Anti-arthritic activity of ethanol extract of Claoxylon indicum on Freund’s complete adjuvant-induced arthritis in mice

Male Kunming mice weighing 25–30 g were purchased from Slac Laboratory Animal Co. Ltd. (Shanghai, China). All the procedures were approved by Animal Ethical Council of Nanjing University of Chinese Medicine. The mice were maintained under laboratory conditions at 25 °C under a normal 12 h/12 h light/dark cycle with humidity of 55% and fed with food and water ad libitum. The mice were allowed 7 days to adapt to the laboratory environment before experiments.

Samples of the dried root of CI were collected from Guangxi Province in October 2013 and identified by Prof. Jianwei Chen (Nanjing University of Chinese Medicine, Jiangsu, China). A voucher specimen was deposited at the herbarium of the Nanjing University of Chinese Medicine College of Pharmacy, Jiangsu (No. 102). The dried stem and root of CI (6.5 kg) was extracted by reflux method thrice with 95% ethanol. The ethanol filtrate was concentrated under reduced pressure at 55 °C by a vacuum rotary evaporator (RE-52A, Shanghai Yarong, China) and further dried in a vacuum drying oven (DZF-6090, Shanghai Jinghong, China) to yield a solid mass of weight 450 g (yield: 6.9%). The dried extract, CIE was freshly emulsified with 0.5% sodium carboxyl methyl cellulose (CMC-Na) and prepared with normal saline before use.

All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals in China [Permit: SCXK (Shanghai) 2007–0005], and animal welfare and experimental procedures were strictly in accordance with the guide for the care and use of laboratory animals (National Research Council of USA, 1996). Adjuvant arthritis was induced on day 0 of the experiment by a single subcutaneous injection of 0.02 mL of complete Freund’s adjuvant (CFA) [containing 5.0 mg of dry, heat-killed Mycobacterium tuberculosis (strain H37Ra) per 1.0 mL sterile, non-metabolizable oils, Sigma-Aldrich, USA] into the plantar surface of right hind paw of the mice [15]. Equal amount of saline was injected to the left paw. This arthritis model, called adjuvant-induced arthritis (AIA) mice, has been widely used as a model of RA [16]. Another induction by an injection with 0.02 mL CFA in the base of tail was carried out 7 days after the first induction. 0.02 mL saline was injected in both paws and tail of vehicle control animals (10 normal mice). Since the second injection, AIA mice were divided into 3 groups [AIA model, CIE (0.4 g/kg) and CIE (0.8 g/kg) groups] randomly (with 10 mice in each group), and then the mice of CIE groups were received 0.4 and 0.8 g/kg of CIE by gastric intubation daily for 28 days. The mice of AIA model and vehicle control groups were given 0.1 mL/10 g of 0.5% CMC-Na instead.

All results were presented as mean ± S.D. Data were analyzed by student’s t-test, two-way or one-way ANOVA followed by Dunnett’s t-test. Results were regarded statistically significant if the P-values were less than 0.05.

There was no sign of toxicity and mortality in mice treated with all tested CIE. It was observed that the body weight of mice increased gradually and the difference was not obvious in all test groups. As shown in Fig. 1a, the clinical scores of arthritis in AIA mice were dramatically increased compared to vehicle control mice, and were reduced by the treatment with CIE. The index of hind paw oedema (Fig. 1b) of CIE treated (0.4 g/kg and 0.8 g/kg) mice was lower (P < 0.05) than that of AIA mice significantly.

Compared to the vehicle control mice, the spleen index of the AIA mice was increased. Administration with CIE suppressed the hyperplasia dramatically. CIE treated (0.4 g/kg and 0.8 g/kg) mice showed a marked decrease (P < 0.05) of spleen index compared to that of the AIA mice (Fig. 1c).

RA is well-characterized with synovial hyperplasia, pannus formation, cartilage and bone destruction in the joint. On day 28 after the primary treatment, the hind-paw joints of the mice were examined. As shown in Fig. 2b, in the AIA mice, the joints showed the infiltration of inflammatory cells, synovial hyperplasia, cartilage erosion and narrow joint space. Compared to vehicle control mice, morphological change score of AIA model mice was increased (P < 0.001) significantly (Fig. 2e). There was a statistically significant reduction (P < 0.05) in the morphological change score in the mice treated with 0.8 g/kg CIE, while a tendency towards a reduction in 0.4 g/kg CIE-treated mice with no significant difference.

MDA and ALP levels were significantly higher (P < 0.01) in AIA model mice. The levels of MDA and ALP were significantly decreased (P < 0.01) compared with AIA model and CIE-treated mice (Fig. 3).

IL-1β and TNF-α are reported to play critical roles in the pathogenesis of RA [21, 22]. Therefore, we investigated whether the therapeutic effects of CIE treatment were associated with changes in the frequencies of cells producing IL-1β or TNF-α in the arthritic joints by immunohistochemical staining. IL-1β- and TNF-α-positive cells were found distributed throughout the synovium, especially in synovial sublining regions (Fig. 4a-h). Compared to vehicle control group, the immunohistochemistry analysis showed that the expression of IL-1β and TNF-α was increased (P < 0.01) in AIA model mice (Fig. 4i). In addition, compared to AIA model group mice, there was a significant reduction (P < 0.01) of IL-1β and TNF-α expression in 0.8 g/kg CIE-treated mice.