Mouse Model Of Osteoarthritis Research Articles

Identification of EGFR as an essential regulator in chondrocytes ferroptosis of osteoarthritis using bioinformatics, in vivo, and in vitro study

ObjectiveThe mechanisms of chondrocytes ferroptosis in osteoarthritis (OA) have not yet been fully elucidated. This study aimed to identify key ferroptosis related genes (FRGs) involved in chondrocytes ferroptosis. MethodsLASSO, SVM-RFE, and receiver operating characteristic curve (ROC) were performed to screen key differentially expressed FRGs (DEFRGs). Functional analyses were conducted using GO, and KEGG analyses. Unsupervised clustering analysis was used to identify ferroptosis related patterns. The CeRNA network was constructed to predict the upstream miRNAs and lncRNAs. Finally, we validated the role of EGFR in chondrocytes ferroptosis using in vivo and in vitro experiments. ResultsA total of 42 DEFRGs were identified between OA and normal cartilages. GO and KEGG analyses indicated that these DEFRGs were significantly engaged in ferroptosis related biological processes and pathways, such as cellular response to oxidative stress, positive regulation of programmed cell death, MAPK and PI3K-Akt signaling pathways. Moreover, four key DEFRGs, including ACSF2, AURKA, EGFR, and KLHL24, were considered as potential biomarkers of OA. Moreover, two distinct ferroptosis related patterns were determined, and a total of 882 differentially expressed genes were identified which might participate in extracellular matrix degradation and inflammatory response. In addition, the CeRNA network showed that EGFR could be competitively regulated by 3 lncRNAs and 4 miRNAs. Significantly, the expression of EGFR was downregulated in human OA cartilages, OA mouse model, and erastin induced chondrocytes. EGFR inhibition could induce the occurrence of chondrocytes ferroptosis and ECM degradation which could be reversed by the addition of Ferrostatin-1. ConclusionOur study has identified ACSF2, AURKA, EGFR, and KLHL24 as ferroptosis-related biomarkers in OA. Furthermore, we have conducted a preliminary investigation into the role of EGFR in regulating chondrocytes ferroptosis. These findings offer novel insights into the molecular mechanisms underlying OA.

Evaluation of Cartilage Integrity Following Administration of Oral and Intraarticular Nifedipine in a Murine Model of Osteoarthritis.

Osteoarthritis (OA) ranks as the prevailing type of arthritis on a global scale, for which no effective treatments are currently available. Arterial hypertension is a common comorbidity in OA patients, and antihypertensive drugs, such as nifedipine (NIF), may affect the course of OA progression. The aim of this preclinical study was to determine the effect of nifedipine on healthy and OA cartilage, depending on its route of administration. In this study, we used the destabilization of medial meniscus to develop a mouse model of OA. Nifedipine was applied per os or intraarticularly (i.a.) for 8 weeks to both mice with OA and healthy animals. Serum biomarker concentrations were evaluated using the Luminex platform and alterations in the knee cartilage were graded according to OARSI histological scores and investigated immunohistochemically. Nifedipine treatment per os and i.a. exerted protective effects, as assessed by the OARSI histological scores. However, long-term nifedipine i.a. injections induced the deterioration of healthy cartilage. Lubricin, cartilage intermediate layer matrix protein (CILP), collagen type VI (COLVI), CILP, and Ki67 were upregulated by the nifedipine treatment. Serum biomarkers MMP-3, thrombospondin-4, and leptin were upregulated in the healthy groups treated with nifedipine, while only the levels of MMP-3 were significantly higher in the OA group treated with nifedipine per os compared to the untreated group. In conclusion, this study highlights the differential effects of nifedipine on cartilage integrity, depending on the route of administration and cartilage condition.

Role of Syndecan-4 in the Inhibition of Articular Cartilage Degeneration in Osteoarthritis.

