Osteoarthritis In Mice Research Articles

RIP1 inhibition reduces chondrocyte apoptosis through downregulating nuclear factor-kappa B signaling in a mouse osteoarthritis model.

Excessive chondrocyte death is a critical player in the process of osteoarthritis (OA). The present study was aimed to study the role of receptor-interacting serine/threonine kinase (RIP) 1-mediated signaling for programmed cell death in OA. In the present study, RIP1 protein expression was evaluated in mouse OA cartilage and cultured primary murine chondrocytes exposed to tumor necrosis factor-alpha (TNF-α). Protein expression involved in necroptosis and apoptosis and chondrocyte-derived extracellular matrix were examined. Inhibition of RIP1 was conducted using the RNAi technique and pharmacological inhibition. Western blot, immunohistochemistry, and immunofluorescence examination were applied. The protein presence of RIP1, but not RIP3, was increased in the mouse OA tissue and cultured chondrocytes exposed to TNF-α. Knockdown of RIP1 increased protein expression of collagen II and sex-determining region Y-box transcription factor 9, and reduced protein expression of matrix metallopeptidases 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5. Inhibition of RIP1 reduced the phosphorylated NF-κB signals, decreased cell apoptosis, and restored extracellular matrix expression in cultured chondrocytes. Both RNAi and pharmacological inhibition of RIP1 decelerated the progress of OA in mice. RIP1 regulates chondrocyte apoptosis through NF-κB signaling. Inhibition of RIP1 provides a novel therapeutic approach for OA therapy.

Inhibition of Slit3/Robo1 signaling alleviates osteoarthritis in mice by reducing abnormal H-type vessel formation in subchondral bone

Background The aberrant H-type vessel formation was found to be intimately linked to subchondral bone remodeling during osteoarthritis (OA) development. Herein, we investigated the role and mechanism of osteoblast-secreted slit guidance ligand 3 (Slit3) in H-type vessel formation during OA progression. Methods Slit3 protein levels in subchondral bone samples of OA patients were detected. The isolated osteoblasts were transfected with Slit3 overexpression or knockdown plasmids, and their conditioned medium was cultured with endothelial progenitor cells (EPCs). The migration, tube formation, VEGF, and H-type vessel marker protein CD31 and endomucin (EMCN) levels in EPCs were accessed. The interactions between Slit3 and roundabout (Robo) family members were validated by Co-IP assay. Besides, whether the Slit3/Robo signaling affects the transforming growth factor β1 (TGF-β1)/SMADs pathway was determined. Additionally, sh-Slit3 was injected into OA mice, followed by the detection of articular cartilage degradation, subchondral bone remodeling, and H-type vessel formation. Results Slit3 was upregulated in subchondral bone tissues of OA patients. Slit3 overexpression in osteoblasts intensified the migration and H-type vessel formation of EPCs, while Slit3 knockdown showed the opposite results. Slit3 overexpression enhanced Robo1 protein level. Robo1 knockdown abrogated Slit3-mediated migration and H-type vessel formation in EPCs. Slit3 activated the TGF-β1/SMADs pathway in EPCs, which might be associated with H-type vessel formation in EPCs. Additionally, Slit3 silencing restrained articular cartilage degradation, aberrant subchondral bone formation, and H-type vessel formation in OA mice. Conclusion Inhibition of Slit3/Robo1 signaling alleviates osteoarthritis in mice by reducing abnormal H-type vessel formation in the subchondral bone.

β-hydroxybutyrate ameliorates osteoarthritis through activation of the ERBB3 signaling pathway in mice.

The ketogenic diet (KD) has demonstrated efficacy in ameliorating inflammation in rats with osteoarthritis (OA). However, the long-term safety of the KD and the underlying mechanism by which it delays OA remain unclear. We found that while long-term KD could ameliorate OA, it induced severe hepatic steatosis in mice. Consequently, we developed two versions of ketogenic-based diets: KD supplemented with vitamin D and intermittent KD. Both KD supplemented with vitamin D and intermittent KD effectively alleviated OA by significantly reducing the levels of inflammatory cytokines, cartilage loss, sensory nerve sprouting, and knee hyperalgesia without inducing hepatic steatosis. Furthermore, β-hydroxybutyrate (β-HB), a convenient energy carrier produced by adipocytes, could ameliorate OA without causing liver lesions. Mechanistically, β-HB enhanced chondrocyte autophagy and reduced apoptosis through the activation of Erb-B2 receptor tyrosine kinase 3 (ERBB3) signaling pathway; a pathway which was down-regulated in the articular chondrocytes from both OA patients and mice. Collectively, our findings highlighted the potential therapeutic value of β-HB and KD supplemented with vitamin D and intermittent KD approaches for managing OA.

