Chondrocyte Apoptosis Research Articles

SUMOylated GLUT1 inhibited the glycometabolism disorder in chondroctyes during osteoarthritis.

Reduction of glucose transporter 1 (GLUT1), even deletion, may results in cartilage fibrosis and osteoarthritis. This study aims to investigate the SUMOylation of GLUT1 in osteoarthritis through small ubiquitin-like modifier 1(SUMO1), and explore the role of SUMOylated GLUT1 in glycometabolism, proliferation and apoptosis in chondrocytes. Human chondrocytes were incubated with 10 ng/mL of IL-1β to mimic osteoarthritis in vitro. GLUT1, SUMO1 and Chondrocyte-related genes including COL2A1, MMP13 and ADAMTS4 were evaluated using western blot. Cell viability and cell apoptosis of chondrocytes were measured by cell counting kit-8 assay and flow cytometry, respectively. The changes in glycometabolism were evaluated using extracellular acidification rate (ECAR) and glucose uptake assay. Co-immunoprecipitation (Co-IP) was used to verify the interaction between GLUT1 and SUMO1. The stabilization role of SUMO1 in GLUT1 was determined by cycloheximide assay. IL-1β induced the decrease of GLUT1, cell viability, ECAR, glucose uptake and COL2A1 and the increase of cell apoptosis, MMP13 and ADAMTS4 in chondrocytes. However, overexpression of SUMO1 led to the reduction of cell apoptosis, MMP13 and ADAMTS4 and the elevation of GLUT1, cell viability, ECAR, glucose uptake and COL2A1 in IL-1β-stimulated chondrocytes. There was SUMOylation sites on GLUT1. Intriguingly, SUMO1 was significantly enriched in GLUT1 using Co-IP assay, and stabilized GLUT1 in chondrocytes. SUMO1-mediated SUMOylation is capable of stabilizing GLUT1 to inhibit glycometabilsm disorder and cell apoptosis in IL-1β-stimulated chondrocytes.

1,25(OH)2D3 induces chondrocyte autophagy and reduces the loss of proteoglycans in osteoarthritis through inhibiting the NF-κB pathway.

Nuclear transcription factor-κB (NF-κB) activation is a pivotal event in the pathogenesis of osteoarthritis (OA). OA patients frequently exhibit vitamin D (VD) deficiency, which is commonly associated with NF-κB activation. Our study aimed to investigate whether VD could protect against OA by modulating NF-κB pathway and to explore the underlying mechanisms. Proteins levels were assessed by western blot analysis, gene expression was quantified by quantitative real-time polymerase chain reaction (qRT‒PCR) in vivo and in vitro. The expression of phosphorylated-p65 (p-p65) in knee OA rats was detected by immunohistochemistry, and an NF-κB nuclear translocation assay was validated in chondrocytes. Immunoprecipitation was employed to detect the interaction between NF-κB and vitamin D receptor (VDR) in vivo and in vitro. Small interfering RNA (Si-NF-κB and Si-VDR) transfection was used to investigate the role of NF-κB and VDR signaling pathway in knee OA rats under VD influence. Cartilage changes were visualized of knee OA rats using hematoxylin and eosin as well as safranin-O/fast green of staining. Our findings indicated that VD alleviates OA by inhibiting NF-κBpathway, which in turn reduces chondrocyte apoptosis and extracellular matrix (ECM) degradation. Further analysis revealed that VD primarily stabilizes NF-κB through the interaction of VDR and NF-κB, modulating the AMPK/mTOR signaling pathway to enhance autophagy and delay the progression of OA. This study highlights the protective role of VD in OA by stabilization of NF-κB, mainly through the interaction between VDR and NF-κB. This interaction regulates the AMPK/mTOR signaling pathway, promoting autophagy and suggesting a potential therapeutic strategy for OA management. Key Points • VD confers a protective effect on OA by primarily stabilizing NF-κB through the interaction between VDR and NF-κB, which in turn inhibits NF-κB phosphorylation and nuclear translocation. • In chondrocytes, VD helps shield against OA by blocking NF-κB's entry into the nucleus, subsequently regulating autophagy via the AMPK/mTOR signaling pathway.

