Chitosan oligosaccharide suppresses synovial inflammation via AMPK activation: An in vitro and in vivo study

Pharmacological Research 113 (2016) 458–467

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Chitosan oligosaccharide suppresses synovial inflammation via AMPK activation: An in vitro and in vivo study Wanlop Kunanusornchai a , Bhee Witoonpanich b , Rath Pichyangkura c , Varanuj Chatsudthipong a , Chatchai Muanprasat a,d,∗ a

Department of Physiology, Faculty of Science, Mahidol University, Rama VI Road, Rajathevi, Bangkok 10400, Thailand Department of Orthopedics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Rama VI Road, Rajathevi, Bangkok 10400, Thailand c Department of Biochemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Phayathai, Bangkok 10330, Thailand d Excellent Center for Drug Discovery (ECDD), Faculty of Science, Mahidol University, Rama VI Road, Rajathevi, Bangkok 10400, Thailand b

a r t i c l e

i n f o

Article history: Received 15 June 2016 Received in revised form 11 September 2016 Accepted 14 September 2016 Available online 17 September 2016 Keywords: Synovial fibroblast Chitosan oligosaccharide AMPK NF-␬B Osteoarthritis

a b s t r a c t Synovial inflammation plays an important role in the early pathogenesis of osteoarthritis (OA). Chitosan oligosaccharide (COS) has been shown to activate AMPK and suppress inflammatory responses in intestinal epithelial cells. This study aimed to investigate the effect of COS on AMPK activation and synovial inflammation using both primary cultures of synoviocytes and a rabbit model of anterior cruciate ligament (ACL) transection-induced OA. COS induced AMPK activation in both rabbit and human synoviocytes. The mechanism of COS-induced AMPK activation involves an increase in the ADP/ATP ratio but not calcium/calmodulin-dependent protein kinase kinase beta (CaMKK␤). Interestingly, COS suppressed the TNF␣-induced iNOS and COX-2 expression via an AMPK-dependent mechanism in both rabbit and human synoviocytes. Importantly, oral administration of COS (10 mg/kg/day) induced AMPK activation and alleviated signs of inflammation including COX-2 expression in the synovium of a rabbit ACL transection model. Taken together, our results indicate that COS suppresses synovial inflammation in vitro and in vivo via AMPK activation. COS may be useful in the prevention of OA. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction A synovial joint is a stabilized structure composed of articular cartilage, ligaments and synovial membrane. Damage to the joint cartilage due to repetitive use or injury, aging and obesity leads to osteoarthritis (OA), the most common aging-related joint disorder, which is characterized by degenerative articular damage and progressive joint pain, stiffness and functional impairment [1]. Due to the aging of the population, the number of OA cases is expected to rise, and safe and effective pharmacological interventions for prevention of OA are therefore urgently needed [2]. Several pharmacological approaches for OA prevention have been proposed including suppression of synovial inflammation (synovitis) [3,4]. Synovial inflammation is characterized by nuclear factor kappa B (NF-␬B)-mediated production of proinflammatory mediators including tumor necrosis factor alpha (TNF␣), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) by the

∗ Corresponding author at: Department of Physiology, Faculty of Science, Mahidol University, Rama VI Road, Rajathevi, Bangkok 10400, Thailand. E-mail address: [email protected] (C. Muanprasat). http://dx.doi.org/10.1016/j.phrs.2016.09.016 1043-6618/© 2016 Elsevier Ltd. All rights reserved.

synoviocytes [3,5]. Several lines of evidence have suggested that synovitis is involved in the pathogenesis of OA beginning at an early stage of disease [3,6]. The inflamed synoviocytes promote joint cartilage degradation through the effects of their secreted products, which include both proinflammatory and catabolic mediators such as TNF␣, NO, PGE2 and matrix metalloproteases [3,7]. These mediators produce a joint microenvironment that is critical for the development of chondrocyte inflammation and apoptosis as well as cartilage degradation [3]. In addition, the proinflammatory cytokines released from bones and cartilages have been recognized as important mediators of the OA pathogenesis [8,9]. The sources and types of proinflammatory mediators may differ in different subgroups of OA patients [10]. AMP-activated protein kinase (AMPK) plays a vital role in controlling cellular energy levels [11]. Increases in the ratios of AMP or ADP to ATP or an increase in the intracellular Ca2+ level promotes AMPK activation via liver kinase B1 (LKB1)- or Ca2+ /calmodulin-dependent protein kinase kinase beta (CaMKK␤)-mediated phosphorylation of Thr-172 in the AMPK ␣ subunit [11]. Interestingly, AMPK activators including 5aminoimidazole-4-carboxamide riboside (AICAR) and metformin suppress inflammatory responses in both immune and non-

