For the latter, after 1 week following BDL, RA was intraperitoneally injected daily at a dose of 0.1 mg / 25 g body weight until the animals were sacrificed 1 week later for Sirius-red staining morphometry, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) analysis of the livers as described below. HSCs were isolated from normal male Wistar rats, C57Bl/6 and Coll-GFP mice by in situ
digestion of the liver and arabinogalactan gradient ultracentrifugation by the Non-Parenchymal Liver Cell Core of the Southern California Research Center for ALPD and Cirrhosis as described.11, 16 The purity of the cells as determined by phase contrast microscopy and ultraviolet-excited fluorescence microscopy exceeded Idasanutlin nmr 96%, and the viability as determined
by trypan blue exclusion exceeded 94%. In vitro activation of HSC was achieved by culturing rat HSCs in Dulbecco’s Deforolimus in vitro modified Eagle’s medium (DMEM) with 1.0 g/L glucose, 10% fetal bovine serum, and 1% antibiotics on plastic dish for 3, 5, or 7 days. Culture-activated rat primary HSCs were treated with the YGW or starch (control) aqueous extract at 25% (v/v). To obtain the extract, the YGW or starch powder (provided by S.P. Pharmaceutics) was suspended in DMEM at a concentration of 35 mg/mL, mixed thoroughly with a vortex for 5 minutes, and centrifuged at 150g for 30 minutes to collect the supernatant. This supernatant was designated as 100% extract and used after filter-sterilization. RA and BC (Sigma Chemical) were dissolved in dimethyl sulfoxide (DMSO) and tested at a concentration of 67.5 to 270 μM. Two weeks after
BDL or sham operation, nonparenchymal cells (NPCs) were isolated from the Coll-GFP mice and subjected to FACS using FACS AriaII sorter (BD Bioscience) at the USC-CSCRM/NCCC Flow Cytometry Core. GFP expression was analyzed by an argon laser at 488 nm and a 530 nm filter. Vitamin A autofluorescence Cytidine deaminase was analyzed by a solid-state laser at 350 nm and a 450 nm filter. As a negative control for vitamin A autofluorescence, we used the spontaneously immortalized rat HSC line (BSC) established from cholestatic liver fibrosis in rats.20 After 3 days of the extract treatment, the cells were washed with cold phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA). To stain α-smooth muscle actin (SMA), a fluorescein isothiocyanate (FITC)-conjugated antibody (1:200, Sigma, St. Louis, MO) was added as a primary antibody at 4°C overnight. After washing and blocking with 5% nonfat milk, fluorescence images were viewed by a Nikon microscope as described above. For intracellular lipid staining, HSCs treated with the extract for 3 days were cultured with retinol (5 μM) and palmitic acid (100 μM) (Sigma) for 48 hours and fixed with 10% formalin in PBS. Oil Red O (0.5% wt/vol in isopropanol) was diluted with 67% volume of water, filtered, and added to the fixed HSCs.