The role of the NOTCH1 signaling pathway in the maintenance of mesenchymal stem cell stemness and chondrocyte differentiation and its potential in the treatment of osteoarthritis

ObjectiveThis study aims to explore the potential role of mesenchymal stem cells (MSCs) in the treatment of osteoarthritis (OA), particularly the function of the NOTCH1 signaling pathway in maintaining the stemness of MSCs and in chondrocyte differentiation.MethodsUtilizing diverse analytical techniques on an osteoarthritis dataset, we unveil distinct gene expression patterns and regulatory relationships, shedding light on potential mechanisms underlying the disease. Techniques used include the culture of MSCs, induction of differentiation into chondrocytes, establishment of stable cell lines, Western Blot, and immunofluorescence. Through the construction of lentiviruses overexpressing and knocking out NOTCH1, the effects of NOTCH1 on the stemness of MSCs and chondrocyte differentiation were investigated. Additionally, the effects of NOTCH1 on chondrocyte homeostasis and apoptosis were evaluated by adding the EZH2 inhibitor GSK126 and the endoplasmic reticulum stress inducer tunicamycin.ResultsExperimental results demonstrated that NOTCH1 expression can influence the maintenance of MSC stemness and chondrocyte differentiation by regulating EZH2. Knockout of NOTCH1 decreased the expression of chondrocyte markers, while overexpression increased their expression. Under conditions of endoplasmic reticulum stress, NOTCH1 expression helped reduce the expression of stress-related proteins, maintain chondrocyte homeostasis, and inhibit apoptosis.ConclusionThe NOTCH1 signaling pathway plays a crucial role in maintaining the stemness of MSCs, differentiating into chondrocytes, and in the treatment of osteoarthritis. NOTCH1 influences the differentiation fate of MSCs and the homeostasis of chondrocytes by regulating EZH2 and other related genes, offering new targets and strategies for the treatment of diseases like osteoarthritis.

NEK2 promotes the progression of osteoarthritis by stabilizing ATF2 through phosphorylation at Ser-112 and inhibiting autophagy.

NEK2 (NIMA-related kinase 2) has recently gained attention for its potential role in osteoarthritis (OA) chondrocytes, however, its specific involvement remains unclear. This study aimed to investigate the role of NEK2 in OA progression and the underlying molecular mechanisms. Primary mouse knee chondrocytes were stimulated with IL-1β to establish an in vitro OA model, followed by the knockdown of NEK2 or ATF2. The results indicated that silencing NEK2 or ATF2 impeded the IL-1β-induced decrease in cell proliferation and increase in inflammation, extracellular matrix (ECM) degradation, and apoptosis in chondrocytes. NEK2 or ATF2 knockdown restored IL-1β-induced autophagy defects. Mechanistically, NEK2 interacts with ATF2 to reduce its ubiquitylation level and enhance its stability by phosphorylating ATF2 at Ser-112. Consistently, ATF2 overexpression reversed the protective effect of NEK2 silencing on IL-1β-induced autophagy defects and chondrocyte injury. Additionally, a mouse OA model was established using medial meniscus destabilization (DMM) surgery, and NEK2 was knocked down by intra-articular injection of an adenovirus-mediated NEK2 interference vector. Downregulation of NEK2 mitigated cartilage degradation andautophagy defects ina mouse OA model. In conclusion, NEK2 promoted OA progression by enhancing ATF2 stability by phosphorylating it at Ser-112.

LncRNA PTS-1 Protects Against Osteoarthritis Through the miR-8085/E2F2 Axis.

