Figure 3
4-MP administration inhibits maternal liver injury and impaired fetal development. The mice were exposed to a liquid ethanol diet for 2 weeks, and simultaneously, 4-MP (10 mg/kg, i.p., n = 8 dams/group) was administered three times per week for 2 weeks. (a) Representative Western blots using an antibody against CYP2E1 in total lysates of the maternal livers of mice at E0 and E15.5 of gestation (top) and densitometry quantification relative to β-actin (bottom). Effects of 4-MP at the E0 (left) and E15.5 (right) period: *p < 0.05 vs. pair-fed mice with saline at E0, **p < 0.01 vs. ethanol-fed mice with saline at E0 or E15.5, respectively, ***p < 0.001 and #p < 0.001 vs. pair-fed mice with saline at E15.5, §p < 0.001 vs. pair-fed mice with 4-MP at E0 and E15.5, respectively, p < 0.001 vs. EtOH-fed mice with saline at E0 (n = 8 dams/group). (b) IHC analysis of CYP2E1 in the livers of E15.5 mice (scale bar, 50 μm) (left) and staining hepatocytes were scored (right). (c) Western blotting for 4-HNE (top panel) and measuring malondialdehyde (MDA) levels (bottom panel) were performed in the livers of E15.5 mice. (d) H&E staining (scale bar, 50 μm) of liver sections after exposure to ethanol and/or 4-MP was performed, and the steatosis area (%) was measured. (e) The inhibitory effects of 4-MP on serum levels of AST, ALT, hepatic triglyceride, and cholesterol at E15.5. (f) Restoring of average embryonic number by 4-MP (n = 6 to 9 pups/mouse). The numbers (blue letters) at the bottom of the bar graph represent the average embryo number. (b–f) *p < 0.05 vs. pair-fed mice with saline, **p < 0.01 vs. ethanol-fed mice with saline. (g) 4-MP reduces the number of offspring with fused toes (Ft) increased in ethanol-fed mice. Differences between categorical variables were analyzed by Fisher’s exact test (p < 0.05; n = 8 dams/group; the number of offspring= 68 normal healthy and 0 Ft for 4-MP-exposed pair-fed mice and 59 normal healthy and 1 Ft for ethanol-fed mice). The numbers (blue letters) at the top of the bar graph represent the rate of fused toes in offspring obtained in each group. (h) 4-MP ameliorates the alterations in postnatal body weight at P0 and P21. Values of postnatal body weight are the mean of pups (n = 6 to 9 pups/mouse; n = 8 dams/group). Effects of 4-MP on macrosomia at P0 (left): *p < 0.05 vs. pair-fed mice with saline, **p < 0.01 vs. ethanol-fed mice with saline; effects of 4-MP on growth retardation at P21 (right): &p < 0.001 vs. pair-fed mice with saline, #p < 0.001 vs. ethanol-fed mice with saline. Data are expressed as the mean ± SEM. Data in a–f and h were analyzed by two-way ANOVA with Tukey’s post hoc multiple comparison test. Data in g (the number and rate) were analyzed by Fisher’s exact test and unpaired t-test. P, Pair-fed; E, Ethanol-fed.
