英-GSPE保护关节炎

Immunology Letters 124 (2009) 102–110 Contents lists available at ScienceDirect Immunology Letters journa l homepage: www.e lsev ier . Grape seed proanthocyanidin extract (GSPE) atte collagen-induced arthritis Mi-La Ch -Sil Yun-Ju W un- The Rheumatis ity of K a r t i c l Article history: Received 23 M Received in re Accepted 3 Ma Available onlin Keywords: Grape seed proanthocyanidin extract (GSPE) Oxidative stress Autoimmune arthritis Collagen-indu Antioxidant ntho n-ind intrap l par resis and bone-marrow cells cultured with the receptor activator of nuclear factor B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Intracellular levels of hydrogen peroxide were deter- mined using carboxy-dichlorodihydrofluorescein diacetate. GSPE treatment significantly attenuated the severity of CIA in a dose-dependent manner and reduced the histology scores for synovial inflamma- tion, cartilage erosion, bone erosion, and the number of TRAP+ osteoclasts. GSPE treatment significantly 1. Introdu Reactive (O2−), hydr continuous act as a ph and are inv ing prolifer [1]. Howev teins, DNA, scavenged b oxide dismu reductase.N ∗ Correspon Medicine, Coll (RhRC), Catho Korea, South K E-mail add 1 These auth 0165-2478/$ – doi:10.1016/j.i ced arthritis (CIA) reduced the numbers of tumor necrosis factor alpha (TNF-�)- or interleukin 17 (IL-17)-producing cells in the synovial tissue and the spontaneous production of TNF-� and IL-17 by splenocytes compared with those in the control mice. The serum levels of type-II-collagen-specific IgG2a and plasma levels of 8-isoprostane in the GSPE-treated mice were significantly lower than those in the control mice. GSPE dose-dependently suppressed osteoclastogenesis in vitro. GSPE significantly reduced hydrogen perox- ide production by anti-CD3-monoclonal-antibody-stimulated CD4+ splenocytes. These results indicate that intraperitoneal injection of GSPE attenuated CIA in mice. GSPE may be useful in the treatment of rheumatoid arthritis. © 2009 Elsevier B.V. All rights reserved. ction oxygen species (ROS) such as the superoxide anion ogen peroxide (H2O2), and the hydroxyl radical, are ly generated during normal cell metabolism. ROS can ysiological defense system against microbial infection olved in maintaining normal cellular functions, includ- ation, apoptosis, and intracellular signal transduction er, excessive amounts of ROS can damage lipids, pro- and the extracellular matrix [2]. Therefore, ROS must be y antioxidants. Enzymatic antioxidants include super- tase, glutathione peroxidase, catalase, and thioredoxin onenzymatic antioxidants includeglutathione, vitamin ding author at: Division of Rheumatology, Department of Internal ege of Medicine, Holy Family Hospital, Rheumatism Research Center lic Research Institute of Medical Science, The Catholic University of orea. Tel.: +82 32 340 7016; fax: +82 32 340 2669. ress: rmin6403@hanmail.net (J.-K. Min). ors contributed equally to this work. A, vitamin C, and vitamin E [3]. Oxidative stress refers to the cellular state in which the production of ROS is elevated and/or the levels of antioxidants reduced. Oxidative stress is considered to be involved in the pathogenesis of atherosclerosis, cancer, neurodegenera- tive disorders, diabetes, ageing, and autoimmune rheumatological diseases, including rheumatoid arthritis (RA), systemic lupus ery- thematosus, and systemic sclerosis [3]. RA is a chronic inflammatory autoimmune disease character- ized by synovitis, bone destruction with pannus formation, and the degradation of the articular cartilage. The cause of RA is unknown, but several studies have suggested that oxidative stress is asso- ciated with the pathogenesis of RA. Epidemiological studies have demonstrated an inverse correlation between the dietary intake of antioxidants and the incidence of RA [4]. Inverse associations between serum antioxidant levels and the risk of developing RA havebeen reported [5]. Thedisease activity also