Chondrocyte Degradation Research Articles

Specific Clearance of Senescent Synoviocytes Suppresses the Development of Osteoarthritis based on Aptamer‐Functionalized Targeted Drug Delivery System

AbstractAge‐related diseases are among the leading causes of morbidity worldwide currently. Therefore, there is an urgent need to develop effective interventions to reduce aging. In mice, the selective elimination of senescent cells via senolytics extends the median lifespan and attenuates various age‐related diseases including osteoarthritis (OA). However, most classified senolytics may also affect non‐senescent cells because of their suboptimal specificity for senescent cells, which hampers the translation of senolytic therapies to human diseases. Precise targeting of senolytics to specific senescent cell types may help circumvent these potential hurdles. In the joints of OA, abundant senescent fibroblast‐like synoviocytes (FLSs) are observed in the synovium region, which can promote the degradation of chondrocytes. Here, an aptamer‐functionalized liposome (LS) is developed, which encapsulates with senolytics (dasatinib and quercetin, DQ), termed as CX3‐LS‐DQ. This CX3‐LS‐DQ exhibits high affinity for FLS, a reasonably long circulation time, minimal toxicity, and strong clearance ability in senescent FLS. In the OA mouse model, intra‐articular injection of CX3‐LS‐DQ effectively attenuates cartilage degradation in vivo. Overall, the FLS‐specific aptamer‐functionalized drug delivery system could act as a novel carrier for targeted drug delivery to the synovium, providing a foundation for potential clinical translation in OA therapy.

Effects of quercetin on apoptosis and extracellular matrix degradation of chondrocytes induced by oxidative stress-mediated pyroptosis.

Quercetin has been preliminarily proven to serve as a potential agent for the treatment of osteoarthritis (OA). However, its effects and potential mechanisms on the pathological process of OA are not very clear. This study aimed to study the protective effect of quercetin on OA. Lipopolysaccharide (LPS)-treated chondrocytes (C28/I2 cell) and anterior cruciate ligament transection with partial medial meniscectomy-treated Wistar rats were used as models of OA in vitro and in vivo. Cell counting kit-8 (CCK-8 kit), flow cytometry, enzyme-linked immunosorbent assay (ELISA) kit, western blot, dichlorodihydrofluorescein diacetate (DCFH-DA) kit, thiobarbituric acid (TBA) test, toluidine blue staining, Hematoxylin eosin (HE) staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining were used to evaluate cell viability, cell apoptosis, inflammatory cytokines level, protein expression, reactive oxygen species (ROS) level, malondialdehyde (MDA) content, morphological changes, and chondrocyte apoptosis of cartilage, respectively. Results showed that quercetin could reduce LPS-induced C28/I2 cell apoptosis, extracellular matrix (ECM) degradation, and cell pyroptosis, and overexpression of nucleotide-binding domain and leucine-rich-repeat-containing (NLR) family, pyrin domain-containing 3 (NLRP3) revealed that quercetin reduced chondrocyte apoptosis and ECM degradation by inhibiting NLRP3-mediated pyroptosis. Furthermore, quercetin could reduce chondrocyte apoptosis and ECM degradation, and inhibit NLRP3-mediated pyroptosis through blocking oxidative stress. It was further confirmed in the rat OA model that quercetin alleviated OA by blocking oxidative stress, reduces chondrocyte pyroptosis, apoptosis, and ECM degradation. In conclusion, quercetin inhibited OA via blocking oxidative stress-induced chondrocyte pyroptosis in models of OA in vitro and in vivo.

Hyperglycemia-induced accumulation of advanced glycosylation end products in fibroblast-like synoviocytes promotes knee osteoarthritis

Osteoarthritis (OA) is significantly associated with diabetes, but how hyperglycemia induces or aggravates OA has not been shown. The synovium plays a critical role in cartilage metabolism and substance exchange. Herein, we intended to investigate whether and how hyperglycemia affects the occurrence and progression of OA by influencing the synovium. In patients with knee OA and diabetes (DM OA), we found a more severe inflammatory response, higher endoplasmic reticulum stress (ERS) levels, and more advanced glycosylation end products (AGEs) accumulation in the synovium than in patients without diabetes. Subsequently, we found similar results in the DM OA group in a rat model. In the in vitro cocultivation system, high glucose-stimulated AGEs accumulation, ERS, and inflammation in rat fibroblast-like synoviocytes (FLSs), which resulted in chondrocyte degeneration due to inflammatory factors from FLSs. Furthermore, in the synovium of the DM OA group and FLSs treated with high glucose, the expression of glucose transporter 1 (GLUT1) and its regulatory factor hypoxia-inducible factor (HIF)-1α was increased significantly. Inhibitors of HIF-1α, GLUT1 or AGEs receptors attenuated the effect of high glucose on chondrocyte degradation in the FLS-chondrocyte coculture system. In summary, we demonstrated that hyperglycemia caused AGEs accumulation in FLSs via the HIF-1α-GLUT1 pathway, which increases the release of inflammatory factors from FLSs, subsequently inducing chondrocyte degradation and promoting OA progression.

