I n f o r m a t i o n s m a t e r i a l v o m 1 7 . 0 1 . 2 0 0 8 Zysten, Fisteln und Co. Der 18. November 1686 war der aufregendste Tag im Leben des Chirurgen Charles-François Félix (1653-1703). Ein Jahr lang hatte sich sein Patient der von ihm vorge-schlagenen Operation seiner Analfistel verweigert. Nun reckte er ihm den After ent-gegen, bereit, einige schmerzhafte Schnitte zu ertragen, denn mit Betäubungsmitteln war es damals nicht weit her. Der Patient war Schmerzen gewöhnt, zeitlebens hatten ihn unzählige Leiden geplagt. Doch ein Misslingen dieser Operation, wo-
Jaseng.co.krHindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2012, Article ID 730907, 11 pagesdoi:10.1155/2012/730907 Research Article
Effect of GCSB-5, a Herbal Formulation, on Monosodium
Iodoacetate-Induced Osteoarthritis in Rats
Joon-Ki Kim,1 Sang-Won Park,1 Jung-Woo Kang,1 Yu-Jin Kim,2 Sung Youl Lee,2
Joonshik Shin,3 Sangho Lee,3 and Sun-Mee Lee1
1 School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, Republic of Korea2 Business Development, Green Cross Corporation, Yongin, Gyeonggi-do 446-770, Republic of Korea3 Jaseng Hospital, 635 Sinsa-Dong, Gangnam-Gu, Seoul, Republic of Korea Correspondence should be addressed to Sun-Mee Lee, [email protected] Received 1 April 2011; Revised 9 November 2011; Accepted 16 November 2011 Academic Editor: Andrew Scholey Copyright 2012 Joon-Ki Kim et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Therapeutic eﬀects of GCSB-5 on osteoarthritis were measured by the amount of glycosaminoglycan in rabbit articular cartilageexplants in vitro, in experimental osteoarthritis induced by intra-articular injection of monoiodoacetate in rats in vivo. GCSB-5was orally administered for 28 days. In vitro, GCSB-5 inhibited proteoglycan degradation. GCSB-5 significantly suppressed thehistological changes in monoiodoacetate-induced osteoarthritis. Matrix metalloproteinase (MMP) activity, as well as, the levels ofserum tumor necrosis factor-α, cyclooxygenase-2, inducible nitric oxide synthase protein, and mRNA expressions were attenuatedby GCSB-5, whereas the level of interleukin-10 was potentiated. By GCSB-5, the level of nuclear factor-κB p65 protein expressionwas significantly attenuated but, on the other hand, the level of inhibitor of κB-α protein expression was increased. These resultsindicate that GCSB-5 is a potential therapeutic agent for the protection of articular cartilage against progression of osteoarthritisthrough inhibition of MMPs activity, inflammatory mediators, and NF-κB activation.
complete therapeutic eﬀects by blocking the activity ofone or two cytokines. Developing therapeutics from herbal Osteoarthritis (OA) is a degenerative joint disease charac- sources may reduce the risk of toxicity or adverse eﬀects terized by joint pain and a progressive loss of articular carti- when the drug is clinically used  and may exert strong, lage. It has been suggested that biochemical alterations occur multifunctional anti-inflammatory eﬀect like many natural within the articular cartilage resulted in imbalance between products do. Therefore, eﬀorts are being made to elucidate synthetic and degradative pathways . A key step in the the role of natural products for the treatment of OA.
pathophysiology of OA is breakdown of extracellular matrix GCSB-5 is a purified extract from a mixture of 6 oriental of articular cartilage by tissue proteinases, enzymes whose herbs which are the ingredients of Chung-Pa-Juhn used in expression is upregulated by inflammatory stimuli, such as Jaseng Hospital (Seoul, Korea) and that have been used in primary inflammatory cytokines . Nonsteroidal anti-in- traditional medicine to treat inflammatory diseases and bone flammatory drugs (NSAIDs) are eﬀective in the management disorders. Ledebouriellae Radix is reported to have anti- of OA inflammation. However, the adverse events secondary inflammatory eﬀects on Freund's adjuvant-induced arthritis to NSAIDs was focused on upper gastrointestinal tolerability in rats . Cimifugin, a major active component of Lede- . In recent years, gene therapy targeted at cytokines of- bouriellae Radix, exhibits inhibitory eﬀects on the synthesis fers new hope to OA treatment, and the current focus is of NO induced by LPS in macrophage cell line RAW 264.7 on the use of biological agents that block the activity of in- . Achyranthis Radix shows anti-inflammatory property flammatory cytokines . Since there are many proinflam- and inhibits free radicals, such as ONOO−, HOCl, and OH matory cytokines, oxidants and other factors exerting action radical . 20-Hydroxyecdysone, which is a major active in initiation and development of OA, it is hard to obtain compound of Achyranthis Radix, has beneficial eﬀects on Evidence-Based Complementary and Alternative Medicine joint and bone in ovariectomized rats . Acanthopanacis 2.3. Animals. Male Sprague-Dawley rats (200–220 g) and Cortex is known to show antiarthritic activity , and male New Zealand white rabbits (2.0–2.2 kg) were obtained Cibotii Rhizoma is known for its analgesic property  from Dae Han Biolink Ltd. (Eumseong, Korea) and housed along with osteoclast formation inhibition . Glycine in solid bottom cages with pellet food and water available ad Semen is eﬀective in reducing swelling  and genistin, libitum. All animal procedures were approved by the Sung- an active compound from Glycine Semen, shows beneficial kyunkwan University Animal Care Committee and were per- eﬀect on bone loss . Eucommiae Cortex exhibits strong formed in accordance with the guidelines of the National analgesic eﬀect  and geniposide from its extract shows Institutes of Health.
