Chondroprotective Effects Research Articles

Herbal Remedies as Potential in Cartilage Tissue Engineering: An Overview of New Therapeutic Approaches and Strategies.

It is estimated that by 2023, approximately 20% of the population of Western Europe and North America will suffer from a degenerative joint disease commonly known as osteoarthritis (OA). During the development of OA, pro-inflammatory cytokines are one of the major causes that drive the production of inflammatory mediators and thus of matrix-degrading enzymes. OA is a challenging disease for doctors due to the limitation of the joint cartilage’s capacity to repair itself. Though new treatment approaches, in particular with mesenchymal stem cells (MSCs) that integrate the tissue engineering (TE) of cartilage tissue, are promising, they are not only expensive but more often do not lead to the regeneration of joint cartilage. Therefore, there is an increasing need for novel, safe, and more effective alternatives to promote cartilage joint regeneration and TE. Indeed, naturally occurring phytochemical compounds (herbal remedies) have a great anti-inflammatory, anti-oxidant, and anabolic potential, and they have received much attention for the development of new therapeutic strategies for the treatment of inflammatory diseases, including the prevention of age-related OA and cartilage TE. This paper summarizes recent research on herbal remedies and their chondroinductive and chondroprotective effects on cartilage and progenitor cells, and it also emphasizes the possibilities that exist in this research area, especially with regard to the nutritional support of cartilage regeneration and TE, which may not benefit from non-steroidal anti-inflammatory drugs (NSAIDs).

Anti-Inflammatory and Chondroprotective Effects of Vanillic Acid and Epimedin C in Human Osteoarthritic Chondrocytes.

In osteoarthritis (OA), inhibition of excessively expressed pro-inflammatory cytokines in the OA joint and increasing the anabolism for cartilage regeneration are necessary. In this ex-vivo study, we used an inflammatory model of human OA chondrocytes microtissues, consisting of treatment with cytokines (interleukin 1β (IL-1β)/tumor necrosis factor α (TNF-α)) with or without supplementation of six herbal compounds with previously identified chondroprotective effect. The compounds were assessed for their capacity to modulate the key catabolic and anabolic factors using several molecular analyses. We selectively investigated the mechanism of action of the two most potent compounds Vanillic acid (VA) and Epimedin C (Epi C). After identification of the anti-inflammatory and anabolic properties of VA and Epi C, the Ingenuity Pathway Analysis showed that in both treatment groups, osteoarthritic signaling pathways were inhibited. In the treatment group with VA, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling was inhibited by attenuation of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) phosphorylation. Epi C showed a significant anabolic effect by increasing the expression of collagenous and non-collagenous matrix proteins. In conclusion, VA, through inhibition of phosphorylation in NF-κB signaling pathway and Epi C, by increasing the expression of extracellular matrix components, showed significant anti-inflammatory and anabolic properties and might be potentially used in combination to treat or prevent joint OA.

AB0068 NOVEL CHONDROPROTECTIVE AND ANTI-INFLAMMATORY EFFECTS OF THE SELECTIVE HUMAN MELANOCORTIN MC3 RECEPTOR AGONIST PG-990 ON SNAP ACTIVATED CHONDROCYTES

