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Anti-inflammatory and analgesic effects and molecular mechanisms of JCICM-6, a purified extract derived from an anti-arthritic Chinese herbal formula.

Abstract

The anti-inflammatory and anti-nociceptive effects and the molecular mechanisms of JCICM-6 purified extract derived from an anti-arthritic Chinese herbal formula composed of Caulis Sinomenii, Aconiti laterralis Preparata, Rhizoma Curcumae longae, Radix Paeoniae albae, and Cortex Moutan, were examined for the first time. JCICM-6 was prepared using pharmaceutical extraction technology, purified by Amberlite XAD-7HP polymeric resin. Pharmacologically, in carrageenan-induced edema and carrageenan-evoked thermal hyperalgesia in paws of rats, the oral administration of JCICM-6 at dosages of 0.4, 0.8, and 1.6g/kg demonstrated significant inhibition with a dose-dependent manner. Mechanistic studies showed that JCICM-6 effectively decreased the production of the proinflammatory cytokines of IL-6 and IL-1 [beta] and expression of COX-2 and iNOS proteins, and simultaneously elevated the level of anti-inflammatory cytokine IL-4 in the carrageenan-injected rat paw tissues and exudates. The positive reference drug, indomethacin at a dosage of 10mg/kg, demonstrated inhibitory potency in both rat models, but it could not augment the production of IL-4, indicating JCICM-6 and indomethacin might possess different pharmacological properties and molecular mechanisms although both have anti-inflammatory and analgesic effects in rats. These results suggest that JCICM-6 would be a valuable candidate for further investigation as a new anti-arthritic drug.

[c] 2008 Elsevier GmbH. All rights reserved.

Keywords: JCICM-6; Herbal formula; Anti-inflammation; Anti-nociception; Cytokines; COX; iNOS

Introduction

In treating inflammatory and arthritic diseases, medical doctors have been commonly prescribing nonsteroidal anti-inflammatory drugs (NSAIDs) and immunosuppressants. However, some of them can cause serious adverse effects such as gastric mucosal damage, water and salt retention (phenylbutazone, oxyphenbutazone) and even, possibly, carcinomas (Suleyman and Buyukokuroglu, 2001). Thus, alternative agents with less severe side effects are required, and botanical products are important candidates (Verpoorte, 1998).

The anti-inflammatory effect of these drugs is believed to result from their ability to inhibit the formation of prostaglandins by cyclooxygenases (COXs). Two iso-forms of cyclooxygenase, COX-1 and COX-2, have been identified. COX-1 constitutively expressed in normal tissues functions as necessarily physiological activities including protection of the gastric mucosal lining; while COX-2 is overproduced in the sites of inflammation (Appleby et al., 1994; Smith et al., 1998). In clinical use, some NSAIDs have been proven to block both COX-1 and COX-2 activities resulting in induction of gastric ulcerization and kidney failure. Moreover, although the marketing of new COX-2 inhibitors has emphasized the advantages of not blocking the necessary COX-1 pathway, gastrointestinal problems from the use of those drugs are still being reported (Silverstein et al., 2000).

Recently, it has been suggested that nitric oxide (NO) is involved in inflammatory process. Excessive and prolonged inducible NOS (iNOS)-mediated NO generation can act as a cytotoxic agent in inflammatory disorders (Bogdan, 2001). Inhibition of iNOS is beneficial for the treatment of inflammatory diseases (Bogdan, 2001; Kroncke et al., 1998).

It is well-known that cytokines play a pivotal role in inflammation (Dinarello, 2000). Those cytokines related to inflammation (Dinarello, 2000). Those cytokines related to inflammation include pro- and anti-inflammatory cytokines. The classic pro-inflammatory cytokines include IL-1[beta], IL-6, IL-2, GM-CSF, and TNF-[alpha] that can induce both acute and more chronic inflammation. However, anti-inflammatory cytokines like IL-4, IL-10, and IFN-[gamma] can block the inflammatory process (Cassatella et al., 1993; Orino et al., 1992). Accordingly, a "balance" between the effects of pro- and anti-inflammatory cytokines is thought to determine the outcomes of induction and severity of inflammatory diseases as well as the therapeutic efficacies of agents. Taken together, COX-2 protein expression, the balance of pro- and anti-inflammatory cytokines and NO activity are considered to be used as molecular targets of drugs in screening of the anti-inflammatory and anti-arthritic compounds.

