Rheumatoid arthritis (RA) is a chronic inflammatory dis-ease characterized by acute arthritis affecting several jointsand accompanying synovial hyperplasia, ultimately leadingto joint destruction and deformity, which reduce the qualityof life severely. Recently, the mechanism underlying the boneremodeling and bone loss has become increasingly clear, al-though the etiology and pathogenesis of RA have not yetbeen fully understood. The presence of osteoclasts at sites offocal bone erosion in RA and in animal models of arthritissuggests that osteotropic factors leading to osteoclast differ-entiation and activation may play a critical role in the patho-genesis of the erosion in joints affected with RA.1,2) Severallines of evidence indicate that some cytokines and hormonesincluding macrophage colony-stimulating factor (M-CSF),3,4)

transforming growth factor (TGF)-b ,5) tumor necrosis factor(TNF)-a,6)interferon (IFN)-g ,7) interleukin (IL)-1a ,8) IL-6,9)

IL-7,10) IL-11,11) IL-12,12) IL-17,13) calcitonin,14) estrogen15)

regulate osteoclast differentiation and activation. In addition,an essential factor for osteoclast differentiation, receptor acti-vator of nuclear factor-kB ligand (RANKL), has recentlybeen identified16,17) and demonstrated to play a critical role inthe pathogenesis of bone erosion in inflammatory arthritis. Adecoy receptor of RANKL, osteoprotegerin (OPG), was alsoidentified and found to suppress bone resorption associatedwith osteoclast development.18) Collagen-induced arthritis(CIA) is an experimental model for RA, and has many mor-phological features similar to those of human RA, includingsynovitis, pannus formation, and erosion of cartilage andbone.19) The expression of RANKL, RANK, and OPG in theCIA model has been demonstrated to play an important rolein developing osteolytic lesions in local subarticular bone aswell as in periarticular osteopenia and systemic osteoporo-sis.20,21) These findings suggest that the RANKL-RANK sig-

naling pathway and the factors involved in the regulation ofRANKL, RANK, and OPG expression can be novel targetsfor the treatment of RA to protect joints from bone destruc-tion.

Important issues concerning RA therapy are the ability tocontrol symptoms and signs of the disease for prolonged pe-riods as well as the capacity to retard the damaging effect ofrheumatoid inflammation on articular cartilage and bone. Forthe remedy of RA, disease modifying antirheumatic drugs(DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs),and steroids are clinically common therapeutic agents, andrecently TNF-a-neutralizing therapy in combination withmethotrexate provided sustained clinical benefit.22) However,the validity for long-term treatment with these medicines hasnot yet been proven, and further adverse events were reportedwith extremely high frequency, which limits their use early inthe disease process and interfere with prolonged administra-tion. Rheumatoid bone destruction, attributed to activatedsynoviocytes and bone-resorbing osteoclasts, can not be eas-ily controlled by inhibiting only one of the factors involvedin the multiple pathological process. Taking into considera-tion the state of RA therapy and the intricate pathogenesis ofRA, the combination therapy using plural therapeutic agentsor the therapy using agents containing plural constituentsmay be useful to suppress inflammatory arthritis.23,24)

Traditional Japanese herbal medicines (Kampo medicines)usually consist of several medicinal plants, and are applied tochronic diseases depending on the degree of development ofthe disease and the condition of the patient. We have so fartested the effectiveness of eight Kampo medicines againstarthritis model mice and found that four Kampo medicines(Makyo-yokkan-to, Dai-bofu-to, Keishi-ka-jutsubu-to, andKanzo-bushi-to) effectively suppressed the severity of arthri-

* To whom correspondence should be addressed. e-mail: inoue@phar.nagoya-cu.ac.jp © 2004 Pharmaceutical Society of Japan

Suppressive Effect of Kanzo-bushi-to, a Kampo Medicine, on Collagen-Induced Arthritis