Novel NFκB Inhibitor SC75741 Mitigates Chondrocyte Degradation and Prevents Activated Fibroblast Transformation by Modulating miR-21/GDF-5/SOX5 Signaling.

Osteoarthritis (OA) is a common articular disease manifested by the destruction of cartilage and compromised chondrogenesis in the aging population, with chronic inflammation of synovium, which drives OA progression. Importantly, the activated synovial fibroblast (AF) within the synovium facilitates OA through modulating key molecules, including regulatory microRNAs (miR’s). To understand OA associated pathways, in vitro co-culture system, and in vivo papain-induced OA model were applied for this study. The expression of key inflammatory markers both in tissue and blood plasma were examined by qRT-PCR, western blot, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assays. Herein, our result demonstrated, AF-activated human chondrocytes (AC) exhibit elevated NFκB, TNF-α, IL-6, and miR-21 expression as compared to healthy chondrocytes (HC). Importantly, AC induced the apoptosis of HC and inhibited the expression of chondrogenesis inducers, SOX5, TGF-β1, and GDF-5. NFκB is a key inflammatory transcription factor elevated in OA. Therefore, SC75741 (an NFκB inhibitor) therapeutic effect was explored. SC75741 inhibits inflammatory profile, protects AC-educated HC from apoptosis, and inhibits miR-21 expression, which results in the induced expression of GDF-5, SOX5, TGF-β1, BMPR2, and COL4A1. Moreover, ectopic miR-21 expression in fibroblast-like activated chondrocytes promoted osteoblast-mediated differentiation of osteoclasts in RW264.7 cells. Interestingly, in vivo study demonstrated SC75741 protective role, in controlling the destruction of the articular joint, through NFκB, TNF-α, IL-6, and miR-21 inhibition, and inducing GDF-5, SOX5, TGF-β1, BMPR2, and COL4A1 expression. Our study demonstrated the role of NFκB/miR-21 axis in OA progression, and SC75741’s therapeutic potential as a small-molecule inhibitor of miR-21/NFκB-driven OA progression.

Role of the hippo signaling pathway in the extracellular matrix degradation of chondrocytes induced by fluoride exposure

To identify the role of the Hippo signaling pathway in the extracellular matrix degradation of chondrocytes induced by fluoride exposure. Environmental response genes (ERGs) of bone injury induced by fluoride exposure were obtained from the Comparative Toxicogenomics Database, and annotated by STRING for KEGG pathway enrichment analysis. The CCK-8 kit was used to measure the proliferation of ATDC5 cells. The malondialdehyde (MDA), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-PX) levels in ATDC5 cells were measured using oxidative stress detection kit. Western blot analysis was used to measure the p-MST1/2, p-LATS1/2, and p-YAP/YAP1 expression levels in the Hippo pathway and the COL2A1, ACAN and MMP13 expression levels in the cartilage matrix. Localizations of YAP1 and COL2A1 proteins in chondrocytes were performed using cell immunofluorescence. Continuous data from the multiple groups were compared using one-way analysis of variance, and then the differences between groups were tested with Dunnett's t-test, with the test level α = 0.05. The 145 ERGs of bone injury induced by fluoride exposure were identified, and KEGG enrichment analysis revealed Hippo signaling pathways significantly related to bone injury. A CCK-8 assay revealed that the viability of the ATDC5 cells was significantly decreased with increased fluorine concentration. The MDA content in 20 mg/L sodium fluoride (NaF) exposure group was significantly higher than that in the control group, the T-SOD, T-AOC and GSH-PX activities in 15 and 20 mg/L NaF exposure groups were significantly lower than those in the control group (P < 0.05). Western blot results showed the protein levels of p-MST1/2, p-LATS1/2 and p-YAP1 in 15 and 20 mg/L NaF exposure groups were significantly lower than those in the control group, while the YAP1 protein level in 20 mg/L NaF group was significantly higher than that in the control group. The COL2A1 and ACAN proteins in 20 mg/L NaF group were significantly decreased, while the MMP13 protein level in 15 and 20 mg/L NaF groups were significantly increased (P < 0.05). It was observed that the expression of YAP1 protein expression level in the cytoplasm decreased with the increased fluoride exposure, whereas that the expression level of YAP1 protein in the nucleus increased. Fluoride inhibited the proliferation of ATDC5 cells, induced oxidative stress, inhibited the activity of the Hippo pathway, and eventually led to cartilage matrix degradation.