In the current study, an anti-arthritic Chinese herbal formula that is often used for treatment of joint pain and rheumatoid arthritis (RA) by Chinese doctors was employed. The formula is composed of five herbs including Caulis Sinomenii, Radix Aconiti lateralis Preparata, Rhizoma Curcumae longae, Radix Paeoniae albae, and Cortex Moutan. All of these five herbs have well-established histories of use for treatment of rheumatic and arthritic diseases in Asian countries including China and Japan (Chen and Chen, 2001).

Unfortunately, laboratory research must make certain assumptions about the efficacy of herbal formulas. In the clinical setting, the doctor typically writes out a prescription based on a particular formula. Herbs are boiled for drink as the medicine. Thus, the actual medicine consumed contains untold thousands of chemical-impractical to analyze chemically. Paradoxically, this chemical complexity gives the herbs their value. The practical solution to this problem is to determine the bioactive constituent of each herb, and let that constituent represent the herb's potency.

In this herbal formula, certain bioactive chemicals have been previously identified. Among these are: sinomenine, curcumin, paeoniflorin, and paeonol. Pharmacological studies have showed that these single compounds have marked anti-inflammatory, analgesic, anti-arthritic, and immunosuppressive activities (Chou, 2003; Liu et al., 1994, 1996; Rafatullah et al., 1990; Tsai et al., 2001). In addition, Radix Aconiti lateralis Preparata contains very small amounts of the aconitum alkaloids, such as aconitine, mesaconitine, and hypaconitine, which are highly toxic but can be hydrolyzed to the more active but less toxic alkaloids, including benzoylaconine, benzoylmesaconine, and benzoylhypaconine that have significant anti-inflammatory and antinociceptive activities (Suzuki et al., 1994).

Recently, JCICM-6, a purified extract deriving from this Chinese herbal formula was obtained by using advanced pharmaceutical extraction and purification technologies. In the present work, the anti-inflammatory and analgesic effects of JCICM-6 in carrageenan-induced rat paw edema and carrageenan-evoked thermal hyperalgesia in paws of rats were studied. The possible effective mechanisms were investigated with regard to the COX-1, COX-2, and iNOS protein expression and the production of pro- and anti-inflammatory cytokines in paw tissues and exudates.

Materials and methods

Plant materials

Caulis Sinomenii was purchased form Zhong-Yue Herbal Pharmaceutical Union Company in China. Radix Aconiti lateralis Preparata and Rhizoma Curcumae longae, cultivated in the Good Agricultural Practice (GAP) bases at Jiangyou Country and Shuangliu Country of Chengdu City, respectively, were purchased in the wholesale market of Chengdu City, Shichuan Province, China. Radix Paeoniae albae and Cortex Moutan, cultivated at the GAP bases at Bozhou City and Tongning City of Anhui Province, respectively, were purchased in the wholesale market of Bozhou City, Anhui Province, China. Prof. Lai Xiao Ping from the Guangzhou University of Traditional Chinese Medicine authenticated all herbs. The authenticated voucher specimens are kept in the School of Chinese Medicine, the Hong Kong Baptist University.

Preparation of JCICM-6 from the herbal formula

JCICM-6 was prepared and its chemical consistency was verified with the methods as described before (Zhou et al., 2006). As described, the five herbs were reduced to coarse powder by pulverization. Caulis Sinomenii (25 g), Radix Aconiti lateralis Preparata (15 g), and Radix Paeoniae alba (30 g) were refluxed together with 80% ethanol and concentrated to produce Extract 1. Cortex Moutan (15 g) and Rhizoma Curcumae longae (15 g) were firstly extracted with supercritical [CO.sub.2] (21.7 L/h) to produce Extracts 2 and 3, respectively. Then, the residues of the two herbs after [CO.sub.2] extraction were separately refluxed with 80% ethanol and concentrated to produce Extracts 4 and 5, respectively. The mixture of Extracts 1, 4 and 5 was further purified with Amberlite XAD-7HP polymeric resin (Rohm and Haas Company, USA), and then combined with Extracts 2 and 3 to obtain JCICM-6. Three batches of JCICM-6 were produced and employed for the current experiments in the animal studies.

