Osteoarthritis (OA) is the most prevalent degenerative arthritis. It is characterized by a progressive destruction of articular cartilage accompanying phenotypes of whole-joint disease, such as cartilage destruction, synovitis, osteophyte formation, and subchondral bone sclerosis (1). In the cartilage, chondrocytes are unique resident cells that can produce cartilage-specific extra-cellular matrix (ECM) components as well as various catabolic and anabolic factors. OA pathogenesis is mainly caused by an imbalance between anabolic and catabolic factors due to de-differentiation and apoptosis of chondrocytes (2).
Various biochemical signals in chondrocytes can lead to the imbalance by modulating proinflammatory cytokine production, the cessation of ECM synthesis, and the irreversible degradation of the ECM by the action of matrix-metalloproteinases (MMPs) (3, 4). Aging and mechanical stresses are potential OA-causing factors that can promote the expression of pro-inflammatory cytokines as well as pro-inflammatory mediators such as cyclo-oxygenase-2 (COX-2) encoded by
Numerous signaling pathways are involved in inflammatory responses and ECM degradation in OA pathogenesis. It has been reported that phosphorylated levels of mitogen-activated protein (MAP) kinases, including extracellular signal-regulated kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK), are up-regulated in osteoarthritic cartilage (7). In fact, MAP kinases play a crucial role in OA pathogenesis by promoting cytokine-mediated MMP expression. For instance, IL-1β can stimulate the induction of MMP-3 and MMP-13 via activation of p38 kinase and JNK in articular chondrocytes (7). ERK activity is predominantly required for the expression of MMP-1 and MMP-3 in IL-1β-stimulated SW1353 human chondrosarcoma cells (8). It has been reported that IL-1β expression in osteoarthritic cartilage and synovial fluids is mediated by nuclear factor-κB (NF-κB) (9). NF-κB dimers are sequestered by an inhibitor of κB (IκB) in the cytoplasm in an inactive form. Signal-induced degradation of IκB initiates the activation of NF-κB, leading to its translocation into the nucleus and up-regulation of inflammation-related genes, including NOS2 and COX-2 (9, 10).
Avenanthramides (Avns) are unique polyphenolic compounds exclusively found in oats (11). It has been scientifically verified that oats (
In the current study, we determined the potential of using Avn-C to prevent OA pathogenesis. Avn-C markedly blocked cartilage destruction by restraining the expression and activities of MMPs in articular chondrocytes. The inhibitory effects of Avn-C on cartilage destruction were mediated by p38 kinase and JNK signaling. Results of this study suggest possible functions and underlying molecular mechanisms of Avn-C in alleviating OA pathogenesis.
To determine effects of Avn-C on OA pathogenesis, we induced an
We attempted to determine
Next, to examine the
To determine the molecular mechanisms involved in the protective effect of Avn-C on the pathogenesis of arthritis, we examined signaling pathways involved in IL-1β mediated MMP expression. It has been reported that MAP kinases including ERK, p38 kinase, and JNK are activated by IL-1β and the activation of these signaling pathways is involved in the pathogenesis of arthritis, including degradation of cartilage ECM, inflammation, and apoptosis of chondrocytes (20). The maximal activation of all three MAP kinases in 15-30 min after IL-1β treatment was verified in current experiments by determining phosphorylation on Thr202/Tyr204 of ERK, Thr180/Tyr182 of p38 kinase, and Thr183/Tyr185 of JNK (Fig. 4A). Pre-treatment with Avn-C significantly blocked p38 kinase and JNK signaling induced by IL-1β, but not ERK signaling (Fig. 4B). NF-κB is a major transcription factor that controls gene expression involved in the production of cytokines such as IL-6 and TNF-α (21). IL-1β also activated NF-κB signaling in mouse chondrocytes as determined by the degradation of IκB in 15-30 min after IL-1β stimulation (Fig. 4A). However, degradation of IκB was sustained by the addition of Avn-C in IL-1β stimulated articular chondrocytes, indicating that Avn-C did not modulate IL-1β-mediated NF-κB signaling (Fig. 4B). Next, we examined whether IL-1β-induced p38 kinase and JNK signaling regulated the expression of MMP-3, -12, and -13. Treatment with SB203580, a specific inhibitor of p38 kinase, blocked IL-1β-induced expression of
OA is typically characterized by progressive degradation and loss of articular cartilage resulting from the action of mechanical and biochemical factors (1, 22). Although various therapeutic approaches have been attempted to prevent OA progression, no effective treatment is currently available. The use of natural health product by OA patients to alleviate symptoms is rising globally. In this study, we evaluated the efficacy of Avn-C extracted from oats as a promising candidate to prevent OA progression. Here, we found that Avn-C prevented cartilage destruction in experimental OA mouse model induced by DMM surgery and inhibited the expression of MMPs, including MMP-3, -12, and -13, by regulating p38 kinase and JNK signaling.
