CMZ reversed chronic ethanol-induced disturbance of PPAR-α possibly by suppressing oxidative stress and PGC-1α acetylation, and activating the MAPK and GSK3ß pathway.

BACKGROUND: Cytochrome P4502E1 (CYP2E1) has been suggested to play critical roles in the pathogenesis of alcoholic fatty liver (AFL), but the underlying mechanisms remains unclear. The current study was designed to evaluate whether CYP2E1 suppression by chlormethiazole (CMZ) could suppress AFL in mice, and to explore the underlying mechanisms. METHODS: Mice were treated with or without CMZ (50 mg/kg bw, i.p.) and subjected to liquid diet with or without ethanol (5%, w/v) for 4 weeks. Biochemical parameters were measured using commercial kits. The protein and mRNA levels were detected by western blot and qPCR, respectively. Histopathology and immunohistochemical assay were performed with routine methods. RESULTS: CYP2E1 inhibition by CMZ completely blocked AFL in mice, shown as the decline of the hepatic and serum triglyceride levels, and the fewer fat droplets in the liver sections. Chronic ethanol exposure led to significant decrease of the mRNA and protein levels of peroxisome proliferator-activated receptor α (PPAR-α), which was blocked by CMZ co-treatment. CMZ co-treatment suppressed ethanol-induced oxidative stress, overproduction of tumor necrosis α (TNF-α), and decrease of protein levels of the PPAR-α co-activators including p300 and deacetylated PGC1-α. Furthermore, CMZ co-treatment led to the activation of AMP-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK), and PI3K/Akt/GSK3ß pathway. However, chronic ethanol-induced decline of acyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) protein levels was partially restored by CMZ, while the activation of autophagy appeared to be suppressed by CMZ. CONCLUSION: These results suggested that CMZ suppressed chronic ethanol-induced oxidative stress, TNF-α overproduction, decline of p300 protein level and deacetylation of PGC1-α, and activated AMPK, MAPK, and PI3K/Akt/GSK3ß pathway, which might contribute to the activation of PPAR-α and account for the protection of CMZ against AFL.

PI3K/Akt pathway activation was involved in acute ethanol-induced fatty liver in mice.

Accumulating evidences support the important roles of sterol regulatory element-binding protein-1 (SREBP-1) activation in ethanol-induced fatty liver, but the underlying mechanisms for its activation are not fully understood. Recent studies have demonstrated that phosphatidylinositol 3 kinase (PI3K)/Akt pathway activation could enhance SREBP-1 activity. The current study was designed to investigate the potential roles of PI3K/Akt pathway in acute ethanol-induced fatty liver in mice. In the first experiment, mice were treated with ethanol (2.5 or 5 g/kg bw) or isocaloric/isovolumetric maltose-dextrin solution, and sacrificed at several time points after ethanol exposure. As expected, ethanol dose-dependently increased the hepatic triglyceride (TG) levels and the protein levels of the mature form of SREBP-1 (n-SREBP-1). The phosphorylation of Akt and glycogen synthase kinase-3ß (GSK-3ß) was significantly increased in mice treated with ethanol (5 g/kg bw), while the protein levels of PI3K-p85 were significantly reduced. To confirm the roles of PI3K/Akt pathway, mice were then pretreated with wortmannin (0.7 or 1.4 mg/kg bw), a specific PI3K/Akt pathway inhibitor, before exposure to ethanol. Interestingly, a dual effect of wortmannin was observed. Low dose of wortmannin significantly reduced the hepatic TG levels, while high dose of wortmannin aggravated ethanol-induced fatty liver. The ratio of LC3II/LC3I of wortmannin (1.4 mg/kg bw) group mice was significantly increased, while the p62 protein level was significantly decreased compared to those of ethanol group, which indicated that wortmannin (1.4 mg/kg bw) might suppress the lipid degradation by autophagy. These results supported the hypothesis that PI3K/Akt activation might be involved in acute ethanol-induced fatty liver, and PI3K/Akt inhibitors might have therapeutic potential for the treatment of ethanol-induced fatty liver.

Garlic oil alleviated ethanol-induced fat accumulation via modulation of SREBP-1, PPAR-α, and CYP2E1.