Osteoarthritis (OA) is a leading cause of pain, disability, and reduced mobility worldwide, characterized by metabolic imbalances in chondrocytes, extracellular matrix (ECM), and subchondral bone. Emerging evidence highlights the critical role of long non-coding RNAs (lncRNAs) in OA pathogenesis. This study focuses on lncRNA PTS-1, a novel lncRNA, to explore its function and regulatory mechanisms in OA progression. The expression profile of lncRNAs was assessed using RNA sequencing and qRT-PCR. The expression of lnc-PTS-1 was further validated by qRT-PCR in degenerated cartilage tissues, degenerative primary chondrocytes, and IL-1β-treated C28/I2 cells. Cell viability, proliferation, and apoptosis rates, along with the mRNA and protein levels of apoptosis-related markers (cleaved Caspase 3, cleaved Caspase 9, Bcl-2, Bax), ECM metabolism markers (MMP-3, MMP-13, aggrecan, collagen II), and inflammation-related markers (IL-1β, IL-6, TNF-α) were evaluated using Cell Counting Kit-8, Toluidine Blue staining, Alcian Blue staining, flow cytometry, qRT-PCR, immunofluorescence, and Western Blot. The interaction between miR-8085 and lnc-PTS-1 or E2F2 was investigated through dual luciferase reporter assays and RNA immunoprecipitation (RIP) analyses. Lnc-PTS-1 expression was significantly downregulated in degenerated cartilage tissues, IL-1β-induced degenerative primary chondrocytes and C28/I2 cells. Functional experiments showed that lnc-PTS-1 knockdown aggravated IL-1β-induced ECM degradation, chondrocyte apoptosis, and inflammation, while its overexpression provided protective effects. Mechanistically, lnc-PTS-1 acted as a competing endogenous RNA (ceRNA) by sponging miR-8085, thereby upregulating E2F2 expression. Notably, miR-8085 upregulation diminished the protective effects of lnc-PTS-1 on ECM degradation, apoptosis, and inflammation, while E2F2 upregulation partially alleviated IL-1β-induced damage. However, these mitigating effects were reversed by miR-8085 overexpression. These findings identify lnc-PTS-1/miR-8085/E2F2 axis as a novel regulatory mechanism in OA pathogenesis, providing theoretical basis and experimental evidence for the potential clinical application of new lncRNA molecules in the treatment of OA.

Identification of Key Chondrocyte Apoptosis-Related Genes in Osteoarthritis Based on Weighted Gene Co-Expression Network Analysis and Experimental Verification.

Osteoarthritis (OA) is the primary cause of disability worldwide. Chondrocyte apoptosis has important implications for OA onset and progression. This work was designed to explore the mechanisms of chondrocyte apoptosis in OA and identify key chondrocyte apoptosis-related genes (CARGs). GSE32317 and GSE55235 datasets were acquired from the Gene Expression Omnibus (GEO) database. OA-associated module genes were determined via weighted gene co-expression network analysis (WGCNA) in GSE32317. CARGs were acquired from public databases. ClusterProfiler was employed for GO and KEGG analyses. Protein-protein interaction (PPI) network establishment was realized via the STRING database and Cytoscape, and the hub genes were screened by MCC, MNC, and DMNC algorithms of cyto-Hubba. The diagnostic values of the hub CARGs in OA in GSE55235 were verified via receiver operating characteristic (ROC) curve analysis. C28/I2 cells were stimulated with IL-1Β to establish the in vitro OA model. WGCNA identified 9,141 OA-related genes and 248 CARGs, resulting in 75 CARGs in OA. GO and KEGG analyses demonstrated that the 75 CARGs were primarily enriched in response to lipopolysaccharide, transcription regulator complex, and DNA-binding transcription factor binding, along with NF-kappa B and TNF signaling pathways. NFKB1 and ICAM1 were identified as the hub CARGs in OA through the three algorithms, which showed favorable prognostic values for OA. Notably, both bioinformatics analysis and in vitro assays revealed upregulated NFKB1 and ICAM1 expression in OA. NFKB1 and ICAM1 were the hub CARGs in OA, and might serve as diagnostic signatures and therapeutic targets for OA therapy.

Interaction Between YTH Domain-Containing Family Protein 2 and SET Domain-Containing Lysine Methyltransferase 7 Suppresses Autophagy in Osteoarthritis Chondrocytes, Exacerbating Cartilage Damage.