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immune cells by inhibiting NF-␬B signaling [12–14]. In addition, several anti-inflammatory drugs including aspirin and fenamates mediate their anti-inflammatory action via AMPK activation [15,16]. Therefore, AMPK is considered to be a drug target for inflammatory disorders including OA [17]. Chitosan oligosaccharide (COS), an oligomer of d-glucosamine, is usually prepared by deacetylation of chitin, which is found in the exoskeleton of arthropods and considered to be the second most abundant natural polymer after cellulose [18]. COS holds great promise for pharmaceutical applications due to its biocompatibility, non-toxicity, intestinal permeability, and therapeutically relevant biological activities [18,19]. In particular, COS exerts antiinflammatory effects in several cell types and experimental models [20]. COS inhibits the NF-␬B-mediated inflammatory responses in endothelial, epithelial and immune cells [20]. Interestingly, our research group demonstrated that COS at a molecular weight (MW) of ∼5000 Da with a degree of deacetylation (DD) of ∼90% reduced the mortality rate and suppressed the intestinal inflammation and the associated mucosal damage in mouse models of inflammatory bowel disease by inhibiting NF-␬B signaling in intestinal epithelial cells (IEC) [21]. In addition, COS stimulates AMPK in IEC and suppresses the colitis-associated colorectal cancer development in mice [22]. Likewise, COS stimulates AMPK in the skeletal muscle of rats [23]. Therefore, we hypothesized that COS (MW ∼5000 Da; DD ∼90%) may activate AMPK in synoviocytes and ameliorate the synovial inflammation in OA. This study aimed to investigate the effects of COS on AMPK activation and the inflammatory responses in synoviocytes that were isolated from rabbits and OA patients. In addition, the in vivo efficacy of COS in activating AMPK and suppressing synovitis was evaluated in a rabbit model of anterior cruciate ligament (ACL) transection-induced OA. 2. Material and methods 2.1. Chemical reagents and antibodies STO-609, compound C and metformin hydrochloride were purchased from Sigma-Aldrich (Saint Louis, MO, USA). TNF␣ was purchased from Calbiochem (Darmstadt, Germany). COS (MW ∼5000 Da; DD ∼90%) was prepared as previously described [21]. Lyophilized COS was diluted in 1% acetic acid and stored at −20 ◦ C. The antibodies against AMPK phosphorylated at Thr-172 (pAMPK), AMPK␣, phosphorylated acetyl-CoA carboxylase (p-ACC), ACC, COX-2, phosphorylated I␬B (p-I␬B) and ␤-actin were purchased from Cell Signaling Technology (Boston, MA, USA). The antibodies against ␣-tubulin and iNOS were from Calbiochem (Darmstadt, Germany) and R&D Systems (Minneapolis, MN, USA), respectively. 2.2. Isolation and culture of rabbit and human synoviocytes The isolation and culture of synoviocytes were performed as previously described with slight modifications [24]. Briefly, infrapatellar fat pads (0.5 × 0.5 cm) were isolated from the knee joints of New Zealand White rabbits. The tissue was gently washed three times with sterile phosphate buffered saline (PBS), chopped into small pieces and transferred to a 25-cm2 flask containing 1 mL of F-12K Ham’s (Kaighn’s) medium (Gibco, Carlsbad, CA, USA) supplemented with 30% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 2% penicillin/streptomycin/fungizone (PSF) (Gibco, Carlsbad, CA, USA). One milliliter of complete medium (20% FBS and 1% PSF) was then added daily for five consecutive days. On day 6, the tissue was removed, and the cells were fed with complete medium until confluence. Cells were grown in 5% CO2 incubator at 37 ◦ C and 3rd to 7th passage were used in the experiments. The

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cells displayed characteristics of synoviocytes including hyaluronic acid secretion and no non-specific esterase staining. This study was approved by the Institutional Animal Care and Use Committee of the Faculty of Science, Mahidol University (MUSC56-010-272), and was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, U.S.A. Using the same protocol, primary human synoviocytes were prepared from synovium of OA patients undergoing arthroplasty operations. The isolated cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% FBS and 1% PSF. All protocols were approved by the Human Ethical Committee, Faculty of Medicine Ramathibodi Hospital, Mahidol University (ID 01-57-46).

2.3. Cell viability assay The synoviocytes were plated (15,000 cells/well) in 96-well plates and cultured overnight, followed by a 24-h exposure to various concentrations of COS (10 ␮g/mL, 50 ␮g/mL, 100 ␮g/mL, 200 ␮g/mL and 500 ␮g/mL), metformin (2 mM), compound C (10 ␮M) or STO-609 (15 ␮M). The cell viability was measured using MTT assays [25]. Briefly, cells were incubated for 4 h with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma-Aldrich, Saint Louis, MO, USA). The formazan was dissolved with dimethyl sulfoxide (DMSO). The absorbance was measured at 535 nm using a microplate reader (Victor 2V, PerkinElmer, Waltham, MA, USA).

2.4. Assays of intracellular ADP/ATP ratio The ADP/ATP ratio was determined using a luminescence-based kit (Abcam, Cambridge, MA, USA) in which luminescent light was released from the luciferase-mediated reaction between ATP and D-luciferin. The synoviocytes were plated in 96-well plates (15,000 cells/well) and grown overnight before a 24-h incubation with vehicle, COS (100 ␮g/mL) or metformin (2 mM). The level of ATP was measured first (value A). After conversion of the ADP to ATP using ADP-converting enzymes, a second measurement of ATP was performed. This latter value represents the sum of ADP and ATP levels (value B). The ADP to ATP ratio was estimated from (B-A)/A.