Background:Osteoarthritis (OA) is a degenerative joint disease that affects over 250 million people worldwide [1] with treatments focussing on the symptoms rather than the cause of the pathology [2, 3]. Thus, this degenerative joint disease requires novel treatment options [3, 4].Therefore, the melanocortin system [4] could provide a novel avenue to explore given its ability to exert anti-inflammatory effects and chondroprotection [5], although the receptor subtype involved is unclear.Objectives:This study aims to assess the chondroprotective and anti-inflammatory effects of the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and the selective human MC3 receptor agonist PG-990 on S-Nitroso-N-acetyl-DL-penicillamine (SNAP) activated chondrocytes.Methods:The human chondrocytic cell-line C-20/A4 was seeded at 25.0 x 106viable cells/ml (5 μl droplet was transferred into individual wells of a 96-well plate). Micromass cultures [6] were stimulated with SNAP (1.0 mM) and after 2h treated with Dexamethasone (1.0 μM), selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride (10.0 μg/ml) or selective human melanocortin MC3 receptor agonist PG-990 (10.0 μg/ml) for 6h. Cell viability was determined by MTT assay, Caspase -3 and -7 activity determined by Caspase-Glo 3/7 apoptosis assay. Glycosaminoglycan (GAG) content determined by alcian blue staining and anti-inflammatory heme-oxygenase-1 (HO-1) protein expression was determined by western blot. Data are expressed as Mean ±S.E.M ofn=4 samples repeated in triplicate. #p≤0.05vscontrol or *p≤0.05vsstimulus.Results:Cell viability analysis showed SNAP stimulation caused a maximal cell death of 23% (#p≤0.05), Dexamethasone, BMS-470539 dihydrochloride and PG-990 inhibited cell death by 2%, 98% and 129% respectively (*p≤0.05). SNAP stimulation caused a significant increase in Caspase -3 and -7 activity, which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 8%, 5% and 19% respectively (*p≤0.05). GAG content was significantly reduced by SNAP by 29% (#p≤0.05), which was inhibited by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by 1%, 3% and 14% respectively (*p≤0.05). SNAP also caused a significant decrease in HO-1 protein expression, which was increased by Dexamethasone, BMS-470539 dihydrochloride and PG-990 by a 1.0-fold, 1.1-fold and 2.1-fold increase respectively (*p≤0.05).Conclusion:The selective human melanocortin MC3 receptor agonist PG-990 exhibited enhanced chondroprotection and modulation of inflammatory and tissue destructive mediators following SNAP activation compared to Dexamethasone and the selective human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride. This suggests that melanocortin peptides display enhanced chondroprotective and anti-inflammatory effects at the MC3 receptor sub-type in this cell line.

Citrulline cooperatively exerts an anti-inflammatory effect on synovial cells with glucosamine and N-acetylglucosamine.

The aim of the present study was to evaluate the anti-inflammatory effects of citrulline (Cit), glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) on synovial cells, which are primarily involved in inflammatory joint diseases. The combined effect of Cit, GlcN and GlcNAc on synovial cell inflammation was assessed by measuring IL-1β-induced IL-6 production. GlcN and GlcNAc (0.5 mM each) alone did not suppress IL-6 production, whereas Cit (0.5 mM) did significantly suppress IL-6 production. Furthermore, the combined effect of Cit, GlcNAc and GlcN was examined; Cit + GlcN and Cit + GlcNAc significantly suppressed not only IL-6 production, but also phosphorylation of ERK1/2. Similarly, combination of GlcN + GlcNAc significantly suppressed IL-6 production and phosphorylation of ERK1/2. These observations suggest that among Cit, GlcNAc and GlcN, the combination of Cit with GlcN or GlcNAc exerts a synergistic anti-inflammatory effect on synovial cells, thereby possibly exhibiting chondroprotective effects and alleviating inflammatory joint diseases.

Exosomal miR-9-5p secreted by bone marrow-derived mesenchymal stem cells alleviates osteoarthritis by inhibiting syndecan-1.

Mesenchymal stem cells (MSCs) have been demonstrated to serve as targets for the treatment of osteoarthritis (OA) and exosomes derived from MSCs also display chondroprotective effects. This study aims to investigate the regulatory role of exosomal microRNA-9-5p (miR-9-5p) secreted by bone marrow-derived MSCs (BM-MSCs) on OA in a rat model induced by anterior cruciate ligament/medial collateral ligament transection. Luciferase reporter assay was conducted to verify the putative miR-9-5p binding sites to 3'UTR of syndecan-1 (SDC1). Additionally, an intra-articular injection of miR-9-5p carried by BM-MSC-derived exosomes or liposomes into rats with OA-like damage was performed to ascertain the role of exosomal miR-9-5p and a gain-of-function study of SDC1 was carried out to explore the potential mechanism in relation to SDC1. Subsequently, the expression of SDC1 was determined and the levels of inflammatory factors (IL-1, IL-6, TNF-α and CRP) and oxidative stress injury indicators (NO, MDA, iNOS, COX2 and SOD), the contents of AKP as well as the levels of OA-related factors (MMP-13, COMP and OCN) were measured. Injection of miR-9-5p-contained exosomes resulted in an alleviation of inflammation and OA-like damage, which was evidenced by downregulated levels of inflammatory factors, reduced oxidative stress injury and decreased OCN, MMP-13, COMP and AKP levels. As a target gene of miR-9-5p, the upregulation of SDC1 led to aggravation of inflammation and OA-like damage, which is opposite to exosomal miR-9-5p. To conclude, these findings suggest the anti-inflammatory and chondroprotective effects of BM-MSC-derived exosomal miR-9-5p on OA via regulation of SDC1.

