Destabilization Of The Medial Meniscus Model Research Articles

Inhibition of CC chemokine receptor 1 ameliorates osteoarthritis in mouse by activating PPAR-γ

BackgroundOsteoarthritis (OA) is a degenerative joint disease characterized by cartilage destruction and inflammation. CC chemokine receptor 1 (CCR1), a member of the chemokine family and its receptor family, plays a role in the autoimmune response. The impact of BX471, a specific small molecule inhibitor of CCR1, on CCR1 expression in cartilage and its effects on OA remain underexplored.MethodsThis study used immunohistochemistry (IHC) to assess CCR1 expression in IL-1β-induced mouse chondrocytes and a medial meniscus mouse model of destabilization of the medial meniscus (DMM). Chondrocytes treated with varying concentrations of BX471 for 24 h were subjected to IL-1β (10 ng/ml) treatment. The levels of the aging-related genes P16INK4a and P21CIP1 were analyzed via western blotting, and senescence-associated β-galactosidase (SA-β-gal) activity was measured. The expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), aggrecan (AGG), and the transcription factor SOX9 were determined through western blotting and RT‒qPCR. Collagen II, matrix metalloproteinase 13 (MMP13), and peroxisome proliferator-activated receptor (PPAR)-γ expression was analyzed via western blot, RT‒qPCR, and immunofluorescence. The impact of BX471 on inflammatory metabolism-related proteins under PPAR-γ inhibition conditions (using GW-9662) was examined through western blotting. The expression of MAPK signaling pathway-related molecules was assessed through western blotting. In vivo, various concentrations of BX471 or an equivalent medium were injected into DMM model joints. Cartilage destruction was evaluated through Safranin O/Fast green and hematoxylin–eosin (H&E) staining.ResultsThis study revealed that inhibiting CCR1 mitigates IL-1β-induced aging, downregulates the expression of iNOS, COX-2, and MMP13, and alleviates the IL-1β-induced decrease in anabolic indices. Mechanistically, the MAPK signaling pathway and PPAR-γ may be involved in inhibiting the protective effect of CCR1 on chondrocytes. In vivo, BX471 protected cartilage in a DMM model.ConclusionThis study demonstrated the expression of CCR1 in chondrocytes. Inhibiting CCR1 reduced the inflammatory response, alleviated cartilage aging, and retarded degeneration through the MAPK signaling pathway and PPAR-γ, suggesting its potential therapeutic value for OA.Graphical

Integrated Network Pharmacology and Experimental Validation Approach to Investigate the Mechanisms of Radix Rehmanniae Praeparata - Angelica Sinensis - Radix Achyranthis Bidentatae in Treating Knee Osteoarthritis.

Knee osteoarthritis (KOA) is a persistent degenerative condition characterized by the deterioration of cartilage. The Chinese herbal formula Radix Rehmanniae Praeparata- Angelica Sinensis-Radix Achyranthis Bidentatae (RAR) has often been used in effective prescriptions for KOA as the main functional drug, but its underlying mechanism remains unclear. Therefore, network pharmacology and verification experiments were employed to investigate the impact and mode of action of RAR in the treatment of KOA. The destabilization of the medial meniscus model (DMM) was utilized to assess the anti-KOA effect of RAR by using gait analysis, micro-computed tomography (Micro-CT), and histology. Primary chondrocytes were extracted from the rib cartilage of a newborn mouse. The protective effects of RAR on OA cells were evaluated using a CCK-8 assay. The antioxidative effect of RAR was determined by measuring reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) production. Furthermore, network pharmacology and molecular docking were utilized to propose possible RAR targets for KOA, which were further verified through experiments. In vivo, RAR significantly ameliorated DMM-induced KOA characteristics, such as subchondral bone sclerosis, cartilage deterioration, gait abnormalities, and the degree of knee swelling. In vitro, RAR stimulated chondrocyte proliferation and the expression of Col2a1, Comp, and Acan. Moreover, RAR treatment significantly reduced ROS accumulation in an OA cell model induced by IL-1β and increased the activity of antioxidant enzymes (SOD and GSH). Network pharmacology analysis combined with molecular docking showed that Mapk1 might be a key therapeutic target. Subsequent research showed that RAR could downregulate Mapk1 mRNA levels in IL-1β-induced chondrocytes and DMM-induced rats. RAR inhibited extracellular matrix (ECM) degradation and oxidative stress response via the MAPK signaling pathway in KOA, and Mapk1 may be a core target.