Osteoarthritis (OA) is characterized by progressive cartilage degeneration mediated by various molecular pathways, including inflammatory and autophagic processes. SET domain-containing lysine methyltransferase 7 (SETD7), a methyltransferase, has been implicated in OA pathology. This study investigates the expression pattern of SETD7 in OA and its role in promoting interleukin-1 beta (IL-1β)-induced chondrocyte injury through modulation of autophagy and inflammation. The expression of SETD7 in cartilage tissues from OA patients and healthy controls was quantified using quantitative reverse transcription PCR and Western blot analysis. Small interfering RNA targeting SETD7 (si-SETD7) was transfected into human articular chondrocytes (HACs) treated with IL-1β to examine its impact on cellular viability, apoptosis, inflammatory responses, and autophagy. Functional assays including Cell Counting Kit-8, flow cytometry, enzyme-linked immunosorbent assay, and commercial kits were employed to assess biochemical changes. Interaction between YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) and SETD7 was explored using RNA immunoprecipitation and co-immunoprecipitation assays. SETD7 was overexpressed in OA cartilage compared with controls and increased further upon IL-1β treatment. Knockdown of SETD7 in IL-1β-treated HACs improved cellular viability, decreased apoptosis, and reversed the adverse effects on lactate dehydrogenase release and inflammatory markers (tumor necrosis factor-alpha and interleukin-6) while enhancing antioxidant enzymes (catalase, malondialdehyde, and superoxide dismutase). Additionally, autophagy was restored, as evidenced by changes in the levels of autophagy related 5, Beclin1, and sequestosome 1. Interfering with autophagy using chloroquine negated the protective effects of SETD7 knockdown. Furthermore, YTHDF2 was found to stabilize SETD7 mRNA, influencing its expression and enhancing IL-1β-induced chondrocyte injury. SETD7 plays a critical role in the pathogenesis of OA by modulating chondrocyte survival, apoptosis, inflammation, and autophagy. The interaction between YTHDF2 and SETD7 exacerbates chondrocyte injury under inflammatory conditions, highlighting potential therapeutic targets for OA treatment. The YTHDF2/SETD7 axis offers a novel insight into the molecular mechanisms governing cartilage degeneration in OA.

Methimazole disrupted skeletal ossification and muscle fiber transition in Bufo gargarizans larvae.

Methimazole (MMI) is an emerging endocrine disrupting chemical (EDC) due to its increasing use in the treatment of thyrotoxicosis (hyperthyroidism), but its potential impact on amphibian development remains largely unexplored. In the present study, the effects of 8 mg/L MMI and 1 μg/L thyroxine (T4) exposure on skeletal ossification and muscle development in Bufo gargarizans tadpoles were comprehensively investigated by double skeletal staining, histological analysis and RNA sequencing. Our results indicated that MMI treatment down-regulated the expression levels of ossification-related genes (e.g., BMPs, MMPs, and Wnt9a) in cartilage, thereby delaying chondrocyte apoptosis and inhibiting hindlimb ossification. Muscle sarcomere was elongated in both the MMI and T4 treatment groups, which may lead to muscle weakness and consequently affect land motion. Additionally, we evaluated the expression levels of fast muscle-related genes (TNNI2 and TNNT3) and slow muscle-related genes (TNNI1 and TNNT1), revealing an opposite trend in the transition from fast to slow muscle after T4 and MMI exposures. In conclusion, these findings fill the data gap regarding MMI contamination in aquatic environments by revealing the negative effects of MMI on amphibian bone and muscle development. Future studies should address the toxicity of EDCs to wildlife and inform aquatic ecosystem conservation strategies.