Despite its widespread existence, there are relatively few drugs that can inhibit the progression of osteoarthritis (OA). Syndecan-4 (SDC4) is a transmembrane heparan sulfate proteoglycan that modulates cellular interactions with the extracellular matrix. Upregulated SDC4 expression in articular cartilage chondrocytes correlates with OA progression. In the present study, we treated osteoarthritic cartilage with SDC4 to elucidate its role in the disease's pathology. In this in vitro study, we used real-time polymerase chain reaction (PCR) to investigate the effects of SDC4 on anabolic and catabolic factors in cultured chondrocytes. In the in vivo study, we investigated the effect of intra-articular injection of SDC4 into the knee joints of an OA mouse model. In vitro, SDC4 upregulated the expression of tissue inhibitor of metalloproteinase (TIMP)-3 and downregulated the expression of matrix metalloproteinase (MMP)-13 and disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 in chondrocytes. Injection of SDC4 into the knee joints of OA model mice prevented articular cartilage degeneration 6 and 8 weeks postoperatively. Immunohistochemical analysis 8 weeks after SDC4 injection into the knee joint revealed decreased ADAMTS-5 expression and increased TIMP-3 expression. The results of this study suggest that the treatment of osteoarthritic articular cartilage with SDC4 inhibits cartilage degeneration.

N6-methyladenosine (m6A) methyltransferase WTAP-mediated miR-92b-5p accelerates osteoarthritis progression

The study was design to investigate the functional roles of Wilms tumor 1-associated protein (WTAP), an enzyme catalyzes m6A modification, in the pathogenesis of osteoarthritis (OA) and further elucidate its possible regulatory mechanism. Herein, we discovered that WTAP was outstandingly upregulated in chondrocyte stimulated with Lipopolysaccharide (LPS) and cartilage tissue of patients with OA. Functional studies have demonstrated that WTAP knockdown enhances proliferation ability, suppresses apoptosis, and reduces extracellular matrix (ECM) degradation in an LPS—induced OA chondrocyte injury model and ameliorates cartilage damage in a destabilizing the medial meniscus (DMM)—induced OA mice model. Conversely, overexpression of WTAP contributes to the opposite effects. Mechanistically, our data has demonstrated that m6A modification mediated by WTAP promotes the maturation of pri-miR-92b to miR-92b-5p, thereby enhancing the targeted inhibitory function of miR-92b-5p on TIMP4. Furthermore, we have discovered that WTAP can directly facilitate the degradation of TIMP4 mRNAs in a YTHDF2-dependent manner. In a nutshell, our findings suggested that WTAP knockdown alleviated OA progression by modulating the miR-92b-5p/TIMP4 axis in an m6A-dependent manner. Our study disclosed that WTAP-mediated m6A modification displayed a crucial role in OA development and suggested that targeting WTAP could be a promising preventive and therapeutic target for patients with OA.7QG3J4kX7R4LACdpcrQ_FqVideo

Platelet-Derived Exosomes Alleviate Knee Osteoarthritis by Attenuating Cartilage Degeneration and Subchondral Bone Loss.

Osteoarthritis (OA) is the most prevalent chronic degenerative joint disease among the aged population. However, current treatments for OA are limited to alleviating symptoms, with no therapies that prevent and regenerate cartilage deterioration. To assess the effects of platelet-derived exosomes (Plt-exos) on OA and then to explore the potential molecular mechanism. Controlled laboratory study. Exosomes derived from human apheresis platelets were isolated and identified. The effects of Plt-exos in protecting chondrocytes under interleukin 1β stimulation were evaluated by analyzing the proliferation and migration in human primary chondrocytes. RNA sequencing was later performed in vitro for primary chondrocytes to reveal the underlying mechanisms of Plt-exo treatment. Anterior cruciate ligament transection was used to construct an OA mice model, and intra-articular injection of Plt-exos was given once a week for 6 weeks. Mice were sacrificed 4 weeks after the last injection. Histologic and immunohistochemistry staining and micro-computed tomography analysis were performed to assess alterations of articular cartilage and subchondral bone. Plt-exos significantly promoted proliferation and migration of chondrocytes within a dose-dependent manner, as well as dramatically promoted cartilage regeneration and attenuated abnormal tibial subchondral bone remodeling, thus slowing the progression of OA. After being treated with Plt-exos, 1797 genes were differentially expressed in chondrocytes (923 upregulated and 874 downregulated genes). Functional enrichment results and hub genes were mainly involved in anti-inflammatory effects, mediating cell adhesion, stimulating cartilage repair, promoting anabolism, and inhibiting catabolism. Our results demonstrated that Plt-exos promoted chondrocyte proliferation and migration in vitro, as well as attenuated cartilage degeneration, improved the microarchitecture of subchondral bone, and retarded OA progression in vivo. Our study illustrated that the administered Plt-exos could alleviate knee OA by attenuating cartilage degeneration and subchondral bone loss, possibly serving as a novel promising treatment for OA in the future.

