Correspondence should be addressed to Ali Mobasheri,

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1 Evidene-Based Complementary and Alternative ediine Volume 2012, Artile ID , 16 pages doi: /2012/ Researh Artile Botanial Extrats from Rosehip (Rosaanina), Willow Bark (Salix alba), and Nettle Leaf (Urtia dioia) Suppress -Indued NF-κB Ativation in Canine Artiular Chondroytes ehdi Shakibaei, 1 David Allaway, 2 Simone Nebrih, 1 and Ali obasheri 3 1 usuloskeletal Researh Group, Institute of Anatomy, Ludwig-aximilian-University unih, unih, Germany 2 Nutrition and etabolism Researh Group, WALTHA Centre for Pet Nutrition, Waltham on the Wolds, elton owbray, Leiestershire LE14 4RT, UK 3 usuloskeletal Researh Group, Division of Veterinary ediine, Shool of Veterinary ediine and Siene, Faulty of ediine and Health Sienes, University of Nottingham, Sutton Bonington Campus, Sutton Bonington, Leiestershire LE12 5RD, UK Correspondene should be addressed to Ali obasheri, ali.mobasheri@nottingham.a.uk Reeived 2 July 2011; Revised 27 Otober 2011; Aepted 10 November 2011 Aademi Editor: Virginia S. artino Copyright 2012 ehdi Shakibaei et al. This is an open aess artile distributed under the Creative Commons Attribution Liense, whih permits unrestrited use, distribution, and reprodution in any medium, provided the original work is properly ited. The aim of this study was to haraterize the anti-inflammatory mode of ation of botanial extrats from rosehip (Rosa anina), willow bark (Salix alba),and nettle leaf (Urtiadioia)in an in vitro model of primary anine artiular hondroytes. ethods. The biologial effets of the botanial extrats were studied in hondroytes treated with for up to 72 h.expression of ollagen type II, artilage-speifi proteoglyan (CSPG), β1-integrin, SOX-9, COX-2, and P-9 and P-13 was examined by western blotting. Results. The botanial extrats suppressed -indued NF-κB ativation by inhibition of IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nulear transloation. These events orrelated with downregulation of NF-κB targets inluding COX-2 and Ps. The extrats also reversed the -indued downregulation of ollagen type II, CSPG, β1-integrin, and artilage-speifi transription fator SOX-9 protein expression. In high-density ultures botanial extrats stimulated new artilage formation even in the presene of. Conlusions. Botanial extrats exerted anti-inflammatory and anaboli effets on hondroytes. The observed redution of -indued NF-κB ativation suggests that further studies are warranted to demonstrate the effetiveness of plant extrats in the treatment of OA and other onditions in whih NF-κB plays pathophysiologial roles. 1. Introdution Osteoarthritis (OA) is a joint disease involving not only artiular artilage but also the synovial membrane, subhondral bone and periartiular soft tissues [1]. OA may our following traumati injury to the joint, subsequent to an infetion of the joint or simply as a result of aging. The symptoms and signs harateristi of OA in the most frequently affeted joints are heat, swelling, pain, stiffness and limited mobility. Other sequelae inlude osteophyte formation and joint malalignment. These manifestations are highly variable, depending on joint loation and disease severity [2]. OA is grossly haraterized by aberrant synthesis of extraellular matrix, gradual hypoellularity, eventual fragmentation and degradation of artilage, new bone formation in the periartiular region (osteophytosis), dereased, then inreased, subhondral bone density, and variable synovial inflammation [3]. In OA, mehanial stress initiates artilage lesions by altering hondroyte-matrix interation and metaboli responses in the hondroytes [4]. The interation between hondroytes and matrix proteins is mediated largely by the β1-integrin reeptors [5]. This

2 2 Evidene-Based Complementary and Alternative ediine interation plays a ruial role in regulating several biologial phenomena, inluding ell morphology, gene expression, and ell survival. β1-integrins are transmembrane signal transdution reeptors mediating ell-matrix interations in artilage [6]. An important signal transdution pathway ativated by β1-integrin reeptors is the APKinase pathway [7, 8]. Furthermore, disruption of ell matrix ommuniation by inhibition of the APKinase pathway has been shown to lead to aspase-3 ativation, leavage of Poly(ADP)Ribose polymerase, and hondroyte apoptosis [8]. There are initial inreases in the amounts of water and proteoglyans assoiated with the observed transient hondroyte proliferation of early OA. Proliferating hondroytes appear in lusters and are aompanied by a hange in ellular morphology and phenotype, indiating a hypertrophi differentiation proess. At the moleular level, OA is haraterized by loss of artilage matrix omponents, partiularly type II ollagen and aggrean due to an imbalane between extraellular matrix destrution and repair [9]. Although OA hondroytes have inreased expression of both anaboli and ataboli matrix genes [10], their ataboli ability is onsidered to dominate their anaboli apaity resulting in artilage loss. As OA progresses from mild to severe, there is a derease in transription of ollagen, failure to maintain the proteoglyan matrix, and redued ability of the hondroytes to regulate apoptosis [11]. In ontrast, ollagen type X, whih is normally produed by terminally differentiated hypertrophi hondroytes, has been demonstrated in surrounding hondroyte lusters in OA artilage [12]. Chondroyte proliferation (loning) is onsidered to be an attempt to repair and ounterat artilage degradation. However, disease progression and seondary inflammation indiate that this is generally unsuessful. The short-lived hyperplasia (hondroyte loning) is followed by hypoellularity and apoptosis [13]. Cataboli events responsible for artilage matrix degradation inlude (i) the release of ataboli ytokines suh as,il-6,andtnf-α [14]; (ii) the prodution of matrix degrading enzymes suh as matrix metalloproteinases (Ps), mainly, stromelysin-1 (P-3) and ollagenase-3 (P-13); (iii) reative oxygen speies (ROS) prodution by hondroytes in OA [4, 14]. Imbalane between Ps and tissue inhibitors of Ps (TIPs) ours, resulting in ative Ps and onsequent artilage matrix degradation. However, may also ontribute to the depletion of artilage matrix by dereasing synthesis of artilage speifi proteoglyans and ollagen type II [4, 15]. The proinflammatory effets of and TNF-α in OA are regulated by the transription fator nulear transription fator κb (NF-κB) [16]. The subunits of NF-κB (p65 and