Pulsed Electromagnetic Field Stimulation of Bone Healing and Joint Preservation: Cellular Mechanisms of Skeletal Response

The US FDA has approved pulsed electromagnetic fields (PEMFs) as a safe and effective treatment for nonunions of bone. Despite its clinical use, the mechanisms of action of electromagnetic stimulation of the skeleton have been elusive. Recently, cell membrane receptors have been identified as the site of action of PEMF and provide a mechanistic rationale for clinical use. This review highlights key processes in cell responses to PEMF as follows: (1) signal transduction through A2A and A3 adenosine cell membrane receptors and (2) dose-response effects on the synthesis of structural and signaling extracellular matrix (ECM) components. Through these actions, PEMF can increase the structural integrity of bone and cartilage ECM, enhancing repair, and alter the homeostatic balance of signaling cytokines, producing anti-inflammatory effects. PEMFs exert a proanabolic effect on the bone and cartilage matrix and a chondroprotective effect counteracting the catabolic effects of inflammation in the joint environment. Understanding of PEMF membrane targets, and of the specific intracellular pathways involved, culminating in the synthesis of ECM proteins and reduction in inflammatory cytokines, should enhance confidence in the clinical use of PEMF and the identification of clinical conditions likely to be affected by PEMF exposure.

Effects of Joint Lavage with Dimethylsulfoxide on LPS-Induced Synovitis in Horses-Clinical and Laboratorial Aspects.

Several studies in human and equine medicine have produced controversial results regarding the role of dimethylsulfoxide (DMSO) as a therapeutic agent. This study aimed to evaluate the effect of joint lavage with different DMSO concentrations on biomarkers of synovial fluid inflammation and cartilage degradation in joints with lipopolysaccharide (LPS)-induced synovitis. Twenty-six tibiotarsal joints of 13 horses were randomly distributed into four groups (lactated Ringer’s solution; 5% DMSO in lactated Ringer’s; 10% DMSO in lactated Ringer’s; and sham). All animals were evaluated for the presence of lameness, and synovial fluid analyses were performed at 0 h, 1 h, 8 h, 24 h, and 48 h (T0, T1, T8, T24, and T48, respectively). The white blood cell counts (WBC), total protein (TP), urea, prostaglandin E2 (PGE2), interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor-α (TNF-α), hyaluronic acid (HA), and chondroitin sulfate (CS) concentrations were measured. The WBC counts and PGE2, IL-1β, IL-6, and TP concentrations increased in all groups at T8 compared to baseline values (p < 0.05). At T48, only the 5% DMSO and 10% DMSO groups showed a significant decrease in WBC counts (p < 0.05). Furthermore, the 10% DMSO group had lower concentrations of PGE2 and IL-1β at T48 than at T8 (p < 0.05) and presented lower IL-6 levels than the5% DMSO and lactated Ringer’s groups at T24. All groups showed an increase in CS concentration after LPS-induced synovitis. Joint lavage with 10% DMSO in lactated Ringer’s has anti-inflammatory but not chondroprotective effects.

