Circular RNA SLTM as a miR-421-competing endogenous RNA to mediate HMGB2 expression stimulates apoptosis and inflammation in arthritic chondrocytes

Osteoarthritis (OA) is the most common age-related joint disease characterized by chronic inflammation, progressive articular cartilage destruction, and subchondral sclerosis. Accumulating evidence suggests that circular RNAs (circRNAs) play key roles in OA, but the function of circSLTM in OA remains greatly unknown. Therefore, this study focused on interleukin-1β (IL-1β)-treated primary human chondrocytes as well as a rat model to investigate the expression pattern and functional role of circSLTM in OA in vitro and in vivo. CircSLTM and high mobility group protein B2 (HMGB2) were upregulated in IL-1β-induced chondrocytes, whereas miR-421 was downregulated. Knockdown of circSLTM or overexpression of miR-421 ameliorated IL-1β-induced chondrocyte apoptosis and inflammation. The regulatory relationship between circSLTM and miR-421, as well as that between miR-421 and HMGB2, was predicted by bioinformatics and then verified by the RNA immunoprecipitation experiment and dual-luciferase reporter gene assay. Furthermore, silencing of circSLTM increased cartilage destruction and decreased cartilage tissue apoptosis rate and inflammation in a rat model of OA. Taken together, our findings demonstrate the fundamental role of circSLTM in OA progression and provide a potential molecular target for OA therapy.     

Hypoxia-inducible factor-2α regulates Fas-mediated chondrocyte apoptosis during osteoarthritic cartilage destruction

Apoptosis of articular chondrocytes is associated with the pathogenesis of osteoarthritis (OA). Recently, we demonstrated that hypoxia-inducible factor (HIF)-2α, encoded by Epas1, causes OA cartilage destruction by regulating the expression of various matrix-degrading enzymes. Here, we investigated the involvement of HIF-2α in chondrocyte apoptosis and OA cartilage destruction. HIF-2α levels in human and mouse OA chondrocytes were markedly elevated in association with increased apoptosis of articular chondrocytes. Overexpression or knockdown of HIF-2α alone did not cause chondrocyte apoptosis. However, HIF-2α expression markedly increased chondrocyte apoptosis in the presence of an agonistic anti-Fas (CD95) antibody. HIF-2α enhanced Fas expression and potentiated downstream signaling pathways, increasing the activity of initiator and executioner caspases. Overexpression of HIF-2α in mouse cartilage tissue, either by intra-articular injection of Epas1 adenovirus (Ad-Epas1) or in the context of chondrocyte-specific Epas1 transgenic mice, increased chondrocyte apoptosis and cartilage destruction. In contrast, chondrocyte-specific knockout of Epas1 in mice suppressed DMM (destabilization of the medial meniscus)-induced chondrocyte apoptosis and inhibited OA cartilage destruction. Moreover, Fas-deficient mice exhibited diminished chondrocyte apoptosis and OA cartilage destruction in response to Ad-Epas1 injection or DMM surgery. Taken together, our results demonstrate that HIF-2α potentiates Fas-mediated chondrocyte apoptosis, which is associated with OA cartilage destruction.     

Midkine promotes articular chondrocyte proliferation through the MK-LRP1-nucleolin signaling pathway

Osteoarthritis (OA) is the most common disease of joint tissues; unfortunately, there are currently no curative therapies available for OA. Chondrocytes, the only cell type residing in cartilage, secrete many types of collagen (the mainly one is type II collagen) and aggrecan, which are the main components of the cartilage matrix. Chondrocyte apoptosis can lead to OA degenerative progression. We previously indicated that recombinant human midkine (rhMK), as a chondrocyte growth factor has a significant reparative effect on cartilage injury animal models. However, the molecular mechanism of this restorative function remains under investigation. Herein, we focused on the molecular mechanism underlying the role of MK in promoting the proliferation of chondrocytes cultured in vitro. Chondrocytes from rats and OA patients were successfully isolated by the digestion of articular cartilage using type II collagenase, and their proliferation was evaluated by a CCK8 assay and flow cytometry. rhMK stimulated the proliferation of chondrocytes from both OA patients and rats. Furthermore, qRT-PCR, shRNA-mediated knockdown, Western blot and immunoprecipitation (IP) assays were performed to identify the receptor and key elements responsible for the role of MK in promoting chondrocyte proliferation. Low-density lipoprotein receptor-related protein 1 (LRP1) was identified as the dominant MK receptor in chondrocytes that, as a translocator, mediates the endocytosis of MK. After being transferred into chondrocytes, MK was shown to form a complex with nucleolin that interacts with the active form of K-Ras. Upon the activation of ERK1/2, cyclin D1 expression was upregulated, promoting the chondrocyte cell cycle. Our data reveal for the first time the role of the MK-LRP1-nucleolin signaling pathway in facilitating MK-induced chondrocyte proliferation, thus providing a strong theoretical foundation for the further use of MK in OA clinical therapy.     

