Chondrocyte Extracellular Matrix Research Articles

Britanin alleviates chondrocyte ferroptosis in osteoarthritis by regulating the Nrf2-GPX4 axis

BackgroundOsteoarthritis (OA) is a chronic joint disease characterized by degeneration of the articular cartilage or inflamed joints. Britanin is a guaiacyl-type sesquiterpene lactone possessing anti-inflammatory and antioxidant activities. MethodsIn this study, western blotting, Reverse-transcription PCR (RT-PCR), and immunofluorescent staining method were used to clarifying the molecular mechanisms of britanin in alleviating chondrocyte ferroptosis. The effect of britanin on reactive oxygen species (ROS) and lipid peroxidation levels in chondrocytes was examined by Dihydroethidium (DHE) stain and fluorescent dye BODIPY581 / 591 C11. The destabilized medial meniscus (DMM) model was used to mimic OA, then britanin was injected into the knee articular cavity. The morphological analysis was performed by hematoxylin and eosin (H&E) staining, Safranin O-Fast green staining and Micro-computed tomography (Micro-CT) analysis. Immunohistochemical analysis and Immunofluorescence assay was conducted to verify the effect of britanin in vivo. ResultsThe results revealed that britanin treatment regulates the expression of chondrocyte extracellular matrix–related factors, such as ADAMTS5, MMP13, SOX9, aggrecan, and COL2A1. Britanin also significantly reversed the influence of interleukin (IL)-1β on the levels of reactive oxygen species and lipid peroxidation and upregulated glutathione peroxidase 4 (GPX4) expression in IL-1β-induced chondrocytes. Erastin could alleviate the effects of britanin on chondrocytes. Moreover, britanin could prevent IL-1β-induced oxidative stress and ferroptosis in chondrocytes. Britanin also upregulated the expression of nuclear factor E2–related factor 2 (Nrf2) in IL-1β-induced chondrocytes, and Nrf2 activator played a similar role as that of britanin in IL-1β-induced chondrocytes. Furthermore, injecting britanin into the knee joints of OA rats alleviated cartilage destruction caused by surgical resection. ConclusionsThese findings confirmed that britanin can inhibit ferroptosis in OA chondrocytes through the Nrf2-GPX4 pathway. Thus, britanin has the potential to be developed as a drug for OA therapy.

Modulation of Ras signaling pathway by exosome miRNAs in T-2 toxin-induced chondrocyte injury

This study aims to investigate the impact of T-2 toxin on the regulation of downstream target genes and signaling pathways through exosome-released miRNA in the development of cartilage damage in Kashin-Beck disease (KBD). Serum samples from KBD patients and supernatant from C28/I2 cells treated with T-2 toxin were collected for the purpose of comparing the differential expression of exosomal miRNA using absolute quantitative miRNA-seq. Target genes of differential exosomal miRNAs were identified using Targetscan and Miranda databases, followed by GO and KEGG enrichment analyses. Validation of key indicators of chondrocyte injury in KBD was conducted using Real-time quantitative PCR (RT-qPCR) and Immunohistochemical staining (IHC). A total of 20 exosomal miRNAs related to KBD were identified in serum, and 13 in chondrocytes (C28/I2). The identified exosomal miRNAs targeted 48,459 and 60,612 genes, primarily enriched in cell organelles and membranes, cell differentiation, and cytoskeleton in the serum, and the cytoplasm and nucleus, metal ion binding in chondrocyte (C28/I2). The results of the KEGG enrichment analysis indicated that the Ras signaling pathway may play a crucial role in the pathogenesis of KBD. Specifically, the upregulation of hsa-miR-181a-5p and hsa-miR-21–3p, along with the downregulation of hsa-miR-152–3p and hsa-miR-186–5p, were observed. Additionally, T-2 toxin intervention led to a significant downregulation of RALA, REL, and MAPK10 expression. Furthermore, the protein levels of RALA, REL, and MAPK10 were notably decreased in the superficial and middle layers of cartilage tissues from KBD. The induction of differential expression of chondrocyte exosomal miRNAs by T-2 toxin results in the collective regulation of target genes RALA, REL, and MAPK10, ultimately mediating the Ras signaling pathway and causing a disruption in chondrocyte extracellular matrix metabolism, leading to chondrocyte injury.

