Introduction
Osteoarthritis (OA) is a common cause of functional deterioration in the joints of older adults and confers a significant burden on the health of the elderly population (1). OA is characterized by the progressive destruction of cartilage. The current understanding of the molecular events during joint destruction suggests that activated synoviocytes are important in the pathogenesis of OA (2–3). Inflammatory mediators, including interleukin (IL)-1β, infiltrate the synovial membrane, resulting in further inflammation. (4). These mediators amplify and sustain the disease process in OA by inducing inflammatory mediators involved in cartilage degradation, such as matrix metalloproteinases (MMPs) (5). Thus, inflammatory mediator-activated pathways have received increasing attention as potential targets for the treatment of human OA in recent years.
Endogenous glucocorticoid concentrations are determined by the activity of the hypothalamic-pituitary-adrenal axis, and tissue and intracellular exposure are further augmented through the activity of the 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD1), which converts inactive cortisone to the active glucocorticoid, cortisol (6). The ability of 11β-HSD1 to elevate cellular glucocorticoid levels has been postulated to be involved in the modulation of a number of metabolic and inflammatory diseases (7,8). Therefore, the identification of mechanisms underlying 11β-HSD1 activity may improve the understanding of the pathogenesis of certain diseases. Tumor necrosis factor-α (TNF-α) and IL-1β are associated with 11β-HSD1 activity, which are also key mediators in OA pathogenesis (9). The present study aimed to identify the signaling pathways involved in 11β-HSD1-mediated inflammatory responses by human OA fibroblast-like synoviocytes (FLSs) and to determine whether this process is inhibited by the use of a selective 11β-HSD inhibitor, BVT-2733.
Materials and methods
Reagents
Rabbit monoclonal antibodies against mouse bone morphogenetic protein-2 (BMP-2), Dickkopf homolog 1 (DKK1), signal transducer and activation of transcription 3 (STAT3) and phospho-STAT3 (p-STAT3) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Mouse monoclonal antibodies against GAPDH were obtained from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). All other chemicals were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA).
Animals
Female C57BL/6 mice were obtained from the Jackson Laboratory (Shandong University, Jinan, China). Mice were ten- to twelve-weeks-old when the experiments were performed (n=10 mice per group). They were housed in filter-top cages and received food and water ad libitum. Animal studies were approved by the Institutional Review Board of the Animal Experiment Committee of Shandong University and were performed according to the Animal Care and Use Codes of Practice of Jinan Central Hospital Affiliated to Shandong University (Jinan, China).
Collagenase-induced osteoarthritis
Mouse OA was induced by intra-articular injection of 1 unit bacterial collagenase (Sigma-Aldrich, St. Louis, MO, USA) into the right knee on days -2 and 0 prior to starting treatment, as previously described (10). On day 36, mice were weighed and sacrificed, under general anaesthesia and left and right knee joints were isolated and processed for histological analysis. Animals were also bled to obtain serum samples in order to determine the levels of the cytokines IL-1β, IL-6, IL-17 and vascular endothelial growth factor (VEGF).
BVT-2733 treatment
BVT-2733 (Collaborative Bio-Engineering Corporation, Nanjing, China) was dissolved in 12% β-hydroxypropylcyclodextrin (Fluka Chemi, Buchs, Switzerland) and 0.3% sodium chloride (Merck Millipore, Darmstadt, Germany). The compound and vehicle were administered by oral gavage twice daily (11). CIOA mice received BVT-2733 (100 mg/kg, orally) or vehicle twice daily for five weeks, starting from day 0 following the last collagenase immunization. Control animals were injected with vehicle alone.
Hematoxylin and eosin (H&E) preparation
Isolated knee joints were fixed in 4% buffered formalin, and subsequently decalcified in formic acid and embedded in paraffin. Eight representative sections (6-μm) from each joint at depths of 4–6 μm were stained with H&E or safranin O-fast green (Sigma)-Aldrich) for histological analysis using a IX71 microscope (Olympus, Shanghai, China) (12). Sections were randomly coded and scored in a blinded manner by two independent investigators.
Histological analysis
The degree of cell infiltration was graded as 0, normal; 1, mild infiltration; 2, moderate infiltration; 3, marked infiltration; and 4, severe infiltration.
