Prompt Description

1. Animal Model Construction and Group Administration 1.1 Preparation of Modeling Reagents Bovine Type II Collagen: Collagen with an immunogenic native structure, lyophilized powder with purity > 95%. Complete Freund's Adjuvant Glacial Acetic Acid: Analytical grade, collagen solvent. 1.2 Animal Selection and Ethics Wistar female rats, 6-8 weeks old, weighing 160-200g, were selected (Wistar rats are more sensitive to collagen-induced arthritis and have a high incidence rate. If Wistar rats are unavailable, SD rats can be used as a substitute). 10-12 rats per group, with the possibility of model failure, the number of modeling groups can be increased appropriately. SPF-grade animal room, temperature 22±2°C, relative humidity 55±5%, 12h light/dark cycle, adaptive feeding for 7 days. Ethical approval must be obtained before the start of animal experiments. 1.3 CIA Model Induction Protocol a. Preparation of collagen solution: Under sterile conditions, dissolve bovine type II collagen lyophilized powder in 0.05 M or 0.1 M glacial acetic acid to a final concentration of 2 mg/mL. This step should be performed at 4°C with slow stirring overnight, avoiding vigorous shaking to prevent collagen denaturation. The dissolved collagen solution should be clear and transparent or slightly opalescent. b. Emulsion preparation: Under ice bath conditions, mix the collagen solution with an equal volume of Complete Freund's Adjuvant and emulsify (using a high-speed tissue homogenizer or repeatedly pushing and pulling with two glass syringes through a three-way valve). c. Initial immunization: After the rats are anesthetized by inhalation (isoflurane), the back is shaved and disinfected. Injection site: intradermal multi-point injection at the dorsal side of the tail root. d. Booster immunization: A booster immunization is performed 7 days after the initial immunization, with the same dose as the initial immunization, avoiding the original injection site. 1.4 Grouping and Administration Protocol Rats begin to show symptoms of joint redness and swelling 10-14 days after the initial immunization. At this time, rats are randomly grouped according to arthritis score or body weight to ensure baseline consistency across groups. Gentian macrophylla extract has shown significant anti-inflammatory effects at a dose of 100 mg/kg. To observe a clear dose-response relationship and cover the threshold of metabolic regulation, three gradients of 100, 200, and 400 mg/kg were designed. The extract needs to be suspended in 0.5% sodium carboxymethyl cellulose solution and administered from the 14th day after modeling, continuing for 28 days until the 42nd day. Group Sample Size Drug Dosage Administration Route/Frequency Blank Control Group 10 0.9% Saline or 0.5% CMC-Na Equal Volume Gavage/Once Daily Model Group 12 Same as above Equal Volume Gavage/Once Daily Positive Control Group 10 Methotrexate 3mg/kg Gavage/Twice Weekly Low-Dose Gentian macrophylla Group 10 Gentian macrophylla ethanol extract 100mg/kg Gavage/Once Daily Medium-Dose Gentian macrophylla Group 10 Gentian macrophylla ethanol extract 200mg/kg Gavage/Once Daily High-Dose Gentian macrophylla Group 10 Gentian macrophylla ethanol extract 400mg/kg Gavage/Once Daily 2. Pharmacodynamic Evaluation Section 2.1 Arthritis Score and Swelling Degree (Macroscopic) a. Observation Frequency: From