anti-inflammatory eﬀect on rheumatoid arthritis rats and enhances the osteoblast-like cell proliferation and inhib- 2.4. Cartilage Glycosaminoglycan Assay. Rabbit knee articu- ited osteoclast . We reported strong antinociceptive and lar cartilage explants were obtained according to the method anti-inflammatory properties of GCSB-5 [11, 13]. Recently, described by Sandy et al. . Briefly, 200–220 mg articu- GCSB-5 reduces the development of acute and chronic in- lar surfaces per joint were dissected and submerged into flammation, and its anti-inflammatory property is likely due complete medium of DMEM supplemented with heat-inac- to inhibition of inducible nitric oxide synthase (iNOS) and tivated 5% FBS, penicillin (100 U/mL), and streptomycin cyclooxygenase (COX)-2 expression via downregulation of (100 μg/mL). After stabilization in incubator, the medium the Akt signal pathway and inhibition of nuclear factor-κB was replaced with basal medium made of DMEM supple- (NF-κB) activation . In phase III clinical study, GCSB-5 mented with heat-inactivated 1% FBS, 10 mM HEPES, peni- was shown to exert therapeutic eﬀects and acted to reduce cillin (100 U/mL), and streptomycin (100 μg/mL). Cartilage OA severity and improved functional recovery without pieces (50–60 mg; 2 × 3 × 0.35 mm/piece) were placed in apparent hepatic or renal toxicity (unpublished data).
24-well cell culture plates and treated with GCSB-5 at 1 × In this study, we examined the chondroprotective and 10−3, 1 × 10−2, and 1 × 10−1 mg/mL or 30 μM diclofenac anti-inflammatory eﬀects of GCSB-5 on monoiodoacetate (Sigma-Aldrich, St. Louis, MO, USA). After 1 h of GCSB-5 or (MIA)-induced OA animal model, both in vitro and in vivo.
diclofenac pretreatment, 5 ng/mL of rhIL-1α (R&D Systems,Minneapolis, MN, USA) was added and further incubated 2. Materials and Methods
at 37◦C in a humidified 5% CO2/95% air incubator. Theamount of glycosaminoglycan (GAG) in the medium was de- 2.1. Preparation and Composition of GCSB-5. GCSB-5 was termined by the 1,9-dimethyl-methylene blue method using prepared by the Hanpoong Pharmaceutical Co., Ltd., Jeonju, the Blyscan Sulfated GAG Assay kit (Biocolor Ltd., County Republic of Korea. The mixture of six crude drugs (Lede- Antrim, UK) according to the manufacturer's instructions.
bouriellae Radix (4.444 g), Achyranthis Radix (4.444 g),Acanthopanacis Cortex (4.444 g), Cibotii Rhizoma (2.778 g), 2.5. MIA-Induced OA. Rats were anesthetized with diethyl Glycine Semen (2.778 g), and Eucommiae Cortex (1.389 g)) ether and given a single intra-articular injection of 3 mg MIA was powdered and boiled for 3 h in distilled water (1 L).
(Sigma-Aldrich, St. Louis, MO, USA) through the infrap- The resulting extract was subjected to ultrafiltration, and atellar ligament of the left knee . MIA was dissolved in the components with molecular weight over 10,000 were physiological saline and administered in a 50 μL volume. Rats excluded. The filtrate was lyophilized as powder and kept were treated with saline, with 300 or 600 mg/kg of GCSB-5 at 4◦C until use. GCSB-5 was administered orally at a dose or with 5 mg/kg of diclofenac by oral administration once of 300 and 600 mg/kg in saline (1 kg/10 mL), and the same daily, for 2, 7, and 28 days since MIA injection. These GCSB- volume of saline was used as a vehicle control group. The 5 doses and MIA injection volume were selected based on validation of GSCB-5 was performed by high-performance previous evaluations .
liquid chromatography analysis of each ingredient ex-tract using six indicator biological components: cimifu-gin for Ledebouriellae Radix, 20-hydroxyecdysone (0.311- 2.6. Gross Observation. After MIA injection, all experimental 0.312 mg/g) for Achyranthis Radix, acanthoside D (0.577- rats were weighed and carefully inspected every 2 days to as- 0.578 mg/g) for Acanthopanacis Cortex, onitin-4-O-β-D- sess knee joint swelling and gait disturbances under natural glucopyranoside for Cibotii Rhizoma, genistin (0.0426- conditions in the cages, where they moved freely. Swelling 0.0427 mg/g) for Glycine Semen, and geniposide (0.431- and limping were classified as no change, mild, and severe on 0.432 mg/g) for Eucommiae Cortex. GCSB-5 was further the basis of severity , and inspection was conducted by an standardized for quality control according to the regulations inspector blinded to treatment details throughout the study.
imposed by Korea Food and Drug Administration (KFDA).
2.7. Roentgenographic Examination and Histopathological 2.2.Chemicals. Dulbecco's modified Eagle's medium(DMEM), Analysis. Seven and 28 days following MIA injection, rats penicillin/streptomycin (10,000 U/mL, 10,000 μg/mL, resp.), were checked with roentgenography to assess chronic mor- and fetal bovine serum (FBS) were obtained from Gibco phological changes of knee articular bones for narrowing, BRL, Life Technologies (Grand Island, NY, USA). All the loss of joint region, cartilage erosion, and osteophyte forma- other materials required for culturing of tissue were pur- tion . For histological analysis, knee joints were removed chased from Sigma Chemical Company (St. Louis, MO, and fixed in 10% neutral buﬀered formalin, decalcified with 10% formic acid, and embedded in paraﬃn. Five micrometer Evidence-Based Complementary and Alternative Medicine Table 1: RT-PCR primers used in study.