Syringic acid promotes cartilage extracellular matrix generation and attenuates osteoarthritic cartilage degradation by activating TGF-β/Smad and inhibiting NF-κB signaling pathway.

Osteoarthritis (OA) is a common chronic degenerative disease which is characterized by the disruption of articular cartilage. Syringic acid (SA) is a phenolic compound with anti-inflammatory, antioxidant, and other effects including promoting osteogenesis. However, the effect of SA on OA has not yet been reported. Therefore, the purpose of our study was to investigate the effect and mechanism of SA on OA in a mouse model of medial meniscal destabilization. The expressions of genes were evaluated by qPCR or western blot or immunofluorescence. RNA-seq analysis was performed to examine gene transcription alterations in chondrocytes treated with SA. The effect of SA on OA was evaluated using destabilization of the medial meniscus model of mice. We found that SA had no obvious toxic effect on chondrocytes, while promoting the expressions of chondrogenesis-related marker genes. The results of RNA-seq analysis showed that extracellular matrix-receptor interaction and transforming growth factor-β (TGF-β) signaling pathways were enriched among the up-regulated genes by SA. Mechanistically, we demonstrated that SA transcriptionally activated Smad3. In addition, we found that SA inhibited the overproduction of lipopolysaccharide-induced inflammation-related cytokines including tumor necrosis factor-α and interleukin-1β, as well as matrix metalloproteinase 3 and matrix metalloproteinase 13. The cell apoptosis and nuclear factor-kappa B (NF-κB) signaling were also inhibited by SA treatment. Most importantly, SA attenuated cartilage degradation in a mouse OA model. Taken together, our study demonstrated that SA could alleviate cartilage degradation in OA by activating the TGF-β/Smad and inhibiting NF-κB signaling pathway.

Supramolecular self-assembly of EGCG-selenomethionine nanodrug for treating osteoarthritis

Osteoarthritis (OA) has emerged as a significant health concern among the elderly population, with increasing attention paid to ferroptosis-induced OA in recent years. However, the prolonged use of nonsteroidal anti-inflammatory drugs or corticosteroids can lead to a series of side effects and limited therapeutic efficacy. This study aimed to employ the Mannich condensation reaction between epigallocatechin-3-gallate (EGCG) and selenomethionine (SeMet) to efficiently synthesize polyphenol-based nanodrugs in aqueous media for treating OA. Molecular biology experiments demonstrated that EGCG-based nanodrugs (ES NDs) could effectively reduce glutathione peroxidase 4 (GPX4) inactivation, abnormal Fe2+ accumulation, and lipid peroxidation induced by oxidative stress, which ameliorated the metabolic disorder of chondrocytes and other multiple pathological processes triggered by ferroptosis. Moreover, imaging and histopathological analysis of the destabilization of the medial meniscus model in mice confirmed that ES NDs exhibiting significant therapeutic effects in relieving OA. The intra-articular delivery of ES NDs represents a promising approach for treating OA and other joint inflammatory diseases.

IĸB Protein BCL3 as a Controller of Osteogenesis and Bone Health.

IĸB protein B cell lymphoma 3-encoded protein (BCL3) is a regulator of the NF-κB family of transcription factors. NF-κB signaling fundamentally influences the fate of bone-forming osteoblasts and bone-resorbing osteoclasts, but the role of BCL3 in bone biology has not been investigated. The objective of this study was to evaluate BCL3 in skeletal development, maintenance, and osteoarthritic pathology. To assess the contribution of BCL3 to skeletal homeostasis, neonatal mice (n=6-14) lacking BCL3 (Bcl3-/- ) and wild-type (WT) controls were characterized for bone phenotype and density. To reveal the contribution to bone phenotype by the osteoblast compartment in Bcl3-/- mice, transcriptomic analysis of early osteogenic differentiation and cellular function (n=3-7) were assessed. Osteoclast differentiation and function in Bcl3-/- mice (n=3-5) was assessed. Adult 20-week Bcl3-/- and WT mice bone phenotype, strength, and turnover were assessed. A destabilization of the medial meniscus model of osteoarthritic osteophytogenesis was used to understand adult bone formation in Bcl3-/- mice (n=11-13). Evaluation of Bcl3-/- mice revealed congenitally increased bone density, long bone dwarfism, increased bone biomechanical strength, and altered bone turnover. Molecular and cellular characterization of mesenchymal precursors showed that Bcl3-/- cells displayed an accelerated osteogenic transcriptional profile that led to enhanced differentiation into osteoblasts with increased functional activity, which could be reversed with a mimetic peptide. In a model of osteoarthritis-induced osteophytogenesis, Bcl3-/- mice exhibited decreased pathological osteophyte formation (P < 0.05). Cumulatively, these findings demonstrate that BCL3 controls developmental mineralization to enable appropriate bone formation, whereas in a pathological setting, it contributes to skeletal pathology.

