A total of 30 male Wistar rats (weight, 100–120 g; age, 4 weeks), were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China). Rats were housed in temperature-controlled animal cages at 25±1°C with a relative humidity of 55±5%, under a standard 12-h light/dark cycle (8:00 a.m.-8:00 p.m.), with free access to food and water. All experiments were performed between 8:00 a.m. and 4:00 p.m. All animals were treated in accordance with the regulations of the State Science and Technology Commission of China for the care and use of laboratory animals (State Science and Technology Commission order no. 2, 1988), and the protocols were approved by the Ethics Committee of Zhejiang Chinese Medical University (Hangzhou, China).

Bovine type II collagen (CII; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved overnight in 0.1 mol/l acetic acid (2 mg/ml) at 4°C and emulsified with incomplete Freund's adjuvant (IFA; Sigma-Aldrich; Merck KGaA) to a final concentration of 1 mg/ml. Rats were anesthetized with 10% chloral hydrate (350 mg/kg, intraperitoneal) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and placed on a heated small animal operating table (Harvard Apparatus, Cambridge, USA) in a prone position. No rats exhibited signs of peritonitis. Bovine CII (2 mg/kg) was intradermally injected into six sites at the base of the tail and back of the rats.

In the experiment, a CIA model was successfully established in total of 22 rats. These 22 CIA rats were randomly divided into the CIA + saline group and CIA + β-endorphin group (n=11 per group). β-Endorphin (Bachem, Bubendorf, Switzerland) was dissolved in saline at a concentration of 5 nmol/ml. Rats were intraperitoneally injected with 1 ml β-endorphin once every other day from day 18 after the injection of CII until day 28. The rats in the CIA + saline group were intraperitoneally injected with 1 ml saline.

The TFL was assessed to determine thermal hyperalgesia using a tail-flick apparatus (Ugo Basile, Comerio, Italy). Rats were immobilized except for free tail movement. Heat from an infrared source was administered to the tail with a radiation intensity of 30 mW/cm². The TFL was defined as the time of heat exposure until withdrawal of the tail, which was recorded by a single blinded observer. In order to avoid tissue damage, a cut-off time of 10 sec was implemented, with this duration being defined as representing the maximum analgesic effect. The average TFL was obtained from three consecutive trials with an interval of 3 min performed on 1/3 of the tail at the distal end.

Paw arthritic signs characterized by edema and erythema were inspected daily following the CII/IFA injection. To evaluate the incidence and severity of arthritis, lesions on all four paws of each rat were graded by using an arthritic scoring system (13). Lesions were graded using a scale from 0 to 4 according to the extent of edema and erythema of the periarticular tissues; 16 was the potential maximum of the combined arthritic scores per animal. The severity scores were defined as follows: 0, no evidence of erythema and swelling; 1, erythema and mild swelling confined to the mid-foot (tarsals) or ankle joint; 2, erythema and mild swelling extending from the ankle to the foot; 3, erythema and moderate swelling extending from the ankle to the metatarsal joints; and 4, erythema and severe swelling encompassing the ankle, foot and digits.

The paw volume was measured by a water displacement plethysmometer (Ugo Basile). The hind paws were respectively immersed in an electrolyte solution up to the boundary between the hairy and non-hairy skin, and the volume displacement in the chamber was determined electronically. The percentage of the paw swelling was calculated by the following formula: % Swelling=(Vt-V0)/V0×100, where Vt represents the paw volume after saline/β-endorphin injection and V0 represents the basal paw volume.

Rats were anesthetized with 10% (w/v) chloral hydrate at a dose of 350 mg/kg (intraperitoneal) on day 28 after the injection of CII, the synovial tissues of rat knees were isolated and removed and subsequently frozen in liquid nitrogen and immediately stored at −80°C. No rats exhibited signs of peritonitis. After homogenization of synovial tissue, total RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and purified with the Prime Script^® RT reagent Kit with gDNA Eraser (cat. no. PR0474A; Takara Bio Inc., Tokyo, Japan). Total RNA (1 µg) was then reverse-transcribed into complementary DNA using the abovementioned kit. Gene expression of TNF-α, IL-1β and IL-6 was analyzed by real-time qPCR using the CFX96™ real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH served as an internal control for detecting the expression of target genes. The primers for TNF-α, IL-1β, IL-6 and GAPDH were designed with Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) (primer sequences are listed in Table I) and were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The PCR mixture of 20 µl contained 10 µl SsoFast EvaGreen supermix (Bio-Rad Laboratories, Inc.), 0.3 µl sense and anti-sense primers (400 nM), 1 µl template complementary DNA and 8.4 µl RNase/DNase-free water. The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 min, followed by 40 cycles of 10 sec at 95°C and 30 sec at 61.9°C. Each sample was measured in triplicate. The generation of specific PCR products was confirmed by melting curve and gel analysis. The expression ratio was calculated using the 2^−∆∆Cq method (14).

Samples were pulverized in liquid nitrogen, resolved in cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing protease inhibitor cocktail (10% v/v; Bio Basic Inc., Markham, ON, Canada), sonicated on ice (5×5 sec), stored for 1 h at 4°C, and then centrifuged at 13,201 × g for 30 min at 4°C to obtain the protein extract. TNF-α, IL-1β and IL-6 were determined using a Rat TNF-α ELISA Kit (cat. no. ELR-TNFalpha-001), Rat IL-1β ELISA Kit (cat. no. ELR-IL1beta-001) and Rat IL-6 ELISA Kit (cat. no. ELR-IL6-001; all Raybiotech Corp., Atlanta, GA, USA), respectively.

All values are expressed as mean ± standard error of the mean, and differences between groups were analyzed using the independent-samples t-test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were conducted using SPSS version 18.0 (SPSS Inc., Chicago, IL, USA).

The mean TFL in the CIA + saline group and CIA + β-endorphin group is presented in Fig. 1. No significant difference in the TFL between the two groups was identified prior to β-endorphin injection. The TFL was significantly increased in the CIA + β-endorphin group when compared with that in the CIA + saline group on days 22 and 28 (P<0.01).

As presented in Fig. 2, no significant difference in the arthritis index was present between the CIA + saline group and the CIA + β-endorphin group on day 18 after the injection of CII prior to β-endorphin treatment. The arthritis index was significantly reduced in the CIA + β-endorphin group when compared with that in the CIA + saline group on days 22 and 28 (P<0.05 and P<0.01, respectively).

The results on the percentage of paw swelling are displayed in Fig. 3. Prior to β-endorphin treatment (day 18), no significant difference in paw swelling was noted between the CIA + saline group and the CIA + β-endorphin group. Of note, on days 22 and 28, paw swelling in the CIA + β-endorphin group was significantly suppressed when compared with that in the CIA + saline group (P<0.05).

As presented in Fig. 4, the mRNA levels of TNF-α and IL-1β in synovial tissue of the CIA + β-endorphin group were significantly downregulated when compared with those in the CIA + saline group (P<0.05) on day 28. However, β-endorphin treatment had no significant effect on the IL-6 mRNA level of CIA rats.

As presented in Fig. 5, the protein levels of TNF-α and IL-1β in inflammatory paw tissue of the CIA + β-endorphin group were downregulated when compared with those in the CIA + saline group (P<0.01). No significant difference in the protein levels of IL-6 was identified between the CIA + saline group and the CIA + β-endorphin group.