Cirsium japonicum var. maackii and apigenin block Hif-2α-induced osteoarthritic cartilage destruction

2.1 Reagents and treatment

Samples of the dried aerial part of Cirsium japonicum var. maackii (CJM) were obtained commercially from Imsil Herbal Medicine (Imsil-gun, Jeollabuk-do, Korea). The dried samples were mixed with 30% ethanol and the mixture was refluxed at 65°C for 3 hours. Total CJM extract was obtained as the filtrate after vacuum filtration at 25°C. Apigenin, cirsimarin and cirsimaritin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pro-inflammatory cytokines (IL-1β, IL-6, IL-17 and TNF-α) were purchased from GenScript (Piscataway, NJ, USA). Mouse articular chondrocytes were treated with IL-1β (1 ng/mL), IL-6 (100 ng/mL), IL-17 (10 ng/mL) or TNF-α (50 ng/mL) and co-treated with CJM extract (10, 50 or 100 μg/mL), apigenin (10, 25 or 50 μmol/L), cirsimarin (10, 25 or 50 μmol/L) or cirsimaritin (10, 25 or 50 μmol/L) for 24 h before they were harvested.

2.2 HPLC analysis of cirsimarin, cirsimaritin and apigenin

Quantitative analysis of cirsimarin, cirsimaritin and apigenin in the CJM extract was performed with a Waters Breeze System HPLC (Waters Co., Milford, MA, USA) equipped with a 250 mm × 4.6 mm (df = 5 μm) C18 INNO column and a UV/VIS detector (set at 270 nm). The concentrations of cirsimaritin, cirsimarin and apigenin in the extract were determined from corresponding calibration curves. Figure 4A shows representative HPLC spectra of the CJM extract and standard.

2.3 Culture of mouse articular chondrocytes and chondrocyte viability assay

Articular chondrocytes were isolated from femoral condyles and tibial plateaus of post-natal day 5 mice. Cartilage tissues were digested with 0.2% collagenase type II, as previously described.23 For the chondrocyte viability assay, the chondrocytes were seeded in a 96-well dish (9 × 10³ cells/well) for 48 hours prior to treatments. CJM or apigenin was added at various concentrations and incubated for 24 hours in DMEM without foetal bovine serum. Cytotoxicity was assessed with the EZ-CyTox Cell Viability Assay kit (Dogen, Seoul, South Korea) and an LDH Colorimetric assay kit (BioVision Inc, Milpitas, CA, USA) following the manufacturer's manual. Briefly, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) solution mixed with serum-free DMEM (1:100, v/v) was added to the cultured cells and incubated for 3 hours. To analyse LDH activity, cell viability was normalized to that of untreated samples (100% viability) and samples treated with Triton X-100 (0% viability). Assays were performed on the supernatants of chondrocytes at time points of 36 hours post-treatment of CJM and apigenin at various concentrations. % cytotoxicity was calculated by the formula (Sample LDH − Negative Control)/(Max LDH − Negative Control) × 100. The absorbance was measured using a microplate reader (VICTOR X3; PerkinElmer, Waltham, MA) at 450 and 595 nm for WST-1 and LDH assays respectively.

2.4 Reverse transcription-polymerase chain reaction and quantitative RT-PCR

Total RNA was isolated from articular chondrocytes using TRIzol (Molecular Research Center Inc, Cincinnati, OH, USA). Total RNA was reverse transcribed and the resulting cDNA was amplified by PCR (iTaq, Intron Biotechnology, Gyeonggi-do, South Korea). The PCR primers are summarized in Table S1. The transcript levels of target genes were quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using SYBR^® premix Ex Taq (TaKaRa Bio, Shiga, Japan). For each target gene, the transcript level was normalized to that of Gapdh and expressed as a fold change relative to the indicated control.

