Treatment with CDAA or CDAA+CCl₄ induces liver injury and expression of markers of hepatic fibrogenesis in mice.

<p>A) The total degree of liver injury (hepatic steatosis and necroinflammation) was determined according to the NAS score. B) The number of apoptotic cells was measured with TUNEL and expressed as apoptotic cell/field. C–E) qRT-PCR shows a progressive increase, in a time dependent manner, of the amount of TIMP-1 (C), CTGF (D) and TGFβ (E) mRNA expression in the group of CDAA, with the evidence of a promoting effect exerted by CCl₄ starting from month 3. Data represent mean ± SD; ò p<0.05 vs CSAA 1 month; *p<0.05 vs CSAA 3 months; §p<0.05 vs CSAA 6 months; #p<0.05 vs CSAA 9 months; ç p<0.05 vs CDAA 6 months; ù p<0.05 vs CDAA 9 months.</p

Treatment with CDAA+CCl₄ induces development of HCC after 9 months.

<p><b>A</b>) Mice sacrificed after 1 and 3 months from the beginning of the treatment do not show any nodular lesions in the parenchyma. Conversely, mice sacrificed after 6 months of treatment show few nodular lesions compatible with HCC. After 9 months of treatment mice show a liver parenchyma completely damaged. B) Histochemical staining for H&E and Sirius Red show respectively no fat deposition and absence of collagen content into the nodules. C) After 6 months of treatment, about 30% of mice show HCC development both with CDAA and CDAA+CCl₄. After 9 months of treatment, 100% of mice from the CDAA+CCl₄ group show at least one nodule into the liver parenchyma. D) After 6 months, the average of nodules dimension in the group of CDAA mice is significantly lower in comparison to that of the group of CDAA+CCl₄ mice. After 9 months, the average of nodules dimension in the group of CDAA+CCl₄ mice results of 9 mm, in comparison to 3 mm of the group of mice treated with CDAA alone. Data represent mean ± SD; *p<0.05 vs CDAA.</p

Treatment with CDAA or CDAA+CCl₄ induces hepatic fibrosis in mice.

<p>A–B) qRT-PCR expression shows a progressive increase, in a time dependent manner, of the amount of Collagen α1(I) (A) and αSMA (B) mRNA in the group of CDAA, with the evidence of a promoting effect exerted by CCl₄ starting from month 6. Conversely, mice treated with control diet (CSAA) do not show any time-dependent increase neither in the level of Collagen α1(I) nor of αSMA, not even after chronic CCl₄ injection. C–D) Morphometric analysis shows increased collagen deposition by Sirius Red staining (C) and increased HSC activation by αSMA Immunohistochemistry (D) in the liver of CDAA treated mice in comparison to CSAA treated mice. This condition is even enhanced in the liver of mice treated with CDAA+CCl₄. Data represent mean ± SD; *p<0.05 vs CSAA; #p<0.05 vs CSAA+ CCl₄; ò p<0.05 vs CDAA.</p

Gene expression of elements involved in carbohydrate and lipid metabolism.

<p>qRT-PCR expression shows a progressive decrease of the amount of PEPCK (A), G6Pase (B), ChREBP (D), ACOX-1 (E) and CPT1A (F) mRNA expression in the groups of CDAA or CDAA+ CCl₄, while only a slight decrease is observed for mRNA expression of SREBP-1c (C). Data represent mean ± SD; ò p<0.05 vs CSAA 1 month; *p<0.05 vs CSAA 3 months; §p<0.05 vs CSAA 6 months; #p<0.05 vs CSAA 9 months; ç p<0.05 vs CDAA 6 months; ù p<0.05 vs CDAA 9 months.</p

Effect of diet treatment on metabolic parameters and body weight.

<p>A–B) Mice treated with CDAA diet for 1 month develop peripheral insulin resistance as shown by the lower glucose uptake vs CSAA-treated mice (M value) with similar levels of FPG. CCl₄ treatment induces an increase in fasting plasma glucose and enhances the condition of insulin resistance, reducing M value. C) Insulin levels measured at 1 month of treatment. D) The graphic shows a gradual increase in body weight in both CDAA and CSAA diet. CCl₄ injection similarly reduces body weight in both diets. E) Epididymal fat/body weight ratio appears to be similar between the two different diets both at 3 and 9 months of treatment, with similar reduction in the presence of CCl₄. Data represent mean ± SD; §p<0.05 vs CSAA; ù p<0.05 vs CDAA; *p<0.05 vs CSAA 3 months; #p<0.05 vs CSAA+CCl₄ 3 months; ò p<0.05 vs CDAA 3 months; ç p<0.05 vs CDAA+CCl₄ 3 months; b p<0.05 vs CSAA+CCl₄ 6 months; c p<0.05 vs CSAA+CCl₄ 9 months; d p<0.05 vs CSAA.</p

Treatment with CDAA or CDAA+CCl₄ induces hepatic inflammation in mice.