Pentraxin 3 deficiency ameliorates the severity of osteoarthritis and alleviates inflammation

BackgroundOsteoarthritis is one of the most common degenerative joint disorders, characterized by articular cartilage breakdown, synovitis, osteophytes generation and subchondral bone sclerosis. Pentraxin 3 (PTX3) is a long pentraxin protein, secreted by immune cells, and PTX3 is identified to play a critical role in inflammation and macrophage polarization. However, the underlying mechanism of PTX3 in osteoarthritis under the circumstance of Ptx3-knockout (KO) mice model is still unknown. MethodsMurine destabilization of the medial meniscus (DMM) OA model was created in Ptx3-knockout (KO) and wildtype mice, respectively. The degenerative status of cartilage was detected by Safranin O, H&E staining, immunohistochemistry (IHC) and micro-CT. OARSI scoring was employed to assess the proteoglycan of cartilage. Serum inflammatory cytokines were examined by ELISA and systematic macrophage polarization in spleen was analyzed by flow cytometry. ResultsSafranin O and H&E staining confirmed that the joint cartilage was mostly with reduced degeneration in both the senior KO mice and the DMM model generated from the KO mice, compared to the WT group. This is also supported by micro-CT examination and OARSI scoring. Immunohistochemistry illustrated an up-regulation of Aggrecan and Collagen 2 and down-regulation of ADAMTS-5 and MMP13 in KO mice in comparison with the WT mice. ELISA indicated a dramatical decrease in the serum levels of TNF-α and IL-6 in KO mice. Polarization of M2-like macrophages was observed in the KO group. ConclusionPentraxin 3 deficiency significantly ameliorated the severity of osteoarthritis by preventing cartilage degeneration and alleviated systematic inflammation by inducing M2 polarization.

Tryptase β regulation of joint lubrication and inflammation via proteoglycan-4 in osteoarthritis

PRG4 is an extracellular matrix protein that maintains homeostasis through its boundary lubricating and anti-inflammatory properties. Altered expression and function of PRG4 have been associated with joint inflammatory diseases, including osteoarthritis. Here we show that mast cell tryptase β cleaves PRG4 in a dose- and time-dependent manner, which was confirmed by silver stain gel electrophoresis and mass spectrometry. Tryptase-treated PRG4 results in a reduction of lubrication. Compared to full-length, cleaved PRG4 further activates NF-κB expression in cells overexpressing TLR2, −4, and −5. In the destabilization of the medial meniscus model of osteoarthritis in rat, tryptase β and PRG4 colocalize at the site of injury in knee cartilage and is associated with disease severity. When human primary synovial fibroblasts from male osteoarthritis patients or male healthy subjects treated with tryptase β and/or PRG4 are subjected to a quantitative shotgun proteomics and proteome changes are characterized, it further supports the role of NF-κB activation. Here we show that tryptase β as a modulator of joint lubrication in osteoarthritis via the cleavage of PRG4.