2.5. Immunoblot analysis Synoviocytes (200,000 cells) were seeded and grown on 60-mm petri dishes. After the treatments, the proteins from the synoviocytes were harvested using RIPA lysis buffer that contained 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM NaVO4 , 1 mM PMSF, 10% Triton-X and protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA). The infrapatellar fat pad was homogenized using a bullet homogenizer in a lysis buffer containing 20 mM TrisHCl, 320 mM sucrose, 1 mM EDTA, 1 mM NaF, 1 mM NaVO4 , 10% Triton-x and a protease inhibitor cocktail. Protein concentrations were determined using the Bradford reagent (BioRad, Philadelphia, PA, USA). The proteins were loaded (45 ␮g/well) and separated using an SDS-PAGE system with an 8% or 10% gel, transferred to nitrocellulose membranes and blocked with 5% non-fat dry milk. The membranes were incubated for 24 h with primary antibodies against p-AMPK, AMPK␣, p-ACC, ACC, COX-2, p-I␬B, iNOS, ␤-actin or ␣-tubulin and then washed and incubated with horseradish peroxidase-labeled secondary antibodies. The bands were visualized using the ECL method, and the band intensity was analyzed using ImageJ software (version 1.48).

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2.6. Quantitative real-time polymerase chain reaction (qPCR) Cells were lysed using TRIzol® reagent (Life Technologies, CA, USA) and RNA was extracted by chloroform-isopropanolethanol method. Complementary DNA (cDNA) was synthesized from 1 ␮g of extracted RNA using iScript cDNA synthesis kit (Bio-rad Laboratories, CA, USA). The relative expressions of interested mRNA were calculated by 2−Ct from 20 ␮L volume reaction of SYBR green mixture (KAPA Biosystem, MA, USA). Primer sequences were used according to the published studies [26,27] i.e., iNOS, 5 to 3 TGA-CGT-CCA-GCG-TAC-AAT-ATC (forward) and GCG-TCT-CCA-GTC-CCA-TCC-T (reverse); COX-2, 5 to 3 CAA-ACT-GCT-CCT-GAA-ACC-CAC-TC (forward) and GCTATT-GAC-GAT-GTT-CCA-GAC-TCC (reverse); and GAPDH, 5 to 3 TCA-CCA-TCT-TCC-AGG-AGC-GA (forward) and CAC-AAT-GCCGAA-GTG-GTC-GT (reverse). All primers were confirmed that they were specific to target genes using primer-BLAST on NCBI website. 2.7. Anterior cruciate ligament (ACL) transection model of OA New Zealand White rabbits (male, 4 weeks old) were used. An analgesic agent, carprofen (Pfizer, Sao Paulo, Brazil), was subcutaneously administered an hour before the operation, and this treatment was repeated daily for 3 days (4 mg/kg/day). Likewise, the antibiotic enrofloxacin (General Drug House Co. Ltd., Bangkok, Thailand) was subcutaneously injected prior to the surgery, and the treatment was continued for 5 days (10 mg/kg/day). Anesthesia during the surgical procedure was achieved by intramuscular injections of ketamine (35 mg/kg; Gedeon Richter Ltd., Budapest, Hungary) and xylazine (5 mg/kg; L.B.S. Laboratory Ltd., Bangkok, Thailand). A medial arthrotomy was performed (1-cm diameter) on the patellofemoral joint of the left knee (operated/OP knees) to expose the ACL. After the left ACL transection, the joint capsule and skin were closed with 4-0 absorbable silk and 3-0 silk sutures, respectively. Post-operative care included cleansing, antiseptic treatment with Betadine and a 1-week use of an Elizabethan

collar. COS or 1% acetic acid (vehicle) was added in 30% apple syrup (30 mL) and orally administered once a day at doses of 5 mg/kg (LCOS) or 10 mg/kg (H-COS) beginning 1 week after the surgery. The animals were sacrificed 10 weeks after the surgery using an intravenous injection of pentobarbital sodium (80 mg/kg; Ceva Santé Animale, Libourne, France). Both the OP and non-operated (nonOP) knees were collected for further analyses.

2.8. Gross morphological and histological analyses After sacrifice, the infrapatellar fat pad located medially to the patellar tendon was removed. The synovium was then isolated, fixed for 72 h in 10% neutral buffered formalin, embedded in paraffin and sectioned before staining with hematoxylin and eosin (H&E). Images were acquired using an inverted microscope. Three samples per rabbit were analyzed. The histological scores of the synovial inflammation were estimated using the criteria in Table 1 [28]. Femoral condyles were kept in normal saline at 4 ◦ C. To analyze the joint surface, India ink was brushed on the surface of femoral condyles. Thirty seconds later, the ink was washed off with cold distilled water at 4 ◦ C. The surface was dried, and the painting and washing steps were repeated once. The degree of knee OA was classified into three levels using the criteria in Table 2 [29]. For the safranin O/fast green straining, the condylar tissues were fixed for 72 h in 10% neutral buffered formalin with 1% cetylpyridinium chloride (CPC; Merck Schuchardt, Hohenbrunn, Germany) before decalcification with 14% EDTA (Loba Chemie, Mumba, India). Complete decalcification was confirmed using X-ray scanning. The femoral condyle was cut at the 5-mm center of the weight-bearing area along the sagittal plane and was embedded in paraffin. Fourmicron sections were cut and stained for the glycosaminoglycan (GAG) content using safranin O/fast green. These sections were evaluated using an inverted microscope, and the images were analyzed using the criteria in Table 3 [30].

