Adjuvant-induced arthritis rat model

Adjuvant-induced arthritis (AIA) of rats was induced by injection of complete Freund’s adjuvant. Briefly, 100 μL of heat‑killed Mycobacterium tuberculosis in mineral oils was intradermally injected into the left hind paw of SD rats (100–120 g) [33–35]. Then, the rats were randomly divided into 7 groups on Day 9 after injection (n = 6), including the normal group (PBS as vehicle), model group (PBS as vehicle), MTX 0.5 mg/kg group (MTX-L, 0.5 mg/kg every 3 days) [34], MTX 7.6 mg/kg group (MTX-H, 7.6 mg/kg per week), TGP group (150 mg/kg per day), TGP (150 mg/kg per day) + N group (100 mg/kg per day), and CET formulation group (containing TGP 150 mg/kg per day +N 100 mg/kg per day).

The body weight, paw volumes, and arthritis index scores were measured every two days. Arthritis severity was graded with clinical scores according to traditional methods [36]. On Day 30 after treatment, the rats were sacrificed, and the right hind paw was amputated for histological examination via hematoxylin and eosin (H&E) staining and micro-CT scanning (SkyScan 1176, Bruker, Belgium) to visualize bone erosion. Rat serum was used to measure cytokines, including TNF-α, IL-6, and IL-1β, according to the manufacturer’s instructions. Lymphocytes isolated from the peripheral blood of two AIA rats were combined into one sample to determine the intracellular paeoniflorin concentration and to perform Western blot assays.