Garlic oil (GO) has been shown to partially attenuate ethanol-induced fatty liver, but the underlying mechanisms remain unclear. The current study was designed to evaluate the protective effects of GO against ethanol-induced steatosis in vitro and in vivo, and to explore potential mechanisms by investigating the sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferators-activated receptor-α (PPAR-α), cytochrome P4502E1 (CYP2E1), and etc. In the in vitro study, human normal cell LO2 was exposed to 100 mM ethanol in the presence or absence of GO for 24 h. We found that ethanol increased the protein levels of n-SREBP-1c and CYP2E1, but decreased the protein levels of PPAR-α, which was significantly attenuated by GO co-treatment. In the in vivo study, male Kun-Ming mice were pretreated with single dose of GO (50-200 mg/kg body weight) at 2 h before ethanol (4.8 g/kg body weight) exposure. The changes of n-SREBP-1c, PPAR-α and CYP2E1 were paralleled well to those of in vitro study. Furthermore, GO significantly reduced the protein levels of fatty acid synthase (FAS), and suppressed ethanol-induced hepatic mitochondrial dysfunction. These results suggested that GO had the potential to ameliorate alcoholic steatosis which might be related to its modulation on SREBP-1c, PPAR-α, and CYP2E1.

[Protective effect of garlic oil given at different time against acute liver injury induced by CCl4].

OBJECTIVE: To observe and compare the protective effect of garlic oil against carbon tetrachloride (CCL)-induced acute liver injury. METHODS: The experiments include 4 preventive groups and 2 therapeutic groups. In every preventive and therapeutic group, the mice were randomized into 6 groups with 15 each, including one negative control group, one solvent control group, one CCl4 model group and 3 garlic oil groups (25, 50, and 100 mg/kg body weight). Before given a single gavage of CCl4 (80 mg/kg), the mice were pretreated with garlic oil by gavage in preventive group 1 (30 days, once daily), preventive group 2 (5 days, once daily), preventive group 3 (ahead of 2 h, once), preventive group 4 (immediately, once) or the vehicle (corn oil, 10 ml/kg) in solvent control group. In therapeutic groups, the mice were gavaged garlic oil 2 h (once, in therapeutic 1) or for 5 days (once daily, in therapeutic 2) after administration CCl. After 24 h of the last administration, blood was collected and centrifuged at 2500 r/min at 4 degrees C for 10 min, and serum was removed to measure ALT and AST activities. The liver was dissected, weighed to calculate the liver coefficient (relative liver weight). At the same time, the liver samples were studied by histological examinations. RESULTS: Compared with negative group, the liver coefficient and the activities of ALT and AST in serum of model group were increased remarkably (P < 0.01). Compared with CCl model group, the liver coefficient and the activities of ALT and AST in serum were decreased significantly (P < 0.01) by garlic oil dose-dependently in each preventive group. Simultaneously, histological assessment showed that garlic oil effectively alleviated hepatocyte injuries induced by CCl4. Comparing the preventive effects of garlic oil in every group, it was better in preventive group 3 than others. However, all indexes and histological examinations in therapeutic group 1 did not show the difference with those of CCl4 model group. In therapeutic group 2, all indexes recovered after 5 d of CCl4 administration. CONCLUSIONS: Garlic oil can prevent acute liver injury induced by CCl4 and the effect is better in ahead of 2 h group than others.

The modulatory effects of garlic oil on hepatic cytochrome P450s in mice.

In order to probe into the effects of garlic oil (GO) on the hepatic CYP2E1, CYP1A2 and CYP3A, male Kun-Ming mice were treated with GO (100 mg/kg body weight) or corn oil for 1 day or consecutive 60 days, respectively, and then the protein expressions and the activities of the enzymes were examined. GO did not alter the physical activities of mice and did not induce lesion to the liver. However, it dramatically inhibited the activities and protein levels of hepatic CYP2E1 and 1A2, but not CYP3A. In addition, we noticed that the inhibition of CYP2E1 and 1A2 by GO was more potent in group of 1 day treatment than those in group of 60 days treatment. Compared with the respective control value, the protein levels of CYP2E1 were decreased by 87.40% (p < .01) and 62.26% (p < .01) by 1 day and 60 days of GO treatment, respectively, while the CYP1A2 protein levels were decreased by 70.76% (p < .01) and 41.49% (p < .01), respectively. These data indicated that the mice could adapt to the prolonged treatment, which might be one reasonable explanation for the conflicting data in the literature. The CYP2E1 and 1A2 suppression might contribute to its hepatoprotection, and data about CYP3A indicated that GO was unlikely to alter the metabolism of the concomitantly used drugs.