2.5 Western blotting and immunohistochemistry

Total proteins were extracted with lysis buffer (150 mmol/L NaCl, 1% NP-40, 50 mmol/L Tris, 0.2% sodium dodecyl sulphate and 5 mmol/L NaF) supplemented with a protease inhibitor and phosphatase inhibitor cocktail (Roche, Madison, WI, USA). MMP3 and MMP13 were detected after trichloroacetic acid precipitation as described previously.23 Each protein was visualized using the SuperSignal West Dura kit (Thermo Scientific, Waltham, MA, USA); total extracellular signal-regulated kinase (ERK) was used as a loading control. Western blot analysis was performed to detect protein levels using the following antibodies: mouse anti-Hif-2α (sc-13596; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Mmp3 (ab52915; Abcam, Cambridge, UK), mouse anti-Mmp13 (ab51072; Abcam), goat anti-COX-2 (sc-1745; Santa Cruz), mouse anti-Erk1/2 (610408; Becton Dickinson, NJ, USA), mouse anti-pErk1/2 (9101; Cell Signaling Technology, Boston, MA, USA) and mouse anti-IκB (9242; Cell Signaling Technology). Anti-Hif-2α antibody (ab8365; Abcam) was used for immunostaining as previously described.14

2.6 Experimental OA models and oral gavage

All animal experiments were approved by the Animal Care and Use Committee of the University of Ajou. For the destabilization of the medial meniscus (DMM)-induced OA model, 12-week-old male C57BL/6 mice were subjected to DMM surgery using a previously described protocol.24 Mouse knee joints were processed for histological analysis 10 weeks after surgery. Experimental OA was also induced in 12-week-old male mice by intraarticular (IA) injection (once weekly for 3 weeks) of Hif-2α-expressing adenovirus (Ad-Hif-2α; 1 × 10⁹ plaque-forming units in a total volume of 10 µL); IA injection of empty adenovirus (Ad-C) was used as a control. The DMM-induced and Ad-Hif-2α-induced OA models received CJM by gavage every other day for 10 weeks and 3 weeks, respectively, and were killed after completion of the gavage feeding.

2.7 Evaluation of cartilage destruction

Cartilage destruction was assessed as previously described.25 Briefly, mouse knee joints were fixed in 4% paraformaldehyde, decalcified with 0.5 mol/L EDTA (pH 8.0) for 2 weeks and embedded in paraffin. The paraffin blocks were sectioned at a thickness of 5 μm and were serially sectioned at 40-μm intervals. The sections were deparaffinized in xylene and hydrated with a graded ethanol series. Cartilage destruction was detected by Safranin O staining and scored using the Osteoarthritis Research Society International (OARSI) grading system.

2.8 Cartilage explants and 1,9-dimethylmethylene blue assay

Mouse femoral head cartilage was dissected from 3-week-old C57BL/6 mice and incubated in DMEM (Gibco-BRL) containing CJM or apigenin with or without IL-1β (10 ng/mL) for 72 hours without changing the medium, as described previously.23 The conditioned medium from each well was collected to quantify aggrecan-release content. The culture medium was mixed with 1,9-dimethylmethylene blue (DMMB) solution (40 mmol/L NaCl, 40 mmol/L Glycine, 46 μmol/L DMMB) at 1:10 and the absorbance was measured at 525 nm within 10 minutes using a microplate reader. Bovine cartilage chondroitin sulphate was used for the standard curve of sulphated glycosaminoglycan (sGAG) content. For mouse knee joints, cartilage explants were obtained and cultured in DMEM (Gibco-BRL) containing CJM or apigenin with or without IL-1β (1 ng/mL) for 72 hours. The accumulation of sGAG was assessed by Alcian blue staining (1 volume of 0.3% Alcian blue 8GX in 70% ethanol, 1 volume of 100% acetic acid and 18 volumes of 100% ethanol), as described previously.26

2.9 Reporter gene assay

The Mmp3, Mmp13, Cox-2 and Adamts4 reporter gene constructs were co-transfected with or without Hif-2α expression vector into mouse articular chondrocytes using LipofectAMINE Plus (Invitrogen, Carlsbad, CA, USA) as described previously.14 The transfected cells were cultured in complete medium for 24 hours. Luciferase activity was determined using an assay kit (Promega, Madison, WI, USA) and subsequently normalized to β-galactosidase activity.