A novel apoferritin nanocage with ECM promoting, ferroptosis suppressing and inflammation targeting property for osteoarthritis therapy

Osteoarthritis (OA) currently affects over 15% of the global population. However, OA management primarily focuses on symptom relief, with a lack of targeted and effective therapeutic strategies. SM04690 (Adavivint, Lorecivivint) represents a small molecule with promising potential as a novel agent for cartilage regeneration and pain relief in OA treatment, Phase III clinical trials are currently underway. This study explores its previously unexamined effects on regulating chondrocyte extracellular matrix (ECM) function. However, its targeted delivery to the inflammatory cells remains a challenge; also, ideal therapeutic agent for OA requires to be multi-functional, e.g. suppressing ferroptosis in chondrocytes. In the current study, we designed an ApoFerritin nanocage loaded with SM04690 (SM04690@ApoFn) for OA therapy. In vitro study showed that SM04690 may have chondro-regenerative effect similar to kartogenin (KGN) but works at much lower concentrations. SM04690@ApoFn demonstrated good biocompatibility both in vitro and in vivo. The following studies revealed that SM04690@ApoFn may effectively promote ECM metabolism in chondrocytes, it may also suppress ferroptosis; meanwhile, it may have the ability to targeting the inflammatory chondrocytes through binding to transferrin receptor (TfR). In vivo study demonstrated that SM04690@ApoFn may alleviate the progression of OA in mice. In conclusion, the multifunctional SM04690@ApoFn nanocage shows great potential for OA therapy by promoting ECM metabolism, suppressing ferroptosis and targeting inflammatory chondrocytes.

GPX7 reduces chondrocyte inflammation and extracellular matrix degradation triggered by IL‑1β, via a mechanism mediated by ferroptosis.

During osteoarthritis (OA), chondrocytes become highly active, with increased matrix synthesis and inflammatory cytokine‑induced catabolic pathways. Early intervention strategies targeting pathological changes may attenuate or halt disease progression. The present study aimed to reveal the role of glutathione peroxidase (GPX)7 in OA. For this purpose, a research model was established by inducing C28/I2 human chondrocytes with interleukin (IL)‑1β, and the expression level of GPX7 was determined. To explore its roles, C28/I2 cells were transfected to gain GPX7 overexpression. The effects of GPX7 overexpression on intracellular inflammation, extracellular matrix (ECM) degradation, apoptosis and ferroptosis were then evaluated. In addition, the cells were treated with the ferroptosis inducer, erastin, and its effects on the aforementioned phenotypes were assessed. The level of GPX7 was decreased in response to IL‑1β treatment, and GPX7 overexpression suppressed cellular inflammation, ECM degradation and apoptosis. Moreover, the reduction of lipid peroxidation, ferrous ions and transferrin indicated that GPX7 overexpression inhibited ferroptosis. Subsequently, inflammation, ECM degradation and apoptosis were found to be promoted in the cells upon treatment with erastin. These findings suggested that the regulatory role of GPX7 may be mediated by a pathway involving ferroptosis. On the whole, the present study revealed that GPX7 reduces IL‑1β‑induced chondrocyte inflammation, apoptosis and ECM degradation partially through a mechanism involving ferroptosis. The results of the present study lay a theoretical foundation for subsequent OA‑related research and may enable the development of translational strategies for the treatment of OA.

CHMP5 attenuates osteoarthritis via inhibiting chondrocyte apoptosis and extracellular matrix degradation: involvement of NF-κB pathway

