In vivo studies with animals
All animal care and experimental protocols for in vivo studies conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH (NIH publication no. 85-23, revised 1996) and approved by the Ethics Committee of the Second Clinical Medical College of Jinan University, Shenzhen People’s Hospital. C57BL/6J wild type mice (8 weeks old) were purchased from the Animal Center of Nanjing University (Nanjing, Jiangsu, China). These mice were maintained at the Animal Center of Shenzhen People’s Hospital with free access to food and water. Based on the previous study, the dose of TEC used to mice was converted as 75 mg/day/kg body weight (mpk). Cholestasis was induced was induced in C57BL/6J mice by administration of ANIT for 48 hr by oral gavage or fed with a diet containing 1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 2 weeks. C57BL/6J mice were treated either with vehicle (corn oil) or ANIT (100mg/kg) by oral gavage, while a subset of C57BL/6J mice were treated with TEC (75 mpk) for 5 days and another subset of C57BL/6J mice treated with TEC by oral gavage 3 days before, at the time of the administration of ANIT, and 24h after. During the DDC feeding, TEC (75 mpk) was administered daily after DDC feeding for one week. All ANIT treated animals were sacrificed 48 hr after gavage, while all 1%DDC treated mice 2 weeks after feeding. For constructed PPARγ knockdown mice, mice were injected i.v. through the tail vein with control adenovirus expressing shorthairpin (sh)RNA against luciferase (Ad-shCtrl) or adenovirus expressing shRNA against PPARγ (Ad-shPPARγ) (0.5–1.5 × 109 active viral particles in 200 μl saline [54 mmol/l NaCl]). At 2 weeks after infection, mice performed experiments as mentioned before. Extraction and determination of total bile acids (TBA) in mouse liver and intestine was performed as previously reported[29].