Bovine type 2 collagen

Six-week-old Sprague Dawley (SD) rats (n = 72, male) were purchased from the Shandong Laboratory Animal Center (China). The breeding and care of the experimental animals were carried out in accordance with the Helsinki Convention on Animal Protection and the Regulations of the People’s Republic of China on the Administration of Experimental Animals. The animals were randomly divided into a normal control (NC) group (n = 24) and a CIA control group (n = 48). Bovine type II collagen (Chondrex, USA) was mixed with complete Freund’s adjuvant (Sigma-Aldrich, USA), and the initial immunization was performed by intracutaneous injection at the tail root. One week later, Bovine type II collagen was mixed with incomplete Freund’s adjuvant (Sigma-Aldrich), and the booster immunization was performed by intracutaneous injection at the tail root. The NC group was injected with an equal volume of phosphate-buffered saline (PBS).

The compound animal model of NAFLD (Zhu et al., 2023 (link)) + collagen-induced RA (CIA) (Wang et al., 2018 (link); Guo et al., 2020 (link)) was established using the following process. Forty unselected Sprague-Dawley rats were grouped as follows: normal (control) group, CIA group, NAFLD model group, and NAFLD + CIA group (10 rats per group). The NAFLD model group and NAFLD + CIA group rats received an HFD (40 kcal% fat, 20 kcal% sucrose, and 2% cholesterol) for 6 weeks to develop NAFLD. The HFD was provided by Xietong Bio-engineering Co., Ltd. Thereafter, 1 g/L of a mixture of CFA and bovine type II collagen (Sigma–Aldrich, MO, United States; ratio of 1:1) in a 0.1-ml emulsifier was subcutaneously injected into the base of the tail in the CIA and NAFLD + CIA groups. The rats were further immunized with 0.1 ml of emulsifier after 7 d via subcutaneous injection into the proximal one-third of the tail. Clinical severity was evaluated by measuring the footpad thickness using a dial-gauge caliper. Paw swelling (ml) was calculated by subtracting the paw volume (left hind paw) at d0 (Figure 1A) (Ahmad Khan et al., 2020 (link)).

Female DBA/1 mice (n = 18), aged 8 weeks, were randomly divided into three groups: L-arginine-deprived feeding group, treatment group, and control group. Mice in the L-arginine-deprived feeding group were fed an arginine-free diet 2 weeks before model induction, while mice in the treatment and control groups were fed a normal diet. The treatment group was given water containing 2 mg/ml D-arginine 7 days prior to bovine type II collagen injection. CIA induction was performed based on reported methods [16 (link)]: bovine type II collagen (Sigma-Aldrich, MO, USA) was dissolved in 0.1 mol/L acetic acid to a final concentration of 2 g/L, mixed and emulsified with complete Freund’s adjuvant (V:V = 1:1) (Sigma-Aldrich, MO, USA) to prepare a type II collagen emulsion. The emulsion was intradermally injected into the tails of mice at primary and secondary immunization on days 0 and 21, respectively.

The animal experiments were carried out under the Guidelines of Ethical and Regulatory for Animal Experiments formulated by the Institute of Basic Theory, China Academy of Chinese Medical Sciences (License Number: SCXK (Beijing) 2016-0011, SYXK (Beijing) 2017-0033). Bovine type II collagen (200 μg, Sigma-Aldrich, Shanghai) emulsified in complete Freund’s adjuvant (200 μg, Sigma-Aldrich, Shanghai) was intradermally injected into male DBA/1J mice (8 weeks old, Medcona) to induce rheumatoid arthritis. Furthermore, 100 μg Bovine type II collagen was added to incomplete Freund’s and administered to mice on day 21 after the primary immunization, to boost the intradermal injection.