miR‐370 and miR‐373 regulate the pathogenesis of osteoarthritis by modulating one‐carbon metabolism via SHMT‐2 and MECP‐2, respectively

The aim of this study was to determine the mechanism underlying the association between one‐carbon metabolism and DNA methylation during chronic degenerative joint disorder, osteoarthritis (OA). Articular chondrocytes were isolated from human OA cartilage and normal cartilage biopsied, and the degree of cartilage degradation was determined by safranin O staining. We found that the expression levels of SHMT‐2 and MECP‐2 were increased in OA chondrocytes, and 3′UTR reporter assays showed that SHMT‐2 and MECP‐2 are the direct targets of miR‐370 and miR‐373, respectively, in human articular chondrocytes. Our experiments showed that miR‐370 and miR‐373 levels were significantly lower in OA chondrocytes compared to normal chondrocytes. Overexpression of miR‐370 or miR‐373, or knockdown of SHMT‐2 or MECP‐2 reduced both MMP‐13 expression and apoptotic cell death in cultured OA chondrocytes. In vivo, we found that introduction of miR‐370 or miR‐373 into the cartilage of mice that had undergone destabilization of the medial meniscus (DMM) surgery significantly reduced the cartilage destruction in this model, whereas introduction of SHMT‐2 or MECP‐2 increased the severity of cartilage destruction. Together, these results show that miR‐370 and miR‐373 contribute to the pathogenesis of OA and act as negative regulators of SHMT‐2 and MECP‐2, respectively.     

Quercetin attenuates oxidative stress-induced apoptosis via SIRT1/AMPK-mediated inhibition of ER stress in rat chondrocytes and prevents the progression of osteoarthritis in a rat model

Apoptosis of chondrocytes are the main initiator of osteoarthritis (OA) and can be explained by oxidative stress and endoplasmic reticulum (ER) stress, thus the pharmacological interventions aimed at inhibiting of these pathways may be a promising approach for the management of OA. Quercetin is a member of the flavonoid family and has antioxidant and anti-inflammatory properties in degenerative diseases. However, its effects and potential mechanisms on the pathological process of OA are not very clear. The present study aimed to investigate the protective effects of quercetin on OA and the underlying mechanisms. The tert-butyl hydroperoxide (TBHP)-stimulated rat chondrocytes and destabilization of the medial meniscus OA rat model was used to explore the protective effects of quercetin. Our results showed that quercetin treatment can attenuate oxidative stress, ER stress, and associated apoptosis. Moreover, quercetin inhibited ER stress through activating the sirtuin1/adenosine monophosphate-activated protein kinase (SIRT1/AMPK) signaling pathway. The protective effects of quercetin were also observed in OA rat model which is evidenced by abolished cartilage degeneration and decreased chondrocytes apoptosis in the knee joints. Our results suggested that quercetin is a promising treatment for OA.     

CircHIPK3 prevents chondrocyte apoptosis and cartilage degradation by sponging miR‐30a‐3p and promoting PON2

Osteoarthritis (OA) is a common joint disease featured by the deterioration of articular cartilage and chondrocyte death. Emerging evidence has indicated that circular RNAs (circRNAs) play an essential role in OA progress. Here, we found that the expression of circHIPK3 was significantly decreased in human and mouse OA cartilage. Knocking down circHIPK3 increased apoptosis and intracellular ROS level in HC‐a chondrocytes. We performed proteomic studies and identified that circHIPK3 regulated chondrocyte apoptosis through the mitochondrial pathway. Results of JC‐1 staining and western blot further confirmed that mitochondrial outer membrane permeabilization was promoted in HC‐a chondrocytes transfected by circHIPK3 siRNA. In terms of mechanism, we showed that PON2 functioned as a potential target of circHIPK3 to regulate chondrocyte apoptosis. Moreover, we revealed that circHIPK3 interacted with miR‐30a‐3p to regulate PON2 expression in chondrocytes. Taken together, our findings suggested that circHIPK3 regulated chondrocyte apoptosis by mitochondrial pathway, and targeting the circHIPK3/miR‐30a‐3p/PON2 axis might be a potential strategy for OA treatment.

The current study revealed the important role of circHIPK3 in regulating chondrocyte apoptosis and maintaining extracellular matrix (ECM) homeostasis. Mechanistically, circHIIPK3 might serve as a sponge of miR‐30a‐3p to regulate PON2 expression. The downregulation of circHIIPK3 resulted in the increased expression of miR‐30a‐3p and decreased expression of PON2, thus leading to mitochondrial pathway apoptosis and ECM destruction.     

