RESUMEN
Fourier transform infrared spectroscopy (FTIRS) can provide rich information on the composition and content of samples, enabling the detection of subtle changes in tissue composition and structure. This study represents the first application of FTIRS to investigate cartilage under microgravity. Simulated microgravity cartilage model was firstly established by tail-suspension (TS) for 7, 14 and 21 days, which would be compared to control samples. A self-developed hollow optical fiber attenuated total reflection (HOF-ATR) probe coupled with a FTIR spectrometer was used for the spectral acquisition of cartilage samples in situ, and one-way analysis of variance (ANOVA) was employed to analyze the changes in the contents of cartilage matrix at different stages. The results indicate that cartilage degenerates in microgravity, the collagen content gradually decreases with the TS time, and the structure of collagen fibers changes. The trends of proteoglycan content and collagen integrity show an initial decrease followed by an increase, ultimately significantly decreasing. The findings provide the basis for the cartilage degeneration in microgravity with TS time, which must be of real significance for space science and health detection.
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Cartílago Articular , Colágeno , Simulación de Ingravidez , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Cartílago Articular/patología , Cartílago Articular/química , Cartílago Articular/metabolismo , Colágeno/análisis , Colágeno/metabolismo , Colágeno/química , Animales , Proteoglicanos/análisis , MasculinoRESUMEN
The rapid advancement of cell therapies underscores the importance of understanding fundamental cellular attributes. Among these, cell fitness-how transplanted cells adapt to new microenvironments and maintain functional stability in vivo-is crucial. This study identifies a chemical compound, FPH2, that enhances the fitness of human chondrocytes and the repair of articular cartilage, which is typically nonregenerative. Through drug screening, FPH2 was shown to broadly improve cell performance, especially in maintaining chondrocyte phenotype and enhancing migration. Single-cell transcriptomics indicated that FPH2 induced a super-fit cell state. The mechanism primarily involves the inhibition of carnitine palmitoyl transferase I and the optimization of metabolic homeostasis. In animal models, FPH2-treated human chondrocytes substantially improved cartilage regeneration, demonstrating well-integrated tissue interfaces in rats. In addition, an acellular FPH2-loaded hydrogel proved effective in preventing the onset of osteoarthritis. This research provides a viable and safe method to enhance chondrocyte fitness, offering insights into the self-regulatory mechanisms of cell fitness.
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Cartílago Articular , Condrocitos , Regeneración , Condrocitos/metabolismo , Condrocitos/citología , Condrocitos/efectos de los fármacos , Animales , Humanos , Cartílago Articular/metabolismo , Ratas , Osteoartritis/metabolismo , Osteoartritis/terapia , Hidrogeles/química , Movimiento Celular/efectos de los fármacosRESUMEN
BACKGROUND: The pathogenesis of osteoarthritis (OA) involves the progressive degradation of articular cartilage. Exosomes derived from mesenchymal stem cells (MSC-EXOs) have been shown to mitigate joint pathological injury by attenuating cartilage destruction. Optimization the yield and therapeutic efficacy of exosomes derived from MSCs is crucial for promoting their clinical translation. The preconditioning of MSCs enhances the therapeutic potential of engineered exosomes, offering promising prospects for application by enabling controlled and quantifiable external stimulation. This study aims to address these issues by employing pro-inflammatory preconditioning of MSCs to enhance exosome production and augment their therapeutic efficacy for OA. METHODS: The exosomes were isolated from the supernatant of infrapatellar fat pad (IPFP)-MSCs preconditioned with a pro-inflammatory factor, TNF-α, and their production was subsequently quantified. The exosome secretion-related pathways in IPFP-MSCs were evaluated through high-throughput transcriptome sequencing analysis, q-PCR and western blot analysis before and after TNF-α preconditioning. Furthermore, exosomes derived from TNF-α preconditioned IPFP-MSCs (IPFP-MSC-EXOsTNF-α) were administered intra-articularly in an OA mouse model, and subsequent evaluations were conducted to assess joint pathology and gait alterations. The expression of proteins involved in the maintenance of cartilage homeostasis within the exosomes was determined through proteomic analysis. RESULTS: The preconditioning with TNF-α significantly enhanced the exosome secretion of IPFP-MSCs compared to unpreconditioned MSCs. The potential mechanism involved the activation of the PI3K/AKT signaling pathway in IPFP-MSCs by TNF-α precondition, leading to an up-regulation of autophagy-related protein 16 like 1(ATG16L1) levels, which subsequently facilitated exosome secretion. The intra-articular administration of IPFP-MSC-EXOsTNF-α demonstrated superior efficacy in ameliorating pathological changes in the joints of OA mice. The preconditioning of TNF-α enhanced the up-regulation of low-density lipoprotein receptor-related protein 1 (LRP1) levels in IPFP-MSC-EXOsTNF-α, thereby exerting chondroprotective effects. CONCLUSION: TNF-α preconditioning constitutes an effective and promising method for optimizing the therapeutic effects of IPFP-MSCs derived exosomes in the treatment of OA.
