International Immunopharmacology 23 (2014) 85–91
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Hinokitiol reduces matrix metalloproteinase expression by inhibiting Wnt/β-Catenin signaling in vitro and in vivo Jin Li, Xin-die Zhou, Kong-he Yang, Teng-di Fan, Wei-ping Chen, Li-feng Jiang, Jia-peng Bao, Li-dong Wu ⁎,1, Yan Xiong ⁎,1 Department of Orthopedics Surgery, The Second Affiliated Hospital of Medical College, Zhejiang University, Hangzhou 310000, China
a r t i c l e
i n f o
Article history: Received 9 April 2014 Received in revised form 7 August 2014 Accepted 13 August 2014 Available online xxxx Keywords: Osteoarthritis Hinokitiol Matrix metalloproteinase β-catenin
a b s t r a c t Objective: In this study, we investigated the effects of hinokitiol on matrix metalloproteinase (MMP)-1, -3, -13, collagen type II (Col2a1) and β-catenin expressions in rat chondrocytes induced by interleukin-1β and in an experimental rat model induced by intra-articular injection of mono-iodoacetate (MIA) into the knee. Methods: Chondrocytes were cultured from the articular cartilage of 2-week-old rats. Passaged chondrocytes were pretreated with hinokitiol for 2 h followed by co-incubation with IL-1β for 24 h. Quantitative real-time polymerase chain reaction and Western blotting were used to assess the expression of MMP-1, -3, -13, Col2a1 and β-catenin. Chondrocytes were also treated with Licl, Dickkopf-1, and/or hinokitiol for 24 h, the MMP-1, -3, -13 and β-catenin protein levels determined by Western blotting. The in vivo effects of hinokitiol were assessed by morphological and histological analyses following MIA injection. Results: Hinokitiol inhibited IL-1β-stimulated MMP-1,-3 and -13 expressions and IL-1β-induced activation of intracellular β-catenin proteins in cultured chondrocytes. In vivo, morphological and histological examinations demonstrated that hinokitiol significantly ameliorated cartilage degeneration. Conclusions: Hinokitiol is an effective anti-inflammatory reagent that acts by inhibiting the Wnt/β-catenin signaling pathway and could be a promising therapeutic agent for the prevention and treatment of osteoarthritis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Osteoarthritis (OA) is the most common type of joint disease [1] and can cause pain and disability in older adults [2]. OA is regarded as an age-related chronic disorder with a complex pathogenesis, especially in older females [3,4], that can be influenced by different factors, such as age, gender, obesity, trauma and prior joint injury [5]. However, the mechanisms mediating the onset and development of OA remain to be elucidated. The joints of OA patients and those of OA animal models have common characteristics, including degradation of articular cartilage and formation of osteophytes [6,7]. Apoptosis of chondrocytes and degradation of extracellular matrix leads to cartilage destruction [7,8]. Most cytological studies have reported excess production of inflammatory cytokines, such as interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) [9]. Among these cytokines, IL-1β has a pivotal role in cartilage matrix degradation, as it can stimulate chondrocytes to induce the expression of matrix metalloproteinases (MMPs), aggrecanases, and other catabolic
⁎ Corresponding authors at: Department of Orthopedics Surgery, The Second Hospital of Medical College, Zhejiang University, Hangzhou 310000, China. Tel./fax: +86 571 87783530. E-mail addresses: [email protected] (L. Wu), [email protected] (Y. Xiong). 1 Li-dong Wu and Yan Xiong contributed equally to this study and share first authorship, so Li-dong Wu and Yan Xiong are two co-corresponding authors.
http://dx.doi.org/10.1016/j.intimp.2014.08.012 1567-5769/© 2014 Elsevier B.V. All rights reserved.
enzymes [10]. MMP-1, -3 and -13 cleave collagen type II, which is directly and indirectly important to the extracellular matrix [11]. Consequently, it has been suggested that modulation of MMP activity may impede the development of OA. Non-steroidal anti-inflammatory drugs (NSAIDs) are the primary treatment for OA; however, NSAIDs ameliorate the clinical symptoms without inhibiting the progression of the disease [12]. Therefore, it is critical that effective agents are identified to cure OA. In the study, we describe the effects of hinokitiol, which has received attention from several investigators recently. Hinokitiol, also known as β-thujaplicin, is a natural tropolone-related compound found in the heartwood of Cupressaceae plants that has a wide range of biochemical and pharmacological activities [13]. It has been used in hair tonics, toothpastes, cosmetics, and food as an antimicrobial agent [14], and several studies have confirmed that it has marked anti-bacterial [15], anti-tumor [16], and neuroprotective activities [17], as well as antioxidant capacities [18]. However, the molecular mechanism responsible for the anti-inflammatory activity of hinokitiol remains poorly understood, and its potential benefits for the treatment and/or prevention of OA have received little attention. The Wnt/β-catenin signaling pathway has been investigated extensively in OA patients and animal models in which β-catenin protein is overexpressed [19]. However, the anti-inflammatory effect of hinokitiol in OA has not been reported. The aim of this study was to evaluate the anti-inflammatory effects of hinokitiol in vitro in cultured chondrocytes
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and in vivo in an animal model of OA. We found that hinokitiol downregulated MMP expression by attenuating Wnt/β-catenin signaling. These results have led us to explore a strategy of developing hinokitiol as a molecular-mechanism-based anti-inflammatory agent for OA.
2. Materials and methods 2.1. Reagents Hinokitiol, IL-1β, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin, penicillin, streptomycin, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and collagenase II were obtained from Gibco BRL (Grand Island, NY, USA). Hinokitiol was dissolved in DMSO and diluted with DMEM for the in vitro experiments and diluted with phosphatebuffered saline (PBS) for the in vivo experiments. Collagenase II was dissolved in DMEM and diluted to 1 mg/ml in DMEM to digest articular cartilage.
2.2. Cell culture Normal articular cartilage was isolated from the knees and femoral heads of 2-week-old rats from Zhejiang Academy of Medical Sciences (Hangzhou, China). Cartilages pieces were digested with 0.25% trypsin for 15 min and incubated with 1 mg/ml collagenase II at 37 °C for 3 h. The resulting cells were plated in 25-cm2 culture flasks in DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C with 5% CO2, designated passage 0 (P0). Confluent chondrocytes were passaged 1:3 with 0.25% trypsin, designated P1, P2 and P3, and the P3 cells were used for quantitative real-time polymerase chain reaction (PCR) and Western blotting analysis.