correlates inversely with antioxidant levels and positively with the presence of oxida- tive stress in patients with RA [6,7]. Increased oxidative enzyme activity has been demonstrated, together with reduced enzymatic and nonenzymatic antioxidant levels, in RA sera and synovial flu- ids [8]. Oxidative damage to proteins, lipids, DNA, cartilage, and see front matter © 2009 Elsevier B.V. All rights reserved. mlet.2009.05.001 o1, Yu-Jung Heo1, Mi-Kyung Park, Hye-Jwa Oh, Jin oo, Ji-Hyeon Ju, Sung-Hwan Park, Ho-Youn Kim, J m Research Center, Catholic Research Institute of Medical Science, The Catholic Univers e i n f o arch 2009 vised form 29 April 2009 y 2009 e 14 May 2009 a b s t r a c t To examine whether grape seed proa dant has therapeutic effect on collage arthritis. Mice were treated with an Clinical, histological, and biochemica genesis were determined by tartrate- com/ locate / nuates Park, Ki Min ∗ orea, Seoul, South Korea cyanidin extract (GSPE) which is known to act as an antioxi- uced arthritis (CIA) in mice, an animal model of rheumatoid eritoneal injection of GSPE (10, 50, or 100mg/kg) or saline. ameters were assessed. The effects of GSPE on osteoclasto- tant acid phosphatase (TRAP) staining of the inflamed joints M.-L. Cho et al. / Immunology Letters 124 (2009) 102–110 103 extracellular collagen has been demonstrated in patients with RA [9]. Some drugs used to treat RA such as methotrexate, etancercept and infliximab are known to play essential roles as antioxidative agents [1]. Lipid peroxidation markers such as serum malondi- aldehyde a collagen-in [10,11]. The tempol, �-l of green tea strated in m Grape se lipids, prot dins are the and are hi or trimers proanthocy activity tha vitamin E, a such as anti vasodilator It has b oxidation-r atheroscler [21]. Thebe lipoprotein chloasma h However, li rheumatoid potentials i on the sev cal, and bio attenuates type-II-coll oxidative st vitro. 2. Materia 2.1. Animal DBA/1J m housed in p (Ralston Pu imental pro Research Et 2.2. Prepara Bovine t Kang of the in its native described p 2.3. Inducti CIA was cations [26 injection of Freund’s ad base of the intraderma sified in inc into thehin tical Compa intraperiton after the se different doses of GSPE (10, 50, or 100mg/kg) five times per 2 days for 2.5 weeks. The control mice were injected with saline. Blood samples were collected from all treated and control mice 8 weeks after the primary immunization and stored at −70 ◦C until use. inica seve ers. T verit mu with dem t or sal b tarsa tire l sum coll ed. T mea trol istolo ind l hydr hen sec mati infla nfiltr ining keni esenc ted w ined term ing cr sing re ex e ero eros mag ous a in th becu tex, , 4 = ular b rtical nd m he re e-res com the ema were eterm .All h d obs easu seru eas nd urine isoprostane are reported to be elevated in duced arthritis (CIA) compared with those in controls beneficial effects of antioxidants, including vitamin E, ipoic acid, N-acetylcysteine, the polyphenolic fraction , and (−)-epigallocatechin-3-gallate, have been demon- ice with CIA [12–17]. ed extract is a natural plant constituent and contains eins, carbohydrates, and polyphenols. Proanthocyani- most abundant phenolic compounds in grape seeds, gh-molecular-weight polymers comprised of dimers of (+)-catechin and (−)-epicatechin [18]. Grape seed anidin extract (GSPE) has more powerful antioxidative n other well-known antioxidants, including vitamin C, nd gallic acid [19]. GSPE has various biological functions bacterial, antiviral, anti-inflammatory, antiallergic, and y actions [20]. een suggested that GSPE is an effective therapy for elated diseases in animal models, including tumors, osis, gastric ulcer, cataract, and diabetic retinopathy neficial effects ofGSPE in reducingoxidized low-density s and postprandial oxidative stress and improving ave been demonstrated in human clinical trials [22–24]. ttle is known about the effects of GSPE on CIA and arthritis. To examine whether GSPE has