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Exosomas , Células Madre Mesenquimatosas , Osteoartritis , Factor de Necrosis Tumoral alfa , Exosomas/metabolismo , Animales , Células Madre Mesenquimatosas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ratones , Osteoartritis/terapia , Osteoartritis/metabolismo , Tejido Adiposo/citología , Ratones Endogámicos C57BL , Masculino , Modelos Animales de Enfermedad , Cartílago Articular/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Células Cultivadas , HumanosRESUMEN
BACKGROUND: Articular cartilage degeneration can result from injury, age, or arthritis, causing significant joint pain and disability without surgical intervention. Currently, the only FDA cell-based therapy for articular cartilage injury is Autologous Chondrocyte Implantation (ACI); however, this procedure is costly, time-intensive, and requires multiple treatments. Mesenchymal stromal cells (MSCs) are an attractive alternative autologous therapy due to their availability and ability to robustly differentiate into chondrocytes for transplantation with good safety profiles. However, treatment outcomes are variable due to donor-to-donor variability as well as intrapopulation heterogeneity and unstandardized MSC manufacturing protocols. Process improvements that reduce cell heterogeneity while increasing donor cell numbers with improved chondrogenic potential during expansion culture are needed to realize the full potential of MSC therapy. METHODS: In this study, we investigated the potential of MSC metabolic modulation during expansion to enhance their chondrogenic commitment by varying the nutrient composition, including glucose, pyruvate, glutamine, and ascorbic acid in culture media. We tested the effect of metabolic modulation in short-term (one passage) and long-term (up to seven passages). We measured metabolic state, cell size, population doubling time, and senescence and employed novel tools including micro-magnetic resonance relaxometry (µMRR) relaxation time (T2) to characterize the effects of AA on improved MSC expansion and chondrogenic potential. RESULTS: Our data show that the addition of 1 mM L-ascorbic acid-2-phosphate (AA) to cultures for one passage during MSC expansion prior to initiation of differentiation improves chondrogenic differentiation. We further demonstrate that AA treatment reduced the proportion of senescent cells and cell heterogeneity also allowing for long-term expansion that led to a > 300-fold increase in yield of MSCs with enhanced chondrogenic potential compared to untreated cells. AA-treated MSCs with improved chondrogenic potential showed a robust shift in metabolic profile to OXPHOS and higher µMRR T2 values, identifying critical quality attributes that could be implemented in MSC manufacturing for articular cartilage repair. CONCLUSIONS: Our results suggest an improved MSC manufacturing process that can enhance chondrogenic potential by targeting MSC metabolism and integrating process analytic tools during expansion.
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Cartílago Articular , Condrocitos , Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Cartílago Articular/metabolismo , Humanos , Condrocitos/metabolismo , Condrocitos/citología , Condrogénesis/efectos de los fármacos , Diferenciación Celular , Células Cultivadas , Proliferación Celular , Trasplante de Células Madre Mesenquimatosas/métodos , AnimalesRESUMEN
Articular cartilage phenotypic homeostasis is crucial for life-long joint function, but the underlying cellular and molecular mechanisms governing chondrocyte stability remain poorly understood. Here, we show that the protein tyrosine phosphatase SHP2 is differentially expressed in articular cartilage (AC) and growth plate cartilage (GPC) and that it negatively regulates cell proliferation and cartilage phenotypic program. Postnatal SHP2 deletion in Prg4+ AC chondrocytes increased articular cellularity and thickness, whereas SHP2 deletion in Acan+ pan-chondrocytes caused excessive GPC chondrocyte proliferation and led to joint malformation post-puberty. These observations were verified in mice and in cultured chondrocytes following treatment with the SHP2 PROTAC inhibitor SHP2D26. Further mechanistic studies indicated that SHP2 negatively regulates SOX9 stability and transcriptional activity by influencing SOX9 phosphorylation and promoting its proteasome degradation. In contrast to published work, SHP2 ablation in chondrocytes did not impact IL-1-evoked inflammation responses, and SHP2's negative regulation of SOX9 could be curtailed by genetic or chemical SHP2 inhibition, suggesting that manipulating SHP2 signaling has translational potential for diseases of cartilage dyshomeostasis.
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Cartílago Articular , Condrocitos , Osteoartritis , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Factor de Transcripción SOX9 , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/genética , Animales , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Condrocitos/metabolismo , Condrocitos/patología , Ratones , Cartílago Articular/metabolismo , Cartílago Articular/patología , Osteoartritis/metabolismo , Osteoartritis/patología , Proliferación Celular , Células Cultivadas , Ratones Endogámicos C57BL , Ratones Noqueados , MasculinoRESUMEN
The extracellular matrix of cartilage primarily constitutes of collagen and aggrecan. Cartilage degradation starts with aggrecan loss in osteoarthritis (OA). Vitamin D (VD) plays an essential role in several inflammation-related diseases and can protect the collagen in cartilage during OA. The present study focused on the role of VD in aggrecan turnover of human articular chondrocytes treated with tumor necrosis factor α (TNF-α) and the possible mechanism. Treatment with different doses of VD and different periods of intervention with TNF-α and TGF-ß1 receptor (TGFßR1) inhibitor SB525334 were investigated. The viability of human chondrocytes and extracellular secretion of TGF-ß1 were measured. The expression of intracellular TGFßR1 and VD receptor was examined. Transcriptional and translational levels of aggrecan and the related metabolic factors were analyzed. The results showed that TNF-α markedly reduced the viability, TGFßR1 expressions and aggrecan levels of human chondrocytes, and increased disintegrin and metalloproteinase with thrombospondin motifs. The alterations were partially inhibited by VD treatment. Furthermore, the effects of VD were blocked by the TGFßR1 inhibitor SB525334 in TNF-α-treated cells. VD may prevent proteoglycan loss due to TNF-α via TGF-ß1 signaling in human chondrocytes.
