after MNX. Joint damage was assessed 3-9 weeks after surgery by histology and micro-CT. The effect of sivelestat was assessed in cartilage explants exposed to macrophage-derived conditioned media. RESULTS: RNA sequencing revealed that SerpinA3N is a major target gene of IL-6 in chondrocytes. The expression of SerpinA3N is increased in OA cartilage. Conditional loss of SerpinA3N in chondrocytes aggravated OA in mice, while intra-articular injection of SerpinA3N limited joint damage. Chondrocytes did not produce serine proteases targeted by SerpinA3N. By contrast, macrophages produced LE on IL-6 stimulation. Sivelestat limited the cartilage catabolism induced by conditioned media derived from IL-6-stimulated macrophages. Additionally, an intra-articular injection of sivelestat is protected against OA in the MNX model. CONCLUSIONS: SerpinA3N protects cartilage against catabolic factors produced by macrophages, including LE. SerpinA3N and LE represent new therapeutic targets to dampen cartilage damage in OA.

Connexin 43 Modulation in Human Chondrocytes, Osteoblasts and Cartilage Explants: Implications for Inflammatory Joint Disorders.

Connexin 43 (Cx43) is crucial for the development and homeostasis of the musculoskeletal system, where it plays multifaceted roles, including intercellular communication, transcriptional regulation and influencing osteogenesis and chondrogenesis. Here, we investigated Cx43 modulation mediated by inflammatory stimuli involved in osteoarthritis, i.e., 10 ng/mL Tumor Necrosis Factor alpha (TNFα) and/or 1 ng/mL Interleukin-1 beta (IL-1ß), in primary chondrocytes (CH) and osteoblasts (OB). Additionally, we explored the impact of synovial fluids from osteoarthritis patients in CH and cartilage explants, providing a more physio-pathological context. The effect of TNFα on Cx43 expression in cartilage explants was also assessed. TNFα downregulated Cx43 levels both in CH and OB (-73% and -32%, respectively), while IL-1ß showed inconclusive effects. The reduction in Cx43 levels was associated with a significant downregulation of the coding gene GJA1 expression in OB only (-65%). The engagement of proteasome in TNFα-induced effects, already known in CH, was also observed in OB. TNFα treatment significantly decreased Cx43 expression also in cartilage explants. Of note, Cx43 expression was halved by synovial fluid in both CH and cartilage explants. This study unveils the regulation of Cx43 in diverse musculoskeletal cell types under various stimuli and in different contexts, providing insights into its modulation in inflammatory joint disorders.

Alpha-ketoglutarate ameliorates age-related and surgery induced temporomandibular joint osteoarthritis via regulating IKK/NF-κB signaling.

Recent studies have shed light on the important role of aging in the pathogenesis of joint degenerative diseases and the anti-aging effect of alpha-ketoglutarate (αKG). However, whether αKG has any effect on temporomandibular joint osteoarthritis (TMJOA) is unknown. Here, we demonstrate that αKG administration improves condylar cartilage health of middle-aged/aged mice, and ameliorates pathological changes in a rat model of partial discectomy (PDE) induced TMJOA. In vitro, αKG reverses IL-1ß-induced/H2O2-induced decrease of chondrogenic markers (Col2, Acan and Sox9), and inhibited IL-1ß-induced/ H2O2-induced elevation of cartilage catabolic markers (ADAMTS5 and MMP13) in condylar chondrocytes. In addition, αKG downregulates senescence-associated (SA) hallmarks of aged chondrocytes, including the mRNA/protein level of SA genes (p16 and p53), markers of nuclear disorders (Lamin A/C) and SA-ß-gal activities. Mechanically, αKG decreases the expressions of p-IKK and p-NF-κB, protecting TMJ from inflammation and senescence-related damage by regulating the NF-κB signaling. Collectively, our findings illuminate that αKG can ameliorate age-related TMJOA and PDE-induced TMJOA, maintain the homeostasis of cartilage matrix, and exert anti-aging effects in chondrocytes, with a promising therapeutic potential in TMJOA, especially age-related TMJOA.