therapeutic n the treatment of RA, we examined the effects of GSPE erity of CIA in DBA/1 mice, using clinical, histologi- chemical parameters. We found that GSPE treatment the severity of CIA in mice, reducing serum levels of agen-specific IgG2a, inflammatory cytokine production, ress, bone destruction in vivo, and osteoclastogenesis in ls and methods s ice (SLC, Inc., Shizuoka, Japan), 6–8 weeks old, were olycarbonate cages and fed with standard mouse chow rina, St Louis, MO, USA) and water ad libitum. All exper- cedures were examined and approved by the Animal hics Committee of the Catholic University of Korea. tion of type II collagen ype II collagenwas kindly providedbyProfessorAndrew University of Tennessee. Type II collagen was extracted form from fetal calf articular cartilage and purified as reviously [25]. on of CIA and GSPE treatment induced as previously described, with minor modifi- ]. Briefly, male DBA/1J mice were given an intradermal 100�g of bovine type II collagen emulsified in complete juvant (1:1,w/v; Arthrogen-CIA, Redmond,WA) into the tail. Two weeks later, the mice were given a booster l injection of 100�g of bovine type II collagen emul- omplete Freund’s adjuvant (1:1, v/v; Difco, Detroit, MI) dpaw.GSPEwaskindlyprovidedbyHanlimPharmaceu- ny (Seoul, Korea). GSPE, dissolved in saline, was given eally to three different groups (10, 50, or 100mg/kg) condary immunization. The mice were injected with 2.4. Cl The observ and se mary im of 0–4 0=no e the foo the tar to the the en as the type II exclud 12. The the con 2.5. H A h fied in were t stained Inflam 0=no some i of the l 3 = thic and pr infiltra determ age de follow ited to 3=mo of bon (0=no at low numer cation, the tra the cor fication trabec ing co bone a file of t Tartrat with a ing to with h nuclei were d al. [30] blinde 2.6. M The were m l assessment of arthritis rity of arthritis was determined by three independent hemicewere observed three times aweek for the onset y of joint inflammation for up to 8 weeks after the pri- nization. The severityof arthritiswasassessedonascale the following criteria, as described previously [27]: a or swelling, 1 = slight edema and erythema limited to ankle, 2 = slight edema and erythema from the ankle to one, 3 =moderate edema and erythema from the ankle l bone, and 4=edema and erythema from the ankle to eg. The arthritic score for each mouse was expressed of the scores of three limbs. The hind paw into which agen+ incomplete Freund’s adjuvant was injected was he highest possible arthritis score for a mouse was thus n arthritis index was used to compare the data among and experimental groups. gical assessment of arthritis eg of each mouse was fixed with 10% formalin, decalci- ochloric acid, and embedded in paraffin. The sections stained with hematoxylin and eosin (H&E). The H&E- tions were scored for inflammation and bone erosion. on was scored according to the following criteria [28]: mmation, 1 = slight thickening of the lining layer or ating cells in the sublining layer, 2 = slight thickening layer plus some infiltrating cells in the sublining layer, ng of the lining layer, influx of cell in the sublining layer, e of cells in the synovial space, and 4= synovium highly ith many inflammatory cells. Cartilage damage was with safranin-O staining. The extent of cartilage dam- ined by safranin-O staining was scored according to the iteria [28]: 0 =no destruction, 1 =minimal erosion, lim- le spots, 2 = slight to moderate erosion in a limited area, tensive erosion, and 4=general destruction. The extent sion was expressed using a scoring system from 0 to 5 ion, 1 = small areas of resorption not readily apparent nification, in the trabecular or cortical bone, 2 =more reas of resorption, not readily apparent at low magnifi- e trabecular or cortical bone, 3 =obvious resorption of lar and cortical bone, without full-thickness defects in loss of some trabeculae, lesions apparent at low magni- full-thickness