| ObjectiveAlcohol is a common toxicant of liver. Excessively alcohol consumption can lead to alcoholic liver disease (ALD). ALD is a gradually progressive liver disease, which includes a cluster of pathological phenotypes from alcoholic fatty liver (AFL) to alcoholic hepatitis, fibrosis, and finally cirrhosis. As the earlier manifestation of ALD, AFL was reversible and was usually considered to be the optimal stage for the prevention and treatment of ALD. Cytochrome P450 2E1 (CYP2E1) is a member of the cytochrome P450 superfamily and also the main member of microsomal ethanol-metabolizing system (MEOS). Activated CYP2E1 can lead to reactive oxygen species (ROS) production. CYP2E1 has been demonstrated to play crucial roles in chronic ethanol-induced fatty liver. Mechnisms studies show that ROS derived from activated CYP2E1 may lead to AFL possibly by influencing peroxisome proliferator-activated receptor-a (PPAR-a), etc.The phenotypes of ethanol-induced liver injury could be influenced by the dose and types of drinking. Binge drinking referes to consumption with large amount of alcohol in a short period of time. Binge drinking can lead to liver injury such as steatosis, hepatitis and liver cell necrosis. Epidemiological studies suggest that the rate of binge drinking in population is increasing worldwide. However, the studies about AFL are most concentrated on chronic ethanol-induced liver injury, while less attention has been paid to binge drinking-induced liver injury. Whether CYP2E1 also plays important roles in acute ethanol-induced fatty liver? whether CYP2E1 inhibitors could attenuated acute ethanol-induced liver injury? These questions are need to be studied.In this study, we established an acute ethanol-induced liver injury mice model, and investigated the effects of chlormethiazole (CMZ, a specific inhibitor of CYP2E1) on acute ethanol-induced fatty liver. We also explore the molecular mechanisms by investigating oxidative stress parameters, mitochondrial injury, and several pathways involved in fatty acid metabolism.MethodsSpecific pathogen Free (SPF) male Kun-Ming mice (18-22 g) were were randomly divided into 3 groups after acclimation for 3 days (n= 10), i.e. control group, ethanol group, and CMZ/ethanol group. The mice in CMZ/ethanol and ethanol groups were treated with single dose of CMZ (50 mg/kg body weight) by intraperitoneal injection and equal volume of sterile saline, respectively, and then exposed to ethanol (5 g/kg body weight,25%, w/v) by gavage in 12 h interval for a total of 3 doses. Mice in control group received equal volume of saline and isocaloric/isovolumetric maltose-dextrin solution. The blood samples were collected by at 4 h after the last dosing of ethanol. The animals were sacrificed, and livers were quickly dissected into several parts for histopathological examination, biochemical assay, western blotting and qRT-PCR, respectively.H&E staining, Sudan III staining, oil red O staining and transmission electron microscopy (TEM) examination were used for histopathological examination. The levels of serum alanine transaminase (ALT), aspartate transaminase (AST) and triglyceride (TG), hepatic malondialdehyde (MDA), hepatic glutathione (GSH) and serum ethanol were measured using commercial assay kits. The serum adiponectin and tumor necrosis factor-a (TNF-a) levels were measured using ELISA kits. The mRNA and protein levels of some factors involved in fatty acid metabolism such as PPAR-a, sterol regulatory element-binding protein-lc (SREBP-lc), AMP-activated protein kinase (AMPK) were measured by western blotting and qRT-PCR, respectively.Results1. CMZ pretreatment attenuated acute ethanol-induced fatty liver jnjury. The levels of serum transaminase activities and hepatic TG levels were significantly increased in ethanol group mice, compared with the control group mice. Compared with those of the ethanol group mice, serum and hepatic TG levels in CMZ/ethanol group mice were decreased by 31.86% and 52%(P<0.05), respectively. The result of histopathological examination showed that excessive fat droplets accumulated in the liver of ethanol group mice, which was greatly blocked by CMZ pretreatment.2. CMZ pretreatment suppressed acute ethanol-induced CYP2E1 activation and the oxidative stress in mice liver. Compared with the control group mice, the protein levels of CYP2E1 and hepatic MDA and 4-HNE levels were all significantly increased in ethanol group mice liver (P<0.05), while the GSH level was significantly decreased (P<0.05). Compared with those of the ethanol group mice, CYP2E1 protein levels and hepatic MDA in CMZ/ethanol group mice were decreased by 28.94% and 37.46%, respectively, while the GSH level was increased by 73.87%(P<0.05) Immunohistochemical staining showed CMZ significantly suppressed the expression of 4-HNE in mice liver.3. CMZ suppressed acute ethanol-induced mitochondria injury. TEM examination showed that acute ethanol induced significant accumulation of fat droplets and also caused significant mitochondrial injury manifesited as the fragmention, malformation and dissolve of mitochondria.These changes were greatly blocked by CMZ pretreatment.4. Effects of CMZ and acute ethanol challenge on PPAR-α pathway. Compared with those of the control group mice, the mRNA levels of PPAR-a, acyl-CoA oxidase (ACOX), and liver fatty acid-binding protein (LFABP) in ethanol group mice liver were decreased by 16.35%,29.39%, and 25.97%(P<0.05), respectively. CMZ restored the mRNA levels of PPAR-a and ACOX. Unexpectedly, the protein levels of PPAR-a, ACOX, and LFABP in the liver of ethanol group mice were all significantly increased compared with those of the control group mice(P<0.05), the protein levels of PPAR-a and LFABP increased were suppressed by CMZ pretreatment (P<0.05).5. Effects of CMZ and acute ethanol challenge on the protein levels of AMPK and acyl-CoA carboxylase (ACC), p-ACC and p-AMPK:No significant changes of the AMPK, p-AMPK, ACC, and p-ACC protein levels in the liver of ethanol group mice compared with those of the control group mice.The protein levels of p-ACC and p-AMPK in CMZ/ethanol group mice liver was slightly decreased compared with that of the ethanol group mice (P<0.05).6. Effects of CMZ and acute ethanol on the SREBP-lc and diacylglycerol acyltransferase 2 (DGAT2). The protein levels of SREBP-lc and and fatty acid synthase (FAS) and DGAT2, the mRNA level of SREBP-lc and FAS were not significantly affected by acute ethanol challenge or CMZ treatment.7. Effects of CMZ and acute ethanol challenge on the activation of autophagy in hepatocyte. Compared with those of the conttol group mice, the ratio of LC3Ⅱ/LC3I was significantly increased in ethanol group mice liver (P<0.05), while the protein level of p62 was significantly decreased (P<0.05). Compared with those of the ethanol group mice, the p62 protein level in CMZ/ethanol group mice was further decreased (P<0.05), while the ratio of LC3Ⅱ/LC3I in CMZ/ethanol group mice was also slightly decreased (P<0.05).8. Effects of CMZ and acute ethanol on the levels of serum adiponectin, TNF-α and ethanol. CMZ significant suppressed acute ethanol-induced decrease of the serum adiponectin level, but did not signifiantly affect the serum ethanol level. CMZ and acute ethanol challenge had no effects on the serum TNF-a levels.Conclusion1. CMZ could attenuate fat accumulation in mice liver induced by acute ethanol.2. CMZ might suppress acute ethanol-induced oxidative stress by inhibiting CYP2E1 activity.3. CMZ might suppress acute ethanol-induced fatty liver by suppressing CYP2E1, activating autophagy, and enhancing adiponection secretion, etc. |
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