Arctiin alleviates knee osteoarthritis by suppressing chondrocyte oxidative stress induced by accumulated iron via AKT/NRF2/HO-1 signaling pathway

Iron overload (IO) was considered to be a risk factor for cartilage degradation in knee osteoarthritis (KOA) advancement. However, few drugs were found to improve cartilage degeneration by alleviating multiple cell death induced by the impaired iron level of the knee joints. We aimed to elucidate that Arctiin (ARC) plays a role in managing KOA caused by accumulated iron levels by restoring chondrocyte apoptosis and ferroptosis. Single-cell RNA sequencing analysis was used to discover the disparities in chondrocytes between KOA patients and non-KOA individuals. CCK-8 assay was performed to detect chondrocyte viability. Annexin V-FITC/PI staining determined the cell apoptosis rate. The fluorescence density reflected the iron content, ROS, lipid-ROS, and mitochondrial membrane potential. Q-RTPCR and Western Blotting were used to detect the expression levels of genes and proteins expression. Micro-CT and Safranin O-Fast Green staining were used to detect the phenotype of the knee joints. ARC increased cell viability and inhibited chondrocyte apoptosis. Further, ARC acts as an anti-ferroptosis effect by reducing the intracellular iron, ROS, and lipid-ROS content and restoring mitochondrial damage. Based on the results of scRNA-seq, we found that ARC can play a role by activating AKT/NRF2/HO-1 signaling pathway. In vivo, ARC can significantly improve the severity of KOA caused by IO. ARC alleviates oxidative stress in chondrocytes via the AKT/NRF2/HO-1 signaling pathway, suggesting the potential application of ARC in KOA.

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.

Modulating Autophagy in Osteoarthritis: Exploring Emerging Therapeutic Drug Targets.

Osteoarthritis (OA) is a degenerative joint disease characterized by the breakdown of cartilage and the subsequent inflammation of joint tissues, leading to pain and reduced mobility. Despite advancements in symptomatic treatments, disease-modifying therapies for OA remain limited. This narrative review examines the dual role of autophagy in OA, emphasizing its protective functions during the early stages and its potential to contribute to cartilage degeneration in later stages. By delving into the molecular pathways that regulate autophagy, this review highlights its intricate interplay with oxidative stress and inflammation, key drivers of OA progression. Emerging therapeutic strategies aimed at modulating autophagy are explored, including pharmacological agents such as AMP kinase activators, and microRNA-based therapies. Preclinical studies reveal encouraging results, demonstrating that enhancing autophagy can reduce inflammation and decelerate cartilage degradation. However, the therapeutic benefits of autophagy modulation depend on precise, stage-specific approaches. Excessive or dysregulated autophagy in advanced OA may lead to chondrocyte apoptosis, exacerbating joint damage. This review underscores the promise of autophagy-based interventions in bridging the gap between experimental research and clinical application. By advancing our understanding of autophagy's role in OA, these findings pave the way for innovative and effective therapies. Nonetheless, further research is essential to optimize these strategies, address potential off-target effects, and develop safe, targeted treatments that improve outcomes for OA patients.

CRNDE alleviates IL-1β-induced chondrocyte damage by modulating miR-31/NF-κB pathway

BackgroundThe long non-coding RNA CRNDE (CRNDE) has been identified as a lncRNA associated with osteoarthritis (OA), playing a role the age-related degeneration of articular cartilage. However, the precise mechanism by which CRNDE affects the physiological functions of OA chondrocytes remains unclear.MethodsTo simulate the inflammatory conditions observed in OA, interleukin (IL)-1β-stimulated chondrocyte C-28/I2 cells were utilized. The expression levels of CRNDE and miR-31 were assessed using reverse transcription-polymerase chain reaction (RT-PCR). Chondrocyte viability and apoptosis were evaluated through CCK-8 assay and flow cytometry, respectively. The levels of IL-6, IL-1β and Tumor necrosis factor (TNF-α) were determined using enzyme-linked immunosorbent assay (ELISA). mRNA expression levels of MMP-13, Aggrecan and COL2A1 were detected by quantitative RT-PCR. Western blot analysis was performed to evaluate the protein levels of factors related to cartilage matrix degradation, including p-p65, p65 and p-IκBα of the NF-κB pathway.ResultsCRNDE expression was downregulated in both OA cartilage tissues and IL-1β-stimulated chondrocytes. Overexpression of CRNDE mitigated IL-1β-stimulated chondrocytes apoptosis, inflammatory responses, and cartilage matrix degradation. Compared with healthy controls, OA tissues exhibited reduced expression of miR-31, which was negatively correlated with the expression of CRNDE. Additionally, overexpression of miR-31 partially reversed the inhibitory effects of CRNDE on apoptosis, inflammation, cartilage matrix degradation, and the inactivation of Nuclear factor (NF)-κB pathway induced by IL-1β stimulation. Moreover, silencing of CRNDE exacerbated IL-1β-induced chondrocytes damage, which was aliviated by the NF-κB pathway inhibitor, Bay 11-7082.ConclusionCRNDE alleviated IL-1β-induced injuries in OA chondrocytes by suppressing the miR-31-mediated NF-κB signaling pathway.