[FIGURE 1 OMITTED]

The average yield rate of JCICM-6 was 6.44 [+ or -] 0.35%, i.e., 100 g plant materials yielded 6.44 g extract. A good chemical consistency of JCICM-6 among the three batches was verified by HPLC fingerprint analysis (Fig. 1) with a correlation coefficient higher than 0.99. The HPLC fingerprint analysis was conducted with a Phenomenex ODS (250 x 4.6 mm I.D.; particle size 5 [micro]m; Phenomenex, Inc., USA) protected by a Security Guard Cartridge (C18, 4 x 3.0 mm I.D.; Phenomenex, Inc., USA) on an Agilent/HP 1100 series LC system (Hewlett Packard, CA, USA) consisting of a G1312A Bin Pump, a G1322A degasser, a G1315A Diode-Array Detector by using acetonitrile (A) and solvent (B) (containing 0.1% phosphoric acid, adjusted with triethylamine to pH 3.5) as mobile phase in a gradient elution matter (percentage of A: 0-25 min 8-23%, 25-30min 23-43%, 30-55%, 43-73%, 55-60 min 73-92%) at a flow-rate of 1.0ml/min. Detection was performed at a wavelength of 240 nm at room temperature.

Experimental animals

Wistar rats weighing 150-200 g were purchased from the Laboratory Animal Services Center, the Chinese University of Hong Kong, Hong Kong. The animals were acclimated for [greater than or equal to]1 week under 12 h light and 12 h dark cycle at room temperature of 22[degrees]C [+ or -] 1[degrees]C. Chow diet and water were provided ad libitum. Animal care and treatment procedures conformed to the Institutional Guidelines and Animal Ordinance (Department of Health, Hong Kong Special Administrative Region). Rats were fasted for 48 h before experiments.

Drugs and reagents

For all experiments, an aqueous solution of JCICM-6 was used at a concentration of 0.07 g/ml, as a stock solution. All dilutions were obtained from the stock solution using a dilution vehicle that consisted of 10% peanut oil, 10% Tween [R]80, and 80% distilled water. Other drugs and reagents, indomethacin, carrageenan and Tween[R]80, were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Induction and assessment of acute inflammation in rat hind paws by carrageenan

The assay was conducted as previously described by Winter et al. (1962). Oral administration was conducted with low (0.4 g/kg), middle (0.8 g/kg), and high (1.6 g/kg) doses of JCICM-6, the reference drug (Indomethacin, 10 mg/kg), or the vehicle, at 1 h prior to inflammation induction. At induction of the paw edema, each rat was injected with 0.1 ml freshly prepared carrageenan (1% w/v) in physiological saline (0.9% w/v NaCl) into subplantar tissues of the right hind paw. The left hind paws without injection were used as controls. The volumes (ml) of both hind paws of each animal were measured using a plethysmometer (plethysmometer 7150, UGO Basile, Italy) at lh before inflammation induction and the time intervals of 1, 2, 3, 4, 6 and 8 h after induction. The rates of increase in paw volume (paw edema) of the right hind paws of rats were calculated by the following equation: the increase rate (%) = (A-- B)/B x 100, where A represents the paw volumes at different time points after injection, and B represents the paw volume before injection. The mean values of the treated animals were compared with the mean values of the control animals, and results were analyzed using statistical methods.

Induction and assessment of carrageenan-evoked thermal hyperalgesia in paws of rats

Similar to that in the induction of acute inflammation by carrageenan, the rats were dosed and injected with 0.1 ml carrageenan (1% w/v) in physiological saline (0.9% w/v NaCl). Hyperalgesia was assessed by placing the hind paw above a light source and measuring the paw-withdrawal latency with a non-noxious heat stimulus, using a commercially available device (Plantar Test, IITC Life Science, CA, USA) as previously described by Hargreaves et al. (1988). The light source was set to 50. Paw-withdrawal latencies were evaluated every 60 min for 240 min, starting 60 min after carrageenan injection and the baseline latencies were assessed before carrageenan injection. The values of withdrawal latency of the treated animals were compared with the values of the control animals, and results were analyzed using statistical methods.

