Inflammation In Chondrocytes Research Articles

Three-dimensional cell culture-derived extracellular vesicles loaded alginate/hyaluronic acid composite scaffold as an optimal therapy for cartilage defect regeneration.

Osteoarthritis (OA) is a chronic musculoskeletal disease characterized by joint inflammation and progressive degeneration of articular cartilage. Currently a definitive cure for OA remains to be a challenge due to the very low self-repair capacity of cartilage, thus development of more effective therapies is needed for cartilage repair. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown great potential as therapeutic agents for stimulating regeneration of articular cartilage. However, a standardized protocol is still lacking for manufacturing of highly active EVs for clinical applications. This study aimed to investigate the efficient production of highly active EVs by 3-dimensional (3D) MSC culture, verify the reparative efficacy of EVs on cartilage defect and elucidate the repair mechanisms. Umbilical cord MSCs were embedded in alginate to form MSC spheroids for 3D culture in human platelet lysate (hPL)-containing medium, which produced 3D culture-derived EVs (3D-EVs) with a significantly improved yield. The 3D-EVs expressed higher level of VEGF, and appeared superior to 2D monolayer MSC culture-derived EVs (2D-EVs) to improve migration and proliferation in MSCs and inflammatory chondrocytes, and to suppress expression of cartilage-degrading factors. Importantly, the 3D-EVs and sodium alginate (SA)-hyaluronic acid (HA) composite hydrogel (3D-EVs/SA-HA) demonstrated significantly improved therapeutic efficacy than 2D-EVs/ SA-HA hydrogel for repair of cartilage defect in vivo. The underlying mechanisms are associated with the concomitant upregulation of type II collagen and cartilage synthesis and downregulation of MMP13 in cartilage tissues. Collectively, these data showed that highly active MSC EVs could be efficiently manufactured by 3D cell culture with hPL-containing medium, and these EVs were superior to 2D-EVs for the repair of articular cartilage defect.

Multifunctional Small RNA Endogenous Modified Exosome‐Loaded Hydrogel Based on Photocrosslinking for Cartilage Repair

AbstractExosomes derived from bone marrow mesenchymal stem cells (BMSCs) hold significant potential in the treatment of osteoarthritis (OA). However, simple exosomes therapy often proves insufficiently effective and challenging to achieve optimal therapeutic outcome. Herein, an innovative injectable multifunctional composite hydrogel (ExoshEZH2@HG) is composed of photocrosslinking partially modified hyaluronic acid with o‐nitrobenzyl alcohol (HA‐NB), gelatin, and shRNA‐EZH2 endogenously modified exosomes (ExoshEZH2), designed for cartilage defects, which are a primary cause of early OA. It exhibits rapid and controllable photo‐induced gelation characteristics, resembling human cancellous bone compressive strength, and boasting excellent biocompatibility. In vitro studies indicate that ExoshEZH2@HG slowly releases ExoshEZH2, promoting the migration and infiltration of BMSCs into surrounding tissues. By inhibiting the overexpression of EZH2, ExoshEZH2 suppresses histone hypermethylation and the nuclear factor kappa‐B (NF‐κB) signaling pathway, thereby inhibiting inflammation and apoptosis in chondrocytes. Simultaneously, it promotes the regeneration of the extracellular matrix and the chondrogenic differentiation of BMSCs. Within a rat cartilage defect model, ExoshEZH2@HG significantly facilitates the regeneration of cartilage tissue resembling hyaline cartilage in the defect area, preventing the progression of OA. In summary, this multifunctional composite hydrogel offers a promising approach for the treatment of cartilage defects and the prevention of OA through multiple synergistic effects.

Upregulating lncRNA GUSBP11 protects chondrocytes from IL-1β-induced inflammatory damage via inhibiting miR-122-5p.