2.3. Cell treatment Before performing the experiments, all cell samples were serumstarved for 12 h. Chondrocytes were seeded in six-well plates at an initial density of 2 × 105/well for gene expression analysis and in 60-mm dishes at an initial density of 5 × 105/well for protein experiments. When chondrocytes reached 70–80% confluency, cells were pretreated with hinokitiol (10, 20, 40, 80 μM) for 2 h and then incubated with 5 ng/mL IL-1β for 24 h. Alternatively, cells were incubated with 80 μM hinokitiol, 20 ng/mL Dickkopf-1 (Dkk-1) or 20 mM Licl for 24 h.
2.4. Cell viability assay To select a suitable range of hinokitiol concentrations for the experiments, the cell viability effect of hinokitiol was evaluated by MTT assay. Cells were plated in three 96-well plates at a density of 6 × 103 cells/well and left to adhere overnight, followed by serum-starvation for 12 h and treatment with various concentrations of hinokitiol, Dkk-1, and Licl for 24, 48 and 72 h. After incubation with the drugs, 5 mg/mL MTT was added (20 μl/well) and cells were incubated for 4 h at 37 °C. The culture medium was then removed and DMSO was added (150 μl/well). The plates were shaken for 3 min to fully dissolve the crystals. Absorbance at 570 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). Six wells without hinokitiol and another six wells without cells were used as the control and blank, respectively. Cell viability at various concentrations was calculated using the following formula: cell viability rate = (average OD of hinokitiol-treated wells − average OD of blank wells) / (average OD of control wells − average OD of blank wells) × 100%, where OD = optical density [20].
2.5. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was isolated from different samples using TRIzol (Invitrogen Inc., Carlsbad, CA, USA). The PrimeScript-RT reagent kit (TaKaRa Biotechnology Co, Ltd., Japan) was used to reverse transcribe 500-ng total RNA to complementary DNA (cDNA). The amplification of MMP-1, -3, -13, Col2a1 and β-catenin was performed using the SYBR Premix Ex Taq kit (TaKaRa Biotechnology Co, Ltd., Japan). The specific primer sequences are shown in Table 1 and were designed by Sangon Biotech (Sangon Biotech (Shanghai) Co, Ltd. China). Sequence specificity was verified using the BLAST algorithm available online at the National Center for Biotechnology Information (NCBI). All experiments were performed according to manufacturer's protocol in triplicate using three independent samples. Quantitative real-time PCR data were calculated by the 2(−ΔΔCT) method [21]. 2.6. Western blotting analysis Samples were washed three times in ice-cold PBS and total proteins were extracted with 40-μl RIPA buffer containing 0.4-μl protease inhibitor for 30 min. Lysates were centrifuged at 13,000 ×g for 15 min and quantified with the Pierce BCA Protein Assay (Thermo Scientific, USA). A quarter volume of 5 × sodium dodecyl sulfate (SDS) loading buffer was mixed with the sample and boiled for 5 min. The samples (40 μg in 20 μl) were separated by SDS-polyacrylamide gel (PAGE) electrophoresis and then transferred to nitrocellulose membranes. After blocking with 5% non-fat milk in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) at room temperature for 1 h, the nitrocellulose membranes were cut into sections based on different protein molecular weights and incubated with diluted primary antibodies against β-actin (Dawen Biotec, Inc., China), MMP-1 (Dawen Biotec, Inc., China), MMP-3 (Proteintech Group, Inc., USA), MMP-13 (Bioworld Technology, Inc., USA), Col2a1 (Sangon Biotech (Shanghai) Co, Ltd. China) and β-catenin (Cell Signaling Technology, Inc., USA) overnight at 4 °C. After washing with TBST, the membranes were incubated with goat anti-mouse or goat anti-rabbit IgG-horseradish peroxidase (HRP)-labeled secondary antibodies (Proteintech Group, Inc., USA) at room temperature for 1 h. After washing with TBST buffer, immunoreactivity was detected with enhanced chemiluminescence (ECL), and densitometry performed using the Quantity One Software (Bio-Rad Laboratories Inc., Munich, Germany). 2.7. Animal study Eighteen 3-week old Sprague Dawley rats weighing 100–120 g were used (Animal Centre of Zhejiang University). All experiments were conducted with approval of the Zhejiang University Animal Care and Use Committee. The rats were divided randomly into A, B and C groups randomly. Group A was the control group, while groups B and C were the OA model groups. The rats were briefly anesthetized by intraperitoneal injection with pentobarbital sodium (3%, 0.1 ml/100 g). The B and C groups received an intra-articular injection of mono-iodoacetate (MIA) [22,23] diluted to 1 mg in 20 μL of 0.9% sterile saline [23]. Group A received a 20-μL injection in 0.9% sterile saline. The injections
Table 1 Primers of targeted genes. Gene 18S MMP-1
Forward
5′-TTGACGGAAGGGCACCA-3′ 5′-GGAACAGATACGAAGAGGAAACA3′ MMP-3 5′-GCATTGGCTGAGTGAAAGAGAC-3′ MMP-13 5′-TGAGAGTCATGCCAACAAATTC-3′ Col2a1 5′-CGAGGCAGACAGTACCTTGA-3′ β-Catenin 5′-CTTACGGCAATCAGGAAAGC-3′
Reverse 5′-CAGACAAATCGCTCCACCAA-3′ 5′-TGTGGGAATCAGAGGTAGAAGA-3′ 5′-ATGATGAACGATGGACAGATGA-3′ 5′-CAGCCACGCATAGTCATGTAGA-3′ 5′-TGCTCTCGATCTGGTTGTTC-3′ 5′-TAGAGCAGACAGACAGCACCTT-3′
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Fig. 1. Effects of hinokitiol on cell viability in normal chondrocytes. Chondrocytes were treated with increasing concentrations of hinokitiol for 24, 48 and 72 h. The cell viability was measured by MTT assay. Values are expressed as means ± SD from three independent experiments. Chondrocytes incubated with culture medium only were used as controls and considered 100% viable. There was no significant difference in cell viability between treatments (P N 0.05).
were repeated once per week for 4 weeks. One month after the first injection, group A received 20-μl 0.9% sterile saline into the articular cavity. Group B received 20-μl 0.1% DMSO in 0.9% sterile saline and group C received 20-μl 80 μM Hinokitiol in 0.9% sterile saline. Then group A was marked as control group, group B was marked as OA
group, and group C was marked as Hinokitiol-treated group. The injections were repeated once per week for 4 weeks. All rats were sacrificed 7 days after the last injection. The knee joints were subjected to assessment in terms of their gross morphology, histology and gene expression levels.