Involvement of increased expression of chemokine C–C motif chemokine 22 (CCL22)/CC chemokine receptor 4 (CCR4) in the inflammatory injury and cartilage degradation of chondrocytes

CCL22, which could induce chondrocyte apoptosis, was identified to be overexpressed in damaged cartilage. This study was conducted with the aim of investigating the effects of CCL22 interference on chondrocyte injury. The osteoarthritis model was established by stimulating chondrocytes with LPS. The expressions of CCL22, CCR4, matrix metallopeptidase (MMP) 3, MMP9, MMP13, (a disintegrin and metalloproteinase with thrombospondin-like motifs) ADAMTS-4, collagen II and inflammatory cytokines were measured using quantitative reverse transcription PCR (RT-qPCR) and western blot. Besides, immunoprecipitation (IP) was employed to verify the binding of CCL22 and CCR4. After CCR4 was overexpressed, cell viability was observed using Cell Counting Kit-8 (CCK-8). Additionally, cell apoptosis as well as its related proteins was detected by TUNEL and western blot, respectively. ng What's more, glycosaminoglycan (GAG) level was detected using GAG kits. CCL22 and CCR4 expression increased noticeably in LPS-stimulated ATDC5 chondrocytes. CCL22 inhibition could suppress the expression of CCR4 in LPS-induced ATDC5 cells. Likewise, CCL22 inhibition could revive the activation of LPS-induced ATDC5 cells by regulating CCR4. In addition, CCL22 knockdown alleviated inflammatory response and cell apoptosis through CCR4. Furthermore, the cartilage degradation of ADTC5 cells could be relieved by CCL22 silence via regulating CCR4. CCL22/CCR4 expression was increased in osteoarthritic cartilage injury and participated in the inflammation and cartilage degradation of chondrocytes.

Zinc finger protein A20 regulates the development and progression of osteoarthritis by affecting the activity of NF-κB p65

Objective To investigate the role of Zinc finger protein A20 in osteoarthritis (OA) by regulating NF-κB p65. Methods A20, MMP1, MMP13 and IL-1β expressions in human OA cartilage samples were detected by qRT-PCR. IL-1β-induced chondrocyte was treated with A20 lentivirus activation particle, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor) with/without A20 siRNA. IL-6, TNF-α, and PGE2 levels were measured by ELISA, and NO production by Greiss reaction. Destabilization of the medial meniscus (DMM) surgery was used to construct the OA models, followed by injection of A20 adenovirus. MMP1 and MMP13 expression was measured by immunohistochemistry. The mRNA and protein expression were performed by qRT-PCR and western blotting, respectively. Results A20 was down-regulated in human OA cartilage samples, and negatively correlated with the expressions of MMP1, MMP13 and IL-1β. The IL-1β-induced chondrocyte manifested decreased A20 with increased NF-κB p65 activity. A20 overexpression suppressed the NF-κB p65 activity in IL-1β-induced chondrocyte. Furthermore, PDTC decreased IL-1β-induced chondrocyte apoptosis with the upregulated COL1A1, COL2A1, COL10A1 and ACAN, as well as the down-regulated MMP1, MMP13, COX2, iNOS, IL-6, TNF-α, NO and PGE2, which was reversed by A20 siRNA. In vivo, OA mice gained higher OARSI score and Mankin’s score, exhibited up-regulations of MMP1 and MMP13, and decreased NF-κB p65 activity, which was improved after injection of A20 adenovirus. Conclusion A20 was reduced in OA cartilage samples, and its overexpression, by suppressing the activity of NF-κB p65, could improve IL-1β-induced chondrocyte degradation and apoptosis in vitro, as well as mitigate the inflammation in OA mice.