Astaxanthin mediated repair of tBHP-Induced cellular injury in chondrocytes

ABSTRACT Objective This study investigates how astaxanthin (AST) counters tert-butyl hydroperoxide (tBHP)-induced cellular damage in C28/I2 chondrocytes, focusing on the circ-HP1BP3/miR-139-5p/SOD1 signaling pathway and its use in sustained-release microspheres for osteoarthritis treatment. Methods We employed a variety of techniques including real-time quantitative PCR, Western blot, ELISA, and dual-luciferase reporter gene assays to explore AST's molecular effects. Additionally, the efficacy of AST-loaded sustained-release microspheres was evaluated in vitro and in a mouse model of osteoarthritis. Results AST significantly enhanced SOD1 expression, reducing apoptosis and inflammation in damaged cells. The AST-loaded microspheres showed promising in vitro drug release, improved cell viability, and reduced oxidative stress. In the osteoarthritis mouse model, they effectively decreased joint inflammation and increased the expression of chondrocyte markers. Conclusion Astaxanthin effectively mitigates oxidative stress and inflammation in chondrocytes via the circ-HP1BP3/miR-139-5p/SOD1 pathway. The development of AST-loaded microspheres offers a novel and promising approach for osteoarthritis therapy, potentially extending to osteoarthritis treatment.

KLF2 Inhibits Ferroptosis and Improves Mitochondrial Dysfunction in Chondrocyte Through SIRT1/GPX4 Signaling to Improve Osteoarthritis.

Osteoarthritis (OA), a disease of articular joints, is the leading cause of disability in the elderly. Repressing ferroptosis and improving mitochondrial function can delay the progression of OA. Kruppel-like factor 2 (KLF2) exerts a protective effect on OA. However, whether KLF2 affects ferroptosis and mitochondrial function during OA remains unknown. The OA in vivo and in vitro models were constructed in this work. The structural damage of knee joint in OA mice was evaluated through Micro-CT scanning. H&E, SOFG, TB, and TUNEL staining were applied for pathological examination of cartilage tissues. ELISA was employed to examine the contents of inflammatory factors. Additionally, iron deposition in cartilage tissues was measured by Prussian blue staining, and the levels of proteins related to ferroptosis were assessed by immunoblotting. Besides, mitochondrial morphology and function were estimated using a transmission electron microscope and JC-1 staining. In interleukin (IL)-1β-treated C28/I2 cells, the levels of inflammatory factors, intracellular ROS, mitochondrial ROS, lipid ROS, and Fe2+ were measured. Mitochondrial function was evaluated by detecting the levels of mitochondrial membrane potential (MMP), ATP, mPTP, and OCR. KLF2 overexpression ameliorated the structural damage of knee cartilage in OA mice. KLF2 upregulation inhibited ferroptosis and alleviated mitochondrial damage in knee cartilage of OA mice and IL-1β-treated C28/I2 cells. Moreover, KLF2 overexpression activated SIRT1/GPX4 signaling in vivo and in vitro. EX527 addition blocked the influences of KLF2 upregulation on ferroptosis and mitochondrial dysfunction in IL-1β-treated C28/I2 cells. Altogether KLF2 inhibits ferroptosis and improves mitochondrial dysfunction in chondrocytes through SIRT1/GPX4 signaling to improve OA.