Fig. 2. Effects of hinokitiol on MMP-1, -3, -13, Col2a1 and β-catenin gene expression in chondrocytes and cartilage. Chondrocytes were pretreated with increasing concentrations of hinokitiol for 2 h, followed by stimulation with 5 ng/ml IL-1β for 24 h. Cartilage from animals in control group, OA group and hinokitiol-treated group was cut into pieces and ground in LN2. Total RNA was isolated and followed by qRT-PCR for determination of relative gene expression levels. Data are expressed as means ± SD. *P b 0.05, **P b 0.01, ***P b 0.001 compared to cells stimulated with IL-1β alone or the OA group.
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2.8. Histological examination
3.2. Effect of hinokitiol on gene expression in chondrocytes and cartilage
Cartilage was harvested from the femoral condyles of each group. The samples were fixed in 4% paraformaldehyde for 4 days and decalcified with 10% buffered formic acid for 2 months [24]. Decalcified specimens were dehydrated in alcohol, embedded in paraffin blocks and sliced into 5-μm sections. The sections were stained with hematoxylin-eosin (HE) and safranin O-fast green. The Mankin scoring system was used for blinded histologic assessment.
The mRNA expression of MMP-1, 3, 13 and β-catenin was up-regulated in chondrocytes treated with IL-1β for 24 h. Hinokitiol significantly inhibited IL-1β stimulation of MMP-1, 3, 13 and β-catenin (P b 0.05) in a dose-dependent manner compared with IL-1β alone. However, that of Col2a1 did not change significantly (P N 0.05; Fig. 2a). There were similar changes in the cartilage. Hinokitiol restrained the expression of the MMP-1, -3, -13 and β-catenin compared with the OA group (P b 0.05), whereas Col2a1 was not up-regulated significantly (P N 0.05; Fig. 2b).
2.9. Statistical analysis All data were expressed as means ± standard deviation (SD) and assessed by one-way analysis of variance (ANOVA) followed by Dunnett's analysis. Differences were considered significant when P b 0.05. 3. Results 3.1. Effects of hinokitiol on chondrocyte viability The viability effect of hinokitiol on normal chondrocytes was assessed by treating cells with increasing concentrations (10–80 μM) for 24, 48 and 72 h and assessing cell viability by MTT assay. OD (optical density) was measured and cell viability was calculated. The results showed no significant cytotoxicity of hinokitiol against normal chondrocytes (P N 0.05; Fig. 1).
Fig. 3. Effects of hinokitiol on MMP-1, -3, -13, Col2a1 and β-catenin protein levels in chondrocytes. Chondrocytes were pretreated with increasing concentrations of hinokitiol for 2 h, followed by stimulation with 5 ng/ml IL-1β for 24 h. Samples were harvested for Western blotting analysis and immunoreactivity was analyzed by densitometry. Data are expressed as means ± SD. **P b 0.01, ***P b 0.001 compared to cells stimulated with IL-1β alone.
3.3. Effects of hinokitiol on protein levels in chondrocytes The MMP-1, 3, 13 and β-catenin protein levels were increased in chondrocytes following a 24-h stimulation with IL-1β. The addition of hinokitiol reduced IL-1β-stimulated production of MMP-1, 3, 13 and β-catenin (P b 0.05); however, Col2a1 protein levels were upregulated significantly (P b 0.05), which did not correlate with Col2a1 mRNA expression. Hinokitiol down-regulated the MMP-1, -3, -13 and β-catenin protein levels and increased the relative expression of Col2a1 (Fig. 3). In addition, the MMP-1, 3, 13 and β-catenin proteins levels were up-regulated by stimulation with Licl and down-regulated by stimulation with Dkk-1 and Licl or hinokitiol and Licl, although the effects of Dkk-1 were more pronounced that those of hinokitiol
Fig. 4. Effects of Licl, Dkk-1 and hinokitiol on MMP-1, 3, 13 and β-catenin protein levels in chondrocytes. Chondrocytes were treated with 20 mM Licl, 20 ng/ml Dkk-1 and/or 80 μM hinokitiol for 24 h and harvested for Western blotting analysis. Immunoreactivity was analyzed by densitometry. Data are expressed as means ± SD. §§§P b 0.001 compared to the normal chondrocytes; **P b 0.01, ***P b 0.001 compared to the cells stimulated with Licl alone; ##P b 0.01, ###P b 0.001 compared to the cells treated with Dkk-1 and hinokitiol with Licl or without.
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(Fig. 4). These results suggest that hinokitiol can reduce the expression of MMP-1, 3 and 13 through inhibiting Wnt/β-catenin signaling, but the effect was not greater than Dkk-1. 3.4. Macroscopic observations Rats in the OA and hinokitiol-treated group exhibited varying degrees of cartilage degradation compared with the control group. In the control group, cartilage from the femoral condyles was glossy with no defects or osteophytes. In the OA group, the cartilage surface was coarse with serious defects that had adhered to other connective tissues. In the hinokitiol-treated group, the cartilage damage was less severe compared to that in the OA group (Fig. 5a). 3.5. Histopathological changes in articular cartilage Histopathological changes in the OA and hinokitiol-treated rats were concentrated on the cartilage surface and cartilage matrix layer. HE staining demonstrated that the articular surface was intact and smooth, and the cartilage matrix was stained purple, while chondrocyte staining was darker. Cartilage lacunas were observed in the control group, while in the OA group, the cartilage structure was badly damaged and the cartilage matrix was thinner or not detected. In the hinokitiol-treated group, the cartilage structure remained complete, although the matrix was thinner, with lighter HE staining (Fig. 5b). Safranin O and fast green staining resulted in crimson staining of the cartilage matrix in the control group that was reduced in the OA and hinokitiol-treated groups. However, cartilage degeneration was not reversed (Fig. 5c). The Mankin scoring system [25] was used in histological analyses to compare the hinokitiol-treated group with the OA group (Table 2).