BackgroundOsteoarthritis (OA), the most common joint disease, is linked with chondrocyte apoptosis and extracellular matrix (ECM) degradation. Charged multivesicular body protein 5 (CHMP5), a member of the multivesicular body, has been reported to serve as an anti-apoptotic protein to participate in leukemia development. However, the effects of CHMP5 on apoptosis and ECM degradation in OA remain unclear.MethodsIn this study, quantitative proteomics was performed to analyze differential proteins between normal and OA patient articular cartilages. The OA mouse model was constructed by the destabilization of the medial meniscus (DMM). In vitro, interleukin-1 beta (IL-1β) was used to induce OA in human chondrocytes. CHMP5 overexpression and silencing vectors were created using an adenovirus system. The effects of CHMP5 on IL-1β-induced chondrocyte apoptosis were investigated by CCK-8, flow cytometry, and western blot. The effects on ECM degradation were examined by western blot and immunofluorescence. The potential mechanism was explored by western blot and Co-IP assays.ResultsDownregulated CHMP5 was identified by proteomics in OA patient cartilages, which was verified in human and mouse articular cartilages. CHMP5 overexpression repressed cell apoptosis and ECM degradation in OA chondrocytes. However, silencing CHMP5 exacerbated OA chondrocyte apoptosis and ECM degradation. Furthermore, we found that the protective effect of CHMP5 against OA was involved in nuclear factor kappa B (NF-κB) signaling pathway.ConclusionsThis study demonstrated that CHMP5 repressed IL-1β-induced chondrocyte apoptosis and ECM degradation and blocked NF-κB activation. It was shown that CHMP5 might be a novel potential therapeutic target for OA in the future.

Sirt1 alleviates osteoarthritis via promoting FoxO1 nucleo-cytoplasm shuttling to facilitate autophagy

This study aims to investigate the role and underlying mechanisms of Sirt1 in the pathophysiological process of OA. Safranine O and HE staining were utilized to identify pathological changes in the cartilage tissue. Immunohistochemistry was employed to evaluate the expression levels of proteins. IL-1β treatment and TamCartSirt1flox/flox mice were utilized to induce OA model both in vitro and in vivo. Key autophagy-related transcription factors, autophagy-related genes, and chondrocyte extracellular matrix (ECM) breakdown enzyme markers were examined using multi assays. Immunofluorescence staining revealed subcellular localization and gene expression patterns. ChIP assay and Co-immunoprecipitation assay were conducted to investigate the interactions between FoxO1 and the promoter regions of Atg7 and Sirt1. Our results demonstrate that Sirt1 deficiency exhibited inhibitory effects on ECM synthesis and autophagy, as well as exacerbated angiogenesis. Moreover, Atg7, Foxo1, and Sirt1 could form a protein complex. Sirt1 was observed to facilitate nuclear translocation of FoxO1, enhancing its transcriptional activity. Furthermore, FoxO1 was found to bind to the promoter regions of Atg7 and Sirt1, potentially regulating their expression. This study provides valuable insights into the involvement of Sirt1-Atg7-FoxO1 loop in OA, opening new avenues for targeted therapeutic interventions aiming to mitigate cartilage degradation and restore joint function.

Oleic and linoleic acids promote chondrocyte apoptosis by inhibiting autophagy via downregulation of SIRT1/FOXO1 signaling

Osteoarthritis (OA) is a complex joint disease that currently has no cure. OA involves metabolic disorders in chondrocytes and an imbalance between autophagy and apoptosis. As a common risk factor for OA, obesity induces changes in the fatty acid composition of synovial fluid, thereby disturbing chondrocyte homeostasis. However, whether unsaturated fatty acids affect the development of OA by regulating chondrocyte autophagy remains unclear. This study aimed to determine the effects of oleic and linoleic acids on chondrocyte autophagy and related mechanisms. Based on the mass spectrometry results, the levels of multiple unsaturated fatty acids, including oleic and linoleic acids, in the synovial fluid of patients with OA and obesity were significantly higher than those in patients with OA only. Moreover, we found that FOXO1 and SIRT1 were downregulated after oleic and linoleic acids treatment of chondrocytes, which inhibited chondrocyte autophagy. Importantly, the upregulation of SIRT1 and FOXO1 expression not only increased the level of autophagy but also improved the expression of chondrocyte extracellular matrix proteins. Furthermore, upregulated SIRT1 and FOXO1 expression alleviated the destruction of the articular cartilage in an OA rat model. Our results suggest that SIRT1/FOXO1 signaling can alleviate oleic acid- and linoleic acid-induced cartilage degradation both in vitro and in vivo and that the SIRT1/FOXO1 pathway may serve as an effective treatment target for inhibiting OA progression.

