Therapeutic Effects of Water Extract of Arisaema Erubescens Tubers on Type II Collagen-induced Arthritis in Rats

Purpose: To investigate the anti-arthritic activity of the water extract of Rhizoma Arisaematis (WERA) using collagen II (CII)-induced arthritis (CIA) rat model. Methods: CIA was induced in male Sprague-Dawley rat by intra-dermal injection of bovine collagen type II (C II) in Freund’s complete adjuvant (cFA). The rats were treated daily for 21 consecutive days with WERA at doses of 100, 200, and 400 mg/kg. Methotrexate (MTX) was used as positive control, and administered at a dose of 3 mg/kg intraperitoneally in two-weekly cycles for 3 weeks. Severity of arthritis was evaluated by arthritic scores, including paw swelling, arthritic score, body weight, thymus index and spleen index. The levels of IL- 1β, IL -6, IL-10 and TNF- α in serum were also measured. Results: The results revealed that WERA significantly inhibited paw edema (p-value < 0.01), decreased arthritis scores (p-value < 0.01) and spleen index (p-value < 0.05), and alleviated the weight loss of CIA rats. Furthermore, the pro-inflammatory cytokines of TNF- α (19.3%, 60.5% and 73.9%, respectively), IL 1β (7.9%, 41.1% and 52.7%, respectively) and IL -6 (26.6%, 48.0% and 72.2%, respectively) were remarkably attenuated in serum of all WERA-treated rats, however, IL-10 (72.4% and 39.1%, respectively) was markedly increased at doses of 200 and 400 mg/kg of WERA. Conclusion: The results demonstrate that WERA exerts therapeutic effects in collagen--induced arthritis of rats by decreasing the levels of TNF- α, IL - 1β, IL -6 and IL-10 in serum, and therefore may be an effective candidate drug for the treatment of human rheumatoid arthritis.


INTRODUCTION
Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation and hyperplasia, cartilage and bone destruction, chronic joint destruction with bone erosion in the extremities (especially the fingers) [1].In addition, RA may also affect multiple organs and tissues, including the heart, lung, and nervous system.It's reported that RA occurs throughout the world with an average morbidity of 1 % and its prevalence is three times more in females than in males [2].It can rapidly progress into multi-system inflammation with irreversible joint damage, and causing premature mortality, disability and compromised quality of life in the industrialized and developing world [3,4].
Currently, the major RA medications used in clinical mainly include these categories: disease modifying anti-rheumatic drugs (DEMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), steroid hormone and biologics (TNF-α antibody and the decoy TNF-α receptor,) [5].Long-term use of these drugs easily leads to immune system weakness, bone marrow suppression, liver and kidney impairment, gastrointestinal discomfort and cartilage degeneration [6].Thus, more effective drugs with low toxicity are needed.In recent years, increasing investigations reported that traditional Chinese medicines (TCMs) has provided effective avenues for discovering new drugs [7,8].In addition, most RA patients are likely to use plant-derived agents for treating RA [9,10].It might be a promising strategy to find new RA drugs from commonly used TCMs.
The present study was aimed to investigate the anti-arthritic effect of the water extract of Rhizoma Arisaematis (WERA) and explore its potential mechanisms.

Bovine collagen type II (CII) and Complete
Freund's Adjuvant (CFA) were purchased from Sigma (Sigma Chemical Co., USA) Methotrexate (MTX) was purchased from Shanghai Sine Pharmaceutical Co., Ltd.(Shanghai, China).TNF-α, IL-1β, IL-6 and IL-10 ELISA kits were purchased from the R&D system (R&D Systems, USA).All the other chemicals and biochemicals used were of the highest grade available.
In the present study, the following instruments were used:

HPLC analysis of WERA
The HPLC separation was performed on the Agilent 1260 HPLC system (Agilent Technologies) with a C18 chromatographic column (250 mm × 4.6 mm, i.d. 5 μm), equipped with a quaternary pump, and an automatic thermostatic column compartment.Separation was performed using gradient elution [H 2 O (A) / methanol (B)] gradient at a flow rate of 0.8 mL/min.Samples were analyzed by using a gradient program as follows: run was commenced with 5 % B, linear gradient to 10 % B within 6 min, followed by linear gradient to 20 % B in 30 min until to 50 min, and finally linear gradient to 30 % B in 60 min..The sample injection volume was 10 μL, the detection wavelength was set at 260 nm, and the column temperature was set at 30 ºC.