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Table 2 Histological score of articular cartilage. Femoral condyle
Control group
OA group
Structural changes Cellular changes Safranin staining Tide mark Sum of score
0.10 0.20 0.50 0.10 0.90
3.83 2.76 2.77 0.58 9.94
± ± ± ± ±
0.04 0.07 0.11 0.04 0.18
± ± ± ± ±
0.29⁎ 0.54⁎ 0.44⁎ 0.29⁎ 1.27⁎
Hinokitiol group 1.88 1.43 1.15 0.20 4.66
± ± ± ± ±
0.25⁎⁎ 0.31⁎⁎ 0.28⁎⁎ 0.25⁎⁎ 0.67⁎⁎
Mankin scoring system was frequently used for evaluating the severity of OA by structural changes, cellular changes, safranin staining and tide mark. The scores were positively correlated with the severity of OA. Values are mean ± SD. ⁎ P b 0.001 compared with control group. ⁎⁎ P b 0.001 compared with OA group.
Mankin scoring system described by Mankin et al. was frequently used in the histopathological classification of the severity of lesions of cartilage through structural changes, cellular changes, safranin staining and tide mark.
4. Discussion OA is regarded as a non-inflammatory arthropathy with symptoms of local inflammation characterized primarily by cartilage degradation [26]. Current medications alleviate the primary symptoms of swelling, pain and muscle tightness but these agents do not cure or reverse the progression of OA. Therefore, the identification of a drug that will prevent or inhibit the progression of OA would be a significant advance in patient care. Evidence has shown that hinokitiol possesses multiple biological functions, including anti-inflammatory effects, via various signaling pathways, such as the mitogen-activated protein kinase
Fig. 5. An in vivo study was performed using Sprague–Dawley rats divided randomly into three groups (n = 6/group). Group A was the control group, group B was the OA group and group C was the hinokitiol-treated group. (a) Representative photographs of macroscopic observations, (b) hematoxylin-eosin staining, (c) cafranin O staining. The original magnification of (b) and (c) was ×100.
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(MAPK) pathway [27]. In this study, we examined whether hinokitiol had anti-osteoarthritic activity on chondrocytes induced by IL-1β and in an OA rat model induced by injection with MIA into the knee joints. The results demonstrated that hinokitiol had significant dosedependent effects on down-regulated MMP-1, -3 and -13 expressions, which associated with inhibition of Wnt/β-catenin activation. MMPs have prominent roles in OA as components of tissue destruction . MMPs are induced by a range of inflammatory cytokines, including IL-1, -4, -6, -10 and TNFα. MMPs are a family of endopeptidases capable of degrading all components of the extracellular matrix. MMP-1 is also known as collagenase-1, while MMP-3 is known as collagenase-3. These two collagenases degrade collagen Type II. MMP-3 cannot cleavage collagen Type II directly but it plays a vital role in activation of the other MMPs, such as MMP-1 and MMP-13, which are pro-MMPs that directly cleave collagenases . Many studies have demonstrated that inhibition of MMP expression inhibit the progression of cartilage degradation in vitro and in vivo. As expected in this study, hinokitiol reduced the synthesis of MMP-1, 3 and 13 induced by IL-1β or Licl in chondrocytes in vitro, and delayed the regression of cartilago articularis in vivo in a OA model caused by MIA injection. Several signaling pathways are involved in the induction of MMPs by IL-1β in chondrocytes, including MAPK, nuclear factor-κB (NF-κB), and Wnt/β-catenin signaling. Among these pathways, Wnt/β-catenin signaling has attracted much attention. Wnt signaling is involved in embryonic development of cartilage and bone homeostasis, and is considered important in joint remodeling [28]. Moreover, data shows that β-catenin was overexpressed in joint tissues from OA patients and in animal models of disease. Dkk-1 is a secreted Wnt inhibitor [29] that binds to lipoprotein-receptor-related proteins 5 and 6 (LRP5/6). LRP5/ 6 stabilizes intracellular β-catenin and single transmembrane protein called Kremen to form a ternary complex that is rapidly internalized and degraded [30]. Licl is used to treat mood disorders and plays a central role in Wnt signaling by inhibiting glycogen synthase kinase-3β (GSK-3β) to activate β-catenin/TCF transcription [31]. Our results demonstrated that hinokitiol reduced the expression of β-catenin induced by IL-1β or Licl. Hence, we investigated whether the anti-inflammatory function of hinokitiol was related to Wnt/β-catenin signaling. Western blotting analysis determined that hinokitiol suppressed MMPs and β-catenin expression stimulated with Licl in chondrocytes and hinokitiol suppressed β-catenin expression in normal chondrocytes. This suggests that the inhibitory effect of hinokitiol on MMPs expression is attributable to inhibition of Wnt/β-catenin signaling, but the inhibitory effect of hinokitiol is weaker than that of Dkk-1. Moreover, we detect that the inhibitory effect of Dkk-1 seems to be slightly recovered by adding hinokitiol (result was not showed). It is hard to explain the result according to available evidence. Further experiments are necessary to discover the mechanism of hinokitiol. We also investigated the protective effect of hinokitiol against cartilage degradation in a rat OA model induced by MIA. Injection of 80 μM hinokitiol into the articular cavity reduced cartilage degeneration during OA progression and inhibited expression of MMP-1, -13 and β-catenin; however, the expression of Col2a1 was increased, which is consistent with the in vitro effects of hinokitiol. In conclusion, the results of our study demonstrate that hinokitiol significantly inhibits the IL-1β-induced expression of MMP-1, -3 and -13 in chondrocytes. These in vitro results indicate that the anti-inflammatory effects of hinokitiol should be brought about by Wnt/β-catenin pathway inhibition. The results suggest that hinokitiol has been promised as a therapeutic drug for OA.
Acknowledgments This study was supported by the National Natural Science Foundation of China (81201429) and the Zhejiang Provincial Natural Science Foundation of China (Y2110448).