Previous papers have reported that Avn-C exhibits anti-inflammatory effects by inhibiting the expression of pro-inflammatory cytokines via NF-κB signaling in skeletal muscle cells (17) and anti-oxidant effects on the inhibition of colon cancer cell growth (15, 18). However, in our current study, Avn-C had no effect on the expression of inflammatory mediators such as
The pro-inflammatory cytokine IL-1β is one of the most critical catabolic factors in the development of OA. It can induce the release of inflammatory mediators and MMPs. Excessive amount of IL-1β is found in cartilage, synovial fluid, the synovial membrane, and the subchondral bone (23). Herein, IL-1β was used to develop an
OA is considered as a whole joint disease with pathological changes, including synovial inflammation, subchondral bone sclerosis, osteophyte formation, and cartilage destruction. Avn-C prevented pathological changes of the whole joint. We scored the grade of cartilage degeneration according to the OARSI system. DMM induced OA cartilage showed an average OARSI grade of 2.4 (P < 0.005), denoting increased subchondral sclerosis and osteophyte formation (Fig. 3B). However, Avn-C injection decreased the severity of OA phenotypes induced by DMM, which showed an OARSI grade 1.1 with thinner subchondral bone plate than in DMM control mice (Fig. 3B).
A previous study has demonstrated that HIF-2α as a critical transcription factor plays an essential role in IL-1β-induced MMP expression during OA pathogenesis (2). Our current results were consistent with results of the previous study showing induction of MMPs except MMP-2, -14, and -15 by IL-1β treatment or HIF-2α overexpression (2). The 5’-flanking regions of
Cartilage destruction in mice was induced by DMM surgery. Sham-operated mice as controls were also prepared. At two weeks after DMM operation, mice were injected with 10 μl of 200 μM Avn-C or DMSO every week for six weeks by intra-articular injection. Knee joints were processed for histological analysis at 8 weeks after DMM surgery. All experiments were approved by Chonnam National University Animal Care and Use Committee.
Mouse joint tissues were decalcified with 0.5 M EDTA (pH 8.0) for two weeks, embedded in paraffin, and sectioned at 5 μm in thickness. Cartilage destruction was evaluated by Safranin-O staining and was scored according to the OARSI grade system. Lateral sections were placed on slides for immunohistochemical staining and incubated in 3% H2O2 for 10 min to block endogenous peroxidase activity. Sections were then incubated with 0.1% trypsin for 30 min at 37°C to retrieve antigen. After blocking with 1% BSA for 30 min, slides were incubated with rabbit anti-MMP-3 (Abcam, Cambridge, UK), rabbit anti-MMP-12 (Epitomics, California, USA), and rabbit anti-MMP-13 (Abcam) followed by staining using the EnVision + System-HRP (Dako, Denmark) and AEC + substrate (Dako). Sections were counter-stained with Hematoxylin (Dako).