Studies have demonstrated that several lncRNAs exhibit abnormal expression levels in patients suffering from osteoarthritis, and in-depth investigation of these aberrantly expressed lncRNAs may pave the way for innovative therapeutic strategies targeting OA. The aim of this study was to examine the expression of glucuronidase beta pseudogene 11 (GUSBP11) in OA patients and to elucidate its potential molecular mechanism. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to detect GUSBP11 levels on cartilage tissues and serum samples obtained from OA patients. To establish an in vitro OA cell model, interleukin-1β (IL-1β) was utilized to induce CHON-001 and ATDC5 cell lines. Cell counting kit-8 (CCK-8) assay and flow cytometry were performed to evaluate cell viability and apoptosis, and enzyme-linked immunosorbent assay (ELISA) was employed to qualify the levels of inflammatory factors. StarBase database predicted that miR-122-5p was the target gene of GUSBP11. Subsequently, luciferase reporter genes were conducted to validate this interaction. Potential target genes of miR-122-5p were predicted, followed by gene function annotation and correlation analysis of these targets. Our findings revealed that GUSBP11 expression was markedly decreased in both the cartilage tissues and serum of OA patients. Diminished levels of GUSBP11 showed high diagnostic accuracy for OA. In the IL-1β-induced OA cell model, GUSBP11 expression was notably reduced, leading to decreased cell viability, an increase in apoptotic cells, and elevated levels of inflammatory factors. Up-regulation of GUSBP11 significantly ameliorated these adverse effects. Luciferase reporter genes confirmed the interaction between GUSBP11 and miR-122-5p, indicating that an increase in miR-122-5p drastically inhibited cell viability while promoting apoptosis and inflammation. In conclusion, within the context of the in vitro OA cell model, GUSBP11 appears to exacerbate IL-1β-induced chondrocyte inflammation through the up-regulation of miR-122-5p. This underscores the potential of GUSBP11 as a novel target and avenue for therapeutic intervention in the treatment of OA.

AM1241 inhibits chondrocyte inflammation and ECM degradation through the Nrf2/HO-1 and NF-κB pathways and alleviates osteoarthritis in mice

BackgroundThis study aimed to investigate the impact of AM1241 on lipopolysaccharide (LPS)-induced chondrocyte inflammation in mice and its potential mechanism for improving osteoarthritis (OA).MethodsThe OA mice model was established employing the refined Hulth method. The impact of different concentrations of AM1241 on mice chondrocyte activity was detected using CCK-8. Changes in the levels of LPS-induced inflammatory factors and cartilage extracellular matrix (ECM) degradation in chondrocytes were determined by western blot, RT-qPCR, ELISA, and immunofluorescence assays, respectively. The specific action modes and binding sites of AM1241 with NEMO/IκB kinases (IKKs) in the NF-κB pathway and Keap1 protein in the Nrf2 pathway were predicted via molecular docking and molecular dynamics simulation, and the NF-κB and Nrf2 pathways were detected using western blot and immunofluorescence. In vivo, the impact of AM1241 on OA mice was analyzed through safranin-fast green staining, IHC staining, Mankin score, and microCT.ResultsAM1241 inhibited the levels of LPS-induced transforming growth factor-β (TGF-β1), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), matrix metalloproteinase-13 (MMP-13), and a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS-5) and diminished the degradation of type II collagen and Aggrecan. For the mechanism, AM1241 regulated the NF-kB and Nrf2/HO-1 signaling pathways by binding to NEMO/IKKβ and Keap1 target proteins and suppressed the activation of the NF-κB signaling pathway by activating the Nrf2 in chondrocytes. In vivo, AM1241 inhibited bone anabolism, mitigated articular cartilage hyperplasia and wear, and reduced the Mankin score in mice, thereby hindering the development of OA.ConclusionAM1241 inhibited activation of the NF-κB signaling pathway via activating Nrf2. It suppressed the expression of inflammation factors and the degradation of ECM in vitro, and improved OA in mice in vivo, suggesting its potential as an effective drug candidate for the treatment of OA. The remarkable efficacy of AM1241 in alleviating murine OA positions it as a potential therapeutic strategy in the clinical management of OA diseases.

Alleviating the IL-1β-stimulated extracellular matrix degradation in osteoarthritis, and chondrocyte inflammation by Morinda officinalis polysaccharide via the SIRT6/NF-κB pathway.

Morinda officinalis polysaccharide (MOP) is a major active component of Morinda officinalis, known for its roles in supporting bone health and reducing oxidation and inflammation. However, no studies to date have specifically examined the effects of MOP on interleukin-1β (IL-1β)-stimulated chondrocyte inflammation or the progression of osteoarthritis (OA). To investigate, cell counting kit-8 assays were performed to evaluate MOP's impact on the viability of human chondrocytes (C28/I2 c...

Alleviating osteoarthritis-induced damage through extracellular vesicles derived from inflammatory chondrocytes.