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Hinokitiol reduces matrix metalloproteinase expression by inhibiting Wnt/β-Catenin signaling in vitro and in vivo Jin Li, Xin-die Zhou, Kong-he Yang, Teng-di Fan, Wei-ping Chen, Li-feng Jiang, Jia-peng Bao, Li-dong Wu ⁎,1, Yan Xiong ⁎,1 Department of Orthopedics Surgery, The Second Affiliated Hospital of Medical College, Zhejiang University, Hangzhou 310000, China
a r t i c l e
i n f o
Article history: Received 9 April 2014 Received in revised form 7 August 2014 Accepted 13 August 2014 Available online xxxx Keywords: Osteoarthritis Hinokitiol Matrix metalloproteinase β-catenin
a b s t r a c t Objective: In this study, we investigated the effects of hinokitiol on matrix metalloproteinase (MMP)-1, -3, -13, collagen type II (Col2a1) and β-catenin expressions in rat chondrocytes induced by interleukin-1β and in an experimental rat model induced by intra-articular injection of mono-iodoacetate (MIA) into the knee. Methods: Chondrocytes were cultured from the articular cartilage of 2-week-old rats. Passaged chondrocytes were pretreated with hinokitiol for 2 h followed by co-incubation with IL-1β for 24 h. Quantitative real-time polymerase chain reaction and Western blotting were used to assess the expression of MMP-1, -3, -13, Col2a1 and β-catenin. Chondrocytes were also treated with Licl, Dickkopf-1, and/or hinokitiol for 24 h, the MMP-1, -3, -13 and β-catenin protein levels determined by Western blotting. The in vivo effects of hinokitiol were assessed by morphological and histological analyses following MIA injection. Results: Hinokitiol inhibited IL-1β-stimulated MMP-1,-3 and -13 expressions and IL-1β-induced activation of intracellular β-catenin proteins in cultured chondrocytes. In vivo, morphological and histological examinations demonstrated that hinokitiol significantly ameliorated cartilage degeneration. Conclusions: Hinokitiol is an effective anti-inflammatory reagent that acts by inhibiting the Wnt/β-catenin signaling pathway and could be a promising therapeutic agent for the prevention and treatment of osteoarthritis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Osteoarthritis (OA) is the most common type of joint disease [1] and can cause pain and disability in older adults [2]. OA is regarded as an age-related chronic disorder with a complex pathogenesis, especially in older females [3,4], that can be influenced by different factors, such as age, gender, obesity, trauma and prior joint injury [5]. However, the mechanisms mediating the onset and development of OA remain to be elucidated. The joints of OA patients and those of OA animal models have common characteristics, including degradation of articular cartilage and formation of osteophytes [6,7]. Apoptosis of chondrocytes and degradation of extracellular matrix leads to cartilage destruction [7,8]. Most cytological studies have reported excess production of inflammatory cytokines, such as interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) [9]. Among these cytokines, IL-1β has a pivotal role in cartilage matrix degradation, as it can stimulate chondrocytes to induce the expression of matrix metalloproteinases (MMPs), aggrecanases, and other catabolic
⁎ Corresponding authors at: Department of Orthopedics Surgery, The Second Hospital of Medical College, Zhejiang University, Hangzhou 310000, China. Tel./fax: +86 571 87783530. E-mail addresses: [email protected] (L. Wu), [email protected] (Y. Xiong). 1 Li-dong Wu and Yan Xiong contributed equally to this study and share first authorship, so Li-dong Wu and Yan Xiong are two co-corresponding authors.
http://dx.doi.org/10.1016/j.intimp.2014.08.012 1567-5769/© 2014 Elsevier B.V. All rights reserved.
enzymes [10]. MMP-1, -3 and -13 cleave collagen type II, which is directly and indirectly important to the extracellular matrix [11]. Consequently, it has been suggested that modulation of MMP activity may impede the development of OA. Non-steroidal anti-inflammatory drugs (NSAIDs) are the primary treatment for OA; however, NSAIDs ameliorate the clinical symptoms without inhibiting the progression of the disease [12]. Therefore, it is critical that effective agents are identified to cure OA. In the study, we describe the effects of hinokitiol, which has received attention from several investigators recently. Hinokitiol, also known as β-thujaplicin, is a natural tropolone-related compound found in the heartwood of Cupressaceae plants that has a wide range of biochemical and pharmacological activities [13]. It has been used in hair tonics, toothpastes, cosmetics, and food as an antimicrobial agent [14], and several studies have confirmed that it has marked anti-bacterial [15], anti-tumor [16], and neuroprotective activities [17], as well as antioxidant capacities [18]. However, the molecular mechanism responsible for the anti-inflammatory activity of hinokitiol remains poorly understood, and its potential benefits for the treatment and/or prevention of OA have received little attention. The Wnt/β-catenin signaling pathway has been investigated extensively in OA patients and animal models in which β-catenin protein is overexpressed [19]. However, the anti-inflammatory effect of hinokitiol in OA has not been reported. The aim of this study was to evaluate the anti-inflammatory effects of hinokitiol in vitro in cultured chondrocytes
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and in vivo in an animal model of OA. We found that hinokitiol downregulated MMP expression by attenuating Wnt/β-catenin signaling. These results have led us to explore a strategy of developing hinokitiol as a molecular-mechanism-based anti-inflammatory agent for OA.
2. Materials and methods 2.1. Reagents Hinokitiol, IL-1β, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin, penicillin, streptomycin, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) and collagenase II were obtained from Gibco BRL (Grand Island, NY, USA). Hinokitiol was dissolved in DMSO and diluted with DMEM for the in vitro experiments and diluted with phosphatebuffered saline (PBS) for the in vivo experiments. Collagenase II was dissolved in DMEM and diluted to 1 mg/ml in DMEM to digest articular cartilage.
2.2. Cell culture Normal articular cartilage was isolated from the knees and femoral heads of 2-week-old rats from Zhejiang Academy of Medical Sciences (Hangzhou, China). Cartilages pieces were digested with 0.25% trypsin for 15 min and incubated with 1 mg/ml collagenase II at 37 °C for 3 h. The resulting cells were plated in 25-cm2 culture flasks in DMEM containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C with 5% CO2, designated passage 0 (P0). Confluent chondrocytes were passaged 1:3 with 0.25% trypsin, designated P1, P2 and P3, and the P3 cells were used for quantitative real-time polymerase chain reaction (PCR) and Western blotting analysis.