Animals and grouping
Male SD rats (weighing 160 -180 g) and KM mice (weighing 18 -22 g) were purchased from Chengdu Da-Shuo Lab Animal, LTD (Chengdu, China).They were kept in an environment with controlled temperature (24 -26 °C) and photoperiod (12:12 h) light-dark cycle.Animals were given standard commercial rat chow and water.Rats were acclimatized for 1 week before experiments and then randomly assigned to 6 groups: the normal group, the RA group, MTX group (3 mg/kg, twice/week), WERA low-dose group (100 mg/kg/d), WERA middle-dose group (100 mg/kg/d) and WERA high-dose group (400 mg/kg/d), and each group consisted 10 animals.Animal experiments were conducted in accordance with current ethical regulations for animal care and use at Chengdu University of Traditional Chinese Medicine.

Acute toxicity of WERA
An acute toxicity study of the WERA was carried out using OECD guidelines [19].The WERA was suspended in water with 0.5 % w/v sodium carboxyl methyl cellulose (Na-CMC) in the doses of 5 mg/kg, 50 mg/kg, 500 mg/kg, 2 g/kg, 5 g/kg and 10 g/kg body weight which were orally administered to the mice.The mortality rates of the mice were observed and recorded within 24 h.

Collagen-induced arthritis (CIA)
CIA model was established according to the methods described previously [20].Collagen type II (2 mg/mL in 0.05 M acetic acid) was emulsified with an equal volume of complete Freund's adjuvant and a final concentration was 1 mg/mL.Rats were injected subcutaneously at the tail root, hind-paw and three places at back (dorsonuchal, located towards the tail about twothirds and one-thirds of the way down its back) with the collagen emulsion (0.5 mg per rat).After one week, a booster injection of the collagen emulsion (0.5 mg per rat) was given at the same places.From 1 to 21 day after the first injected, the WERA group and MTX group were treated with WERA and MTX orally, the normal group and RA group were given an equal volume of the CMC-Na at the same time.

Evaluation of the arthritic score
Arthritis severity of the rats were assessed using the method described previously [21,22].Paws were examined and graded for severity and loci of erythema, swelling and induration using a 5point scale: 0=no signs of disease, 1=signs involving the ankle/wrist, 2=signs involving the ankle plus tarsal of the hind paw and/or wrist plus carpals of the forepaw, 3=signs extending to the metatarsals or metacarpals, and 4=severe disease involving the entire hind or fore paw.The maximum arthritic score per rat was set at 16 (4 points × 4 paws).

Determination of index of thymus and spleen
Treatment of 21 days, the rats were sacrificed under anesthesia (pentobarbital sodium, 40 mg/kg, i.p.).The thymus and spleen were then promptly removed and weighed.The index of thymus and spleen were expressed as the ratio, was divided body weight by thymus and spleen wet weight (mg/g), respectively [23].

Determination of pro-inflammatory cytokines in serum
Rat blood sample was obtained and allowed to clot for 1h at room temperature.Subsequently, serum was recovered and frozen at -20 °C prior to analysis [24].The contents of TNF-α, IL-1β, IL-6 and IL-10 in serum were determined by commercial ELISA kits according to the manufacturer's protocol.

Statistical analysis
Data were presented as Mean ± SD, and analyzed using SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA).Differences between experimental groups were tested using one-way ANOVA, and p < 0.05 was considered statistically significant.

HPLC profile of WERA
In the results of our investigation, three major constituents in the HPLC fingerprint of WERA were identified and recognized as inosine (retention time: 17.237 min), guanosine (retention time: 19.108 min) and adenosine (retention time: 37.762 min) and computed the content was 0.008, 0.051, and 0.042 mg/g, respectively (Fig 1).

Acute toxicity of WERA
According to advance with the OECD guidelines, the test animals were observed individually.In our toxicity study, neither death nor any abnormal neuro behaviors were observed during our test period, indicating that it is safe for WERA administration within the dose of 10 g/kg.