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Agrecanos , Cartílago Articular , Condrocitos , Proteoglicanos , Transducción de Señal , Factor de Crecimiento Transformador beta1 , Factor de Necrosis Tumoral alfa , Vitamina D , Humanos , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Agrecanos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Vitamina D/farmacología , Proteoglicanos/metabolismo , Proteoglicanos/farmacología , Cartílago Articular/metabolismo , Cartílago Articular/efectos de los fármacos , Células Cultivadas , Supervivencia Celular/efectos de los fármacos , Osteoartritis/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Receptores de Calcitriol/metabolismoRESUMEN
Articular cartilage receives nutrients and oxygen from the synovial fluid to maintain homeostasis. However, compared to tissues with abundant blood flow, articular cartilage is exposed to a hypoxic environment (i.e., physioxia) and has an enhanced hypoxic stress response. Hypoxia-inducible factors (HIFs) play a pivotal role in this physioxic environment. In normoxic conditions, HIFs are downregulated, whereas in physioxic conditions, they are upregulated. The HIF-α family comprises three members: HIF-1α, HIF-2α, and HIF-3α. Each member has a distinct function in articular cartilage. In osteoarthritis, which is primarily caused by degeneration of articular cartilage, HIF-1α is upregulated in chondrocytes and is believed to protect articular cartilage by acting anabolically on it. Conversely, in contrast to HIF-1α, HIF-2α exerts a catabolic influence on articular cartilage. It may therefore be possible to develop a new treatment for OA by controlling the expression of HIF-1α and HIF-2α with drugs or by altering the oxygen environment in the joints.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cartílago Articular , Condrocitos , Homeostasis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Osteoartritis , Humanos , Cartílago Articular/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Osteoartritis/metabolismo , Condrocitos/metabolismo , Oxígeno/metabolismo , Hipoxia/metabolismo , Hipoxia/fisiopatologíaRESUMEN
Osteoarthritis (OA) is an age-related disease characterized by inflammation, pain, articular cartilage damage, synovitis, and irreversible disability. Gynostemma pentaphyllum (Thunb.) Makino (GP), a herbal medicine traditionally used in East Asia for its anti-inflammatory properties, was investigated for its potential to modulate OA pathology and symptoms. This study evaluated GP's efficacy in inhibiting pain, functional decline, and cartilage destruction in monosodium iodoacetate-induced OA and acetic acid-induced writhing models. Additionally, the effects of GP on OA-related inflammatory targets were assessed via mRNA and protein expression in rat knee cartilage and lipopolysaccharide-induced RAW 264.7 cells. The GP group demonstrated significant pain relief, functional improvement, and cartilage protection. Notably, GP inhibited key inflammatory mediators, including interleukin (IL)-1ß, IL-6, matrix metalloproteinases (MMP)-3 and MMP-13, cyclooxygenase-2, and prostaglandin E receptor 2, surpassing the effects of active controls. These findings suggest that GP is a promising candidate for disease-modifying OA drugs and warrants further comprehensive studies.
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Analgésicos , Antiinflamatorios , Gynostemma , Osteoartritis , Extractos Vegetales , Animales , Gynostemma/química , Ratones , Osteoartritis/tratamiento farmacológico , Osteoartritis/patología , Osteoartritis/inducido químicamente , Osteoartritis/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Células RAW 264.7 , Ratas , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Analgésicos/farmacología , Analgésicos/uso terapéutico , Masculino , Cartílago Articular/efectos de los fármacos , Cartílago Articular/patología , Cartílago Articular/metabolismo , Modelos Animales de Enfermedad , Ratas Sprague-Dawley , Dolor/tratamiento farmacológicoRESUMEN
BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative disease. We explored the role and regulatory mechanisms of lncRNA-FAS-AS1 in OA progression. METHODS: We exposed human immortalized chondrocytes to IL-1ß for 24 h to induce an OA cell model. The target molecule levels were assessed using western blot and quantitative real-time PCR (RT-qPCR). Cell viability and apoptosis were measured using CCK-8 and flow cytometry. The m6A modification of FAS-AS1 was determined using MeRIP. We examined the binding relationships between FAS-AS1, Fragile X mental retardation 1 (FMR1), and A disintegrin and metalloproteinase 8 (ADAM8) using RIP and RNA pull-down. The OA animal model was established by separating the medial collateral ligament and medial meniscus. Safranin-O staining and Mankin's scale were employed to evaluate pathological changes within the cartilage. RESULTS: FAS-AS1, METTL14, and ADAM8 were upregulated, and the JAK/STAT3 signaling pathway was activated in OA mice and IL-1ß-induced chondrocytes. FAS-AS1 knockdown inhibited extracellular matrix degradation in IL-1ß-induced chondrocytes; however, ADAM8 overexpression reversed this effect. FAS-AS1 maintained the stability of ADAM8 mRNA by recruiting FMR1. METTL14 knockdown repressed FAS-AS1 expression in an m6A-dependent manner. FAS-AS1 overexpression reversed the inhibitory effects of METTL14 knockdown on JAK/STAT3 signaling and cartilage damage in the OA model both in vitro and in vivo. CONCLUSION: METTL14-mediated FAS-AS1 promotes OA progression through the FMR1/ADAM8/JAK/STAT3 axis.