2.3. Cell treatment Before performing the experiments, all cell samples were serumstarved for 12 h. Chondrocytes were seeded in six-well plates at an initial density of 2 × 105/well for gene expression analysis and in 60-mm dishes at an initial density of 5 × 105/well for protein experiments. When chondrocytes reached 70–80% confluency, cells were pretreated with hinokitiol (10, 20, 40, 80 μM) for 2 h and then incubated with 5 ng/mL IL-1β for 24 h. Alternatively, cells were incubated with 80 μM hinokitiol, 20 ng/mL Dickkopf-1 (Dkk-1) or 20 mM Licl for 24 h.
2.4. Cell viability assay To select a suitable range of hinokitiol concentrations for the experiments, the cell viability effect of hinokitiol was evaluated by MTT assay. Cells were plated in three 96-well plates at a density of 6 × 103 cells/well and left to adhere overnight, followed by serum-starvation for 12 h and treatment with various concentrations of hinokitiol, Dkk-1, and Licl for 24, 48 and 72 h. After incubation with the drugs, 5 mg/mL MTT was added (20 μl/well) and cells were incubated for 4 h at 37 °C. The culture medium was then removed and DMSO was added (150 μl/well). The plates were shaken for 3 min to fully dissolve the crystals. Absorbance at 570 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). Six wells without hinokitiol and another six wells without cells were used as the control and blank, respectively. Cell viability at various concentrations was calculated using the following formula: cell viability rate = (average OD of hinokitiol-treated wells − average OD of blank wells) / (average OD of control wells − average OD of blank wells) × 100%, where OD = optical density [20].
2.5. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was isolated from different samples using TRIzol (Invitrogen Inc., Carlsbad, CA, USA). The PrimeScript-RT reagent kit (TaKaRa Biotechnology Co, Ltd., Japan) was used to reverse transcribe 500-ng total RNA to complementary DNA (cDNA). The amplification of MMP-1, -3, -13, Col2a1 and β-catenin was performed using the SYBR Premix Ex Taq kit (TaKaRa Biotechnology Co, Ltd., Japan). The specific primer sequences are shown in Table 1 and were designed by Sangon Biotech (Sangon Biotech (Shanghai) Co, Ltd. China). Sequence specificity was verified using the BLAST algorithm available online at the National Center for Biotechnology Information (NCBI). All experiments were performed according to manufacturer's protocol in triplicate using three independent samples. Quantitative real-time PCR data were calculated by the 2(−ΔΔCT) method [21]. 2.6. Western blotting analysis Samples were washed three times in ice-cold PBS and total proteins were extracted with 40-μl RIPA buffer containing 0.4-μl protease inhibitor for 30 min. Lysates were centrifuged at 13,000 ×g for 15 min and quantified with the Pierce BCA Protein Assay (Thermo Scientific, USA). A quarter volume of 5 × sodium dodecyl sulfate (SDS) loading buffer was mixed with the sample and boiled for 5 min. The samples (40 μg in 20 μl) were separated by SDS-polyacrylamide gel (PAGE) electrophoresis and then transferred to nitrocellulose membranes. After blocking with 5% non-fat milk in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) at room temperature for 1 h, the nitrocellulose membranes were cut into sections based on different protein molecular weights and incubated with diluted primary antibodies against β-actin (Dawen Biotec, Inc., China), MMP-1 (Dawen Biotec, Inc., China), MMP-3 (Proteintech Group, Inc., USA), MMP-13 (Bioworld Technology, Inc., USA), Col2a1 (Sangon Biotech (Shanghai) Co, Ltd. China) and β-catenin (Cell Signaling Technology, Inc., USA) overnight at 4 °C. After washing with TBST, the membranes were incubated with goat anti-mouse or goat anti-rabbit IgG-horseradish peroxidase (HRP)-labeled secondary antibodies (Proteintech Group, Inc., USA) at room temperature for 1 h. After washing with TBST buffer, immunoreactivity was detected with enhanced chemiluminescence (ECL), and densitometry performed using the Quantity One Software (Bio-Rad Laboratories Inc., Munich, Germany). 2.7. Animal study Eighteen 3-week old Sprague Dawley rats weighing 100–120 g were used (Animal Centre of Zhejiang University). All experiments were conducted with approval of the Zhejiang University Animal Care and Use Committee. The rats were divided randomly into A, B and C groups randomly. Group A was the control group, while groups B and C were the OA model groups. The rats were briefly anesthetized by intraperitoneal injection with pentobarbital sodium (3%, 0.1 ml/100 g). The B and C groups received an intra-articular injection of mono-iodoacetate (MIA) [22,23] diluted to 1 mg in 20 μL of 0.9% sterile saline [23]. Group A received a 20-μL injection in 0.9% sterile saline. The injections
Table 1 Primers of targeted genes. Gene 18S MMP-1
Forward
5′-TTGACGGAAGGGCACCA-3′ 5′-GGAACAGATACGAAGAGGAAACA3′ MMP-3 5′-GCATTGGCTGAGTGAAAGAGAC-3′ MMP-13 5′-TGAGAGTCATGCCAACAAATTC-3′ Col2a1 5′-CGAGGCAGACAGTACCTTGA-3′ β-Catenin 5′-CTTACGGCAATCAGGAAAGC-3′
Reverse 5′-CAGACAAATCGCTCCACCAA-3′ 5′-TGTGGGAATCAGAGGTAGAAGA-3′ 5′-ATGATGAACGATGGACAGATGA-3′ 5′-CAGCCACGCATAGTCATGTAGA-3′ 5′-TGCTCTCGATCTGGTTGTTC-3′ 5′-TAGAGCAGACAGACAGCACCTT-3′
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Fig. 1. Effects of hinokitiol on cell viability in normal chondrocytes. Chondrocytes were treated with increasing concentrations of hinokitiol for 24, 48 and 72 h. The cell viability was measured by MTT assay. Values are expressed as means ± SD from three independent experiments. Chondrocytes incubated with culture medium only were used as controls and considered 100% viable. There was no significant difference in cell viability between treatments (P N 0.05).
were repeated once per week for 4 weeks. One month after the first injection, group A received 20-μl 0.9% sterile saline into the articular cavity. Group B received 20-μl 0.1% DMSO in 0.9% sterile saline and group C received 20-μl 80 μM Hinokitiol in 0.9% sterile saline. Then group A was marked as control group, group B was marked as OA
group, and group C was marked as Hinokitiol-treated group. The injections were repeated once per week for 4 weeks. All rats were sacrificed 7 days after the last injection. The knee joints were subjected to assessment in terms of their gross morphology, histology and gene expression levels.