Excessive mechanical stretch‑mediated osteoblasts promote the catabolism and apoptosis of chondrocytes via the Wnt/β‑catenin signaling pathway.

Excessive biomechanical loading is considered an important cause of osteoarthritis. Although the mechanical responses of chondrocytes and osteoblasts have been investigated, their communication during mechanical loading and the underlying molecular mechanisms are not yet fully known. The present study investigated the effects of excessive mechanically stretched osteoblasts on the metabolism and apoptosis of chondrocytes, and also assessed the involvement of the Wnt/β-catenin signaling pathway. In the present study, rat chondrocytes and osteoblasts were subjected to mechanical tensile strain, and an indirect chondrocyte-osteoblast co-culture model was established. Reverse transcription-quantitative PCR and western blotting were performed to determine the expression levels of genes and proteins of interest. An ELISA was performed to investigate the levels of cytokines, including matrix metalloproteinase (MMP) 13, MMP 3, interleukin-6 (IL-6) and prostaglandin E2 (PG E2), released from osteoblasts. Flow cytometry was performed to detect the apoptosis of chondrocytes exposed to stretched osteoblast conditioned culture medium. The levels of MMP 13, IL-6 and PG E2 increased significantly in the supernatants of stretched osteoblasts compared with the un-stretched group. By contrast, the mRNA expression levels of Collagen 1a and alkaline phosphatase were significantly decreased in osteoblasts subjected to mechanical stretch compared with the un-stretched group. The mRNA expression level of Collagen 2a was significantly decreased, whereas the expression levels of MMP 13 and a disintegrin and metalloproteinase with thrombospondin-like motifs 5 were significantly increased in chondrocytes subjected to mechanical stretch compared with the un-stretched group. In the co-culture model, the results indicated that excessive mechanically stretched osteoblasts induced the catabolism and apoptosis of chondrocytes, which was partly inhibited by Wnt inhibitor XAV-939. The results of the present study demonstrated that excessive mechanical stretch led to chondrocyte degradation and inhibited osteoblast osteogenic differentiation; furthermore, excessive mechanically stretched osteoblasts induced the catabolism and apoptosis of chondrocytes via the Wnt/β-catenin signaling pathway.

Activation of autophagy contributes to the protective effects of lycopene against oxidative stress-induced apoptosis in rat chondrocytes.

Oxidative stress is commonly associated with osteoarthritis (OA). Lycopene (LYC), a natural carotenoid compound, is an effective antioxidant with potential cartilage-protecting actions. However, how it affects hydrogen peroxide (H2 O2 )-induced damage to the cartilage is unclear. In this study, an in vitro oxidative stress model was developed via treating primary chondrocytes with H2 O2 . Western blot, immunohistochemistry, and quantitative RT-PCR (qRT-PCR) were used to assess the levels of related factors. Reactive oxygen species (ROS) and apoptosis levels were analyzed by the use of appropriate probes and flow cytometry. The expression and activity of stress-specific enzymes (malondialdehyde, superoxide dismutase, and catalase) were also assessed. The role of autophagy was explored by using the inhibitor, 3-methyladenine (3-MA), as well as monodansylcadaverine staining, western blotting, and red fluorescent protein-green fluorescent protein-light chain 3 lentivirus infection. The result showed LYC exerted significant chondrocyte-protective effects, including reduced inflammation and chondrocyte degradation, increased chondrocyte proliferation, apoptosis inhibition, and reduced ROS production. LYC could effectively induce autophagy in the H2 O2 treatment group, and this effect could be attenuated by 3-MA. In terms of mechanism, LYC played a role in inhibiting MAPK and PI3K/Akt/NF-κB axis, which down-regulates levels of mTOR and had a potential therapeutic significance for cartilage degeneration.

Excavating bioactivities of nanozyme to remodel microenvironment for protecting chondrocytes and delaying osteoarthritis