The role of extracellular vesicles (EVs) derived from inflammatory chondrocytes in EV-based therapy for osteoarthritis (OA) has received little attention. We examined the effects of EVs derived from both normal rat chondrocytes (nEVs) and IL-1β-treated rat chondrocytes (iEVs) on IL-1β-treated rat chondrocytes, macrophages, and osteoblasts, alongside mRNA-seq and miRNA-seq analyses of both them. Additionally, nEVs and iEVs were administered intra-articularly in the joints of rat models subjected to anterior cruciate ligament transection (ACLT), and the morphological alterations across the joints were assessed. These findings indicated that iEVs, compared with nEVs, significantly enhanced collagen II synthesis in IL-1β-treated chondrocytes, accompanied by marked increases in ER stress and autophagy. In comparison to nEVs, iEVs exhibited a greater effect on facilitating M2-type macrophage polarization while simultaneously diminishing M1-type polarization, a process likely mediated by the downregulation of chemotactic cytokines such as Cxcl10, Ccl5, Cxcl9, Cxcl1, and Cxcl11. iEVs exerted a more pronounced influence on the phenotypic characteristics of IL-1β-treated osteoblasts than nEVs. In the ACLT-rat model, iEVs, akin to nEVs, effectively mitigated articular cartilage degradation. However, there was no significant difference in OARSI Scores between the two groups, despite iEVs exerting a greater effect on increasing hyaline cartilage thickness and proteoglycan content. iEVs were superior to nEVs in attenuating synovium inflammation and promoting trabecula formation in the femur subchondral bone. Consequently, iEVs, akin to nEVs, significantly alleviated OA-induced damage. Moreover, iEVs outperformed nEVs in certain aspects, notably in augmenting hyaline cartilage, reducing synovium inflammation, and promoting trabecular formation in the subchondral bone during the early stage of OA.

Microfluidic Synthesis of miR-200c-3p Lipid Nanoparticles: Targeting ZEB2 to Alleviate Chondrocyte Damage in Osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degeneration. Chondrocyte inflammation, apoptosis, and extracellular matrix degradation accelerated OA progression. MicroRNA (miRNA) has the potential to be a therapeutic method for osteoarthritis. However, it is difficult to penetrate the cell to exercise its biological function, and its extracellular effect is unclear. lipo-AgPEI-miR-200c-3p was created by combining miR-200c-3p with silver nitrate polyvinylimine nanoparticles on a microfluidic device. The drug release curve, stability, temperature sensitivity, cytotoxicity, and the impact of lipo-AgPEI-miR-200c-3p on the expression of proteins linked to matrix disintegration, apoptosis, and inflammatory factors were all detected. Results showed that the particle size of Lipo-AgPEI-miR-200c-3p was about 130 nm, the Zeta potential was lowered to 1.08±0.12 mV. Lipo-AgPEI-miR-200c-3p could increase cell viability, prevent cell apoptosis, and decrease the expression levels of TNF-α, IL-6, IL-1β, and MCP-1 in ADTC5 cells following LPS stimulation. MMP3, MMP13, and ADAMTS-4 expression was downregulated whereas collagen II expression was upregulated. The ZEB2 expression was greatly elevated following LPS stimulation and dramatically decreased following transfection of miR-200c-3p. Collagen II expression rose following transfection of si-ZEB2, whereas the expression levels of inflammatory factors, apoptosis-related proteins, MMP3, MMP13, and ADAMTS-4 decreased. The dual luciferase experiment demonstrated that ZEB2 was the target gene of miR-200c-3p. The synergistic effect of AgPEI and miR-200c-3p can inhibit the inflammatory response, apoptosis, and matrix degradation of chondrocytes. Lipo-AgPEI-miR-200c-3p can also improve transfection efficiency and obtain good physicochemical properties of drugs. miR-200c-3p may be crucial in the development of OA and can influence the target gene ZEB2, control the inflammatory response, apoptosis, and chondrocyte matrix breakdown.

Combined single-cell RNA sequencing and mendelian randomization to identify biomarkers associated with necrotic apoptosis in intervertebral disc degeneration