Fig. 2. Effects of hinokitiol on MMP-1, -3, -13, Col2a1 and β-catenin gene expression in chondrocytes and cartilage. Chondrocytes were pretreated with increasing concentrations of hinokitiol for 2 h, followed by stimulation with 5 ng/ml IL-1β for 24 h. Cartilage from animals in control group, OA group and hinokitiol-treated group was cut into pieces and ground in LN2. Total RNA was isolated and followed by qRT-PCR for determination of relative gene expression levels. Data are expressed as means ± SD. *P b 0.05, **P b 0.01, ***P b 0.001 compared to cells stimulated with IL-1β alone or the OA group.
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2.8. Histological examination
3.2. Effect of hinokitiol on gene expression in chondrocytes and cartilage
Cartilage was harvested from the femoral condyles of each group. The samples were fixed in 4% paraformaldehyde for 4 days and decalcified with 10% buffered formic acid for 2 months [24]. Decalcified specimens were dehydrated in alcohol, embedded in paraffin blocks and sliced into 5-μm sections. The sections were stained with hematoxylin-eosin (HE) and safranin O-fast green. The Mankin scoring system was used for blinded histologic assessment.
The mRNA expression of MMP-1, 3, 13 and β-catenin was up-regulated in chondrocytes treated with IL-1β for 24 h. Hinokitiol significantly inhibited IL-1β stimulation of MMP-1, 3, 13 and β-catenin (P b 0.05) in a dose-dependent manner compared with IL-1β alone. However, that of Col2a1 did not change significantly (P N 0.05; Fig. 2a). There were similar changes in the cartilage. Hinokitiol restrained the expression of the MMP-1, -3, -13 and β-catenin compared with the OA group (P b 0.05), whereas Col2a1 was not up-regulated significantly (P N 0.05; Fig. 2b).
2.9. Statistical analysis All data were expressed as means ± standard deviation (SD) and assessed by one-way analysis of variance (ANOVA) followed by Dunnett's analysis. Differences were considered significant when P b 0.05. 3. Results 3.1. Effects of hinokitiol on chondrocyte viability The viability effect of hinokitiol on normal chondrocytes was assessed by treating cells with increasing concentrations (10–80 μM) for 24, 48 and 72 h and assessing cell viability by MTT assay. OD (optical density) was measured and cell viability was calculated. The results showed no significant cytotoxicity of hinokitiol against normal chondrocytes (P N 0.05; Fig. 1).
Fig. 3. Effects of hinokitiol on MMP-1, -3, -13, Col2a1 and β-catenin protein levels in chondrocytes. Chondrocytes were pretreated with increasing concentrations of hinokitiol for 2 h, followed by stimulation with 5 ng/ml IL-1β for 24 h. Samples were harvested for Western blotting analysis and immunoreactivity was analyzed by densitometry. Data are expressed as means ± SD. **P b 0.01, ***P b 0.001 compared to cells stimulated with IL-1β alone.
3.3. Effects of hinokitiol on protein levels in chondrocytes The MMP-1, 3, 13 and β-catenin protein levels were increased in chondrocytes following a 24-h stimulation with IL-1β. The addition of hinokitiol reduced IL-1β-stimulated production of MMP-1, 3, 13 and β-catenin (P b 0.05); however, Col2a1 protein levels were upregulated significantly (P b 0.05), which did not correlate with Col2a1 mRNA expression. Hinokitiol down-regulated the MMP-1, -3, -13 and β-catenin protein levels and increased the relative expression of Col2a1 (Fig. 3). In addition, the MMP-1, 3, 13 and β-catenin proteins levels were up-regulated by stimulation with Licl and down-regulated by stimulation with Dkk-1 and Licl or hinokitiol and Licl, although the effects of Dkk-1 were more pronounced that those of hinokitiol
Fig. 4. Effects of Licl, Dkk-1 and hinokitiol on MMP-1, 3, 13 and β-catenin protein levels in chondrocytes. Chondrocytes were treated with 20 mM Licl, 20 ng/ml Dkk-1 and/or 80 μM hinokitiol for 24 h and harvested for Western blotting analysis. Immunoreactivity was analyzed by densitometry. Data are expressed as means ± SD. §§§P b 0.001 compared to the normal chondrocytes; **P b 0.01, ***P b 0.001 compared to the cells stimulated with Licl alone; ##P b 0.01, ###P b 0.001 compared to the cells treated with Dkk-1 and hinokitiol with Licl or without.
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(Fig. 4). These results suggest that hinokitiol can reduce the expression of MMP-1, 3 and 13 through inhibiting Wnt/β-catenin signaling, but the effect was not greater than Dkk-1. 3.4. Macroscopic observations Rats in the OA and hinokitiol-treated group exhibited varying degrees of cartilage degradation compared with the control group. In the control group, cartilage from the femoral condyles was glossy with no defects or osteophytes. In the OA group, the cartilage surface was coarse with serious defects that had adhered to other connective tissues. In the hinokitiol-treated group, the cartilage damage was less severe compared to that in the OA group (Fig. 5a). 3.5. Histopathological changes in articular cartilage Histopathological changes in the OA and hinokitiol-treated rats were concentrated on the cartilage surface and cartilage matrix layer. HE staining demonstrated that the articular surface was intact and smooth, and the cartilage matrix was stained purple, while chondrocyte staining was darker. Cartilage lacunas were observed in the control group, while in the OA group, the cartilage structure was badly damaged and the cartilage matrix was thinner or not detected. In the hinokitiol-treated group, the cartilage structure remained complete, although the matrix was thinner, with lighter HE staining (Fig. 5b). Safranin O and fast green staining resulted in crimson staining of the cartilage matrix in the control group that was reduced in the OA and hinokitiol-treated groups. However, cartilage degeneration was not reversed (Fig. 5c). The Mankin scoring system [25] was used in histological analyses to compare the hinokitiol-treated group with the OA group (Table 2).