defects in the cortical bone and marked one loss,without distortion of theprofile of the remain- surface, and 5= full-thickness defects in the cortical arked trabecular bone loss, with distortion of the pro- maining cortical surface), as previously described [29]. istant acid phosphatase (TRAP) stainingwas performed mercial kit (cat. no. 387-A, Sigma, St Louis, MO), accord- manufacturer’s instructions, omitting counterstaining toxylin. TRAP+ multinucleate cells with three or more considered osteoclasts. The numbers of osteoclasts ined according to the method described by Bendele et istological assessmentsweremadeby two independent ervers. rement of type-II-collagen-specific antibodies m levels of type-II-collagen-specific IgG2a and IgG1 ured by enzyme-linked immunosorbent assay (ELISA), 104 M.-L. Cho et al. / Immunology Letters 124 (2009) 102–110 as previously described, with minor modifications [31]. Briefly, microtiter plateswere coatedwith type II collagen (4�g/mL in PBS) at 4 ◦C overnight, followed by a blocking step for 30min at room temperature. Serum samples were then diluted 1:8000 in Tris- buffered sa 0.5% Tween which the IgG2a and I Quantitatio tively. Stand in serial dil arbitrary un The absorba reader oper 2.7. Cell pre Eightwe were collec spleens wer ammonium and centrifu resuspende (Corning, N CD4+ T cells of >90%. 2.8. Measur supernatant Isolated � and IL-17 Antibodies biotinylated ture and de (Sigma) wa of cytokine standard cu murine TNF mined with 2.9. Immun Joint sp staining 8 w chemical st previously, joints were bone decalc deparaffiniz Endogenou Immunohis peroxidase kit (Vector instructions serum for had been w � antibody Biotechnolo ulins were u washed, bio The slides w tain ABC ki the diamin The slides mounted. 2.10. Osteoclastogenesis Murine osteoclasts were generated as previously described, with minor modifications [32]. Briefly, bone-marrow cells (BMCs) ormal 6-week-old mice were cultured overnight and the herent BMCswere collected. TheBMCswere culturedwith�- um essential medium containing 10% fetal bovine serum for in the presence of 30ng/mL macrophage colony-stimulating (M-CSF) and 50ng/mL receptor activator of nuclear factor d (RANKL), with or without various concentrations of GSPE. tainingwasperformed. TRAP+ cellswith three ormorenuclei efined as osteoclasts and the numbers of osteoclasts were d. easurement of 8-isoprostane use plasma sampleswere obtained 8weeks after the primary ization and stored at −70 ◦C until analysis. The concen- of 8-isoprostane in the plasma was measured using a ercially available immunoassay kit (Cayman Chemical Co., bor, MI), according to the instructions provided by the man- rer. easu boxy lar o det ncub ere son, son). tatist resu s of d nces ults fects hritis mic epen mic hange rolmi issolve diffe immu g/kg S. The betwe rthriti A repr line (pH 8.0) containing 1% bovine serum albumin and -20, and incubated in the microtiter plates for 1h, after plates were washed five times. The concentrations of gG1 were measured using mouse IgG2a and IgG1 ELISA n Kits (Bethyl Laboratories, Montgomery, TX), respec- ard serum from arthritic mice was added to each plate utions, and a standard curve was constructed to assign its to the levels anti-type-II-collagen IgG2a and IgG1. nce values were determined with an ELISA microplate ating at 450nm. paration and culture eks after the primary immunization, themouse spleens ted for cell preparation and washed twice with PBS. The e minced and the red blood cells were lysed with 0.83% chloride. The cells were filtered through a cell strainer ged at 1300 rpm at 4 ◦C for 5min. The cell pellets were d in RPMI 1640 medium and plated in 24-well plates Y, USA) at a concentration of 1×106 cells/well. Splenic were isolated by magnetic bead separation to a purity ement of TNF-˛ and interleukin 17 (IL-17) in culture s splenocyteswere cultured for 72h. The amounts of TNF- in the culture supernatants were measured by ELISA. directed against