AIM To investigate whether Yiguanjian decoction (YGJ) has an anti-liver cirrhotic

AIM To investigate whether Yiguanjian decoction (YGJ) has an anti-liver cirrhotic effect and whether it regulates hepatic stem cell differentiation. constant temperature environment and supplied with laboratory chow and water intragastric administration once a day. The rats were randomly divided into a 2-AAF/CCl4 group (= 8), an FLSPC group (= 8), an FLSPC + YGJ group (= 8), and an FLSPC + SORA group (= 8). The FLSPC, FLSPC + YGJ, and FLSPC + SORA groups were treated with FLSCs a single intra-splenic injection at the 9th wk. The FLSPC + YGJ and FLSPC + SORA groups were orally administrated at dosages of 3.56 g/kg and 1.0 mg/kg, respectively, once per day for 4 wk. Normal rats (N, = 5) received an equal amount of subcutaneous olive oil and the same volume of oral physiological saline. Isolation, characterization, and transplantation of Dlk-1+ FLSPCs FLSPCs were isolated from ED14/15 fetal livers of pregnant Wistar rats as previously described[21]. The livers were cut into pieces and digested with 0.05% trypsin and 0.05% NB4 for 15 min. Next, a single-cell suspension was collected and stained with an anti-Dlk-1 antibody. Dlk-1 positive cells were sorted using a magnetic bead sorter instrument BMP4 (Miltenyi Biotec). The purity of the Dlk-1 positive cells was analyzed by flow cytometry (BD Accuri C6, BD Biosciences) and was determined to be 60.58%. Odanacatib inhibitor At the beginning of the 9th wk, the rats given FLSPC therapy were transplanted with Dio-stained Dlk-1+ FLSPCs (1 106 cells per rat) intra-splenic injection. Serum chemistry Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were measured using standard Odanacatib inhibitor laboratory methods. Histochemical and immunohistochemical analyses Odanacatib inhibitor of rat livers Paraformaldehyde-fixed (4%) specimens were cut into 4 m sections and stained with 0.1% (w/v) Sirius Red (Direct Red 80; Aldrich, Milwaukee, WI, United States), or hematoxylin and eosin (H&E). Immunostaining was performed according to previously published methods[22]. Briefly, sections were de-paraffinized, washed, and pre-incubated in blocking solution, followed by incubation with anti–SMA (1:200), anti-HNF4 (1:200), and anti-Hep (1:200) antibodies. Next, the sections were incubated with HRP-conjugated secondary antibodies (1:1000), washed, stained with diaminobenzidine (DAB), and counterstained with hematoxylin. A Leica SCN 400 microscope was used to visualize the samples. For immunofluorescent staining, Alexa Fluor 488 and cyanine 3 secondary antibodies (Jackson ImmunoResearch, West Grove, PA, United States) were used with counterstaining. Images were obtained with a confocal laser scanning microscope (FV10i, Olympus, Japan). WB-F344 Odanacatib inhibitor and RAW264.7 cell culture and treatment WB-F344 Odanacatib inhibitor cells (a rat oval cell line that is morphologically and functionally similar to freshly isolated HPCs[23]) and RAW264.7 cells (a murine macrophage cell line) were purchased from the Shanghai Cell Bank (Chinese Academy of Sciences, Shanghai, China). WB-F344 cells were cultured at 37 C in an atmosphere containing 5% CO2 in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mmol/L glutamine, and penicillin/streptomycin (100 mg/mL). Activation of RAW264.7 cells was induced with LPS 100 ng/mL for 8 h at 37 C in an atmosphere containing 5% CO2 in DMEM supplemented with 10% FCS[24], and then co-cultured with WB-F344 cells in Transwell chambers. A total of 2 104 WB-F344 cells were seeded into the upper compartment and 4 104 RAW264.7 cells were seeded into the lower compartment of the Transwell chamber. LPS was added to the culture medium, and the medium was replaced every 48 h for a total culture time of 7 d. RNA preparation and quantitative real-time reverse transcription-PCR The mRNA expression of tumor necrosis factor-alpha (TNF-), transforming growth factor beta 1 (TGF-1), -SMA, collagen type I [Col(1)], CD68, CD163, HNF4, Hep, Wnt-1, -3A, -4, -5A, -5B, -8A, -8B, -10B, -11, -catenin, frizzled (FZD)-1, -2, -3, -4, -5, -6, low-density lipoprotein receptor-related protein (LRP)-5, -6, and GAPDH was quantified using quantitative reverse transcription (RT)-PCR. Total RNA was extracted from the liver tissue using a total RNA purification kit (Lot. 250800) (TOYOBO, Osaka, Japan). RNA was reverse-transcribed to cDNA and gene expression was measured using SYBR Green.