MicroRNA-675-3p regulates IL-1β-stimulated human chondrocyte apoptosis and cartilage degradation by targeting GNG5

Growing evidence has indicated that microRNAs (miRNAs) are modulators of osteoarthritis (OA) development and progression. In this study, we first evaluated the anti-apoptosis and chondroprotective effects of microRNA-675-3p (miR-675-3p) on interleukin-1β (IL-1β)-stimulated human chondrocytes. The overexpression of miR-675-3p inhibited apoptosis and cartilage matrix degradation and promoted cell proliferation in human chondrocytes. Target gene prediction and luciferase reporter assays suggested that G-protein subunit γ 5 (GNG5) may be the target gene of miR-675-3p. The overexpression of miR-675-3p inhibited IL-1β-stimulated chondrocyte apoptosis, and this effect was reversed by the overexpression of GNG5. Finally, we used bioinformatic tools and biological methods to show that the long noncoding RNA X-inactive specific transcript (lncRNA XIST) could bind to miR-675-3p, which affects the expression of GNG5 mRNA. Our findings may substantiate miR-675-3p as a new treatment for OA.

α-Bisabolol suppresses the inflammatory response and ECM catabolism in advanced glycation end products-treated chondrocytes and attenuates murine osteoarthritis

As a chronic musculoskeletal degeneration disease, osteoarthritis (OA) clinically manifests as joint pain, stiffness and a limited range of movement. OA has affected the life quality of at least one-tenth of the population but lacks satisfactory treatments. α-Bisabolol (BISA) is a small oily sesquiterpene alcohol widely found in essential oils of chamomile (Matricaria recutita), salvia and wood of Candeia and has multiple biological properties, particularly an anti-inflammatory effect. The purpose of this study is to assess the anti-inflammatory and chondroprotective effect of BISA in OA progression and explore its underlying mechanism. We isolated human chondrocytes and treated them with advanced glycation end products (AGEs) to imitate OA progression in vitro. BISA pretreatment suppressed the AGE-induced inflammatory reaction and extracellular matrix (ECM) degeneration by blocking nuclear factor kappa B (NF-κB), p38 and c-Jun N-terminal kinase (JNK) signaling. Moreover, a mouse destabilization of the medial meniscus (DMM) model was established by surgery to investigate BISA protection in vivo. BISA administration attenuated DMM-induced radiological and histopathological changes relative to the DMM group and resulted in lower OARSI scores. Taken together, the results of our study indicate the potential of BISA in OA therapy.

Polyphenols suppress inducible oxidative stress in human osteoarthritic and bovine chondrocytes

Reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in chondrocyte senescence and cartilage aging, pathogenesis of osteoarthritis (OA), and rheumatoid arthritis. Naturally occurring polyphenolic compounds (PPCs), such as curcumin (turmeric), resveratrol (grape), and epigallocatechin-3-gallate (EGCG) (green tea), have been known for their anti-inflammatory and chondroprotective effects. However, the potential protective effects of these PPCs against oxidative stress in chondrocytes are unclear. To investigate this, bovine articular chondrocytes and human osteoarthritic chondrocytes were pre-treated with PPCs at varying concentrations, and then exposed to hydrogen peroxide (H2O2) as an ROS inducer or S-nitroso-N-acetylpenicillamine (SNAP) as a NO donor. Alternatively, chondrocytes were co-treated with polyphenols and H2O2. Intracellular ROS/NO were measured using a fluorescent dye technique (H2DCF-DA for ROS; DAF-FM for NO). Our findings showed that PPC pre-/co-treatment inhibited both H2O2-induced ROS and SNAP-induced NO at different concentrations in both bovine chondrocytes and human osteoarthritic chondrocytes. Curcumin also increased glutathione peroxidase activity in the presence of H2O2 in bovine chondrocytes. Taken together, these findings indicate that PPCs are capable of suppressing oxidative stress- induced responses in chondrocytes, which may have potential therapeutic value for OA clinical application.

Standardized extract of Olea europaea leaves suppresses pro-inflammatory cytokines through regulating redox signaling pathways in human chondrocytes

Purpose: The main objective of this study was to investigate the anti-inflammatory, chondroprotective and redox modulatory effects of standardized Olea europaea L. (olive) leaf extract (ZeyEX®) in human osteoarthritic chondrocytes (OACs).