Fig. 1. Cytotoxicity evaluation and effect of COS on AMPK activation in rabbit synoviocytes. (A) Effect of COS on cell viability. (B) Effect of other chemicals used in this study on cell viability. Rabbit synoviocytes were treated for 24 h with the vehicles, COS, metformin (Met; 2 mM), compound C (CC; 10 ␮M) or STO-609 (15 ␮M). Cell viability was measured using MTT assays. (C) Time course of COS-induced AMPK phosphorylation. Cells were treated with COS (100 ␮g/mL) for the indicated incubation periods. Dose-response curves for COS-induced AMPK phosphorylation (D) and ACC phosphorylation (E) are shown. The cells were treated for 24 h with the vehicle, metformin (Met; 2 mM) or COS at the indicated concentrations before sample collection for western blot analysis. The data are expressed as the means ± S.E.M. *, p < 0.05; **, p < 0.01 compared with the value at 0 h or control (one-way ANOVA) (n = 4).

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Table 1 Criteria for scoring histopathology of synovitis (40× magnification). Criteria

Score

Description

Intimal hyperplasia

0 1 2 3 0 1 2 3 0 1

1–2 layers only 3–4 layers, focal ≥5 layers, focal ≥5 layers, diffuse None One focus of infiltration 2–3 foci of infiltration Diffuse infiltration or >5 foci None Light focal collagenous staining up to 30% Heavy focal staining or total light diffuse collagenous staining Heavy diffuse collagenous staining 0–4 vascular elements 5–8 vascular elements 9–12 vascular elements >12 vascular elements Sum of the scores from the four criteria

Lymphocytic/plasmocytic infiltration

Subintimal fibrosis (loose connective tissue area only)

2

3 0 1 2 3 0–12

Vascularity

Summary score

Table 2 Criteria for scoring gross morphology of articular cartilage using India ink staining. Score

Grading

Description

1

Intact surface

2

Minimal fibrillation

3

Overt fibrillation

4

Erosion

Surface normal in disappearance of India ink retention Surface retains Indian ink as elongated specks or light gray patch Areas that are velvety in appearance and retain Indian ink as intense black patches Loss of cartilage to expose the underlying bone

Table 3 Scoring system for analysis of articular cartilage damage at the weight-bearing and lesion areas (100× magnification). Assessment

Score

Observation

Safranin O-fast green staining (GAG content)

0

Full staining throughout the articular cartilage Loss of staining in the superficial hyaline cartilage Loss of staining in the upper 2/3 of the hyaline cartilage Loss of staining in all of the hyaline cartilage Normal Surface irregularities Fissures in <50% of the surface Fissures in>50% of the surface Erosion of 1/3 of the hyaline cartilage Erosion of 2/3 of the hyaline cartilage Full depth erosion of the hyaline cartilage

1 2 3 Structure

0 1 2 3 4 5 6

2.9. Statistical analysis The data are expressed as means ± S.E.M. The significant differences were determined using one-way ANOVA, Kruskal-Wallis one-way ANOVA or Wilcoxon signed-rank test as appropriate, with a p value <0.05 being considered statistically significant. GraphPad Prism software (5th version) was used for all statistical analyses.

Fig. 2. Mechanism of COS-induced AMPK activation. Involvement of CaMKK␤ in mediating COS-induced AMPK phosphorylation (A) and ACC phosphorylation (B). Rabbit synoviocytes were pretreated for 30 min with STO-609 (CaMKK␤ inhibitor, 15 ␮M) before a 24-h incubation with COS (100 ␮g/mL) or vehicle. (C) Effect of COS on the ADP/ATP ratio. Cells were treated for 24 h with the vehicle, COS (100 ␮g/mL) or metformin (Met; 2 mM) before measuring the ADP/ATP ratio using assay kits. The data are expressed as the means ± S.E.M. *, p < 0.05; **, p < 0.01 compared with control (one-way ANOVA) (n = 4).

3. Results 3.1. Effect of COS on AMPK activation in rabbit synoviocytes The effect of COS on AMPK activation and synovial inflammation was investigated in synoviocytes isolated from rabbit knee joints. First, the cytotoxic effects of COS and other compounds used in this study were investigated using MTT assays. As demonstrated in Fig. 1A and B, 24-h incubations with COS (10 ␮g/mL to 500 ␮g/mL), metformin (Met; AMPK activator; 2 mM), compound C (CC; AMPK inhibitor; 10 ␮M) or STO-609 (CaMKK␤ inhibitor; 15 ␮M) had no effect on the viability of the rabbit synoviocytes compared with the vehicle-treated group. To investigate the effect of COS on AMPK activity, phosphorylation of AMPK and its downstream target acetyl Co-A carboxylase

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Fig. 3. Effect of COS on TNF␣-induced I␬B phosphorylation (A), iNOS and COX-2 protein expression (B), and iNOS and COX-2 mRNA expression (C) in rabbit synoviocytes. Cells were pretreated for 24 h with the vehicle, metformin (Met; 2 mM), COS (100 ␮g/mL) or COS (100 ␮g/mL) plus compound C (10 ␮M) (COS + CC) before a 6-h incubation with TNF␣ (10 ng/mL). Protein and mRNA samples were collected for western blot and qPCR analyses. The data are expressed as the means ± S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (one-way ANOVA) (n = 3).