Primer sequences (5 → 3) Product length (bp) Sense: GTA GCC CAC GTC GTA GCA AA Antisense: CCC TTC TCC AGC TGG AAG AC Sense: TTC TTT GCT TCT GTG CTT AAT GCG Antisense: GTT GTT GCT GAA CTT CCA ATC GT Sense: CTG CAT GTG GCT GAT GTC ATC Antisense: AGG ACC CGT CAT CTC CAG GGT AAT C Sense: TGA TGT TCC CAT TAG ACA GC Antisense: GAG GTG CTG ATG TAC CAG TT Sense: CAG TCA GCC AGA CCC ACA T Antisense: GCT CCA CTG CCT TGC TTT Sense: TTG TAA CCA ACT GGG ACG ATA TGG Antisense: GAT CTT GAT CTT CAT GGT GCT AG (5 μm) sections were stained with hematoxylin and eosin 2.11. Total RNA Extraction and Reverse Transcription- (H and E) or safranin-O fast green (SOFG) and observed.
Polymerase Chain Reaction (RT-PCR). Articular cartilage Histopathological changes in each animal were quantitatively samples collected 2 and 28 days after MIA injection were expressed by three grades for each finding . Grading was pulverized in TRI Reagent (Molecular Research Center Inc., done under the authority of Medplan Pathology Laborato- Cincinnati, OH, USA) for RNA extraction. Equal amounts ries, Seoul, Korea.
of RNA from articular cartilages were subjected to reversetranscription using iNtRON RNA PCR kit (iNtRON Biotech-nology Co., Seongnam, Korea) to generate cDNA for RT- 2.8. Gelatinase Assay. Rat articular cartilage samples of MIA- PCR analysis. RT-PCR analysis was performed with the induced OA were harvested 7 and 28 days after MIA injec- GeneAmp PCR system 2700 (Applied Biosystems Co., Foster tion. Gelatinase activities were measured by the gelatin zy- City, CA, USA). The primers used in the RT-PCR are listed mography method described by Dumond et al. . Proteins in Table 1. All PCR reactions included an initial denaturation were extracted from pulverized cartilage tissues and elec- step at 94◦C for 5 min and a final extension at 72◦C for 7 min.
trophoresed on 10% zymogram precast gels. The cleared gels The PCR amplification cycling conditions were as follows: were captured, and the area of each band was quantified with 32 cycles of 94◦C (30 s), 58◦C (30 s), and 72◦C (30 s) for densitometric scanning analysis program (Science Lab 98 TNF-α; 32 cycles of 94◦C (45 s), 65◦C (45 s), and 72◦C Image Gauge, version 3.12, Fuji Photo Film Co. Ltd., Tokyo, (60 s) for iNOS; 40 cycles of 94◦C (45 s), 65◦C (45 s), and 73◦C (60 s) for COX-2; 36 cycles of 94◦C (30 s), 60◦C(30 s), and 72◦C (45 s) for IL-1β; 40 cycles of 94◦C (30 s), 2.9. Serum Cytokine Levels. Commercial tumor necrosis 66◦C (45 s), and 72◦C (45 s) for IL-10; 30 cycles of 94◦C factor (TNF)-α, interleukin (IL)-1β, and IL-10 enzyme- (30 s), 56◦C (30 s), and 72◦C (60 s) for β-actin. After RT- linked immunosorbent assay (ELISA) kits (BD Biosciences PCR, 10 μL samples of the amplified products were resolved Co., CA, USA) were used for quantification of the serum by electrophoresis on 1.5% agarose gels and stained with levels of TNF-α, IL-1β, and IL-10, respectively.
ethidium bromide. The intensity of each PCR product wasevaluated semiquantitatively using a digital camera (DC120; 2.10. Western Blot Immunoassay. 15 μg of whole protein was Eastman Kodak, Rochester, NY, USA) and a densitometric used for determination of the content of COX-2 and iNOS.
scanning analysis program (ID Main; Advanced American 20 μg of nuclear protein was used for determination of the Biotechnology, Fullerton, CA, USA).
content of the NF-κB/p65 subunit. 20 μg of the cytosolicprotein was used for determination of the content of the 2.12. Statistics. All results are presented as mean ± S.E.M.
inhibitor of κB (IκB)-α. ImageQuantTM TL software (Amer- The overall significance of the experimental results was sham Biosciences/GE Healthcare, Piscataway, NJ, USA) was examined by one-way analysis of variance and the two-tail used for densitometric evaluation of visualized immunore- Dunnet's t-test. Diﬀerences between groups were considered active bands. The following primary antibodies were used: significant at P < 0.05 with the appropriate Bonferroni COX-2 (Abcam, Cambridge, UK; 1 : 1000), iNOS (Trans- correction for multiple comparisons.
duction Lab., CA, USA; 1 : 1000), phosphoryl NF-κB/p65(Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 1000), 3. Results
and IκB-α (Santa Cruz Biotechnology; 1 : 5000) were used,and the signals were normalized to that of β-actin (Sigma 3.1. Cartilage Glycosaminoglycan Release. In the control Chemical Co.; 1 : 1000) or lamin B1 (Abcam; 1 : 2500).
group, the level of GAG in the culture medium remained Evidence-Based Complementary and Alternative Medicine Table 2: Quantitative summary of gross observations in MIA-induced osteoarthritic rats treated with GCSB-5.
0.0 ± 0.0 1.7 ± 0.2a 1.0 ± 0.2a,b 1.2 ± 0.2a 1.2 ± 0.1a 0.0 ± 0.0 1.3 ± 0.2a 0.4 ± 0.2c 0.5 ± 0.2c 0.5 ± 0.2c GCSB-5 or diclofenac was treated daily for 14 days after 2 weeks of OA induction by intra-articular injection of MIA.
aDenotes significant diﬀerences (P < 0.01) versus the control group.
b,cDenote significant diﬀerences (P < 0.05, P < 0.01) versus the vehicle-treated MIA group.