TRPM2-mediated feed-forward loop promotes chondrocyte damage in osteoarthritis via calcium-cGAS-STING-NF-κB pathway

IntroductionsOsteoarthritis (OA) is a significant contributor to disability in the elderly population. However, current therapeutic options are limited. The transient receptor potential melastatin 2 (TRPM2) is involved in a range of disease processes, yet its role in OA remains unclear. ObjectivesTo investigate the role of TRPM2 in OA. MethodsCartilage samples were collected from patients with osteoarthritis (OA) and mice with OA to examine TRPM2 expression levels. To investigate the effects of TRPM2 modulation on the destabilization of the medial meniscus (DMM) induced knee OA in mice, we utilized TRPM2 knockout mice and employed adenovirus-mediated overexpression of TRPM2. Furthermore, siRNA-mediated TRPM2 knockdown or plasmid-mediated TRPM2 overexpression was conducted to explore the role of TRPM2 in IL-1β-induced chondrocytes. The regulatory mechanism of IL-1β on TRPM2 expression was screened by signaling pathway inhibitors, and the transcription factors and binding sites of TRPM2 were predicted using the database. The binding of RELA (NF-κB-p65) to the Trpm2 promoter was verified by chip-PCR and ChIP-qPCR. The therapeutic potential of Ca2+ chelation with BAPTA-AM for the treatment of osteoarthritis (OA) was investigated. ResultsAn increased expression of TRPM2 was observed in the cartilage of OA patients and OA mice. Furthermore, mice deficient in Trpm2 exhibited a protective effect against DMM-induced OA progression. In contrast, TRPM2 overexpression resulted in exacerbation of DMM-induced OA and thepromotion of an OA-like phenotype of chondrocytes. TRPM2 was upregulated by IL-1β in an NF-κB-p65-dependent manner. Subsequently, the TRPM2-Ca2+-mtDNA-cGAS-STING-NF-κB axis in the progression of OA was validated. Furthermore, inhibition of the TRPM2-Ca2+ axis with BAPTA-AM effectively attenuated established OA. ConclusionsOur data collectively revealed a pathological feedback loop involving TRPM2, Ca2+, mtDNA, cGAS, STING, and NF-κB in OA chondrocytes. This suggests that disrupting this loop could be a viable therapeutic approach for OA.

Loganin inhibits the ROS-NLRP3-IL-1β axis by activating the NRF2/HO-1 pathway against osteoarthritis

Loganin (LOG), a bioactive compound derived from Cornus officinalis Siebold & Zucc, has been understudied in the context of osteoarthritis (OA) treatment. In this study, we induced an inflammatory response in chondrocytes using lipopolysaccharide (LPS) and subsequently treated these cells with LOG. We employed fluorescence analysis to quantify reactive oxygen species (ROS) levels and measured the expression of NLRP3 and nuclear factor erythropoietin-2-related factor 2 (NRF2) using real-time quantitative polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorescence (IF) techniques. Additionally, we developed an OA mouse model by performing medial meniscus destabilization (DMM) surgery and monitored disease progression through micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, safranin O and fast green (S&F) staining, and immunohistochemical (IHC) analysis. Our results indicate that LOG significantly reduced LPS-induced ROS levels in chondrocytes, inhibited the activation of the NLRP3 inflammasome, and enhanced NRF2/heme oxygenase 1 (HO-1) signaling. In vivo, LOG treatment mitigated cartilage degradation and osteophyte formation triggered by DMM surgery, decreased NLRP3 expression, and increased NRF2 expression. These findings suggest that LOG has a protective effect against OA, potentially delaying disease progression by inhibiting the ROS-NLRP3-IL-1β axis and activating the NRF2/HO-1 pathway.

Increased Rab1a accelerates osteoarthritis by inhibiting autophagy via activation of the mTORC1-S6K pathway

IntroductionCartilage degradation is a critical alteration in the progression of osteoarthritis (OA) due to the disorder of chondrocyte metabolic homeostasis. Autophagy plays an important role in maintaining intracellular homeostasis. Recent investigations have increasingly underscored the importance of autophagy in modulating the pathological mechanisms underlying OA. Ras-related protein Rab-1a (Rab1a) has been illustrated to regulate autophagy in many diseases but not in OA. ObjectivesThis study aims to elucidate whether Rab1a could regulate the development of OA through modulation of chondrocyte autophagy and apoptosis. MethodsProteomic sequencing, Western blotting, and immunohistochemistry were applied to detect the expression level of Rab1a in vitro and in vivo. Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways were rigorously identified. The effects of Rab1a and the interaction between Rab1a, mTORC1, autophagy and apoptosis were explored by qPCR, Western blotting, and immunofluorescence. An experimental mouse OA model was also performed to confirm the role of Rab1a in OA pathogenesis in vivo. Histological analysis was employed to demonstrate cartilage damage. ResultsRab1a expression was significantly upregulated in inflamed chondrocytes and knee OA cartilage. Inhibition of Rab1a partially attenuated the degradation of the extracellular matrix and cell apoptosis both in vitro and in vivo, whereas overexpression of Rab1a intensified cartilage matrix degradation and cellular apoptosis. Additionally, elevated Rab1a levels were observed to suppress autophagy and activate the mTORC1-S6K signaling pathway, thereby aggravating OA pathogenesis. ConclusionThe augmentation of Rab1a expression impairs autophagy and promotes apoptosis through the activation of the mTORC1-S6K signaling pathway, further exacerbating OA pathogenesis. This finding suggests that Rab1a serves as a promising and innovative therapeutic target for the prevention and treatment of OA.