Age-dependent alteration of TGF-β signalling in osteoarthritis

Osteoarthritis (OA) is a disease of articular cartilage, with aging as the main risk factor. In OA, changes in chondrocytes lead to the autolytic destruction of cartilage. Transforming growth factor-β has recently been demonstrated to signal not only via activin receptor-like kinase 5 (ALK5)-induced Smad2/3 phosphorylation, but also via ALK1-induced Smad1/5/8 phosphorylation in articular cartilage. In aging cartilage and experimental OA, the ratio ALK1/ALK5 has been found to be increased, and the expression of ALK1 is correlated with matrix metalloproteinase-13 expression. The age-dependent shift towards Smad1/5/8 signalling might trigger the differentiation of articular chondrocytes with an autolytic phenotype.     

Procyanidin B3 prevents articular cartilage degeneration and heterotopic cartilage formation in a mouse surgical osteoarthritis model

Osteoarthritis (OA) is a common disease in the elderly due to an imbalance in cartilage degradation and synthesis. Heterotopic ossification (HO) occurs when ectopic masses of endochondral bone form within the soft tissues around the joints and is triggered by inflammation of the soft tissues. Procyanidin B3 (B3) is a procyanidin dimer that is widely studied due to its high abundance in the human diet and antioxidant activity. Here, we evaluated the role of B3 isolated from grape seeds in the maintenance of chondrocytes in vitro and in vivo. We observed that B3 inhibited H(2)O(2)-induced apoptosis in primary chondrocytes, suppressed H(2)O(2)- or IL-1?-induced nitric oxide synthase (iNOS) production, and prevented IL-1?-induced suppression of chondrocyte differentiation marker gene expression in primary chondrocytes. Moreover, B3 treatment enhanced the early differentiation of ATDC5 cells. To examine whether B3 prevents cartilage destruction in vivo, OA was surgically induced in C57BL/6J mice followed by oral administration of B3 or vehicle control. Daily oral B3 administration protected articular cartilage from OA and prevented chondrocyte apoptosis in surgically-induced OA joints. Furthermore, B3 administration prevented heterotopic cartilage formation near the surgical region. iNOS protein expression was enhanced in the synovial tissues and the pseudocapsule around the surgical region in OA mice fed a control diet, but was reduced in mice that received B3. Together, these data indicated that in the OA model, B3 prevented OA progression and heterotopic cartilage formation, at least in a part through the suppression of iNOS. These results support the potential therapeutic benefits of B3 for treatment of human OA and heterotopic ossification.     

Avenanthramide C as a novel candidate to alleviate osteoarthritic pathogenesis

Osteoarthritis (OA) is a degenerative disorder that can result in the loss of articular cartilage. No effective treatment against OA is currently available. Thus, interest in natural health products to relieve OA symptoms is increasing. However, their qualities such as efficacy, toxicity, and mechanism are poorly understood. In this study, we determined the efficacy of avenanthramide (Avn)-C extracted from oats as a promising candidate to prevent OA progression and its mechanism of action to prevent the expression of matrix-metalloproteinases (MMPs) in OA pathogenesis. Interleukin-1 beta (IL-1β), a proinflammatory cytokine as a main causing factor of cartilage destruction, was used to induce OA-like condition of chondrocytes in vitro. Avn-C restrained IL-1β-mediated expression and activity of MMPs, such as MMP-3, -12, and -13 in mouse articular chondrocytes. Moreover, Avn-C alleviated cartilage destruction in experimental OA mouse model induced by destabilization of the medial meniscus (DMM) surgery. However, Avn-C did not affect the expression of inflammatory mediators (Ptgs2 and Nos) or anabolic factors (Col2a1, Aggrecan, and Sox9), although expression levels of these genes were upregulated or downregulated by IL-1β, respectively. The inhibition of MMP expression by Avn-C in articular chondrocytes was mediated by p38 kinase and c-Jun N-terminal kinase (JNK) signaling, but not by ERK or NF-κB. Interestingly, Avn-C added with SB203580 and SP600125 as specific inhibitors of p38 kinase and JNK, respectively, enhanced its inhibitory effect on the expression of MMPs in IL-1β treated chondrocytes. Taken together, these results suggest that Avn-C is an effective candidate to prevent OA progression and a natural health product to relieve OA pathogenesis.     