mouse TNF-� and IL-17 and against anti-mouse TNF-� and IL-17 were used as the cap- tection antibodies, respectively. Alkaline phosphatase s used for the chromogenic reaction. The amounts s present in the test samples were determined from rves constructed with serial dilutions of recombinant -� and IL-17 (R&D Systems). The absorbance was deter- an ELISA microplate reader at 405nm. ohistochemical analysis of TNF-˛ and IL-17 ecimens were collected for immunohistochemical eeks after the primary immunization. Immunohisto- aining for TNF-� and IL-17 was performed as described with a minor modification [31]. Briefly, whole ankle fixed in 4% paraformaldehyde, decalcified in EDTA ifier, and embedded in paraffin. The tissue slides were ed with xylene and rehydrated in a gradient of ethanol. s peroxidase activity was quenched with 3% H2O2. tochemistry was performed with the avidin–biotin complex (ABC) technique using the Vectastain ABC Laboratories, USA), according to the manufacturer’s . Nonspecific binding sites were blocked with normal 30min at room temperature. After the tissue slides ashed, they were incubated with primary anti-TNF- (R&D Systems) and anti-IL-17 antibody (Santa Cruz gy) overnight at 4 ◦C. Isotype-matched immunoglob- sed as the negative controls. After the slides had been tinylated secondary antibody was applied for 30min. ere incubated with an avidin–biotin complex (Vectas- t) for 1h. The final color product was developed using obenzidine chromogen (Dako, Carpinteria, CA, USA). were counterstained with Mayer’s hematoxylin and from n nonad minim 5 days factor B ligan TRAP s were d counte 2.11. M Mo immun tration comm Ann Ar ufactu 2.12. M Car Molecu used t then i cells w Dickin Dickin 2.13. S The Group Differe 3. Res 3.1. Ef Art tion of dose-d treated Fig. 1. C thecont GSPE, d to three ondary 50, 100m with PB by GSPE for the a control. rement of intracellular H2O2 -dichlorodihydrofluorescein diacetate (H2DCFDA; Probes), an oxidation-sensitive fluorescent dye, was ect H2O2. The cells were washed twice with PBS and ated with 5�M H2DCFDA for 10min at 37 ◦C. The analyzed with a FACSCalibur flow cytometer (Becton San Jose, CA, USA) using Flowjo software (Becton ical analysis lts are expressed as means± S.D. (or means± S.E.M.). ata were compared using the Mann–Whitney U-test. were considered statistically significant at P<0.05. of GSPE treatment on arthritis scores developed about 3 weeks after the primary immuniza- e with type II collagen (Fig. 1). There was a significant dent reduction of themean arthritis scores of theGSPE- e compared with those of the control mice (Fig. 1). s in the arthritis scores of GSPE-treated mice compared with those of ce. CIAwas inducedaspreviouslydescribed,withminormodifications. d in phosphate-buffered saline (PBS), was given intraperitoneally rent groups (10, 50, or 100mg/kg), (n=6 mice/group) after sec- nization. The mice were injected with different doses of GSPE (10, ) five times per 2 days for 2.5 weeks. The control mice were injected re was a significant dose-dependent reduction in the arthritis scores en days 24 and 35. Values are means± S.E.M.s (n=5–6). Mean values s scores are indicated on the graph. *P<0.01 comparedwith the saline esentative result of at least three independent experiments is shown. M.-L. Cho et al. / Immunology Letters 124 (2009) 102–110 105 Fig. 2. Histolo tive hi (100×), safran mati described in S phs ar result of at lea 3.2. Effects The effe assessed by immunizati In the CIA tory cells, sy cartilage an reduction in or 100mg/k arthritis sco but this wa destr Fig. 3. Effects each group) w *P<0.01 versu gical assessment of arthritis in mice with CIA after treatment with GSPE. Representa in-O (200×), and TRAP (100×) (a). The effects of GSPE treatment on synovial inflam ection 2 (b). The results are expressed as the mean (±S.E.M.) scores. Photomicrogra st three independent experiments is shown. of GSPE treatment on histological findings tilage ct of GSP