, Osaka, Japan) and diluted to 1 mg/ml in physiological saline. Cap was dissolved in 1% ethanol + 1% Tween 20 in physiological saline. LPS (20 mg/kg) was administered intraperitoneally (ip) and 4 mg/kg Cap was administered subcutaneously (sc) to the backs of the mice 5 min after LPS administration. Mice were divided into four groups: vehicle group, LPS group, Cap group, and LPS + Cap group. The animals were sacrificed under anesthesia for the following procedures at 1, 3, 6, 9, and 12 h after LPS administration. Whole blood was taken from the abdominal aorta of the mice.

The samples were centrifuged, and the supernatant was measured. Measurements were performed using Quantikine® Immunoassay Mouse TNF-α,

Quantikine® Immunoassay Mouse sTNFRI, and Quantikine® Immunoassay Mouse sTNFRII (R&D Systems, Inc., MN, USA). Within 30 min, absorbance Enzalutamide was measured at 450 nm and 570 nm using a plate reader (Labsystems Multiscan MS; Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan). The measured value of the vehicle group was defined as the control value. The limits of detection of sTNF, sTNF-R1, and sTNF-R2 levels were 5.1, 5.0, and 5.0 pg/mL, respectively. Measurement of circulating TNF-α, TNF-R1, and TNF-R2 mRNA expression (derived from macrophages) levels in whole blood Whole blood was taken from the abdominal aorta of the mice under anesthesia at 0.5, 1, Fluorouracil molecular weight 3, 6, and 9 h after LPS administration. Total RNA was extracted from 300 μl of whole blood using a total RNA extraction kit (PureLink™ Total RNA Blood Purification Kit for isolating total RNA from whole Blood; Invitrogen Corporation, CA, USA). Synthesis of Silibinin cDNA was performed by reverse transcription using total RNA solution (PrimeScript™ RT reagent Kit; Takara Bio Inc, Shiga, Japan), and mRNA was measured using a thermal cycler (LightCycler®, Roche Diagnostics, Basel, Switzerland). The results were adjusted using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18s rRNA, a housekeeping gene, as the internal

standards. Values are shown as mean ± standard deviation (SD). Statistical analysis was performed using Tukey’s test. A significant difference was determined as P < 0.05. The circulating sTNF level significantly increased in the LPS group 1 h after LPS administration compared to both the vehicle (P < 0.01, Fig. 1A) and LPS + Cap (P < 0.01, Fig. 1A) groups (n = 3-4). There was no significant difference in the circulating sTNF levels between the vehicle and LPS + Cap groups (Fig. 1A). From 3 h until 12 h after LPS stimulation, circulating sTNF levels in the LPS group significantly increased compared to the vehicle group (P < 0.05 or 0.01, Fig. 1A). Both the circulating sTNF-R1 and -R2 levels in the LPS and LPS + CAP groups significantly increased from 0.5 h to 12 h after LPS administration, compared to the vehicle group (P < 0.05 or 0.01, Figs. 1B and C).