(ACC) [31] was determined using western blot analysis. As shown in Fig. 1C and D, COS induced AMPK phosphorylation in time- and concentration-dependent manners. Of particular importance, a significant degree of AMPK phosphorylation was observed 24 h after treatment with COS (100 ␮g/mL). Therefore, this treatment protocol (24-h incubation with 100 ␮g/mL COS) was used in the subsequent experiments. Similarly, COS dose-dependently induced ACC phosphorylation (Fig. 1E), which confirmed that COS activates AMPK in rabbit synoviocytes. Metformin (2 mM), a known AMPK activator, was used as a positive control. 3.2. Mechanisms of COS-induced AMPK activation in rabbit synoviocytes To investigate the involvement of CaMKK␤ in COS-induced AMPK activation, synoviocytes were pretreated with STO-609

(CaMKK␤ inhibitor) for 30 min before a 24-h treatment with COS (100 ␮g/mL). STO-609 pretreatment had no effect on the COS-induced AMPK phosphorylation (Fig. 2A) and ACC phosphorylation (Fig. 2B), which indicated that the mechanism of COS-induced AMPK activation did not require CaMKK␤. A control experiment confirmed that STO-609 effectively suppressed CaMKK␤-mediated AMPK phosphorylation (data not shown). In addition, the effect of COS on the ADP/ATP ratio was investigated using ADP/ATP ratio assay kits. As depicted in Fig. 2C, a 24-h treatment with COS (100 ␮g/mL) resulted in an increase in the ADP/ATP ratio. Metformin was used as a positive control. These findings suggest that COS may activate AMPK in rabbit synoviocytes by modulating the ADP/ATP ratio.

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Fig. 4. Effect of COS on AMPK activation and TNF␣-induced iNOS and COX-2 expression in primary culture of human synoviocytes. The effects of COS on AMPK (A) and ACC (B) phosphorylation. Cells were treated for 24 h with the vehicle, COS (100 ␮g/mL) or metformin (Met; 2 mM) before protein collection for western blot analysis. The effect of COS on TNF␣-induced iNOS (C) and COX-2 (D) expression. Cells were pretreated for 24 h with vehicle, metformin (Met; 2 mM), COS (100 ␮g/mL) or COS (100 ␮g/mL) plus compound C (10 ␮M) (COS + CC) before a 6-h incubation with TNF␣ (10 ng/mL). Protein samples were collected for western blot analysis using antibodies against the indicated proteins. The data are expressed as the means ± S.E.M. *, p < 0.05; **, p < 0.01 compared with the control or as indicated (one-way ANOVA) (n = 4).

3.3. Anti-inflammatory effect of COS in rabbit synoviocytes is AMPK-dependent

3.4. Effect of COS on AMPK activation and synovial inflammation in human synoviocytes

TNF␣ is a proinflammatory cytokine that is involved in the induction of synovial inflammatory responses including iNOS and COX-2 expression during OA pathogenesis [3]. We therefore investigated the effect of COS on the TNF␣-induced iNOS and COX-2 expression in rabbit synoviocytes. The role of AMPK in mediating the anti-inflammatory effect of COS was investigated using pretreatment with compound C, an AMPK inhibitor. In this experiment, rabbit synoviocytes were incubated for 24 h with COS (100 ␮g/mL) with or without pretreatment with compound C, followed by a 6-h exposure to TNF␣. As shown in Fig. 3A and B, COS significantly suppressed the TNF␣-induced protein expression of phosphorylated I␬B (p-I␬B), iNOS and COX-2 in rabbit synoviocytes. Interestingly, compound C significantly abrogated the inhibitory effects of COS on these proinflammatory biomarkers. Similar results were observed from the mRNA expression analysis by qPCR (Fig. 3C). Overall results indicate that COS exerts an anti-inflammatory effect in rabbit synoviocytes via AMPK activation. Metformin, a known AMPK activator, showed a similar degree of anti-inflammatory effects compared to COS.