Yuka ONO,a Makoto INOUE,*,a Hajime MIZUKAMI,a and Yukio OGIHARAb

a Laboratory of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Nagoya City University; 3–1 Tanabe-dori,Mizuho-ku, Nagoya 467–8603, Japan: and b Laboratory of Kampo Medicine, Faculty of Pharmacy, Meijo University; 150Yagotoyama,, Tenpaku-ku, Nagoya 468–8503, Japan.Received February 17, 2004; accepted July 2, 2004; published online July 5, 2004

Kanzo-bushi-to (KBT) is a traditional Japanese herbal medicine (Kampo medicine), which is used in Japanto treat rheumatoid arthritis. In the present study, we investigated the suppressive effect of KBT on collagen-in-duced arthritis (CIA) and further studied the underlying mechanism. CIA was induced in male DBA/1J mice byimmunization with bovine type II collagen, followed by a booster injection 21d later. KBT was given at a dose of430 mg/kg/d from three days before the first immunization to the end of the experiment. KBT suppressed CIAdevelopment effectively and further protected focal bone erosion and bone destruction as evidenced by the re-duced histological score. Histochemical examination revealed that KBT decreased TRAP-positive cells at thesynovium-bone interface and at the sites of focal bone erosion, coincident with the findings that RANKL/OPGmRNA ratio was significantly reduced by KBT treatment. KBT also decreased mRNA levels of M-CSF and iNOSin joints and of iNOS in peritoneal macrophages. In conclusion, KBT prevented osteoclast generation by decreas-ing RANKL/OPG ratio and M-CSF mRNA levels, resulting in reduction in bone erosion and destruction. In ad-dition, KBT has anti-inflammatory effect such as the suppression of iNOS expression in peritoneal macrophagesand joints of CIA mice. These finding suggests that KBT is a potential new therapeutic agent for the treatment ofRA.

Key words Kanzo-bushi-to; Kampo medicine; collagen-induced arthritis; bone destruction; osteoclast

1406 Biol. Pharm. Bull. 27(9) 1406—1413 (2004) Vol. 27, No. 9

tis in an induced-arthritis model, CIA mice.25) Especially,KBT decreased serum anti-type II collagen antibody levelssignificantly,25) although such an effect was not seen in otherKampo medicines. These results suggest that the inhibitionof arthritis by KBT is ascribed to anti-inflammatory and im-munosuppressive effect. We therefore evaluated the effect ofKBT on bone destruction in the joints of CIA model mice, asone of the most important aims of RA therapy is to blockbone destruction. We provided here several lines of evidencethat KBT effectively suppressed bone destruction in arthriticjoints, accompaning the reduction of osteotropic factors.

MATERIALS AND METHODS

Animals Male DBA/1J mice were purchased from Nip-pon Charles River (Kanagawa, Japan). All mice were kept ina temperature-controlled room (23�1 °C) with lighting from6 a.m. to 6 p.m., under specific-pathogen-free conditions andgiven a sterilized commericial diet (CE-2; Nippon Crea Co.,Ltd., Shizuoka, Japan) and water ad libitum at the LaboratoryAnimal Center of Nagoya City University. Mice were used at8 weeks of age. All animal procedures were approved by theinstitutional animal care and use committee of Nagoya CityUniversity.

Preparation of Kanzo-bushi-to (KBT) KBT (dose perperson per day) was prepared as follows. Cinnamomi Cortex(3 g; Tsumura Co. Ltd., Lot No. 18054631), GlycyrrhizaeRadix (2 g; Tsumura Co. Ltd., Lot No. 19022891), Atracty-lodis Lanceae Rhizoma (4 g; Daiko Galenical Co., Ltd., LotNo. OL12), Aconiti Tuber (0.5 g; Mikuni Pharmaceutical In-dustrial Co., Ltd., Lot No. C358) were added to 700 mlwater, decocted for 1 h and concentrated to 300 ml. This de-tection was filtrated through cheese-cloth and lyophilized togive 2.7�0.2 g of powdered extract. Main ingredients arecinnamic aldehyde, glycyrrhizin, atractylon, and aconitine inCinnamomi Cortex, Glyccyrhizae Radix, AtractylodisLanceae Rhizoma, Aconiti Tuber, respectively.