p50) are loated in the ytoplasm as an inative omplex in assoiation with an inhibiting IκBα subunit. In response to phosphorylation, IκBα dissoiates from the omplex and NF-κB transloates to the ell nuleus and binds to target genes of NF-κB [17]. NF-κB might be also responsible for downregulation of the transription fator SOX-9, whih is involved in the regulation of genes for artilage-speifi extraellular matrix (EC) proteins [18]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are urrently the most widely used anti-inflammatory drugs. However, they exhibit numerous undesired side effets and are only temporarily effetive. Therefore, naturally ourring botanial extrats apable of inhibiting NF-κB mediated ataboli ativity may prove to be promising therapeuti agents for the treatment of OA [19]. Plant extrats with antiinflammatory ativity may also help to redue the frequeny of onsumption and dosage of NSAIDs in arthritis patients. In reent years there has been a proliferation of researh into botanial extrats with potential anti-inflammatory properties [20]. The most important fator that drives the interest in botanial extrats is the realization that inflammation plays a entral role in the development of many hroni diseases in humans and ompanion animals. The use of herbal mediine is inreasing among human arthritis patients in the United States and western Europe [21]. Aording to The Arthritis Foundation, almost 45% of patients in North Ameria apply ointments or rubs for OA. A variety of topial and oral preparations are urrently available. any of these are traditional Chinese, Indian, or Korean herbal mediines, whih have been used in the armamentarium of indigenous pratitioners. The beststudied botanials investigated to date inlude rosehip (Rosa anina) [22], Tripterygium wilfordii Hook F extrat [23], Triptolide [24], Devil s Claw (Harpagophytum proumbens) [25], ginger (Zingiber offiinale Ros.) [26], and Withania somnifera (ashwagandha) [27]. In addition, onventional [28] and several systemati reviews [29] have examined published evidene for the effetiveness of botanial antiinflammatory drugs. A few of these have speifially foused on the treatment of OA and hroni low bak pain [30, 31]. The present study was designed to haraterize the effets and mehanism of ation of three botanial extrats; rosehip (Rosa anina), willow bark (Salix alba), and nettle leaf (Urtia dioia) in primary anine artiular hondroytes. Previous studies have reported on the anti-inflammatory ativity of Urtia dioia [17, 30, 31]. In this study, we show that these botanial extrats exhibit a strong apaity for the inhibition of NF-κB and its regulated gene produts in hondroytes in vitro. 2. aterials and ethods 2.1. Antibodies. Antibodies to ollagen type II (AB746), β1-integrin (AB1977), and artilage-speifi proteoglyan antibody (AB2015) were purhased from illipore (Shwalbah, Germany). Seondary antibodies were purhased from Dianova (Hamburg, Germany). Antibodies to β-atin (A5316) were from Sigma (unih, Germany). Antibodies raised against P-9 (AB911) and P-13 were purhased from R&D Systems (Abingdon, UK). Cylooxygenase-2 (COX-2) ( ) antibody was obtained from Cayman Chemial (Ann Arbor, I, USA). onolonal anti- ERK antibody and polylonal anti-sh antibody were purhased from Beton Dikinson (Heidelberg, Germany). Antibodies to p65 (IG-512), phospho-iκbα (IG-156A) and pan-iκbα (IG-127), were obtained from Bioarta

3 Evidene-Based Complementary and Alternative ediine 3 (Hamburg, Germany). Antibodies to NF-κB p65 (RelA) and phospho-speifi ps529 ( ) were obtained from Rokland laboratories (Biomol, Hamburg, Germany). SOX- 9 antibody was purhased from Aris Antibodies GmbH (Hiddenhausen, Germany). Peptide aldehydes and a speifi proteosome inhibitor N-A-Leu-Leu-norleuinal (ALLN) were obtained from Boehringer annheim (annheim, Germany). The TT assay was purhased from Sigma (unih, Germany) Culture edium and Chemials. Culture medium (Ham s F-12/Dulbeo s modified Eagle s medium (50/50) ontaining 10% fetal alf serum (FCS), 25 μg/ml asorbi aid, 50 IU/mL streptomyin, 50 IU/mL peniillin, 2.5 μg/ml amphoteriin B, essential amino aids and L-glutamine) was obtained from Seromed (unih, Germany). Trypsin/EDTA (EC ) was purhased from Sigma (unih, Germany). Epon was obtained from Plano (arburg, Germany). was obtained from Strathman Bioteh GmbH (Hannover, Germany) Preparation of the Botanial Extrats. The botanial extrats from rosehip (Rosa anina), white willow bark (Salix alba) and nettle leaf (Urtia dioia) were supplied as powders from Shamanshop, Camden, NY, USA; atalogue numbers ( C, C, and C, resp.). The botanial extrats were prepared using hloroform, a ommonly used solvent in the laboratory beause it is relatively unreative, misible with most organi liquids, and onveniently volatile. Also, plant material is ommonly extrated with hloroform for pharmaeutial proessing. Chloroform solvent extrations were arried out by Puleva Bioteh, Granada, Spain. Eah botanial extrat ( 10 g) was paked in filter paper and loaded into the main hamber of a Soxhlet extration unit. Chloroform (200 ml) was heated to reflux and inubated for 2 h. Following hloroformevaporation under vauum in a rotary evaporator (plaed in a water bath at 60 C) the soluble botanial extrat was dissolved in dimethyl sulfoxide (DSO) at a stok onentration of 10 mg/ml and stored in aliquots at 80 C. The final onentration of DSO did not, in any ase, exeed 0.1%. Further dilutions were made in ell ulture medium to ahieve the final working onentrations Chondroyte Isolation and Culture. Primary anine artiular hondroytes were isolated from the joints of lientowned dogs undergoing orthopaedi surgery at the Clini of Veterinary Surgery, Ludwig-aximilian-University unih, Germany. Fully informed owner onsent was obtained and the Ethial Review Committees of WALTHA, Ludwig- aximilian-university, and the University of Nottingham approved the projet. Cartilage explants were slied and digested primarily with 1% pronase for 2 h at 37 C and subsequently with 0.2% ollagenase for 4 h at 37 C. Isolated hondroytes were maintained in ulture medium at a density of ells/ml in Petri dishes in monolayer ulture and on glass plates for a period of 24 h at 37 Cwith 5% CO Experimental Design. Serum-starved hondroytes (passage two, ultivated in 3% FCS) were treated with the botanial extrats (10 μg/ml) alone for 24 h (pretreatment) and then otreated with a ombination of botanial extrats (10 μg/ml) and (10ng/mL) for a further 48h in monolayer ultures. Chondroytes treated with the