Identification of BCL3 as a biomarker for chondrocyte programmed cell death in osteoarthritis.

Osteoarthritis (OA) is a condition that is widely prevalent and causes joint pain and disability, with programmed cell death (PCD) playing a role in its pathogenesis. This study aimed to identify biomarkers associated with PCD in OA and explore their potential roles. ThreeRNA-sequencing datasets (GSE114007, GSE51588 and GSE220243) related to OA were analysed. Differential expression and weighted gene co-expression network identified key differentially expressed PCD-related genes (DE-PRMGs). Potential biomarkers were identified and validated through receiver operating characteristic (ROC) curves, correlation analyses, gene set enrichment analysis, single-cell expression and RT-qPCR. A total of 45 DE-PRMGs were identified, affecting pathways like TNF signalling and RNA degradation. BCL3, TREM2 and NRP2 were prioritized as potential OA biomarkers, which are associated with ribosome function and immune cell infiltration and potentially linked to PCD. The functional role of one of the molecules identified, BCL3, was explored further using a cell model of inflammation induced chondrocytes. BCL3 was significantly down regulated in OA samples from the public dataset and clinical samples analysed by RT-qPCR. BCL3 overexpression reduced apoptosis in chondrocytes stimulated with inflammatory cytokines. Thus the functional studies highlighted the anti-apoptotic role of BCL3 in chondrocytes and provide new insights into OA pathogenesis with potential for future therapeutic development.

Robinin protects chondrocytes injury via TLR2/TLR4/NF-κB signaling in osteoarthritis.

Osteoarthritis (OA) is a joint disease closely related to aging and characterized by degeneration of articular cartilage. Robinin is a natural agent with various pharmacological properties. Recently, Robinin has been found to have the potential to improve the bone-related diseases. However, its effect on OA development remained unknown. Here, we discuss the specific role and underlying mechanisms of Robinin in interleukin-1beta (IL-1β)-treated chondrocytes and OA mouse model. Chondrocytes were isolated from the mouse to conduct in vitro assays. We evaluated cell viability and apoptosis using Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis, respectively. Western blotting assessed the levels of proteins related to apoptosis, extracellular matrix (ECM), and signaling pathways. Immunofluorescence staining was used to detect the expression of ECM and signaling markers. ELISA was conducted to assess the levels of inflammatory markers. The OA mice model was established using surgical destabilization of the medial meniscus (DMM), and then H&E staining and Safranin O staining were conducted to observe the histopathological changes in synovial tissues. TUNEL assay was used to detect cell apoptosis in vivo. Real-time RT-PCR was operated to measure mRNA level in vitro and in vivo. We discovered that Robinin reversed the IL-1β-induced decrease in chondrocyte viability. Robinin suppressed IL-1β-induced apoptosis of chondrocytes. The ECM destruction and inflammatory response induced by IL-1β were markedly reversed by Robinin incubation in the mouse chondrocytes. Besides, the upregulated cytokine mRNA levels in IL-1β-treated chondrocytes were reduced by Robinin treatment. The downregulation of COL2A1 level and upregulation of MMP13 and ADAMTS5 levels were counteracted by Robinin treatment. Robinin reduced the protein levels of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) but enhanced the level of phosphorylated p65 (p-p65) in IL-1β-stimulated chondrocytes and OA mice. Robinin mitigated inflammation, cell apoptosis and cartilage destruction in synovial tissues from the OA mice. In conclusion, Robinin alleviated OA development in vitro and in vivo via TLR2/TLR4/NF-κB signaling pathway.