Induction of Collagen-Induced Arthritis (CIA) in MiceMice were randomly separated into three groups: normal,non-immunized mice; control, untreated CIA mice; KBT-treated, KBT-treated CIA mice. CIA was induced and evalu-ated as described previously.25) Briefly, the severity of arthri-tis was evaluated for each paw by scoring method accordingto the degree of inflammation, where grade 0, normal; grade1, swelling of one finger ; grade 2, swelling of more than twofingers; grade 3, swelling of heel; and grade 4, joint defor-mity with ankylosis, resulting in maximum score of 16 peranimal.

Polymerase Chain Reaction Amplification of Reverse-Transcribed mRNA For semiquantitative reverse-tran-scriptase PCR (RT-PCR) analysis, total RNA from peritonealmacrophages was extracted using Trizol reagent (Invitrogen,Carisbad, CA, U.S.A.) according to the manufacturer’s in-structions. The extracted RNA was treated with DNaseI (In-vitrogen, Carisbad, CA, U.S.A.) in order to degrade contami-nating DNA. The RNA was dissolved in DEPC-treated waterand quantified by GeneQuant II (Amersham PharmaciaBiotech, Buckinghamshire, U.K.). To prepare first strandcDNA, 500 ng of total RNA was reverse-transcribed usingRevertra Ace-a (Toyobo Co, Osaka, Japan) according to themanufacturer’s instructions. The resulting cDNA was sub-

jected to PCR amplification with Taq polymerase (Roche,Mannheim, Germany) using specific PCR primers for iNOSand b-actin as shown in Table 1. A thermal cycle of 30 s at94 °C, 30 s at 55 °C, 1 min at 72 °C was applied for 30 cycles.PCR products were analysed on 1.5% agarose gels stainedwith ethidium bromide.

RT-southern Blot Analysis Total RNA from murinejoints was isolated using Trizol reagent and first strandcDNA was prepared as described above. The resulting cDNAwas subjected to hot-start PCR amplification with Ampli Taqpolymerase (Applied Biosystems, Foster City, CA, U.S.A.)using specific PCR primers as shown in Table 1. The numberof cycles necessary to amplify cDNA but remain below satu-ration was determined for each primer set and cell type. Eachthermal cycle of 30 s at 94 °C, 30 s at 55 °C, 1 min at 72 °Cwas applied for 15 cycles (b-actin), 24 cycles (RANK), 25cycles (IL-6), 28 cycles (RANKL and M-CSF), 32 cycles(OPG), and 35 cycles (iNOS and TNF-a). PCR productswere applied to 1.5% agarose gels and then transferred topositively charged nylon membranes. After fixation under ul-traviolet irradiation, the membranes were hybridized withdigoxigenin-labeled (DIG-labeled) cDNA probes and visual-ized using alkaline phosphatase-labeled anti-DIG antibody(Roche, Mannheim, Germany). The density of interestingbands was determined with Lumi-Imager F1 (Roche,Mannheim, Germany).

Histological Assessment Hind and front paws werefixed in 15% phosphate-buffered formalin for 3 d, decalcifiedin 10% EDTA for 14 d at 4 °C, then embedded in paraffin.Serial paraffin sections (7 mm) were stained with hematoxylinand eosin (H&E), and with fast red violet for tartrate-resis-tant acid phosphatase (TRAP) activity. TRAP staining wasperformed according to the manufacture’s instructions at-tached to the kit (Muto Chemical Co., Ltd, Tokyo, Japan).Histopathological changes in joints were scored using thefollowing parameters. 0: normal, 1: infiltration of inflamma-tory cells, 2: synovial hyperplasia, 3: pannus formation, 4:bone erosion, 5: bone destruction.