G-protein receptor kinase 2 inhibition attenuates osteoarthritis progression and promotes parathyroid hormone chondroprotection

Purpose: This study aimed to evaluate the role of GRK2 inhibition in PTH1R resensitization and PTH-mediated chondroprotection in OA. Osteoarthritis (OA) is a progressive degenerative joint disease, highlighted by chondrocyte hypertrophy, cartilage degeneration, subchondral bone remodeling, and synovial inflammation. Current treatments are limited to palliative pain management therapies with little potential to prevent further disease progression. Parathyroid hormone (PTH) has demonstrated chondroprotective and chondroregenerative effects in OA mice.

Proteoglycan 4 expression by superficial zone chondrocytes is regulated in part by JNK and P38 in vitro

Purpose: Osteoarthritis (OA) is a degenerative disease that affects joint homeostasis leading to cartilage degeneration. Inflammatory mediators, including interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), are key factors in this catabolic process that are known to activate the mitogen-activated protein kinases (MAPKs) and have been shown to play a central role in mediating this degradative response by OA chondrocytes. However, these studies typically use chondrocytes from full-thickness cartilage. A study by Rosenzweig et al. showed that the temporal and spatial expression of phosphorylated MAPKs in response to cartilage injury was zone-specific, and observed a chondroprotective effect of activation of the MAPK jun amino-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) in the superficial zone (SZ). The purpose of this study was to investigate the role of JNK and p38 in regulating key aspects of the SZ phenotype including cell morphology, actin polymerization status, expression of the chondroprotective SZ molecule proteoglycan 4 (PRG4), and localization of yes-associated protein and transcriptional co-activator with PDZ-binding motif, which we have previously shown to regulate PRG4 expression in SZ chondrocytes (SZC). Methods: Bovine chondrocytes were isolated from the SZ of the metacarpo-phalangeal joint of calves aged 6-9 months by sequential digestion of the tissue in 0.5% protease (45 minutes) followed by 0.1% collagenase (14-17 hours). SZC were seeded in monolayer in high glucose DMEM supplemented with ITS, 100nM dexamethasone, 40μg/mL proline, 100mM pyruvate, 100μg/mL ascorbic acid. After 24 hours of culture, SZC were treated with either SP600125 (JNK inhibitor (JKNi); 10μM) or SB203580 (p38 inhibitor (p38i); 5μM) and the effects on the SZ phenotype (cell shape, actin polymerization status, and PRG4 expression, YAP/TAZ localization) were assessed after 24 hours of treatment. Cell shape was evaluated by measuring cell circularity and area using Image J software analysis. Actin polymerization status was assessed by visualizing globular and filamentous actin by confocal microscopy and determining the ratio of globular to filamentous actin, which was extracted by differential triton extraction and separated by SDS-PAGE. PRG4 mRNA levels was determined by RT-PCR. YAP/TAZ localization was assessed by fractionating the cellular cytoplasmic and nuclear components (NE-PER Nuclear and Cytoplasmic Extraction Kit; ThermoFisher Scientific) and evaluating them by SDS-PAGE followed by western blot. Experiments were repeated with SZC from 3 independent cell isolations. Statistical analysis was performed using Student’s T-test with significance assigned as p<0.05. Results: SZC at 24 hours maintain their phenotype as demonstrated by their spindle cell shape and secretion of PRG4. To determine whether JNKi and p38i affect chondrocyte morphology and actin organization, SZC were treated with JNKi and p38i and cell shape and actin polymerization status was assessed. Treatment with JNKi did not significantly affect cell area (N=3; p=0.43) but resulted in an increase in cell circularity (N=3; p=0.047). No changes in actin organization were observed with confocal microscopy, and no significant changes in the G-/F-actin ratio were observed (N=4; p=0.5) with JNKi treatment. Treatment with p38i did not affect cell area (N=3; p=0.13) or circularity (N=3; p=0.91). Similarly, no changes in actin organization were observed with confocal microscopy, and no significant changes in the G-/F-actin ratio (N=4; p=0.49) were observed with p38i treatment. Despite no change in actin polymerization status with treatment, a decrease in nuclear TAZ was observed with JNKi (N=5; p=0.03) and p38i (N=4; p=0.009) treatment. No change in levels of cytoplasmic YAP was observed with JNKi treatment; however, treatment with the p38i resulted in an increase in cytoplasmic YAP (N=4; p=0.02). PRG4 mRNA levels decreased with JNKi (N=3; p<0.0001) and p38i (N=3; p<0.001) treatment. Conclusions: The data suggests that JNK and p38 regulates TAZ nuclear localization and PRG4 expression without affecting actin cytoskeleton status. Regulation of the SZ phenotype is complex and likely results from the convergence of several major regulatory pathways. While actin polymerization and nuclear localization of YAP/TAZ have been shown to regulate PRG4 expression, JNK and p38 also appear to regulate PRG4 expression and YAP/TAZ localization; however it has yet to be determined whether JNK and p38 act downstream of actin polymerization or independent of actin polymerization. Understanding the regulatory mechanisms governing the SZ phenotype and the expression of the chondroprotective molecule PRG4 may provide insight into therapeutic treatments to delay the onset or progression of OA.