Identification and Analysis of Neutrophil Extracellular Trap-Related Genes in Osteoarthritis by Bioinformatics and Experimental Verification.

Osteoarthritis (OA) is a common joint disease with long-term pain and dysfunction that negatively affects the quality of life of patients. Neutrophil extracellular traps (NETs), consisting of DNA, proteins and cytoplasm, are released by neutrophils and play an important role in a variety of diseases. However, the relationship between OA and NETs is unclear. In our study, we used bioinformatics to explore the relationship between OA and NETs and the potential biological markers. GSE55235, GSE55457, GSE117999 and GSE98918 were downloaded from the Gene Expression Omnibus (GEO) database for subsequent analysis.After differential analysis of OA expression matrices, intersection with NET-related genes (NRGs) was taken to identify Differentially expressed NRGs (DE-NRGs) in OA processes. Evaluation of immune cell infiltration by ssGSEA and CIBERSORT algorithm. The GSVA method was used to analyze the activity changes of Neutrophils pathway, Neutrophil degranulation and Neutrophil granule constituents pathway. Based on RandomForest (RF), Least Absolute Shrinkage and Selection Operator (LASSO), and Support Vector Machine-Recursive Feature Elimination (SVM-RFE) learning algorithms, five core genes (CRISPLD2, IL1B, SLC25A37, MMP9, and TLR7) were identified to construct an OA-related nomogram model for predicting OA progression. ROC curve results for these genes validated the nomogram's reliability. Correlation analysis, functional enrichment, and drug predictions were performed for the core genes. TLR7 emerged as a key focus due to its high importance ranking in RF and SVM-RFE analyses. Gene Set Enrichment Analysis (GSEA) revealed a strong association between TLR7 and the Neutrophil extracellular trap pathway. Expression of core genes was demonstrated in mice OA models and human OA samples. TLR7 expression in ATDC5 cell line was significantly higher than control after TNFα induction, along with increased IL6 and MMP13. TLR7 may be related to NETs and affects OA.

CircGNB1 drives osteoarthritis pathogenesis by inducing oxidative stress in chondrocytes.

Circular RNAs (circRNAs) have risen to prominence as important regulators of biological processes. This study investigated whether circGNB1 functions as a competitive endogenous RNA to regulate the pathological process of oxidative stress in age-related osteoarthritis (OA). The relationship between circGNB1 expression and oxidative stress/OA severity was determined in cartilages from OA patients at different ages. The biological roles of circGNB1 in oxidative stress and OA progression, and its downstream targets were determined using gain- and loss-of-function experiments in various biochemical assays in human chondrocytes (HCs). The in vivo effects of circGNB1 overexpression and knockdown were also determined using a destabilization of the medial meniscus (DMM) mouse model. Increased circGNB1 expression was detected in HCs under oxidative and inflammatory stress and in the cartilage of older individuals. Mechanistically, circGNB1 sponged miR-152-3p and thus blocked its interaction with its downstream mRNA target, ring finger protein 219 (RNF219), which in turn stabilized caveolin-1 (CAV1) by preventing its ubiquitination at the K47 residue. CircGNB1 inhibited IL-10 signalling by antagonizing miR-152-3p-mediated RNF219 and CAV1 inhibition. Consequently, circGNB1 overexpression promoted OA progression by enhancing catabolic factor expression and oxidative stress and by suppressing anabolic genes in vitro and in vivo. Furthermore, circGNB1 knockdown alleviated the severity of OA, whereas circGNB1 overexpression had the opposite effect in a DMM mouse model of OA. CircGNB1 regulated oxidative stress and OA progression via the miR-152-3p/RNF219/CAV1 axis. Modulating circGNB1 could be an effective strategy for treating OA.

MiR-204 ameliorates osteoarthritis pain by inhibiting SP1-LRP1 signaling and blocking neuro-cartilage interaction.

Osteoarthritis (OA) is a painful degenerative joint disease and is the leading cause of chronic disability among elderly individuals. To improve the quality of life for patients with OA, the primary goal for OA treatment is to relieve the pain. During OA progression, nerve ingrowth was observed in synovial tissue and articular cartilage. These abnormal neonatal nerves act as nociceptors to detect OA pain signals. The molecular mechanisms for transmitting OA pain in the joint tissues to the central nerve system (CNS) is currently unknown. MicroRNA miR-204 has been demonstrated to maintain the homeostasis of joint tissues and have chondro-protective effect on OA pathogenesis. However, the role of miR-204 in OA pain has not been determined. In this study, we investigated interactions between chondrocytes and neural cells and evaluated the effect and mechanism of miR-204 delivered by exosome in the treatment of OA pain in an experimental OA mouse model. Our findings demonstrated that miR-204 could protect OA pain by inhibition of SP1- LDL Receptor Related Protein 1 (LRP1) signaling and blocking neuro-cartilage interaction in the joint. Our studies defined novel molecular targets for the treatment of OA pain.