Assay for Nitric Oxide Produced by PeritonealMacrophages Thioglycolate-elicited peritoneal macropha-ges were prepared as described previously with a minor mod-ification. Briefly, DBA/1J mice were injected intraperi-

September 2004 1407

Table 1. Primer Sequences Used in the Present Study

RANKL forward 5�-CTCCATGAAAACGCAGG-3�RANKL reverse 5�-CGACATACACCATCAGC-3�RANK forward 5�-ATCTCTCTGGTAGTAGTGGCTG-3RANK reverse 5�-TGTGTAGCCATCTGTTGAGTTG-3OPG forward 5�-TGACCACTCTTATACGGACAG-3�OPG reverse 5�-TTCTCTCAATCTCTTCTGGGC-3�M-CSF forward 5�-CTTGGCTTGGGATGATTCTC-3�M-CSF reverse 5�-CTGGTACTTCTCTTGCCCTC-3�IL-6 forward 5�-ACCTGTCTATACCACTTCAC-3�IL-6 reverse 5�-GATGGTCTTGGTCCTTAGCC-3�IL-17 forward 5�-ACTCTCCACCGCAATGAAGAC-3�IL-17 reverse 5�-TGAATCTGCCTCTGAATCCAC-3�TNFa forward 5�-TCATTCCTGCTTGTGGC-3�TNFa reverse 5�-CCATTCCCTTCACAGAG-3�iNOS forward 5�-TGCAAGGAAGGGAACTCTTC-3�iNOS reverse 5�-GAACGTGTTTACCATGAGGC-3�b-Actin forward 5�-CAGGCAGCTCATAGCTCTTCT-3�b-Actin reverse 5�-TGGGTCAGAAGGACTCCTATG-3�

toneally with 2 ml of 3% thioglycolate medium (Difco Labo-ratories, Detroit, MI, U.S.A.) one day after second immuniza-tion in CIA model. Six days later, peritoneal macrophageswere harvested from the abdominal cavity and maintained inRPMI1640 (IrvineScientific Co, Santa Ana, CA, U.S.A.)supplemented with 10% fetal bovine serum (FBS). The re-sulting macrophages were seeded at a concentration of2�106 cells/ml and incubated in the presence or absence oflipopolysaccharide (LPS: Sigma, St. Louis, MO, U.S.A.)1 mg/ml and IFN-g (Pepro Tech, Inc, London, U.K.)100 U/ml for 20 h. Nitric oxide (NO) production was deter-mined by measuring as nitrite concentration in the culturemedium using Griess reagent.26)

Western Blot Analysis Following 24 h incubation in thepresence or absence of LPS (1 mg/ml) and IFN-g (100 U/ml),thioglycolate-elicited peritoneal macrophages were rinsedwith PBS, and then lysed in the lysis buffer containing 20 mM

Tris/HCl pH 8.0/137 mM NaCl/2 mM EDTA/1% glycerol/1%Triton-X-100 on ice. The resulting cell lysate (20 mg of pro-tein) was subjected to 8% SDS-PAGE analysis. After elec-trophoresis, the separated proteins were transferred to immo-bilon-P transfer membrane (Millipore Co, Bedford, MA,U.S.A.) with a wet electrotransfer system (Bio-Rad, Rich-mond, CA, U.S.A.). The membranes were incubated withblocking buffer (Tris–borate–EDTA buffer (TBS), pH 7.5/5%powdered skimmed milk/0.05% Tween-20) for 6 h and incu-bated with a 1 : 3000 dilution of anti-iNOS antibody (SantaCruz Biotechnology, Inc, Santa Cruz, CA, U.S.A.) in TBS,pH 7.5/1% powdered skimmed milk at 4 °C overnight. Themembranes were washed in three changes of wash buffer(0.05% Tween-20 in TBS), and then incubated with a1 : 3000 dilution of alkaline phosphatase-conjugated anti-rab-bit IgG antibody (Bio Lad, Hercules, CA, U.S.A.) at 4 °Covernight. Finally, they were washed in four changes of thewash buffer, and iNOS was detected using CDP-Star (PEBiosystems, Bedford, MS, U.S.A.) as a substrate of alkalinephosphatase. Protein concentration was determined using theBio-Rad protein assay (Bio-Rad, Richmond, CA, U.S.A.).