PROTEIN PHOSPHATASE PPM1A INHIBITION ATTENUATES OSTEOARTHRITIS VIA REGULATION OF TGF-Β/SMAD2 SIGNALLING IN CHONDROCYTES

TGF-β/Smad2 signaling is considered to be one of the important pathways involved in osteoarthritis (OA) and protein phosphatase magnesium-dependent 1A (PPM1A) functions as an exclusive phosphatase of Smad2 and regulates TGF-β signaling, here, we investigated the functional role of PPM1A in OA pathogenesis.PPM1A expressions in both human OA cartilage and experimental OA mice chondrocytes were analyzed immunohistochemically. Besides, the mRNA and protein expression of PPM1A induced by IL-1β treatment were also detected by q-PCR and immunofluorescence in vitro. OA was induced in PPM1A knockout (KO) mice by destabilization of the medial meniscus (DMM), and histopathological examination was performed. OA was also induced in wild-type (WT) mice, which were then treated with an intra-articular injection of a selective PPM1A inhibitor for 8 weeks.PPM1A protein expressions were increased in both human OA cartilage and experimental OA mice chondrocytes. We also found that treatment with IL-1β in mouse primary chondrocytes significantly increased both mRNA and protein expression of PPM1A in vitro. Importantly, our data showed that PPM1A deletion could substantially protect against surgically induced OA. Concretely, the average OARSI score and quantification of BV/TV of subchondral bone in KO mice were significantly lower than that in WT mice 8 weeks after DMM surgery. Besides, TUNEL staining revealed a significant decrease in apoptotic chondrocytes in PPM1A-KO mice with DMM operation. With OA induction, the rates of chondrocytes positive for Mmp-13 and Adamts-5 in KO mice were also significantly lower than those in WT mice. Moreover, compared with WT mice, the phosphorylation of Smad2 in chondrocytes was increased in KO mice underwent DMM surgery. However, articular-injection with SD-208, a selective inhibitor of TGF-β/Smad2 signaling could significantly abolish the chondroprotective phenotypes in PPM1A-KO mice. Additionally, both cartilage degeneration and subchondral bone subchondral bone sclerosis in DMM model were blunted following intra-articular injection with BC-21, a small-molecule inhibitor for PPM1A.Our study demonstrated that PPM1A inhibition attenuates OA by regulating TGF-β/Smad2 signaling. Furthermore, PPM1A is a potential target for OA treatment and BC-21 may be employed as alternative therapeutic agents for the management of OA.

Formononetin improves the inflammatory response and bone destruction in knee joint lesions by regulating the NF-kB and MAPK signaling pathways.

Formononetin (FMN) is a phytoestrogen that belongs to the isoflavone family. It has antioxidant and anti-inflammatory effects, as well as, many other biological activities. Existing evidence has aroused interest in its ability to protect against osteoarthritis (OA) and promote bone remodeling. To date, research on this topic has not been thorough and many issues remain controversial. Therefore, the purpose of our study was to explore the protective effect of FMN against knee injury and clarify the possible molecular mechanisms. We found that FMN inhibited osteoclast formation induced by receptor activator of NF-κB ligand (RANKL). Inhibition of the phosphorylation and nuclear translocation of p65 in the NF-κB signaling pathway plays a role in this effect. Similarly, during the inflammatory response of primary knee cartilage cells activated by IL-1β, FMN inhibited the NF-κB signaling pathway and the phosphorylation of the ERK and JNK proteins in the MAPK signaling pathway to suppress the inflammatory response. In addition, in vivo experiments showed that both low- and high-dose FMN had a clear protective effect against knee injury in the DMM (destabilization of the medial meniscus) model, and the therapeutic effect of high-dose FMN was stronger. In conclusion, these studies provide evidence of the protective effect of FMN against knee injury.

4-Octyl itaconate treatment inhibits mitochondrial dysfunction and ER stress via stimulating SIRT1 expression in vitro and attenuates osteoarthritis process in murine DMM model in vivo

Osteoarthritis (OA), a degenerative disease, has many pathological factors, including oxidative stress (OS), endoplasmic reticulum (ER) stress, apoptosis, and extracellular matrix (ECM) degeneration. OS regulates the pathological progression of OA via mitochondrial dysfunction, and ER stress and by modulating the derivation and elimination of reactive oxygen species (ROS). Though. 4-octyl itaconate (4-OI) is known for its effects on the treatment of various diseases, how it affects OA and its underlying mechanism still needs to be elucidated. Therefore, it is necessary to explore the effects of 4-OI both in vivo and in vitro. In our study, we used H2O2, which can accumulate ROS, OS and ER stress, to imitate the OA model in vitro. Using western blot, real-time quantitative reverse transcription polymerase chain reaction and immunofluorescence staining, we found that 125 μM 4-OI can inhibit apoptosis, ECM degeneration, mitochondrial dysfunction, ER stress and abundant accumulation of ROS in chondrocytes. We also showed that 4-OI can activate the Sirtuin1 gene (SIRT1). We used a SIRT1 inhibitor EX527 to identify the relationships among these pathological factors, and it showed that the 4-OI-mediated effect was achieved by promoting the SIRT1. In vivo, the bilateral medial meniscus was resected to establish the destabilization of the medial meniscus model wherein intraperitoneal injections of 2% and 5% 4-OI postponed the progression of OA. Collectively, this study confirmed that 4-OI can potentially impede the progression of OA by promoting the expression of SIRT1.

Chondroprotective Mechanism of Eucommia ulmoides Oliv.-Glycyrrhiza uralensis Fisch. Couplet Medicines in Knee Osteoarthritis via Experimental Study and Network Pharmacology Analysis.