BackgroundIntervertebral disc degeneration (IDD) is associated with back pain; back pain is a world-wide contributor to poor quality of life, while necroptosis has the characteristics of necroptosis and apoptosis, however, its role in IDD is still unclear. Therefore, the aim of this study was to identify biomarkers associated with necroptosis in IDD. PurposeTo explore biomarkers associated with necroptosis in IDD, reveal the pathogenesis of IDD, as well as provide new directions for the diagnosis and treatment of this disease. Study design/settingsRetrospective cohort study. Our study employs scRNA-seq coupled with MR analysis to investigate the causal relationship between necroptosis and IDD, laying a foundational groundwork for unveiling the intricate pathogenic mechanisms of this condition. MethodsData quality control and normalisation was executed in single-cell dataset, GSE205535. Then, different cell types were obtained by cell annotation through marker genes. Subsequently, chi-square test was employed to assess the distribution difference of different cell types between IDD and control to screen key cells. AUCell was applied to calculate necroptosis-related genes (NRGs) scores of all cell types, further key cells were divided into high and low NRGs groups according to the median AUC scores of different cell types. Afterwards, the differentially expressed genes (DEGs) within the two score groups were screened. Then, the genes that had causal relationship with IDD were selected as biomarkers by univariate and multivariate Mendelian randomization (MR) analysis. Finally, the expression of biomarkers in different cell types and pseudo-time analysis was analyzed separately. ResultsIn GSE205535, 16 different cell populations identified by UMAP cluster analysis were further annotated to 8 cell types using maker genes. Afterwards, 53 DEGs were screened between the high and low NRGs groups. In addition, 9 genes with causal relationship with IDD were obtained by univariate MR analysis, further multivariate MR analysis proved that NT5E and TMEM158 had a direct causal relationship with IDD, which were used as biomarkers in this study. This study not only found that the expression levels of NT5E and TMEM158 were higher in IDD group, but also found that fibrochondrocytes and inflammatory chondrocytes were the key cells of NT5E and TMEM158, respectively. In the end, the biomarkers had the same expression trend in the quasi-time series, and both of them from high to low and then increased. ConclusionNT5E and TMEM158, as biomarkers of necroptotic apoptotic IDD, were causally associated with IDD. clinical significanceThe understanding of chondrocytes as key cells provides new perspectives for deeper elucidation of the pathogenesis of IDD, improved diagnostic methods, and the development of more effective treatments. These findings are expected to provide a more accurate and personalised approach to clinical diagnosis and treatment, thereby improving the prognosis and quality of life of patients with IDD.

Antioxidant taurine inhibits chondrocyte ferroptosis through upregulation of OGT/Gpx4 signaling in osteoarthritis induced by anterior cruciate ligament transection.

The aim of this study was to investigate the potential molecular mechanisms by which taurine protects against cartilage degeneration. The anterior cruciate ligament transection (ACLT) surgery was used to construct an animal model of osteoarthritis (OA). Metabolomics was used to identify characteristic metabolites in osteoarthritic chondrocytes. Transcriptomics and metabolomics were used to explore potential mechanisms by which the small molecule metabolite taurine protects against inflammatory chondrocyte damage. Cell transfection and small molecule inhibitors/agonists were used to validate the molecular mechanisms by which taurine protects inflammatory chondrocytes in vitro. Finally, adeno-associated virus and small molecule inhibitors/agonists were used to validate the molecular mechanisms by which taurine protects against cartilage degeneration in vivo. Metabolomic assays identified taurine as a possible key metabolic molecule in the progression of OA. Transcriptomics and metabolomics revealed that O-GlcNAc transferase (OGT)-dependent O-GlcNAcylation and Gpx4-dependent ferroptosis may mediate the inflammatory protective effects of taurine on chondrocytes, which was further confirmed by gain and loss of function in vitro. Subsequently, further experiments indicated that the possible existence of a direct binding site for Gpx4 and OGT proteins, which provides evidence for the presence of O-GlcNAc modification of Gpx4 protein. Finnaly, we demonstrated that Gpx4-dependent ferroptosis and OGT-dependent O-GlcNAcylation may be potential mechanisms by which taurine protects against cartilage degeneration in vivo. Antioxidant taurine inhibits chondrocyte ferroptosis through upregulation of OGT/Gpx4 signaling. Supplementation with taurine, a safe nonessential amino acid, may be a potential therapeutic strategy for OA.

Comparison of Concentration- and Homology-Dependent Effects of the Proinflammatory Cytokine Interleukin-1β (IL-1β) in a Bovine Chondrocyte Inflammation Model.