<p>A) Immunohistochemistry for pan-macrophage marker F4/80. B) TNFα and (C) MCP-1 mRNA levels are increased in CDAA or CDAA+ CCl₄ already after 1 month of treament. D–F) Markers of Inflammasome activation ASC, Caspase-1 and IL-1β show increased expression in CDAA or CDAA+ CCl₄ groups. Data represent mean ± SD; ò p<0.05 vs CSAA 1 month; *p<0.05 vs CSAA 3 months; §p<0.05 vs CSAA 6 months; #p<0.05 vs CSAA 9 months; ç p<0.05 vs CDAA 6 months; ù p<0.05 vs CDAA 9 months.</p

The expression of pro-carcinogenic genes is modified already after 3 months of treatment.

<p>A) Immunohistochemistry for p-AKT and p-c-Myc shows positive staining in the liver parenchyma of CDAA+CCl₄ mice, in correspondence of HCC foci (20X magnification) B) Glypican-3 Immunohistochemistry shows positive staining in the tumor area (5X and 20X magnification). C) Mice treated with CDAA or CDAA+CCl₄ show, starting at 3 months, increased levels of mRNA for IGF-2 and (D) SPP-1 and (E) decreased mRNA levels of PTEN. Data represent mean ± SD; *p<0.05 vs CSAA; #p<0.05 vs CSAA+CCl₄.</p

Treatment with CDAA determines a progressive increase of hepatic steatosis.

<p>A) Increased levels of liver/body weight fraction are observed in the group of mice treated with CDAA diet, either with or without CCl₄ administration, in comparison to mice treated with control diet, starting at 1 month. B) Histochemical staining and relative morphometric evaluation for Oil Red O and C) hepatic triglyceride measurement show increased deposition of lipids in the liver parenchyma of CDAA and CDAA+CCl₄ treated animals in comparison to CSAA treated mice. Data represent mean ± SD; ò p<0.05 vs CSAA 1 month; *p<0.05 vs CSAA 3 months; §p<0.05 vs CSAA 6 months; #p<0.05 vs CSAA 9 months; ç p<0.05 vs CDAA 6 months; ù p<0.05 vs CDAA 9 months.</p

Effect of prolactin on the phosphorylation of Ca-dependent PKC isoforms

<p><b>Copyright information:</b></p><p>Taken from "Prolactin stimulates the proliferation of normal female cholangiocytes by differential regulation of Ca-dependent PKC isoforms"</p><p>http://www.biomedcentral.com/1472-6793/7/6</p><p>BMC Physiology 2007;7():6-6.</p><p>Published online 19 Jul 2007</p><p>PMCID:PMC1939715.</p><p></p> Immunoblots for PKC-α, PKC-β-I, PKC-β-II and PKC-γ in normal female cholangiocytes stimulated for 90 minutes at 37°C with 0.2% BSA (basal value) or prolactin (100 nM) with 0.2% BSA. When cholangiocytes were treated with prolactin, there was an increase in the phosphorylation of PKCβ-I and a marked decrease in PKCα phosphorylation; no significant changes in the phosphorylation of PKCβ-II and PKCγ were observed in normal female cholangiocytes treated with prolactin or 0.2% BSA. Data are mean ± SEM of 3 experiments. * p < 0.05 vs. corresponding basal values. PKC = protein kinase C

Measurement of the number of top panel PCNA- and lower panel CK-19-positive cholangiocytes in liver sections (5 μm, 3 slides analyzed per group) and c PCNA protein expression in purified female cholangiocytes from NaCl- or prolactin-treated rats

<p><b>Copyright information:</b></p><p>Taken from "Prolactin stimulates the proliferation of normal female cholangiocytes by differential regulation of Ca-dependent PKC isoforms"</p><p>http://www.biomedcentral.com/1472-6793/7/6</p><p>BMC Physiology 2007;7():6-6.</p><p>Published online 19 Jul 2007</p><p>PMCID:PMC1939715.</p><p></p> Administration of prolactin to normal female rats increased the number of PCNA-positive cholangiocytes (arrows) and CK-19-positive cholangiocytes compared with normal rats treated with NaCl. Orig. magn., ×20 (PCNA) and ×10 (CK-19). Data are mean ± SEM of 5 values obtained from the 3 slides evaluated per each group of animal. * p < 0.05 vs. the corresponding value of NaCl-treated rats