Bile acids play a crucial function in liver organ regeneration and
Bile acids play a crucial function in liver organ regeneration and damage, but their function in acetaminophen (APAP)Cinduced liver organ injury isn’t known. mRNA in the intestines. Liver organ regeneration after APAP treatment was considerably faster in CA dietCfed mice after APAP administration supplementary to fast cyclin D1 WASL induction. Used together, these data indicate that bile acids play a crucial function in both recovery and initiation of APAP-induced liver organ injury. Bile acids are flexible biological substances that regulate energy homeostasis, activate nuclear receptors and cell signaling pathways, and control cell proliferation GDC-0879 and inflammatory procedures in the liver organ and gastrointestinal system.1,2 Bile acids maintain their very own homeostasis by activating?a organic signaling GDC-0879 network involving hepatic and intestinal farnesoid X receptor (FXR), little heterodimer partner, and intestinal fibroblast development aspect (FGF) 15 (FGF19 in individual) appearance, culminating in inhibition of the principal bile acidCsynthesizing enzyme, CYP7A1.3C6 Although bile acids are potent signaling substances at pathophysiological concentrations, they trigger apoptosis, necrosis, and oxidative strain.3,7C10 Bile acids are also implicated in stimulation GDC-0879 of liver regeneration.11C14 Studies in recent years indicate that this bile acidCmediated gut-liver signaling axis may play a critical GDC-0879 role in regulation of liver homeostasis.6,15,16 Acetaminophen (APAP) is the most commonly used analgesic and antipyretic agent.17 An overdose of APAP is?the major cause of acute liver failure in the United States.18,19 The mechanisms of APAP-induced liver injury and subsequent liver regeneration are the focus of intense investigation.20C22 In an overdose situation, excess APAP is mainly metabolized by CYP2E1 to a reactive metabolite, = 5 to 8 per group per time point) were used in these studies. All animal studies were approved by and performed in accordance with the Institutional Animal Care and Use Committee at University of Kansas Medical Center (Kansas City). Mice were fed a normal diet, a 2% cholestyramine (CSA)Ccontaining diet, or a 0.2% cholic acid (CA)Ccontaining diet for 1 week. After a 1-week feeding of specific diets, mice were injected i.p. with 400 mg/kg APAP (Sigma, St. Louis, MO) dissolved in warm sterile saline solution. Mice were not fasted before APAP treatment. Mice were sacrificed at 0, 1, 4, 8, 12, and 24 hours after APAP treatment, and livers, intestines, and serum were collected and processed as previously described.14 Histological and IHC Data Paraffin-embedded liver sections (4 m thick) were used for H&E staining and immunohistochemical (IHC) detection of proliferating cell nuclear antigen (PCNA), as previously described.14 H&E-stained slides were used for necrosis scoring, as previously described,24 and PCNA-stained slides were used for PCNA scoring, as previously described.25 Protein Extraction, Microsome Preparation, and Western Blot Analysis Protein extracts were prepared from frozen liver tissues using radioimmunoprecipitation assay buffer, containing fresh protease and phosphatase inhibitors, and used for Western blot analysis, as previously described.14 Primary and secondary antibodies were purchased from Cell Signaling Technologies (Danvers, MA). Microsomes were prepared from murine liver by homogenizing tissue using a Teflon homogenizer in phosphate buffer (pH 7.4) and centrifuged at 9000 for 30 minutes at 4C. The supernatant from this centrifugation was centrifuged at 100,000 for 60 minutes at 4C. The microsomal pellet was resuspended in phosphate buffer with 0.1 mmol/L EDTA (pH 7.4). Microsomes (50 g) were used for CYP2E1 Western blot analysis. Real-Time PCR Analysis Total RNA was extracted from all murine intestines using TRIzol, per the manufacturers protocol (Sigma), and reverse transcribed as previously described.14 FGF15 mRNA levels were determined by real-time PCR assays around the Applied Biosystems Prism 7300 Instrument (Grand Island, NY), as previously described.14 18S RNA levels were used for data normalization. Serum ALT Measurement Serum alanine aminotransferase (ALT) was measured by kinetic assay method using the Infinity ALT kit (Thermo Scientific, Waltham, MA), according to the manufacturers protocol. Bile Acid Analysis Serum, liver, and intestinal total bile acids were measured enzymatically using a Total Bile Acid Assay Kit (BQ kits, San Diego, CA), per the manufacturers protocol. Briefly, in a 96-well plate, 20 L of bile acid standard and murine serum samples were added in triplicate. Diaphorase in phosphate buffer (150?L) was then added and incubated at 37C for 4 minutes. After adding 30 L of 3–hydroxy steroid dehydrogenase in Tris buffer, absorbance was measured at 540 nm kinetically for 5?minutes. Specific GDC-0879 hepatic bile acids were analyzed by Ultra Performance Liquid Chromatography-Mass Spectrometry (Waters, Milford, MA), as previously described.14 Glutathione Analysis Total GSH was measured in liver homogenates by the 5,5-dithiobis-(2- nitrobenzoic acid)-glutathione reductase.