Osteoarthritis (OA) is the main cause of disability in the elderly. Effective intervention in the early and middle stage of osteoarthritis can greatly prevent or slow down the development of the disease, and reduce the probability of joint replacement. However, there is to date no effective intervention for early and middle-stage OA. OA microenvironment mainly destroys the balance of oxidative stress, extracellular matrix synthesis and degradation of chondrocytes under the joint action of biological and mechanical factors. Herein, hollow Prussian blue nanozymes (HPBzymes) were designed via a modified hydrothermal template-free method. The aim of this study was to investigate the effects of HPBzymes on chondrocytes and the progression of OA. The intrinsic bioactivities of HPBzymes were excavated in vitro and in vivo, remodeling microenvironment for significantly protecting chondrocytes and delaying the progression of traumatic OA by inhibiting reactive oxygen species (ROS) and Rac1/nuclear factor kappa-B (NF-κB) signaling in a rat model. HPBzyme significantly diminished interleukin (IL)-1β-stimulated inflammation, extracellular matrix degradation, and apoptosis of human chondrocytes. HPBzyme attenuated the expression of Rac1 and the ROS levels and prevented the release and nuclear translocation of NF-κB. Deeply digging the intrinsic bioactivities of nanozyme with single component to remodel microenvironment is an effective strategy for ROS-associated chronic diseases. This study reveals that excavating the bioactivities of nanomedicine deserves attention for diagnosis and treatment of severe diseases.

CircSERPINE2 weakens IL-1β-caused apoptosis and extracellular matrix degradation of chondrocytes by regulating miR-495/TGFBR2 axis.

The dysregulated circular RNAs (circRNAs) are relevant to the development of osteoarthritis (OA). The circRNA serpin family E member 2 (circSERPINE2) is dysregulated in OA, while the role and mechanism of circSERPINE2 in OA are largely unknown. The aim of our research is to explore how and whether circSERPINE2 regulates interleukin-1β (IL-1β)-caused chondrocyte damage in OA. In the present study, the chondrocytes (CHON-001 cells) were exposed to IL-1β to mimic the injury in OA. CircSERPINE2, microRNA-495 (miR-495) and transforming growth factor-β receptor 2 (TGFBR2) abundances were detected via quantitative reverse-transcription polymerase chain reaction (qRT-PCR) or Western blot. Cell apoptosis was assessed via viability, apoptotic rate and caspase-3 activity. Extracellular matrix was investigated by levels of Sry-type high-mobility-group box 9 (SOX9), collagen type II α 1 (COL2A1) and Aggrecan using Western blot. The interaction among circSERPINE2, miR-495 and TGFBR2 was assessed via dual-luciferase reporter analysis and RNA immunoprecipitation (RIP). The results showed that circSERPINE2 expression was reduced in OA patients and IL-1β-treated chondrocytes. CircSERPINE2 overexpression mitigated IL-1β-caused apoptosis and extracellular matrix degradation. miR-495 was targeted by circSERPINE2 and up-regulated in OA patients and IL-1β-treated chondrocytes. miR-495 up-regulation reversed overexpression of circSERPINE2-mediated inhibition of apoptosis and extracellular matrix degradation. TGFBR2 was targeted by miR-495 and lowly expressed in OA patients and IL-1β-treated chondrocytes. CircSERPINE2 could mediate TGFBR2 expression by binding with miR-495. As a conclusion, circSERPINE2 attenuated IL-1β-caused apoptosis and extracellular matrix degradation of chondrocytes by regulating miR-495/TGFBR2 axis, indicating a new target for OA treatment.

Ball-Bearing-Inspired Polyampholyte-Modified Microspheres as Bio-Lubricants Attenuate Osteoarthritis.

Osteoarthritis, a lubrication dysfunction related disorder in joint, is characterized by articular cartilage degradation and joint capsule inflammation. Enhancing joint lubrication, combined with anti-inflammatory therapy, is considered as an effective strategy for osteoarthritis treatment. Herein, based on the ball-bearing-inspired superlubricity and the mussel-inspired adhesion, a superlubricated microsphere, i.e., poly (dopamine methacrylamide-to-sulfobetaine methacrylate)-grafted microfluidic gelatin methacrylate sphere (MGS@DMA-SBMA), is developed by fabricating a monodisperse, size-uniform microsphere using the microfluidic technology, and then a spontaneously modified microsphere with DMA-SBMA copolymer by a one-step biomimetic grafting approach. The microspheres are endowed with enhanced lubrication due to the tenacious hydration layer formed around the charged headgroups (-N+ (CH3 )2 - and -SO3- ) of the grafted poly sulfobetaine methacrylate (pSBMA), and simultaneously are capable of efficient drug loading and release capability due to their porous structure. Importantly, the grafting of pSBMA enables the microspheres with preferable properties (i.e., enhanced lubrication, reduced degradation, and sustained drug release) that are highly desirable for intraarticular treatment of osteoarthritis. In addition, when loaded with diclofenac sodium, the superlubricated microspheres with excellent biocompatibility can inhibit the tumor necrosis factor α (TNF-α)-induced chondrocyte degradation in vitro, and further exert a therapeutic effect toward osteoarthritis in vivo.