These symptoms gradually aggravated at 21 days (data notshown) and were the most severe at 28 days. Twenty- eight days after MIA injection, swelling and limping were attenuated by both 300 and 600 mg/kg GCSB-5 treatment 3.3. Roentgenographic and Histopathological Analysis. Seven μg/mg car days after MIA injection, rats underwent the first roentgeno- graphic examination. Their roentgenographic examinations revealed degenerative changes, such as irregularity or osteo-phytes on the surface of the cartilage and subchondral bone(data not shown). At 28 days, rats underwent the second roentgenographic examination (Figures 2(a)–2(d)). Mor- phological changes were more significant, showing rough edges of cartilage and the tendency of patellar displace-ment. These changes were attenuated by GCSB-5 600 mg/kg Figure 1: GAG release in rabbit articular cartilage explant cultures treatment. Twenty-eight days after MIA injection, H and E at 24, 48, and 72 h. Rabbit articular cartilage explants were staining revealed irregular surface accompanied by ulcera- stimulated with rhIL-1α (5 ng/mL). The amount of GAG release tion, fibrillation, and loss of cartilage tissue (Figures 2(e)– stimulated by rhIL-1α (◦) increased approximately 3.6 timescompared to control (•) at 72 h. GCSB-5 (1.0 × 10−3 () and 2(h)). However, these cartilage damages were attenuated by 1.0 × 10−2 () mg/mL) and diclofenac (30 μM ()) eﬃciently GCSB-5 600 mg/kg treatment. SOFG staining also revealed inhibited the GAG release. However, a high concentration of GCSB- clearly diﬀused PG depletion in joint cartilage tissues of 5 (1.0×10−1 () mg/mL) slightly inhibited it. Each value represents MIA-injected rats (Figures 2(i)–2(l)). This loss of PG was the mean ± S.E.M. from 6 articular cartilage explants cultures per attenuated by GCSB-5 600 mg/kg treatment. Summation of group. ∗∗Significantly diﬀerent (P < 0.01) from control. + and all histopathologic finding scores in vehicle-treated MIA ++Significantly diﬀerent (P < 0.05, P < 0.01) from rhIL-1α.
group and in 300 and 600 mg/kg GCSB-5-treated MIAgroups were 24.5 ± 1.3, 16.1 ± 1.4, and 12.5 ± 1.1, respectively(Table 3).
constant at approximately 1.5 μg/mg cartilage throughoutthe experiment. In the rhIL-1α-treated group, on the other 3.4. Gelatinase Assay. Seven days after MIA injection, the hand, the level of GAG in the culture medium dramatically activities of matrix metalloproteinase (MMP)-2 and -9 in- increased to approximately 4 times the control values.
creased to 2.7 and 2.4 times that in the control group, GCSB-51 × 10−3 and 1 × 10−2 mg/mL treatments attenuated respectively. Similarly, 28 days after MIA injection, the the elevation in GAG release at 72 h (Figure 1).
activities of MMP-2 and -9 increased to 2.3- and 2.8-foldhigher than the control level, respectively. On day 7, GCSB-5 3.2. Gross Observation. In the MIA-injected groups, swelling and diclofenac treatment showed no significant modulation and limping were first observed 7 days after MIA injection.
on MMP activities (data not shown). However, on day 28, They subsided transiently and then reappeared at 14 days.
GCSB-5 300 mg/kg treatment exhibited significant MMP-2 Evidence-Based Complementary and Alternative Medicine Figure 2: Roentgenography and histopathological features of osteoarthritic lesion in the knee joint of rats 28 days after intra-articularinjection of MIA (H and E staining, ×100; SOFG staining, ×100). Control (a) represents intact normal joint feature. Vehicle-treated MIA(b) shows a severely damaged joint with rough edges around the tibia and femur, indicative of bone lysis, swelling, and tendency of patellardisplacement. However, these damages were reduced significantly by treatment with 600 mg/kg GCSB-5 (c) and 5 mg/kg diclofenac (d).
SOGF-stained control (e) represents normal cartilage PG staining, whereas vehicle-treated MIA (f) represents severely damaged cartilageshowing marked fibrillation and the depletion of SOFG staining with separation of cartilage from subchondral bone. 600 mg/kg GCSB-5 (g)and 5 mg/kg diclofenac (h) treatments significantly reduced cartilage damage. H and E stained control (i) represents the normal status ofjoint cartilage, whereas vehicle-treated MIA (j) represents severely damaged cartilage showing widespread cell necrosis and inflammation.
However, treatment with 600 mg/kg GCSB-5 (k) and 5 mg/kg diclofenac (l) treatments significantly reduced joint cartilage damage.
and -9 activities attenuation (79.6%, P < 0.01 and 81.2%, The levels of TNF-α, IL-1β, IL-10, COX-2, and iNOS P < 0.01, resp.), while GCSB-5 600 mg/kg treatment did not mRNA expression increased 5.3, 2.1, 1.3, 7.8 and 8.8 times aﬀect the MMP-2 and -9 activities (91.0% and 91.6%, resp.) in the vehicle-treated MIA groups, compared to those in the control group 2 days after MIA injection, respectively(Figures 4 and 6). Increase in TNF-α, COX-2, and iNOS 3.5. Inflammatory Mediators. The serum levels of TNF-α, IL- mRNA expression was significantly suppressed by treatment 1β, and IL-10 were 30.0 ± 4.5 pg/mL, 29.1 ± 3.7 pg/mL, and with GCSB-5. However, GCSB-5 did not aﬀect the level of IL- 27.8 ± 0.6 pg/mL in the control. 2 days after MIA injection, 1β mRNA expression. Interestingly, increase of IL-10 mRNA the serum levels of TNF-α, IL-1β, and IL-10 increased to 2.8-, expression was significantly potentiated by GCSB-5. At 7 and 3.4- and 2.2-fold higher than the control level, respectively.