ROR1/STAT3 positive feedback loop facilitates cartilage degeneration in Osteoarthritis through activation of NF-κB signaling pathway.

Osteoarthritis (OA) is a chronic joint disorder with a serious impact on society. The main pathological change in OA is articular cartilage degeneration, which is directly associated with imbalance of anabolic and catabolic activities in chondrocytes. To evaluate the expression and biological effects of ROR1 in OA cartilage and determine whether knockdown of ROR1 attenuates cartilage degeneration. ROR1 expression in OA clinical specimens was evaluated by western blotting and immunohistochemistry. The effects of ROR1 on anabolic and catabolic activities were evaluated in Wnt5a-treated human primary chondrocytes by western blotting, immunofluorescence, and luciferase assay. The effects of ROR1 knockdown on cartilage degeneration in a surgical OA mouse model were examined by X-ray imaging and Safranin O-Fast Green histological staining. ROR1 was considerably upregulated in cartilage tissues of OA patients. ROR1 knockdown alleviated the activation of the NF-κB signaling pathway and reversed the suppression of collagen II and aggrecan by Wnt5a, as well as upregulation of ADAMTS-5 and MMP-13 in chondrocytes. In addition, ROR1 knockdown significantly reduced Wnt5a-induced STAT3 nuclear translocation. STAT3 binding to the ROR1 promoter indicated a positive feedback loop between ROR1 and STAT3. ROR1 knockdown was confirmed to dramatically alleviate cartilage degradation in the DMM induced-OA mouse model. Increased expression of ROR1 in OA cartilage tissues leads to a positive feedback loop with STAT3, which activates the NF-κB signaling pathway, resulting in an imbalance between chondrocyte anabolism and catabolism. These results indicate a potential new therapeutic target for the treatment of OA.

The surgical destabilization ofthe abductor muscle leads to development ofinstability-associated hip osteoarthritis inmice.

Osteoarthritis (OA) of the hip is a common and debilitating painful joint disease. However, there is paucity of surgically induced hip OA models in small animals that allow scientists to study the onset and progression of the disease. A growing body of evidence indicates a positive association between periarticular myotendinous pathology and the development of hip OA. Thus, in the current study, we aimed to establish a novel mouse instability-associated hip OA model via selective injury of the abductor complex around the hip joint. C57BL6/J mice were randomized to sham surgery or abductor injury, in which the myotendinous insertion at the third trochanter and greater trochanter were surgically detached. Mice were allowed free active movement until they were sacrificed at either 3 weeks or 20 weeks post-injury. Histologic analyses and immunohistochemical staining of the femoral head articular cartilage were performed, along with microCT (µCT) analysis to assess subchondral bone remodeling. We observed that mice receiving abductor injury exhibited significantly increased instability-associated OA severity with loss of proteoglycan and type II collagen staining compared to sham control mice at 20 weeks post-surgery, while comparable matrix metalloproteinase 13 expression was observed between injury and sham groups. No significant differences in subchondral bone remodeling were found after 3 or 20 weeks following injury. Our study further supports the link between abductor dysfunction and the development of instability-associated hip OA. Importantly, this novel surgically induced hip OA mouse model may provide a valuable tool for future investigations into the pathogenesis and treatment of hip OA.

TAF15 regulates the BRD4/GREM1 axis and activates the gremlin-1-NF-κB pathway to promote OA progression