Ruboxistaurin maintains the bone mass of subchondral bone for blunting osteoarthritis progression by inhibition of osteoclastogenesis and bone resorption activity

Osteoarthritis (OA) is a common degenerative disease with a series of changes occurring in aging cartilage, such as increased oxidative stress, decreased markers of healthy cartilage and alterations in the autophagy pathway. And increasing evidence indicates that osteoarthritis affects the whole joint, including both cartilage and subchondral bone. The agents that can effectively suppress chondrocyte degradation and subchondral bone deterioration are crucial for the prevention and treatment of OA. Ruboxistaurin (RU), an orally active protein kinase C inhibitor, can reduce macrophage adhesion to endothelial cells and relieve the local inflammation when applicating in diabetes and kinds of aging-related vasculopathy, which were realized by its effects on decreasing inflammatory cytokines' expression and increasing cell anti-oxidative stress ability. However, whether ruboxistaurin protects against OA remains unknown. In this study, we investigated the therapeutic effects of ruboxistaurin in an anterior cruciate ligament transection (ACLT)-induced OA model by preventing the bone mass loss of subchondral bone. We found that ruboxistaurin can effectively alleviate ACLT-induced osteoarthritis, as demonstrated by the phenomenon of correcting pathological bone loss caused by osteoclasts overactivated in the early stage of osteoarthritis and protecting against articular cartilage degeneration. Moreover, we found that ruboxistaurin inhibited osteoclast formation and resorption activity by suppressing the expressions of osteoclast‐related genes and (PKCδ/MAPKs) signaling cascade. Taken together, these results show that ruboxistaurin may be a potential therapeutic agent for rescuing abnormal subchondral bone deterioration and cartilage degradation in OA and reverses the vicious cycle related to osteoarthritis.

Jumonji domain containing-3 (JMJD3)inhibition attenuatesIL-1β-inducedchondrocytes damage in vitro and protects osteoarthritis cartilage in vivo.

This study aimed to explore the effects and relative mechanism of JMJD3 on knee osteoarthritis (OA). In this study, we first analyzed the expression of JMJD3 in OA cartilage using western blot and immunohistochemistry. In an in vitro study, the effects of GSK-J4, JMJD3 inhibitor, on ATDC-5 chondrocytes were evaluated by CCK-8 assay. Real-time PCR and western blot were used to examine the inhibitory effect of GSK-J4 on the inflammation and ECM degradation of chondrocytes. NF-κB p65 phosphorylation and nuclear translocation were measured by western blot and immunofluorescence. In the animal study, twenty mice were randomized into four experimental groups: sham group, DMM-induced OA + DMSO group, OA + low-dose GSK-J4 group, and OA + high-dose GSK-J4 group. After the treatment, hematoxylin-eosin and safranin O/fast green staining were used to evaluate cartilage degradation of knee joint, with OARSI scores for quantitative assessment of cartilage damage. Our results revealed that JMJD3 was overexpressed in OA cartilage and GSK-J4 could suppress the IL-1β-induced production of pro-inflammatory cytokines and catabolic enzymes, including IL-6, IL-8, MMP-9 and ADAMTS-5. Consistent with these findings, GSK-J4 could inhibit IL-1β-induced degradation of collagen II and aggrecan. Mechanistically, GSK-J4 dramatically suppressed IL-1β-stimulated NF-κB signal pathway activation. In vivo, GSK-J4 prevented cartilage damage in mouse DMM-induced OA model. This study elucidates the important role of JMJD3 in cartilage degeneration in OA, and our results indicate that JDJM3 may become a novel therapeutic target in OA therapy.

The promoting effect of MMP13 on mediating the development of HFLS-RA by the target of miR-19a through IL-17 signaling pathway.