Knee osteoarthritis (KOA) is the primary prevalent disabling joint disorder among osteoarthritis (OA), and there is no particularly effective treatment at the clinic. Traditional Chinese medicine (TCM) herbs, such as Eucommia ulmoides Oliv. and Glycyrrhiza uralensis Fisch. (E.G.) couplet medicines, have been reported to exhibit beneficial health effects on KOA, exact mechanism of E.G. nevertheless is not fully elucidated. We assess the therapeutic effects of E.G. on KOA and explore its underlying molecular mechanism. UPLC-Q-TOF/MS technique was used to analyze the active chemical constituents of E.G. The destabilization of the medial meniscus model (DMM) was employed to evaluate the chondroprotective action of E.G. in KOA mice using histomorphometry, μCT, behavioral testing and immunohistochemical staining. Additionally, network pharmacology and molecular docking were used to predict potential targets for anti-KOA activities of E.G., which was further verified through in vitro experiments. In vivo studies have shown that E.G. could significantly ameliorate DMM-induced KOA phenotypes including subchondral bone sclerosis, cartilage degradation, gait abnormality and thermal pain reaction sensibility. E.G. treatment could also promote extracellular matrix synthesis to protect articular chondrocytes, which was indicated by Col2 and Aggrecan expressions, as well as reducing matrix degradation by inhibiting MMP13 expression. Interestingly, network pharmacologic analysis showed that PPARG might be a therapeutic center. Further study proved that E.G.-containing serum (EGS) could up-regulate PPARG mRNA level in IL-1β-induced chondrocytes. Notably, significant effects of EGS on the increment of anabolic gene expressions (Col2, Aggrecan) and the decrement of catabolic gene expressions (MMP13, Adamts5) in KOA chondrocytes were abolished due to the silence of PPARG. E.G. played a chondroprotective role in anti-KOA by inhibiting extracellular matrix degradation, which might be related to PPARG.

Accuracy of in vivo microCT imaging in assessing the microstructural properties of the mouse tibia subchondral bone.

Osteoarthritis (OA) is one of the most common musculoskeletal diseases. OA is characterized by degeneration of the articular cartilage as well as the underlying subchondral bone. Post-traumatic osteoarthritis (PTOA) is a subset of OA caused by mechanical trauma. Mouse models, such as destabilization of the medial meniscus (DMM), are useful to study PTOA. Ex vivo micro-Computed Tomography (microCT) imaging is the predominant technique used to scan the mouse knee in OA studies. Nevertheless, in vivo microCT enables the longitudinal assessment of bone microstructure, reducing measurement variability and number of animals required. The effect of image resolution in measuring subchondral bone parameters was previously evaluated only for a limited number of parameters. The aim of this study was to evaluate the ability of in vivo microCT imaging in measuring the microstructural properties of the mouse tibia trabecular and cortical subchondral bone, with respect to ex vivo high resolution imaging, in a DMM model of PTOA. Sixteen male C57BL/6J mice received DMM surgery or sham operation at 14 weeks of age (N=8 per group). The right knee of each mouse was microCT scanned in vivo (10.4μm voxel size) and ex vivo (4.35μm voxel size) at the age of 26 weeks. Each image was aligned to a reference image using rigid registration. The subchondral cortical bone plate thickness was measured at the lateral and medial condyles. Standard morphometric parameters were measured in the subchondral trabecular bone. In vivo microCT imaging led to significant underestimation of bone volume fraction (-14%), bone surface density (-3%) and trabecular number (-16%), whereas trabecular thickness (+3%) and separation (+5%) were significantly overestimated. Nevertheless, most trabecular parameters measured in vivo were well correlated with ex vivo measurements (R2 = 0.69-0.81). Degree of anisotropy, structure model index and connectivity density were measured in vivo with lower accuracy. Excellent accuracy was found for cortical thickness measurements. In conclusion, this study identified what bone morphological parameters can be reliably measured by in vivo microCT imaging of the subchondral bone in the mouse tibia. It highlights that this approach can be used to study longitudinal effects of diseases and treatments on the subchondral cortical bone and on most subchondral trabecular bone parameters, but systematic over- or under-estimations should be considered when interpreting the results.

Stevioside attenuates osteoarthritis via regulating Nrf2/HO-1/NF-κB pathway.

