Histopathological analysis. Sections of formalin-fixed, paraffin-embedded Crizotinib livers were stained with: 1) hematoxylin and eosin to assess for histological features of steatohepatitis or 2) picrosirius red stain to evaluate for hepatic collagen deposition. The OilRed O tissue staining method on OCT-embedded frozen sections was used to quantify the steatosis. Liver sections were also subject to immunohistochemical staining for macrophages with monoclonal F4/80 antibody (Abcam, Cambridge, MA) and ��-smooth muscle actin (��-SMA) with a monoclonal antibody against ��-SMA (Lab Vision, Fremont, CA) using a labeled streptavidin-biotin immunoenzymatic antigen detection system (UltraVision Mouse Tissue Detection System Anti-Mouse-HRP/DAB; Lab Vision). Biochemical assays.
Serum alanine aminotransferase (ALT) was determined using a kinetic method [ALT Liquid, Advanced Diagnostics (South Plainfield, NJ) and D-TEK (Bensalem, PA)]; liver thiobarbituric acid reactive substances (TBARS) and triglycerides were measured as previously described, using commercial kits (39). Serum endotoxin was quantified using LAL assay (detection limit 0.1 EU/ml; Cambrex, Walkersville, MD). Isolation of liver mononuclear cells and flow cytometry analysis. Animals received anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg); the livers were perfused with Hank’s balanced saline solution (HBSS) followed by in vivo digestion with 0.33 mg/ml Liberase RI Enzyme (F. Hoffmann-La Roche, Basel, Switzerland) in HBSS.
The liver mononuclear cells (LMNCs) were purified from whole liver cell suspension obtained after tissue disruption using centrifugation at slow speed (500 g) and subsequent isolation in Percoll 40/70 gradient density at 800 g; LMNC were harvested from the gradient interface. The cells were further washed in saline supplemented with 2% FBS, stimulated with a cocktail of PMA (50 ng/ml), ionomycin (1 ��g/ml), and brefeldin A (10 ��g/ml) in RPMI 1640 + 10% FBS for 4 h, and stained for surface CD68 and intracellular TNF-�� using specific fluorescent-labeled antibodies and CytoFix/CytoPerm Kit (BD Bioscience, San Jose, CA). The cells were gated by size and granularity, and their fluorescence was analyzed using the LSR flow cytometer. Cytokine measurements. Serum TNF-�� level was determined using the Pierce Multiplex Cytokine Array (Pierce, Woburn, MA). mRNA analysis.
Total RNA extraction from liver tissue and mRNA quantification using SYBR Green-based real-time quantitative polymerase chain reaction was performed as previously described (39). All specific mRNA levels were normalized against the housekeeping gene, 18S, in the same sample. The specific PCR primer sequences for target Carfilzomib genes 18S, p22phox, p47phox, p67phox, and gp91phox have been published previously (27); additional genes studied here are listed in Table 1. Table 1.