The Role Of Ceramide In The Pathogenesis Of Alcoholic Liver Disease

Objective:Alcoholic liver disease(ALD) is a chronic disease with a histological spectrum ranging from hepatic steatosis, steatohepatitis, fibrosis, and ultimately to cirrhosis. Due to the incomplete understanding of its pathogenesis, there is little progress has been made in ALD treatment. Growing evidence supports that ceramide accumulation plays a key role in the pathogenesis and progression of ALD. Adenosine monophosphate activated protein kinase(AMPK) is central to the pathogenesis of alcohol-induced hepatic steatosis. We concluded that ethanol activated acid sphingomyelinase(ASMase), thus hydrolysing sphingomyelin to promote ceramide accumulation, and then activating protein phosphatase 2A(PP2A) activity, which may be an important mechanism in the inhibitory effect of ethanol on AMPK phosphorylation. It is known that AMPK can regulate lipid metabolism including the fatty acid oxidation and its synthesis, when AMPK phosphorylation is blocked, the fatty acid synthesis increased and its oxidation decreased, which contributes the progression of steatosis. We attempt to discuss the function of ceramide, acid sphingomyelinase and protein phosphatase 2A in the pathogenesis of alcoholic liver disease.Methods:50 male Wistar rats weighting 180-220 g were fed for 7 days normally, and then 10 of them were randomized into the normal control group. The other part of rats were gavaged with alcohol of increasing concentration(concentration 30%-60%, alcohol 5-9g/Kg/d), developing to the rat model of ALD. At the end of 4th, 8th, 12 th and 16 th week, 1 0, 9, 8 and 8 rats were sacrificed separately. Portions of rat livers were fixed in neutral-buffered formalin overnight, and then were embedded in paraffin. The rest was snap-frozen in liquid nitrogen immediately, then stored at-80°C fridge for subsequent protein, lipid and RNA studies.RT-qPCR was applied to determine the mRNA expression of ASMase and PP2 A. The protein levels of ASMase and PP2 A were determined by Western-blot. The content of ceramide in liver tissue was quantificated by Liquid chromatography tandem mass spectrometry.Results:1 Histopathological changes of hepatic tissue: In the 4th week, we observed that mild steatosis developed in pericentral to midzonal regions of hepatic lobules. With the progression of ALD, the steatosis and fibroplasias became more serious. In the 16 th week model, it showed that diffuse microvesicular adipose degeneratio and fibrosis septa in the portal area.Nonsteatosis was observed in the hepatic tissue of normal control group.2 The expression of ASMase and PP2 A protein by immune-histochemistry staining: In the consumption of ethanol groups(4W-16W), the expression of ASMase and PP2 A in hepatic tissue of rats were up-regulated, compared with the normal control group.3 The mRNA expression of ASMase and PP2 A by real-time PCR: The mRNA expression of ASMase and PP2 A in hepatic tissue of rats were up-regulated in the consumption of ethanol groups(4W-16W), compared with the normal control group(P<0.05).4 The expression of ASMase and PP2 A protein by western blot: The levels of ASMase and PP2 A in the liver of rats were elevated in the consumption of ethanol groups(4W-16W), compared with those of the normal control group(P<0.01).5 The content of ceramide in hepatic tissue was quantificated by Liquid chromatography tandem mass spectrometry: With the progression of hepatic steatosis, the content of ceramide was increased in the consumption of ethanol groups(4W-16W), compared with the normal control group(P<0.05).Conclusions:1 The model of rats with ALD can be established by gavaged with alcohol of increasing concentration. The pathological features, such as steatosis, inflammation and fibrosis in hepatic tissue of rats, can basically reflect the clinical pathological changes of alcoholic liver disease.2 The expression of ceramide, ASMase and PP2 A were all proved to be increased with the progression of ALD, implying that they may play a role in the pathogenesis of ALD.