Osteoarthritis (OA) is a chronic disease that may cause articular cartilage degeneration, and synovial inflammation, resulting in considerable pain, poor quality of life, and functional limitations. Previous research has shown that ECM degradation and inflammation are involved in the progression of OA. Stevioside (STE), a naturally diterpenoid glycoside, is isolated from the Stevia rebaudiana (Bertoni), which has been exerted a variety of pharmacological activities, involving anti-inflammatory, anti-oxidative, and neuroprotective effects. However, STE's effects on OA and its mechanism still need further research. In the present study, we examined the anti-inflammatory effects of STE (STE) in both mouse chondrocytes and OA model induced by destabilization of the medial meniscus (DMM). In vitro, the mouse chondrocytes were treated with STE (0, 10, 20, 40​M, 24​h) after stimulated with IL-1β (10​ng/mL, 24​h). The expression of ant-inflammation-relative mediators iNOS and Cox-2 were detected by Western blot and RT-PCR. The catabolic factors (MMP-13, ADAMTS-4) and cartilage matrix constituent (Aggrecan, Collagen II) were measured by Western blot and Immunofluorescence staining. The Nrf2/HO-1/NF-κB signaling molecules were detected by Western blot. In vivo, histological analysis was used to evaluate the severity of mouse OA models. STE remarkably inhibited the IL-1β-induced expression of iNOS and Cox-2, generation of MMP-13, ADAMTS-4 and degradation of Aggrecan and Collagen II. Furthermore, we found that the chondroprotective effect of STE via Nrf2/HO-1/NF-κB signaling pathway. In vivo, the cartilage treated with STE displayed attenuated degeneration, low OARIS scores compared with DMM group. In conclusion, we considered that STE might be a promising therapeutic agent for the treatment of OA in future. Our findings indicated that STE can ameliorate the development of OA via inhibiting the inflammation. The underlying mechanism may be related to the Nrf2/HO-1/NF-κB signaling pathway. Moreover, the treatment of STE significantly relieves the progression in the mouse DMM model. All of the results demonstrated the therapeutic of STE in OA treatment. This study demonstrates a more efficient and safe application of STE in treating osteoarthritis, provide a new concept for the cartilage targeted application of natural compounds.

Integrin αVβ3 Signaling in the Progression of Osteoarthritis Induced by Excessive Mechanical Stress

Osteoarthritis (OA) is believed to be linked with cartilage degeneration, subchondral bone sclerosis, and synovial inflammation that lead to joint failure, and yet treatment that can effectively reverse the pathological process of the disease still not exists. Recent evidence suggests excessive mechanical stress (eMS) as an essential role in the pathogenesis of OA. Increased levels of integrin αVβ3 have been detected in osteoarthritic cartilage and were previously implicated in OA pathogenesis. However, the role of integrin αVβ3 in the process of eMS-induced OA remains unclear. Here, histologic and proteomic analyses of osteoarthritic cartilage in a rat destabilization of the medial meniscus model demonstrated elevated expression of integrin αVβ3 as well as more serious cartilage degeneration in the medial weight-bearing area. Furthermore, results of in vitro study demonstrated that eMS led to a significant increase of integrin αVβ3 expression and phosphorylation of downstream signaling molecules such as FAK and ERK, as well as upregulated expressions of inflammatory and degradative mediators. In addition, we found that inhibition of integrin αVβ3 could alleviate chondrocyte inflammation triggered by eMS both in vivo and in vitro. Our findings suggest a central role for upregulation of integrin αVβ3 signaling in OA pathogenesis and demonstrate that activation of integrin αVβ3 signaling in cartilage contributes to inflammation and joint destruction in eMS-induced OA. Taken together, our data presented here provide a possibility for targeting integrin αVβ3 signaling pathway as a disease-modifying therapy.

Stabilization of hypoxia-inducible factor-1α alleviates osteoarthritis via interacting with Per2 and resetting the circadian clock