botanial extrats alone over the entire period served as treatments and those treated with were used as inflammatory ontrols. In addition, untreated hondroytes (i.e., ells only exposed to serum-starved medium) served as untreated ontrols. For investigation of NF-κB transloation and IκBα phosphorylation, hondroytes were treated either with IL- 1β (10 ng/ml) or otreated with a ombination of botanial extrats (10 μg/ml) and (10 ng/ml) for 0, 15, 30, and 60 min and nulear/ytoplasmi extrats were prepared TT Assay. Chondroytes were seeded in 96-well plates with 5000 ells/well and inubated overnight in ulture medium ontaining 10% FCS. Positive ontrol ells were left untreated or were treated with the ompounds alone. Negative ontrols were ells treated with alone. Additionally, hondroytes were inubated only with the same quantity of DSO in serum starved medium as in working solutions (without the botanial extrats). For every ontrol and experimental treatment, three wells were used. For measurements after 0, 24, 48, and 72 h, the medium (with or without botanial extrats) was replaed with serum-starved medium and TT (10 μl) was added. After inubation for 4h at 37 C TT solubilization solution was added and ells were inubated at 37 C until TT formazan rystals were ompletely dissolved. Absorbane was measured at a wavelength of 550 nm with a spetrophotometer Immunofluoresene irosopy. Cells were ultivated on glass plates and inubated for 24 h. The ells were then washed three times and preinubated for 1 h with serumstarved medium before stimulation with 10 ng/ml or 10 μg/ml botanial extrats alone or otreated with 10 μg/ml botanial extrats and 10 ng/ml for 30 min in serum-starved (3% FCS) medium. Cells on the glass plates were washed three-times in Hanks solution before methanol fixation for 10 min at ambient temperature (AT), and rinsing with phosphate-buffered saline (PBS). Cell and nulear membranes of hondroytes were permeabilized by treatment with 0.1% Triton X-100 for 1 min on ie. Cells were washed with bovine serum albumin (BSA) for 10 min at AT, rinsed with PBS, and inubated with primary antibodies (p65, phospho-p65, 1 : 30 in PBS). They were gently washed several times with PBS before inubation with seondary antibody (goat-anti-rabbit immunoglobulin onjugated with FITC, diluted 1 : 50 in PBS). Glass plates were finally washed three-times with PBS, overed with fluoromount mountant, and examined under a light mirosope (Axiophot 100, Zeiss, Germany) Isolation of Nulear and Cytoplasmi Chondroyte Extrats. Chondroytes were trypsinized and washed twie in ie-old PBS (1 ml). The supernatant was removed and ell

4 4 Evidene-Based Complementary and Alternative ediine pellets were resuspended in hypotoni lysis buffer (400 μl) ontaining protease inhibitors. After inubation on ie for 15 min, 10% NP-40 (12.5 μl) was added and the ell suspension was vigorously mixed for 15 se. The extrats were entrifuged for 1.5 min. The supernatants (ytoplasmi extrats) were frozen at 70 C. Ie-old nulear extration buffer (25 μl) was added to the pellets and inubated for 30 min with intermittent mixing. Extrats were entrifuged and the supernatant (nulear extrats) transferred to prehilled tubes for storage at 70 C High-Density Cultures. The high density mass ulture was performed on a steel grid bridge as previously desribed [5]. Briefly, a ellulose filter was plaed on the bridge onto whih a ell suspension (8 μl), ontaining approximately 1 million ells, was plaed. Culture medium was in ontat with the filter and the ells were maintained at the filtermedium interfae through diffusion. After one day in ulture, ells formed a three-dimensional pellet on the filter. Culture medium was hanged every three days Transmission Eletron irosopy (TE). Cells were fixed for 1 h with Karnovsky s fixative (paraformaldehydeglutaraldehyde) followed by postfixation in 1% OsO 4 solution (0.1 phosphate buffer), as previously desribed [32]. onolayer ell pellets were rinsed and dehydrated in an asending alohol series before being embedded in Epon and ut on a Reihert-Jung Ultraut E (Darmstadt, Germany). Ultrathin setions were ontrasted with 2% uranyl aetate/lead itrate. A transmission eletron mirosope (TE 10, Zeiss, Jena, Germany) was used to examine the ultures Western Blot Analysis. Chondroyte monolayers were washed three times with Hank s balaned salt solution (HBSS) and whole ell proteins were extrated by inubation with lysis buffer (50 m Tris/HCl, ph 7.2, 150 m NaCl, l% (v/v) Triton X-100, 1 m sodium orthovanadate, 50 m sodium pyrophosphate, 100 m sodium fluoride, 0.01% (v/v) aprotinin, 4 μg/ml pepstatin A, 10μg/mL leupeptin, 1 m PSF) on ie for 30 min, and ell debris was removed by entrifugation. Supernatants were stored at 70 C. Total protein onentration of whole ell, nulear and ytoplasmi extrats was determined aording to the biinhonini aid system (Uptima, Interhim, ontluon, Frane) using BSA as a standard. After adjusting the equal amounts (50 μg of protein per lane) of total protein, proteins were separated by SDS-PAGE (5, 7.5% gels) under reduing onditions. The separated proteins were transferred onto nitroellulose membranes. embranes were preinubated in bloking buffer (5% (w/v) skimmed milk powder in PBS/0.1% Tween-20) for 30 min and inubated with primary antibodies (1 h, AT). embranes were washed three times with bloking buffer and inubated with alkaline phosphatase onjugated seondary antibodies for 30 min. They were finally washed three times in 0.1 Tris ph 9.5 ontaining 0.05 gcl 2 and 0.1 NaCl. Nitro blue tetrazolium and 5-bromo-4-hloro-3-indoylphosphate (p-toluidine salt; Piere, Rokford, IL, USA) were used as substrates to reveal alkaline phosphatase-onjugated speifi antigen-antibody omplexes Statistial Analysis. The results are expressed as the means ± SD of a representative experiment performed in tripliate. The means were ompared using student s t-test assuming equal varianes and P < 0.05 was onsidered statistially signifiant. 