Klf10 is involved in extracellular matrix calcification of chondrocytes alleviating chondrocyte senescence

Osteoarthritis (OA) is a chronic degenerative disease resulting joint disability and pain. Accumulating evidences suggest that chondrocyte extracellular matrix calcification plays an important role in the development of OA. Here, we showed that Krüppel-like factor 10 (Klf10) was involved in the regulation of chondrocyte extracellular matrix calcification by regulating the expression of Frizzled9. Knockdown of Klf10 attenuated TBHP induced calcification and reduced calcium content in chondrocytes. Restoring extracellular matrix calcification of chondrocytes could aggravate chondrocyte senescence. Destabilization of a medial meniscus (DMM) mouse model of OA, in vivo experiments revealed that knockdown Klf10 improved the calcification of articular cartilage and ameliorated articular cartilage degeneration. These findings suggested that knockdown Klf10 inhibited extracellular matrix calcification-related changes in chondrocytes and alleviated chondrocyte senescence.

Biological functions and applications of LncRNAs in the regulation of the extracellular matrix in osteoarthritis.

Osteoarthritis (OA) is a major cause of disability, characterized by chronic pain, irreversible destruction, and loss of function of the articular cartilage. The integrity and arrangement of the composition and structure of the extracellular matrix (ECM) are essential for maintaining the elasticity, integrity, and mechanical support function of the cartilage tissue. Osteoarthritis causes substantial changes in the ECM, driving the progression of the disease. Recent studies have shown that the ECM plays a critical role in the development of cartilage tissue as well as the occurrence and development of osteoarthritis by directly or indirectly regulating chondrocyte proliferation, apoptosis, differentiation, and gene expression. Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs derived from large transcripts. Mutations and disorders of lncRNAs are closely related to the development of osteoarthritis. Abnormal expression of lncRNAs in osteoarthritic cartilage regulates the synthesis and decomposition of the cartilaginous ECM. Therefore, the use of lncRNAs as nucleic acid drugs that regulate their targets may reduce ECM degradation, thereby delaying the pathological progression of osteoarthritis. In this review, the regulatory effects of lncRNAs on ECM in different cell behaviors related to OA are summarized. The roles of lncRNAs in the proliferation, apoptosis, differentiation, and ECM-related gene activity of chondrocytes, as well as the application of lncRNAs as potential gene therapy drugs for the repair and regeneration of osteoarthritic tissue, are also reviewed. A better understanding of the roles of lncRNAs in guiding chondrocyte behavior and ECM metabolism is critical for their future applications in osteoarthritis therapy and regenerative medicine.

Gossypol Acetic Acid alleviates the Ferroptosis of Chondrocytes in Osteoarthritis by Inhibiting GPX4 Methylation.

Osteoarthritis (OA) is a chronic joint disease, usually accompanied by degeneration of the articular cartilage, fibrosis, bone hyperplasia around the joint, and damage to the entire articular surface. Gossypol is a natural phenolic compound isolated from the seed of cotton plants, and gossypol acetic acid (GAA) is a medicinal form of Gossypol. Recently, various biological activities of GAA, including anti-inflammatory and anti-tumor effects, have been widely reported. However, its effect on chondrocytes in OA has yet to be determined. In this study, we investigated the effect of GAA on ferroptosis in OA chondrocytes. The effect of GAA on the cell viability and cytotoxicity of chondrocytes in rat cells was investigated using CCK8. Western blotting, Reverse-transcription PCR (RT-PCR), and immunofluorescence staining were used to elucidate the molecular mechanisms and signaling pathways of GAA inhibition of ferroptosis in OA chondrocytes. The effect of GAA on reactive oxygen species (ROS) production and lipid peroxidation levels in chondrocytes was examined using dihydroethidium (DHE) staining and fluorescent dye BODIPY581/591 C11. in vivo, micro-CT imaging, hematoxylin and eosin staining, Safranin O-Fast staining, and immunohistochemistry were performed to evaluate the effects of GAA on OA cartilage. The results showed that GAA treatment regulated the expression of chondrocyte extracellular matrix (ECM) related factors, including ADAMTS5, MMP13, SOX9, Aggrecan, and COL1A2 and reduced the ROS and lipid peroxidation levels. Besides, Erastin could reverse the effects of GAA on chondrocytes. Similar to GAA, 5-AZA caused the reduction of ROS and lipid peroxidation levels and reversed the effect of IL-1β on the expression of ECM-related factors in OA chondrocytes. The above results clarified that GAA alleviated the ferroptosis of chondrocytes in OA by inhibiting GPX4 methylation. Our findings revealed that GAA might be developed as a drug for treating OA clinically.