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Proteínas ADAM , Condrocitos , Progresión de la Enfermedad , Proteínas de la Membrana , ARN Largo no Codificante , Factor de Transcripción STAT3 , Transducción de Señal , Regulación hacia Arriba , Animales , Humanos , Masculino , Ratones , Proteínas ADAM/metabolismo , Proteínas ADAM/genética , Adenosina/análogos & derivados , Apoptosis , Artritis Experimental/metabolismo , Artritis Experimental/genética , Artritis Experimental/patología , Cartílago Articular/metabolismo , Cartílago Articular/patología , Línea Celular , Condrocitos/metabolismo , Condrocitos/patología , Modelos Animales de Enfermedad , Interleucina-1beta/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Ratones Endogámicos C57BL , Osteoartritis/metabolismo , Osteoartritis/genética , Osteoartritis/patología , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/genética , Osteoartritis de la Rodilla/patología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genéticaRESUMEN
Osteoarthritis is a degenerative joint disease with joint pain as the main symptom, caused by fibrosis and loss of articular cartilage. Due to the complexity and heterogeneity of osteoarthritis, there is a lack of effective individualized disease-modifying osteoarthritis drugs in clinical practice. Chondrocyte senescence is reported to participate in occurrence and progression of osteoarthritis. Here we show that small molecule 10-hydroxy-2-decenoic acid suppresses cartilage degeneration and relieves pain in the chondrocytes, cartilage explants from osteoarthritis patients, surgery-induced medial meniscus destabilization or naturally aged male mice. We further confirm that 10-hydroxy-2-decenoic acid exerts a protective effect by targeting the glycosylation site in the Asp_Arg_Hydrox domain of aspartyl ß-hydroxylase. Mechanistically, 10-hydroxy-2-decenoic acid alleviate cellular senescence through the ERK/p53/p21 and GSK3ß/p16 pathways in the chondrocytes. Our study uncovers that 10-hydroxy-2-decenoic acid modulate cartilage metabolism by targeting aspartyl ß-hydroxylase to inhibit chondrocyte senescence in osteoarthritis. 10-hydroxy-2-decenoic acid may be a promising therapeutic drug against osteoarthritis.
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Cartílago Articular , Senescencia Celular , Condrocitos , Ácidos Grasos Monoinsaturados , Osteoartritis , Animales , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/patología , Masculino , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoartritis/tratamiento farmacológico , Osteoartritis/prevención & control , Ratones , Senescencia Celular/efectos de los fármacos , Humanos , Ácidos Grasos Monoinsaturados/farmacología , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Cartílago Articular/patología , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , FemeninoRESUMEN
Osteoarthritis (OA) is the most common form of arthritic disease, and phenotypic modification of chondrocytes is an important mechanism that contributes to the loss of cartilage homeostasis. This study identified that Fascin actin-bundling protein 1 (FSCN1) plays a pivotal role in regulating chondrocytes phenotype and maintaining cartilage homeostasis. Proteome-wide screening revealed markedly upregulated FSCN1 protein expression in human OA cartilage. FSCN1 accumulation was confirmed in the superficial layer of OA cartilage from humans and mice, primarily in dedifferentiated-like chondrocytes, associated with enhanced actin stress fiber formation and upregulated type I and III collagens. FSCN1-inducible knockout mice exhibited delayed cartilage degeneration following experimental OA surgery. Mechanistically, FSCN1 promoted actin polymerization and disrupted the inhibition of Decorin on TGF-ß1, leading to excessive TGF-ß1 production and ALK1/Smad1/5 signaling activation, thus, accelerated chondrocyte dedifferentiation. Intra-articular injection of FSCN1-overexpressing adeno-associated virus exacerbated OA progression in mice, which was mitigated by an ALK1 inhibitor. Moreover, FSCN1 inhibitor NP-G2-044 effectively reduced extracellular matrix degradation in OA mice, cultured human OA chondrocytes, and cartilage explants by suppressing ALK1/Smad1/5 signaling. These findings suggest that targeting FSCN1 represents a promising therapeutic approach for OA.