BackgroundAnterior cruciate ligament (ACL) injury is recognized as a risk factor for osteoarthritis (OA) progression. Herein, the function of TAF15 in ACL injury-induced OA was investigated. MethodsOA cell model and OA mouse model were established by interleukin-1 beta (IL-1β) stimulation and ACL transection administration, respectively. The pathological changes were analyzed by histopathology. The mRNA and protein expressions were assessed using qRT-PCR, Western blot and IHC. Chondrocyte viability and apoptosis were examined by CCK8 assay and TUNEL staining, respectively. The interactions between TAF15, BRD4 and GREM1 were analyzed by RIP or ChIP assay. ResultsTAF15 expression was markedly elevated in OA, and its knockdown suppressed IL-1β-induced chondrocyte apoptosis and ECM degradation in vivo and cartilage pathological changes in vitro. TAF15 promoted BRD4 mRNA stability, and TAF15 silencing's repression on chondrocyte apoptosis and ECM degradation induced by IL-1β was abrogated following BRD4 overexpression. BRD4 promoted GREM1 expression by directly binding with GREM1. In addition, the GREM1/NF-κB pathway functioned as the downstream pathway of BRD4 in promoting OA progression. ConclusionTAF15 upregulation facilitated chondrocyte apoptosis and ECM degradation during OA development by acting on the BRD4/GREM1/NF-κB axis, which provided a theoretical basis for the development of novel therapies for OA.

GPRC5B protects osteoarthritis by regulation of autophagy signaling.

Osteoarthritis (OA) is one of the most common chronic diseases in the world. However, current treatment modalities mainly relieve pain and inhibit cartilage degradation, but do not promote cartilage regeneration. In this study, we show that G protein-coupled receptor class C group 5 member B (GPRC5B), an orphan G-protein-couple receptor, not only inhibits cartilage degradation, but also increases cartilage regeneration and thereby is protective against OA. We observed that Gprc5b deficient chondrocytes had an upregulation of cartilage catabolic gene expression, along with downregulation of anabolic genes invitro. Furthermore, mice deficient in Gprc5b displayed a more severe OA phenotype in the destabilization of the medial meniscus (DMM) induced OA mouse model, with upregulation of cartilage catabolic factors and downregulation of anabolic factors, consistent with our invitro findings. Overexpression of Gprc5b by lentiviral vectors alleviated the cartilage degeneration in DMM-induced OA mouse model by inhibiting cartilage degradation and promoting regeneration. We also assessed the molecular mechanisms downstream of Gprc5b that may mediate these observed effects and identify the role of protein kinase B (AKT)-mammalian target of rapamycin (mTOR)-autophagy signaling pathway. Thus, we demonstrate an integral role of GPRC5B in OA pathogenesis, and activation of GPRC5B has the potential in preventing the progression of OA.

MATN3 delivered by exosome from synovial mesenchymal stem cells relieves knee osteoarthritis: Evidence from in vitro and in vivo studies

BackgroundSynovial mesenchymal stem cell (SMSC) exerts chondroprotective effects in osteoarthritis (OA) clinical models. However, the regulatory potentials of SMSC-derived exosomes (SMSC-Exo) in OA still need to be discovered, which attracted our attention. MethodsThe destabilization of the medial meniscus surgery was performed on the knee joints of a mouse OA model, followed by injection of SMSC-Exo. In addition, SMSC-Exo was administrated to mouse chondrocytes to observe the functional and molecular alterations. ResultsBoth of SMSC-Exo and overexpression of Matrilin-3 (MATN3) alleviated cartilage destruction and suppressed degradation of extracellular matrix (ECM) in the OA rat model. In addition, assays concerning the in vitro OA model induced by IL-1β showed that SMSC-Exo could promote chondrocyte viability and inhibit autophagy defects. Furthermore, SMSC-Exo achieved the chondroprotective effects through the delivery of MATN3/IL-17A, and MATN3 could suppress the activation of PI3K/AKT/mTOR signaling through IL-17A. ConclusionSMSC-Exo exerts beneficial therapeutic effects on OA by preventing ECM degradation and autophagy defects by delivering MATN3/IL-17A. The Translational Potential of this ArticleThe translational potential of this study is not only limited to the treatment of knee osteoarthritis but also provides new insights for the treatment of other joint diseases by exploring the mechanism of MATN3. In addition, SMSCExo, as a novel drug carrier, has great potential for treating and diagnosing other diseases. With further research, these findings will provide new directions for developing personalized and innovative treatment options.

Small extracellular vesicles derived from human adipose-derived mesenchymal stromal cells cultured in a new chemically-defined contaminate-free media exhibit enhanced biological and therapeutic effects on human chondrocytes in vitro and in a mouse osteoarthritis model.