Immunoblotting analysis
Frozen joint tissues were cut into sections (4–6 μm) and incubated for 10 min at 4°C in lysis buffer (20 mM HEPES, pH 7.4; 2 mM ethylene glycol tetraacetic acid; 50 mM β-glycerophosphate; 0.1% Triton X-100; 10% glycerol; 1 mM dithiothreitol; 1 μg/ml leupeptin; 5 μg/ml aprotinin; 1 mM phenylmethylsulfonyl fluoride; and 1 mM sodium orthovanadate). Sections were then removed, incubated on ice for a further 10 min and centrifuged at 17,000 × g for 30 min at 4°C. The supernatants were subjected to SDS-PAGE, blotted onto nitrocellulose membranes (Sigma-Aldrich) and blocked by incubation with 5% bovine serum albumin in phosphate-buffered saline (PBS) for 1 h at room temperature. They were then incubated overnight at 4°C with the appropriate primary antibodies diluted in PBS (1:2,000). Following three washes with PBS containing 0.1% Tween-20, samples were incubated for 1 h at room temperature with the corresponding monoclonal rabbit horseradish peroxidase-conjugated antibodies (Merck Millipore) diluted in PBS. Bound antibodies were detected using an enhanced chemiluminescence detection kit (Merck Millipore) and exposure to photographic film (Merck Millipore). Quantitative data were obtained using a densitometer with an Image-Pro Plus image analysis software system (Eastman Kodak Co., Rochester, NY, USA).
Milliplex<sup>®</sup> cytokine assay
To quantify cytokine production by FLSs, culture supernatants were analyzed with a Milliplex multi-analyte panel kit (Millipore Corporation, St. Charles, MO, USA), which allows the simultaneous quantification of cytokines IL-1β, IL-6, IL-17 and VEGF. The data were analyzed using Bio-Plex Manger 4.1.1 software (Bio-Rad Laboratories, Hercules, CA, USA).
Statistical analysis
Data are expressed as the mean ± standard error of the mean and were analyzed statistically using one-way analysis of variance. When ANOVA showed significant differences between groups, Tukey’s post hoc test was used to determine the specific pairs of groups with statistically significant differences. P<0.05 was considered to indicate a statistically significant difference. The data were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).
Results
BVT-2733 treatment reduces cartilage and bone destruction in CIOA mice
The results revealed chronic synovitis with evident inflammatory infiltration along with an increase of cell influx, 5 weeks after treatment (day 36; Fig. 1A and B). The degree of cell infiltration was graded as 0, normal; 1, mild infiltration; 2, moderate infiltration; 3, marked infiltration; and 4, severe infiltration. The histological score was 2.32±0.65 for mice with CIOA treated with vehicle compared with 0.63±0.29 for CIOA mice treated with BVT-2733 (P<0.001). Although BVT-2733 treatment resulted in an absence of changes in bone and cartilage (Fig. 1C and F), the vehicle-treated mice showed near complete loss of proteoglycans in the cartilage compared with the control group (Fig. 1E and D).
BVT-2733 reduces the expression of bone markers
The effect of BVT-2733 treatment on the expression of bone markers was investigated by western blotting on day 36 of CIOA. A low level of expression of DKK1 was established in the joints of normal control and BVT-2733-treated mice, compared with a higher rate in the vehicle-treated CIOA mice (Fig. 2). By contrast, a marked increase in the expression levels of BMP-2 was observed in the joints of arthritic mice compared with those of control mice (Fig. 2), BVT-2733-treated CIOA mice also exhibited a low level of BMP-2 expression (Fig. 2).
BVT-2733 suppresses STAT3 phosphorylation
STAT3 phosphorylation was measured in CIOA mice treated with BVT-2733 or vehicle on day 36 (Fig. 3). BVT-2733 was shown to reduce levels of the p-STAT3 protein compared with the vehicle-treated group.
BVT-2733 treatment inhibits the release of proinflammatory cytokines in CIOA mice
CIOA is characterized by a marked increase in the expression of pro-inflammatory cytokines. To ascertain whether BVT-2733 inhibits this process, BVT-2733-treated and vehicle-treated mice with CIOA were bled at day 36 following the initiation of treatment. Serum IL-1β, IL-6, IL-17 and VEGF levels were measured by an enzyme-linked immunosorbent assay. The vehicle-treated mice had higher serum levels of IL-1β, IL-6, IL-17 and VEGF than that of BVT-2733-treated mice (Fig. 4). These results suggest that BVT-2733 may reduce the severity of CIOA by inhibiting the production of inflammatory cytokines.