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Proteínas Portadoras , Condrocitos , Proteínas de Microfilamentos , Osteoartritis , Animales , Humanos , Masculino , Ratones , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Cartílago Articular/metabolismo , Cartílago Articular/patología , Condrocitos/metabolismo , Condrocitos/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Osteoartritis/patología , Osteoartritis/metabolismo , Osteoartritis/genética , Fenotipo , Receptores Odorantes , Transducción de SeñalRESUMEN
The study aims to elucidate the therapeutic mechanism of Baicalin (BAI) in alleviating cartilage injury in osteoarthritic (OA) rat models, concentrating on its regulation of the miR-766-3p/AIFM1 axis. An OA rat model was developed with unilateral anterior cruciate ligament transection (ACLT). Interventions comprised of BAI treatment and intra-articular administration of miR-766-3p inhibitor. For evaluation, histopathological staining was conducted to investigate the pathological severity of knee cartilage injury. The levels of oxidative stress (OS) indicators including MDA, SOD, and GSH-Px, were quantified using colorimetric assays. Inflammatory factors (IFs; TNF-?, IL-1?, and IL-6) in knee joint lavage fluids were assessed using ELISA, while RT-PCR was employed to quantify miR-766-3p expression. TUNEL apoptosis staining was utilized to detect chondrocyte apoptosis, and western blotting examined autophagy-related markers (LC3, Beclin, p62), extracellular matrix (ECM) synthesis-associated indices (COL2A, ACAN, MMP13), and apoptosis-inducing factor mitochondrion-associated 1 (AIFM1). Histological examination revealed a marked amelioration of cartilage injury in the BAI-treated OA rat models compared to controls. BAI treatment significantly reduced inflammation and OS of knee joint fluid, activated autophagy, and decreased chondrocyte apoptosis and ECM degradation. Interestingly, the inhibitory effects of BAI on these pathological markers were significantly decreased by the miR-766-3p inhibitor. Further assessment revealed that BAI efficiently promoted miR-766-3p expression while inhibiting AIFM1 protein expression. BAI potentially mitigates articular cartilage injury in OA rats, likely through modulation of miR-766-3p/AIFM1 axis. Keywords: Baicalin, microRNA, AIFM1, Osteoarthritisv, Rat.
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Flavonoides , MicroARNs , Ratas Sprague-Dawley , Animales , Flavonoides/farmacología , Flavonoides/uso terapéutico , MicroARNs/metabolismo , MicroARNs/genética , MicroARNs/biosíntesis , Ratas , Masculino , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Osteoartritis/patología , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Cartílago Articular/patología , Factor Inductor de la Apoptosis/metabolismo , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacosRESUMEN
OBJECTIVE: The aim of this study was to investigate whether ASA VI controls osteoarthritis (OA) by regulating mitochondrial function. METHODS: Primary chondrocytes were isolated and cultured from rat knee joints. The chondrocytes were treated with ASA VI and interleukin-1ß (IL-1ß) to simulate the inflammatory environment of OA. Cell viability, apoptosis, inflammatory cytokine levels, and extracellular matrix (ECM) component levels were assessed. Mitochondrial function, including ATP levels, mitochondrial membrane potential, reactive oxygen species (ROS) levels, and mitochondrial DNA content, was evaluated. The expression of Sirtuin 3 (Sirt3), a key regulator of mitochondrial homeostasis, was examined. Additionally, a rat OA model was established by destabilizing the medial meniscus, and the effects of ASA VI on cartilage degeneration were assessed. RESULTS: ASA VI treatment improved cell viability, reduced apoptosis, and decreased IL-6 and TNF-α levels in IL-1ß-induced chondrocytes. ASA VI also upregulated Collagen II and Aggrecan expression, while downregulating ADAMTS5 and MMP-13 expression. Furthermore, ASA VI mitigated IL-1ß-induced mitochondrial dysfunction by increasing ATP levels, restoring mitochondrial membrane potential, reducing ROS production, and preserving mitochondrial DNA content. These effects were accompanied by the activation of Sirt3. In the rat OA model, ASA VI treatment increased Sirt3 expression and alleviated cartilage degeneration. CONCLUSION: ASA VI exerts chondroprotective and anti-inflammatory effects on IL-1ß-induced chondrocytes by improving mitochondrial function through Sirt3 activation. ASA VI also attenuates cartilage degeneration in a rat OA model. These findings suggest that ASA VI may be a potential therapeutic agent for the treatment of osteoarthritis by targeting mitochondrial dysfunction.