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Table 2 Histological score of articular cartilage. Femoral condyle
Control group
OA group
Structural changes Cellular changes Safranin staining Tide mark Sum of score
0.10 0.20 0.50 0.10 0.90
3.83 2.76 2.77 0.58 9.94
± ± ± ± ±
0.04 0.07 0.11 0.04 0.18
± ± ± ± ±
0.29⁎ 0.54⁎ 0.44⁎ 0.29⁎ 1.27⁎
Hinokitiol group 1.88 1.43 1.15 0.20 4.66
± ± ± ± ±
0.25⁎⁎ 0.31⁎⁎ 0.28⁎⁎ 0.25⁎⁎ 0.67⁎⁎
Mankin scoring system was frequently used for evaluating the severity of OA by structural changes, cellular changes, safranin staining and tide mark. The scores were positively correlated with the severity of OA. Values are mean ± SD. ⁎ P b 0.001 compared with control group. ⁎⁎ P b 0.001 compared with OA group.
Mankin scoring system described by Mankin et al. was frequently used in the histopathological classification of the severity of lesions of cartilage through structural changes, cellular changes, safranin staining and tide mark.
4. Discussion OA is regarded as a non-inflammatory arthropathy with symptoms of local inflammation characterized primarily by cartilage degradation [26]. Current medications alleviate the primary symptoms of swelling, pain and muscle tightness but these agents do not cure or reverse the progression of OA. Therefore, the identification of a drug that will prevent or inhibit the progression of OA would be a significant advance in patient care. Evidence has shown that hinokitiol possesses multiple biological functions, including anti-inflammatory effects, via various signaling pathways, such as the mitogen-activated protein kinase
Fig. 5. An in vivo study was performed using Sprague–Dawley rats divided randomly into three groups (n = 6/group). Group A was the control group, group B was the OA group and group C was the hinokitiol-treated group. (a) Representative photographs of macroscopic observations, (b) hematoxylin-eosin staining, (c) cafranin O staining. The original magnification of (b) and (c) was ×100.
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(MAPK) pathway [27]. In this study, we examined whether hinokitiol had anti-osteoarthritic activity on chondrocytes induced by IL-1β and in an OA rat model induced by injection with MIA into the knee joints. The results demonstrated that hinokitiol had significant dosedependent effects on down-regulated MMP-1, -3 and -13 expressions, which associated with inhibition of Wnt/β-catenin activation. MMPs have prominent roles in OA as components of tissue destruction . MMPs are induced by a range of inflammatory cytokines, including IL-1, -4, -6, -10 and TNFα. MMPs are a family of endopeptidases capable of degrading all components of the extracellular matrix. MMP-1 is also known as collagenase-1, while MMP-3 is known as collagenase-3. These two collagenases degrade collagen Type II. MMP-3 cannot cleavage collagen Type II directly but it plays a vital role in activation of the other MMPs, such as MMP-1 and MMP-13, which are pro-MMPs that directly cleave collagenases . Many studies have demonstrated that inhibition of MMP expression inhibit the progression of cartilage degradation in vitro and in vivo. As expected in this study, hinokitiol reduced the synthesis of MMP-1, 3 and 13 induced by IL-1β or Licl in chondrocytes in vitro, and delayed the regression of cartilago articularis in vivo in a OA model caused by MIA injection. Several signaling pathways are involved in the induction of MMPs by IL-1β in chondrocytes, including MAPK, nuclear factor-κB (NF-κB), and Wnt/β-catenin signaling. Among these pathways, Wnt/β-catenin signaling has attracted much attention. Wnt signaling is involved in embryonic development of cartilage and bone homeostasis, and is considered important in joint remodeling [28]. Moreover, data shows that β-catenin was overexpressed in joint tissues from OA patients and in animal models of disease. Dkk-1 is a secreted Wnt inhibitor [29] that binds to lipoprotein-receptor-related proteins 5 and 6 (LRP5/6). LRP5/ 6 stabilizes intracellular β-catenin and single transmembrane protein called Kremen to form a ternary complex that is rapidly internalized and degraded [30]. Licl is used to treat mood disorders and plays a central role in Wnt signaling by inhibiting glycogen synthase kinase-3β (GSK-3β) to activate β-catenin/TCF transcription [31]. Our results demonstrated that hinokitiol reduced the expression of β-catenin induced by IL-1β or Licl. Hence, we investigated whether the anti-inflammatory function of hinokitiol was related to Wnt/β-catenin signaling. Western blotting analysis determined that hinokitiol suppressed MMPs and β-catenin expression stimulated with Licl in chondrocytes and hinokitiol suppressed β-catenin expression in normal chondrocytes. This suggests that the inhibitory effect of hinokitiol on MMPs expression is attributable to inhibition of Wnt/β-catenin signaling, but the inhibitory effect of hinokitiol is weaker than that of Dkk-1. Moreover, we detect that the inhibitory effect of Dkk-1 seems to be slightly recovered by adding hinokitiol (result was not showed). It is hard to explain the result according to available evidence. Further experiments are necessary to discover the mechanism of hinokitiol. We also investigated the protective effect of hinokitiol against cartilage degradation in a rat OA model induced by MIA. Injection of 80 μM hinokitiol into the articular cavity reduced cartilage degeneration during OA progression and inhibited expression of MMP-1, -13 and β-catenin; however, the expression of Col2a1 was increased, which is consistent with the in vitro effects of hinokitiol. In conclusion, the results of our study demonstrate that hinokitiol significantly inhibits the IL-1β-induced expression of MMP-1, -3 and -13 in chondrocytes. These in vitro results indicate that the anti-inflammatory effects of hinokitiol should be brought about by Wnt/β-catenin pathway inhibition. The results suggest that hinokitiol has been promised as a therapeutic drug for OA.
Acknowledgments This study was supported by the National Natural Science Foundation of China (81201429) and the Zhejiang Provincial Natural Science Foundation of China (Y2110448).
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