3. Results This in vitro study was undertaken to investigate the antiinflammatory effet of three botanial extrats on the signaling pathway leading to the ativation of the transription fator NF-κB and a seletion of its target gene produts, namely, proteins important to hondroyte funtion. Chondroytes treated with botanial extrats (10 μg/ml) showed no signs of ytotoxiity at the light and eletron mirosopi (ultrastrutural) levels. was used to examine the effet of botanial extrats on the NF-κB ativation pathway, beause the pathway ativated by this ytokine is relatively well understood Botanial Extrats Suppress -Indued Chondroyte Cytotoxiity. To test -inhibited hondroyte proliferation an TT assay was performed to study the effets of botanial extrats on the viability and proliferation of hondroytes treated with or without. The TT assay is based on the ability of living ells to redue the TT salt, whereas dead ells or those with impaired mitohondrial ativity are unable to do so. Chondroytes were ultured in a 96-well plate and treated with, botanial extrats, and botanial extrats then treated with for the indiated times. The viability and proliferation of the hondroytes ultivated only in the presene of was signifiantly lower ompared to those of hondroytes treated with botanial extrats, botanial extrats and, or left untreated (Figure 1). The results showed a positive effet of three botanial extrats with regard to ell viability and proliferation on inhibiting -indued ytotoxiity on hondroytes Botanial Extrats Blok -Indued Cellular/Ultrastrutural Changes and Apoptosis in Chondroytes. Control monolayer hondroytes after 24 (not shown), 48 (Figure 2(a)), and 72 h (not shown) showed a typial flattened shape with small ytoplasmi proesses, a large, mostly euhromati nuleus with nuleoli and a well-strutured ytoplasm. -treatment of hondroyte monolayer ultures for 24 (data not shown) and 48 h (Figure 2(b)) leadto degenerative hanges suh as multiple vauoles, swelling of rough ER, lustering of swollen mitohondria, and degeneration of other ell organelles. After longer inubation periods (72 h) (data not shown) more severe features of ellular degeneration were seen in response to treatment. These inluded areas of ondensed heterohromatin in the ell

5 Evidene-Based Complementary and Alternative ediine 5 Absorbane Untreated ontrol DSO ontrol RC SA Treatment UD RC SA UD Figure 1: Effet of botanial extrats and on the proliferation of hondroytes in vitro. Serum-starved hondroytes were exposed to (10 ng/ml) for 48 h, botanial extrats (10 μg/ml) for 72h, otreated first with botanial extrats (10μg/mL eah) for 24 h, and then with (10 ng/ml) for 48 h, treated with DSO (as ontrol) for 72 h or left untreated for 72 h. Cell viability was examined by TT assay. The TT assay is a spetrophotometri measurement of the ell viability as a funtion of the mitohondrial ativity. This assay was performed in tripliate and the results are provided as mean values with standard deviations from three independent experiments. Treatments: Untreated ontrol; ; RC (Rosa anina); SA (Salix alba); UD (Urtia dioia). nulei and multiple ytoplasmi vauoles. The flattened monolayer hondroytes beame inreasingly rounded and apoptoti (Figure 2(b)). Chondroytes pretreated with any of the botanial extrats (10μg/mL) (24 h) and then otreated with and the same botanial extrats (10 μg/ml) for 48 h showed less severe ellular degeneration on the ultrastrutural level (Figures 2() 2(e)). The hondroytes remained a flattened shape with numerous mirovilli-like ytoplasmi proesses. Chondroytes treated with botanial extrats alone (eah at 10 μg/ml) showed no signs of ytotoxi effets on the viability of ells at the light mirosopi and ultrastrutural levels (Figures 2(f) 2(h)). Taken together, these results indiate that all three botanial extrats have antiapoptoti effets and ounterat indued apoptosis in hondroytes Botanial Extrats Inhibit -Indued Downregulation of Extraellular atrix and Signaling Proteins in Chondroytes. Serum-starved hondroytes were treated with IL- 1β (10 ng/ml) alone or were preinubated with three different botanial extrats (10 μg/ml eah) for 24 h and then otreated with (10 ng/ml) for 24, 48, and 72 h. As shown in Figure 3, hondroytes stimulated with IL- 1β alone showed downregulation of synthesis of ollagen type II (Figure 3(I)), artilage-speifi proteoglyan (CSPG) (Figure 3(II)), and β1-integrin (Figure 3(III)). In ontrast to hondroytes stimulated with alone, pretreatment with all botanial extrats resulted in a signifiant upregulation of synthesis of ollagen type II (Figure 3(I)), CSPG, (Figure 3(II)) and β1-integrin (Figure 3(III)). In untreated and in positive ontrol ultures, expression of ollagen type II, CSPG, and β1-integrin were equally strong in hondroytes (Figures 3(I) 3(III)). Synthesis of the housekeeping protein β-atin remained unaffeted in hondroytes exposed to botanials (Figure 3(IV)) Botanial Extrats Inhibit -Indued Upregulation of NF-κB-Dependent ProInflammatory Enzymes and atrix Degrading Gene Produts in Chondroytes. stimulation ativates COX-2 and Ps expression in hondroytes [33]. To investigate whether the three botanial extrats were able to inhibit -indued expression of these proteins, the following experiment was performed. Serum-starved hondroytes were exposed to (10 ng/ml) alone or were preinubated with three different botanial extrats (10μg/mL eah) for 24 hours and then o-treated with (10 ng/ml) for 24, 48 and 72 h. The whole ell extrats were prepared and analyzed by western blotting for the presene of COX-2, P-9 and P-13 (Figures 4(I) 4(III)). As shown in Figure 4, hondroytes showed up-regulation of synthesis of COX-2 (Figure 4(I)), P-9 (Figure 4(II)) and P-13 (Figure 4(III)) in response to (10ng/mL). In ontrast to hondroytes stimulated with alone, pretreatment with all botanial extrats and o-treatment with led to a derease in COX-2, P-9 and P-13 expression (Figures 4(I) 4(III)). In untreated and positive ontrol ultures, expression of COX-2, P-9, and P- 13 was not detetable in hondroytes (Figures 4(I) 4(III)). Synthesis of the housekeeping protein β-atin remained unaffeted (Figure 4(IV)) Botanial Extrats Inhibit the -Indued Downregulation of Adaptor Protein Sh, Signaling Protein P-ERK1/2, and Cartilage-Speifi Transription Fator SOX-9 Expression in Chondroytes. The APKinase pathway plays an important role in hondroyte differentiation and stimulates the hondrogeni fator SOX-9 in hondroytes [7, 8]. SOX- 9 is a transription fator that ontrols the expression of hondroyte-speifi EC protein genes and plays a pivotal role in hondroyte differentiation, thus it was seleted for this study. Additionally, the APKinase signaling pathway, the adaptor protein Sh and the extraellular regulated kinase (Erk1/2) were evaluated. To test the hypothesis that botanial extrats are able to stimulate SOX-9 prodution in hondroytes, monolayer ultures were either left untreated or treated with or botanial extrats alone or were pretreated with botanial extrats (10 μg/ml) for 24 h and then stimulated with for 24 h. The ell lysates were analyzed by immunoblotting. In untreated and in positive ontrol ultures, expression of Sh, ERK1/2, and SOX-9 were equally strong in hondroytes (Figures 5(I) 5(III)). The results demonstrated that treatment with the three botanial extratsinhibited the -indued derease in Sh, ERK1/2 and SOX-9 expression (Figures 5(I) 5(III)). Data shown are representative of three independent experiments. Synthesis of the housekeeping protein β-atin remained unaffeted (Figure 5(IV)) Botanial Extrats Inhibit -Indued NF-κB Ativation in Chondroytes. To examine if botanial extrats blokthe -induedativationofnf-κb, nulear protein