Western blot analysis of iNOS, COX-1, and COX-2 protein expressions

Paw soft tissues were removed from individual rats and homogenized in RIPA buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF), 10 [micro]g/ml aprotinin, 1 [micro]M pepstatin, and 10 [micro]M leupeptin. The homogenates were centrifuged at 12,000 g for 20 min, and 50 [micro]g of protein from the supernatants was then separated on 8% sodium dodecylsulphate-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Milipore, MA, USA). Following the transfer, the membranes were blocked overnight at 4[degrees]C with 5% skim milk in Tris-buffered saline (TBS, 20 mM Tris, 500 mM NaCl, pH 7.5), and then incubated with rabbit polyclonal anti-COX-2 (3 [micro]g/ml, Lab Vision, Fremont, CA, USA), mouse monoclonal anti-COX-1 (5 [micro]g/ml, Cayman, Ann Arbor, MI, USA), or mouse monoclonal anti-iNOS (Sigma Chemical Co., St. Louis, MO, USA) in TBS for 1 h at room temperature. The membranes were washed six times with Tris-buffered saline-Tween (TBST, 20 mM Tris, 500 mM NaCl, pH 7.5, 0.1% Tween 20). The membranes were then incubated with 1:10,000 dilution anti-rabbit (Zymed, South San Francisco, CA, USA) or anti-mouse IgG secondary antibodies (Zymed, South San Francisco, CA, USA) conjugated to horseradish peroxidase in 2% skim milk in TBST for 1 h at room temperature. The membranes were washed six times and the immunoreactive proteins were detected by enhanced chemiluminescence (ECL) using hyperfilm and ECL reagent (Amersham International plc., Buckinghamshire, UK). The relative optical density of bands was quantified by densitometric scanning of the Western blots.

Measurement of cytokines in paw tissue exudates

To obtain paw tissue exudates, rats were sacrificed by diethyl ether asphyxiation or by exsanguination. Then, each hind paw was cut at the level of the calcaneus bone and centrifuged at 400g for 15 min twice at 4[degrees]C to collect tissue exudates (edema fluid). The levels of IL-1[beta], IL-4, IL-6, IL-10, GM-CSF, IFN-[gamma], and TNF-[alpha] in the paw tissue exudates at 4 h after carrageenan injection were measured by using Bio-Plex rat cytokines assay following manufacturer's instructions. Briefly, beads coated with capture antibodies (5000 beads per cytokine) were incubated with premixed standards or sample supernatants (50 [micro]l) in 96-well filter plates. Plates were shaken for 30 s at 1000 rpm and then incubated at room temperature for 30 min at 300 rpm. After incubation, premixed detection antibodies (1 [micro]g/ml) were added, and plates were shaken and incubated as before. After washing, streptavidin-phycoerythrin (2 [micro]g/ml) was added to the wells, and the plates were incubated for 10 min at room temperature with shaking. After washing, beads were resuspended in 125 [micro]1 of Bio-Plex cytokine assay buffer and read by the Bio-Plex array reader. Data were analyzed with Bio-Plex Manager software version 2.0. Results were expressed as pg of cytokines per mg of protein.

Statistical analysis

All values are expressed as mean [+ or -]SEM. Statistical significance of the differences was assessed by ANOVA, followed by post hoc test with LSD methods. P values lower than 0.05 were considered significant.

[FIGURE 2 OMITTED]

Results

Inhibition of the paw edema of rats by oral treatment of JCICM-6

Fig. 2 shows the effect of JCICM-6 on inhibition of the acute paw edema in rats evoked by carrageenan injection into the subplantar tissues of the right hind paws. It can be seen that the maximum phlogistic response of carrageenan was observed at 4-6h after the injection in the control animals. The paw volumes from JClCM-6-treated animals with dosages of 0.8 and 1.6 g/ kg at 1, 2, 3, 4, 6, and 8 h after induction of paw edema showed marked decrease in comparison with the data of non-treated animals at the same time points, even lower than those of the indomethacin-treated animals. While in JCICM-6-treated animals with low dose of 0.4 g/kg, only at 1, 2, 3, 4, and 6 h after carrageenan injection were the paw volumes significantly lower than those of the control animals. These results indicate that the antiacute inflammatory effect of JCICM-6 in rats was dose dependent.

Inhibition of the hyperalgesic response to carrageenan in rats by oral treatment of JCICM-6

In the carrageenan-evoked thermal hyperalgesia experiments, the baseline latencies for all treated and non-treated groups before carrageenan injection were similar with basal paw-withdrawal latencies as 15.16 [+ or -] 0.32s. Injection of carrageenan into the subplantar tissues of the right hind paws of rats evoked a significant thermal hyperalgesia, showing as marked decreases of the paw-withdrawal latency that began from 1 h but reached the lowest value of 5.77 [+ or -] 3.91s at 4 h after carrageenan injection. Oral administration of JCICM-6 1 h before the carrageenan injection significantly inhibited the paw thermal hyperalgesia in a dose-dependent manner, showing that the paw-withdrawal latencies in all of the JCICM-6- and indomethacin-treated rats were significantly longer than those of the vehicle-treated animals at all of the four time points tested. Moreover, the analgesic potency of JCICM-6 at dosage of 1.6 g/kg was comparable to the effect of indomethacin (Fig. 3).