Primary chondrocytes were isolated from femoral condyles and tibial plateaus of postnatal day 5 mice. The articular cartilage was pre-incubated for 2 h at 37°C with 0.2% trypsin and 0.2% type II collagenase and further digested with 0.2% type II collagenase for an additional 90 min. Chondrocytes were main-tained as a monolayer in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and appropriate antibiotics. On culture day 3, cells were treated as indicated for each experiment. Chondrocytes were treated with indicated concentration of IL-1β (GeneScript, Piscataway, NJ, USA) for 24 h with or without Avn-C (SI-033-053-1, Sigma-Aldrich, St Louis, MO, USA) dissolved in DMSO, SB203580 (p38 inhibitor), or SP6000125 (JNK inhibitor) (Santa Cruz, CA, USA).
Total RNA was isolated from cultured articular chondrocytes using TRI reagent (TR118, Molecular Research Center Inc., Cincinnati, OH, USA). The RNA was reverse transcribed to cDNA. The resulting cDNA was amplified by PCR and quantified by qRT-PCR. qRT-PCR was performed using SYBR premix Ex Taq (RR420, TaKaRa, Japan). All qRT-PCR reactions were performed in duplicate and the threshold cycle value of each gene was normalized against that of
Cultured mouse articular chondrocytes were washed with cold PBS and lysed in lysis buffer containing 50 mM Tris-HCl, pH8.0, 150 mM NaCl, 5 mM NaF, 1% NP-40, 0.2% SDS, 0.5% deoxycholate, a protease inhibitor cocktail, and a phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Cell lysates were loaded and separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes. After blocking with 5% skim milk at room temperature for 1 h, membranes were incubated with primary antibodies overnight at 4°C with indicated antibodies. The following antibodies were used: rabbit anti-MMP-3 (Abcam), rabbit anti-MMP-12 (Epitomics), rabbit anti-MMP-13 (Abcam), mouse anti-ERK1 (BD Biosciences, NJ, USA), rabbit anti-phospho-Erk1/2 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-JNK (Cell Signaling Technology), mouse anti-phospho-JNK (Cell Signaling Technology), rabbit anti-p38 (Santa Cruz), mouse anti-phospho-p38 (Cell Signaling Technology), and rabbit anti-IκBα (Santa Cruz). Membranes were then incubated with horse-radish peroxidase conjugated secondary antibodies, anti-mouse (Sigma-Aldrich) or anti-rabbit IgG (Sigma-Aldrich), and detected using an ECL solution (GE Healthcare, Pittsburgh, PA, USA). Images were detected using ImageSaver6 software via an EZ-capture MG system (ATTO, Tokyo, Japan). Protein level was normalized against the level of β-actin (Sigma-Aldrich) as a loading control.
To quantify activities of MMP-3, -12, and -13 in the culture medium, primary chondrocytes were cultured without serum. They were then treated with IL-1β and indicated dose of Avn-C. Supernatants were collected after 24 h and stored at −80°C until ELISA. ELISA kits #71130, #71157 and #71156 for analyzing substrate degrading activities of MMP-3, -12, and -13, respectively, were purchased from Anaspec (CA, USA). ELISAs were performed according to the manufacturer’s specifications using duplicate wells for each sample.
All experiments were repeated at least three times. Data obtained from qRT-PCR and enzymatic activity assays were first tested for conformation to a normal distribution using the Shapiro-Wilk test, followed by analysis with Student’s t-test (pairwise com-parisons) or analysis of variance with post-hoc tests (multiple comparisons) as appropriate. The n-value is the number of independent experiments or mice. Threshold for significance was set at the 0.05 level of probability (P < 0.05).
This work was supported by grants (NRF-2019R1A5A2027521 and 2021R1A2C300572711) of the National Research Foundation of Korea (NRF) funded by the Korea government (MSIT), the Korea Healthcare Technology R&D Project of the Korea Health Industry Development Institute (HR14C0008), and Chonnam National University Hospital Biomedical Research Institute (BCRI18012).
The authors have no conflicting interests.