Comparison of post-traumatic osteoarthritis mouse models

Comparison of post-traumatic osteoarthritis mouse models

Differential effects of alendronate on chondrocytes, cartilage matrix and subchondral bone structure in surgically induced osteoarthritis in mice

Bisphosphonates (BP) are considered a treatment option for osteoarthritis (OA) due to reduction of OA-induced microtrauma in the bone marrow, stabilization of subchondral bone (SB) layer and pain reduction. The effects of high-dose alendronate (ALN) treatment on SB and articular cartilage after destabilization of the medial meniscus (DMM) or Sham surgery of male C57Bl/6J mice were analyzed. We performed serum analysis; histology and immunohistochemistry to assess the severity of OA and a possible pain symptomatology. Subsequently, the ratio of bone volume to total volume (BV/TV), epiphyseal trabecular morphology and the bone mineral density (BMD) was analyzed by nanoCT. Serum analysis revealed a reduction of ADAMTS5 level. The histological evaluation displayed no protective effect of ALN-treatment on cartilage erosion. NanoCT-analysis of the medial epiphysis revealed an increase of BV/TV in ALN-treated mice. Only the DMM group had significantly higher SB volume accompanied by decreased subchondral bone surface. Furthermore Nano-CT analysis revealed an increase in trabecular density and number, a decreased BMD and reduced osteophyte formation in the ALN mice. ALN treatment affected bone micro-architecture by reducing osteophytosis with simultaneous increasing subchondral bone plate thickness, trabecular thickness and BMD. Accordingly, ALN cannot be considered as a potential treatment strategy in general, however in a subgroup of patients with high bone turnover in an early-stage of OA, ALN might be an option when applied during a restricted time frame.

Targeting Synovial Macrophages with Astaxanthin-Loaded Liposomes for Antioxidant Treatment of Osteoarthritis.

Osteoarthritis (OA) is a chronic joint disease highly associated with an imbalance in the network of inflammatory factors and typically characterized by oxidative stress and cartilage damage. Moreover, the specificity of the joint structure makes it difficult for drugs to achieve good penetration and effective enrichment in the joint cavity. Therefore, therapeutic strategies that increase the specific targeting of drugs to inflammatory joint and incorporate antioxidative stress effects are important to improve the efficacy of OA. Here, we developed a folic acid-modified liposomal nanoparticle (AST@Lip-FA) loaded with the antioxidant astaxanthin (AST) to enhance the water solubility and stability of AST and to target the delivery of AST to the site of OA, leading to a significant improvement in therapeutic efficacy. In vitro experiments demonstrated that, due to the recognition by FA of the receptor folate receptor β on the surface of activated macrophages, the cellular uptake efficiency of AST@Lip-FA was increased. Meanwhile, intracellularly overexpressed inflammatory mediators such as reactive oxygen species and nitric oxide were efficiently removed by AST@Lip-FA. In addition, in the ACLT-induced OA mouse model, AST@Lip-FA was precisely enriched in the inflamed joints and achieved long-term retention, fully utilizing the anti-inflammatory, antioxidant, and cartilage-protecting effects of AST to effectively alleviate the progression of OA. In summary, AST@Lip-FA has an important prospect as a potential and effective therapeutic strategy for OA.