To investigate the potential application of COS in the alleviation of synovitis in humans, the effects of COS on AMPK activation and TNF␣-induced iNOS and COX-2 expression were investigated in primary synoviocytes derived from OA patients. As shown in Fig. 4A and B, COS treatment (100 ␮g/mL; 24 h) resulted in AMPK and ACC phosphorylation. Importantly, the 24-h treatment with COS (100 ␮g/mL) prevented the TNF␣-induced iNOS and COX-2 expression in an AMPK-dependent manner (Fig. 4C and D). Metformin was used as a positive control in these experiments. These results indicate that COS inhibits the TNF␣-induced inflammatory responses in human synoviocytes via AMPK activation. 3.5. Amelioration by COS of synovitis in the rabbit ACL transection model The in vivo efficacy of COS on the alleviation of synovial inflammation was investigated in an ACL transection model of knee OA in rabbits [29]. ACL transection was performed in the left knees. ACL

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Fig. 5. Characterization of articular cartilage damage in the ACL transection rabbit model. Ten weeks after ACL transection, macroscopic and microscopic changes of the articular cartilage in operated knees (OP) and non-operated knees (non-OP) were analyzed. (A) Macroscopic score analysis of India ink-stained samples. (B) Representative images of the articular surfaces stained with safranin O/fast green (100× magnification). The thin arrow indicates loss of GAG content. The thick arrow indicates fissure. (C) Histopathological scores of GAG content (safranin O-fast green staining) and cartilage surface structure. NS, non-significant. The data were analyzed using the Wilcoxon signed-rank test (n = 7).

transection causes joint instability, which leads to a malalignment of the force distribution and cartilage damage at the weight-bearing area, e.g., the surface of the medial femoral condyle. Macroscopic and histopathological analyses of the joints were performed 10 weeks after ACL transection. Macroscopic score analysis of the articular cartilage surface using India ink staining reveals that cartilage erosion tended to increase in the operated (OP) knees, whereas it was absent in the non-operated (non-OP) knees (Fig. 5A). In addition, histopathological analysis of cartilage sections at the weight-bearing areas or those containing lesions was performed using safranin O/fast green staining of glycosaminoglycan (GAG) content. As shown in Fig. 5B, loss of GAG content and formation of fissures were noted in the OP knees. Analysis of the histopathological scores including the safranin O/fast green staining scores and the surface structure scores indicated that the OP knees tended to have higher GAG loss and articular cartilage damage compared with non-OP knees (Fig. 5C). Interestingly, OP knees exhibited signs of synovitis including increased cell layers in intimal layers, increased cell infiltration, and increased angiogenesis and fibrosis in the subintimal layers (Fig. 6A and B). These results indicate that this ACL transection model (at 10 weeks after surgery) represented early-stage OA in which mild-to-moderate cartilage fibrillation, GAG loss and synovial inflammation were observed without full cartilage erosion, cluster formation, and bone spur formation [32]. To investigate the in vivo anti-inflammatory efficacy of COS and its ability to activate AMPK in the synovial membrane, COS at one of two doses (5 mg/kg/day (L-COS) or 10 mg/kg/day (HCOS)) was orally administered to the rabbits beginning 1 week

after ACL surgery. These doses were selected based on their effectiveness in exerting an anti-inflammatory effect in our previous study [33]. As shown in Fig. 6A and B, histological score analysis demonstrated that oral administration of COS at 10 mg/kg/day significantly reduced synovitis in the arthritic knees. In addition, expression of COX-2, a proinflammatory mediator involved in OA pathogenesis, in the synovium was significantly suppressed by 10 mg/kg/day COS (Fig. 6C). Consistent with these results, oral administration of COS at 10 mg/kg/day also caused a significant increase in AMPK and ACC phosphorylation in synovial membranes from both OP and non-OP knees (Fig. 6D and E). These findings indicate that oral administration of COS at 10 mg/kg/day effectively alleviates synovial inflammation and activates AMPK in a rabbit model of ACL transection-induced OA.

4. Discussion The present study demonstrated that COS stimulates AMPK activity, which leads to a suppression of TNF␣-induced NF-␬B signaling and COX-2 and iNOS expression in both rabbit and human synoviocytes. The mechanism by which COS activates AMPK in synoviocytes may involve an alteration of the ADP/ATP ratio. Importantly, oral administration of COS led to AMPK activation and suppression of synovitis and COX-2 expression in the synovium of a rabbit model of ACL transection-induced knee OA. The biological activities of COS depend on its molecular weight (MW) and degree of deacetylation (DD). In this study, we demonstrated that COS with a MW of 5000 Da and DD of ∼90% exerts

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Fig. 6. Effect of oral administration of COS on synovial inflammation and AMPK activation in synovial tissues in the rabbit ACL transection model. One week after ACL transection, rabbits were orally administered 30% apple syrup alone or with COS at 5 mg/kg/day (L-COS) or 10 mg/kg/day (H-COS). Ten weeks after ACL transection, rabbits were sacrificed and the infrapatellar fat pads from the non-operated knees (non-OP) and operated knees (OP) were collected for histological and western blot analyses. (A) Representative images of synovial membranes stained with H&E (40×). (B) Histopathological scores of synovitis including intimal hyperplasia, immune cell infiltration, subintimal fibrosis and angiogenesis. The scores are the averages from three samples per animal. Statistical analyses were performed using the Wilcoxon signed-rank test between non-OP and OP in the same animal and Kruskal-Wallis one-way ANOVA among the OP in each group. *, p < 0.05 compared with non-OP; #, p < 0.05; ##, p < 0.01 compared with vehicle-treated OP (n = 7). (C) Effect of oral administration of COS on COX-2 expression in the synovial membranes. *, p < 0.05 (one-way ANOVA) (n = 4). (D) Effect of oral administration of COS on AMPK phosphorylation in the synovium. *, p < 0.05 (one-way ANOVA) (n = 6). (E) Effect of oral administration of COS on ACC phosphorylation in the synovium. *, p < 0.05; **, p < 0.01 (one-way ANOVA) (n = 4).