ALGX‐XC20, a Novel Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonist Attenuates Joint Pain and Articular Cartilage Damage in an Osteoarthritis Rat Model

Currently, there are no approved disease‐modifying drugs for osteoarthritis (OA). OA patients depend upon analgesic drugs such as nonsteroidal anti‐inflammatory drugs (NSAIDs) and opioid analgesics for pain management. Despite existing therapies, pain relief in OA remains largely inadequate. Recently, the functional role for TRPA1 receptor‐channels which are expressed on nociceptor sensory nerves and other non‐neuronal cells within the joint has been identified, although studies of the role of TRPA1 in the pathogenesis of OA pain are limited. TRPA1 represents a promising target for the development of novel therapeutic agent for managing OA pain. This study investigated the effects of ALGX‐XC20, a highly selective and potent TRPA1 antagonist with a long in‐vivo half‐life on pain behaviors and cartilage damage in a rat model of monosodium iodoacetate (MIA)‐induced OA. The analgesic effect of a single dose (30 mg/kg; oral) of ALGX‐XC20 was evaluated for a period of 14 days post‐OA induction and compared to celecoxib (30 mg/kg; oral), a selective cyclooxygenase‐2 (COX‐2) inhibitor by using pain‐related behavior assessments, including hindlimb weight distribution and von Frey tests. The histological scoring of cartilage damage was also determined according to the Mankin grading system. The MIA‐induced OA rat group displayed a large reduction in the weight bearing distribution on the ipsilateral hindlimb (p&lt;0.01–0.05) and hind paw mechanical withdrawal thresholds compared to saline control group, which was maintained for 14 days. The MIA‐evoked reduction in weight bearing distribution was significantly reversed (~50%) in the MIA‐induced group treated with ALGX‐XC20 compared to the MIA‐treated group for the 2‐week duration, while the antiallodynic effect of ALGX‐XC20 was less pronounced. In addition, histopathology studies showed that ALGX‐XC20 was also effective in reducing the severe cartilage damage in the MIA‐injected knee joint as shown by a significant improvement in the Mankin score in the ALGX‐XC20 treated group at 14 days (p&lt;0.05). The magnitude of the effect on spontaneous weight bearing behavior due to ALGX‐XC20 treatment is closely comparable to the effects observed for the MIA‐celecoxib group and reported for TRPA1‐KO animals. The positive analgesic effects in this OA model due to pharmacological inhibition of TRPA1 by ALGX‐XC20 occur without any adverse behavioral effects. These in vivo data suggest that ALGX‐XC20 may be used as a novel therapeutic analgesic/anti‐inflammatory drug for the treatment of chronic arthritis, such as OA pain. This TRPA1 antagonist drug may also represent a new class of disease‐modifying drug by providing a chondroprotective effect on articular cartilage.