[FIGURE 3 OMITTED]

Inhibition of COX-1 and COX-2 protein expressions in the paw tissues of rats by treatment of JCICM-6

It can be seen in Fig. 4 that JCICM-6 dose-dependently attenuated the protein expression of COX-2 in carrageenan-injected paw tissues. Around 60%, 40%, and 30% reduction was achieved by treatment with JCICM-6 at dosages of 1.6, 0.8, and 0.4 g/kg, respectively. Examination of COX-1 protein expression showed that the level of expression was not suppressed by administration of JCICM-6 at any dosage. These results suggest that JCICM-6 may have a selective inhibitory effect on COX-2 protein expression in the paw edema tissues of rats. However, indomethacin at a dosage of 10 mg/kg demonstrated significant inhibition on both COX-2 and COX-1 protein expressions.

Inhibition of iNOS protein expression in the paw tissues of rats by treatment of JCICM-6

Fig. 5 shows that the protein expression of iNOS in carrageenan-injected paw tissues was dose-dependently attenuated in JCICM-6-treated rats. Only did JCICM-6 at 1.6 g/kg significantly inhibit the iNOS protein expression and around 50% reduction was achieved by the treatment of JCICM-6 at this dosage. Indomethacin at a dosage of 10 mg/kg also demonstrated significant inhibition in iNOS protein expression.

Inhibition of pro-inflammatory cytokine production by treatment of JCICM-6

The pro-inflammatory cytokines IL-1[beta], IL-6, GM-CSF, and TNF-[alpha] in the carrageenan-injected paw tissue exudates of rats were examined by Bio-Plex cytokine assay. As seen in Fig. 6, the level of IL-6 in the paw tissue exudates of JCICM-6-treated rats was significantly and dose-dependently reduced in comparison with that in the paw tissue exudates of the control animals. However, only had the high dose of JCICM-6 a significant inhibitory effect on the production of IL-1[beta] With regard to the production of TNF-[alpha] and GM-CSF, JCICM-6 demonstrated some degrees of inhibition but without dose dependence. Indomethacin showed a significant inhibition on the production of IL-6 and GM-CSF.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Augmentation of the anti-inflammatory cytokine production by treatment of JCICM-6

Fig. 7 shows that the level of IL-4 in the carrageenan-injected paw tissue exudates of the rats treated with JCICM-6 at dosage of 1.6 g/kg was significantly increased in comparison with that of the control animals, while the levels of IL-10 and IFN-[gamma] showed some degrees of inhibition or augmentation but without dose dependence. Meanwhile, the results demonstrated that indomethacin significantly inhibited the production of IFN-[gamma], but with little effect on the production of IL-4 and IL-10.

Discussion

Chinese herbs are commonly prescribed as combined herbal formulae to achieve sufficient treatment in complex conditions such as RA through possible mechanisms known as multiple components directing to multiple pathophysiological targets. Because individual Chinese herb per se already contains multiple components, let alone the combination of herbs, therefore the most important issue to be considered prior to the pharmacological study of an herbal extract coming from a formula is to keep the extract chemically consistent and identify the main chemical components inside, and thus to make the pharmacological results more reliable, repeatable, comparable, and most importantly meaningful. In this study, we utilized qualified plant materials from GAP bases and conducted rigorous in-process control to achieve this goal (Fig. 1). By using this extract with consistent quality, our current study showed how JCICM-6 inhibited experimental inflammation and underlying mechanism.

[FIGURE 6 OMITTED]

RA is a chronic, inflammatory autoimmune disorder characterized by joint pain and inflammation, pannus formation, even cartilage destruction, bone erosion and joint dysfunction in the late stages. It has a very complicated pathogenesis, and multiple pathological changes are implicated (Weyand, 2000). Various pro-inflammatory cytokines and inflammatory mediators are involved in the generation and development of RA (Smolen and Steiner, 2003). Theoretically, an ideal treatment or drug for RA should have multiple pharmacological potencies, including anti-inflammation, analgesia, immunomodulation, and cartilage and bone protection. The combinative remedy using herbal medicine may provide such multiple potencies. Our previous study showed that the crude extract of this anti-arthritic Chinese herbal formula exhibited significant suppressive effects on adjuvant-induced and collagen II-induced experimental arthritides of rats (Cai et al., 2005, 2006). In the current study, we demonstrated that treatment of JCICM-6 of the herbal formula purified by Amberlite XAD-7HP polymeric resin significantly inhibited carrageenan-induced rat paw edema and thermal hyperalgesia in paws of rats. These results indicate that JCICM-6 has a potent suppressive effect on acute inflammation and inflammation-mediated pain of joints, which are two of the most important targets of drugs in the treatment of inflammatory arthritic diseases.