28 days, there were no significant diﬀerences in the level of Increase in TNF-α level was significantly suppressed by inflammatory mediators mRNA expression among any of the treatment with GCSB-5, while increase in IL-10 level was experimental groups (data not shown).
significantly potentiated by GCSB-5. However, GCSB-5 didnot aﬀect the serum level of IL-1β (Table 4). The levels of 3.6. Nuclear NF-κB and Cytosolic IκB-α Immunoblot Assay. COX-2 and iNOS protein expression increased 3.3 and 12 The nuclear localization of NF-κB was measured by the pro- times in the vehicle-treated MIA groups, compared to those tein level of NF-κB p65 subunit in the nucleus. Cytosolic IκB- in the control group 2 days after MIA injection, respectively α was also examined from cytosol fraction as an endogenous (Figure 5). Increase in COX-2 and iNOS protein expression NF-κB inhibitor. The level of nuclear NF-κB p65 protein was significantly suppressed by treatment with GCSB-5.
expression increased 2.5 times, whereas the level of cytosolic Evidence-Based Complementary and Alternative Medicine Table 3: Summary of microscopic findings.
Structural changes in the joint Surface irregularities Average pathology score Average pathology score Fibrillation of cartilage surface Average pathology score Disorganization of chondrocytes Average pathology score Exposure of subchondral bone Average pathology score Cellular changes of chondrocyte Average pathology score Average pathology score Inflammatory cell infiltration in synovial tissue Average pathology score Synovial cell proliferation Average pathology score Safranin-O staining Reduction of staining in cartilage Average pathology score Total pathology score (average ± S.E.M) 24.5 ± 1.3 16.1 ± 1.4a 12.5 ± 1.1b 13.4 ± 2.2b +: mild, ++: moderate, and +++: severe.
a,bDenote significant diﬀerences (P < 0.05, P < 0.01) versus the vehicle-treated MIA group. N = 4.
Evidence-Based Complementary and Alternative Medicine Table 4: Eﬀect of GCSB-5 on serum TNF-α, IL-1β, and IL-10 levels in MIA-induced osteoarthritic rats.
30.0 ± 4.5 29.1 ± 3.7 27.8 ± 0.6 85.4 ± 6.6b 99.5 ± 11.2b 60.4 ± 7.2b 60.0 ± 4.6a,c 102.6 ± 10.4b 91.4 ± 8.8b,d 55.3 ± 6.2c 93.9 ± 9.0b 84.5 ± 8.5b,c Diclofenac 5 mg/kg 50.1 ± 4.0d 49.5 ± 7.5d 86.1 ± 9.2b,c The serum concentration of TNF-α, IL-1β, and IL-10 was determined usingenzyme-linked immunosorbent assay. The results are presented as mean ± S.E.M. of 6 rats per group.
a,bDenote significant diﬀerences (P < 0.05, P < 0.01) compared with controlgroup.
c,dDenote significant diﬀerences (P < 0.05, P < 0.01) compared with vehicle-treated MIA group.
IκB-α protein expression decreased 2.0-fold in the vehicle- MIA + GCSB-5 300 mg/kg treated MIA group, compared to that in the control group MIA + GCSB-5 600 mg/kg 2 days after MIA injection. These changes were significantly MIA + diclofenac 5 mg/kg inhibited by GCSB-5 treatment (Figure 7).
Figure 3: Activities of MMP-2 (Gelatinase A) and MMP-9 (Gelati-nase B) assessed by zymography in knee joint cartilages obtained 28 days after MIA injection. The latent and active amounts ofgelatinase were combined to give a total value for each gelatinase.
Today, cure for OA remains elusive. The management of OA Each value represents the mean ± S.E.M. from 6 rats per group.
is largely palliative focusing on the alleviation of symptoms.
aSignificantly diﬀerent (P < 0.01) from control. bSignificantly Current recommendations for the management of OA diﬀerent (P < 0.05) from vehicle-treated MIA.
include a combination of nonpharmacological (weight loss,education programs, and exercise) and pharmacologicalinterventions (paracetamol, NSAIDs, etc.) . Of the phar- model in which lesions resembling some aspects of human macological intervention available, analgesics and NSAIDs OA produced quickly and has been suggested as a model have been proven to be highly eﬀective in controlling the for the study of chondroprotective drugs . In the present symptoms and signs of OA. However, they have potential study, we investigated GCSB-5 on the clinical and behavioral gastrointestinal (GI) adverse eﬀects. Herbal medicinal prod- changes associated with MIA-induced OA. Swelling and ucts (HMPs) are not yet among the recommended treatment limping were apparent as early as 7 days after MIA injection, options, although they are used in a variety of oral and top- after which they became transiently subsided. At 14 days, ical forms in the treatment of OA. The mechanism of action there was a second period of knee joint swelling and limping of HMPs is broader than that of NSAIDs and/or analgesics that was progressively aggravated until day 28. Administra- in current use for symptomatic OA. Although the exact tion of GCSB-5 once daily for 28 days significantly reduced mechanisms of action have not yet been elucidated, there is the severity of swelling and limping. These results suggest no doubt that all herbal medicines act via several pathways, that GCSB-5 may have potential as a treatment for OA.
including inhibition of COX and/or lipoxygenase, inhibition Roentgenographic and histological observations strongly of cytokine release, inhibition of elastase or hyaluronidase, supported the behavioral changes following MIA injection and induce antioxidative activity . On the basis of this as well as the protective eﬀect of GCSB-5.
knowledge, our experimental herbal extract, GCSB-5, con- Cartilage comprises an extracellular matrix consisting sisted of various herbs known to exhibit antiarthritic, anti- of PGs, collagens (types II, IX, XI, and others), and water.
inflammatory and analgesic eﬀects, is expected to show ther- Cartilage PGs consist of a protein core with GAG side chains apeutic activity against OA.
