Paeoniflorin

A first set of mice was used to identify the dosage of IFN-α required to establish a model of IFN-α induced depression. These mice were given daily subcutaneous (s.c.) injections of either sterile saline solution or recombinant human IFN-α2b (Kawin Technology, Beijing, China), diluted with sterile saline, at doses from 0.06×10⁶ to 15×10⁶ IU/kg for 4 successive weeks [38 ]. A second set of mice was given IFN-α s.c. at a constant volume of 15×10⁶ IU/kg daily at every 10:30 for 4 successive weeks, followed by daily intragastric (i.g.) administration of paeoniflorin (purity 99%; Chengdu Must Bio-Technology Co., Ltd, China) at 10 mg/kg (low dose), 20 mg/kg (medium dose), or 40 mg/kg (high dose) dissolved in saline [30 ]; or escitalopram (10 mg/kg i.g.; H. Lundbeck, Denmark), 30 min before IFN-α administration. Appropriate vehicle (saline)-treated groups were also assessed simultaneously. The details of these two experimental designs are shown in Figures 1A and 2A.

HPLC fingerprint analysis was used to identify the main chemical components of the WTD extract. The extract was dissolved in water at a concentration of 1.92 g/ml (w/v) and further diluted to 0.96 g/ml (w/v) with methanol-water (50:50). Samples were passed through an Acclaim™ 120 C18 column (4.6 mm × 250 mm, 5 μm) at a flow rate of 1.0 ml/min with the mobile phases of methanol (A) and 0.1% phosphoric acid (B). HPLC signals were measured at a detection wavelength of 240 nm with gradient elution as detailed in Table 1. Ephedrine hydrochloride, pseudoephedrine hydrochloride, paeoniflorin, verbasil-7-O-glucoside, glycyrrhizin, benzoylaconitine, benzoylneoaconitine, benzoylhypoaconitine, and glycyrrhizinate were purchased from Chengdu MUST Biotechnology Co., Ltd. (Chengdu, China). The TCM compound solution was evenly mixed by vortexing, 200 µL of the solution was accurately measured, and 1 ml of methanol:water (8:2, V/V) was added, and the mixture was vortexed. After centrifugation for 10 min at 4°C and 20,000×g, the supernatant was filtered through a 0.22 µm filter membrane and the filtrate was collected for analysis.

Hydroxysafflor yellow A (batch no. MUST-13121913), paeoniflorin (batch no. MUST-13113009), calycosin-7-O-β-D-glucoside (batch no. MUST-14031314), calycosin (batch no. MUST-13081501), and formononetin (batch no. MUST-14030710) were purchased from Chengdu Must Bio-technology Co. Ltd. (Sichuan, China). Ononin (batch no. 130507) was obtained from Chengdu Pufei De Biotech Co. Ltd. (Sichuan, China). The purity of all the above chemical reference substances were determined higher than 98 %. Rabbit anti-rat neuron specific nuclear protein (NeuN) and mouse anti-rat glial fibrillary acidic protein (GFAP) were bought from Millipore Co. Ltd (USA). Rabbit anti-rat CD34 was purchased from Abcam (UK).