Extracellular Vesicles in Infrapatellar Fat Pad from Osteoarthritis Patients Impair Cartilage Metabolism and Induce Senescence.

Infrapatellar fat pad (IPFP) is closely associated with the development and progression of knee osteoarthritis (OA), but the underlying mechanism remains unclear. Here, it is find that IPFP from OA patients can secret small extracellular vesicles (sEVs) and deliver them into articular chondrocytes. Inhibition the release of endogenous osteoarthritic IPFP-sEVs by GW4869 significantly alleviated IPFP-sEVs-induced cartilage destruction. Functional assays in vitro demonstrated that IPFP-sEVs significantly promoted chondrocyte extracellular matrix (ECM) catabolism and induced cellular senescence. It is further demonstrated that IPFP-sEVs induced ECM degradation in human and mice cartilage explants and aggravated the progression of experimental OA in mice. Mechanistically, highly enriched let-7b-5p and let-7c-5p in IPFP-sEVs are essential to mediate detrimental effects by directly decreasing senescence negative regulator, lamin B receptor (LBR). Notably, intra-articular injection of antagomirs inhibiting let-7b-5p and let-7c-5p in mice increased LBR expression, suppressed chondrocyte senescence and ameliorated the progression of experimental OA model. This study uncovers the function and mechanism of the IPFP-sEVs in the progression of OA. Targeting IPFP-sEVs cargoes of let-7b-5p and let-7c-5p can provide a potential strategy for OA therapy.

Network pharmacology integrated with experimental validation revealed the mechanism of Fengshi Gutong Capsule in the treatment of osteoarthritis

Ethnopharmacological relevanceFengshi Gutong Capsule (FSGTC) is a commonly used Chinese medicine for the treatment of joint pain caused by osteoarthritis (OA). However, the mechanism of action of FSGTC for OA remains unclear. Aim of the studyThis study aimed to explore the alleviating effects and potential mechanisms of action of FSGTC for OA through data mining, network pharmacology, and in vitro experiments. Materials and methodsHigh-performance liquid chromatography (HPLC) was performed to establish the fingerprints of FSGTC and detect the components of FSGTC absorbed in the blood. The effects of FSGTC on inflammation, immunity, and liver and kidney functions in patients with OA were evaluated by mining clinical data. The potential targets and pathways of FSGTC were screened using network pharmacology. Subsequently, CCK-8 assay, flow cytometry, western blotting, RT-qPCR, ELISA, and immunofluorescence were performed in IL-1β-stimulated chondrocytes for further validation. ResultsEighty-seven common peaks and 10 components were identified using the HPLC fingerprints of 12 batches of samples, and the similarity was in the range of 0.973–0.998. Retrospective clinical analysis demonstrated a significant reduction in inflammatory response levels among patients with OA who received FSGTC treatment. Network pharmacology analysis revealed that FSGTC potentially targeted processes related to inflammation, oxidative stress, and apoptosis. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT), The nuclear factor-κB (NF-κB), and janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathways were predicted to be the main pathways involved in the therapeutic effects of FSGTC in OA. In vitro, FSGTC-containing serum aided the proliferation of chondrocytes stimulated by IL-1β, while concurrently mitigating apoptosis, suppressing the expression of inflammatory cytokines and oxidative molecules, and inhibiting the degradation of the chondrocyte extracellular matrix (ECM). ConclusionsFSGTC alleviates the inflammatory response in patients with OA. This therapeutic effect was attributed to its anti-inflammatory and antioxidant properties, and its ability to promote IL-1β-induced chondrocyte proliferation, inhibit apoptosis, and prevent the degradation of extracellular matrix. These favorable results were associated with the inhibition of the PI3K/AKT, NF-κB, and JAK2/STAT3 signaling pathways.