. When cartilage is damaged by inflammatory mediators Articular cartilage destruction is a key pathological char- such as rhIL-1α, PGs degrade and consequently release GAG, acteristic of OA. MIA is an inhibitor of glyceraldehyde-3- which is a typical clinical symptom of OA. GCSB-5 did not phosphate dehydrogenase activity, and therefore an inhibitor inhibit GAG release at low concentrations, but showed an of glycolysis shown to induce chondrocyte death in vitro .
inhibitory eﬀect at moderate-to-high concentrations. This Intra-articular injection of MIA induces chondrocyte death analysis reflected the histochemical appearance of cartilage.
in the articular cartilage of rodent and nonrodent species SOFG staining showed significant PG loss and lesion devel- . Injection of MIA into the knees of rats provides a opment in subchondral bone which were induced by MIA Evidence-Based Complementary and Alternative Medicine MIA + GCSB-5 300 mg/kgMIA + GCSB-5 600 mg/kg MIA + GCSB-5 300 mg/kgMIA + GCSB-5 600 mg/kg MIA + diclofenac 5 mg/kg MIA + diclofenac 5 mg/kg Figure 5: COX-2 and iNOS protein expressions in cartilage from Figure 4: TNF-α, IL-1β, and IL-10 mRNA expressions in cartilage knee joints of rats at day 2 after MIA injection. Each value represents from knee joints of rats at day 2 after MIA injection. Each value the mean ± S.E.M. from 6 rats per group. a,bSignificantly diﬀerent represents the mean ± S.E.M. from 6 rats per group. a,bSignificantly (P < 0.01, P < 0.05) from control. c,dSignificantly diﬀerent (P < diﬀerent (P < 0.01, P < 0.05) from control. c,dSignificantly diﬀerent 0.01, P < 0.05) from vehicle-treated MIA.
(P < 0.01, P < 0.05) from vehicle-treated MIA.
MIA injection. Our results indicate that GCSB-5 inhibits injection. These cartilage damages were attenuated by GCSB- collagen degradation through inhibition of MMP-2 and -9 5 treatment. Our data suggest that GCSB-5 may protect activities in late stage of OA.
articular cartilage from degradation.
Matrix turnover is solely dependent on chondrocytes, MMPs are a family of proteinases that together can de- which are believed to be the main site of inflammatory grade all extracellular matrix components. Type IV colla- mediators production in human OA . Overexpression genases (gelatinases) are members of the MMP family and of MMPs is induced by several cytokines, such as TNF- are thought to play an important role in the degradation of α, IL-1, IL-17, and IL-10. TNF-α and IL-1β drive the extracellular components. The gelatinase subclass can be di- catabolic processes in OA, leading to cartilage degradation.
vided into gelatinase-A (MMP-2) and gelatinase-B (MMP- In this study, the levels of TNF-α, COX-2, and iNOS mRNA 9), which is capable of degrading types IV and V collagens, expression were significantly increased 2 days after MIA elastin, and gelatin . MMP-2 is known to be produced by injection and returned to control level at 28 days (data osteoblasts and tissue structural cells, including fibroblasts not shown). These increases were attenuated by GCSB-5 and endothelial cells, whereas MMP-9 is produced by in- treatment. On the other hand, a critical function of IL- flammatory cells such as macrophages, neutrophils, and eos- 10 is to limit inflammatory responses . This cytokine inophils [33, 34]. These MMPs are secreted as latent pre- inhibits IL-1β and TNF-α expression and is present in cursors and can be activated by limited proteolysis. The in- OA chondrocytes, where it may counteract their catabolic creased expression of MMP-2 and -9 in the synovium of eﬀects . Interestingly, GCSB-5 treatment significantly patients with arthritic eﬀusions superiorly reflects the potentiated this increase. Our results indicate that GCSB- inflammatory condition of the joints, and a positive corre- 5 shows a significant anti-inflammatory action in the early lation between MMP-9 production and rapid destruction of stage of OA.
the hip joint has been described in OA . Although GCSB- Inappropriate regulation of NF-κB activity has been 5 did not aﬀect MMP-2 and -9 activities 7 days after MIA implicated in the pathogenesis of inflammatory diseases, injection (data not shown), GCSB-5 especially at a dose of such as rheumatoid arthritis and OA . NF-κB signal- 300 mg/kg suppressed MMP-2 and -9 activities 28 days after ing pathways mediate critical events in the inflammatory Evidence-Based Complementary and Alternative Medicine NF-κB p65 NF-κB p65 MIA + GCSB-5 300 mg/kg MIA + GCSB-5 300 mg/kg MIA + GCSB-5 600 mg/kg MIA + GCSB-5 600 mg/kg MIA + diclofenac 5 mg/kg MIA + diclofenac 5 mg/kg Figure 7: Nuclear NF-κB p65 and cytosolic IκB-α protein expres- Figure 6: COX-2 and iNOS mRNA expressions in cartilage from sions in cartilage from knee joints of rats at day 2 after MIA knee joints of rats at day 2 after MIA injection. Each value represents injection. Each value represents the mean ± S.E.M. from 6 rats per the mean ± S.E.M. from 6 rats per group. aSignificantly diﬀerent group. a,bSignificantly diﬀerent (P < 0.05, P < 0.01) from control.
(P < 0.05) from control. bSignificantly diﬀerent (P < 0.05) from c,dSignificantly diﬀerent (P < 0.05, P < 0.01) from vehicle-treated response by chondrocytes, leading to progressive extracellu-lar matrix damage and cartilage destruction. NF-κB mediates This paper was supported by Green Cross Corporation, fibronectin fragment-induced chondrocyte activation and Korea, and Jaseng Hospital, Korea.
increased expression of proinflammatory cytokines, che-mokines as well as MMPs such as IL-6, IL-8, MCP-1, growth-related oncogene-α, -β, -γ, and MMP-13 by human articular chondrocytes [40, 41]. In this study, we showed that GCSB-  M. B. Goldring and S. R. Goldring, "Osteoarthritis," Journal of 5 inhibits nuclear translocation of NF-κB/p65 subunit and Cellular Physiology, vol. 213, no. 3, pp. 626–634, 2007.
degradation of IκB-α.
