Nrf2 and Snail-1 in the prevention of experimental liver fibrosis by caffeine

Figure 1 Macroscopic and histological differences and fibrosis index with caffeine treatment. A: Liver fibrosis percentage of healthy rats, caffeine (CFA)-treated, thioacetamide (TAA)-intoxicated, TAA-intoxicated treated with CFA, bile duct ligation (BDL) rats, BDL rats treated with CFA. Sections, 4 μm thick, stained with Masson’s trichrome, × 10. White arrows show the extracellular matrix (ECM) (fibrosis); B: Fibrosis quantification. Fibrosis percentages are shown, they were obtained by computer-assisted morphometric analysis (Software Image pro plus 6.3); C: Knodell Index for fibrosis, sections 4 μm thick, stained with Masson’s trichrome, × 10; D: Inflammatory infiltrate amount. Sections 4 μm thick, stained with hematoxylin and eosin, × 40. Black arrows show inflammatory cells; E: Knodell Index for fibrosis, sections 4 μm thick, stained with hematoxylin and eosin, × 40.

Figure 2 Expression of fibrogenic and antioxidant genes in liver. Reverse transcription-polymerase chain reactions were performed for connective tissue growth factor (CTGF) (A), collagen I (Col-1) (B), and transforming growth factor β1 (TGF-β1) (C), superoxide dismutase (SOD) (D) and catalase (CAT) (E). Gene amplification was normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. CAT (F) and SOD antioxidant activity (G) were analyzed by zymography in acrylamide gels. TAA: Thioacetamide; BDL: Bile duct ligation; CFA: Caffeine.

Figure 3 Expression of inflammatory genes in liver. A: Immunohistochemistry for CD11b, sections 4 μm thick, stained with Masson’s trichrome, × 40. Results for CD11b positive area are shown as percentage; B: Tumor necrosis factor alpha (TNF-α) liver levels in different groups of treatment, performed by enzyme-linked immunosorbant assay; C-E: Reverse transcription-polymerase chain reaction were performed for TNF-α, interleukin-1β (IL-1β), IL-6. Gene amplification was normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. TAA: Thioacetamide; BDL: Bile duct ligation. CFA: Caffeine.

Figure 4 Expression of transcriptional factors. Western-blots were performed for the transcriptional factor Nrf2 (A) and Snail-1 (B). Densitometric values were normalized against the constitutive protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and represented like fold increase respect to healthy animals. TAA: Thioacetamide; BDL: Bile duct ligation. CFA: Caffeine.