6 6 Evidene-Based Complementary and Alternative ediine (a) (b) () (d) (e) (f) (g) (h) Figure 2: Effet of botanial extrats on -indued ell degradation and apoptosis. Serum-starved hondroytes were either left untreated, (a) exposed to (10 ng/ml) alone (b), or to botanial extrats alone (f h) for 1, 12, 24, 48, and 72 h or pretreated for 24 h with botanial extrats (10 μg/ml) before being otreated with (10 ng/ml) and botanial extrats (10 μg/ml) ( e) and evaluated with TE. Chondroytes treated with (10 ng/ml) exhibited harateristi features of degeneration: annular hromatin ondensation at the nulear envelope of hondroytes, swelling of mitohondria, and rough ER in a time-dependent manner (b). Chondroytes that were pretreated with botanial extrats and then otreated with and botanial extrats (panels e) showed less severe ell degeneration at the ultrastrutural level. In ontrol ultures (a) and treated with botanial extrats alone (panels f h) showed no ultrastrutural hanges. A : 5000; Bar = 1 μm. Treatments: Rosa anina, panel (); Salix alba, panel (d); Urtia dioia, panel (e); Rosa anina without, panel (f); Salix alba without, panel (g); Urtia dioia without, panel (h). extrats from serum-starved hondroytes were probed for the phosphorylated form of p65 NF-κB-subunit after pretreatment with botanial extrats (10 μg/ml eah) for 4 hours followed by otreatment with 10 ng/ml and botanial extrats for 1 h. Some hondroyte ultures remained either untreated or were treated with 10 μg/ml botanial extrats (eah alone) or with 10 ng/ml alone for 1 h (Figure 6(I)). Results indiate that botanial extrats inhibited -indued NF-κB ativation (Figure 6(I)). The synthesis of the PARP protein remained unaffeted (Figure 6(II)) Botanial Extrats Inhibit -Stimulated Nulear- Transloation of NF-κB in Chondroytes. Immunofluoresene mirosopy was employed to reveal transloation of phosphorylated NF-κB from the hondroyte ytoplasm to the nuleus in response to. Chondroytes remained either unstimulated (Figure 7(a)) or were treated with 10 μg/ml botanial extrats (eah alone) or with 10 ng/ml alone for 10 min (Figure 7(b)) or were otreated with 10 μg/ml botanial extrats (eah alone) 10 min and then 10 ng/ml for 1h (Figures 7() 7(e)) before indiret immunolabeling with anti-nf-κb antibody. Control hondroytes and hondroytes treated with the botanial extrats alone (not shown) showed only ytoplasmi labeling of NF-κB (Figure 7(a)). -stimulated ells revealed lear and intensive ytoplasmi and nulear staining for NF-κB (Figure 7(b)). Cotreatment of hondroytes with botanials and resulted in inhibition of nulear transition of ativated phosphor-p65 and dereased ytoplasmi staining for this protein and showed a derease in ativation of

7 Evidene-Based Complementary and Alternative ediine 7 I Type II ollagen II CSPG III β1-integrin IV β-atin C RC RC C SA SA C UD UD Figure 3: Effets of botanial extrats on -indued downregulation of extraellular matrix and signaling proteins in hondroytes. Serum-starved hondroytes ( ells/ml) were ultured for 24 h and then treated with 10 ng/ml for 48 h, botanial extrats (eah 10 μg/ml) for 72 h, or pretreated with botanial extrats (10 μg/ml eah) for 24 h and then otreated with 10 ng/ml for 48 h or left untreated and evaluated after 72 h. Western blot analysis revealed down-regulation of ollagen type II (I), CSPG (II) and β1-integrin (III) in hondroytes by. Co-treatment of hondroytes preinubated with botanial extrats and suppressed the -indued inhibition of ollagen type II, CSPG and β1-integrin (I, II, III). In untreated and in botanial extrats alone treated ontrol ultures, expression of ollagen type II, CSPG and β1-integrin were equally strong in hondroytes (I III). Expression of β-atin was not affeted by and/or botanial extrats (IV). Data shown are representative of three independent experiments. Treatments: C (untreated ontrol); ;RC(Rosa anina); SA (Salix alba); UD (Urtia dioia). NF-κB (Figures 7() 7(e)). These immunomorphologial findings were onsistent with the NF-κB inhibition observed by western blotting Botanial Extrats Inhibit -Indued IκBα Degradation in Chondroytes. In this study, botanial extrats inhibited -indued ativation of NF-κB and its transloation to the hondroyte nuleus. An important prerequisite for the ativation of NF-κB is the phosphorylation and degradation of IκBα, the natural bloker of NF-κB [34]. To examine whether inhibition of -indued NF-κB ativation ours through inhibition of IκBα degradation, some hondroyte ultures were treated with (10 ng/ml) for the indiated times (Figures 8(I) 8(III)) and other hondroyte ultures were first treated with three botanial extrats (10 μg/ml eah) for 4 h followed by o-treatment with (10 ng/ml) for the indiated time periods. ould not indue IκBα degradation in hondroytes when o-treated with botanial extrats (Figures 8(I) 8(III)). Considering, -indued IκBα degradation in untreated ultures is an indiator of NF-κB ativation, the results suggest that the botanial extrats blok -indued IκBα degradation Botanial Extrats Inhibit -Dependent IκBα Phosphorylation in Chondroytes. To determine if the botanial extrats are able to inhibit the -indued phosphorylation of IκBα, serum-starved hondroytes were treated with for 1h and examined by western blot analysis using an antibody that reognizes the phosphorylated form of IκBα.It is known that phosphorylation of IκBα leads to its degradation [16], and that the phosphorylation and degradation of IκBα are inhibited by a speifi proteosome inhibitor N-A- Leu-Leu-norleuinal (ALLN) [35]. As shown in Figures 9(I) 9(III), was still able to phosphorylate some IκBα in ells pretreated with the inhibitor and IκBα phosphorylation was signifiantly higher ompared to ontrol ells. Interestingly,