Wnt5a manipulate the progression of osteoarthritis via MMP-13 dependent signaling pathway.

The object of this study was to propose a Wnt5a-matrix metalloproteinase (MMP)-13 dependent signaling axis for osteoarthritis (OA) progression. To this end, the chondrocytes were isolated from both OA patients and normal controls. The chondrocytes were treated with diverse concentrations of Wnt5a (0, 50, 100, and 200 ng/mL), respectively. The expression levels of Wnt5a, MMP-13, and Collagen type II were examined using reverse transcription-polymerase chain reaction and western blotting. At the same time, the cell proliferation and cell apoptosis of chondrocytes were also observed. Compared with control tissues, the activities of Wnt5a and MMP-13 were significantly enhanced in chondrocytes of OA patients. Treated with different concentrations of Wnt5a (0, 50, 100, and 200 ng/mL), chondrocyte cell proliferation was clearly downregulated. At the same time, the chondrocyte cell apoptosis was obviously accelerated. The expression pattern of Collagen type II was same as cell proliferation manner. Co-treatment of MMP-13 siRNA could significantly compensate the functions of Wnt-5a administration, suggesting MMP-13 was a direct target of Wnt-5a. Collectively, the study speculated a novel Wnt5a-MMP-13 molecular mechanism for OA progression and shed an innovative signaling axis for the disorder.

Long-term hypoxic atmosphere enhances the stemness, immunoregulatory functions, and therapeutic application of human umbilical cord mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are usually cultured in a normoxic atmosphere (21%) in vitro, while the oxygen concentrations in human tissues and organs are 1% to 10% when the cells are transplanted in vivo. However, the impact of hypoxia on MSCs has not been deeply studied, especially its translational application. In the present study, we investigated the characterizations of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in hypoxic (1%) and normoxic (21%) atmospheres with a long-term culture from primary to 30 generations, respectively. The comparison between both atmospheres systematically analyzed the biological functions of MSCs, mainly including stemness maintenance, immune regulation, and resistance to chondrocyte apoptosis, and studied their joint function and anti-inflammatory effects in osteoarthritis (OA) rats constructed by collagenase II. We observed that long-term hypoxic culture surpassed normoxic atmosphere during hUC-MSCs culture in respect of promoting proliferation, anti-tumorigenicity, maintaining normal karyotype and stemness, inhibiting senescence, and improving immunoregulatory function and the role of anti-apoptosis in chondrocytes. Furthermore, we demonstrated that the transplantation of long-term hypoxic hUC-MSCs (Hy-MSCs) had a better therapeutic effect on OA rats compared with the hUC-MSCs cultured in the normoxic atmosphere (No-MSCs) in terms of the improved function and swelling recovery in the joints, and substantially inhibited the secretion of pro-inflammatory factors, which effectively alleviated cartilage damage by reducing the expression of matrix metallopeptidase 13 (MMP-13). Our results demonstrate that Hy-MSCs possess immense potential for clinical applications via promoting stemness maintenance and enhancing immunoregulatory function.

Single-cell profiling uncovers synovial fibroblast subpopulations associated with chondrocyte injury in osteoarthritis