Amentoflavone maintaining extracellular matrix homeostasis and inhibiting subchondral bone loss in osteoarthritis by inhibiting ERK, JNK and NF-κB signaling pathways

Amentoflavone (AF), a plant biflavone isolated from Selaginella sinensis ethanol extract, is characterized by anti-inflammatory and anti-oxidant properties. According to previous studies, inflammation and oxidative stress are closely related to the pathophysiology of osteoarthritis (OA). However, the effects and mechanisms of AF on OA have not been elucidated.To investigate the inhibitory effects and its molecular mechanism of AF on extracellular matrix (ECM) degradation stimulated by IL-1β as well as subchondral bone loss induced by RANKL in mice chondrocytes. Quantitative PCR was used to detect the mRNA expression of genes related to inflammation, ECM, and osteoclast differentiation. Protein expression level of iNOS, COX-2, MMP13, ADAMTS5, COL2A1, SOX9, NFATc1, c-fos, JNK, ERK, P65, IκBα was measured by western blotting. The levels of TNF-α and IL-6 in the supernatants were measured by ELISA. The amount of ECM in chondrocytes was measured using toluidine blue staining. The levels of Aggrecan and Col2a1 in chondrocytes were measured using immunofluorescence. Tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining and immunofluorescence were used to detect the effect of AF on osteoclast differentiation and bone resorption. The effect of AF on destabilization of the medial meniscus (DMM)-induced OA mice can be detected in hematoxylin–eosin (H&E) staining, Safranin O green staining and immunohistochemistry.AF might drastically attenuated IL-1β-stimulated inflammation and reduction of ECM formation by blocking ERK and NF-κB signaling pathways in chondrocytes. Meanwhile, AF suppressed the formation of osteoclasts and the resorption of bone function induced by RANKL. In vivo, AF played a protective role by stabilizing cartilage ECM and inhibiting subchondral bone loss in destabilization of the medial meniscus (DMM)-induced OA mice, further proving its protective effect in the development of OA. Our study show that AF alleviated OA by suppressing ERK, JNK and NF-κB signaling pathways in OA models in vitro and DMM-induced OA mice, suggesting that AF might be a potential therapeutic agent in the treatment of OA.

Extracellular vesicles from inflammatory-stimulated BMSCs ameliorate osteoarthritis via Rpl14 mediated synovial macrophage polarization

Bone marrow mesenchymal stem cells (BMSCs) and extracellular vesicles (EVs) were considered promising methods for the treatment of diverse diseases, including osteoarthritis (OA). However, the role of inflammatory-stimulated BMSCs driven EVs (iBMSC-EVs) in OA remains unclear. This study aims to investigate the potential function of iBMSC-EVs in ameliorating OA and its underlying molecular mechanism. In this study, EVs were isolated from inflammatory-stimulated BMSCs and destabilization of the medial meniscus (DMM) surgery was performed to induce knee OA model in mice. Micro-CT, H&E Staining, Safranin O-Fast Green Staining and immunofluorescence were used to examine the in vivo therapeutic effect of iBMSC-EVs on OA. Public single-cell RNA-seq data was downloaded and bulk RNA-seq data was acquired to identify critical genes promoted by iBMSC-EVs in macrophage polarization during OA. Real-time quantitative polymerase chain reaction (RT-qPCR), western blot, enzyme-linked immunosorbent assay (ELISA), cell counting kit-8 (CCK-8), transwell and scratch assays were used to further confirm the in vitro effect of iBMSC-EVs on macrophage polarization and chondrocyte proliferation, migration, as well as ECM anabolism and catabolism. In vivo experiments demonstrated that iBMSC-EVs could alleviate OA in mice and promote the reprogramming of M1 macrophages towards an M2 phenotype. RNA-Seq and scRNA-Seq analyses indicated that iBMSC-EVs altered the expression profiles of OA-related genes, with Rpl14 identified as a critical gene regulating macrophage reprogramming in OA. In vivo and in vitro experiments further confirmed that iBMSC-EVs, by downregulating Rpl14 expression, promoted M1 macrophage reprogramming to M2, facilitating chondrocyte proliferation and migration, inhibiting ECM degradation and ultimately alleviating OA progression. In conclusion, our data revealed the substantive role of iBMSC-EVs in ameliorating OA progression via Rpl14 mediated synovial macrophage polarization, presenting a promising therapeutic method for the treatment of OA.

New role of calcium-binding fluorescent dye alizarin complexone in detecting permeability from articular cartilage to subchondral bone.