Inflammation models with the proinflammatory cytokine interleukin-1β (IL-1β) are widely used in the in vitro investigation of new therapeutic approaches for osteoarthritis (OA). The aim of this study was to systematically analyze the influence of IL-1β in a 3D chondral pellet culture model. Bovine articular chondrocytes were cultured to passage 3 and then placed in pellet culture. Titration of IL-1β (100-0.1 ng/mL) was performed with both human and bovine recombinant protein in chondrocyte culture for 2 weeks. Gene expression of anabolic (collagen 2, aggrecan, cartilage oligomeric protein (COMP), proteoglycan-4 (PRG-4)), catabolic matrix metallo proteinases (MMP-3, MMP-13), dedifferentiation (collagen 1) markers and inflammatory cytokines IL-6 and IL-8 was determined. Analysis of the cell culture medium was performed for the inflammatory markers IL-6 and nitric oxide (NO). In general, the influence of IL-1β was shown by a decrease in the expression of anabolic markers (collagen 2, aggrecan, PRG-4), whereas the catabolic markers MMP-3 and MMP-13 as well as the inflammatory markers IL-6 and IL-8 were significantly increased. This was observed both at the early time point (day 4) and at the late time point (day 14). The described inflammatory effects were confirmed by increased concentration-dependent release of NO and IL-6. The threshold concentration for a detectable effect compared to control differed between groups, but was reached earlier by homologous application of IL-1β. This study provides a systematic evaluation of IL-1β-specific effects on chondrocytes in a 3D pellet culture model, which is highly relevant for comparisons of studies in OA-specific drug development.

Circ-PDE1C/miR-766-3p/SGTB axis regulates the IL-1β-induced apoptosis, inflammation and oxidative stress in human chondrocytes

Abstract Background Osteoarthritis (OA) is a common degenerative joint disease. Circular RNA Phosphodiesterase 1 C (circ-PDE1C, hsa_circ_0134111) has participated in the IL-1β-induced chondrocyte damages. The objective of our study was to explore the molecular mechanism of circ-PDE1C. Methods Circ-PDE1C, microRNA-766-3p (miR-766-3p) or Small Glutamine Rich Tetratricopeptide Repeat Co-Chaperone Beta (SGTB) expression was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell counting kit-8 (CCK-8) assay and flow cytometry were used to analyze proliferation and apoptosis, respectively. Western blotting assay was performed for protein detection. The inflammatory cytokines were measured by Enzyme-linked immunosorbent assay (ELISA). Oxidative stress was assessed by commercial kits. Target analysis was conducted by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Results Circ-PDE1C was abnormally overexpressed in OA tissues and IL-1β-exposed chondrocytes. Downregulation of circ-PDE1C alleviated the IL-1β-induced cell apoptosis, inflammation, extracellular matrix degradation and oxidative stress. Circ-PDE1C could interact with miR-766-3p to serve as miRNA sponge. The function of si-circ-PDE1C was attributed to the inhibition of miR-766-3p. Additionally, miR-766-3p directly targeted the 3’UTR of SGTB. The miR-766-3p upregulation impeded the IL-1β-triggered cell damages through reducing the level of SGTB. Moreover, SGTB expression was regulated by circ-PDE1C via binding to miR-766-3p in IL-1β-induced chondrocytes. Conclusion Altogether, circ-PDE1C enhanced the IL-1β-induced dysfunction in chondrocytes via upregulating SGTB by targeting miR-766-3p.

The Potential Role of Bone Morphogenetic Protein-2/-4 in Excessive Mechanical Overloading-Initiated Joint Degeneration.

Excessive mechanical overloading of articular cartilage caused by excessive exercise or severe trauma is considered a critical trigger in the development of osteoarthritis (OA). However, the available clinical theranostic molecular targets and underlying mechanisms still require more elucidation. Here, we aimed to examine the possibility that bone morphogenetic proteins (BMPs) serve as molecular targets in rat cartilages and human chondrocytes under conditions of excessive mechanical overloading. Two rat models involving high-intensity running training and surgery for destabilization of medial meniscus, along with a cell model subjected to cyclic tensile strain, were established to simulate and investigate excessive mechanical overloading effects on cartilages/chondrocytes. We employed various methods, including immunohistochemistry, real-time polymerase chain reaction, western blot analysis, and enzyme-linked immunosorbent assay, to evaluate the expression, secretion, phosphorylation, and nuclear translocation of mRNA/proteins in cartilages and chondrocytes. Our findings revealed a simultaneous upregulation of BMP-2 and downregulation of BMP-4 in degenerated and inflamed cartilages and chondrocytes under excessive mechanical overloading. Furthermore, toll-like receptor 2 and nuclear factor kappa B-p50/p65 subunits signaling were identified as regulators governing this distinct expression pattern. Treatment with recombinant BMP-2 and/or BMP-4 proteins significantly ameliorated cartilage degeneration and chondrocyte inflammation induced by excessive mechanical overloading. These results strongly suggest that BMP-2 upregulation and BMP-4 downregulation might represent mechanisms for self-rescue and degeneration in damaged cartilage/chondrocytes, respectively. Our findings advance new insights that BMP-2/-4 might be potential molecular targets for excessive mechanical overloading-caused OA development and should be taken into account in future clinical applications.