Chondrocyte targeting gold nanoparticles protect growth plate against inflammatory damage by maintaining cartilage balance

Cartilage destruction caused by inflammation is a clinical challenge. Many studies have investigated cartilage destruction in adults, but little research was conducted on children. In this study, the protective effect of gold nanoparticles (AuNPs) on the cartilage of children was realized by counteracting chondrocyte apoptosis and extracellular matrix (ECM) degradation in a young mouse model of lipopolysaccharide (LPS)-induced growth plate (GP) cartilage damage. Initially, engineered AuNPs can be efficiently absorbed by chondrocytes, approximately 20 times the amount absorbed by macrophages, resulting in a 29% ± 0.05% increase in chondrocyte viability. Then, AuNPs exposure significantly reduced the release of inflammatory cytokines and secretion of ECM degradation factors induced by LPS. Subsequently, AuNPs were applied to resist LPS-induced cartilage destruction in young mice. AuNPs inhibited the formation of gaps, without chondrocytes and extracellular matrix, between the proliferative and hypertrophy zones of the GP cartilage, and the gaps were noticeable in the LPS group. This finding can be attributed to the capability of AuNPs to reduce the LPS-induced apoptosis rate of mouse chondrocytes by 72.38% and the LPS-induced ECM degradation rate by 70.89%. Further analysis demonstrated that remission is partly due to AuNPs’ role in maintaining the balance of catabolic and anabolic factors in the ECM. Altogether, these findings indicate that AuNPs can partially protect the cartilage of children from inflammatory damage by suppressing chondrocyte apoptosis and ECM degradation.

ALKBH5-mediated m6A demethylation of HS3ST3B1-IT1 prevents osteoarthritis progression

HS3ST3B1-IT1 was identified as a downregulated long noncoding RNA in osteoarthritic cartilage. However, its roles and mechanisms in the pathogenesis of osteoarthritis (OA) are unclear. In this study, we demonstrated that the expressions of HS3ST3B1-IT1 and its maternal gene HS3ST3B1 were downregulated and positively correlated in osteoarthritic cartilage. Overexpression of HS3ST3B1-IT1 significantly increased chondrocyte viability, inhibited chondrocyte apoptosis, and upregulated extracellular matrix (ECM) proteins, whereas HS3ST3B1-IT1 knockdown had the opposite effects. In addition, HS3ST3B1-IT1 significantly ameliorated monosodium-iodoacetate-induced OA invivo. Mechanistically, HS3ST3B1-IT1 upregulated HS3ST3B1 expression by blocking its ubiquitination-mediated degradation. Knockdown of HS3ST3B1 reversed the effects of HS3ST3B1-IT1 on chondrocyte viability, apoptosis, and ECM metabolism. AlkB homolog 5 (ALKBH5)-mediated N6-methyladenosine (m6A) demethylation stabilized HS3ST3B1-IT1 RNA. Together, our data revealed that ALKBH5-mediated upregulation of HS3ST3B1-IT1 suppressed OA progression by elevating HS3ST3B1 expression, suggesting that HS3ST3B1-IT1/HS3ST3B1 may serve as potential therapeutic targets for OA treatment.

Ajugol's upregulation of TFEB-mediated autophagy alleviates endoplasmic reticulum stress in chondrocytes and retards osteoarthritis progression in a mouse model

BackgroundOsteoarthritis (OA), a degenerative disease with a high global prevalence, is characterized by the degradation of the extracellular matrix (ECM) and the apoptosis of chondrocytes. Ajugol, a extract derived from the herb Rehmannia glutinosa, has not yet been investigated for its potential in modulating the development of OA.MethodsWe employed techniques such as western blotting, immunofluorescence, immunohistochemistry, X-ray imaging, HE staining, and SO staining to provide biological evidence supporting the role of Ajugol as a potential therapeutic agent for modulating OA. Furthermore, in an in vivo experiment, intra-peritoneal injection of 50 mg/kg Ajugol effectively mitigated the progression of OA following destabilization of the medial meniscus (DMM) surgery.ResultsOur findings revealed that treatment with 50 μM Ajugol activated TFEB-mediated autophagy, alleviating ER stress-induced chondrocyte apoptosis and ECM degradation caused by TBHP. Furthermore, in an in vivo experiment, intra-peritoneal injection of 50 mg/kg Ajugol effectively mitigated the progression of OA following destabilization of the medial meniscus (DMM) surgery.ConclusionThese results provide compelling biological evidence supporting the role of Ajugol as a potential therapeutic agent for modulating OA by activating autophagy and attenuating ER stress-induced cell death and ECM degradation. The promising in vivo results further suggest the potential of Ajugol as a treatment strategy for OA progression.