8 8 Evidene-Based Complementary and Alternative ediine I COX-2 II P-9 III P-13 IV β-atin C RC RC C SA SA C UD UD Figure 4: Effets of botanial extrats on -indued upregulation of proinflammatory enzymes in hondroytes. Serum-starved hondroytes ( ells/ml) were ultured for 24 h and then treated with 10 ng/ml for 48 h, botanial extrats (eah 10 μg/ml) for 72 h, or pretreated with botanial extrats (10 μg/ml eah) for 24 h and then otreated with 10 ng/ml for 48 h or left untreated and evaluated after 72 h. -stimulation leads to an inrease in synthesis of COX-2, P-9, and P-13 (I, II, III). However, COX- 2, P-9, and P-13 upregulation was bloked in hondroytes pre-inubated with botanial extrats (10 μg/ml eah) for 24 h and then o-treated with (10 ng/ml) for 48 h (I, II, III). In untreated and in botanial extrats alone treated ontrol ultures, expression of COX-2-, P-9 and P-13 were not seen in hondroytes (I III). Expression of the housekeeping gene β-atin was not affeted by treatment with and/or botanial extrats (IV). Data shown are representative of three independent experiments. Treatments: C (untreated ontrol); ;RC(Rosa anina); SA (Salix alba); UD (Urtia dioia). all botanial extrats were able to inhibit the phosphorylation of IκBα indued by in the presene or absene of the inhibitor Botanial Extrats Inhibit -Indued Effets in a 3- Dimensional (High-Density) Culture odel of Chondroytes. To test whether hondroytes from monolayer ultures with or without and/or botanials were able to produe artilage-speifi EC and artilage, high-density ultures were prepared from hondroytes in monolayer ulture. These onsisted of untreated ontrol ells and ells treated with botanial extrats (10μg/mL) or (10 ng/ml) alone for 24 h before being treated with (10 ng/ml) and ultivated for 7 days under idential onditions. As shown in Figure 10, ontrol ultures of hondroytes formed blastema-like nodules and made tight ontats. They exhibited round to oval shapes, large euhromati nulei, free ytoplasmi ribosomes, mitohondria and endoplasmi retiulum (ER), as well as vauoles. The ells appeared as viable hondroytes exhibiting harateristi morphologial features and formed a regular fibrillar extraellular matrix (Figure 10(a)). In ontrast, hondroytes underwent apoptosis when treated with (10 ng/ml) for 7 days (Figure 10(b)). Chondroytes o-treated with botanial extrats and (eah 10 μg/ml) showed well-developed artilage nodules (Figures 10() 10(e)). Pre-treatment with botanial extrats (eah 10μg/mL) alone resulted in welldeveloped artilage nodules with viable ells and organized organelles; the ells formed a dense and regular EC (Figures 10(f) 10(h)). 4. Disussion The goal of this study was to haraterize the effet and mode of ation of three botanial extrats derived from plants with previously reported anti-inflammatory ativity on NF-κB expression in primary anine hondroytes in vitro. Under the experimental onditions, (1) botanial extrats inhibited the -mediated suppression of key extraellular matrix and signaling proteins in hondroytes; (2) botanial extrats antagonized the -dependent upregulation of P-9, P-13, and COX-2; (3) aused phosphorylation and nulear transloation of the p65 NF-κB subunit; (4) aused phosphorylation and subsequent degradation of the inhibitory subunit of NF-κB: IκBα;(5)-indued NF-κB ativation and IκBα degradation was inhibited by botanials; (6) finally, in ontrast to -treated ells, the ells treated with botanial extrats redifferentiated into hondroytes after transfer to high-density ulture and produed a artilage-speifi matrix, that is, ollagen type II, even when otreated with. Therefore, the results obtained strongly suggest that the botanial extrats inhibit -indued upregulation of P-9, P-13, and COX-2 by preventing, at least in part, IκBα degradation and NF-κB ativation. The shemati in Figure 11 summarizes the possible mode of ation of the botanial extrats. Systemati reviews of linial studies show little evidene to support botanial remedies as effiaious the treatment for OA [36]. However, the three plants from whih extrats have been analyzed in this study have been used in traditional mediines for many enturies and all had laims related to treatment for OA. Whilst patients ontinue to seek

9 Evidene-Based Complementary and Alternative ediine 9 I Sh II ERK-1/2 III SOX-9 IV β-atin C RC RC C SA SA C UD UD Figure 5: Effet of botanial extrats on signaling proteins and artilage-speifi transription fator SOX-9 in hondroytes. Serum-starved hondroytes ( ells/ml) were ultured for 24 h and then treated with 10 ng/ml for 48 h, botanial extrats (eah 10 μg/ml) for 72 h, or pretreated with botanial extrats (10 μg/ml eah) for 24 h and then otreated with 10 ng/ml for 48 h or left untreated and evaluated after 72 h. Results of western blot analysis revealed down-regulation of Sh (I), ERK1/2 (II), and SOX-9 (III) in hondroytes with. Cotreatment of hondroytes preinubated with botanial extrats and relieved the -indued inhibition of Sh (I), ERK1/2 (II), and SOX-9 (III). In untreated and in positive ontrol ultures, expression of Sh, ERK1/2, and SOX-9 were equally strong in hondroytes (I III). Expression of β-atin was not affeted by and/or botanial extrats (IV). Data shown are representative of three independent experiments. Treatments: C (untreated ontrol); ; RC(Rosa anina); SA (Salix alba); UD (Urtia dioia). Nulear extrats I NF-κB II PARP C RC RC C SA SA C UD UD Figure 6: Botanial extrats blok the -indued phosphorylation and nulear transloation of p65 in hondroytes. Western blot analysis of -treated nulear extrats. Serum-starved hondroytes ( ells/ml) were pretreated with botanial extrats (10 μg/l eah) for 4 hours followed by otreatment with 10 ng/ml and botanial extrats for 1 h. Some hondroyte ultures remained either untreated or were treated with 10 μg/ml botanial extrats (eah alone) or with 10 ng/ml alone for 1 h. Nulear extrats were probed for phospho p65, (I) by western blot analysis using antibodies to p65, phospho-speifi p65, and PARP (II, ontrol). Treatment of hondroytes with (10 ng/ml) revealed a lear inrease in expression of phospho-p65 in the nulear extrats (I). Co-treatment of hondroytes with botanial extrats (all three) ompletely abolished the -dependent ativation of phospho p65 in the nuleus (I). Synthesis of PARP remained unaffeted in nulear extrats (II). Data shown are representative of three independent experiments. Treatments: C (untreated ontrol); ;RC(Rosa anina); SA (Salix alba); UD (Urtia dioia). natural remedies to help treat themselves or their ompanion animals, it is important to provide insights into whether, and if so how, suh botanials may work. As suh, in vitro models provide an objetive benhmark to indiate potential modes of ation if translated in vivo. The three botanial