Chondroitin Sulfate Nanovectorized by LC-PUFAs Nanocarriers Extracted from Salmon (Salmo salar) by Green Process with Decreased Inflammatory Marker Expression in Interleukin-1β-Stimulated Primary Human Chondrocytes In Vitro Culture.

Chondroitin sulfate (CS), a glycosaminoglycan, supports health through various physiological functions, including tissue protection, bone growth, and skin aging prevention. It also contributes to anticoagulant or anti-inflammatory processes, with its primary clinical use being osteoarthritis treatment. This study presents the results of the valorization of lipids and CS, both extracted from salmon co-products through enzymatic processes. The polar lipids, naturally rich in long-chain fatty acids (docosahexaenoic acid DHA C22:6 n-3 and eicosapentaenoic acid EPA C20:5 n-3), and the CS, primarily located in the nasal cartilage, were separated and concentrated before being characterized using various techniques to determine functional and lipid composition. These compounds were then used to formulate liposomes of 63 to 95 nm in size composed of 19.38% of DHA and 7.44% of EPA and encapsulating CS extract with a Δdi-4S/Δdi-6S ratio of 0.53 at 2 weight masses (10-30 kDa and >30 kDa) or CS standard all at two different concentrations. Liposomes were tested on human chondrocytes in inflamed conditions. Thus, compatibility tests, the expression of various inflammation markers at transcriptional and molecular levels, nitrites, and the amount of collagenase produced were analyzed. The results showed that CS, in synergy with the liposomes, played a positive role in combating chondrocyte inflammation even at a low concentration.

Triptolide attenuates LPS-induced chondrocyte inflammation by inhibiting inflammasome activation via the Wnt/β-catenin and NF-κB signaling pathways.

Osteoarthritis (OA) is a common form of arthritis characterized by subchondral bone proliferation and articular cartilage degeneration. Recently, the Nod-like receptor pyrin domain 3 (NLRP3) inflammasome has gained attention due to its association with synovial inflammation in OA. Triptolide (TP), known for its immunosuppressive and anti-inflammatory effects, has been studied in various diseases. However, the specific impact of TP on OA and its underlying mechanism remains largely unexplored. In this study, chondrocytes were treated with a specific concentration of TP, and subsequent analysis through Western blotting and immunofluorescence staining revealed decreased expression levels of MMP-13, NLRP3, Caspase-1, ASC, β-catenin, p-p65, and IκB compared to the model group. ELISA results demonstrated significantly lower levels of IL-1β, IL-18, and TNF-α in the TP treatment group compared to the model group. In addition, triptolide ameliorates the degradation of the extracellular matrix (ECM) by enhancing the expression of collagen-II. In conclusion, our findings suggest that TP exhibits anti-inflammatory effects on chondrocytes in the presence of LPS-induced inflammation by inhibiting the activation of the NLRP3 inflammasome via the Wnt/β-catenin and NF-κB pathway. These results contribute to a better understanding of TP's potential therapeutic benefits in managing OA.

WTAP mediates IL-1β-induced chondrocyte injury by enhancing CA12 mRNA stability depending on m6A modification

BackgroundOsteoarthritis (OA) poses a significant risk to the mobility of patients. Carbonic anhydrase 12 (CA12) can boost apoptosis and inflammation in several cancers, but its role in OA is unknown.MethodsDifferentially expressed genes in OA were analyzed using the GEO database (GSE169077). RT-qPCR and western blot estimated relative mRNA and protein levels of CA12. Cell viability and apoptosis were estimated by cell counting and flow cytometry assays. Oxidative stress (OxS) was determined by detecting with ROS and MDA levels, as well as CAT and SOD activities. Cytokine levels of IL-6 and TNF-α were detected by ELISA. Parameters associated with apoptosis and extracellular matrix (ECM) were detected by western blot. The m6A modification profile was determined by methylated RNA immunoprecipitation assays.ResultsRelative CA12 and wilms’ tumor 1-associating protein (WTAP) mRNA and protein levels were overexpressed in OA articular cartilages and IL-1β-challenged chondrocytes CHON-001. CA12 silencing impaired IL-1β-induced cell apoptosis, inflammation, OxS, and ECM degradation in chondrocytes. Yet, CA12 overexpression exerted an opposing function. WTAP reinforced the stability of CA12 mRNA depending on the m6A modification. Furthermore, WTAP knockdown weakened cell apoptosis, inflammation, OxS, and ECM degradation in chondrocytes induced by IL-1β, but these changes were impaired after CA12 overexpression. In addition, WTAP knockdown mitigates cartilage degeneration in DMM-induced mouse models.ConclusionIL-1β-induced WTAP enhances CA12 mRNA stability depending on m6A modification, thus promoting chondrocyte apoptosis, inflammatory response, OxS, and ECM degradation, providing evidence to support the possibility of WTAP and CA12 as potential targets for OA treatment.