 J. Saklatvala, "Inflammatory signaling in cartilage: MAPK and Although the results of present study provided clues for NF-κB pathways in chondrocytes and the use of inhibitors further studies on the pharmacological mechanisms of for research into pathogenesis and therapy of osteoarthritis," GCSB-5, the relationship between the eﬀects and its active Current Drug Targets, vol. 8, no. 2, pp. 305–313, 2007.
components remains to be clarified. Therefore, the detailed  F. E. Silverstein, G. Faich, J. L. Goldstein et al., "Gastrointesti- molecular mechanisms of GCSB-5 and further studies of nal toxicity with Celecoxib vs nonsteroidal anti-inflammatorydrugs for osteoarthritis and reumatoid arthritis: the CLASS anti-inflammatory properties of the active ingredients study: a randomized controlled trial," Journal of the American should be elucidated.
Medical Association, vol. 284, no. 10, pp. 1247–1255, 2000.
 L. X. Chen, L. Lin, H. J. Wang et al., "Suppression of early experimental osteoarthritis by in vivo delivery of the adenovi- ral vector-mediated NF-κBp65-specific siRNA," Osteoarthritisand Cartilage, vol. 16, no. 2, pp. 174–184, 2008.
These results indicate that GCSB-5 improves OA-induced  S. W. Park, C. H. Lee, S. H. Kim et al., "General pharmaco- cartilage damage, which inhibits MMP activities, down- logical study of GCSB-5, a herbal formulation," The Journal of regulates the expression of inflammatory mediators, and Applied Pharmacology, vol. 14, pp. 194–201, 2006.
suppresses NF-κB activity, suggesting that GCSB-5 may be  H. W. Kim, Y. B. Kwon, T. W. Ham et al., "The antinociceptive a potential therapeutic agent for OA.
and anti-inflammatory eﬀect of ethylacetate extracts from Evidence-Based Complementary and Alternative Medicine Bang-Poong (Radix ledebouriellae) on the Freund's adjuvant- explant culture and collagenase-induced rabbit osteoarthritis induced arthritis in rats," Journal of Veterinary Science, vol. 3, model," Osteoarthritis and Cartilage, vol. 10, no. 6, pp. 471– no. 4, pp. 343–349, 2002.
 B. Zhao, X.-B. Yang, X.-W. Yang et al., "Intestinal permeability  Y. Sakano, N. Terada, H. Ueda et al., "Histological study of of the constituents from the roots of saposhnikovia divaricata articular cartilage in experimental rat knee arthritis induced in the human caco-2 cell monolayer model," Planta Medica, by intracapsular injection of cationic polyethyleneimine," vol. 77, no. 13, pp. 1531–1535, 2011.
Medical Electron Microscopy, vol. 33, no. 4, pp. 246–257, 2000.
 Y. Ida, Y. Satoh, M. Katsumata et al., "Two novel oleanolic acid  K. Kobayashi, R. Imaizumi, H. Sumichika et al., "Sodium io- saponins having a sialyl Lewis X mimetic structure from doacetate-induced experimental osteoarthritis and associated Achyranthesfauriei root," Bioorganic and Medicinal Chemistry pain model in rats," Journal of Veterinary Medical Science, vol.
Letters, vol. 8, no. 18, pp. 2555–2558, 1998.
65, no. 11, pp. 1195–1199, 2003.
 D. Seidlova-Wuttke, D. Christel, P. Kapur, B. T. Nguyen, H.
 H. Dumond, N. Presle, P. Pottie et al., "Site specific changes in Jarry, and W. Wuttke, "β-Ecdysone has bone protective but no gene expression and cartilage metabolism during early experi- estrogenic eﬀects in ovariectomized rats," Phytomedicine, vol.
mental osteoarthritis," Osteoarthritis and Cartilage, vol. 12, no.
17, no. 11, pp. 884–889, 2010.
4, pp. 284–295, 2004.
 H. C. Kim, S. I. Lee, and D. K. Ahn, "Eﬀect of acanthopanacis  L. G. Ameye and W. S. S. Chee, "Osteoarthritis and nutrition.
cortex on the IL-8 production in human monocyte as a From nutraceuticals to functional foods: a systematic review rheumatoid arthritis remedy," Journal of Herbalogy, vol. 10, no.
of the scientific evidence," Arthritis Research and Therapy, vol.
1, pp. 49–59, 1995.
8, no. 4, article R127, 2006.
 C. H. Lee, S. H. Kim, J. S. Lee et al., "Evaluation of the antino- ciceptive properties of GCSB-5, a herbal formulation," Korean  M. Cameron, J. J. Gagnier, C. V. Little, T. J. Parsons, A. Bl¨umle, Journal of Pharmacognosy, vol. 36, no. 4, pp. 299–304, 2005.
and S. Chrubasik, "Evidence of eﬀectiveness of herbal medici-  X. C. Nguyen, V. M. Chau, V. K. Phan et al., "Inhibitors of os- nal products in the treatment of arthritis—Part 1: osteoarthri- teoclast formation from rhizomes of Cibotium barometz," tis," Phytotherapy Research, vol. 23, no. 11, pp. 1497–1515, Journal of Natural Products, vol. 72, no. 9, pp. 1673–1677, 2009.
 S. H. Kim, C. H. Lee, J. S. Lee et al., "Anti-inflammatory ac-  C. Cournil, B. Liagre, L. Grossin et al., "Overexpression and tivities of a herbal preparation GCSB-5 on acute and chronic induction of heat shock protein (Hsp) 70 protects in vitro and inflammation," Korean Journal of Pharmacognosy, vol. 36, no.
in vivo from mono-iodoacetate (MIA)-induced chondrocytes 4, pp. 311–317, 2005.
death," Arthritis Research and Therapy, vol. 3, supplement 1, p.
 R. W. Wong and A. B. Rabie, "Eﬀect of genistin on bone for- mation," Frontiers in Bioscience, vol. 2, pp. 764–770, 2010.
 J. Dunham, S. Hoedt-Schmidt, and D. A. Kalbhen, "Prolonged  N. D. Hong, Y. S. Rho, J. W. Kim, D. H. Won, N. J. Kim, and eﬀect of iodoacetate on articular cartilage and its modification B. S. Cho, "Studies on the general pharmacological activities by an anti-rheumatic drug," International Journal of Experi- of Eucommia ulmoides Oliver," Korean Journal of Pharmacog- mental Pathology, vol. 74, no. 3, pp. 283–289, 1993.
nosy, vol. 19, pp. 102–110, 1998.
