Table 1.
Gene-specific primers used in quantitative real-time PCR.
Figure 1.
CMZ prevented ethanol-induced activation of CYP2E1 in mice liver.
(a) Protein levels of CYP2E1 were detected by western blot; (b) CYP2E1 activity; (c) Immunohistochemical staining of CYP2E1. Data were presented as mean ± SD. **P<0.01, compared with control group; ##P<0.01, compared with ethanol group.
Figure 2.
CYP2E1 inhibition by CMZ completely blocked chronic ethanol-induced lipid accumulation in mice liver.
Mice were treated with liquid diet with or without ethanol in the presence/absence of CMZ for 4 weeks. (a) H&E staining; (b) Sudan III staining; (c) Oil red O staining; (d) Ultrastructural examination.
Table 2.
Effects of CMZ and ethanol on the levels of serum ALT, AST, TG, ethanol, TNF-α, adiponectin, and hepatic TG, MDA, GSH.
Figure 3.
CMZ prevented the decrease of protein levels of PPAR-α, p300 and Sirt-1, and inhibited the increased acetylation of PGC-1α induced by ethanol.
Total protein samples were prepared using RIPA buffer, and protein levels of PPAR-α, RXR-α, p300, PGC-1α, and Sirt-1 were detected by western blot. The acetylation of PGC-1α was determined by immunoprecipitation analysis. The mRNA levels of PPAR-α, RXR-α, and PGC-1α were measured using qPCR. (a) Protein levels of PPAR-α, RXR-α, p300, and PGC-1α; (b) Protein levels of Sirt-1; (c) Acetylation of PGC-1α; (d) mRNA levels of PPAR-α, RXR-α, and PGC-1α. Data were presented as mean ± SD from at least 3 independent experiments, and expressed as the percentage of the control. *P<0.05, **P<0.01, compared with control group; #P<0.05, ##P<0.01, compared with ethanol group.
Figure 4.
The phosphorylation of AMPK was increased in the liver of CMZ/ethanol group mice.
(a) Representative western blot bands for phosphor-AMPK and AMPK; (b) Quantitative data analyses. Data were presented as mean ± SD from at least 3 independent experiments, and expressed as the percentage of the control. *P<0.05, **P<0.01, compared with control group; #P<0.05, compared with ethanol group.
Figure 5.
CMZ co-treatment enhanced the phosphorylation of Erk1/2 and p38MAPK.
(a) Representative western blot bands for phospho-Erk1/2, Erk1/2, phospho- JNK, JNK, phospho-p38MAPK, and p38MAPK; (b) Quantitative data analyses. Data were presented as mean ± SD from at least 3 independent experiments, and expressed as the percentage of the control. **P<0.01, compared with control group; #P<0.05, ##P<0.01, compared with ethanol group.
Figure 6.
CMZ co-treatment increased protein levels of GSK-3β and phospho-GSK3β in mice liver.
Total protein samples were prepared using RIPA buffer, and GSK-3β and phospho-GSK-3β protein levels were detected by western blot. (a) Representative western blot bands; (b) Quantitative data analyses. Data were presented as mean ± SD from at least 3 independent experiments, and expressed as the percentage of the control. *P<0.05, **P<0.01, compared with control group; #P<0.05, ##P<0.01, compared with ethanol group.
Figure 7.
CMZ co-treatment led to the increased phosphoryaliton and activation of Akt.
Total protein samples were prepared using RIPA buffer, and protein levels of phospho-Aktser473, phospho-Aktthr308, and the total Akt were detected by western blot. (a) Representative western blot band; (b) Quantitative data analyses. Data were presented as mean ± SD from at least 3 independent experiments, and expressed as the percentage of the control. **P<0.01, compared with control group; #P<0.05, ##P<0.01, compared with ethanol group.
Figure 8.
CMZ co-treatment significantly increased the protein levels of p50, the regulatory subunit of PI3K.
Total protein samples were prepared by using RIPA buffer, and protein levels of the regulatory subunits of PI3K (p85, p55, p50) and the catalytic subunit (p110) were detected by western blot. (a) Representative western blot bands; (b) Quantitative data analyses. Data were presented as mean ± SD from at least 3 independent experiments, and expressed as the percentage of the control. **P<0.01, compared with control group; ##P<0.01, compared with ethanol group.
Figure 9.
Effects of ethanol and CMZ on the mRNA and protein levels of n-SREBP-1, phospho-ACCser79, ACC, FAS, and DGAT2.
(a) Representative western blot bands for n-SREBP-1, phospho-ACCser79, ACC, FAS, and DGAT2. (b) Quantitative data analyses. (c) The mRNA levels of SREBP-1, ACC, and FAS. Data were presented as mean ± SD from at least 3 independent experiments, and expressed as the percentage of the control. *P<0.05, **P<0.01, compared with control group; #P<0.05, ##P<0.01, compared with ethanol group.
Figure 10.
Effects of ethanol and CMZ on the protein levels of LC3 and P62.
Total protein samples were prepared using RIPA buffer, and protein levels two biomarkers of autophagy, i.e. LC3 and P62, were detected by western blot. Data were presented as mean ± SD from at least 3 independent experiments, and expressed as the percentage of the control. **P<0.01, compared with control group; ##P<0.01, compared with ethanol group.
Figure 11.
A possible scheme for the protective effects of CMZ against chronic ethanol-induced fatty liver.
Ethanol-induced CYP2E1 activation can lead to the suppression of PPAR-α, which may be related with the decline of the p300 protein level, the increase of PGC-1α acetylation, and the disturbance of several protein kinases including AMPK, MAPK, and GSK3β. CYP2E1 activation can also result in oxidative stress, which will lead to the overproduction of TNF-α by activating kupffer cells. In contrast, the protective effects of CMZ against AFL might not be associated with the SREBP-1 mediated lipogenesis and autophagy pathway, which are needed to be confirmed in future studies.