Human small extracellular vesicles (sEVs) derived from adipose-derived mesenchymal stromal cells (ASC) have been reported to suppress the progression of osteoarthritis (OA) in animal studies and subsequently, translation of this potential to assess their clinical efficacy is anticipated. However, fabrication protocols for sEVs to eliminate potential contamination by culture medium-derived components need to be established prior to their clinical use. The purpose of the present studies was to elucidate the influence of medium-derived contaminants on the biological effects of sEVs, and to establish isolation methods for sEVs using a new clinical grade chemically-defined media (CDM). The quantity and purity of ASC-derived sEVs cultured in four different CDMs (CDM1, 2, 3 and 4) were evaluated. The concentrates of the four media incubated without cells were used as the background (BG) control for each set of sEVs. The biological effect of sEVs fabricated in the four different CDMs on normal human articular chondrocytes (hACs) were evaluated in vitro using a variety of methodological assessments. Finally, the sEVs with the highest purity were tested for their ability to suppress the progression of knee OA mouse model. Analysis of the BG controls revealed that CDM1-3 contained detectable particles, while there was no visible contamination of culture media-derived components detected with CDM4. Accordingly, the sEVs fabricated with CDM4 (CDM4-sEVs) exhibited the highest purity and yield. Notably, the CDM4-sEVs were the most efficient in promoting the cellular proliferation, migration, chondrogenic differentiation, and anti-apoptotic activity of hACs. Furthermore, CDM4-sEVs significantly suppressed the osteochondral degeneration in vivo model. Small EVs derived from ASCs cultured in a CDM without detectable contaminants demonstrated enhanced biological effects on hACs and the progression of OA. Thus, sEVs isolated with CDM4 most optimally meet the requirements of efficacy and safety for assessment in their future clinical applications.

MicroRNA-224-5p nanoparticles balance homeostasis via inhibiting cartilage degeneration and synovial inflammation for synergistic alleviation of osteoarthritis

MicroRNAs play a crucial role in regulating cartilage extracellular matrix (ECM) metabolism and are being explored as potential therapeutic targets for osteoarthritis (OA). The present study indicated that microRNA-224-5p (miR-224-5p) could balance the homeostasis of OA via regulating cartilage degradation and synovium inflammatory simultaneously. Multifunctional polyamidoamine dendrimer with amino acids used as efficient vector to deliver miR-224-5p. The vector could condense miR-224-5p into transfected nanoparticles, which showed higher cellular uptake and transfection efficiency compared to lipofectamine 3000, and also protected miR-224-5p from RNase degradation. After treatment with the nanoparticles, the chondrocytes showed an increase in autophagy rate and ECM anabolic components, as evidenced by the upregulation of autophagy-related proteins and OA-related anabolic mediators. This led to a corresponding inhibition of cell apoptosis and ECM catabolic proteases, ultimately resulting in the alleviation of ECM degradation. In addition, miR-224-5p also inhibited human umbilical vein endothelial cells angiogenesis and fibroblast-like synoviocytes inflammatory hyperplasia. Integrating the above synergistic effects of miR-224-5p in regulating homeostasis, intra-articular injection of nanoparticles performed outstanding therapeutic effect by reducing articular space width narrowing, osteophyte formation, subchondral bone sclerosis and inhibiting synovial hypertrophy and proliferation in the established mouse OA model. The present study provides a new therapy target and an efficient intra-articular delivery method for improving OA therapy. Statement of significanceOsteoarthritis (OA) is the most prevalent joint disease worldwide. Gene therapy, which involves delivering microRNAs, has the potential to treat OA. In this study, we demonstrated that miR-224-5p can simultaneously regulate cartilage degradation and synovium inflammation, thereby restoring homeostasis in OA gene therapy. Moreover, compared to traditional transfection reagents such as lipofectamine 3000, G5-AHP showed better efficacy in both microRNA transfection and protection against degradation due to its specific surface structure. In summary, G5-AHP/miR-224-5p was developed to meet the clinical needs of OA patients and the high requirement of gene transfection efficiency, providing a promising paradigm for the future application and development of gene therapy.