Rapamycin protects chondrocytes against IL-18-induced apoptosis and ameliorates rat osteoarthritis.

Interleukin 18 (IL-18) promotes inflammation and apoptosis in chondrocytes, thereby contributing to the development and progression of osteoarthritis (OA). Here, we investigated the effects of IL-18 treatment and inhibition in rat chondrocytes in vitro and in vivo. We used RT-PCR and Western blotting to measure the mRNA and protein levels of the chondrocyte-specific genes Collagen II and Aggrecan as well as the protein levels of apoptosis-related (Bax, Bcl2, Caspase3/9), autophagy-related (Atg5, Atg7, Beclin1, LC3), and mTOR pathway-related genes (PI3K, Akt, mTOR). We observed a decrease in Collagen II and Aggrecan mRNA and protein levels, upregulation of chondrocyte apoptosis, downregulation of chondrocyte autophagy, and activation of the PI3K/Akt/mTOR pathway upon IL-18 treatment. PI3K/Akt/mTOR pathway activation and inhibition tests using rat 740Y-P (PI3K activator), SC79 (AKT activator), 3BDO (mTOR activator), or LY294002 (PI3K inhibitor) revealed that activation of the PI3K/Akt/mTOR pathway enhances chondrocyte-specific gene degradation induced by IL-18, while its inhibition has protective effects on chondrocytes. We also found that treatment with rapamycin (a selective mTOR inhibitor) also exerts chondro-protective effects that ameliorate OA by promoting autophagy. These results suggest that inhibition of the mTOR pathway could be exploited for therapeutic benefits in the treatment of OA.

Metformin limits osteoarthritis development and progression through activation of AMPK signalling

ObjectivesIn this study, we aim to determine the effect of metformin on osteoarthritis (OA) development and progression.MethodsDestabilisation of the medial meniscus (DMM) surgery was performed in 10-week-old wild type and...

The CRD of Frizzled 7 exhibits chondroprotective effects in osteoarthritis via inhibition of the canonical Wnt3a/β-catenin signaling pathway

Osteoarthritis (OA) is a chronic inflammatory joint disease without effective drugs. Frizzled 7 (FzD7) binds its ligand Wnt3a through an extracellular cysteine-rich domain (CRD) to transduce the canonical Wnt/β-catenin signaling pathway, which has been strongly implicated in OA pathogenesis. Effects of recombinant protein of FzD7 CRD on Wnt/β-catenin signaling and chondral destruction was evaluated in this study. Firstly, increased protein levels of FzD7, Wnt3a and β-catenin were detected in human OA cartilage implying that the canonical Wnt/β-catenin signaling mediated by Wnt3a and FzD7 executes an essential role in OA. Then we showed that FzD7 CRD antagonized the Wnt3a/β-catenin signaling pathway in a dose-dependent manner by binding Wnt3a. In addition, FzD7 CRD increased the expression of glycosaminoglycans (GAGs), Collagen II, aggrecan and reduced the expression of matrix metalloproteinase (MMP)-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) in Wnt3a-stimulated human chondrocytes. Furthermore, a single intra-articular injection of the FzD7 CRD was efficacious in destabilization of the medial meniscus (DMM) mouse OA model, significantly improving Osteoarthritis Research Society International (OARSI) histology scores compared to mice treated with PBS. The results indicate that the FzD7 CRD exhibits chondroprotective effects by binding Wnt3a to suppress the Wnt3a/β-catenin signaling. Targeting the FzD7 CRD may be a novel therapy for the treatment of OA.

Chondro-protective effects of celastrol on osteoarthritis through autophagy activation and NF-κB signaling pathway inhibition.