Statistical Analysis Data were represented as mean�S.E. of the number of animals described in the legends. Sta-tistical significance was determined by non-paired Student’st-test, or Mann–Whitney U-test using Stat View software. p values less than 0.05 were considered significant.

RESULTS

Suppressive Effect of KBT on the Development of CIAWe first determined the effect of KBT on the development ofarthritis using CIA model mice. KBT was administered at adose of 0.43 g/kg/d from 3 d before the first immunizationwith bovine type II collagen until the end of the experiment.The dose of KBT corresponds to a concentration of 10 timesthe daily human dose and suppressed macrophage functioneffectively in ex vivo study in our previous study.25) Figure 1shows that KBT treatment reduced the severity of joint in-flammation significantly and delayed the onset of arthritisfrom 21.8 to 26.8 d after the first immunization. The inci-dence of arthritis reached to 100% in the end of experimentand did not vary between KBT-treated and control CIAgroups. During the course of the experiment, the change inbody weight and other changes in the appearance were notobserved in KBT-treated group. Although data were notshown, immunosuppressant, FK506 (10 mg/kg/d, p.o.),markedly reduced the arthritis severity (average score:2.0�0.2 at day 36), and prolonged the onset to 29.3 d. How-ever, FK506 decreased the body weight of mice severely,which was considered as an adverse effect.

Reduction of Joint Destruction by KBT Five weeksafter the first immunization, the tarsocrural joints of KBT-treated, control and normal mice were examined histologi-cally after H&E staining of joint sections. No synovitis, pan-nus formation, or focal bone erosion was seen in normalmouse joints (Fig. 2C), while in control CIA mice infiltratinginflammatory cells were observed in the hyperplastic syn-ovium, and periarhthritis, osteomyelitis, and focal bone ero-sion was prominent (Fig. 2A). In KBT-treated CIA mice, theseverity of arthritis was markedly ameliorated, although thehyperplasia of synovium and focal bone erosion were seen intheir joints (Fig. 2B). When the inflammation in joints wasassessed by histological scoring, as described in Materialsand Methods, KBT decreased the histological score signifi-cantly, relative to control CIA group (Fig. 2D).

It is well known that osteoclasts play a critical role in boneresorption and can be observed at the sites of bone erosion inRA patients. We therefore stained the sections of tarsocruraljoint with TRAP, which stains tartrate-resistant acid phos-phatase-positive cells. In control CIA mice, TRAP-positivecells were seen at the sites of focal bone erosion (Fig. 3A)and within erosive pits in the bone (data not shown). On the

1408 Vol. 27, No. 9

Fig. 1. Effect of KBT on the Severity (A), Incidence (B), and Onset (C) of CIA

DBA/1J mice were immunized intradermally with CII on day 0 and given a booster by intraperitoneal injection with CII on day 21. KBT were given to the mice from 3 d beforethe first immunization. Arthritis was evaluated daily by a clinical score as described in Materials and Methods. The onset of arthritis in CIA mice in Fig. 1B was taken as the daywhen erythema and/or swelling were first observed. Open circle and column, KB-treated CIA group (n�8); solid circle and closed column, control CIA group (n�8). Values repre-sent mean�S.E.M. * and a) p�0.05, ** p�0.01 vs. control group (Mann–Whitney U-test in A and Student’s t-test in C).

other hand, TRAP-positive cells were extensively reduced inKBT-treated mice, coincident with the decrease in bone ero-sion (Figs. 3B, C).