Extracts of Oldenlandia diffusa protects chondrocytes via inhibiting apoptosis and associated inflammatory response in osteoarthritis

Ethnopharmacological relevanceOsteoarthritis (OA) is a type of joint disorder that is marked by the gradual breakdown of cartilage and persistent inflammation of the synovial membrane, and is a leading cause of disability among elderly people worldwide. Oldenlandia diffusa (OD) is a member of the Rubiaceae family, and various researches have revealed that it possesses antioxidant, anti-inflammatory, and anti-tumor properties. Extracts of Oldenlandia diffusa is commonly used in traditional oriental medicine to treat various illnesses, including inflammation and cancer. Aim of the studyThis study is aimed at investigating the anti-inflammatory and anti-apoptosis effects of OD and its potential mechanisms on IL-1β-induced mouse chondrocytes, as well as its characteristics in a mouse osteoarthritis model. Materials and methodsIn this study, the key targets and potential pathways of OD were determined through network pharmacology analysis and molecular docking. The potential mechanism of OD in osteoarthritis was verified by in vitro and in vivo studies. ResultsThe results of network pharmacology showed that Bax, Bcl2, CASP3, and JUN are key candidate targets of OD for the treatment of osteoarthritis. There is a strong correlation between apoptosis and both OA and OD. Additionally, molecular docking results show that β-sitosterol in OD can strongly bind with CASP3 and PTGS2. In vitro experiments showed that OD pretreatment inhibited the expression of pro-inflammatory factors induced by IL-1β, such as COX2, iNOS, IL-6, TNF-α, and PGE2. Furthermore, OD reversed IL-1β-mediated degradation of collagen II and aggrecan within the extracellular matrix (ECM). The protective effect of OD can be attributed to its inhibition of the MAPK pathway and inhibition of chondrocyte apoptosis. Additionally, it was found that OD can alleviate cartilage degradation in a mouse model of knee osteoarthritis. ConclusionOur study showed that β-sitosterol, one of the active components of OD, could alleviate the inflammation and cartilage degeneration of OA by inhibiting chondrocyte apoptosis and MAPK pathway.

Chondrocyte Thrombomodulin Protects against Osteoarthritis.

Osteoarthritis (OA) is a prevalent form of arthritis that affects over 32.5 million adults worldwide, causing significant cartilage damage and disability. Unfortunately, there are currently no effective treatments for OA, highlighting the need for novel therapeutic approaches. Thrombomodulin (TM), a glycoprotein expressed by chondrocytes and other cell types, has an unknown role in OA. Here, we investigated the function of TM in chondrocytes and OA using various methods, including recombinant TM (rTM), transgenic mice lacking the TM lectin-like domain (TMLeD/LeD), and a microRNA (miRNA) antagomir that increased TM expression. Results showed that chondrocyte-expressed TM and soluble TM [sTM, like recombinant TM domain 1 to 3 (rTMD123)] enhanced cell growth and migration, blocked interleukin-1β (IL-1β)-mediated signaling and protected against knee function and bone integrity loss in an anterior cruciate ligament transection (ACLT)-induced mouse model of OA. Conversely, TMLeD/LeD mice exhibited accelerated knee function loss, while treatment with rTMD123 protected against cartilage loss even one-week post-surgery. The administration of an miRNA antagomir (miR-up-TM) also increased TM expression and protected against cartilage damage in the OA model. These findings suggested that chondrocyte TM plays a crucial role in counteracting OA, and miR-up-TM may represent a promising therapeutic approach to protect against cartilage-related disorders.

Evaluation of expanded peripheral blood derived CD34+ cells for the treatment of moderate knee osteoarthritis.

Knee osteoarthritis (OA) is a degenerative joint disease of the knee that results from the progressive loss of articular cartilage. It is most common in the elderly and affects millions of people worldwide, leading to a continuous increase in the number of total knee replacement surgeries. These surgeries improve the patient's physical mobility, but can lead to late infection, loosening of the prosthesis, and persistent pain. We would like to investigate if cell-based therapies can avoid or delay such surgeries in patients with moderate OA by injecting expanded autologous peripheral blood derived CD34+ cells (ProtheraCytes®) into the articular joint. In this study we evaluated the survival of ProtheraCytes® when exposed to synovial fluid and their performance in vitro with a model consisting of their co-culture with human OA chondrocytes in separate layers of Transwells and in vivo with a murine model of OA. Here we show that ProtheraCytes® maintain high viability (>95%) when exposed for up to 96 hours to synovial fluid from OA patients. Additionally, when co-cultured with OA chondrocytes, ProtheraCytes® can modulate the expression of some chondrogenic (collagen II and Sox9) and inflammatory/degrading (IL1β, TNF, and MMP-13) markers at gene or protein levels. Finally, ProtheraCytes® survive after injection into the knee of a collagenase-induced osteoarthritis mouse model, engrafting mainly in the synovial membrane, probably due to the fact that ProtheraCytes® express CD44, a receptor of hyaluronic acid, which is abundantly present in the synovial membrane. This report provides preliminary evidence of the therapeutic potential of CD34+ cells on OA chondrocytes in vitro and their survival after in vivo implantation in the knee of mice and merits further investigation in future preclinical studies in OA models.