Osteoarthritis (OA) is a degenerative articular cartilage disease accompanied by superfluous apoptosis of chondrocytes in the elderly. Celastrol is a potent bioactive medicine which can exert anti-inflammatory and anti-oxidative effects in various diseases. This study aimed to elucidate the possible role of celastrol in OA as well as the specific mechanism of celastrol in vitro and in vivo. Autophagy-related biomarkers and apoptotic molecules were evaluated by PCR, Western blot and immunofluorescence staining. The level of autophagy was assessed by MDC staining and transmission electron microscopy. To study the downstream signaling pathway, nuclear factor kappa B (NF-κB) signaling pathway-related proteins were examined by Western blot. Moreover, an anterior cruciate ligament transection (ACLT) rat model was established to observe the protective effect of celastrol on rat cartilage. We found celastrol ameliorated IL-1β-induced chondrocyte apoptosis and increased the expression of LC3-II and Beclin-1. In addition, the suppression of celastrol-induced autophagy by 3-methyladenine (3MA) prevented the protective effect of celastrol in chondrocytes. Moreover, celastrol decreased the IL-1β-stimulated phosphorylation degree of IκBα and P65. We also found PDTC (a known NF-κB pathway inhibitor) can promote the activation of autophagy and attenuate the apoptosis of chondrocytes. Meanwhile, the results of rat ACLT model revealed the same effect as in vitro experiments. In summary, celastrol protected against chondrocyte apoptosis by promoting autophagy and inhibiting NF-κB signaling pathway in vitro and in vivo.

Topical Application of Wogonin Provides a Novel Treatment of Knee Osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease characterized by inflammatory degradation of articular cartilage and subchondral bone. Wogonin, a compound extracted from the plant Scutellaria baicalensis (colloquially known as skullcap), has previously been shown to have direct anti-inflammatory and antioxidative properties. We examined the pain-reducing, anti-inflammatory, and chondroprotective effects of wogonin when applied as a topical cream. We validated the efficacy of delivering wogonin transdermally in a cream using pig ear skin in a Franz diffusion system. Using a surgical mouse model, we examined the severity and progression of OA with and without the topical application of wogonin. Using a running wheel to track activity, we found that mice with wogonin treatment were statistically more active than mice receiving vehicle treatment. OA progression was analyzed using modified Mankin and OARSI scoring and direct quantification of cyst-like lesions at the chondro-osseus junction; in each instance we observed a statistically significant attenuation of OA severity among mice treated with wogonin compared to the vehicle treatment. Immunohistochemistry revealed a significant decrease in protein expression of transforming growth factor β1 (TGF-β1), high temperature receptor A1 (HTRA1), matrix metalloprotease 13 (MMP-13) and NF-κB in wogonin-treated mice, further bolstering the cartilage morphology assessments in the form of a decrease in inflammatory and OA biomarkers.

The Secretome Derived From Mesenchymal Stromal Cells Cultured in a Xeno-Free Medium Promotes Human Cartilage Recovery in vitro.

Osteoarthritis (OA) is a disabling joint disorder causing articular cartilage degeneration. Currently, the treatments are mainly aimed to pain and symptoms relief, rather than disease amelioration. Human bone marrow stromal cells (hBMSCs) have emerged as a promising paracrine mechanism-based tool for OA treatment. Here, we investigate the therapeutic potential of conditioned media (CM) and extracellular vesicles (EVs) isolated from hBMSC and grown in a xeno-free culture system (XFS) compared to the conventional fetal bovine serum-culture system (FBS) in an in vitro model of OA. First, we observed that XFS promoted growth and viability of hBMSCs compared to FBS-containing medium while preserving their typical phenotype. The biological effects of the CM derived from hBMSC cultivated in XFS- and FBS-based medium were tested on IL-1α treated human chondrocytes, to mimic the OA enviroment. Treatment with CM derived from XFS-cultured hBMSC inhibited IL-1α-induced expression of IL-6, IL-8, and COX-2 by hACs compared to FBS-based condition. Furthermore, we observed that hBMSCs grown in XFS produced a higher amount of EVs compared to FBS-culture. The hBMSC-EVs not only inhibit the adverse effects of IL-1α-induced inflammation, but play a significant in vitro chondroprotective effect. In conclusion, the XFS medium was found to be suitable for isolation and expansion of hBMSCs with increased safety profile and intended for ready-to-use clinical therapies.