extrats in this study are derived from different parts of the plant and previous publiations havelaimed different ative omponents. The data provided in this study may be used to suggest that there are atives in all three botanials that an inhibit the -indued inflammatory proess upstream of the IκBα phosphorylation step. Whilst the exat mehanism of ation remains unknown, the data may also be used to indiate that IL- 1β-suppressing ativities are ommon to a number of plant speies and tissues. With a growing number of publiations laiming speifi atives, it may be an appropriate time to onsider ommonalities of botanial extrats rather than fous attention on unique attributes of a multitude of speifi plants, whih may lead to onfusion and skeptiism toward the area of phytotherapy. All three botanial extrats had a positive effet on hondroyte viability, differentiation and funtion as well as having inhibitory effets on -indued suppression of proliferation and viability. Furthermore, the three botanials enhaned mitohondrial ativity in hondroytes, as measured using the TT assay. Reports have observed botanials that interfere with the assay through antioxidant (e.g., thiols and flavonoids) redution of TT [37], and we have also

10 10 Evidene-Based Complementary and Alternative ediine UD SA edium RC (a) () (b) (d) Figure 7: Botanial extrats inhibit -indued nulear transloation of NF-κB in hondroytes. Chondroyte ultures either served as ontrols (a) or were treated with alone for 10 min (b) or otreated with botanial extrats for 10 min and then otreated with for 1 h ( e) before immunolabeling with NF-κB antibodies and FITC-oupled seondary antibodies. In ontrol ells anti-nf-κb labeling was restrited to the ytoplasm (a). Cells treated with alone revealed nulear transloation of NF-κB (b) that was partly inhibited by otreatment with botanial extrats ( e). (a e): 160. Data shown are representative of three independent experiments. (e) observed signifiant negative and insignifiant responses with other botanials (data not shown). Whilst the exat reason for the enhaned mitohondrial ativity was not investigated, it was onsidered an indiator of enhaned metabolism and, as suh, was onsidered a potentially positive attribute. Cell-matrix interations in artilage are essential for the proliferation, differentiation and survival of ells and this interation is mediated by speifi surfae reeptors, for example, integrins [6, 7, 38]. β1-integrins are able to organize ell surfae mehanoreeptor omplexes [39] and funtion as signal transdution moleules [40] stimulating APkinase pathways [7, 8]. Several studies have already shown that redued ell-matrix interations lead to inhibition of Erk1/2 signaling and stimulate the apoptoti pathway in hondroytes [8]. In this in vitro model system, indued downregulation of ollagen type II, CSPG, and integrin expression in hondroytes. These findings are in agreement with previous in vitro studies [18]. Treatment with three botanials prevented the -indued inhibition of ollagen type II, CSPG, and integrin expression in stimulated hondroytes. In this study, indued upregulation of P-9, P-13, and COX-2. Cell-matrix interation requires a permanent remodeling of extraellular matrix proteins exeuted by Ps, a group of zin-dependent endopeptidases that leave EC moleules [41] and high levels of Ps (P-1, P-3, P-9, and P-13) are found in the synovium and serum of OA and RA patients [42, 43]. COX-2 is an important mediator of pain and inflammation in OA joints [44] ausing PGE 2 and thromboxane prodution [45]. PGE 2 indues many other pathologial ataboli effets in artilage suh as dereased proliferation of hondroytes and inhibition of EC synthesis [45]. We suggest that the downregulation of Ps and COX-2 by botanials is regulated, at least in part, via NF-κB inhibition, beause the expression of these enzymes is regulated by NF-κB [33, 46 48]. Cytokine-indued P and COX-2 upregulation is regulated by ativation of the ubiquitous transription fator NF-κB [49]. This transription fator plays an important role during the pathogenesis of OA, by mediating the expression of ataboli and inflammation-related genes. Interestingly, inhibitors of NF-κB have anti-inflammatory and antidegradative effets in animal models of OA [50].

11 Evidene-Based Complementary and Alternative ediine 11 Cytoplasmi extrats edium RC treatment (min) I IκBα edium SA treatment (min) II IκBα edium UD treatment (min) III IκBα IV β-atin Figure 8: Effets of botanial extrats on the kinetis of IκBα by in hondroytes. Effet of botanial extrats on -indued degradation of IκBα. Western blot analysis with -treated ytoplasmi extrats. Serum-starved hondroytes ( ells/ml) were treated with (10 ng/ml) for 0, 10, 30 and 60 min. Other ultures were initially treated with botanial extrats (10 μg/ml) for 4 h and then otreated with (10 ng/ml) for the indiated times or left untreated (medium ontrols). The ytoplasmi extrats were prepared, frationated on SDS-PAGE, and eletroblotted onto nitroellulose membranes. Western blot analysis was performed with anti-iκbaand antiβ-atin (ontrol). aused IκBα degradation in ultures as early as 10 min after treatment. In otreated hondroytes with eah botanial extrats the degradation of IκBα was not observed (I III). Synthesis of β-atin remained unaffeted (IV). Data shown are representative of three independent experiments. Treatments: medium (ontrols); ; RC(Rosa anina); SA (Salix alba); UD (Urtia dioia). Cytoplasmi extrats edium RC ALLN I p-iκbα edium SA ALLN II p-iκbα edium UD ALLN III p-iκbα IV IκBα Figure 9: Effet of botanial extrats on the phosphorylation of IκBα by in hondroytes. Western blot analysis with -treated ytoplasmi extrats. Serum-starved hondroytes ( ells/ml) were pretreated with ALLN (100 μg/ml) for 30 min and otreated with botanial extrats (eah 10 μg/ml)for4handstimulatedwith (10 ng/ml) for the final 1 h. The ytoplasmi extrats were prepared, frationated on SDS-PAGE, and eletroblotted onto nitroellulose membranes. Western blot analysis was performed using anti-iκbα and p-iκbα antibodies. Treatment of hondroytes with (10 ng/ml) revealed an inrease in the phosphorylated IκBα-forminytoplasmi extrats. In the presene of the inhibitor phosphorylation of IκBα was signifiantly inreased (I III). Phosphorylation of IκBα was inhibited in hondroytes otreated with botanial extrats in the presene or absene of the inhibitor (I III). Data shown are representative of three independent experiments. Treatments: medium (ontrols); ;RC(Rosa anina); SA (Salix alba); UD (Urtia dioia).