Substrate stiffness regulates the proliferation and inflammation of chondrocytes and macrophages through exosomes.

Osteoarthritis (OA) progression is characterized by decreased cartilage stiffness and degradation of the extracellular matrix (ECM), which significantly influence cartilage behavior and fate. In contrast, processes such as chondrocyte calcification and aging often result in increased stiffness. Despite extensive studies on how ECM stiffness regulates cellular functions, the impact of substrate stiffness on the cartilage microenvironment and intercellular communications remains not well understood. Using tunable stiffness Gelatin methacryloyl (GelMA) hydrogel, we demonstrated that a potential optimal substrate stiffness can promote maximal chondrocyte proliferation and exosome secretion. The exogenous addition of stiffness-tuned exosomes induced significant changes in chondrocyte morphology, proliferation, migration, and inflammation. Notably, blocking Yes-associated protein (YAP) synthesis negated the proliferation enhancement induced by exosomes from chondrocytes cultured on medium stiffness substrates (ExoMedium), confirming that substrate stiffness regulates cell proliferation through exosomes by modulating YAP expression and its nuclear localization. Moreover, our study revealed that exosomes from medium stiffness substrates-mimicking normal cartilage stiffness-not only reduce inflammation in chondrocytes but also shift macrophage polarization from M1 to M2. Conversely, exosomes from soft stiffness substrates, akin to osteoarthritic tissue, exacerbate chondrocytes inflammation and M1 macrophage polarization. These findings highlight the crucial role of stiffness-tuned exosomes in OA progressing, affecting chondrocyte proliferation, migration, inflammation and macrophage polarization, and provide new insights into the potential novel treatment strategies using engineered scaffolds and exosomes. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by decreased cartilage stiffness and degradation of the extracellular matrix (ECM). While some studies suggest that increased substrate stiffness enhances cell proliferation, others have reported the opposite effect. Whether there exists an optimal matrix stiffness that promotes chondrocytes anabolism and how matrix stiffness regulates the cartilage microenvironment and intercellular communications remain unclear. Utilizing tunable stiffness Gelatin methacryloyl (GelMA) hydrogel, this study demonstrated that a potential optimal substrate stiffness can maximize chondrocyte proliferation and exosome secretion. The introduction of stiffness-tuned exosomes induced significant changes in chondrocyte and macrophage proliferation, migration, and inflammation, offering new insights into OA progression and highlighting their potential as a promising therapeutic strategy for osteoarthritis treatment and tissue regeneration.

Chondroitin sulfate alleviated lipopolysaccharide-induced arthritis in feline and canine articular chondrocytes through regulation of neurotrophic signaling pathways and apoptosis

Osteoarthritis (OA) is a pervasive degenerative joint disease affecting companion animals, characterized by chronic inflammation and cartilage degradation. However, the effectiveness of chondroitin sulfate (CS) in treating OA in dogs and cats remains controversial. This study aimed to determine the therapeutic effects and molecular mechanisms of CS on lipopolysaccharide (LPS)-induced inflammation in feline and canine articular chondrocytes (FAC and CAC) at the cellular level in vitro. Our findings demonstrated that CS treatment (800 µg/mL) significantly enhanced cell viability and reduced oxidative stress in FAC and CAC, as evidenced by decreased levels of reactive oxygen species and increased activities of antioxidant enzymes. Furthermore, CS treatment effectively suppressed LPS-induced secretion of pro-inflammatory cytokines, including interleukin-1, tumor necrosis factor-α, interleukin-8, interleukin-10, and matrix metalloproteinases-3, and reduced apoptosis, as confirmed by fluorescence staining and flow cytometry. Transcriptomic analysis revealed that CS upregulated neurotrophic signaling pathways, promoting cell survival and proliferation. Metabolomic analysis indicated that CS treatment upregulated metabolites associated with glycerophospholipid and purine metabolism, suggesting enhanced membrane integrity and energy metabolism. Conversely, pathways involved in protein catabolism and arachidonic acid metabolism were downregulated, indicating a reduction in inflammatory mediators. Collectively, these findings elucidate the multifaceted role of CS in modulating chondrocyte metabolism and inflammatory responses, highlighting its potential to alleviate OA.