MiR-5581 Contributes to Osteoarthritis by Targeting NRF1 to Disturb the Proliferation and Functions of Chondrocytes

Chondrocyte survival is critical for the preservation of a healthy cartilage matrix. Limited chondrocyte function and survival can result in articular cartilage failure, thereby contributing to osteoarthritis (OA). In this study, miR-5581 was significantly up-regulated in OA samples, and miR-5581-associated genes were enriched in Kras signaling. miR-5581 up-regulation was observed in clinical OA samples and IL-1β-stimulated chondrocytes. miR-5581 inhibition attenuated IL-1β-induced chondrocyte proliferation suppression, extracellular matrix (ECM) synthesis suppression and degradation, and IL-1β-suppressed Kras signaling activation. miR-5581 was targeted to inhibit NRF1. In IL-1β-treated chondrocytes, NRF1 overexpression attenuated IL-1β-induced cellular damage and partially abolished the effects of miR-5581 overexpression on IL-1β-stimulated chondrocytes. NRF1 was down-regulated in knee joint cartilage of OA mice. In conclusion, miR-5581, which was up-regulated in OA samples and IL-1β-stimulated chondrocytes, inhibited chondrocyte proliferation and ECM synthesis, and promoted ECM degradation through targeting NRF1, whereby Kras signaling might be involved.

Intermittent cyclic mechanical compression promotes endplate chondrocytes degeneration by disturbing Nrf2/PINK1 signaling pathway-dependent mitophagy.

An abnormal mechanical load is a pivotal inducer of endplate cartilage degeneration, which subsequently promotes intervertebral disc degeneration. Our previous study indicated that intermittent cyclic mechanical compression (ICMC) promotes endplate chondrocyte degeneration, but the mechanism underlying this effect is unclear. In this study, we investigated PTEN-induced kinase 1(PINK1) dependent mitophagy during ICMC-induced endplate chondrocyte degeneration. Furthermore, we determined whether NF-E2-related factor 2 (Nrf2) activation correlated with PINK1-dependent mitophagy regulation and increased oxidation resistance of endplate chondrocytes under ICMC application. First, we generated a mechanical compression-induced endplate chondrocyte degeneration model in vitro and in vivo. ICMC was found to promote endplate chondrocyte extracellular matrix degradation. PINK1-mediated mitophagy was suppressed in the ICMC-stimulated endplate chondrocytes, while increased mitochondrial reactive oxygen species generation suggested that mitophagy is involved in the protective effect of mechanical strain on endplate chondrocytes. Moreover, Nrf2 expression, interaction with Kelch-like ECH-associated protein (Keap1), and nuclear translocation were inhibited by ICMC. Nrf2 overexpression inhibited reactive oxygen species production and reversed ICMC-induced endplate chondrocyte degeneration. Transfection with PINK1 shRNA abolished this effect and partially blocked Nrf2-induced mitophagy. Our findings suggested that ICMC could inhibit the Nrf2/PINK1 signaling pathway to reduce the mitophagy levels which significantly promote oxidative stress and thereby endplate chondrocyte degeneration. Therapeutic regulation of the Nrf2/PINK1 signaling pathway may be an efficient anabolic strategy for inhibiting this process.

Function and regulation of nuclear factor 1 X-type on chondrocyte proliferation and differentiation

Nuclear factor 1 X-type (Nfix) is a transcription factor related to mental and physical development. However, very few studies have reported the effects of Nfix on cartilage. This study aims to reveal the influence of Nfix on the proliferation and differentiation of chondrocytes, and to explore its potential action mechanism. We isolated primary chondrocytes from the costal cartilage of newborn C57BL/6 mice and with Nfix overexpression or silencing treatment. We used Alcian blue staining and found that Nfix overexpression significantly promoted ECM synthesis in chondrocytes while silencing inhibited ECM synthesis. Using RNA-seq technology to study the expression pattern of Nfix in primary chondrocytes. We found that Nfix overexpression significantly up-regulated genes that are related to chondrocyte proliferation and extracellular matrix (ECM) synthesis and significantly down-regulated genes related to chondrocyte differentiation and ECM degradation. Nfix silencing, however, significantly up-regulated genes associated with cartilage catabolism and significantly down-regulated genes associated with cartilage growth promotion. Furthermore, Nfix exerted a positive regulatory effect on Sox9, and we propose that Nfix may promote chondrocyte proliferation and inhibit differentiation by stimulating Sox9 and its downstream genes. Our findings suggest that Nfix may be a potential target for the regulation of chondrocyte proliferation and differentiation.