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Condrocitos , Homeostasis , Mitocondrias , Osteoartritis , Saponinas , Sirtuina 3 , Animales , Masculino , Ratones , Ratas , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Apoptosis/efectos de los fármacos , Cartílago Articular/patología , Cartílago Articular/metabolismo , Cartílago Articular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Interleucina-1beta/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Osteoartritis/patología , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 3/metabolismo , Sirtuina 3/genética , Saponinas/farmacologíaRESUMEN
Synovial fluid lubricates articular joints by forming a hydrated layer between the cartilage surfaces. In degenerative joint diseases like osteoarthritis (OA), the synovial fluid is compromised, which leads to less effective innate lubrication and exacerbated cartilage degeneration. Studies over the years have led to the development of partially or fully synthetic biolubricants to reduce the coefficient of friction with cartilage in knee joints. Cartilage-adhering, hydrated lubricants are particularly important to provide cartilage lubrication and chondroprotection under high normal load and slow speed. Here, we report the development of a hyaluronic acid (HA)-based lubricant functionalized with cationic branched poly-L-lysine (BPL) molecules that bind to cartilage via electrostatic interactions. We surmised that the electrostatic interactions between the BPL-modified HA molecules (HA-BPL) and the cartilage facilitate localization of the HA molecules to the cartilage surface. The number of BPL molecules on the HA backbone was varied to determine the optimal grafting density for cartilage binding and HA localization. Collectively, our results show that our HA-BPL molecules adhered readily to cartilage and were effective as a lubricant in cartilage-on-cartilage shear measurements where the modified HA molecules significantly reduce the coefficient of friction compared to phosphate-buffered saline or HA alone. This proof-of-concept study shows how the incorporation of cartilage adhering moieties, such as cationic molecules, can be used to enhance cartilage binding and lubrication properties of HA.
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Cartílago Articular , Cationes , Ácido Hialurónico , Lubrificación , Polilisina , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Adsorción , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Cationes/química , Animales , Polilisina/química , Polilisina/farmacología , Bovinos , Lubricantes/química , Lubricantes/farmacología , Fricción/efectos de los fármacos , Líquido Sinovial/metabolismo , Líquido Sinovial/química , Líquido Sinovial/efectos de los fármacosRESUMEN
In orthopedic research, many studies have applied vitamin E as a protective antioxidant or used tert-butyl hydroperoxide to induce oxidative injury to chondrocytes. These studies often support the hypothesis that joint pathology causes oxidative stress and increased lipid peroxidation that might be prevented with lipid antioxidants to improve cell survival or function and joint health; however, lipid antioxidant supplementation was ineffective against osteoarthritis in clinical trials and animal data have been equivocal. Moreover, increased circulating vitamin E is associated with increased rates of osteoarthritis. This disconnect between benchtop and clinical results led us to hypothesize that oxidative stress-driven paradigms of chondrocyte redox function do not capture the metabolic and physiologic effects of lipid antioxidants and prooxidants on articular chondrocytes. We used ex vivo and in vivo cartilage models to investigate the effect of lipid antioxidants on healthy, primary, articular chondrocytes and applied immuno-spin trapping techniques to provide a broad indicator of high levels of oxidative stress independent of specific reactive oxygen species. Key findings demonstrate lipid antioxidants were pro-mitochondrial while lipid prooxidants decreased mitochondrial measures. In the absence of injury, radical formation was increased by lipid antioxidants; however, in the presence of injury, radical formation was decreased. In unstressed conditions, this relationship between chondrocyte mitochondria and redox regulation was reproduced in vivo with overexpression of glutathione peroxidase 4. In mice aged 18 months or more, overexpression of glutathione peroxidase 4 significantly decreased the presence of pro-mitochondrial peroxisome proliferation activated receptor gamma and deranged the relationship between mitochondria and the redox environment. This complex interaction suggests strategies targeting articular cartilage may benefit from adopting more nuanced paradigms of articular chondrocyte redox metabolism.
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Condrocitos , Peroxidación de Lípido , Mitocondrias , Oxidación-Reducción , Estrés Oxidativo , Condrocitos/metabolismo , Condrocitos/efectos de los fármacos , Animales , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/farmacología , Antioxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Cartílago Articular/metabolismo , Ratones , Células CultivadasRESUMEN
Osteoarthritis (OA) is a common joint disease associated with the aging of the population, and it reduces the quality of life of patients. It is characterized by the destruction of articular cartilage and the secretion of inflammatory cytokines. Owing to the unclear pathogenesis of OA, current treatment methods have significant limitations. Oxidative stress has been revealed to play an important role in the development of OA. Our experiments indicated that the levels of GSH decreased and the level of MDA increased in chondrocytes, which induced ferroptosis in chondrocytes in OA. We also revealed that ferroptosis was the main mechanism of cartilage destruction caused by the addition of the ferroptosis activator erastin and the ferroptosis inhibitor ferrostatin-1. NOX1 is the main modulator of oxidative stress by increasing the generation of reactive oxidative species (ROS). We suppressed the expression of NOX1 in chondrocytes through cell transfection. The expression of collagen II and MMP13, and the secretion of IL-1ß and TNF-α were reversed. An increase in the mitochondrial membrane potential and a decrease in the level of intracellular ROS indicate an improvement in oxidative damage. Additionally, we determined the effect of the Nrf2/HO-1 pathway on NOX1-mediated chondrocyte injury. We found that NOX1 inhibited the expression of Nrf2/HO-1, but the activation of Nrf2 improved the oxidative damage to chondrocytes in vivo and vitro. This study revealed that NOX1-mediated oxidative stress induces chondrocyte ferroptosis by inhibiting the Nrf2/HO-1 pathway. Our findings contribute to revealing the pathogenesis of OA, providing targets for drug design and optimizing the clinical treatment of OA.