By investigating the expression profiles of miR-19a andmetalloproteinases (MMP13) in human fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) and HFL cells lines, this study intends to confirm the directly target connection between them and reveal the effect of suppressing MMP13 on HLFS-RA migration, invasion and apoptosis. After screening the abnormal expressed messenger RNAs and microRNAs in synovial tissues of patients with RA, the underlying pathway was determined by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The HFLS-RA cell line was transfected for the following experiments with pcDNA3.1(+) served as vector. The directly target association between miR-19a and MMP13 was confirmed by Luciferase reporter assay. Microarray analysis suggested that MMP13 was upregulated while miR-19a was downregulated in HFLS of RA tissues compared with the healthy control group. MMP13 was related to many proteins in protein-protein interaction network, which might be the main influencing factor of RA. KEGG pathway analysis identified that interleukin (IL)-17 pathway was activated in the regulation of MMP13 in the development of RA. Through observing the alteration of luciferase activity, miR-19a could indeed bind to the 3'UTR of the downstream of MMP13, the target association was then confirmed. The proliferation and invasion of HFLS-RA were promoted by overexpressing MMP13 protein. miR-19a could function as a suppressor of MMP13 and thereby retard the severity of RA. The results showed that miR-19a could regulate the expression of MMP13 in HFLS-RA by mediating the proliferation and invasion of HFLS-RA through IL-17 signaling pathway, thereby participating in the degradation of chondrocytes in the progression of RA.

LncRNA MEG3 Inhibits the Degradation of the Extracellular Matrix of Chondrocytes in Osteoarthritis via Targeting miR-93/TGFBR2 Axis.

As a degenerative joint disease, osteoarthritis (OA) is characterized by articular cartilage degradation. Long noncoding RNAs (lncRNAs) act critical roles in the regulation of OA development, including affecting the proliferation, apoptosis, extracellular matrix (ECM) degradation, and inflammatory response of chondrocytes. The current study's aim was to investigate the regulatory function and the underlying molecular mechanism of lncRNA MEG3 in ECM degradation of chondrocytes in OA. In the current study, chondrocytes were induced by interleukin-1β (IL-1β) to simulate OA condition, and further assessed cell viability, lncRNA MEG3 and miR-93 expression levels. Overexpression or knockdown of lncRNA MEG3 in chondrocytes treated with IL-1β were performed to investigate the function of MEG3 in regulating cell proliferation, apoptosis and ECM degradation using EdU assay, flow cytometry, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blot. The interaction between MEG3 and miR-93 was assessed using qRT-PCR. Furthermore, overexpression of miR-93 was performed as recovery experiment to explore the functional mechanism of MEG3. MEG3 was significantly downregulated in chondrocytes treated with IL-1β, whereas miR-93 was upregulated concomitantly. Overexpression of MEG3 induced the proliferation, suppressed the apoptosis, and relieved the degradation of ECM in IL-1β-induced chondrocytes. By contrast, knockdown of MEG3 suppressed the proliferation, promoted the apoptosis, and aggravated ECM degradation in IL-1β induced chondrocytes. In addition, MEG3 was found to relieve the inhibitive expression of TGFBR2 as a competitive endogenous RNA (ceRNA) of miR-93, and then activated transforming growth factor-β (TGF-β) signaling pathway, regulated chondrocytes ECM degradation in IL-1β induced chondrocytes subsequently. LncRNA MEG3 targeted miR-93/TGFBR2 axis, regulated the proliferation, apoptosis and ECM degradation of chondrocytes in OA.

Ginsenoside Rd inhibits IL-1β-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1β, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1β by suppressing the increase in IL-1β, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1β-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1β to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1β-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Frontline Science: Reprogramming COX-2, 5-LOX, and CYP4A-mediated arachidonic acid metabolism in macrophages by salidroside alleviates gouty arthritis.

Cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and cytochrome P450 (CYP) 4A-mediated arachidonic acid (AA) metabolism play an essential role in human inflammatory disorders. Blocking COX-2 pathway would shunt AA metabolism to the other pathway, thereby decreasing the efficacy and exacerbating adverse effects. Here we demonstrated that reprogramming COX-2, 5-LOX, and CYP4A-mediated AA metabolism in macrophages by salidroside (Sal) ameliorates monosodium urate (MSU) crystal-induced inflammation. Compared with COX-2 inhibitor celecoxib, Sal (80mg/kg) presented a superior anti-arthritic profile in MSU crystal-treated rats, accompanied with the decreased expression of COX-2, 5-LOX, and CYP4A and production of prostaglandin E2 (PGE2 ), leukotriene B4 (LTB4 ), and 20-hydroxyeicosatetraenoic acid (20-HETE) in the synovial fluid macrophages. Sal decreased representative M1 marker (iNOS and CD86, etc.) expression and M1 cytokine (TNF-α and IL-1β) production, whereas it increased M2 marker (CD206 and Arg-1) expression and M2 cytokine (TGF-β and IL-10) production. The injection of conditioned medium from MSU crystal-treated macrophages into the ankle joint of rats reproduced the gouty inflammation, which was attenuated by Sal. Mechanistically, down-regulation of COX-2, 5-LOX, and CYP4A in the RAW264.7 and NR8383 macrophages by Sal skewed macrophage polarization away from the M1 phenotype, and thereby prevented neutrophil migration and chondrocyte degradation with STAT1 and NF-κB inactivation. Conversely, overexpression of COX-2, 5-LOX, CYP4A or STAT1, or exogenous addition of IL-1β or TNF-α partially abolished these effects. Together, inhibition of COX-2, 5-LOX, and CYP4A in macrophages by Sal ameliorates MSU crystal-induced inflammation through decreasing TNF-α and IL-1β production, and may serve as a novel therapeutic strategy.