Expression of RANKL, RANK, and OPG In view ofthe evidence that RANKL, RANK, and OPG are critically in-volved in osteoclast differentiation and activation, we evalu-ated the effect of KBT on their expression in affected jointsby determining their mRNA expression. Total RNA was ex-tracted from the joints of CIA mice 5 weeks after the first im-

munization, applied to RT-PCR, and then detected withcDNA probes labeled with digoxigenin (RT-southern analy-sis). RANKL and RANK mRNA levels were higher in thejoints of CIA mice than those of normal mice, while their ex-pression was suppressed by KBT treatment (Figs. 4A, B).This result was supported by the evidence that the decreasedexpression of RANKL and RANK was observed in the jointsof KBT-treated CIA mice as detected by immunohistochem-istry (data not shown). On the other hand, although the ex-

September 2004 1409

Fig. 3. Effect of KBT on TRAP-Positive Cells in Inflamed Joints

The sections of tarsocrural joints of control CIA (A, original magnification �20) and KB-treated CIA (B, original magnification �20) CIA mice were stained with TRAP on day14 after the second immunization. Representative joints with an average arthritic score in each group are shown. (C) The number of TRAP-positive cells (Arrows indicated some ofTRAP-positive cells.) in a constant view area are represented as mean�S.E.M. of 8 mice per group. * p�0.05 vs. control group (Student’s t-test).

Fig. 2. Effect of KBT on Joint Destruction in CIA

The sections of tarsocrural joints were stained with hematoxylin and eosin (original magnification, �20) on day 14 after the second immunization. Representative joints with anaverage arthritc score in control CIA (A), KB-treated CIA (B), and normal group (C) are shown. (D) Histological changes were scored on scale of 0—5 according to the methoddescribed in Materials and Methods. Arrows indicated the lesion areas. Values are mean�S.E.M. of 8 mice in each group. * p�0.05 vs. control group (Mann–Whitney U-test).

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Fig. 4. Effect of KBT on RANKL, RANK, and ORG mRNA Expression in Arthritic Joints of CIA Mice

Total RNA was extracted from the joints of carpal bones and digital bones of hands on day 14 after the second immunization. The mRNA was reverse-transcribed, amplifiedusing respective specific primers for RNAKL, RANK, and OPG, and detected with digoxigenin-labeled cDNA probes. Each band shown in Fig. 4A represents a mouse. Densito-metric quantitative analysis of the bands in Fig. 4A was performed in Fig. 4B. The ratio of RANKL to OPG mRNA levels was represented in Fig. 4C. Values are mean�S.E.M. of6 mice. Statistical significance was evaluated by Student’s t-test.

Fig. 5. Effect of KBT on mRNA Expression of Cytokines Involved in Bone-Resorbing in Arthritic Joints of CIA Mice

Total RNA was extracted from the joints of carpal bones and digital bones of hands on day 14 after the second immunization. The mRNA was reverse-transcribed, amplifiedusing respective specific primers for TNF-a , IL-6, and M-CSF and detected with digoxigenin-labeled cDNA probes. Each band shown in Fig. 5A represents a mouse. Densitomet-ric quantitative analysis of the bands in Fig. 5A was performed in Fig. 5B. Values are mean�S.E.M. of 6 mice. Statistical significance was evaluated by Student’s t-test.

pression of OPG was slightly enhanced by induction of CIA,it was not influenced by KBT treatment. Considering the im-portance of RANKL/OPG balance in osteoclast develop-ment, the significant decrease in RANKL/OPG ratio afterKBT treatment indicated that KBT effectively suppressed os-teoclast formation (Fig. 4C).

Effect of KBT on Osteotropic Factors A number of os-teotropic factors induces osteoclastogenesis by enhancing os-teoclast differentiation, activation, or survival. Among them,some cytokines such as TNF-a , M-CSF, and IL-6 were inti-mately implicated in joint destruction of RA. We thereforeassessed the effect of KBT on the mRNA expression of os-teotropic factors in affected joints. In control CIA mice, allthe cytokines that we studied were markedly induced by CIAcompared with normal mice (Fig. 5A). When the effect ofKBT was examined, KBT markedly suppressed mRNA ex-pression of M-CSF, in addition to slight inhibition of IL-6mRNA expression. However, TNF-a mRNA levels were notsignificantly affected by KBT treatment (Fig. 5B).