ObjectiveThe study aimed to establish whether HIF1α entrains the core clock in chondrocytes and how the HIF1α affects the circadian rhythm. MethodsWe subjected primary chondrocytes to chronic circadian desynchrony (CCD) and subsequently treated with oxygen rhythm and dimethyloxalylglycine (DMOG). Circadian oscillations were analyzed with a real-time monitoring system of Per2 promoter activity and qPCR. Chondrocytes were assayed core clock genes expression patterns and extracellular matrix metabolism. HIF1α siRNA was used to knock down HIF1α. ChIP-qPCR and dual-luciferase reporter assay were used to validate that Per2 was the target gene of HIF1α. The surgical model of osteoarthritis was induced by destabilization of the medial meniscus (DMM). The chronic jet lag model was established light/dark cycle advance shift. Joint degeneration was measured using histological staining, immunological assays, and micro-CT scanning. ResultsWe report that different patterns of clock genes expression between healthy and osteoarthritic cartilage tissues and oxygen rhythms and DMOG reset the molecular clockwork which was dampened after CCD culture in a HIF1α-dependent manner. HIF1α increased the amplitude of oscillation by directly binding to the HRE-like and E-box-like elements located on the Per2 promoter. The rhythmic circadian clock significantly enhanced extracellular matrix production. Our study also demonstrates that DMOG ameliorated the increased OA severity caused by jet lag in the DMM model. ConclusionsThis study shows that circadian clock resetting caused by DMOG is at least partially mediated by the HIF1α through interaction with the Per2 promoter and proposes DMOG as supportive therapy for OA.

Can CD200R1 Agonists Slow the Progression of Osteoarthritis Secondary to Injury?

Post-traumatic knee osteoarthritis is characterized by cartilage degeneration, subchondral bone remodeling, osteophyte formation, and synovial changes. Therapeutic targeting of inflammatory activity in the knee immediately post injury may alter the course of osteoarthritis development. This study aimed to determine whether CD200R1 agonists, namely the protein therapeutic CD200Fc or the synthetic DNA aptamer CCS13, both known to act as anti-inflammatory agents, are able to delay the pathogenesis of injury-associated knee osteoarthritis in a murine model. Ten week old male C57BL/6 mice were randomized and surgical destabilization of the medial meniscus (DMM) to induce knee arthritis or sham surgery as a control were performed. CCS13 was evaluated as a therapeutic treatment along with CD200Fc and a phosphate-buffered saline vehicle control. Oligonucleotides were injected intra-articularly beginning one week after surgery, with a total of six injections administered prior to sacrifice at 12 weeks post-surgery. Histopathological assessment was used as the primary outcome measure to assess cartilage and synovial changes, while µCT imaging was used to compare the changes to the subchondral bone between untreated and treated arthritic groups. We did not find any attenuation of cartilage degeneration or synovitis in DMM mice with CD200Fc or CCS13 at 12 weeks post-surgery, nor stereological differences in the properties of subchondral bone. The use of CD200R1 agonists to blunt the inflammatory response in the knee are insufficient to prevent disease progression in the mouse DMM model of OA without anatomical restoration of the normal joint biomechanics.

4-octyl Itaconateinhibits lipopolysaccharide (LPS)-induced osteoarthritis via activating Nrf2 signalling pathway.

Small molecule drug intervention for chondrocytes is a valuable method for the treatment of osteoarthritis (OA). The 4‐octyl itaconate (OI) is a cellular derivative of itaconate with sound cell permeability and transformation rate. We attempted to confirm the protective role of OI in chondrocytes and its regulatory mechanism. We used lipopolysaccharide (LPS) to induce chondrocyte inflammation injury. After the OI treatment, the secretion and mRNA expression of Il‐6, Il‐10, Mcp‐1 and Tnf‐α were detected by ELISA and qPCR. The protective effect of OI on articular cartilage was further verified in surgical destabilization of the medial meniscus model of OA. Cell death and apoptosis were evaluated based on CCK8, LDH, Typan blue staining, Annexin V and TUNEL analyses. The small interfering RNAs were used to knockout the Nrf2 gene of chondrocytes to verify the OI‐mediated Nrf2 signalling pathway. The results revealed that OI protects cells from LPS‐induced inflammatory injury and attenuates cell death and apoptosis induced by LPS. Similar protective effects were also observed on articular cartilage in mice. The OI activated Nrf2 signalling pathway and promoted the stable expression and translocation of Nrf2 into the nucleus. When the Nrf2 signalling pathway was blocked, the protective effect of OI was significantly counteracted in chondrocytes and a mouse arthritis model. Both itaconate and its derivative (i.e., OI) showed important medical effects in the treatment of OA.

Metformin reduces chondrocyte pyroptosis in an osteoarthritis mouse model by inhibiting NLRP3 inflammasome activation.