an anti-inflammatory effect in synoviocytes via AMPK activation. This result is consistent with the previous observation that COS possesses several biological activities including an anti-tumor effect, a radical scavenging activity, an anti-inflammatory effect and the ability to protect against ultraviolet B-induced cell damage [18,20]. Although the anti-inflammatory effect of COS is well documented, its underlying mechanism remains unclear. Recently, our research group demonstrated that COS activates AMPK in intestinal epithelial cells (IEC) via a mechanism that involves a calciumsensing receptor-induced elevation of intracellular calcium levels and CaMKK␤-mediated AMPK phosphorylation [22]. However, in this study, our results showed that a CaMKK␤ inhibitor had no effect on COS-induced AMPK activation and that COS induced an increase in ADP/ATP ratio in rabbit synoviocytes. These findings suggest that the mechanism of COS-induced AMPK activation in synovio-

cytes is distinct from that in IEC. Our data also suggest that COS may activate AMPK in synoviocytes by modulating cellular energy balance, a mechanism similar to that of metformin. Indeed, COS and glucosamine, a monomer of COS, have previously been shown to inhibit adipogenesis in adipocytes through AMPK-dependent mechanisms [34]. Glucosamine affects hexose metabolism by utilizing ATP for glucosamine-6-phosphate production. This leads to a decrease in the intracellular ATP level and increased intracellular ADP and AMP levels [35]. In addition, glucosamine stimulates hyaluronic acid production in the synovium [36]. This anabolic process utilizes ATP, which can lead to an increase in the ADP/ATP ratio. Intestinal absorption of COS occurs through endocytotic pathways, and eukaryotic cells have the ability to degrade COS into small oligomers and/or monomers (glucosamine) via lysozyme-mediated mechanisms [19,37]. We therefore speculate that AMPK activation

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following COS treatment in synoviocytes may be a consequence of the modulation of cellular energy balance by COS or its metabolites (e.g., glucosamine), which leads to LKB1-mediated AMPK phosphorylation. This possibility warrants future investigation. It should also be noted that administration of high-MW chitosan (MW of 560 kDa; DD of 90%) has previously been shown to inhibit high fat diet-induced lipogenesis in livers and adipose tissues of obese rats via AMPK activation [38]. Since chitosan has been reported to be degraded into COS in rats [39,40], it is conceivable that COS may mediate the AMPK activation induced by high-MW chitosan administration. Furthermore, the effect of chitosan with different MW and DD on AMPK activation warrants further studies. Upon activation, AMPK suppresses the NF-␬B-mediated inflammatory responses in several cell types [20]. In this study, we demonstrated that COS inhibited TNF␣-induced I␬B phosphorylation and COX-2 and iNOS expression in both rabbit and human synoviocytes in an AMPK-dependent manner. Interestingly, COS inhibited TNF␣-induced NF-␬B and inflammatory responses in IEC via an AMPK-independent mechanism. This suggested that COS exerts its inhibitory effects on NF-␬B-mediated inflammatory responses via different mechanisms in different cell types. Indeed, previous studies have revealed that the mechanisms by which COS inhibits NF-␬B include inhibition of p38 mitogenactivated protein kinase (MAPK), extracellular regulated protein kinase 1/2 (ERK1/2), and the binding of LPS to toll-like receptor 4 [21,41,42]. Furthermore, AMPK inhibits NF-␬B signaling via several downstream mediators including sirtuin 1 (SIRT1), Forkhead box O (FoxO) family members, and peroxisome proliferator-activated receptor ␥ co-activator 1␣ (PGC-1␣) [43]. These downstream mediators directly or indirectly inactivate the p65 subunit of NF-␬B, which suppresses the NF-␬B-mediated inflammatory responses [43]. Our data showed that an AMPK inhibitor completely abolished the inhibitory effect of COS on TNF␣-induced p-I␬B, iNOS and COX-2 expression. These results indicated that COS exerts an anti-inflammatory effect via AMPK activation in rabbit and human synoviocytes. Interestingly, metformin, a known AMPK activator, has been shown to suppress experimental arthritis via mechanisms involving AMPK activation, mammalian target of rapamycin inhibition and autophagy-mediated suppression of NF-␬B signaling [44]. Therefore, it is possible that COS may exert the anti-inflammatory action via autophagy signaling network. TNF␣ plays an important role in inducing synovial inflammation that is involved in the early stages of OA pathogenesis [45]. Interestingly, TNF␣ is detected in synovial fluid and is implicated as a cause of OA. In addition, TNF␣ in synovial fluid is associated with an increased risk of OA in the knees [6,46], and TNF␣ gene polymorphisms are associated with an increased risk of OA [47]. Furthermore, TNF␣ stimulates the production of matrix metalloproteinases (MMPs) in chondrocytes via NF-␬B-dependent pathways, which leads to the degradation of cartilage extracellular matrix and cartilage erosion [3,48]. Our in vitro results in human synoviocytes suggest that COS may be beneficial in the prevention of OA progression. The rabbit ACL transection model is a commonly used OA model. Its characteristics resemble several features of human OA including cartilage erosion, synovitis and other pathological markers [29,49]. In this study, macroscopic and microscopic analyses of OA severity were performed 10 weeks after ACL transection, at which time synovitis and mild-to-moderate cartilage erosion were observed. This model therefore represents an early stage of OA. Interestingly, oral administration of 10 mg/kg/day of COS significantly reduced the signs of synovitis and COX-2 expression in synovial tissues. Indeed, it has previously been demonstrated that the carboxymethylated chitin and chitosan reduced expression of matrix metalloproteases, which is involved in cartilage erosion, in a model of OA in rabbits [50,51]. A dose conversion based on body surface area reveals