 R. A. Barve, J. C. Minnerly, D. J. Weiss et al., "Transcriptional  J. Zhu, X. Gao, W. L. Xie, Y. Z. Jin, and W. J. Sun, "Eﬀect of gen- profiling and pathway analysis of monosodium iodoacetate- iposide on serum IL-1β and TNF-α of rheumatoid arthritis induced experimental osteoarthritis in rats: relevance to rats," Zhongguo Zhongyao Zazhi, vol. 30, no. 9, pp. 708–711, human disease," Osteoarthritis and Cartilage, vol. 15, no. 10, pp. 1190–1198, 2007.
 H. Ha, J. Ho, S. Shin et al., "Eﬀects of Eucommiae Cortex on  A. L. Stevens, C. A. Wheeler, S. R. Tannenbaum, and A. J.
osteoblast-like cell proliferation and osteoclast inhibition," Grodzinsky, "Nitric oxide enhances aggrecan degradation by Archives of Pharmacal Research, vol. 26, no. 11, pp. 929–936, aggrecanase in response to TNF-α but not IL-1β treatment at a post-transcriptional level in bovine cartilage explants,"  H. J. Chung, H. S. Lee, J. S. Shin et al., "Modulation of acute Osteoarthritis and Cartilage, vol. 16, no. 4, pp. 489–497, 2008.
and chronic inflammatory processes by a traditional medicine  R. Trelstad and P. Kemp, Matrix Glycoproteins and Proteogly- preparation GCSB-5 both in vitro and in vivo animal models," cans, WB Saunders, Philadelphia, Pa, USA, 1993.
Journal of Ethnopharmacology, vol. 130, no. 3, pp. 450–459,  K. Kusano, C. Miyaura, M. Inada et al., "Regulation of matrix metalloproteinases (MMP-2,-3,-9, and-13) by interleukin-1  J. D. Sandy, H. L. G. Brown, and D. A. Lowther, "Degradation and interleukin-6 in mouse calvaria: association of MMP of proteoglycan in articular cartilage," Biochimica et Biophysica induction with bone resorption," Endocrinology, vol. 139, no.
Acta, vol. 543, no. 4, pp. 536–544, 1978.
3, pp. 1338–1345, 1998.
 S. E. Bove, S. L. Calcaterra, R. M. Brooker et al., "Weight bearing as a measure of disease progression and eﬃcacy of  G. Murphy and A. J. Docherty, "The matrix metalloprotein- anti-inflammatory compounds in a model of monosodium ases and their inhibitors," American Journal of Respiratory Cell iodoacetate-induced osteoarthritis," Osteoarthritis and Carti- and Molecular Biology, vol. 7, no. 2, pp. 120–125, 1992.
lage, vol. 11, no. 11, pp. 821–830, 2003.
 S. C. Chu, S. F. Yang, K. H. Lue, Y. S. Hsieh, T. Y. Hsiao, and  M. J. Yeom, H. C. Lee, G. H. Kim, I. Shim, H. J. Lee, and D.
K. H. Lu, "The clinical significance of gelatinase B in gouty H. Hahm, "Therapeutic eﬀects of Hominis placenta injection arthritis of the knee," Clinica Chimica Acta, vol. 339, no. 1-2, into an acupuncture point on the inflammatory responses in pp. 77–83, 2004.
subchondral bone region of adjuvant-induced polyarthritic  C. Melchiorri, R. Meliconi, L. Frizziero et al., "Enhanced and rat," Biological and Pharmaceutical Bulletin, vol. 26, no. 10, pp.
coordinated in vivo expression of inflammatory cytokines 1472–1477, 2003.
and nitric oxide synthase by chondrocytes from patients with  J. H. Choi, J. H. Choi, D. Y. Kim et al., "Eﬀects of SKI 306X, osteoarthritis," Arthritis and Rheumatism, vol. 41, no. 12, pp.
a new herbal agent, on proteoglycan degradation in cartilage 2165–2174, 1998.
Evidence-Based Complementary and Alternative Medicine  K. W. Moore, R. De Waal Malefyt, R. L. Coﬀman, and A.
O'Garra, "Interleukin-10 and the interleukin-10 receptor,"Annual Review of Immunology, vol. 19, pp. 683–765, 2001.
 F. Iannone, C. De Bari, F. Dell' Accio et al., "Interleukin-10 and interleukin-10 receptor in human osteoarthritic and healthychondrocytes," Clinical and Experimental Rheumatology, vol.
19, no. 2, pp. 139–145, 2001.
 A. S. Baldwin, "The transcription factor NF-κB and human disease," Journal of Clinical Investigation, vol. 107, no. 1, pp.
 C. B. Forsyth, A. Cole, G. Murphy, J. L. Bienias, H. J. Im, and R. F. Loeser Jr., "Increased matrix metalloproteinase-13 pro-duction with aging by human articular chondrocytes inresponse to catabolic stimuli," Journals of Gerontology A, vol.
60, no. 9, pp. 1118–1124, 2005.
 J. I. Pulai, H. Chen, H. J. Im et al., "NF-κB mediates the stim- ulation of cytokine and chemokine expression by human ar-ticular chondrocytes in response to fibronectin fragments,"Journal of Immunology, vol. 174, no. 9, pp. 5781–5788, 2005.
Urticaire M. Vigan (Praticien hospitalier)* *Auteur correspondant : Unité fonctionnelle d'allergologie, département de dermatologie, Hôpital Saint-Jacques, 25030 Besançon cedex, France E-mail : [email protected] Téléphone : 01 40 40 40 40 – Fax : 01 40 40 41 41 Le médecin généraliste est souvent le premier consulté lors de la survenue d'une urticaire. Il