Osteoarthritis (OA) is a chronic degenerative joint disorder characterized by the progressive deterioration of articular cartilage and concomitant alterations in subchondral bone architecture. However, the precise mechanisms underlying the initiation and progression of OA remains poorly understood. In the present study, we explored whether the calcification in the articular cartilage occurred in the early stage of mouse OA model, generated by the surgery destabilization of the medial meniscus (DMM), via the intra-articular injection of alizarin complexone due to its anionic nature for binding calcium-containing crystals. Although we did not observe the calcification in the articular cartilage of early stage of DMM mice, we unexpectedly identified alizarin complexone had the diffusion capacity for detecting the permeability from the articular cartilage to subchondral bone. Our data showed that the diffusion of alizarin complexone from the articular cartilage to calcified cartilage was greater in the early stage of DMM mice than that in sham controls. Additionally, we observed enhanced penetration of alizarin complexone through the periosteum in DMM mice compared to sham mice. In summary, we developed a novel imaging method that offers a valuable tool for further exploration of biochemical communication underlying OA development. Our findings provided new evidence that increased molecular interactions between the articular cartilage and subchondral bone is involved in the pathogenesis of OA progression.

Targeted Therapy of Osteoarthritis via Intra-Articular Delivery of Lipid-Nanoparticle-Encapsulated Recombinant Human FGF18 mRNA.

Fibroblast growth factor 18 (FGF18) emerges as a promising therapeutic target for osteoarthritis (OA). In this study, a novel articular cavity-localized lipid nanoparticle (LNP) named WG-PL14 is developed. This optimized formulation has a nearly 30-fold increase in mRNA expression as well as better articular cavity enrichment compared to commercial lipids MC3 when performing intra-articular injection. Then, a mRNA sequence encoding recombinant human FGF18 (rhFGF18) for potential mRNA therapy in OA is optimized. In vitro assays confirm the translation of rhFGF18 mRNA into functional proteins within rat and human chondrocytes, promoting cell proliferation and extracellular matrix (ECM) synthesis. Subsequently, the therapeutic efficacy of the LNP-rhFGF18 mRNA complex is systematically assessed in a mouse OA model. The administration exhibits several positive outcomes, including an improved pain response, upregulation of ECM-related genes (e.g., AGRN and HAS2), and remodeling of subchondral bone homeostasis compared to a control group. Taken together, these findings underscore the potential of localized LNP-rhFGF18 mRNA therapy in promoting the regeneration of cartilage tissue and mitigating the progression of OA.

Forsythoside A mitigates osteoarthritis and inhibits chondrocyte senescence by promoting mitophagy and suppressing NLRP3 inflammasome via the Nrf2 pathway

BackgroundChondrocyte senescence and inflammation are hallmarks of osteoarthritis (OA). Forsythiaside A (FTA), a phenylethanol glycoside isolated from air-dried fruits of Forsythia, has been reported to have significant anti-inflammatory and antioxidant properties. However, its protective effects against OA have not been elucidated. PurposeWe explored the therapeutic efficacy of FTA in inhibiting chondrocyte senescence and inflammation during OA, as well as the potential underlying mechanisms. Study designThis study aimed to investigate the novel mechanism of FTA in alleviating OA in both cell and animal models. MethodsThe protective effect of FTA against tert-butyl hydroperoxide-induced chondrocyte damage was assessed, and the effects of FTA on cartilage aging and OA progression were evaluated using a medial meniscus (DMM)-induced knee OA mouse model. The regulatory effects of FTA on the NLRP3 Inflammasome, mitophagy, and the PKC/Nrf2 pathway were also explored. ResultsIn vitro, FTA improved mitochondrial function, enhanced mitophagy, suppressed NLRP3 inflammasome activation, and inhibited chondrocyte senescence; however, these chondroprotective effects were partially reversed after mitophagy inhibition, NLRP3 inflammasome activation, and Nrf2 pathway inhibition. Furthermore, we found that FTA directly interacts with Nrf2 and enhances its phosphorylation by protein kinase C (PKC). In vivo, FTA attenuated the pathological signs of knee OA in a DMM-model mouse model, which was partially reversed by ML385. ConclusionFTA inhibited chondrocyte senescence and OA progression by activating the PKC–Nrf2 pathway. Thus, FTA is a potential novel therapeutic agent for OA.

Knockdown of nuclear protein 1 delays pathological pro-gression of osteoarthritis through inhibiting chondrocyte ferroptosis.