Inhibition of NO Production and NO Synthase (iNOS)Expression in Peripheral Macrophages and Joints of CIAMice To study the inflammatory effect of KBT, thioglycol-late-elicited macrophages, which were prepared from CIAmice, were stimulated with LPS/IFN-g and then NO produc-tion, iNOS, and iNOS mRNA were determined by Griessreagent method, western blot analysis, and semiquantitative

RT-PCR, respectively. As shown in Fig. 6A, KBT suppressedNO production significantly, resulting from the decrease iniNOS protein (Fig. 6B) and mRNA expression (Fig. 6C). Inaddition, when mRNA expression in the affected joints ofCIA mice was examined with RT-southern method, iNOSmRNA expression was markedly reduced in KBT-treatedmice (Figs. 6D, E).

DISCUSSION

Bone destruction in RA patients is considered to be theconsequence of chronic synovial inflammation. However,bone destruction is not able to start, unless osteoclasts areformed and activated, evidenced by that c-fos-deficient mice,which completely lack osteoclasts, were fully protectedagainst bone destruction despite the presence of synovial in-flammation.27) There is ample evidence that joint inflamma-tion and destruction can be uncoupled.28) The balance of de-struction and protective mediators determines the relativeerosive nature of a given arthritis, rather than the bulk of theinflammation mass.29,30) Therefore, in addition to the use ofanti-inflammatory therapy, suppression of osteoclast differen-tiation and activation could be beneficial for the treatment ofRA. Osteoclasts differentiate from osteoclast progenitor cellsby two uniquely essential singals provided by osteoblasts.One is mediated by M-CSF through its cognate receptor c-

September 2004 1411

Fig. 6. Effect of KBT on NO Production and iNOS Expression in Peritoneal Macrophages Prepared from CIA Mice, and on iNOS mRNA Levels in theJoints of CIA Mice

(A) Macrophages were stimulated with LPS and IFN-g for 20 h and NO production was assessed by using griess reagent. (B) Pooled macrophages were stimulated with LPS andIFN-g for 24 h. Total protein was extracted and subjected to SDS-PAGE, followed by western blot analysis. (C) Pooled macrophages were stimulated with LPS and IFN-g for 6 hand total RNA were extracted. iNOS mRNA expression was detected using RT-PCR. (D) Total RNA was extracted from the joints of carpal bones and digital bones of hands on day14 after the second immunization. The mRNA was reverse-transcribed, amplified using respective specific primers for iNOS and detected with digoxigenin-labeled cDNA probes.Each band shown in Fig. 6D represents a mouse. Densitometric quantitative analysis of the bands in Fig. 6D was performed in Fig. 6E. Values are mean�S.E.M. of 6 mice. Statisti-cal significance was evaluated by Student’s t-test.

fms, expressed on osteoclast progenitor cells.3) The other istransmitted by RANKL through RANK expressed on osteo-clast progenitor cells, mature osteoclasts, and chondrocytes.Thus, M-CSF and RANKL are together essential and suffi-cient for osteoclast production, and promote multinucleationof pre-fusion osteoclasts and survival of nascent osteoclasts.On the other hand, OPG, a decoy receptor of RANKL, isknown to reduce osteoclast numbers and prevent bone ero-sion in CIA.31) OPG is steadily expressed in normal jointsand is induced by TGF-b ,32) IL-1a ,33) and TNF-a ,34) whichare produced in inflamed joints. This suggests that the bal-ance between RANKL and OPG is important to ultimatelydetermine whether osteoclastogenesis is activated or inhib-ited. In the present study, we found that KBT reducedRANKL expression detected by RT-southern analysis andimmunohistochemical study (data not shown), and that KBTalso diminished M-CSF mRNA levels in joints affected byCIA. In contrast, we did not detect the change in OPG ex-pression between control CIA and KBT-treated CIA mice.These results suggest that KBT treatment interferes with notonly RANKL/RANK, but also M-CSF/c-fms signaling path-ways, finally leading to the reduction in osteoclast formation.RANKL is expressed on osteoblasts, activated T cells, andsynovial fibroblasts at the sites of bone erosion and synoviumbone interface in inflamed joints, and is known to be regu-lated by many osteotropic factors. M-CSF is also producedby osteoblasts/stroma cells in inflamed joints. Taken together,the effect of KBT may be in part due to decrease in os-teotropic factor production, abrogation of signal transductionof osteotropic factors in osteoblasts to induce RANKL, orsuppression of T cell activation.