Effect of WERA on paw swelling, arthritis score and weight growth
No redness or joint swelling was observed in normal group (Figure 2 and 3A).The rats injected collagen exhibited peripheral paw edema in the first 24 h.With 21 days treatment, the paw edema in WERA and MTX treated animals were almost disappeared In our results, WERA treatment significantly reduced the paw edema (p-value < 0.05) compared with rats in RA group during 10 to 21 days after immunization (Figures 2, 3B, D, E and F).MTX (3 mg/kg) also showed significant (p < 0.05) inhibitory effect on the paw edema (Figures 2 and 3C).
WERA at both 200 and 400 mg/kg doses significantly decrease arthritic scores of rats compared with the RA model group from the 13th day.WERA at 100 mg/kg dose decreased arthritic scores from day 13 to 18, however it did not show significant difference with RA model group on day 21.RA rats showed marked weight loss compared with normal group on 5th day (p-value < 0.05).During the next two weeks, the results showed the weight loss of RA rats were reversed by administration of WERA at 200, 400 and 100 mg/kg (Figure 5).Although the weight gain in WERA-treated rats was lower than it had been initially, it was still much higher than that of MTX group rats.When compared with the normal group, animals treated with WERA did not show significant weight loss during the experiment.

Effect of WERA on the index of thymus and spleen
As shown in Fig. 6, both thymus index and spleen index in RA group were markedly increased compared with the normal group.The indices of spleen of the WERA treated animals were significantly decreased, and this situation can be reversed by both MTX (3 mg/kg) and WERA (100, 200 and 400 mg/kg).In addition, MTX (3 mg/kg) and WERA (400 mg/kg) also decreased thymus index.

DISCUSSION
In the present study, we have confirmed for the first time that WERA attenuate joint swelling of RA rats induced by type II collagen, and WERA  Collagen-induced arthritis in experimental animal model for RA in which the symptoms similar to human RA from genetic linkage to pathology and clinic manifestations.Thus, collagen-induced arthritis model is widely used to explore the pathogenesis and therapy principle of RA [26].Therefore, arthritis model of rats induced by collagen was selected in our present investigation to study the anti-arthritic activity of WERA in vivo.That elimination of clinical symptoms of patients is a crucial index for evaluating the therapeutic effects of RA treatment [27].Arthritic scores and paw swelling have usually been used for measurement of the anti-arthritic activity.Our study showed that orally treated WERA significantly relieved joint swelling and redness at the dose of 100, 200 and 400 mg/kg.Treatment with WERA can decreased the swelling paw volume and arthritic scores.In addition, WERA can also suppress the weight loss in CII-induced RA rats.
It has been reported that pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 are documented as being critically important in development of RA [28,29].TNF-α has been considered to be on top of a cytokine cascade, which can increase the releases of IL-6 and IL-1β, and stimulate cartilage matrix degradation.In addition, IL-1β and IL-6 have also been reported to contribute to the development of arthritis [30,31].IL-10 played a vitally important role in protecting the integrality of joint tissues; inhibit cytokine production, and inhibit IL-18 mRNA expression in pathological process of RA [32,33].In this study, WERA significantly decreased the serum levels of TNF-α, IL-1β and IL-6, whereas the level of IL-10 was markedly increased at doses of 200 and 400 mg/kg.Thus, the mechanism for the therapeutic effect of WERA on RA may be involved in both down-regulating the level of TNF-α, IL-1β and IL-6 and upregulating the expression of IL-10.
Thymus and spleen are the two major organs of the body immune organs, and the relative weights of thymus and spleen are usually used to evaluate the drugs immunoregulatory activity.After administration of WERA, the RA rats exhibited a marked reduction of index of spleen and thymus (400 mg/kg).This result indicated that WERA might affect immune function on RA rats.
By HPLC fingerprint analysis, we found that the main components of WERA were nucleosides.Nucleosides were previously found to be immunostimulatory activity agents [34], therefore, according to our results, we can deduct that the nucleosides in WERA, may be responsible for the observed anti-arthritic activity.

CONCLUSION
The results of the present study suggest that the water extract of Rhizoma arisaematis is therapeutically effective in CII-induced arthritis in rats.Its effects may be attributed to its ability to decrease spleen index, down-regulate TNF-α, IL-1β and IL-6 levels, and up-regulate concentration of IL-10 levels, suggesting that the plant can potentially be developed for the treatment or prevention of RA.

Figure 3 :
Figure 3: Representative paws of rats photographed on day 21.A-F represent the rats in normal group, RA model group, WERA at 100 mg/kg, 200 mg/kg, 400 mg/kg, and MTX groups, respectively

Figure 6 :
Figure 6: Effect of WERA on the indexs of thymus and spleen.Data are expressed as mean ± SD (n = 10); * p < 0.05, ** p < 0.01, compared to control group