2.10 Statistical analysis

All experiments were performed independently at least three times. Statistical comparisons of two independent groups were made using two-tailed independent t tests. Multiple comparisons were made using ANOVA with a post-hoc Bonferroni test. Data based on ordinal grading systems, such as OARSI grade and subchondral bone thickness, were analysed using a non-parametric Mann–Whitney U test. A P-value of 0.05 was considered significant.

3.1 CJM inhibits Mmp3, Mmp13 and Cox-2 expression under in vitro conditions mimicking OA

We first determined whether CJM demonstrates cytotoxicity towards chondrocytes. Treatment of the mouse articular chondrocytes with different concentrations of CJM for 36 hours resulted in no observable cytotoxicity as determined by WST-1 assay, LDH assay and CJM-induced catabolic factor expression (Figure S1).

Cartilage degradation and inflammation lead to OA development and progression.1, 2 We postulated that CJM could block cartilage degradation and inflammation via regulation of Mmp3, Mmp13, Adamts4, Adamts5 and Cox-2 expression. To test the effect of CJM on mouse articular chondrocytes, the expression levels of Mmp3, Mmp13, Adamts4, Adamts5 and Cox-2 were assessed by biochemical analysis. As shown in Figure 1A and 1 and Figure S2A, the expression levels of IL-1β-induced Mmp3, Mmp13, Adamts4, Adamts5 and Cox-2 were gradually down-regulated by CJM in a concentration-dependent manner.

Although IL-1β is an important OA-related pro-inflammatory cytokine, other pro-inflammatory cytokines (eg, IL-6, IL-17 and TNF-α) have recently been found to play important roles in OA development.5, 6, 14 Notably, CJM reduced IL-6-, IL-17- or TNF-α-stimulated Mmp3, Mmp13, Adamts4 and Adamts5 expression (Figure 1C–E, Figure S2C–E). These results indicate that CJM can alleviate cartilage degradation and inflammation under in vitro OA-mimetic conditions and, therefore, potentially protect cartilage from destruction during OA progression and development.

3.2 Oral administration of CJM protects against cartilage destruction in the DMM-induced OA model and ex vivo organ culture system

To evaluate the role of CJM under in vivo conditions, we tested whether administration of CJM protects against osteoarthritic cartilage destruction in the DMM-induced OA model. The mice were orally administered either PBS or PBS containing CJM every other day for 10 weeks, starting at DMM (Figure 2A). Compared to that of the PBS control group, oral administration of CJM resulted in dramatic protection against OA development (Figure 2B) as indicated by significantly lower OARSI scores and subchondral bone plate thickness (Figure 2C). Moreover, CJM restored the accumulation of extracellular sulphated proteoglycans and inhibited aggrecan release in IL-1β-stimulated cartilage explants (Figure S3A and B). Therefore, these results indicate that CJM may activate cartilage regeneration to facilitate OA development.

3.3 Hif-2α expression is regulated by CJM in mouse articular chondrocytes

Hif-2α plays a central role in catabolic factor expression and works as a master regulator for inducing OA development.6, 14, 27 The level of Hif-2α was increased by in vitro conditions that mimicked OA and Hif-2α-overexpressing chondrocytes demonstrated up-regulated Mmp3, Mmp13, Adamts4 and COX-2 expression (Figure S4). Therefore, we determined whether Hif-2α levels or Hif-2α-induced Mmp3, Mmp13, Adamts4 and Cox-2 expressions were regulated by CJM in mouse articular chondrocytes. Treatment of chondrocytes with IL-1β up-regulated Hif-2α and this up–regulation was attenuated by CJM in a concentration-dependent manner as determined by RT-PCR and qRT-PCR (Figure 3A). Furthermore, Mmp3, Mmp13, Adamts4 and Cox-2 induction by Hif-2α overexpression was reduced by CJM treatment in cultured mouse articular chondrocytes (Figure 3B, Figure S5A).