Suramin ameliorates osteoarthritis by acting on the Nrf2/HO-1 and NF-κB signaling pathways in chondrocytes and promoting M2 polarization in macrophages

Osteoarthritis (OA)—the most prevalent of arthritis diseases—is a complicated pathogenesis caused by cartilage degeneration and synovial inflammation. Suramin has been reported to enhance chondrogenic differentiation. However, the therapeutic effect of suramin on OA-induced cartilage destruction has remained unclear. Suramin is an anti-parasitic drug that has potent anti-purinergic properties. This study investigated the protective effects and underlying mechanisms of suramin on articular cartilage degradation using an in vitro study and mice model with post-traumatic OA. We found that suramin markedly suppressed the IL-1β increased expression of matrix destruction proteases—such as ADAMT4, ADAMTS5, MMP3, MMP13, and inflammatory mediators—including the iNOS, COX2, TNFα, and IL-1β; while greatly enhancing the synthesis of cartilage anabolic factors—such as COL2A1, Aggrecan and SOX9 in IL-1β-induced porcine chondrocytes. In vivo experiments showed that intra-articular injection of suramin ameliorated cartilage degeneration and inhibited synovial inflammation in an anterior cruciate ligament transection (ACLT)-induced OA mouse model. In mechanistic studies, we found that exogenous supplementation of suramin can activate Nrf2, and accordingly inhibit the nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κB) and mitogen-activated protein kinase (MAPK) pathways, thereby alleviating the inflammation and ECM degeneration of chondrocytes stimulated by IL-1β. In addition, suramin also repolarized M1 macrophages to the M2 phenotype, further reducing the apoptosis of chondrocytes. Collectively, the results of the study suggests that suramin is a potential drugs which could serve as a facilitating drug for the application of OA therapy toward clinical treatment.

Structure-based virtual screening for discovery of paederosidic acid from Paederia scandens as novel P2Y14R antagonist

BackgroundThe activation of P2Y14 receptor (P2Y14R) promotes osteoclast formation and causes neuropathic pain, exhibiting possible link to osteoarthritis (OA). Given lack of P2Y14R antagonist, the present study aims to search a novel P2Y14R antagonist with low toxicity and high activity from natural products as a possible drug candidate in treatment of OA. MethodsThe role of P2Y14R on OA was verified using P2Y14R knockout (KO) rats. Molecular docking virtual screening strategy and activity test in P2Y14R stably-expressed HEK293 cells were used to screen target compound from natural product library. The MM/GBSA free energy calculation/decomposition technique was used to determine the principal interaction mechanism. Next, the binding of target compound to P2Y14R was examined using cellular thermal shift assay and drug affinity responsive target stability test. Finally, the therapeutic effect of target compound was performed in monosodium iodoacetate (MIA)-induced OA mouse model. To verify whether the effect of target compound was attributed to P2Y14R, we establish the osteoarthritis model in P2Y14R KO mice to perform pharmacodynamic evaluation. Importantly, to investigate the potential mechanism by which target compound attenuate OA, expressions of the major transcription factors involved in osteoclast differentiation were detected by western blot, while markers of nerve damage in dorsal root ganglion (DRG) were evaluated by RT-qPCR and immunofluorescence techniques. ResultsDeficiency of P2Y14R alleviated pain behavior and cartilage destruction in MIA-induced OA rats. 14 natural compounds were screened by Glide docking-based virtual screening, among which paederosidic acid exhibited the highest antagonistic activity to P2Y14R with IC50 of 8.287 μM. As a bioactive component extracted from Paederia scandens, paederosidic acid directly interacted with P2Y14R to enhance the thermostability and decrease the protease sensitivity of target protein, which significantly inhibited receptor activator for nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis. More importantly, paederosidic acid suppressed osteoclast formation by downregulating expressions of NFAT2 and ATP6V0D2, as well as relieved neuropathic pain by decreasing expressions of CGRP, CSF1 and galanin in DRG. ConclusionsPaederosidic acid targeted P2Y14R to improve OA through alleviating osteoclast formation and neuropathic pain, which provided an available strategy for developing novel drug leads for treatment of OA.