Asunto(s)
Condrocitos , Ferroptosis , Hemo-Oxigenasa 1 , NADPH Oxidasa 1 , Factor 2 Relacionado con NF-E2 , Osteoartritis , Estrés Oxidativo , Especies Reactivas de Oxígeno , Transducción de Señal , Condrocitos/metabolismo , Ferroptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Animales , NADPH Oxidasa 1/metabolismo , NADPH Oxidasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Ratones , Osteoartritis/metabolismo , Osteoartritis/patología , Especies Reactivas de Oxígeno/metabolismo , Ciclohexilaminas/farmacología , Masculino , Cartílago Articular/metabolismo , Cartílago Articular/patología , Humanos , Proteínas de la Membrana , FenilendiaminasRESUMEN
Temporomandibular joint osteoarthritis (TMJOA) is considered to be a low-grade inflammatory disease involving multiple joint tissues. The crosstalk between synovium and cartilage plays an important role in TMJOA. Synovial cells are a group of heterogeneous cells and synovial microenvironment is mainly composed of synovial fibroblasts (SF) and synovial macrophages. In TMJOA, SF and synovial macrophages release a large number of inflammatory cytokines and extracellular vesicles and promote cartilage destruction. Cartilage wear particles stimulate SF proliferation and macrophages activation and exacerbate synovitis. In TMJOA, chondrocytes and synovial cells exhibit increased glycolytic activity and lactate secretion, leading to impaired chondrocyte matrix synthesis. Additionally, the synovium contains mesenchymal stem cells, which are the seed cells for cartilage repair in TMJOA. Co-culture of chondrocytes and synovial mesenchymal stem cells enhances the chondrogenic differentiation of stem cells. This review discusses the pathological changes of synovium in TMJOA, the means of crosstalk between synovium and cartilage, and their influence on each other. Based on the crosstalk between synovium and cartilage in TMJOA, we illustrate the treatment strategies for improving synovial microenvironment, including reducing cell adhesion, utilizing extracellular vesicles to deliver biomolecules, regulating cellular metabolism and targeting inflammatory cytokines.
Asunto(s)
Microambiente Celular , Condrocitos , Osteoartritis , Membrana Sinovial , Articulación Temporomandibular , Humanos , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoartritis/terapia , Condrocitos/metabolismo , Condrocitos/patología , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Animales , Articulación Temporomandibular/metabolismo , Articulación Temporomandibular/patología , Trastornos de la Articulación Temporomandibular/metabolismo , Trastornos de la Articulación Temporomandibular/patología , Trastornos de la Articulación Temporomandibular/terapia , Células Madre Mesenquimatosas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Citocinas/metabolismo , Macrófagos/metabolismo , Cartílago Articular/metabolismo , Cartílago Articular/patologíaRESUMEN
BACKGROUND: Cartilage metabolism dysregulation is a crucial driver in knee osteoarthritis (KOA). Modulating the homeostasis can mitigate the cartilage degeneration in KOA. Curcumenol, derived from traditional Chinese medicine Curcuma Longa L., has demonstrated potential in enhancing chondrocyte proliferation and reducing apoptosis. However, the specific mechanism of Curcumenol in treating KOA remains unclear. This study aimed to demonstrate the molecular mechanism of Curcumenol in treating KOA based on the transcriptomics and metabolomics, and both in vivo and in vitro experimental validations. MATERIALS AND METHODS: In this study, a destabilization medial meniscus (DMM)-induced KOA mouse model was established. And the mice were intraperitoneally injected with Curcumenol at 4 and 8 mg/kg concentrations. The effects of Curcumenol on KOA cartilage and subchondral was evaluated using micro-CT, histopathology, and immunohistochemistry (IHC). In vitro, OA chondrocytes were induced with 10 µg/mL lipopolysaccharide (LPS) and treated with Curcumenol to evaluate the proliferation, apoptosis, and extracellular matrix (ECM) metabolism through CCK8 assay, flow cytometry, and chondrocyte staining. Furthermore, transcriptomics and metabolomics were utilized to identify differentially expressed genes (DEGs) and metabolites. Finally, integrating multi-omics analysis, virtual molecular docking (VMD), and molecular dynamics simulation (MDS), IHC, immunofluorescence (IF), PCR, and Western blot (WB) validation were conducted to elucidate the mechanism by which Curcumenol ameliorates KOA cartilage degeneration. RESULTS: Curcumenol ameliorated cartilage destruction and subchondral bone loss in KOA mice, promoted cartilage repair, upregulated the expression of COL2 while downregulated MMP3, and improved ECM synthesis metabolism. Additionally, Curcumenol also alleviated the damage of LPS on the proliferation activity and suppressed apoptosis, promoted ECM synthesis. Transcriptomic analysis combined with weighted gene co-expression network analysis (WGCNA) identified a significant downregulation of 19 key genes in KOA. Metabolomic profiling showed that Curcumenol downregulates the expression of d-Alanyl-d-alanine, 17a-Estradiol, Glutathione, and Succinic acid, while upregulating Sterculic acid and Azelaic acid. The integrated multi-omics analysis suggested that Curcumenol targeted KDM6B to regulate downstream protein H3K27me3 expression, which inhibited methylation at the histone H3K27, consequently reducing Succinic acid levels and improving KOA cartilage metabolism homeostasis. Finally, both in vivo and in vitro findings indicated that Curcumenol upregulated KDM6B, suppressed H3K27me3 expression, and stimulated collagen II expression and ECM synthesis, thus maintaining cartilage metabolism homeostasis and alleviating KOA cartilage degeneration. CONCLUSION: Curcumenol promotes cartilage repair and ameliorates cartilage degeneration in KOA by upregulating KDM6B expression, thereby reducing H3K27 methylation and downregulating Succinic Acid, restoring metabolic stability and ECM synthesis.