that 10 mg/kg/day of COS in rabbits is equivalent to 3.24 mg/kg/day of COS in adult humans [52]. This dose of COS corresponds to a daily ingestion of approximately 200 mg COS. Importantly, it has previously been shown that ingestion of chitosan (3 g/day) for 24 weeks was not associated with significant adverse effects or toxicity [53]. Oral administration of COS for OA prevention in humans is therefore possible. Evaluation of the effectiveness of COS in the prevention of OA in humans warrants future investigations. One of the unique features of COS is its ability to be structurally modified to produce a variety of COS derivatives or composites with distinctive physicochemical and biological properties. This makes possible the development of COS derivatives/composites specifically targeted to the joint stromal compartment, which may be proven to be safer than the use of current immunosuppressive agents. 5. Conclusions In conclusion, COS acts as an AMPK activator in both rabbit and human synoviocytes. COS suppresses synovial inflammation in vitro and in vivo via AMPK-dependent mechanisms. Further studies to support the development of COS may provide a safe and effective strategy for prevention of OA. Acknowledgements This work was supported by the Thailand Research Fund and Mahidol University through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0369/2552 to WK and VC). Financial support from the Thailand Research Fund and Mahidol University (grant BRG5980008), the National Research Council of Thailand (NRCT), the Faculty of Science, Mahidol University, and the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative are gratefully acknowledged. References [1] D.J. Hunter, M. Nevitt, E. Losina, V. Kraus, Biomarkers for osteoarthritis: current position and steps towards further validation, Best Pract. Res. Clin. Rheumatol. 28 (2014) 61–71. [2] E.M. Roos, N.K. Arden, Strategies for the prevention of knee osteoarthritis, Nat. Rev. Rheumatol. 12 (2016) 92–101. [3] J.P. Pelletier, J. Martel-Pelletier, S.B. Abramson, Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets, Arthritis Rheum. 44 (2001) 1237–1247. [4] W.H. Robinson, C.M. Lepus, Q. Wang, H. Raghu, R. Mao, T.M. Lindstrom, J. Sokolove, Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis, Nat. Rev. Rheumatol. 12 (10) (2016) 580–592. [5] S.B. Abramson, M. Attur, A.R. Amin, R. Clancy, Nitric oxide and inflammatory mediators in the perpetuation of osteoarthritis, Curr. Rheumatol. Rep. 3 (2001) 535–541. [6] M.D. Smith, S. Triantafillou, A. Parker, P.P. Youssef, M. Coleman, Synovial membrane inflammation and cytokine production in patients with early osteoarthritis, J. Rheumatol. 24 (1997) 365–371. [7] M.G. Attur, I.R. Patel, R.N. Patel, S.B. Abramson, A.R. Amin, Autocrine production of il-1 beta by human osteoarthritis-affected cartilage and differential regulation of endogenous nitric oxide, il-6, prostaglandin e2, and il-8, Proc. Assoc. Am. Phys. 110 (1998) 65–72. [8] R.F. Loeser, S.R. Goldring, C.R. Scanzello, M.B. Goldring, Osteoarthritis: a disease of the joint as an organ, Arthritis Rheum. 64 (2012) 1697–1707. [9] M.B. Goldring, M. Otero, Inflammation in osteoarthritis, Curr. Opin. Rheumatol. 23 (2011) 471–478. [10] J.W. Bijlsma, F. Berenbaum, F.P. Lafeber, Osteoarthritis: an update with relevance for clinical practice, Lancet 377 (2011) 2115–2126. [11] D.G. Hardie, Ampk: positive and negative regulation, and its role in whole-body energy homeostasis, Curr. Opin. Cell Biol. 33C (2014) 1–7. [12] S.J. Koh, J.M. Kim, I.K. Kim, S.H. Ko, J.S. Kim, Anti-inflammatory mechanism of metformin and its effects in intestinal inflammation and colitis-associated colon cancer, J. Gastroenterol. Hepatol. 29 (2014) 502–510. [13] N.L. Huang, S.H. Chiang, C.H. Hsueh, Y.J. Liang, Y.J. Chen, L.P. Lai, Metformin inhibits tnf-alpha-induced ikappab kinase phosphorylation, ikappab-alpha degradation and il-6 production in endothelial cells through pi3k-dependent ampk phosphorylation, Int. J. Cardiol. 134 (2009) 169–175.

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