To assess the protective effect of CJM in Hif-2α-induced OA development, cartilage destruction and Hif-2α expression were detected by Safranin O staining and immunohistochemistry respectively. As shown in Figure 3C and 3, although IA injection of Ad-Hif-2α promoted cartilage destruction in a mouse model, oral administration of CJM ameliorated this cartilage destruction. Moreover, Hif-2α expression in the cartilage tissue of mice after IA injection with Ad-Hif-2α was dramatically reduced by CJM administration.

These results clearly indicate that Hif-2α overexpression-induced catabolic factors and cartilage destruction were regulated by CJM through Hif-2α suppression in both cultured articular chondrocytes and mice.

3.4 Apigenin from CJM is a key component for regulating Hif-2α expression

Although several compounds have been identified previously from CJM, their effects on the Hif-2α regulation of OA development remain largely unknown. Therefore, we designed an in vitro analysis to identify the suppressive effects of several individual components of CJM on Hif-2α regulation. The contents of cirsimaritin, cirsimarin and apigenin were determined by HPLC separation of total CJM extract (Figure 4A). Although cirsimarin (Figure 4B) and cirsimaritin (Figure 4C) reduced Cox-2 expression, they did not effectively suppress IL-1β-induced Hif-2α, Mmp3 and Mmp13 up-regulation in articular chondrocytes. However, apigenin gradually attenuated IL-1β-induced Hif-2α up-regulation, as well as that of Mmp3, Mmp13, Adamts4 and Cox-2, in a concentration-dependent manner (Figure 4D, Figure S2B). Apigenin also restored the accumulation of extracellular sulphated proteoglycans and inhibited aggrecan release in IL-1β-stimulated cartilage explants (Figure S3C and D). Furthermore, Hif-2α-induced Mmp3, Mmp13, Adamts4 and Cox-2 expressions were dramatically reduced by apigenin treatment (Figure 4E, Figure S5B). These data strongly suggest that apigenin attenuates the production of Mmp3, Mmp13, Adamts4 and Cox-2 and is a key component of CJM for reducing Hif-2α expression in articular chondrocytes.

3.5 Apigenin directly regulates Mmp3, Mmp13, Adamts4 and Cox-2 transcriptional activity and modulates NF-κB and JNK signalling pathways for Hif-2α regulation

To elucidate the effect of CJM and apigenin on the direct regulation of catabolic factors by Hif-2α, we examined Hif-2α transactivation of the mouse promoters with or without CJM and apigenin in reporter gene assays. Transfection of a Hif-2α expression vector in mouse articular chondrocytes elevated transcriptional activities from the promoters of Mmp3, Mmp13, Adamts4 and Cox-2 (Figure 5A-D). However, these elevated transcriptional activities were decreased by CJM and apigenin in a concentration-dependent manner. These results show that CJM and apigenin affect Hif-2α-mediated transcription of Mmp3, Mmp13, Adamts4 and Cox-2 mRNA. Moreover, upstream NF-κB and JNK signalling pathways regulate Hif-2α expression and IL-1β, a known activator of NF-κB and JNK signalling, increase Hif-2α expression in chondrocytes.14, 27 Because IL-1β-mediated NF-κB and JNK signalling pathways are involved in OA pathogenesis,27-29 we further examined whether apigenin inhibits these signalling pathways. Mouse articular chondrocytes were pre-incubated for 24 hours either with or without apigenin and were then exposed to IL-1β (1 ng/mL) for 10 minutes. NF-κB and JNK signalling pathways were analysed by Western blotting. IκB degradation and JNK phosphorylation induced by IL-1β stimulation were prevented by apigenin preincubation (Figure 6A and 6). Collectively, our results clearly indicate that Hif-2α expression is suppressed by apigenin treatment via the inhibition of NF-κB and JNK signalling pathways during OA development.