Osteoarthritis (OA) is an age-related degenerative disease, and its incidence is increasing with the ageing of the population. Metformin, as the first-line medication for the treatment of diabetes, has received increasing attention for its role in OA. The purpose of the present study was to confirm the therapeutic effect of metformin in a mouse model of OA and to determine the mechanism underlying the resultant delay in OA progression. The right knees of 8-week-old C57BL/6 male mice were subjected to destabilization of the medial meniscus (DMM). Metformin (200 mg/kg) was then administered daily for 4 or 8 weeks. Safranin O-fast green staining, H&E staining and micro-CT were used to analyse the structure and morphological changes. Immunohistochemical staining was used to detect type II collagen (Col II), matrix metalloproteinase 13 (MMP-13), NOD-like receptor protein 3 (NLRP3), caspase-1, gasdermin D (GSDMD) and IL-1β protein expression. Reverse transcription-quantitative PCR was used to detect the mRNA expression of NLRP3, caspase-1, GSDMD and IL-1β. Histomorphological staining showed that metformin delayed the progression of OA in the DMM model. With respect to cartilage, metformin decreased the Osteoarthritis Research Society International score, increased the thickness of hyaline cartilage and decreased the thickness of calcified cartilage. Regarding the mechanism, in cartilage, metformin increased the expression of Col II and decreased the expression of MMP-13, NLRP3, caspase-1, GSDMD and IL-1β. In addition, in subchondral bone, metformin inhibited osteophyte formation, increased the bone volume fraction (%) and the bone mineral density (g/cm3), decreased the trabecular separation (mm) in early stage of osteoarthritis (4 weeks) but the opposite in an advanced stage of osteoarthritis (8 weeks). Overall, metformin inhibited the activation of NLRP3 inflammasome, decreased cartilage degradation, reversed subchondral bone remodelling and inhibited chondrocyte pyroptosis.

Effect of Suppression of Rotational Joint Instability on Cartilage and Meniscus Degeneration in Mouse Osteoarthritis Model.

ObjectiveJoint instability and meniscal dysfunction contribute to the onset and progression of knee osteoarthritis (OA). In the destabilization of the medial meniscus (DMM) model, secondary OA occurs due to the rotational instability and increases compressive stress resulting from the meniscal dysfunction. We created a new controlled abnormal tibial rotation (CATR) model that reduces the rotational instability that occurs in the DMM model. So, we aimed to investigate whether rotational instability affects articular cartilage degeneration using the DMM and CATR models, as confirmed using histology and immunohistochemistry.DesignTwelve-week-old male mice were randomized into 3 groups: DMM group, CATR group, and INTACT group (right knee of the DMM group). After 8 and 12 weeks, we performed the tibial rotational test, safranin-O/fast green staining, and immunohistochemical staining for tumor necrosis factor (TNF)-α and metalloproteinase (MMP)-13.ResultsThe rotational instability in the DMM group was significantly higher than that of the other groups. And articular cartilage degeneration was higher in the DMM group than in the other groups. However, meniscal degeneration was observed in both DMM and CATR groups. The TNF-α and MMP-13 positive cell rates in the articular cartilage of the CATR group were lower than those in the DMM group.ConclusionsWe found that the articular cartilage degeneration was delayed by controlling the rotational instability caused by meniscal dysfunction. These findings suggest that suppression of rotational instability in the knee joint may be an effective therapeutic measure for preventing OA progression.

Associations between biomarkers and histological assessment in individual animals in a destabilization of the medial meniscus (DMM) model of osteoarthritis (OA).

To date, the use of biomarkers for assessing individual severity of osteoarthritis (OA) is limited, and the correlation of histological scores with biomarkers for individual animals in the destabilization of the medial meniscus (DMM) model of OA has not been well investigated. Accordingly, this study investigated how well representative biomarkers in the DMM model reflected specific changes in individual animals. Rats were randomly divided into the OA group and the sham group. OA model was established by destabilization of the medial meniscus (DMM). After 2,4,6,8,10 and 12 weeks (n=14, each week), the concentrations of CTXII, COMP, C2C, and OC in serum were measured, and cartilage degeneration, osteophytes, and synovial membrane inflammation, typical of OA, were scored using Osteoarthritis Research Society International (OARSI) scoring system. Additionally, the correlation between each biomarker and the specific changes in osteoarthritis was analyzed for individual animals using the Generalized Estimating Equation (GEE). Statistical analysis showed a low correlation between CTXII and osteophyte score of the medial femur (coefficient = -0.0088, p= 0.0103), COMP and osteophyte score of the medial tibia (coefficient = -0.0911, p= 0.0003), and C2C and synovial membrane inflammation scores of the medial femoral (coefficient = 0.054, p= 0.0131). These results suggest that representative OA bio- markers in individual animals in the DMM model did not reflect histological scores well.