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.
Plant materials and preparation of extract
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.
Adjuvant-induced arthritis in mice and administration
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 . 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 . 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.
Measurement of paw edema
Toxic symptoms, such as irritability, drowsiness, dyspnoea, ataxia and reflexes, and death during administration period in mice were observed and recorded. The disease recovery was evaluated by hind paws oedema index after sacrifice by cervical dislocation. The right hind paw of each mouse was dissected and weighed. The index of hind paws oedema was expressed as the ratio (mg/g) of hind paw weight versus body weight [9, 17, 18].
The severity of arthritis was assessed by three independent observers. The mice were observed periodically for the verity of joint inflammation every 3 days after the second induction until sacrificed. The severity of arthritis was graded on a scale of 0–4 with the following criteria : 0 = no edema or swelling, 1 = slight edema and limited erythema, 2 = slight edema and erythema from the ankle to the tarsal bone, 3 = moderate edema and erythema from the ankle to the tarsal bone, and 4 = edema and erythema from the ankle to the entire leg. The arthritis score for each mouse was the sum of severity in all four limbs (maximum 16 points for individual mice).
Measurement of MDA and ALP
The whole blood of mice in all the groups was collected through fossa orbitalis vein at day 28 after treatments. The serum was separated and divided into aliquots at 4 °C. The levels of malondialdehyde (MDA) and alkaline phosphatase (ALP) in serum were investigated by commercially available colorimetric assay kits (Jian Cheng Bioengineering Institute, China) according to the manufacturer instructions.
Spleen index determination
The mice of all the groups were sacrificed at day 28 after treatments, and the spleen were dissected and weighed. The index of spleen was calculated as the ratio (mg/g) of spleen wet weight versus body weight .
The specimens of knee joints were taken and fixed in 10% formalin and analyzed for pathological changes with hematoxylin and eosin. The severity of the arthritis was evaluated based on the changes in pathology, inflammation and erosion . Morphological changes was graded on a scale of 0–4 with the following criteria: 0 = normal cartilage, articular cavity and synovial tissues, and no subcutaneous tissue inflammation, 1 = normal cartilage and articular cavity, slight synovial tissues thickening and inflammation, and slight subcutaneous tissue inflammation, 2 = normal cartilage and articular cavity, mild synovial tissues thickening and inflammation, and mild subcutaneous tissue inflammation, 3 = normal cartilage and articular cavity, moderate synovial tissues thickening and inflammation, and moderate subcutaneous tissue inflammation, 4 = normal articular cavity, damage cartilage, narrow joint space, severe synovial tissues thickening and inflammation, and severe subcutaneous tissue inflammation.
IL-1β and TNF-α expression by immunohistochemistry
Immunohistochemical detection of interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) of the specimens of knee joints was performed by standard immunohistochemical techniques with primary antibodies against IL-1β and TNF-α (Bioworld, Shanghai, China), and horseradish peroxidase (HRP)-Polymer anti-Mouse/Rabbit IHC Kit (Mainxin-Bio, Fuzhou, China). Average density was measured with Motic Image Advanced 3.2 software (Motic China Group Co., LTD.).
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.