12 12 Evidene-Based Complementary and Alternative ediine (a) (b) () (d) (e) (f) (g) (h) Figure 10: Cultivation of hondroytes in a 3-dimensional ulture system (High-density ultures) in vitro in the presene of botanial extrats. Chondroytes were treated with botanial extrats (10 μg/ml) or (10 ng/ml) alone for 24 h before being treated with (10 ng/ml) for 7 days in high-density ultures. Control ultures of hondroytes showed well-developed artilage nodules (a). Treatment with resulted in ell destrution after 7 days (b). After the otreatment of high-density ultures with botanial extrats and (panels e) or in the absene of (panels f h) the hondroytes exhibited well-developed artilage nodules. 4000; bars: 1 μm. Treatments: Rosa anina, panel (); Salix alba, panel (d); Urtia dioia, panel (e); Rosa anina without, panel (f); Salix alba without, panel (g); Urtia dioia without, panel (h). In the present study, inreased phosphorylation of p65 in response to was demonstrated. This phosphorylation event, in turn, leads to its degradation and subsequent release of ativated NF-κB. The results also showed that IκBα was ompletely abolished in the ytoplasmi extrats in hondroyte ultures treated with alone, indiating that this ytokine indued its degradation. This indiates NFκB ativation. Treatment of the hondroyte ultures with the botanial extrats resulted in high onentrations of IκBα in the ytoplasm and dereased levels of phosphorylated p65 in nulear extrats. These results strongly suggest that the botanial extrats inhibit -indued downregulation of artilage speifi EC ompounds, APK-signaling proteins, artilage-speifi transription fators and upregulation of proinflammatory and degrading enzymes through NF-κB ativation by preventing, at least in part, IκBα phosphorylation and degradation. The artilage-speifi transription fator SOX-9 plays an important role in the expression of artilage-speifi extraellular matrix genes [51]. In this study, a redution in ollagen type II and SOX-9 expression in hondroytes after treatment with was observed, in agreement with another study [52]. Other investigators have shown that ytokines partially redue SOX-9 protein levels through a NF-κB-dependent, posttransriptional mehanism in mouse hondroytes [18]. However, by treating ells with the botanial extrats, inhibition of the -indued NF-κBdependent downregulation of ollagen type II and SOX-9 expression was observed. The results of this study suggest that the botanial extrats markedly suppressed ytokineindued ativation and upregulation of proinflammatory enzymes suh as Ps and COX-2, transription fator NF-κB and downregulation of artilage-speifi matrix omponents and important signaling proteins in hondroytes.

13 Evidene-Based Complementary and Alternative ediine 13 R Plasma membrane Cytoplasm P Rosa anina Salix alba Urtia dioia p65 p50 IκBα P p65 p50 IκBα P p65 p50 U ALLN P IκBα U Nuleus P Transription p65 p50 COX-2 Ps VEGF EC Figure 11: Inhibitory effets of botanial extrats on -indued NF-κB ativation in hondroytes in vitro. stimulates the IL- 1β reeptor, initiating an intraellular signal transdution asade, whih ativates the ytoplasmi IκBα kinases (Iκκ)-α, Iκκ-β, and Iκκ-γ. These kinases phosphorylate inative IκBα. Phosphorylated IκBα is then ubiquitinated and degraded by the proteasome and ative NFκB is released. NF-κB transloates to the nuleus, where it ativates proinflammatory and proapoptoti gene prodution. In hondroytes, botanial extrats inhibit the NF-κB signal transdution pathway, ubiquitination of phosphorylated IκBα and blok transloation of ativated NF-κB to the nuleus. Whilst botanial extrats may exhibit multiple modes of ation, based on the IκBα phosphorylation data, it is probable that inhibition of the signaling pathway upstream of IκBα phosphorylation is likely to be the major ause of the anti-inflammatory ativity observed in this study. onolayer ultures of hondroytes appear to be a valid model for investigating the mode of ation of plant extrats with potential anti-inflammatory properties. However, in vivo hondroytes exist within a three-dimensional extraellular matrix. Therefore, studies were also performed using highdensity ultures, whih indiated that the botanial extrats do inhibit -indued inflammation and apoptosis, allowing the ells in high-density ultures to redifferentiate bak into hondroytes. 5. Conlusion The three botanial extrats used in this study were derived from different parts of the plants used in traditional mediine. Previous publiations have laimed different ative omponents and several onstituents whih have antiinflammatory effets. In this study similar in vitro effets of these three different botanial extrats are important observations and an be used to support the view that they may be potential hondroprotetive agents. However, further investigations are needed to haraterize the biologial entities present within the extrats, to eluidate their subellular targets in vitro and to determine whether they are apable of any similar ativity or synergism in vivo. Abbreviations ALLN: Proteasome inhibitor N-A-Leu-Leu-norleuinal AT: Ambient temperature CSPG: Cartilage-speifi proteoglyans COX-2: Cylooxygenase-2 DSO: Dimethyl sulfoxide ERK 1/2: Extraellular regulated kinases 1 and 2 FCS: Fetal alf serum IKK: LκB kinase : Interleukin-1β APK: itogen-ativated protein kinase P: atrix metalloproteinase NF-κB: Nulear fator-κb TT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NSAIDs: Nonsteroidal anti-inflammatory drugs

14 14 Evidene-Based Complementary and Alternative ediine OA: Osteoarthritis; PARP (poly(adp-ribose) polymerase) PBS: Phosphate-buffered saline Sh: sr homology ollagen TIPS: Tissue inhibitors of Ps. Conflit of Interests The authors delare that they have no ompeting interests nor ommerially support or endorse Shamanshop, the soure of the botanial extrats used in this study. Authors Contribution S. Nebrih arried out the experimental work and data olletion.. Shakibael, D. Allaway, and A. obasheri oneived the study design, oordinated the studies, data interpretation, and paper preparation. All authors have read and approved the final paper. Aknowledgments This study was supported by grants to. Shakibaei and A. obasheri from the WALTHA Centre for Pet Nutrition (ars). A.obasheri wishes to aknowledge the finanial support of the Biotehnology and Biologial Sienes Researh Counil (BBSRC; Grant no. BBS/S//2006/13141), The Wellome Trust (Grant no. CVRT VS 0901), and startup funding from the University of Nottingham. The authors would like to aknowledge the exellent tehnial assistane of rs. Christina Pfaff and s. Ursula Shwikowski and the additional sientifi support provided by Dr. Constanze Buhrmann. We would like to aknowledge Puleva Bioteh for the botanial extrations. The authors thank Professor Dr. Ulrike atis, from the Clini of Veterinary Surgery at Ludwig-aximilian-University unih, Germany, for the generous supply of anine artiular artilage samples. Referenes [1]. B. Goldring and S. R. Goldring, Osteoarthritis, Journal of Cellular Physiology, vol. 313, pp , [2] Y. Henrotin, C. Sanhez, and. Balligand, Pharmaeutial and nutraeutial management of anine osteoarthritis: present and future perspetives, Veterinary Journal, vol. 170, no. 1, pp , [3] J. A. Bukwalter, J. artin, and H. J. ankin, Synovial joint degeneration and the syndrome of osteoarthritis, Instrutional Course Letures, vol. 49, pp , [4]. B. 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