Discussion
Despite the identification of 11β-HSD1 more than half a century ago, there has been little investigation into its physiological role until relatively recently (13). It is notable that the severity of acute inflammation is greater in 11β-HSD1-deficiency or inhibition. However, in certain conditions in which inflammation is invovled, such as obesity or diabetes, 11β-HSD1-deficiency or inhibition appears to be beneficial and has been shown to reduce inflammation (13). Data have shown that IL-1β induces the expression of 11β-HSD1 mRNA (14). A recent study suggested that 11β-HSD1 inhibition regulates proinflammatory gene expression and may attenuate atherosclerosis in mice (15). Modulation of 11β-HSD1 has been proposed as a strategy by which to reduce vascular inflammation (16). Notably, 11β-HSD1 was found to be aberrantly expressed in rheumatoid arthritis (RA) (17). However, the functional role of 11β-HSD1 in OA remains unclear. In the present study, BVT-2733 was used as a novel selective inhibitor of 11β-HSD1. BVT-2733 is a relatively novel inhibitor of 11β-HSD1 and has been used in previous studies (18) The data indicated that inhibition of 11β-HSD1 by BVT-2733 prevented the development of arthritis and joint destruction in CIOA. In addition, the protective effects of BVT-2733 were shown to be mediated by the inhibition of certain inflammatory signaling pathways.
Furthermore, the expression of bone markers in CIOA was examined by western blotting. DKK1 has been shown to promote angiogenic responses and cartilage matrix proteinase secretion in synovial fibroblasts from osteoarthritic joints. The blockade of this molecule therefore has the potential to reduce OA-induced synovitis and joint deterioration (19). In the current study, a high level of expression of DKK1 was observed in the joints of vehicle-treated mice, compared with significantly lower levels in normal control mice and BVT-2733-treated CIOA mice. In addition, BMP-2 is known to promote transforming growth factor β3-induced chondrogenesis of human osteoarthritic synovium-derived stem cells (20). BVT-2733 treatment also significantly reduced the level of BMP-2 expression in CIOA mice compared with the vehicle-treated group. Furthermore, STAT3 phosphorylation in CIOA mice at day 36 following the initiation of treatment with BVT-2733 was significantly reduced compared with the vehicle control group. p-STAT3 is known to upregulate the cellular immune response in human adult articular chondrocytes (21).
Proinflammatory cytokines are important in the pathogenesis of OA. IL-1β promotes inflammation and destruction of the synovial tissue, bone, cartilage and joints of patients with RA (22). IL-1β is a mediator of the generation of synovial inflammation and pannus formation (23). IL-1β is also involved in inducing expression of other inflammatory cytokines, such as TNF-α and IL-6 (24). A previous study showed that an increase in levels of IL-1β in synovial tissue (25) was correlated with the histological features of arthritis. IL-17 promotes inflammation by enhancing the production of cytokines, such as IL-1β, TNF-α and IL-6 (26). Growing evidence shows that IL-17 contributes to inflammation in the pathogenesis of arthritis (27). In CIOA, the IL-17 level is known to be elevated in the inflamed synovium and may act on osteoblasts to induce the expression of receptor activator for NF-κB ligand (RANKL), which may lead to osteoclastogenesis (28). IL-17 deficiency prevents the induction of CIOA in mice (29). In accordance with previous findings, the present study showed that BVT-2733 treatment caused a marked increase in the expression of IL-1β, IL-6, IL-17 and VEGF in the serum of CIOA mice. In addition, histopathological analysis indicated that BVT-2733 treatment reduced the synovial inflammation and bone destruction in CIOA mice.
In conclusion, the current study suggests that inhibition of 11β-HSD1 by BVT-2733 ameliorates the joint inflammation and cartilage destruction observed in the development and progression of CIOA. These effects were associated with the inhibition of proinflammatory cytokines, a reduction in the expression of bone markers and an increase in p-STAT3 levels in joints and serum of CIOA mice. This implies that 11β-HSD1 may be a therapeutic target in OA. 11β-HSD1 blockade may thus be developed as one of a number of strategies for use in the treatment of OA.