The Effect And Mechanism Of Chronic Intermittent Hypoxia On Hepatic Lipid Accumulation In Alcoholic Liver Disease In Mice

Background and objectiveAlcoholic liver disease(ALD)is a collective term for a range of liver diseases caused by prolonged excessive alcohol consumption.It is one of the diseases with high morbidity and mortality worldwide.Unfortunately,there is still a lack of an effective therapy for ALD.Alcoholic steatosis is a reversible and self-limiting early stage of ALD and is considered as a principal target for intervention in ALD.A deeper understanding of its pathogenesis is important for alleviating the pathological process of ALD and developing more effective treatments.Obstructive sleep apnea(OSA)is a respiratory disorder typically characterized by chronic intermittent hypoxia(CIH)during sleep.OSA has been shown to promote the development of Nonalcoholic fatty liver disease(NAFLD).Although clinical data suggest that chronic alcoholics are often characterized by snoring(European respiratory journal.2000),the effect of CIH on hepatic steatosis in ALD and its underlying mechanisms remain to be elucidated.This study first replicates the Gao-binge(Chronic-plus-single-binge ethanol feeding)mouse model of ALD commonly used in the field of ALD.The CIH exposure methods was conducted by exposing mice to chambers with automated,programcontrolled O2 concentration exchanging systems.We thus investigated the effects of CIH on hepatic steatosis and liver injury in ALD mice,as well as its underlying mechanisms.Methods and results1.CIH alleviates hepatic lipid accumulation and alcoholic liver injury in ALD mice.Male C57BL/6J mice(aged 8 weeks,weighing 20-25 g)were randomly divided into four groups:the control group(Ctrl),Gao-binge ALD group(EtOH),CIH intervention ALD group(EtOH+CIH),and the CIH intervention Ctrl group(CIH alone).The results showed that the survival rate of mice in the EtOH+CIH group was significantly higher compared to that in the EtOH group.CIH intervention had no significant effect on diet intake or body weight of ALD mice,but significantly inhibited the increased liver weight and liver weight to body weight ratio,as well as the increased serum ALT and AST levels in the EtOH group.Hepatic triglyceride levels and H&E and Oil Red O staining indicated that lipid accumulation in the liver was lower in the EtOH+CIH group than those in the EtOH group.In addition,both lipid peroxidation and liver fibrosis were lower compared to those in the EtOH group.CIH did not have a significant effect on these indicators in mice fed control liquid diet.2.CIH may reduce hepatic lipid accumulation by inhibiting lipid uptake and enhancing lipophagy in hepatocyte.The expressions of genes and proteins related to lipid synthesis were not significantly different in the liver tissues between the EtOH group and the EtOH+CIH group.The expression of PPAR-α,a transcription factor regulating fatty acid β-oxidation,was reduced in the liver tissues from the EtOH group compared to the Ctrl group,no significant change after CIH intervention.The expression levels of Acox1,Cpt1α and Ehhadh,key genes in fatty acid β-oxidation,were not significantly different between the EtOH group and the EtOH+CIH group.The expression levels of Cd36 and Slc27α1,key genes in lipid uptake,were significantly lower in the liver tissues from the EtOH+CIH group than those from the EtOH group.In addition,the expression level of Glycine N-Methy ltransferase(GNMT),an important regulator of lipophagy,was significantly higher in liver tissues from the EtOH+CIH group compared to that from the EtOH group.3.CIH may alleviate liver injury by downregulating hepatic CYP2E1 expression.The expression levels of ethanol dehydrogenase,acetaldehyde dehydrogenase and peroxidase proteins involved in the oxidative metabolism of alcohol were not significantly different in the liver tissues of all groups of mice.Compared with the Ctrl group,cytochrome P450 2E1(Cytochrome P450 family 2 subfamily E member 1,CYP2E1)was significantly up-regulated in the liver tissues of mice in the EtOH group,but CIH intervention significantly down-regulated the protein level of CYP2E1.4.HIF-2α(but not HIF-1α)mediates the inhibiting effect of CIH on hepatic lipid accumulation.HIF-1α protein expressions were not significantly changed in the cytoplasm or nucleus of the liver from the EtOH group and the EtOH+CIH group,HIF-2α protein expression levels were significantly increased in the cytoplasm and nucleus of mouse hepatocytes in the EtOH group,CIH intervention significantly reduced nuclear HIF-2α protein levels as well as its downstream target genes in the EtOH group.Similar to CIH intervention,the HIF-2α inhibitor PT2385(20 mg/kg)significantly reduced hepatic lipid accumulation and liver injury in the EtOH group.PT2385 also reversed the up-regulated CYP2E1 protein level in the liver tissues from the EtOH group.Conclusions and significanceThe present study suggests that,contrary to the results reported in the literature on CIH aggravating lipid accumulation in NAFLD,CIH may reduce hepatic lipid accumulation and alleviate liver injury in ALD mice by inhibiting the protein expression and transcriptional activities of HIF-2α:On the one hand,CIH may reduce fatty acid uptake by hepatocytes and promote lipophagy by down-regulating HIF-2αprotein expression and transcriptional activities.On the other hand,CIH can inhibit CYP2E1 expression and reduce ROS accumulation through down-regulation of HIF2α transcription,thereby alleviating liver injury in ALD mice.The above results provide novel experimental evidence for understanding the effect of snoring on lipid metabolism and liver injury in alcoholics.Furthermore,the present study provides new strategies for the clinical use of HIF-2α inhibitors in the prevention and treatment of ALD.