CircTBX5 knockdown modulates the miR-558/MyD88 axis to alleviate IL-1β-induced inflammation, apoptosis and extracellular matrix degradation in chondrocytes via inactivating the NF-κB signaling

BackgroundIt has been widely shared that the dysregulation of circular RNA (circRNA) may contribute to the progression of osteoarthritis (OA). OA is characterized by persistent chondrocyte injury. We aimed to clarify the role of circTBX5 in IL-1β-induced chondrocyte injury.MethodsThe expression of circTBX5, miR-558 and MyD88 mRNA was measured using quantitative real-time PCR (qPCR). Cell viability, proliferation and apoptosis were assessed by CCK-8, EdU or flow cytometry assay. The protein levels of extracellular matrix (ECM)-associated markers, MyD88, IkBα, p65 and phosphorylated IkBα were measured by western blot. The release of inflammatory factors was assessed by ELISA. The targets of circTBX5 were screened by RIP and pull-down assay. The putative binding between miR-558 and circTBX5 or MyD88 was validated by dual-luciferase reporter assay.ResultsCircTBX5 and MyD88 were enhanced, while miR-558 was downregulated in OA cartilage tissues and IL-1β-treated C28/I2 cells. IL-1β induced C28/I2 cell injury by impairing cell viability and proliferation and promoting cell apoptosis, ECM degradation and inflammatory response, while circTBX5 knockdown alleviated IL-1β induced injury. CircTBX5 bound to miR-558 to regulate IL-1β induced cell injury. In addition, MyD88 was a target of miR-558, and circTBX5 targeted miR-558 to positively regulate MyD88 expression. MiR-558 enrichment attenuated IL-1β induced injury by sequestering MyD88 expression. Moreover, circTBX5 knockdown weakened the activity of NF-κB signaling, while miR-558 inhibition or MyD88 overexpression recovered the activity of NF-κB signaling.ConclusionCircTBX5 knockdown modulated the miR-558/MyD88 axis to alleviate IL-1β induced chondrocyte apoptosis, ECM degradation and inflammation via inactivating the NF-кB signaling pathway.

Circular RNA CircDHRS3 Aggravates IL-1β-induced ECM Degradation, Apoptosis, and Inflammatory Response via Mediating MECP2 Expression.

Previous studies have reported that circular RNA hsa_circ_0010024 (circDHRS3), microRNA (miR)-193a-3p, and Methyl CpG binding protein 2 (MECP2) are unconventionally expressed in osteoarthritis (OA) cartilage samples. However, the regulatory mechanisms among circDHRS3, miR-193a-3p, and MECP2 in OA pathogenesis are unclear. Changes of circDHRS3, miR-193a-3p, and MECP2 mRNA were detected by qRT-PCR. Several protein levels were evaluated using western blotting. Cell proliferation was analyzed by 5-Ethynyl-2'-deoxyuridine (EdU) and cell counting assays. Cell apoptosis was determined by flow cytometry assay. Detection of pro-inflammatory cytokines was conducted using ELISA. The relationship between circDHRS3 or MECP2 and miR-193a-3p was validated by dual-luciferase reporter assay. We verified that circDHRS3 and MECP2 were overexpressed in OA cartilage samples, whereas miR-193a-3p was downregulated. CircDHRS3 silencing weakened IL-1β-induced chondrocyte cartilage extracellular matrix (ECM) degradation, apoptosis, and inflammatory response. CircDHRS3 adsorbed miR-193a-3p to modulate MECP2 expression. Also, silencing of miR-193a-3p impaired circDHRS3 silencing-mediated suppression on IL-1β-induced chondrocyte injury. Also, MECP2 overexpression alleviated miR-193a-3p mimic-mediated inhibition on IL-1β-prompted chondrocyte injury. CircDHRS3 silencing reduced MECP2 expression via sponging miR-193a-3p, thereby weakening IL-1β-induced chondrocyte ECM degradation, apoptosis, and inflammatory response.