Transcription factor 7-like 2 controls matrix degradation through nuclear factor κB signaling and is repressed by microRNA-155 in nucleus pulposus cells.

TCF7L2, a key transcription factor in the canonical Wnt pathway, plays a vital role in the matrix degradation of chondrocytes. However, it is unknown whether TCF7L2 is important in the matrix metabolism of inner gel-like nucleus pulposus (NP) cells; thus, the aim of this study was to explore the effect and mechanism of TCF7L2 in this process. Western blotting and immunofluorescence analyses were used to observe TCF7L2 expression in rat and human NP tissues. Real-time PCR and western blotting were performed to detect the expression of TCF7L2 stimulated by inflammatory cytokines. Dual luciferase reporter assay, real-time PCR, western blotting and knockdown experiments were performed to demonstrate the role of NF-κB signaling in matrix regulation by TCF7L2 and the regulation of TCF7L2 by miR-155 in intervertebral disc degeneration. TCF7L2 is present in rat and human NP tissues and is expressed in the nucleus of NP cells. TCF7L2 is refractory to stimulation of rat and human NP cells with the inflammatory cytokines TNF-α and IL-1β, in contrast to the results in other cell types. Loss-of-function experiments using TCF7L2 siRNA or lentiviral shTCF7L2 showed that TCF7L2 knockdown suppresses matrix degradation through p65/NF-κB signaling in the absence and presence of TNF-α. In addition, TCF7L2 expression is repressed by miR-155 overexpression and promoted by miR-155 inhibition. Overall, these results demonstrate that the suppression of TCF7L2, which is modulated by miR-155, inhibits matrix degradation through p65/NF-κB signaling. TCF7L2 suppression may have therapeutic potential in intervertebral disc degeneration.

Oxymatrine exerts protective effects on osteoarthritis via modulating chondrocyte homoeostasis and suppressing osteoclastogenesis.

Osteoarthritis (OA) is a common degenerative disease characterized by the progressive destruction both articular cartilage and the subchondral bone. The agents that can effectively suppress chondrocyte degradation and subchondral bone loss are crucial for the prevention and treatment of OA. Oxymatrine (OMT) is a natural compound with anti‐inflammatory and antitumour properties. We found that OMT exhibited a strong inhibitory effect on LPS‐induced chondrocyte inflammation and catabolism. To further support our results, fresh human cartilage explants were treated with LPS to establish an ex vivo degradation model, and the results revealed that OMT inhibited the catabolic events of LPS‐stimulated human cartilage and substantially attenuated the degradation of articular cartilage ex vivo. As subchondral bone remodelling is involved in OA progression, and osteoclasts are a unique cell type in bone resorption, we investigated the effects of OMT on osteoclastogenesis, and the results demonstrated that OMT suppresses RANKL‐induced osteoclastogenesis by suppressing the RANKL‐induced NFATc1 and c‐fos signalling pathway in vitro. Further, we found that the anti‐inflammatory and anti‐osteoclastic effects of oxymatrine are mediated via the inhibition of the NF‐κB and MAPK pathways. In animal studies, OMT suppressed the ACLT‐induced cartilage degradation, and TUNEL assays further confirmed the protective effect of OMT on chondrocyte apoptosis. MicroCT analysis revealed that OMT had an attenuating effect on ACLT‐induced subchondral bone loss in vivo. Taken together, these results show that OMT interferes with the vicious cycle associated with OA and may be a potential therapeutic agent for abnormal subchondral bone loss and cartilage degradation in osteoarthritis.