Asunto(s)
Condrocitos , Curcuma , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Osteoartritis de la Rodilla , Ácido Succínico , Animales , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Ratones , Masculino , Curcuma/química , Osteoartritis de la Rodilla/tratamiento farmacológico , Osteoartritis de la Rodilla/metabolismo , Ácido Succínico/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Sesquiterpenos/farmacología , Simulación del Acoplamiento Molecular , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , HumanosRESUMEN
Connexin 43 (Cx43) is crucial for the development and homeostasis of the musculoskeletal system, where it plays multifaceted roles, including intercellular communication, transcriptional regulation and influencing osteogenesis and chondrogenesis. Here, we investigated Cx43 modulation mediated by inflammatory stimuli involved in osteoarthritis, i.e., 10 ng/mL Tumor Necrosis Factor alpha (TNFα) and/or 1 ng/mL Interleukin-1 beta (IL-1ß), in primary chondrocytes (CH) and osteoblasts (OB). Additionally, we explored the impact of synovial fluids from osteoarthritis patients in CH and cartilage explants, providing a more physio-pathological context. The effect of TNFα on Cx43 expression in cartilage explants was also assessed. TNFα downregulated Cx43 levels both in CH and OB (-73% and -32%, respectively), while IL-1ß showed inconclusive effects. The reduction in Cx43 levels was associated with a significant downregulation of the coding gene GJA1 expression in OB only (-65%). The engagement of proteasome in TNFα-induced effects, already known in CH, was also observed in OB. TNFα treatment significantly decreased Cx43 expression also in cartilage explants. Of note, Cx43 expression was halved by synovial fluid in both CH and cartilage explants. This study unveils the regulation of Cx43 in diverse musculoskeletal cell types under various stimuli and in different contexts, providing insights into its modulation in inflammatory joint disorders.
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Condrocitos , Conexina 43 , Interleucina-1beta , Osteoartritis , Osteoblastos , Factor de Necrosis Tumoral alfa , Humanos , Conexina 43/metabolismo , Conexina 43/genética , Condrocitos/metabolismo , Osteoblastos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacología , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoartritis/genética , Líquido Sinovial/metabolismo , Cartílago Articular/metabolismo , Cartílago Articular/patología , Células Cultivadas , Anciano , Persona de Mediana Edad , Inflamación/metabolismo , Inflamación/genética , Inflamación/patología , Cartílago/metabolismo , Cartílago/patología , Artropatías/metabolismo , Artropatías/patología , Artropatías/genéticaRESUMEN
Senescent chondrocytes or signaling mechanisms leading to senescence are promising new therapeutic approaches for ameliorating cartilage degradation. Herein, we show that the transactive response DNA/RNA-binding protein (TDP-43) regulates chondrocyte senescence and ameliorates cartilage degradation. First, a significant decrease in TDP-43 was observed in 16-month-old mice compared with younger mice. Immunohistochemistry (IHC) analysis of mouse articular cartilage showed that p21, p16, p53, and matrix metalloprotein-13 (MMP13) were increased, but laminB1 and Collagen type II alpha1 1 chain (Col2a1) were decreased in 16-month-old mice. Furthermore, TDP-43 levels were decreased in vivo following D-galactose (D-gal) induction. Therefore, we investigated the role of TDP-43 in the senescent chondrocytes. ATDC5 cells were induced to overexpress TDP-43. Western blot analysis showed increased expression of laminB1, Ki67, and PCNA but decreased expression of p21, p16, p53, and MMP13. Senescence-associated-ß-galactosidase (SA-ß-Gal) assay, γH2AX staining, and EdU were performed to assess changes in chondrocytes, showing weaker SA-ß-Gal and γH2AX staining but stronger EdU and Alican Blue staining. However, TDP-43 deficiency had opposing effects, and similar to D-gal stimulation results. Taken together, our data verified that TDP-43 negatively correlated with senescence markers, positively correlated with cell proliferation markers, and could alleviate cartilage degradation induced by D-gal. This may be an essential mechanism of cellular senescence and cartilage degradation.