Therapeutic effects of bone marrow mesenchymal stem cells-derived exosomes on osteoarthritis

Mesenchymal stem cells (MSCs) have shown chondroprotective effects in clinical models of osteoarthritis (OA). However, effects of MSC-derived exosomes on OA remain unclear. The study aimed to investigate the therapeutic potential of exosomes from human bone marrow MSCs (BM-MSCs) in alleviating OA. The anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) surgery were performed on the knee joints of a rat OA model, followed by intra-articular injection of BM-MSCs or their exosomes. In addition, BM-MSC-derived exosomes were administrated to primary human chondrocytes to observe the functional and molecular alterations. Both of BM-MSCs and BM-MSC-derived exosomes alleviated cartilage destruction and subchondral bone remodelling in OA rat model. Administration of BM-MSCs and exosomes could reduce joint damage and restore the trabecular bone volume fraction, trabecular number and connectivity density of OA rats. In addition, in vitro assays showed that BM-MSCs-exosomes could maintain the chondrocyte phenotype by increasing collagen type II synthesis and inhibiting IL-1β–induced senescence and apoptosis. Furthermore, exosomal lncRNA MEG-3 also reduced the senescence and apoptosis of chondrocytes induced by IL-1β, indicating that lncRNA MEG-3 might partially account the anti-OA effects of BM-MSC exosomes. The exosomes from BM-MSCs exerted beneficial therapeutic effects on OA by reducing the senescence and apoptosis of chondrocytes, suggesting that MSC-derived exosomes might provide a candidate therapy for OA treatment.     

Chondroptosis: An immunohistochemical study of apoptosis and Golgi complex in chondrocytes from human osteoarthritic cartilage

The Golgi complex is thought to play an important role in the apoptotic process of osteoarthritic (OA) chondrocytes. However, the exact relationship between modifications of the Golgi complex and apoptosis in human OA cartilage requires to be established. We compared the patterns and immunolabeling intensities for anti-Golgi 58 K protein with apoptosis markers such as TUNEL and caspase-2L in OA cartilage removed from patients during knee total replacement surgery. We observed important modifications in labeling of the Golgi 58 K protein in OA chondrocytes compared with normal cell. Immunohistochemical analysis revealed co-localization between 58 K protein and caspase-2L, suggesting that this enzyme was localized in Golgi complex of OA chondrocytes. In addition, these cells labeled positive with the TUNEL technique, but in different proportions to caspase-2L. Our results support the concept, previously reported, that apoptosis in OA cartilage (chondroptosis) might be a variant of the classical apoptosis.     

Pentraxin 3 regulated by miR-224-5p modulates macrophage reprogramming and exacerbates osteoarthritis associated synovitis by targeting CD32

Emerging evidence has shown an imbalance in M1/M2 macrophage polarization to play an essential role in osteoarthritis (OA) progression. However, the underlying mechanistic basis for this polarization is unknown. RNA sequencing of OA M1-polarized macrophages found highly expressed levels of pentraxin 3 (PTX3), suggesting a role for PTX3 in OA occurrence and development. Herein, PTX3 was found to be increased in the synovium and articular cartilage of OA patients and OA mice. Intra-articular injection of PTX3 aggravated, while PTX3 neutralization reversed synovitis and cartilage degeneration. No metabolic disorder or proteoglycan loss were observed in cartilage explants when treated with PTX3 alone. However, cartilage explants exhibited an OA phenotype when treated with culture supernatants of macrophages stimulated with PTX3, suggesting that PTX3 did not have a direct effect on chondrocytes. Therefore, the OA anti-chondrogenic effects of PTX3 are primarily mediated through macrophages. Mechanistically, PTX3 was upregulated by miR-224-5p deficiency, which activated the p65/NF-κB pathway to promote M1 macrophage polarization by targeting CD32. CD32 was expressed by macrophages, that when stimulated with PTX3, secreted abundant pro-inflammation cytokines that induced severe articular cartilage damage. The paracrine interaction between macrophages and chondrocytes produced a feedback loop that enhanced synovitis and cartilage damage. The findings of this study identified a functional pathway important to OA development. Blockade of this pathway and PTX3 may prevent and treat OA.Subject terms: Osteoarthritis, Extracellular signalling molecules     

In vitro inhibition of IL-1beta catabolic effects on cartilage: a mechanism involved on diacerein anti-OA properties

Osteoarthritis (OA) is a progressive joint disease which represents a combination of several disorders leading to cartilage degradation. The main characteristic of OA is an imbalance between chondrocyte anabolic and catabolic activities. Cytokines produced by the synovium and chondrocytes, especially interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), play a significant role in the degradation of cartilage. They stimulate the production of nitric oxide (NO), which is involved in cartilage catabolism and also may induce the apoptosis of chondrocytes. The IL-1beta produced in activated chondrocytes or synovium may modulate disease progression in OA and should therefore be considered a potential target for therapeutic interventions. Drug and non-drug treatments are used to relieve pain and/or swelling in OA. Diacerein is a slow-acting drug that may slow down the breakdown of cartilage and relieve pain and swelling. It is not clear whether diacerein works but it has been proposed that diacerein acts as a symptom-modifying and perhaps disease-structure modifying drug.