To investigate the effect of nuclear protein 1 (Nupr1) on the pathological progression of osteoarthritis and its relationship with ferroptosis of chondrocytes. Chondrocytes of mouse knee were divided into small interfering RNA (siRNA) control group, small interfering RNA targeting Nupr1 (siNupr1) group, siRNA control+IL-1β group (siRNA control interference for 24 h followed by 10 ng/mL IL-1β) and siNupr1+IL-1β group (siNupr1 interference for 24 h followed by 10 ng/mL IL-1β). The protein and mRNA expressions of Nupr1 were detected by Western blotting and real-time RT-PCR. Cell proliferation viabilities were measured using the cell counting kit-8 method. The levels of ferrous ions were detected by FerroOrange staining. Lipid peroxidation levels were detected by C11-BODIPY-591 fluorescence imaging. The contents of malondialdehyde (MDA) and glutathione (GSH) were detected by enzyme-linked immunosorbent assay. The protein expressions of long-chain acyl-coenzyme A synthetase 4 (ACSL4), tumor protein 53 (P53), glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 gene (SLC7A11) were detected by Western blotting. The osteoarthritis model was constructed by destabilization of the medial meniscus (DMM) surgery in 7-week-old male C57BL/6J mice. The mice were randomly divided into 4 groups with 10 animals in each group: sham surgery (Sham)+adeno-associated virus serotype 5 (AAV5)-Short hairpin RNA (shRNA) control group, Sham+AAV5-shRNA targeting Nupr1 (shNupr1) group, DMM+AAV5-shRNA group, and DMM+AAV5-shNupr1 group. Hematoxylin and eosin staining and Safranin O-Fast Green staining were used to observe the morphological changes in cartilage tissue. The Osteoarthritis Research Society International (OARSI) osteoarthritis cartilage histopathology assessment system was used to evaluate the degree of cartilage degeneration in mice. The mRNA expressions of Nupr1, matrix metallopeptidase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), cyclooxygenase 2 (COX2), and glutathione peroxidase 4 (GPX4) were detected by real-time RT-PCR. In vitro experiments showed that knocking down Nupr1 alleviated the decrease of chondrocyte proliferation activity induced by IL-1β, reduced iron accumulation in mouse chondrocytes, lowered the lipid peroxidation, downregulated ACLS4 and P53 protein expression and upregulated GPX4 and SLC7A11 protein expression (all P<0.01), thereby inhibiting ferroptosis in mouse chondrocytes. Meanwhile, in vivo animal experiments demonstrated that knocking down Nupr1 delayed the degeneration of articular cartilage in osteoarthritis mice, improved the OARSI score, slowed down the degradation of the extracellular matrix in osteoarthritis cartilage, and reduced the expression of the key ferroptosis regulator GPX4 (all P<0.01). Knockdown of Nupr1 can delay the pathological progression of osteoarthritis through inhibiting ferroptosis in mouse chondrocytes.

Molybdenum nanodots act as antioxidants for photothermal therapy osteoarthritis

Osteoarthritis (OA) manifests as the degradation of cartilage and remodeling of subchondral bone. Restoring homeostasis within the joint is imperative for alleviating OA symptoms. Current interventions primarily target singular aspects, such as anti-aging, inflammation inhibition, free radical scavenging, and regeneration of cartilage and subchondral bone. Herein, we developed molybdenum nanodots (MNDs) as bionic photothermal nanomaterials to mimic the antioxidant synthase to concurrently protected cartilage and facilitate subchondral bone regeneration. With near-infrared (NIR) irradiation, MNDs effectively eliminate reactive oxygen and nitrogen species (ROS/RNS) from OA chondrocytes, thereby reversed mitochondrial dysfunction, mitigating chondrocyte senescence, and simultaneously suppresses inflammation, hence preserving the inherent homeostasis between cartilage matrix synthesis and degradation while circumventing safety concerns. RNA sequencing of OA chondrocytes treated with MNDs-NIR revealed the reinstatement of chondrocyte functionality, activation of antioxidant enzymes, anti-aging properties, and regulation of inflammation. NIR irradiation induces thermogenesis and synergistically promotes subchondral bone regeneration via MNDs, as validated through histological assessments and microcomputed tomography (Micro-CT) scans. MNDs-NIR effectively attenuate cellular senescence and inhibit inflammation in vivo, while also remodeling mitochondrial dynamics by upregulating fusion proteins and inhibiting fission proteins, thereby regulating the oxidative stress microenvironment. Additionally, MNDs-NIR exhibited remarkable therapeutic effects in alleviating articular cartilage degeneration in an OA mouse model, evidenced by a 1.67-fold reduction in subchondral bone plate thickness, an 88.57% decrease in OARSI score, a 5.52-fold reduction in MMP13 expression, and a 6.80-fold increase in Col II expression. This novel disease-modifying approach for OA utilizing MNDs-NIR offers insight and a paradigm for improving mitochondrial dysfunction by regulating the accumulation of mitochondrial ROS and ultimately alleviating cellular senescence. Moreover, the dual-pronged therapeutic approach of MNDs-NIR, which addresses both cartilage erosion and subchondral bone lesions in OA, represents a highly promising strategy for managing OA.