DDX3X Activates Chondrocyte Pyroptosis to Promote Osteoarthritis Progression.

The RNA-binding protein DDX3X is associated with several biological processes including inflammation and immunity. However, the role of DDX3X in the pathology of inflammation-related osteoarthritis (OA) remains unclear. This study was to explore the action of DDX3X in the progression of OA as well as the underlying mechanisms by using RNA immunoprecipitation (RIP), Immunohistochemical (IHC) and DDX3X knockout mice, etc. We found that DDX3X expression was upregulated in cartilage tissue of OA patient. The in vitro study also showed upregulation of DDX3X in the inflammatory chondrocytes stimulated by LPS. DDX3X overexpression reduced cell viability by inducing pyroptosis in chondrocytes. Knockdown of DDX3X rescued LPS-induced chondrocytes pyroptosis through regulating NLRP3 signaling. In addition, DDX3X deletion attenuates osteoarthritis in vivo. In conclusion, DDX3X promotes OA progression by regulating chondrocytes pyroptosis via the activation of NLRP3 signaling.

Inhibition of CRLF1 expression by miR-8485 alleviates IL-1β-induced chondrocyte inflammation, apoptosis, and extracellular matrix degradation

The aim of this study was to investigate the impact of differentially expressed miR-8485 on chondrocyte inflammation in osteoarthritis (OA) and its underlying pathological mechanisms. MiR-8485, which was downregulated in OA, was identified by microarray analysis, and was also found to be decreased in IL-1β-induced C28/I2 cells. miR-8485 down-regulation or IL-1β treated of C28/I2 cells induces a decrease in cellular activity, an increase in apoptosis, an elevation in Cleaved caspase-3, MMP13, and ADAMTS5 protein levels, a decrease in Collagen II and Aggrecan levels, and an increase in the levels of pro-inflammatory factors TNF-α and IL-6. CRLF1 was identified to be a downstream target gene of miR-8485 using bioinformatics prediction and dual luciferase reporter gene assays. CRLF1 was shown to be increased in IL-1β-treated C28/I2 cells, and CRLF1 overexpression partially abrogated the suppressive effect of upregulated miR-8485 on chondrocyte inflammation. In addition, miR-8485 was able to inhibit MAPK/ERK and PI3K/AKT signaling activation by inhibiting CRLF1. In conclusion, miR-8485 was able to inhibit CRLF1 expression and thus inhibit IL-1β-triggered inflammation in chondrocytes, potentially through the inhibition of MAPK/ERK and PI3K/AKT signaling pathways.

Extracellular Vesicles Derived Ectonucleoside Triphosphate Diphosphohydrolase 3 Alleviates Mitochondrial Dysfunction of Osteoarthritis Chondrocytes via Ectonucleotide Pyrophosphatase/Phosphodiesterase 1-Induced Suppression of the AKT/Notch2 Pathway.

Osteoarthritis (OA) is the most common joint disease that usually starts from joint cartilage injury. Notch2, a versatile signaling in human development and diseases, was recently uncovered to be an important regulator in chondrocyte damage. However, in OA chondrocytes, how Notch2 activation is dysregulated is largely unknown. Here, integrated bioinformatic analysis was performed on GEO datasets (GSE199193 and GSE224255) to search potential extracellular vesicles (EVs) derived regulators of Notch2 in OA chondrocytes. Ectonucleoside triphosphate diphosphohydrolase 3 (Entpd3), a most differentially expressed gene both in LPS-induced macrophage EV and Notch2 mutant chondrocytes, was screened as the candidate regulator of Notch2 in OA chondrocytes. Gain-of-function experiments in cultured human chondrocytes revealed that recombinant Entpd3 protein and macrophage EV both had a protective effect on LPS-induced inflammation, oxidative stress, apoptosis, and collagen loss in chondrocytes. In terms of mechanism, Entpd3 directly interacted with ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and suppressed AKT/Notch2-mediated mitochondrial dysfunction. Finally, we verified that either macrophage EV administration or Entpd3 overexpression was able to alleviate osteoarthritis in mice in vivo. In conclusion, Entpd3 is identified as a new regulator in OA, which alleviates mitochondrial dysfunction induced chondrocyte damage via ENPP1-induced suppression of the AKT/Notch2 pathway.