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].