Rosmarinic acid promotes cartilage regeneration through Sox9 induction via NF-κB pathway inhibition in mouse osteoarthritis progression

BackgroundThe natural polyphenolic compound known as Rosmarinic acid (RosA) can be found in various plants. Although its potential health benefits have been extensively studied, its effect on osteoarthritis (OA) progression and cartilage regeneration function still needs to be fully elucidated in OA animal models. This study elucidated the effect of RosA on OA progression and cartilage regeneration. MethodsIn vitro assessments were conducted using RT-PCR, qRT-PCR, Western blotting, and ELISA to measure the effects of RosA. The molecular mechanisms of RosA were determined by analyzing the translocation of p65 into the nucleus using immunocytochemistry (ICC). Histological analysis of cartilage explant was performed using alcian blue staining and immunohistochemistry (IHC). For in vivo analysis, the destabilization of the medial meniscus (DMM)-induced OA mouse model was utilized to evaluate cartilage destruction through Safranin-O staining. The expression of catabolic and anabolic factors in mice knee joints was quantified by immunohistochemistry. ResultsThe expression of catabolic factors in chondrocytes was significantly impeded by RosA. It also suppressed the NF-κB signaling pathway by decreasing phosphorylation of p65 and reducing degradation of IκB protein. In ex vivo experiments, RosA protected sulfated proteoglycan erosion triggered by IL-1β and suppressed the catabolic factors in cartilage explant. RosA treatment in animal models resulted in preventing cartilage destruction and reducing catabolic factors in the cartilage. RosA was also found to promote the expression of Sox9, Col2a1, and Acan in vitro, ex vivo, and in vivo analyses. ConclusionsRosA attenuated the OA progression by suppressing the catabolic factors expression. These effects were facilitated through the suppression of the NF-κB signaling pathway. Additionally, it promotes cartilage regeneration by inducing anabolic factors. Therefore, RosA shows potential as an effective therapeutic agent for treating OA.

Natural synergy: Oleanolic acid-curcumin co-assembled nanoparticles combat osteoarthritis

Curcumin (Cur) is a natural polyphenol that is one of the most valuable natural products. However, its use as a functional food is limited by low water solubility, chemical instability and poor bioavailability. In this study, a supramolecular co-assembly strategy was used to construct an oleanolic acid-curcumin (OLA-Cur) co-assembly composite nano-slow-release treatment system. As a co-assembled compound, OLA is a widely present pentacyclic triterpenoid compound with multiple biological activities in the plant kingdom, which is expected to jointly alleviate the damaging effects of papain-induced mouse osteoarthritis model. The OLA-Cur NPs shows the solid core-shell structure, which can effectively improve the water solubility of Cur and OLA, and has good stability and sustained release characteristics. The analysis results show that the two compounds are mainly assembled through hydrogen bonding interactions, hydrophobic interactions, and π - π stacking interactions. The OLA-Cur NPs can inhibit the release of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β induced by LPS in RAW264.7 mouse macrophages, promote the secretion of anti-inflammatory cytokine IL-10, and improve the oxidative stress index of hydrogen peroxide induced human rheumatoid arthritis synovial fibroblasts. In addition, it has a certain improvement effect on cartilage and subchondral bone damage in mouse osteoarthritis models. These findings suggest that constructing co-assembled composite nanoparticles based on pure natural compounds may break through the limitations of a variety of important nutritional ingredients in functional foods.