In the past two decades, a number of botanical-derived medicines have been developed as anti-inflam-matory agents but only a few of them have been studied with the goal of elucidating the molecular mechanisms of their actions (Surh et al., 2001). To address this issue, we firstly evaluated the anti-inflammatory and analgesic properties of JCICM-6, and then determined the molecular mechanisms relevant to these actions, focusing on several key molecular targets in RA, including COX-2, iNOS, and cytokines.

[FIGURE 7 OMITTED]

As far as the COXs are concerned, it is widely accepted that COX-2 is the predominant cyclooxygenase isoform in all stages of inflammation, including facilitation of the production of pro-inflammatory prostanoids and of inflammatory prostaglandins (Vane and Botting, 1998). Overproduced prostaglandins can induce or potentiate inflammation (Harris et al., 2002), while selective inhibitors of COX-2 can suppress inflammatory conditions through inhibition of the inflammatory prostaglandins (Dannenberg et al., 2001). Salvemini et al. have proposed that NO stimulates COX activity in macrophages through direct interaction with active sites of the COX enzyme. Inhibition of NO production by the suppression of the enzyme activity of iNOS is one of the pathways for anti-inflammatory effect (Salvemini et al., 1993). Furthermore, they reported that NO produced by iNOS is involved in maintenance of the carrageenan-evoked inflammatory response, while peripheral or central administration of iNOS inhibitors could effectively inhibit carrageenan-induced hyperalgesia in rats (Salvemini et al., 1996). In the present study, we demonstrated that JCICM-6 of this anti-arthritic herbal formula can significantly suppress the de novo expressions of inducible NOS and COX-2 enzymes, but it cannot inhibit the COX-1 enzyme, which is believed to be one of the mechanisms by which JCICM-6 reduces carrageenan-induced paw edema of rats.

It is widely accepted that pro-inflammatory cytokines are the classic potent mediators of inflammatory conditions and inflammatory pain both in experimental animal models and in humans. Our current studies on carrageenan-induced rat hind paw edema, a model frequently employed to study inflammation, provides evidence that carrageenan can stimulate the release of TNF-[alpha], which, in turn induces release of IL-1[beta] and IL-6 thus stimulating the production of COX products (Cunha et al., 1992). The release of IL-1[beta] by the stimulation of TNF-[alpha] can further induce the expression of iNOS, while IL-6 is an intermediate in this process. This scenario has been postulated in cytokine-mediated pyrogenic responses in rats in which pro-inflammatory stimuli induce the production of IL-6 in periphery sites (secondary to the production of TNF-[alpha]) and that IL-6 then induces production of IL-1 in the brain (Rothwell, 1991). In addition, GM-CSF is thought to be able to contribute to the development of human RA because in vitro it stimulates the proliferation of synoviocytes while in vivo it exacerbates symptoms of acute inflammatory arthritis (Bischof et al., 2000). More convincing evidence is the observation that treatment with antibodies directed against GM-CSF can reduce inflammation and cartilage destruction in arthritic mice (Cook et al., 2001); while the GM-CSF-deficient mice were partially protected from collagen-induced arthritis (Campbell et al., 1998).