BackgroundChondrocytes and synovial cells participate in the pathogenesis of osteoarthritis (OA). Nonetheless, the interactions and correlations between OA synovial cells and chondrocytes remain unclear. This study aims to elucidate the interactions and correlations between OA synovial cells and chondrocytes, so as to deepen understanding of OA pathogenesis.MethodsSingle-cell sequencing analysis was employed to analyze clusters of synovial and chondrocyte cells within the OA dataset. Through cell interaction analysis, the potential interactions between these two cell types were further explored. Differential gene expression analysis was used to examine the differences among synovial-related cell clusters.ResultsThe study identified specific characteristics of synovial fibroblasts through single-cell sequencing analysis. Subsequent cell interaction analysis revealed interactions and correlations between synovial fibroblast clusters and cell clusters in both damaged and non-damaged cartilages. CILP+ fibroblasts showed significant interactions with non-damaged chondrocytes, while POSTN+ fibroblasts exhibited significant interactions with damaged chondrocytes. Furthermore, differential gene expression analysis revealed that genes such as PRELP, CLU, COMP, TNFRSF12A, INHBA, CILP, and SERPINE2, were significantly upregulated in CILP+ fibroblasts. These genes are involved in promoting cell proliferation, inhibiting inflammatory pathways, and stabilizing cell structure, thereby exerting reparative and protective effects on chondrocytes. In contrast, COL6A3, COL6A1, COL1A2, COL1A1, COL3A1, TGF-β1, MMP2, AEBP1, SPARC, FNDC1, and POSTN were upregulated in POSTN+ fibroblasts. These genes may contribute to chondrocyte damage and further degeneration by promoting chondrocyte catabolism, driving inflammation, activating inflammatory pathways, and facilitating chondrocyte apoptosis and destruction.ConclusionOur study elucidated the interactions and correlations between OA synovial cells and chondrocytes. CILP+ synovial fibroblasts may exert reparative and protective effects on chondrocytes of patients with OA by promoting cell proliferation, inhibiting inflammation, and stabilizing cellular structures, thereby potentially mitigating the progression of cartilage lesions in affected patients. In contrast, POSTN+ synovial fibroblasts may exacerbate chondrocyte deterioration in patients with OA by enhancing degradation, inflammation, and apoptosis, thereby exacerbating cartilage lesions. Investigating the underlying molecular mechanisms between OA synovial cells and chondrocytes refines the understanding of OA pathogenesis and provides valuable insights for the clinical diagnosis and treatment of 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.

Regulation of chondrocyte apoptosis in osteoarthritis by endoplasmic reticulum stress.

Osteoarthritis(OA), a common degenerative joint disease, is characterized by the apoptosis of chondrocytes as a primary pathophysiological change, with endoplasmic reticulum stress(ERS) playing a crucial role. It has been demonstrated that an imbalance in endoplasmic reticulum(ER) homeostasis can lead to ERS, activating three cellular adaptive response pathways through the unfolded protein responseto restore ER homeostasis. Mild ERS exerts a protective effect on cells, while prolonged ERS that disrupts the self-regulatory balance of the ER activates apoptotic signaling pathways, leading to chondrocyte apoptosis and hastening OA progression. Hence, controlling the ERS signaling pathway and its apoptotic factors has become a critical focus for preventing and treating OA. This review aims to elucidate the key mechanisms of ERS pathway-induced apoptosis, associated targets, and regulatory pathways, offering valuable insights to enhance the mechanistic understanding of OA. It also reviews the mechanisms studied for ERS-related drugs or compounds for the treatment of OA.

Enhancing retention and permeation of rapamycin for osteoarthritis therapy using a two-stage drug delivery system

Osteoarthritis (OA) remains a challenging degenerative joint disease, largely associated with chondrocyte apoptosis during its development. Preserving chondrocytes stands as a promising strategy for OA treatment. Rapamycin (RP) exhibits chondrocyte protection by fostering autophagy. Nevertheless, the swift clearance of intra-articular injections and the dense cartilage extracellular matrix (ECM) hinder RP from effectively reaching chondrocytes. Herein, we developed a "two-stage" drug delivery system (RP@PEG-PA@P-Lipo). This system comprises primary nanoparticles (P-Lipo), liposomes modified with a collagen II targeting peptide (WYRGRLC), and secondary nanoparticles (RP@PEG-PA), PEG-modified PAMAM encapsulating rapamycin (RP). RP@PEG-PA@P-Lipo demonstrates adherence to the cartilage surface with WYRGRLC, substantially prolonging retention within the joint cavity. Subsequently, released RP@PEG-PA can effectively penetrate the cartilage and deliver RP to chondrocytes through small size and charge-driven forces. In vitro and in vivo experiments corroborate its notable therapeutic effects on OA. This study holds promise in offering a novel approach for clinical drug delivery and OA treatment.