Several lines of evidence suggest that RANKL and RANKinteraction is not the sole pathway that leads osteoclast prog-enitor cells to differentiation into osteoclasts. IL-1 inducesthe multinucleation and bone-resorbing activity of osteo-clasts even in the absence of osteoblasts/stromal cells.8) TNF-a stimulates osteoclast differentiation by a mechanism inde-pendent of the RANKL-RANK interaction,6) although thereis an opposite report that TNF-a acts directly on the osteo-clast precursor to potentiate RANKL-induced osteoclastoge-nesis, even if basal levels of RANKL are not upregulated.35)

However, although the pathophysiological importance ofRANKL-independent pathway is presently obscure in osteo-clastogenesis of RA patients, the finding that KBT did not af-fect TNF-a expression suggests that KBT cannot regulateRANKL-independent pathway effectively.

Inflammatory conditions such as RA, which are character-ized by local osteolysis, are associated with activation ofiNOS. The intimate implication of NO in the pathogenesis ofarthritis was indicated by the evidence that NOS inhibitorsreduce NO levels and ameliorate arthritis in streptococcalcell wall-induced36) and adjuvant-induced arthritis.37) Thepresent study represented that KBT effectively reduced iNOSmRNA levels in affected joints, and decreased iNOS and NOinduction in addition to iNOS mRNA levels in thioglycollate-elicited peritoneal macrophages of CIA mice. This resultsuggests that KBT is likely to exert anti-inflammatory effecton not only local NO-producing cells in joints, includingchondrocytes, synovial cells and macrophages, but also sys-temic macrophages represented by peritoneal macrophages.In addition, NO is known to serve as an essential and rela-

tively specific downstream mediator in the signaling pathwayof IL-1-induced bone resorption, and iNOS-deficient miceexhibit profound defects of IL-1-induced osteoclastic boneresorption.38) These findings may indicate that inhibition ofNO production by KBT is likely to suppress bone destructionindirectly. In this respect, NO is a new target for the treat-ment of RA, and KBT with inhibitory activity of NO produc-tion may be beneficial for the remedy of RA.

Considering a potential of KBT as a therapeutic agent forRA, KBT protects bone destruction in joints affected withCIA, to say the least, by two distinct ways. One is that KBTinsulates RANKL–RANK interaction by reducing RANKL/OPG balance in osteoblasts and activated T cells, which maybe caused by the blockade of signaling pathway or produc-tion of osteotropic factors. The other is that KBT may pre-vent the differentiation and activation of osteoclast progenitorcells to mature osteoclast by reducing production of os-teotropic factors or modulating signaling pathway of RANK.In brief, these effects of KBT may be attributed to anti-in-flammatory effect on osteotropic factor production and directeffect on osteoblasts. However, we should determine whetherthe joint-protective effect results from anti-inflammatory ef-fect on the early stage of RA or direct effect on osteoblasts orother cells.

Traditional Japanese herbal medicine (Kampo medicine) isthe representative of combination formulas, for example,KBT, which we studied in the present study, consists of fourmedicinal crude drugs: Cinnamomi Cortex, GlycyrrhizaeRadix, Atractylodis Lanceae Rhizoma, and Aconiti Tuber. Inaddition, its characteristic is to have plural points of action,which are ingeniously integrated to improve the condition ofpatient and cure a disease. Actually, the evidence presentedhere indicated that KBT has at least two points of action,anti-inflammatory and anti-osteoclastogenic actions. In con-clusion, although the precise mechanism by which KBT sup-presses the severity of arthritic joints is now under investiga-tion, KBT is a candidate for novel therapeutic agents of RA.

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