In contrast, some cytokines demonstrate antagonistic action in the process of inflammation. For instance, IL-4, a T cell-derived cytokine, appears to play a role in coordinating anti-inflammatory effects by suppressing IL-1, TNF-[alpha], and GM-CSF production and up-regulating the production of IL-1 receptor antagonist (IL-1ra) (Fenton et al., 1992). IL-10, another anti-inflammatory cytokine produced by Th2 lymphocytes and monocytes, exerts an anti-inflammatory effect through inhibition of the induction of nitric oxide synthase, the COX-2 expression and the production of inflammatory cytokines produced by murine Th1 lymphocytes (Fiorentino et al., 1989). Similar to IL-4, IL-10 can also induce the formation of IL-1 receptor antagonist (lL-lra) (Jenkins et al., 1994). Furthermore, injection of monoclonal antibodies of mouse IL-4 and IL-10 can potentiate the carrageenan- and TNF-[alpha]-evoked inflammatory pain (Cunha et al., 1999), suggesting that IL-4 and IL-10 may have therapeutic potentials in acute and chronic inflammatory conditions. In addition, IFN-[gamma] displays protective anti-inflammatory function, including suppression of the spontaneous intracellular production of IL-1 of macrophages in synovial fluid of RA patients (Schindler et al., 1990). Treatment with IFN-[IFN-[gamma] was associated with significant reduction of leukocyte influx into the synovium and the amelioration of synovial hyperplasia and erosion of RA patients. Notably, some beneficial effects of IFN-[gamma] administration have also been observed in an RA clinical trial (Wahl et al., 1991). In the current study, we demonstrated that JCICM-6 significantly inhibited the production of pro-inflammatory cytokines of IL-6 and IL-1 and augmented the production of IL-4 in carrageenan-injected paw tissue exedates of rats, thus tending to "injected paw tissue exudates of rats, thus tending to "balance" the pro- and anti-inflammatory cytokines in tissues, and this may contribute to - or explain - the anti-inflammatory effect of JCICM-6. However, indomethacin showed only inhibition of the pro-inflammatory cytokines without augmentation of the anti-inflammatory cytokines. These results indicate that the pharmacological properties and the effective mechanisms of JCICM-6 and indomethacin might be different, although both of them demonstrated significant inhibition of carrageenan-induced rat paws edema. With regard to the analgesic mechanism of JCICM-6, it is considered to be also associated with the inhibition of pro-inflammatory cytokines in carragee-nan-evoked thermal hyperalgesia in rats. In addition, as for the inhibition of the production of GM-CSF, it was shown that JCICM-6 was more effective at a low dosage (0.4g/kg) than at higher doses (0.8 and 1.6 g/kg). Similar phenomena were also found in the studies of classic NSAIDs as well as of COX-2 selective inhibitors. Indeed, it is reported that some of them, such as flurbiprofen and naproxen, did not decrease but rather increase the release of GM-CSF due to a reduction of COX activity (Calatayud et al., 2001).

In conclusion, the anti-inflammatory and anti-nociceptive effects of JCICM-6, an Amberlite XAD-7HP resin purified extract from an anti-arthritic Chinese herbal formula, have been demonstrated in experimental models of carrageenan-induced paw edema and thermal hyperalgesia in paws of rats for the first time. The molecular mechanisms by which JCICM-6 exerts its anti-inflammatory effects are believed to involve the inhibition of the production cascade of pro-inflamatory cytokines IL-6 and IL-1 and suppression of expression of the proteins iNOS and COX-2, as well as augmentation of the production of anti-inflammatory cytokine IL-4. These results suggest two effective therapeutic strategies in treating arthritis: one would be to inhibit the production of the pro-inflammatory cytokines IL-6 and IL-1, a second would be to augment the production of the anti-inflammatory cytokine IL-4. All those results indicate that the JCICM-6, the purified extract of this particular herbal formula, would be a valuable candidate for further investigation as a novel anti-arthritic botanical drug.

Acknowledgments

This research was funded by the Hong Kong Jockey Club Charities Trust (JCICM-6-02) and the Faculty Research Grants of Hong Kong Baptist University (FRG/05-06/II-85). The authors thank Drs. Zhong Qiu Liu and Xiong Cai, Mr. Zhi Xiong Feng for their technical supports and Dr. Martha Dahlen for her professional English editing of this paper.

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Y.F. Wong (a), (l), H. Zhou (a), (l), J.R. Wang (a), Y. Xie (a), H.X. Xu (b), L. Liu (a), *

(a) School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China

(b) Hong Kong Jockey Club Institute of Chinese Medicine Limited, 7IF, 2 Science Park West Avenue, Hong Kong Science Park, Pak Shek Kok, Shatin, New Territories, Hong Kong, China

* Corresponding author. Tel.: +8523411 2457; fax: +85234112461 E-mail address: liulianguriikbu.edu.hk (L. Liu).

(1) The authors share the first authorship.

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Author:Wong, Y.F.; Zhou, H.; Wang, J.R.; Xie, Y.; Xu, H.X.; Liu, L.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jun 1, 2008
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