Animal/Liver Weights and Liver/BodyWeight(BW)Ratio(final) at Necropsy.

<p>Liver/BW ratio  = [mice liver weight(g)/mice body weight (g)]×100%;</p><p>*Significantly different from ethanol+ DEN group, <i>P</i><0.05.</p

Primer sequences for real-time RT-PCR analysis.

<p>Primer sequences for real-time RT-PCR analysis.</p

The expression level of Nrf2 in mice liver at 3-week and 35-week.

<p>A: Real-time RT-PCR analysis of Nrf2 gene expression. B–C: Western blot analysis of protein level of Nrf2 at 3-week and 35-week. lane 1.Control; lane 2.Maotai; lane 3.Ethanol; lane 4.Maotai+DEN; lane 5.Ethanol+DEN; lane 6.DEN; D: Quantification of Nrf2 level by western blot analysis. E–G, H–J: Light microscopy of Nrf2 expression through Immunohistochemistry in control group, Maotai+ DEN group and ethanol+ DEN group at 3-week and 35-week (400× magnification, allows indicate Nrf2 expression cytolymph and nucleus positive cells); K: Quantification of Nrf2 expression in Immunohistochemistry. Maotai + DEN treatment always enhanced the mRNA and protein expression of Nrf2 at both 3-week and 35-week (<i>p</i><0.05), but ethanol + DEN treatment only transiently increased the mRNA level of Nrf2 at 3-week and the expression level of Nrf2 in this group showed no difference with that in control group at 35-week (<i>p</i>>0.05). *Significantly different in Maotai+ DEN group and ethanol+ DEN group from control group, ^#Significantly different in Maotai+ DEN group from ethanol+ DEN group, <i>p</i><0.05.</p

Serum ALT/AST and MDA in liver homogenate in each group in different periods(mean±SD).

†<p>Significantly different from Control group, ^#Significantly different in Maotai+ DEN group and DEN group from ethanol+ DEN group, <i>P</i><0.05.</p

The expression level of GCLM in mice liver at 3-week and 35-week.

<p>A: Real-time RT-PCR analysis of GCLM gene expression. B–C: Western blot analysis of protein level of GCLM at 3-week and 35-week. lane 1.Control; lane 2.Maotai; lane 3.Ethanol; lane 4.Maotai+DEN; lane 5.Ethanol+DEN; lane 6.DEN; D: Quantification of GCLM level by western blot analysis. Maotai + DEN could upregulate the mRNA level and protein level of GCLM at 3-week(<i>p</i><0.05), but no any increased expression of GCLM could be seen either in Maotai+ DEN group or ethanol+ DEN group at 35-week (<i>p</i>>0.05). *Significantly different in Maotai+ DEN group and ethanol+ DEN group from control group, ^#Significantly different in Maotai+ DEN group from ethanol+ DEN group, <i>p</i><0.05.</p

Representative images of the mice liver specimen at 35-week.

<p>A: Control group; B:Maotai+ DEN group; C: Ethanol+ DEN group. No obvious macroscopic hepatic pathological changes was seen in control group and Maotai+ DEN group. The liver in ethanol+ DEN treated group almost showed swelling, cirrhosis, brown and produced unequal sized gray nodules(arrows indicate the tumor node).</p

Pathological changes of liver in 3 groups mice at 35-week.

<p>A–C: Light microscopy of H&E staining in control group, Maotai+ DEN group and ethanol+ DEN group (400× magnification). Hepatocyte ballooning degeneration, edema and regeneration could be seen in Maotai+ DEN group, The lobular structure of hepatocytes in ethanol+ DEN group was destroyed with fatty degeneration, bridging necrosis, hepatocytes abnormal fission, atypical hyperplasia in ethanol+ DEN group; D–F: Light microscopy of Masson staining in control group, Maotai+ DEN group and ethanol+ DEN group (100×magnification).As the arrow indicate, significant liver fibrosis and pseudolobule formation in ethanol+ DEN group, but only moderate fibrosis formation in Maotai+ DEN group; G–I: Light microscopy of reticulin fiber staining in control group, Maotai+ DEN group and ethanol+ DEN group (400×magnification).Arrows indicate liver tissue structure obvious change and “floating trabeculae” in ethanol+ DEN group, but was normal in Maotai+ DEN group.</p

The expression level of MTs in mice liver at 3-week and 35-week.

<p>A-B: Real-time RT-PCR analysis of MT-1/2 gene expression. Both the Maotai +DEN group and ethanol+ DEN group produced a significant induction of MT-1 in mRNA level at 3-week. But Maotai +DEN group still produced a dramatic induction of MT-1/MT-2 at 35-week, it was about 19 times of ethanol+ DEN group in MT-1 (<i>p</i><0.01). C–D: Western blot analysis of MTs protein level at 3-week and 35-week. lane 1.Control; lane 2.Maotai; lane 3.Ethanol; lane 4.Maotai+DEN; lane 5.Ethanol+DEN; lane 6.DEN; E: Quantification of MTs level by western blot analysis. The specific up-regulation of MT-1/2 was confirmed through western blotting assay. Maotai+ DEN always had a higher MTs protein level. but it was generally lower in ethanol+ DEN group. F-H, I-K: Light microscopy of MTs expression through Immunohistochemistry in control group, Maotai+ DEN group and ethanol+ DEN group at 3-week and 35-week (400× magnification, allows indicate MTs expression cytolymph and nucleus positive cells); L: Quantification of MTs expression in Immunohistochemistry. Immunohistochemical assay also showed similar MTs protein expression with western blotting assay (<i>p</i><0.01). *Significantly different in Maotai+ DEN group and ethanol+ DEN group from control group, ^#Significantly different in Maotai+ DEN group from ethanol+ DEN group, <i>p</i><0.05.</p

GPC3 expression of liver tissue in mice through Immunohistochemistry (400× magnification, allows indicate GPC3 expression cytolymph positive cells).

<p>A: The negative expression of GPC3 expression in control group mice liver; B: The small amount of positive expression of GPC3 was be seen in Maotai+ DEN group mice liver; C:The remarkable positive expression of GPC3 in ethanol+ DEN group mice liver.</p

The evolution of hepatic fibrosis in each group of mices

<p>*Significantly different from ethanol+ DEN group, <i>P</i><0.05.</p